A Comprehansive Study on Quality And Safety of Raw Shell Eggs From Commercial Layer Farms and Different Markets, CB Madhavprasad

Tags: Salmonella Typhimurium, shell eggs, Salmonella Enteritidis, Salmonella, Salmonella spp, Campylobacter spp, Paraffin oil, PHMG, shell egg, Maharashtra Animal and Fishery Sciences University, Veterinary Public Health, Bombay Veterinary College, Advisory Committee, Associate Dean, Dr. A. Samad Associate Dean, commercial layer, quality, safety, BVC, Dept. of Medicine and Pharmacology & Toxicology, MADHAVAPRASAD C. B. Enrolment, Mumbai, Nil Nil Nil Nil NA NA NA NA NA NA NA NA, Salmonella Typhimurium suspension, penetration rate, Isolated Salmonella, Salmonella Typhi, Salmonella Heidelberg, cracked egg, Total Viable Count, Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA
Content: A COMPREHENSIVE STUDY ON QUALITY AND SAFETY OF RAW SHELL EGGS FROM COMMERCIAL LAYER FARMS AND DIFFERENT MARKETS. THESIS Submitted in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY IN VETERINARY PUBLIC HEALTH BY MADHAVAPRASAD C. B. Enrolment No : V/06/288 Bombay Veterinary College, Mumbai MAHARASHTRA ANIMAL AND FISHERY SCIENCES UNIVERSITY, NAGPUR- 440 006. (INDIA) 2009
DECLARATION OF STUDENT I hereby declare that the experimental Research work and interpretation of the thesis entitled "A comprehensive study on quality and safety of raw shell eggs from commercial layer farms and different markets." or part thereof has not been submitted for any other degree or diploma of any University, nor the data have been derived from any thesis/publication of any University or scientific organization. The sources of materials used and all assistance received during the course of investigation have been duly acknowledged.
Date : Counter signed by
Signature (Madhavaprasad C. B.) Enrolment No:V/06/288
Chairman, Advisory Committee with date
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Declaration of Advisory Committee Shri Madhavaprasad C.B. has satisfactorily prosecuted his course of research for a period of not less than two semesters (for Ph.D.) and that the thesis entitled, " A comprehensive study on quality and safety of raw shell eggs from commercial layer farms and different markets" submitted by him is the result of research work is sufficient to warrant its presentation to the examination in the subject of Veterinary Public Health for the award of Doctor of Philosophy degree by the Maharashtra Animal and Fishery Sciences University, Nagpur. We also certify that the thesis or part there of has not been previously submitted by him for a degree of any other University.
Place : Mumbai.
Signature
Date :
(Dr. A. M. Paturkar) Advisor/Guide Professor and Head Dept. of Veterinary Public Health Advisory Committee
Name and Designation
Signature
1) Dr. A. Samad Associate Dean, BVC, Mumbai
2) Dr. A. S. Ranade H.O.D. Poultry Science, LPM, AGB etc BVC, Mumbai
3) Dr. A. S. Banalikar H.O.D. Microbiology, VPH, Pathology etc BVC, Mumbai
4) Dr. D.V. Keskar Professor and Head, Dept. of Medicine and Pharmacology & Toxicology, BVC, Mumbai
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CERTIFICATE This is to certify that the thesis entitled, "A comprehensive study on quality and safety of raw shell eggs from commercial layer farms and different markets" submitted by Shri Madhavaprasad C.B. to the Maharashtra Animal and Fishery Sciences University in partial fulfillment of the requirement for the degree of Doctor of Philosophy has been approved by the Student's Advisory Committee after examination in collaboration with the External Examiner.
Name & Signature of External Examiner
Signature with Seal Head of Department
Name & Signature Advisor/Guide
Advisory Committee Name and Designation 1) Dr. A. Samad Associate Dean, BVC, Mumbai 2) Dr. A. S. Ranade H.O.D., of Poultry Science, LPM, AGB etc, BVC, Mumbai 3) Dr. A. S. Banalikar H. O. D. of Microbiology, VPH, Pathology etc BVC, Mumbai. 4) Dr. D.V. Keskar Professor and Head, Dept. of Medicine and Pharmacology & Toxicology, BVC, Mumbai
Signature
Signature with seal Dean/Associate Dean (Name)
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ACKNOWLEDGEMENTS Living in an ecosystem of an educational institute is a pleasure in itself. It is an ecosystem, which is dynamic, vibrant, full of enthusiasm and activity, turbulent at times, deep and tranquil most of the times and leaves a lasting impact on the minds and lives of students. I consider myself fortunate to be the part of an old, beautiful, dynamic ecosystem of Bombay Veterinary College as a student to pursue my Ph. D programme in one of the finest Veterinary Public Health laboratories in India. I remain grateful that such an opportunity has come my way. Dr. A. M. Paturkar, Professor and University Head, Veterinary Public Health, my teacher and guide is a man of supreme abilities and confidence. To work under him as a student is a matter of great pleasure and marked by lively learning experiences. I have been greatly benefited by his insight and approach into the subject of veterinary public health, quick decisions, meticulous planning, affectionate guidance, adherence to the plan and timeline of research, invaluable support and care through out my study. I respect him as a teacher, scientist and above all a nice human being. And for all that, I just say, thank you sir. I am grateful to Dr. A. Samad, Associate Dean, Bombay Veterinary College, for his keen interest, critical evaluation, valuable support, constant encouragement and passion for research. An extraordinary teacher of epidemiology and I always love to listen to his lectures. I sincerely thank, Dr. A.S. Banalikar, H.O.D, Microbiology, Veterinary Public Health, Pathology and Parasitology for his nobility, valuable time, critical evaluation and scientific approach. It is a pleasure to interact with him. My sincere thanks to Dr. A. S. Ranade, H.O.D, Poultry Science, LPM, AGB and Extension for his valuable time, critical suggestions, support, encouragement and co-operation throughout my research work. I am thankful to Dr. D. V .Keskar, Professor and Head, Dept. of Medicine for his ever willing help, friendly attitude, good guidance and constant encouragement. I am grateful to Dr. A. T. Sherikar, Ex.Vice-chancellor of MAFSU, for his affectionate enquiries, guidance and support throughout my study period. v
I extend my sincere thanks to Dr. M.A. Baig, Professor, LPM for his help and cooperation. I sincerely thank, all the course teachers of minor subjects, Dr. (Mrs) S.B.Mazee, Associate Professor of microbiology, Dr. (Mrs) Gatne.M.M. Professor of Pharmacology, Dr. A.M.Das, Professor(retd.)Microbiology, Dr. Dighe, Associate Professor, Medicine for their dedicated and intellectually stimulating lectures. My special and affectionate thanks to Dr. R.J. Zende, Associate Professor,VPH, for his help, valuable suggestions, constant motivation and support during my research work. I extend my heartfelt thanks to Dr. Vilas Vaidya, Asst.Prof. Dr. Nanavare, Dr. Mahesh Javale, Research Assistants, Dr. Chandrakanth, Asst.prof. VPH department for their help, support and encouragement. The invaluable help, co-operation, support and some interesting lessons on basics of pesticide residue analysis in GC by Dr. Amila Kadam and Trupthi Padalkar is gratefully acknowledged and remembered for ever. I do remember Tushara, Deepa, Manisha, and Archana for their nice interaction and support. The three years of (very long...) study went quite meaningfully and tirelessly with the associations of my senior Ph.D. colleagues Vijay Jadhav, and Rajkumar Barge and my classmates Rajendra Velhanikar, Nilesh Kumar Pagruth, Jayanth Patil and A.D. Patil. I thank them all with utmost pleasure and sincerity. I fondly remember my association and memorable interaction with all post-graduate scholars of our discipline, Swami, Abhijeet, Vikram, Deepthi, Pravin, Deepali, Sonali, Santosh, Amit, Viraj, Shilpa and Praful, who made my work easier, took special care, valued my interferences and made my life here buoyant and cheerful. I thank each one of them with humility and grace. I sincerely wish them all the success in life and career they choose. I sincerely thank all the faculty members of Veterinary Public Health, Maharashtra Animal and Fishery Sciences University and specially Dr. N.N. Zade, Professor, VPH, Nagpur, for help, co operation and words of encouragement during my stay over here. I am grateful to Dr. Kavithkar, Associate Prof. for his help and co operation. I greatfully acknowledge the help and the hospitality provided by Dr. Ninad Korde and family. vi
My sincere thanks are extended to Ramesh Jadhav, Dhopate, Mrs. Meena Dabhekar, Mrs. Sunita Malap, Dilip, Tambe, Auti, Kamble and Hatankar for their care and assistance during my research work. I sincerely thank all the authorities of Karnataka Veterinary, Animal and Fisheries Sciences University, for granting the external deputation facility for three years which enabled me to complete and concentrate on my studies and helped me to sail without any financial difficulties. I am grateful to all the staff members of Veterinary College, Bidar for their help and cooperation. My sincere thanks are extended to Staff of Vista Food Processing Pvt. Ltd. Specially, Mr. Bhupinder Singh, Mr. Atul and Dr. Dinkar for their involvement and full cooperation throughout the project work. I also thank Dr. Dhanjay, Mr. Manjunath and all staff of Vista for their kind help and hospitality. I extend my sincere thanks to Dr. Pal EOS Laboratories for timely supply of media and chemicals. The familial support and the value system provided by my parents, brothers, sisters, brother-in laws, sister-in laws, is something phenomenal, which nurtured the person in me and acted as a source of energy and inspiration to move forward in life. I am ever indebted to them and seek their blessings always. I am grateful to my parent-in laws, Suresh and Ashwini for their cooperation, help and support. Shivashankar bhava, a special person whose benevolent and selfless blessings were always with me to progress further in matters dear to me. I am just grateful to him. The beautiful, unassuming and ever cheerful minds our home Sriranjini, Shami, Swathi, Samahitha , Pranav, Abhi and Adithya acted as catalysts in reaching the target and I simply adore them. The words cease to mean to express my feelings towards my wife Viju and son Samahitha. I can only understand and request them to forgive me if ever possible as I have no right and reason to be away from them and put them into emotional hardship. But they stood by me with utmost responsibility and maturity. I am ever indebted to them. vii
I have some friends who care, dare and share but don't spare in matters of life and career. I know them well and they know me very well. They are with me both in good and bad times and are beyond thanks. I wish them all the success in life. There are innumerable persons and circumstances that helped me to evolve as an individual and I thank them all.
Place: Mumbai Date: 01/10/2009
(Dr. Madhavaprasad C.B.)
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TABLE OF CONTENTS
Chapter
Page
I) INTRODUCTION ... ... ... ... ... 1
II) REVIEW OF LITERATURE ... ... ... ... 7
III) MATERIALS & METHODS ... ... ... ... 43
IV) RESULTS & DISCUSSION ... ... ... ... 74
V) SUMMARY & CONCLUSION(S) ... ... ... 143
VI) PROPOSED AREA OF FUTURE RESEARCH ... 150
A) BIBLIOGRAPHY ... ... ... ... ... i ­ xx
B ) VITA
... ... ... ... ... ... xxi
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LIST OF TABLES
Table No. 3.1. 3.2. 3.3 3.4 3.5 3.6 3.7 3.8 3.9 3.10 4.1 4.2 4.2(a) 4.2(b) 4.3. 4.3(a) 4.3(b) 4.3(c) 4.4
Title of Table Total number of samples collected from all sources for physicochemical, microbiological, pesticide and antimicrobial residue analysis Total number of raw shell egg samples collected for sanitation, shell penetration and recovery and shelf life studies from commercial layer farm Total number of raw shell egg samples procured from farms and markets for physico-chemical quality study Number of samples collected from commercial layer farms and markets for detection of Salmonella and Campylobacter species Effect of different sanitizers on surface inoculation of SalmonellaTyphimurium on raw shell eggs Number of raw shell egg samples analyzed for shelf life study after treatment with paraffin oil alone and in combination with different sanitizers Number of samples analyzed for shell penetration and recovery study of SalmonellaTyphimurium in whole egg contents treated with different sanitizers Number of samples analyzed for shell penetration and recovery of artificially inoculated SalmonellaTyphimurium in eggs good versus poor quality shells Number of samples of raw shell eggs for pesticide and antimicrobial residues Name of antimicrobials and detection level in ppb by Premi test. Physico-chemical and microbiological analysis of raw shell eggs from commercial layer farms and market samples Comparison of weight of raw shell eggs and shell thickness between commercial layer farms and market Analysis of variance for egg weights between commercial layer farms and market Analysis of variance for shell thickness between commercial layer farms and market Comparison of internal physico-chemical qualities of shell eggs between commercial layer farms and market Analysis of variance for average albumin heights of eggs between commercial layer farms and market Analysis of variance for average Haugh units of eggs between commercial layer farms and market Analysis of variance for average albumin pH values of eggs between commercial layer farms and market Comparison of Total Viable Count (log CFU/ml) of raw shell eggs between commercial layer farms and market.
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4.4(a) 4.4(b) 4.5 4.6 4.7 4.7(a) 4.7(b) 4.7(c) 4.7(d) 4.8 4.8(a) 4.8(b) 4.8(c) 4.8(d) 4.9 4.10 4.11 4.12 4.13 4.14
Analysis of variance for Total Viable Count from external surface of eggs between commercial layer farms and markets Analysis of variance for Total Viable Count from internal contents of eggs between commercial layer farms and markets Percent prevalence of Salmonella spp. and Campylobacter spp. in commercial layer farms and market samples Effect of various sanitizers on artificially inoculated Salmonella Typhimurium and Total Viable Count on surface of shell eggs. Efficacy of various sanitizers on Salmonella Typhimurium and Total Viable Count on the surface of shell eggs Analysis of variance for effect of sanitizers on pre inoculated Salmonella Typhimurium on surface of shell eggs Analysis of variance for effect of sanitizers on Total Viable Count on surface of shell eggs (Pre-inoculated) Analysis of variance for effect of sanitizers on post- inoculated Salmonella Typhimurium on surface of shell eggs Analysis of variance for effect of sanitizers on Total Viable Count on surface of shell eggs (post-inoculated) Comparison of effect of different sanitizers on log reductions of Salmonella Typhimurium and Total Viable Count on surface of shell eggs Analysis of variance for effect of sanitizers on log reductions of pre inoculated SalmonellaTyphimurium on the surface of eggs Analysis of variance for effect of sanitizers on log reductions of Total Viable Count (pre-inoculated) on the surface of eggs Analysis of variance for effect of sanitizers on log reductions of postinoculated Salmonella Typhimurium on the surface of eggs Analysis of variance for effect of sanitizers on log reductions of Total Viable Count (post-inoculated) on the surface of eggs Shell penetration and recovery of Salmonella Typhimurium in whole egg contents after surface inoculation of raw shell eggs and treated with different sanitizers stored at refrigeration temperature. Fixing of performance criteria for raw shell eggs to meet Food Safety Objectives based on in vitro studies of sanitation and penetration of Salmonella Typhimurium (log CFU/ml) in shell eggs. Shell penetration and recovery of Salmonella Typhimurium in whole egg contents after surface inoculation of good and poor quality shell eggs stored at refrigeration temperature Shelf-life of raw shell eggs treated with paraffin oil alone and in combination with various sanitizers stored at ambient (28 - 330C) temperature Shelf-life of raw shell eggs treated with paraffin oil alone and in combination with various sanitizers at refrigeration storage (3-7 0C) temperature Average loss of egg weight (g) of fresh raw shell eggs stored at ambient (28-33 0C) and refrigeration (3-7 0C) temperature
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LIST OF FIGURES
Figure No. Figure : 1 Figure : 2 Figure : 3 Figure : 4 Figure : 5 Figure : 6 Figure : 7
Title of Figures Comparison of external quality parameters of shell eggs from commercial layer farms and markets Comparison of internal quality parameters of shell eggs from commercial layer farms and markets Comparison of microbiological quality of raw shell eggs from commercial layer farms and markets Percent prevalence of Salmonella spp. and Campylobacter spp. from different sources Albumin height (mm) of raw shell eggs stored at ambient temperature treated with paraffin oil alone and in combination with various sanitizers Haugh unit of raw shell eggs stored at ambient temperatures treated with paraffin oil alone and in combination with various sanitizers Percent weight loss of shell eggs stored at ambient and refrigeration temperature
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ABBREVIATIONS
Abbreviation Aldrin ALOP AOAC BAM BGSA BHC CAC CFU Chloropyriphosethyl Cypermethrin Deltamethrin ECD EEC Endosulfan Ethion FAO Fenvalerate FSO FTD GC GC-MS GEP GMPs HCB HCHs Heptachlor Heptachlor epoxide HU ICMSF
Full Name 1R, 4S, 4aS, 5S, 8R, 8aR- 1,2,3,4,10, 10- hexachloro-1,4, 4a,5,8, 8a-hexahydro- 1,4,5,8-dimethanonaphthalene Appropriate Level Of Protection Association of Official Analytical Chemists Bacteriological Analytical Manual Brilliant Green Sulfa Agar Benzene Hexachloride Codex Alimentarius Commission Colony Forming Unit O,O,-diethyl O-3,5,6--trichloro-2-pyridylphosphorothioate (RS)--cyano 3-phenoxybenzyl (1 RS, 3RS; 1RS, 3SR)-3(2,2-dichlorovinyl)-2,2-Dimethylcyclopropanecarboxylate (S)--cyano-3-phenoxybenzyl (1R, 3R)-3-2,2-dibromovinyl)2,2-dimethylcyclopropane carboxylate Electron Capture Detector European Economic Commission 1,4,5,6,7,7-hexachloro-8,9,10-trinoborn-5-enylene-2,3bismetyylene sulfite O,O,O',O'- tetraethyl S,S'-methylenebis(phosphorodithioate) Food and Agriculture Organization of United Nations (RS)- -cyano-3-phenoxybenzyl (RS)-2-(4-chlorophenyl)-3methylbutyrate Food Safety Objective Flame Thermionic Detector Gas Chromatography Gas Chromatography- Mass Spectrometry Good Egg production Practices Good Manufacturing Practices Hexachlorobenzene Hexachlorocyclohexanes 1,4,5,6,7,10,10-heptachloro-2,3, 3a, 4,7, 8,9,-tetrahydro-4,7enodomethyleneindene 2,3,-epoxy-1,4,5,6,7,8,8-heptachloro-2,3, 3a, 4,7, 7ahexahydro-4,7-endomethanoindane Haugh Unit InterNational Commission on Microbiological Specifications
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IEC IFT LOD Malathion mCCDA MRL NSS o,p'- DDT o,p'-DDD o,p'-DDE OC OIE OP p,p'-DDD p,p'-DDE p,p'-DDT Parathion methyl PFA Phenthoate PHMG PO ppb ppm Profenofos R.V.M SP TSP TVC USDA US-FDA WHO XLD - HCH - HCH - HCH - HCH g l
for Foods International Egg Commission Institute of Food Technologists Limit of Detection Diethyl (dimethoxyphosphoinothioylthio) succinate modified Cefeperazone Charcoal Agar Maximum Residue Limit Normal Saline Solution 1-(2-chlorophenyl)-1-4-(4-chlorophenyl)-2,2,2- trichloroethane 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethylene Organochlorine Office International des Epizootes Organophosphorous 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane 1,1, 1-trichloro-2,2-bis(4-chlorophenyl)ethane 1,1,1-trichloro-2-2-bis(4-chlorophenyl)ethane O,O-dimethyl O-4-nitrophenyl phosphorothioate Prevention of Food Adulteration Act S--ethoxycarbonylbenzyl O,O-dimethylphosphorodithioate Polyhexamethylene Guanidine Performance Objectives Parts Per Billion Parts Per Million (RS)-(O-4-bromo-2-chlorophenyl O-ethyl S-propyl Rappaport-Vassiliadis Media Synthetic Pyrethroid Trisodium Phosphate Total Viable Count United States Department of Agriculture United States-Food and Drug Administration World Health Organization Xylose Lysine Deoxycholate 1, 2, 3, 4, 5, 6- Hexachlorocyclohexane 1, 2, 3, 4, 5, 6- Hexachlorocyclohexane 1, 2, 3, 4, 5, 6- Hexachlorocyclohexane 1, 2, 3, 4, 5, 6- Hexachlorocyclohexane Micro-grams Micro-liter
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INTRODUCTION The shell eggs from hens (Gallus domesticus) are one of the few foods that are consumed or used throughout the world without any religious or cultural bias and constitute an integral segment of the world food industry. The world egg industry is primarily based on chicken (Gallus domesticus) eggs though the shell eggs from other species of birds like ducks and quails are found in the market places (Stadelman and Cotterill, 2002).The global egg production in the past three decades (1970 to 2006) increased from 19.5 million tons to 61 million tons or by 213% and more remarkable is that the share of Asian countries rose from mere 23.7% in 1970 to 60.8% in 2006 which is expected to increase further as more and more Asian countries are in the phase of commercialization and mechanization of the layer farms with a flock size of more than 1,00,000 birds. The shell eggs are an important commodity in the international trade and India rank's second in the world egg production today with a market share of 7% where as China rank's first with a market share of 68.2% that changed the pattern of global egg trade and had impact on pattern of egg exports and imports (Windhorst, 2008). The per capita egg consumption in India as on 2006 was 1.5 kg or 28 eggs and expected to increase to 2.2 kg or 40 eggs (IEC, 2007). Within the egg industry, egg products such as frozen liquid eggs, frozen whites and yolks, dried whole eggs etc constitute important market products. The recent trend of establishment of egg-breaking or egg processing plants in developing Asian countries clearly signal's the expansion or scope of the egg industry as a whole. The growth of new markets, shift in the patterns of trade brings along with it the issue of shell egg quality and safety to the forefront, because ignoring the issue amounts to ignoring the trade and opportunity and risk to poultry and human health. The quality of the shell eggs is very good immediately after lay as it is very fresh and freshness makes a major contribution to the quality of egg and egg products as variability in freshness is perceived as lack of quality by the consumers. The loss of fresh-ness or the decline of quality occurs thereafter very fast if collection, handling and storage practices are not adequate and proper (Karoui et al. 2006). Generally the loss of quality occurs from the time egg is laid and continue to progress with the passage of time. As the egg ages, the loss of 1
quality of albumin and yolk becomes evident and the freshness reduces. This phenomenon is attributed to movement or exchange of carbon dioxide and moisture from within the egg through the shell and is aided by factors like temperature, humidity and handling. The rate of diffusion of gases and moisture depends on the quality or thickness of the shell and density of pores. The control of these factors especially the temperature and humidity of storage of eggs with better hygiene and handling practices help to maintain freshness and quality of raw eggs for longer time and consumer acceptability of eggs in the market. To market the eggs of consistently superior quality is a real challenge in all seasons especially in summer without any quality control or assurance program in place. The quality of the eggs can be preserved by several methods such as oiling (Homler and Stadelman, 1963; Beyer, 2005), thermo stabilization (Funk, 1943, Scott and Vickery, 1954), flash heat treatment with boiling water (Romanoff and Romanoff, 1944) refrigeration, treatment with beta or gamma irradiation (Ma et al 1990) etc. and which results in reduced spoilage and increased shelf life. The preservation of shell eggs through refrigeration or below the temperature of 10 0C extends the shelf life of eggs to several months and is practiced in developed countries. It is one of the best methods available for preservation of eggs for extended periods of time. The sanitation or disinfection of shell eggs are principally followed to remove the inadequacies in hygiene during production, storage and distribution which help to destroy the potential spoilage and pathogenic organisms which may be present on the fresh shell eggs. As it is impracticable to produce very clean eggs on large scales without automation, the use of effective, safe sanitizers and shell egg sanitation becomes a necessity. Though the established sanitizers like chlorine, quaternary ammonium compounds, alcohol etc. are used for disinfection, a constant search for newer chemicals with better sanitizing abilities and long term action always remains a dear subject of research in food sanitation. Shell eggs are usually fried, boiled, or baked and these cooking methods may or may not reach appropriate temperature to destroy any pathogen that may be present especially Salmonellae. Eggs are boiled or cooked long enough to solidify the yolk and approximately 10 min of boiling is known to inactivate Salmonellae, but other cooking procedures that leave the yolk in a liquid state 2
such as soft boiled and fried eggs ("over easy") are not always sufficient to inactivate Salmonella spp (ICMSF, 1998). Liquid eggs, white, and yolk that do not contain chemical additives are usually pasteurized at temperatures that vary from 55.6 °C to 69 °C at processing time that varies from 10 to 1.5 min. Lower temperature and shorter processing time increases the risk of survival of Salmonella spp., whereas higher temperature and longer processing time increases damage to the functional properties of the egg (ICMSF, 1998; FDA (USDA), 2004). Food processing treatments such as washing, peeling, canning or cooking, baking, bread making, dairy product manufacture, drying, thermal processing, fermentation, freezing, infusion, juicing, malting, milling, parboiling, storage and milling had a desirable effect of significant reduction of pesticide residues in the prepared food, particularly through washing, peeling and cooking operations ( Geetanjali et al. 2009). The thermo stability of Oxytetracycline, Tetracycline and Doxycycline residues is found to be less ( more degradation to an extent of more than 98%) at lower temperature and long time treatments than at higher temperatures and short time(less degradation to an extent of 50­90%) in foods ( Hassani et al 2008). The Benzyl penicillin get inactivated to 39% at 141єC for 7.5 min of heating and 90% inactivation at 10 min of heating (Sanz et al 2003). Food safety as an essential public health function is advocated by many international bodies viz, WHO, OIE and CAC and is an issue of increasing concern worldwide. Safety of shell eggs is one of the components of public health, and world wide the pathogens of concern as risk to the consumers include bacteria such as Salmonella, Campylobacter, Staphylococcus aureus, Listeria etc. and all are recognized causes of food borne illness (OIE, 2002). Biological hazards in poultry might be arising directly from diseased birds, unhygienic conditions at farm, unhealthy employees, use of contaminated water source etc. (Coutts and Wilson, 1990; Stadelman and Cotterill, 2002). Although significant reduction has been recorded in incidence of food borne human salmonellosis, in some countries with extensive pre-harvest Salmonella control programmes but still there is an increase in incidence of some emerging food borne pathogens like Salmonella Enteritidis PT 4, Campylobacter species, E.coli serotypes in chicken eggs and disease prevalent flocks (WHO, 2002; IFT, 2002). Many infected birds are intestinal carriers and infection may be carried through 3
faeces, fomites and on egg shells. Vertical transmissions in eggs occur due to shell contamination or internal transovarian contamination of yolk. Chemical contaminants such as veterinary drugs and pesticide residues appearing in these products may cause serious health concerns amongst consumers. The chemical contamination of raw shell eggs with pesticides may occur through consumption of contaminated feed, contaminated drinking water, contaminated feed ingredients which are grown on pesticide contaminated soil or used in layer houses for the control of pests. The residues of antimicrobials appear because of indiscriminate use of antibiotics/anticoccidials for therapeutic and/or for growth promotion. Some of the pesticides and persistent organic pollutants have a tendency for bioaccumulation and biomagnifications resulting in higher concentrations in foods we eat while antibiotics show sequestration properties in egg yolk and delay or unpredictable time of residue appearance in shell eggs causing health hazards of different severities in human beings. Modern shell egg production involves number of generic segments in the human food chain like primary production of eggs in commercial layer farms, egg processing plants, wholesale and retail markets, transport, processed or finished products etc. which serve as multiple sources or links in the contamination of the products. The detection and identification of the agents or hazards both biological and chemical and their sources by standard microbiological and analytical methods provide sufficient scope for improvement in shell egg production, processing and preservation practices to provide safe and wholesome eggs for human consumption. An emerging or recent concept evolving in the area of food safety is the use of Food Safety Objectives (FSOs) and Performance Objectives (PO) by National Governments and industry which is in essence a "build on operation" which effectively utilizes Good Hygienic Practices (GHPs), Good Manufacturing Practices (GMPs), Hazard Analysis Critical Control Point (HACCP), risk analysis, performance criteria etc as a tool for achieving desired public health goal or Appropriate Level Of Protection(ALOP) at national level (ICMSF, 2005). FSOs and POs serve a purpose different from a microbiological criterion, which describes sampling and testing of foods for acceptance or rejection where as assessing processing and preservation parameters is the preferred option to check that an FSO or a PO is achieved or not. The POs give consideration to the 4
initial level of hazard before any treatment, as well as the decrease and possible increase of that hazard level prior to consumption and therefore act as additional tools for the food industry to build food safety in to their products. The scientific work on detection of pathogens and chemical contaminants in food products of animal origin are carried out with more emphasis on quality and safety of meat and very less work was carried out on quality and safety of shell eggs from the point of view of consumers which is more or less true for Indian scenario. The shell eggs being an important nutrient rich dietary source for human consumption and one of the foods coming under semi-perishable category, a study in this direction may help in knowing the quality and safety of the eggs which are being produced and marketed as well as possibly may help in defining performance criteria for sanitary process for raw shell eggs for food industry to meet PO or FSO. Therefore, the surveillance of raw shell egg quality, pathogens and chemical contaminants from different sources mainly from commercial layer farm and market samples becomes relevant for assuring safety and quality of shell eggs. Hence, the study was proposed with the following objectives ­ 1. To evaluate the physico-chemical quality of raw shell eggs from commercial layer farms and markets. 2. To assess the safety of fresh raw shell eggs from commercial layer farms and markets for the presence of selected enteric pathogens with special emphasis on Salmonella spp. and Campylobacter spp. by cultural methods. 3. To study the effect of sanitizers' viz., 70% alcohol, 65 ppm polyhexamethylene guanidine and 200 ppm chlorine on artificially inoculated Salmonella Typhimurium on raw shell eggs collected from layer farm. 4. To study the shelf life of raw shell eggs stored at ambient temperature (28-330C) and refrigeration temperature (5 ± 20C) after treatment with different sanitizers. 5
5. To assess the safety of raw shell eggs for selected pesticides viz. organochlorines, organophosphates and synthetic pyrethroids by Gas Chromatography and for antimicrobial residues. 6
REVIEW OF LITERATURE Review of literature on different aspects of quality and safety of raw shell eggs is covered in this chapter. The information from the literatures are included to cover the aspects on quality such as physico-chemical and microbiological, the safety aspects like prevalence of Salmonella spp. and Campylobacter spp, residues of pesticides and antimicrobials in shell eggs. The review of literature also includes information or research work done on shell egg sanitation, shell penetration and recovery of Salmonella spp. from whole egg contents and shelf life shell eggs at ambient and refrigeration storage temperatures. In addition to this, the review also includes other relevant literatures useful to the present research work on quality and safety of shell eggs. 2.1 Physico-chemical quality of shell eggs Nys et al (2001) carried out a project for developing tools for grading of shell eggs and improving quality and safety of hen eggs in production system and supply chain. They observed that egg quality (bacterial load, cracked eggs) was inferior in furnished cages than in control cages but brown medium heavy genotypes showed more cracked eggs than white light hybrids. However, in both the systems the levels of bacterial counts remained low. Several workers used non-invasive technique for measuring egg quality (Lin et al 2004, and Schwдgele et al 2004) and observed that the non-destructive assessment of the internal egg quality showed reasonable correlation with albumen pH (8.5) and Haugh units (79), which are conventional commercial and research methods accepted to evaluate freshness of the egg. Moreover, the method was non destructive and the measurement required only few seconds. Berardinelli et al (2003) studied the effect of mechanical vibrations due to transport and measured the egg quality indices from the packing house to market with Power Spectral Density (PSD) values and vibrating table of an electro-dynamic shaker to simulate the stress on packaged eggs in cardboard boxes. Egg quality traits like Haugh unit, vitelline membrane strength and air cell 7
height were determined after 8 days of storage at 18°C and their results showed a significant decrease in Haugh unit (28%) in the sample stressed with a PSD profile obtained at the highest level of the column, in comparison with a nonvibrated sample. Variations in the resistance of vitelline membrane (18%) were also observed among the samples which were vibrated with different PSD profiles Silversides and Budgell (2004) conducted experiments to study the relationship between egg albumen height, pH and whipping volume. The study included shell eggs from Brown Leghorn hens (commercial brown egg layer) and Babcock hens (commercial white egg layer) and fresh versus 5 and 10 days of stored eggs. They measured egg weight, albumen height, pH, and volume after whipping for 80 sec. and observed that egg weight and the weights of the 3 principal components of the egg are increased with increasing age of the hen, along with yolk weights increasing proportionately more. With storage, egg and albumen weights decreased, whereas yolk weight increased. Eggs from Brown Leghorn hens were smallest but had proportionately the largest yolks. Albumen height decreased with time in storage, and albumen pH and whipping volume increased. Casiraghi et al (2005) studied the commercial shell eggs quality with special emphasis on shell breakage both at packaging and transport with different European commercial weight grades such as S, M, L, and XL and all from Grade A eggs marketed in retail out lets. XL eggs showed the highest average percentage of cracked eggs and least shell thickness and they further concluded that several significant correlations between breaking strength and various egg geometrical and structural parameters were existent. Effects of Indian cow urine on the egg production and quality in layers was studied by Garg et al (2005) in India. The experimental design comprised of 50 layer birds of 20-22 weeks of age kept under hygienic conditions in poultry farm and were divided into 2 equal groups. Group I (25 birds) were untreated control and group II (25 birds) was Treatment Group which were given distilled cow urine of Sahiwal breed @1 ml/ bird daily dosing with water for 105 days. They recorded egg quality parameters like daily egg production, egg weight, egg length and egg width, albumin index, yolk index, shell thickness, shell weight, albumin height and observed increased egg weight by 16.08%, shape index by 8
4.82%, shell thickness by 10.26%, shell weight by 15.51%, yolk height 7.21%, albumin weight by 14% in treated group. They concluded that treatment had shown significant differences in egg quality and production from that of the control. A survey of consumers from Spain, Germany and other European countries was carried out by Hernandez (2005) with the objective of validating and ranking the shell eggs quality attributes. Results showed that safety and freshness were the most important quality factors followed by nutritional and sensory characteristics. Consumers defined egg quality as related to shell strength, albumen consistency, and intense yolk colour. Measurement of eggshell quality over the past 40 years of research was reviewed by Hunton (2005) and detailed the developments in egg shell research that started with concept of measuring the whole-egg specific gravity, shell thickness followed by the development of measuring the egg shell strength by Brooks and Hale in1955. A further advancement or invention in 1962 by Schoorl and Boersma where non-destructive instrument for measuring shell quality was considered as significant break ­through. Later this equipment was modified by Hamilton and coworkers in 1979 and named Ottawa eggshell strength tester and was considered as an invaluable addition. Hunton concluded that the evolution of several methods over the years contributed immensely to the egg shell research and for better defining of commercial crack incidence in shell eggs and dietary supplementation of calcium to birds. Jones and Northcutt (2005) performed a survey in US for recording the common practices in shell egg processing facilities and reported that over 90 % of the processing plants performed daily sanitation. Most facilities did not attempt to recycle water from their process and 65.8 % utilized wells as their primary source of water. Only 19.2 % of the facilities discharged water to city sewers. Sheikh and Younis (2005) evaluated the influence of hen layer age and storage periods at room temperature (33.2°C) of white and brown hy-line strains during summer conditions. Eggs from white and brown Hy-line hens at 43 and 63 weeks of age were sampled in their study immediately after lay and after periods of storage of 7, 14, 21 and 28 days and egg quality traits were measured for yolk and albumen weight, egg weight, shell thickness, pH and microbiology. They reported that the eggs from the brown strain were 3.73% heavier, had 4.21% 9
more eggshell, and had a higher specific gravity than the white strain eggs with1.079 and 1.073. They further noted that increased egg weight with increasing hen age while the yolk increased more in size than did the shell and albumen. The albumen height of eggs from Hy-line brown was greater than that of Hy-line white hens, and it decreased as the hen age increased and with increasing of storage time. The pH of the albumen was affected by strain and hen age, but it increased with the time in storage. Longer periods of storage resulted in significantly (P<0.05) lower albumen weight and albumen height and higher albumen pH across the two age groups. The loss in weight from shell, albumen and yolk was increased with increasing storage period and age of hens. They further observed that, the shelf life for white Hy-line strains and younger egg age was up to 28 days while the maximum shelf life for brown and older eggs did not exceed 21 days of storage. Strahle et al (2005) used air cell height as the criteria for measurement of freshness of shell eggs and conducted experiments to test whether the air-cell height is correlated with the traits of internal egg quality like albumen height and Haugh units. The eggs in their study stored at 15°C and the egg quality parameter obtained by them were air cell height 3.03 +/- 8.29, albumen height 4.80 +/- 1.00, Haugh units 65.42 +/- 11.52. None of the eggs recorded more than 6mm of air cell height, which is the critical for the freshness of the egg and were significantly influenced by age of the egg, age of the hen and genetic origin of the hen. They concluded that the air cell height had a very low correlation with albumen height which was not good enough to measure the internal egg quality like albumen height. They further observed that the age of the egg and the age of the hen and even the interaction between both the traits were highly significant of the albumen height. Studies were conducted to provide realistic commercial guidelines on egg internal quality and egg shell quality for the Australian egg industry by Roberts (2005). Study conducted comprised both longitudinal and cross sectional surveillance on eggs from commercial flocks that comprised flock followed throughout their laying life and but across a range of ages of flocks. The data or results of the study indicated that considerable variation existed among flocks in egg and egg shell quality measurements with little difference among the three main brown egg layer strains. However, consistent findings in relation to bird age 10
with an increase in egg weight and shell weight up to 45 and 40 weeks of age was obtained, respectively, after which these weights remained relatively constant. Also the findings were consistent with reduced shell colour, shell breaking strength, deformation, percentage shell, and reduced albumen height and Haugh Units. They concluded that both temperature and humidity influenced the loss of weight from stored eggs whereas Haugh Units were affected mainly by temperature. Parmar et al (2006) evaluated the egg quality traits in indigenous Kadaknath breed of poultry from two field survey centers [Jhabua-I (Meghnagar) and Jhabua-II (Jobat)] of district Jhabua (Madhya Pradesh) and found that the overall average dark brown shell colour pooled for two centers was 67.87 percent, followed by light brown shell color, which was 32.12 percent. They also recorded that 65.39 percent of eggs were strong shelled, 32.06 percent medium and 2.56 percent brittle shelled. The mean shell thickness, shape index, albumin index, Haugh unit, yolk index, yolk weight and albumin weight pooled for both the centers were found to be 0.31mm, 73.93%, 7.03, 73.77, 37.07%, 14.77g and 20.74 g, respectively. Most of the egg quality traits studied at farmers door were found to be inferior when compared with Kadaknath birds maintained at poultry breeding farm, Veterinary College, Jabalpur and Government Poultry breeding farm, Jhabua, which indicated that better management practices could improve the egg quality characteristics of birds reared by farmers under field conditions Chatterjee et al (2007) Studied the comparative growth, production, egg and carcass traits of different crosses of Brown Nicobari with White Leghorn under intensive and extensive management systems in Andaman, India and observed that the average egg weight was found to be 51g and that of shape index, albumen weight, yolk weight, yolk height, shell weight, albumen, yolk and shell percentages differed significantly (P<0.05) between the studied genetic groups except the egg weight. They also found that the albumen pH was alkaline, while yolk pH was acidic in nature. 11
2.2 Microbiological quality of shell eggs, layers and environmental samples Nabbut et al (1982) isolated Salmonella serotypes from farm animals, animal feed, sewage and sludge in Saudi Arabia and found that out of 65 different serotypes, six serotypes most frequently recovered or isolated were Livingstone, Concord, Schottmuelleri, Lille, Typhimurium and Cerro. Clark and Bueschkens (1985) infected chicken eggs with Campylobacter jejuni and employed temperature or pressure differential methods of inoculation and found that more than 90% of the eggs carried C. jejuni when iron was present in the inoculum and the percentage declined rapidly until by day 8 and less than 10% of the eggs were detectably infected. They recovered organisms in hatched chicks up to 11% but recovery from infertile eggs was found to be poor. Clark and Bueschkens (1986) infected the chicken eggs and stored for 5.5 days before incubation to study the effect of egg storage upon the survival of Campylobacter jejuni in laboratory-infected fertile poultry eggs and showed that the rate of infection of the eggs had decreased up to 20% or less and no infected chicks were hatched. Similarly they found that 8 days of storage also failed to yield infected chicks. Shane et al (1986) demonstrated in their experiment that contamination of commercial table eggs with a fecal suspension containing 4.4 x 106 CFU/g Campylobacter jejuni resulted in shell penetration in 3/70 eggs and recovery of the organism in homogenized egg contents was found to be 1/70 eggs where as viability of C. jejuni on the shell surface was retained for only 16 hours. They also observed from their field survey of three commercial laying farms and their associated egg-packing plants that hens demonstrated to be fecal shedders of C. jejuni (12% to 62% incidence} but did not produce infected eggs. The organism could not be detected in the environment of the packing plant, including grading machinery and effluent. Contamination of shell eggs by Aeromonas, Alcaligenes, Enterobacter, Flavobacterium, Micrococcus, Proteus, Pseudomonas and Serratia spp. occur commonly and as reported by several workers including contamination by pathogens such as Campylobacter, Salmonella and Staphylococcus spp. etc 12
that cause food borne illness or disease (Baker, 1990; Jay, 1996; and ICMSF 1998). Davies and Wray (1997) studied the distribution of Salmonella contamination in ten animal feed mills and found that the Salmonella isolation rate were ranged from 1.1% to 41.7% of the samples tested and the most contaminated mills were those where the inside of the cooling systems for pellet or mash had been colonized by Salmonella. A wide range of Salmonella serotypes were isolated which included Salmonella Typhimurium and Salmonella Enteritidis. Limited sampling for every two weeks for an 18-month period in another animal feed mill showed marked variation in the contamination rate of samples and range of Salmonella serotypes were found. Contamination of ingredient intake pits and out loading gantries for finished products by wild bird droppings containing Salmonella was also found in four mills during their study. Boer (1998) found that for isolation and detection of Salmonella from foods, motility enrichment in Modified Semisolid Rappaport­Vassiliadis (MSRV) medium showed equal or better results than others and addition of nitrofurantoin to diagnostic semisolid Salmonella agar and xylose lysine deoxycholate agar favoured the isolation of Salmonella Enteritidis. Takase et al (1999) conducted experiments to study the growth of Salmonella Typhimurium and Salmonella Enteritidis in egg yolks from highly immunized hens with oil adjuvanted bacterin of Salmonella Enteritidis for four week interval to specifically observe the suppression of growth of Salmonella serovars. They concluded that there was no difference in the growth titer between the antibody-positive yolk and the negative yolk samples and suggested that the antibodies in the yolk did not influence the growth of serovars of Salmonella organism, even if the hens were hyper immunized. Allen and Griffiths (2001) used luminescent Campylobacter jejuni ATCC 33291 to assess eggshell colonization and penetration in fresh and retail eggs and found that C. jejuni colonized both fresh and retail eggs at 37, 40, and 42°C under microaerophilic conditions. Also they observed that fresh eggs were more heavily colonized than retail eggs, and under aerobic conditions, fresh eggs were colonized at similar levels for all three temperatures. They found that C. jejuni was able to penetrate the eggshell in 2 of 48 (4.2%) fresh eggs assessed in their study. 13
Barbour et al (2001) made preliminary attempts towards production of table eggs free from Salmonella Enteritidis (SE) contamination, by application of pasteurization and dry heat and employed three highly virulent strains of SE known for potential food borne illness in humans. They reported that treatment of inoculated intact shell eggs in a hot water bath at 57°C for 25 min, followed by application of hot air at 55°C for another 57 min resulted in the highest reduction in SE count/ml of the egg content for strain 1 (average 33.33 cfu), followed by strain 2 (average of 0.73Ч104 cfu), and strain 3 (average of 1.60Ч104 cfu). All counts after treatment were significantly less than the initial count before treatment (P<0.05). Decreasing the density of the SE in the three inocula by about 13 logs to be 1.80Ч106 cfu/ml of egg content for strain 1, 2.34Ч106 for strain 2, and 2.20Ч106 for strain 3 (P>0.05) and applying the same pasteurization and dry heat treatment on inoculated eggs resulted in complete absence of viability in the three strains, averaging zero cfu/ml of egg content. The pasteurization and dry heat treatment of intact shell-table eggs resulted in significant reduction of the high initial count of SE strains, regardless of their plasmid profile or density of contamination. It was concluded from their study that the production of table eggs completely clean of SE could be obtained by the same treatment when the initial contamination was low (around 106 cfu/ml of egg content. Nys et al (2001) studied quality and safety of hen eggs in different production systems and found that the bacterial counts from furnished cages as well as controlled cages was low but eggs from older hens showed not vulnerable to bacterial penetration or it was occurred with less frequency. Also it was observed that the feed allocated to the hens however influenced the bacterial penetration of the eggs. Hens kept at higher temperature (32°C) in the hen house laid eggs that were more prone to contamination by eggshell penetration as frequently as non-stressed hens. The shell contamination at the end of storage was however highly correlated with Salmonella Enteritidis penetration rather than the external characteristics of shell eggs. Stadelman and Cotteril (2002) reported that the initial level of contamination of the egg shell and egg contents, the integrity of the shell, the storage conditions and the age of the egg were critical to the microbial or pathogen contamination. Bulk breakage and separation of eggs under 14
commercial conditions resulted in contamination of egg product by microorganisms which were present on the egg shell surface. Drociuk et al (2003) in their study observed that 78.0% of the out-breaks with Salmonella Enteritidis in USA were associated with consumption of shell eggs and the decrease in their incidence largely attributed to farm based control measures and cautioned that Salmonella Enteritidis could survive boiling water temperature if the yolk was not completely solidified. They recommended that one of the important control measures was microbiological testing of hen houses for the presence of SE and surveillance of SE outbreaks necessary to detect changes in trends of SE infection in the region. Howard (2003) investigated the invasion of avian reproductive tissues by Salmonella Typhimurium (ST) and Salmonella Enteritidis (SE) so as to know the new route of infection by transovarian means as compared to the more traditional path where SE originated from fecal contamination through the shell. He inoculated the shell eggs with both Salmonella Enteritidis and Salmonella Typhimurium onto the vitelline membrane and incubated for 24 hrs and samples from the interior of the egg membrane, the albumen of the egg and the membrane were analyzed for growth of the organism. He found that the albumen and membrane were more hospitable environments to bacterial growth with increased storage times and experiments with migration of bacteria in to pre ovulatory tissues with the removal of follicular sacks indicated that follicles with intact follicular sacks were more susceptible to bacterial colonization than other treatment groups. Lu et al (2003) studied the association of Salmonella enterica serovar Enteritidis with resistance to chicken egg albumen and identified YafD gene, the over expression of which conferred enhanced resistance of Salmonella serovars viz., Salmonella Enteritidis and Typhimurium to egg albumen. They observed that the disruption of this gene in Salmonella enterica serovar Enteritidis rendered the organism more susceptible to egg albumen and proposed that the YafD provides a survival advantage to Salmonella enterica serovar Enteritidis in eggs by repairing DNA damage caused by egg albumen and that it might be one of the biologic determinants that contributed to the epidemiological association of Salmonella enterica serovar Enteritidis with egg products. 15
Newell and Fearnley (2003) studied the sources of Campylobacter colonization in chickens and observed that Campylobacters are ubiquitous in the environment and could be readily carried into the house by a number of vehicles, including human activity associated with routine flock management. The dry conditions of feed and fresh litter were found to be lethal to C. jejuni. as the organisms were not isolated from clean dry litter and a fact that feed, feed additives, and fresh litter were not potential sources of infection . Several epidemiological studies where the relationships between water source (well or mains water) and broiler flock Campylobacter positivity were considered, the results of the most studies found that the water source was found to be low-risk factor. Sahin (2003) studied the ecology of campylobacter colonization in poultry and role of maternal antibodies in protection and sources of flock infection. He detected high levels of specific antibodies in egg yolks, sera of broiler breeders, and young broiler chicks, as maternally-derived antibodies. He further observed that antibodies were against multiple outer membrane components of Campylobacter, and were active in antibody dependent complement-mediated destruction of C. jejuni. He further challenged commercial broilers with culture of C. jejuni and shown that colonization occurred much sooner in birds challenged at the age of 21-days which were naturally negative for Campylobacter antibody than it did in the birds inoculated at 3-days of age which were naturally positive for Campylobacter maternal antibody and results indicated that Campylobacter maternal antibodies played a partial role in protecting young chickens against colonization by C. jejuni Detection and survival of Campylobacter in chicken eggs was studied by Sahin et al (2003) to determine vertical transmission of C.jejuni via eggs by adopting temperature differential method and it was observed that Campylobacter had limited ability to penetrate the eggshell. It was also demonstrated that the organism was able to survive for up to 14 days when C. jejuni was directly inoculated into the egg yolk and the eggs were stored at18 oC and viability of C. jejuni was dramatically shortened when injected into the albumen or the air sac. When they examined freshly laid eggs from Campylobacter-inoculated specific pathogen-free (SPF) layers, C. jejunicontamination was detected in three of 65 pooled whole eggs (5­10 eggs in 16
each pool) via culture and PCR and the organism was not detected from any of the 800 eggs (80 pools), collected from the same SPF flock, but kept at18 oC for 7 days before testing. Likewise, Campylobacter was not recovered from any of 500 fresh eggs obtained from commercial broiler-breeder flocks that were actively shedding Campylobacter in faeces and none of the 1000 eggs from broiler breeders obtained from a commercial hatchery. The results of their study suggested that vertical transmission of C. jejuni through the egg probably a rare event and did not play a major role in the introduction of Campylobacter to chicken flocks. Gurtler and Fehlhaber (2004) studied the Growth of Salmonella Enteritidis in yolk from eggs laid by immunized hens which were specific pathogen free. In their study specific pathogen-free (SPF)-laying hens were immunized with Salmonella Enteritidis followed by egg collection, the yolk separation and the quantitative determination of concentration of Salmonella Enteritidis antibodies by using the enzyme-linked immunosorbent assay (ELISA), the radial immunodiffusion and the bicinchoninic acid protein assay. Further, they inoculated the yolks with approximately 10, 100 or 1000 Salmonella Enteritidis cells/ml and incubated at 15, 20 and 30 °C for 0, 2, 6 and 24 h. It was observed from their study that the highest level of antibody concentration was found in the hyper immunized group and there was no difference in the generation times of Salmonella Enteritidis between the antibody-positive yolk and the negative yolk at the three different incubation temperatures. The results suggested that antibodies in the yolk did not influence the growth of Salmonella Enteritidis, even if the hens were highly immunized. Hutchison et al (2004) conducted studies for an assessment of the microbiological risks involved with egg washing under commercial conditions as it was perceived in European Union that wetted eggs were prone to spoilage and water loss. The study included the effects of spray jet washing under various processing conditions to shell surface counts of Salmonella (Salmonella Enteritidis PT4 or Salmonella Typhimurium DT104) and the presence of bacteria in egg contents. The results of their study indicated that washing of eggs under optimum conditions resulted in a more than 5-log reduction of Salmonella counts from the shell surface and Salmonella was not isolated from the egg of the yolk or albumen. However, they found that contamination did occur if strict control 17
was not maintained over the wash and rinse water temperatures and facilitated the entry of both Salmonella Enteritidis and Salmonella Typhimurium to the egg contents. Different Salmonella enterica serovars, including Enteritidis, were tested for growth at 20°C in separated albumen upon inoculation with 39 cfu /ml by Messens and his co- researchers (2004) and observed that the pronounced growth occurred more frequently and up to a one-log unit higher level in fresh albumen than in albumen stored prior to inoculation. They observed that the growth of Salmonella inoculated at a level of 8 cfu in the albumen of fresh and stored whole shell eggs, growth occurred more frequently in the albumen of fresh eggs compared to eggs stored prior to inoculation and concluded that cooling practices would help to prevent Salmonella growth in eggs. Saleha (2004) carried out epidemiological studies on the colonization of chickens with Campylobacter in broiler farms in Malaysia and considered several factors for his study like farm location and chicken house structure, water source, rearing practice and hygiene management and collected environmental samples among others such as water, feed, wood shavings, flies and chicken house environment. He observed that none of the feed, wood shavings, flies and chicken house environment was positive for Campylobacter while only 1.5% of untreated water supplies were found positive for Campylobacters. He postulated that prevalence of Campylobacters' colonization was possibly associated with untreated water, presence of other animals and unhygienic management practices as well as flying birds as they were found to harbour Campylobacters. Code of hygienic practice for egg and egg products as outlined by Codex Alimentarius Commission (CAC) (2005) emphasized that the microbiological status of the egg is a key determinant of the quality and safety of the product and commences at the farm. Contamination of eggs is influenced by the method of collection, cleaning, storage and processing. The structures of the shell eggs though act as a significant physical and chemical barrier to pathogen invasion and growth, the drying period of the cuticle that occurs within 3 min. of lay is most vulnerable for contamination from faecal microorganisms according to Sparks and Board (1985). Reitsma et al (2005) carried out studies on Campylobacter isolation methods from fresh poultry products and employed Preston, Cefeperazone 18
Charcoal Deoxycholate Broth (CCDB) and Bolton broths for enrichment and Cefeperazone Charcoal Deoxycholate Agar (CCDA) and Karmali agar for plating and observed that not one method was able to detect all positive samples (79 out of 89; 89%) but Preston broth + CCDA resulted in 74% positive samples. De Reu et al (2005) employed total aerobic counts and Gram-negative flora as tool for quality assurance in the production chain of consumption eggs. They found positive correlation between the initial bacterial eggshell contamination and the concentration of bacteria in the air of the poultry houses. Results of the study suggested that the total count of aerobic bacteria and the total count of Gram-negative bacteria on the eggshell could be used as a tool to detect the critical contamination points in the egg production chain. In their study it was also observed that metal mat was the critical control point in cage system of production and nest boxes for organic production system. Sheikh and Younis (2005) studied the effect of layer age, room temperature storage period and strain of hen on egg quality and egg microbiology during summer. The study included the eggs from of white and brown Hy-line strains of layers. They found that the microbial contents of egg during the 28 days of storage influenced the shelf life. It was also observed that the shelf life for white Hy-line eggs and younger age continued through 28 days, while the maximum shelf life for brown and older eggs had shelf life of only 21 days. Braden (2006) reported in his epidemiological study on incidence of Salmonella enterica serotype Enteritidis through consumption of shell eggs in USA that there was a steady increase in the incidence of Salmonella Enteritidis infection in human beings since 1970s and the internal contamination of shell eggs by transovarian route was the major source of contamination. He advocated that the farm based control programs, sustained refrigeration of shell eggs, and education of consumers and food workers about the risk of consuming raw or undercooked eggs would bring down the incidence of Salmonella Enteritidis infection. Clavijo et al (2006) identified the genes associated with survival of Salmonella enterica Serovar Enteritidis in chicken egg albumen as the organism was able to persist in the chicken eggs. The results of their study indicated that the genes involved in cell wall structural and functional integrity, and nucleic acid 19
and amino acid metabolism were important for Salmonella enterica serovar Enteritidis to persist in egg albumen. Guan et al (2006) designed an in vitro study of Salmonella Enteritidis and Salmonella Typhimurium Definitive Type 104 and its survival in egg albumen and penetration through the vitelline membrane and employed eight strains of Salmonella Enteritidis and 24 SalmonellaTyphimurium DT104 strains. They observed that the concentration of the organisms declined more rapidly when incubated at 42°C than at 37°C and dropped to non detectable levels within 96 hrs at the higher, but not at the lower, temperature. Like wise, the results of another experiment with 3 Salmonella Enteritidis and 3 Salmonella Typhimurium DT104 strains inoculated onto the vitelline membranes of egg yolks, which were then submerged in the original albumen and incubated for 24 hrs at 42°C showed that the organisms were survived in egg albumen but did not penetrate the vitelline membrane while penetration did occur when the samples were incubated at 30°C for 72 hrs. Howard et al (2006) conducted experiments to identify the proximate timelines for bacterial invasion of the egg by observing growth and survival of Salmonella Typhimurium which were inoculated on to yolk membrane stored under refrigerated conditions for long term. It was established from the study that Salmonella Typhimurium net growth occurred in albumen by the second week and continued from 4 to 8 weeks while in yolk net growth only occurred at week 5 and 7. Salmonella Typhimurium net growth on vitelline membranes occurred by 2 weeks and continued from 4 to 8 weeks while yolk samples showed no net increases in Salmonella Typhimurium populations over the 8 week period. Kusum et al (2006) discovered new Salmonella serovar, Salmonella Lamphun from animal feeds in Thailand, in 2003, which belonged to group C, with antigenic formula 6, 8: y: 1, 2... The pulsed field gel electrophoresis pattern of both isolates comprises 11 DNA fractions sized 48, 65, 77, 105, 110, 170, 244, 330, 337, 453 and 1,135 kbp. and reported that up to April 2005, no human or animal infection by the new Salmonella serovar was occurred. Wagenaar et al (2006) studied the occurrence of Campylobacter in primary animal production and observed that all types of poultry (broilers, layers, turkeys, ducks, fowl, quail, and ostriches) could become colonized with Campylobacter including wild birds and the incidence in positive flocks may vary, 20
depending on the country (i.e. continent and climate zone), and shows a strong seasonality in the infection rate. Wales et al (2006) evaluated a semi quantitative technique for the enumeration of Salmonella in the environment of layer flocks and compared the findings with those of a standard qualitative technique. They collected the samples from faeces, floor dust, and dust on cages, feeders and egg belts and cultured the organism in buffered peptone water, carried out enrichment and plating on semisolid selective and solid isolation media. The results indicated that the numbers of Salmonella detected for a site or sample type did not correlate closely with the prevalence of positive samples. Bolton (2007) observed that thermophilic or thermotolerant species of Campylobacters such as C.jejuni and C.coli were found in the intestinal tract of animals and wild birds including poultry and hence serve as major zoonotic pathogens and majority of the infections were attributed to food borne transmissions. He also observed that Campylobacters won't multiply outside the host but could survive for long time in the environment and enough to cause infection. Organisms found to be susceptible for sanitizers, thermal treatments and irradiation. Gast et al (2007) studied in vitro penetration of egg yolks by Salmonella Enteritidis and Salmonella Heidelberg strains during 36 hour ambient temperature storage at 20-300C and found that after inoculation onto the exterior surface of the vitelline.membrane, all 8 Salmonella strains penetrated to the yolk contents (at a mean frequency of 45.1%), and most strains grew to significantly higher levels (with a mean log10 bacterial concentration of 2.2 cfu/ml) during incubation at 30°C. They also found significant differences in penetration frequency and yolk multiplication between individual strains and between serotypes (Salmonella Enteritidis > Salmonella Heidelberg for both parameters). Penetration and multiplication were significantly less frequent during incubation at 20°C. Their results demonstrated that controlling ambient temperatures during pre refrigeration storage might be an important adjunct to prompt refrigeration for limiting Salmonella growth in eggs. The safety of shell eggs from commercial egg processing centers was assessed by Musgrove et al (2008) in USA by selecting marker organisms from Enterobacteriaceae and related organisms. They collected and analyzed 21
samples from three commercial egg processing plants and from different sites from the plants along the processing line such as pre wash rinse, first washer, second washer, sanitizer rinse, dryer, oiler, check detection scales, egg grader/packer etc and found that the greatest numbers of isolates were identified were those collected from eggs during preprocessing (accumulator, pre wash rinse) or from eggs judged as dirty (rewash belt entrance or exit) and smallest number of isolates were from during or at the end of processing. The isolates frequently obtained were Escherichia coli and Enterobacter spp. Cedecea, Citrobacter, Erwinia, Hafnia, Klebsiella, Kluyvera, Leclercia, Morganella, Proteus, Providencia, Rahnella, Salmonella, and Serratia either in all or some of the plants. Non-Enterobacteriaceae isolated and identified included Aeromonas, Chryseomonas, Listonella, Pseudomonas, Sphingobacterium, Vibrio, and Xanthomonas. They concluded that all of the genera and species which were recovered less frequently from fully processed eggs than from unwashed eggs, which indicated that shell eggs were less contaminated with bacteria as a result of commercial washing procedures. Mas et al (2008) Compared three environmental sampling methods for detecting Salmonella in commercial laying flocks in UK namely the European Union (EU) baseline survey method (five faeces and two dust samples), an inhouse (Veterinary Laboratories Agency, (VLA) 'wet' method that involved collecting 10 dust and 10 faeces samples into jars with buffered peptone water; and a method that had two samples of pooled faeces and one of dust (cultured as one sample of each type), which had been adopted for the National Control Program(NCP) for laying flocks across the EU and concluded that the wet method was the most sensitive, and the NCP the least, although individual NCP samples were found to be the most sensitive ones. Ozbey et al (2008) attempted for isolation of salmonella spp. from faecal samples of cracked egg fed hens and other diets from different protein sources like control diet soybean meal, experimental diets namely soybean meal + cracked egg (3.25%), soybean meal + cracked egg (7.50% ) and they analyzed three feed and 30 faecal samples. The results of their study suggested that the Salmonella spp. were isolated and identified in 2 out of the 3 diets (soybean meal + cracked egg 7.50% and soybean meal + cracked egg 3.25%), as well as in the faeces of 5 hens fed on ration soybean meal + cracked egg (7.50%) and 3 22
hens fed on ration soybean meal + cracked egg (3.25%) which further indicated that an increased prevalence of Salmonella spp. in the faeces of hens which were fed with diets supplemented with different amounts of cracked egg. Hoorebeke et al (2009) revealed that in all European Union Member States, Salmonella monitoring in poultry flocks is obligatory and in these monitoring programmes, a limited number of pooled faeces and/or dust samples were collected to determine Salmonella's presence in the flocks. They verified the sampling protocols that were adopted to detect expected low within-flock prevalences of an intermittently shed pathogen was sufficiently sensitive or not and they made comparison between different sampling procedures for the assessment of the between- and within-flock prevalence of Salmonella in laying hens. In total, 19 farms were sampled in their study using a comparable sampling methodology as in the official surveillance programmes and found that Salmonella could not be detected in any of the flocks. After transportation of the hens to the laboratory and subsequent analysis of cloacal swabs and caecal contents, Salmonella Enteritidis was detected in laying hens from five of 19 farms. They observed within-flock prevalence ranged from 1% to 14%. Based on the results of the study, depending on the sampling procedure, different estimates of the prevalence of Salmonella could be obtained and the proportion of Salmonella infected flocks was underestimated based on the results of the official monitoring programme. Koyuncu and Haggblom (2009) compared the ability of the standard method used for isolation of Salmonella in feed in the Nordic countries, the NMKL71 method (Nordic Committee on Food Analysis) to that of Modified Semisolid Rappaport Vassiliadis method (MSRV) and the International Standard Method (EN ISO 6579:2002) with five different feed materials namely wheat grain, soybean meal, rapeseed meal, palm kernel meal, pellets of pig feed and also scrapings from a feed mill elevator and four different levels of the Salmonella serotypes viz., S. Typhimurium, S. Cubana and S. Yoruba which were added to each feed material. The results of their study indicated that three methods showed no differences in detection levels, with an accuracy and sensitivity of 65% and 56%, respectively. However, Muller-Kauffmann tetrathionate-novobiocin broth (MKTTn), performed less well due to many false negative results on Brilliant Green agar (BGA) plates. Compared to other feed 23
materials, palm kernel meal showed a higher detection level with all serotypes and methods tested. They concluded that the accuracy, Sensitivity and specificity of the investigated cultural methods were equivalent but the detection levels for different feed and feed ingredients varied considerably. Orji et al (2009) Isolated Salmonella from poultry droppings and other environmental sources in Awka region, Nigeria and were able to get isolates of different Salmonella serotypes from all the sources. Salmonella Paratyphi A had an isolation rate of 12.5% from poultry droppings, 4.2% from fresh beef, and 2.1% and 4.2% from meat retailers' aprons and tables, respectively. Other serotypes isolated from the sources included SalmonellaTyphimurium, Salmonella Enteritidis, Salmonella Gallinarum, Salmonella Pullorum, Salmonella Typhi and Salmonella Agama. SalmonellaTyphi was not isolated from poultry droppings throughout their study. 2.3 Effect of sanitizers on surface inoculated Salmonella Typhimurium on raw shell eggs, shell penetration and recovery in internal egg contents Padron (1995) evaluated the dipping of shell eggs in hydrogen peroxide solution to eliminate Salmonella Typhimurium from egg shell membranes and found that dipping Salmonella Typhimurium-contaminated eggs twice in 6% hydrogen peroxide solution reduced the average number of organisms in eggshell membranes by 95% and the number of Salmonella Typhimuriumpositive eggs by 55% compared with the infected untreated group. He observed that dipping the eggs in 6% hydrogen peroxide solution did not adversely affect hatchability. Schoeni et al (1995) studied the growth and penetration of Salmonella Enteritidis, Salmonella Heidelberg and Salmonella Typhimurium inoculated into yolks and albumen of shell eggs and showed that all strains increased 3 logs or more in number in one day when incubated at 25 °C. with maximum numbers of Salmonella ranged from 108 to 1010cfu/g. However, they stated that all strains grew at 10 °C, but peak numbers were lower and occurred later than those at 25 24
°C. They also noted that at 25 °C, all three Salmonella strains penetrated the shell in 3 days, but at 4 °C, only Salmonella Typhimurium was found in one membrane sample and all Salmonella-positive samples were detected by enrichment. Bacterial penetration into eggs washed with three commercial eggwashing chemicals and stored at different temperatures and times was studied by Hong and Slavik (1998) namely quaternary ammonium compound (QAC, pH 7.5), sodium carbonate (Na2CO3, pH 12) and sodium hypochlorite (NaOCl, 100 ppm, pH 7.5). Their experiment included 150 intact-shell eggs which were washed at 43.3°C with each of three chemicals and control group was washed with tap water (H2O, pH 7.0). The washed eggs then were inoculated by immersion for 3 min into an aqueous suspension of Salmonella Enteritidis at 104 colony-forming units/ml and dried for 30 min. They kept washed and inoculated eggs at 4°C and 23°C. They further checked the bacterial penetration at 0, 1, 7, 14, and 21-day intervals. The results of microbial tests showed that both QAC and sodium hypochlorite treatments reduced bacterial penetration to less than 3.4% and 6.7%, respectively, on day 1 and 16.7% on day 21. The sodium carbonate treatment facilitated bacterial penetration during egg storage to less than 30% on day 1 and 76.7% on day 21. The eggs washed with tap water had a bacterial penetration rate of less than 6.7% on day 1 and 20% on day 21. As the storage intervals increased to 21 days, the bacterial penetration rate was found to be increased. It was also observed that the different storage temperatures (4°C and 23°C) did not cause a significant difference in bacterial penetration rates within a 21-day interval. The results of electron microscopy showed that QAC and sodium hypochlorite at 100 ppm resulted in microbiologically clean eggs and did not destroy egg shell surfaces but protected the eggs against future bacterial recontamination. The alkaline sodium carbonate produced visually clean eggs but altered the eggshell surface, which allowed bacterial recontamination. A study by Miyamoto et al (1998) with respect to Salmonella penetration through egg shell associated with freshness of laid eggs and refrigeration showed that there was no significant difference between Salmonella Enteritidis and Salmonella Typhimurium in ability to penetrate through eggshell. Also Salmonella penetration was significantly decreased by cooling the eggs at 4 0C 25
for 15 min prior to immersing them in Salmonella Enteritidis or Salmonella Typhimurium suspension. The results suggested that Salmonella cells readily penetrated through the shell of freshly laid eggs, but that this penetration was suppressed by cooling the eggs before they were exposed to Salmonella suspensions. For their experiment, the eggs of 0.25 to 3 h, 3.25 to 6 h, 1 day, and 7 days old held at two temperatures were immersed in Salmonella Enteritidis or Salmonella Typhimurium suspensions containing 103 or 106 CFU/ml at 25 0C for 10 min. Cox et al (1999) reviewed bacterial penetration of the egg shell and shell membranes of the chicken hatching egg and observed that the most likely area on the egg to be penetrated was the air cell end, especially when temperature differential and moisture were favorable and the natural defenses of the egg was not enough to protect the egg completely against such attack from microorganisms. Himathongkham et al (1999) carried out experiments on efficacy of disinfection of shell eggs externally contaminated with Salmonella Enteritidis and observed that contamination of the surface of shell eggs by dipping in a culture of Salmonella Enteritidis resulted in the presence of Salmonella Enteritidis in/on the shells as well as shell membranes but not in the egg content. They observed that disinfection with Lugol's solution, chlorhexidine, ethanol, quarternary ammonium solutions or flaming after dipping in ethanol failed to achieve complete decontamination of the shell and membranes with resulting false positives when eggs were broken for culturing of the content whereas dipping eggs for three seconds in boiling water resulted in complete destruction of Salmonella Enteritidis in shells and membranes but sometimes caused the eggs to crack and concluded that the factors which accounts for presence of Salmonella Enteritidis in shell eggs made shell egg screening as a necessity. Favier et al (2000) devised an experiment to study the effect of washing of shell eggs with surfactants on Yersinia enterocolitica, mesophilic aerobic bacteria and shell microstructure. The effectiveness of Extran, Tergitol type 08, a sodium hypochlorite solution containing 100 ppm of free chlorine and a combination of Tergitol and sodium hypochlorite (100 ppm of free chlorine in wash solutions) for hen eggs contaminated with potentially pathogenic strain of Yersinia enterocolitica O:9 was assessed. Also the effect of these chemicals on 26
the mesophilic aerobic flora and the presence of Yersinia and Salmonella were investigated. The results of their study indicated that the reduction of the Y. enterocolitica populations on eggshell with Tergitol/100 ppm was highest followed by chlorine, Extran 100 ppm and combination of two surfactants with reduction of 4·27, 3·11, 3·08 and 2·38 log cycles, respectively. The alterations produced on the eggshell microstructure by the used chemicals were observed by scanning electron microscopy and blue lake penetration. They observed that the Tergitol/100 ppm chlorine combination caused the most marked alterations. Experiments by Cogan et al (2001) on the effects of inoculum size and suspending media on growth of Salmonella Enteritidis in artificially contaminated eggs showed that the growth of the organism was markedly affected by the size of the inoculum and the composition of the medium. The high levels of growth was seen when inoculum size was more than or equal to 25 cells per egg or 250 cells per egg and stored at 20 °C and at 30 °C than for 2 cells at 20 °C. Buffered peptone water was found to be the most suitable medium for suspension. Favier et al (2001) conducted experiments to asses the effectiveness of chlorine 100ppm, 3%sodium chloride, 1, 5, and 12% trisodium phosphate, and 4,573 W/cm2 ultraviolet radiation on the reduction of Yersinia enterocolitica and mesophilic aerobic bacteria from eggshell surface. The results of their study showed that 100 ppm chlorine was highly effective and comparable with UV exposure for more than 25 min. with 1.28 and 1.60 log reductions of mesophilic aerobic bacteria as against control eggs with 4.55 log CFU/egg. They obtained highest reductions of the average bacterial count (7.35 log CFU/egg) on Y. enterocolitica-inoculated eggs with 5 and 12% TSP and 100 ppm of chlorine and the reductions achived with 12% TSP (3.74-log reduction) was significantly higher (P < 0.05) than those obtained with the remaining treatments. They also found that the Y. enterocolitica was more resistant to UVR than the eggshell natural mesophilic aerobic microflora, except when low inoculum (4.39 log CFU/egg) was assayed Knape et al (2001) studied response of food borne Salmonella Enteritidis and Salmonella Typhimurium as marker strains inoculated on to egg shell surfaces to disinfectants that were iodine based and performed under simulated industry egg processing conditions with a commercial egg washer used for the sanitizer delivery system. Shell eggs were sanitized by distilled deionized water; 27
Iodine based disinfectant and chlorine 200 ppm with re-circulated egg washer water containing 1.40--2.85 g/l total dissolved solids. The results of their study indicated that all treatments significantly (p < 0.05) decreased Salmonella spp. populations on the shell compared to dry (no spray) egg controls. However, efficacy of egg sanitizers appeared to be dependent on the level of total dissolved solids in the egg wash water. Surface pasteurization of shell eggs was tried by James et al (2002) for decontamination of table eggs which might be able to eliminate the drawbacks of other methods of sanitation especially egg washing technology that facilitated the penetration of bacteria into whole egg contents. They applied four different heat treatments viz., hot air, hot water, infra-red radiation, and atmospheric steam for the surface pasteurization and concluded from their results that temperatures of different heat treatments were sufficient to achieve significant reductions in Salmonella numbers which could be attained on the outside of an egg without raising the interior temperatures and avoid coagulation of the egg contents. Davies and Breslin (2003) carried out investigations for possible alternative decontamination methods to inactivate Salmonella Enteritidis on the surface of table eggs by ionized water, exposure to ozone in a dry as well as humid atmosphere and commercial herbal antibacterial product. Results of their study suggested that commercial ionized water anolyte was highly effective while ozone in a humid environment was only partially effective in eliminating Salmonella Enteritidis from shell egg surface. Russell (2003) carried out a study on effect of sanitizers BioSentry 904 and Biox H applied by electrostatic spraying on pathogenic and indicator bacteria such as Salmonella Enteritidis, Staphylococcus aureus, and Listeria monocytogenes and the indicator bacterium Escherichia coli which were applied to eggs and allowed to attach for 1 h on to the surface of eggs. It was observed that BioSentry 904 completely eliminated all Salmonella Enteritidis on 100, 93, and 60% of eggs in experiments 1, 2, and 3, respectively where as BioSentry Biox H combination completely eliminated all Salmonella Enteritidis on 100, 93, and 93% of eggs in experiments 1, 2, and 3, respectively. Similarly, BioSentry 904 completely eliminated all Staph. aureus on 100, 87, and 100% of eggs in experiments 1, 2, and 3, respectively. BioSentry Biox H combination completely 28
eliminated all Staph. aureus on 100, 100, and 80% of eggs in experiments 1, 2, and 3, respectively. BioSentry 904 completely eliminated all L. monocytogenes on 100% of eggs in experiments 1, 2, and 3. BioSentry Biox H combination completely eliminated all L. monocytogenes on 93, 87, and 73% of eggs in experiments 1, 2, and 3, respectively whereas BioSentry 904 completely eliminated all E. coli on 93, 93, and 60% of eggs in experiments 1, 2, and 3, respectively. BioSentry 904 and Biox H completely eliminated all E. coli on 93% of eggs in experiments 1, 2, and 3, respectively. Russell concluded that the sanitizers BioSentry 904 and Biox H were found to be extremely effective when used in conjunction with electrostatic spraying for eliminating pathogenic and indicator populations of bacteria from eggshell surfaces. Cole (2004) discussed the aspects of Food Safety Objectives (FSO) and Performance Objectives (PO) for industry and Governments, their concepts and current status for wider applications. He proposed a mathematical equation from which process or product performance criteria could be established to achieve the stated Food Safety Objectives or Appropriate Level of Protection (ALOP). Similar views were expressed by (Buchanan 2004, and ICMSF 2005). Soljour et al (2004) conducted experiments on efficacy of egg cleaning and/or compounds such as sodium carbonate, sodium hypochlorite, and potassium hydroxide and evaluated for bactericidal activity at pH values of 10, 11, and 12 against various concentrations (102, 104, or 106 CFU/ml) of Salmonella Enteritidis inoculated onto the eggshell surface. The results of their study indicated that none of the chemicals applied at the recommended manufacturer's concentrations (sodium carbonate 36 ppm, other treatments 200 ppm) could eliminate Salmonella Enteritidis from eggshells artificially contaminated with the highest bacterial concentrations of 104 or 106 CFU/ml. They also suggested that higher concentrations of each product, at least 5 to 20 times greater than recommended doses, were needed to destroy the bacteria on egg surfaces. For both shell and suspension assays, inactivation of Salmonella Enteritidis occurred at lower concentrations at pH 12 than at pH 11 and pH 10. However, neither pH nor contact time influenced Salmonella Enteritidis inactivation when the initial bacterial numbers on eggshells were high. 29
Hutchison et al (2005) employed small scale bucket washer for shell egg washing and its implications on the microbiological qualities of the eggs and showed that the bucket washer reduced the total bacterial numbers on the surface of cage-produced eggs under manufacturer-recommended conditions on average by 1.5 log CFU per egg. The washing of soiled or dirty eggs and without replacing the wash water led to increase the levels of bacteria on the surfaces of the shells of visibly clean eggs. In one of the batches of the washed eggs, the bacteria could be detected in 10% of the pooled content of the eggs after two weeks of storage at 15єC as against 0% in unwashed egg contents. Jones et al. (2005) conducted a study to examine the effects of cool water washing on the microbial quality of shell eggs. They employed six dual tank wash water temperature schemes for their ability to reduce naturally occurring aerobic bacteria and inoculated Salmonella Enteritidis (SE). The wash water schemes consisted of T1 (48.9oC), T2 (48.9oC, 23.9oC), T3 (48.9 0C, 15.6oC), T4 (23.9oC), T5 (15.6oC) and T6 (23.9oC, 15.6oC) and pH range 10.511.5 throughout the study. Eggs were exposed to the wash water temperature schemes in a pilot egg washer with recirculation of wash water tanks. The total period of time eggs were exposed to the wash water combinations was 60 sec. Washed eggs were sprayed at 48.9oC, with 200 ppm chlorine rinse solution. Eggs were stored and sampled for 9 wks. They observed that the external aerobic populations were lowest for T1 (typical U.S. wash water configuration), followed by T2 and T3. Aerobic surface contamination was greatest in T5 eggs. All treatments reduced Salmonella Enteritidis levels in a similar manner as detected by shell and membrane emulsion and egg contents pools after enrichment. De Reu et al (2005) assessed the Influence of eggshell condensation and heat stress for laying hens on the bacterial eggshell penetration and the whole egg contamination with Salmonella enterica serovar Enteritidis by agar filled and whole egg inoculation technique with 104 CFU Salmonella enterica serovar Enteritidis. They kept one half of the eggs at 20°C and 60% relative humidity (RH) for 21 days and the other half was stored at 6°C and 85% RH for 24 hrs and immediately kept at 20°C and 60% RH for 20 days. Their study indicated that the bacterial eggshell penetration increased significantly from 50% for the control group to 64% for the group of eggs where condensation on the eggshell 30
took place but did not result in increase of whole egg contamination and it was between 9 and 11% for condensed and non condensed egg shell, respectively. The influence of heat stress in laying hens on bacterial eggshell penetration and whole egg contamination with Salmonella Enteritidis was studied for two breeds viz., Isabrown Warren and Lohmann Brown by the same workers De Reu et al (2005). They kept one group of each breed of the birds under heat stress for 9 days at 32 °C at the beginning, middle and end of laying period while the control group remained at standard conditions of 20°C. The results of their study indicated that at the beginning of laying period, differences between all groups were found to be non significant either the breed or the heat stress affected the shell penetration or the whole egg contamination where as at the middle of laying period, the breed influenced the shell penetration by bacteria significantly. They also observed that Isabrown Warren eggshells were penetrated by bacteria up to 23% while Lohmann Brown eggshells were penetrated up to 42%. They concluded that, the beginning and the middle of laying periods showed that the whole egg contamination of eggs produced by heat stressed hens was lower (not significant) compared to eggs from the control. Romo and Yousef (2005) tried ozone and UV radiation for inactivation of Salmonella enterica Serovar Enteritidis on shell eggs for effective egg sanitization treatments. Shell eggs were exposed to Salmonella enterica serovar Enteritidis at the dose rate of 8.03x 105 to 4.03 x106 CFU/g of eggshell and were treated with gaseous ozone (O3) at 0 to 15 lb/in2 gauge for 0 to 20 min, UV radiation at 100 to 2,500 mW/cm2 for 0 to 5 min and combined treatment of UV (1,500 to 2,500 mW/cm2) for 1 min, followed by ozone at 5 lb/in2 gauge for 1 min. The results of the study indicated that all treatments were effective and, significantly (P, 0.05) reduced Salmonella on shell eggs with 5.9 to 4.3 log reductions. Guan et al (2006) conducted an experiment on survival of Salmonella Typhimurium DT104 and Salmonella Enteritidis in chicken egg albumin and yolk under in vitro conditions and found that the both organisms were capable of surviving in albumin and yolk at 24 hrs of incubation at 420C and concluded that both the organisms could be found in albumen during the formation of egg and survive for longer period and were influenced by storage conditions. 31
Egg shell factors influencing egg shell penetration and whole egg contamination by different bacteria, including Salmonella Enteritidis was investigated by De Reu et al (2006) with 7 selected bacterial strains namely Staphylococcus warneri, Acinetobacter baumannii, Alcaligenes sp., Serratia marcescens, Carnobacterium sp., Pseudomonas sp. and Salmonella Enteritidis which were recovered from egg contents. They attempted to correlate bacterial eggshell penetration with various eggshell characteristics and bacterial strains and to assess the contamination of whole eggs with the bacterial strains. The results of their study suggested that the eggshell characteristics such as area of the egg shell, shell thickness and number of pores did not influence the bacterial egg shell penetration but for each individual bacterial strain the mean cuticle deposition was lower for penetrated as compared to non-penetrated eggshells. The whole egg contamination was not influenced by either the area of the eggshell or the porosity of the eggshell. A significant finding of their study showed Gram-negative, motile and non-clustering bacteria penetrated the egg shell most frequently. Pseudomonas sp. (60%) and Alcaligenes sp. (58%) were found to be the primary invaders followed by Salmonella Enteritidis (43%) and all strains were able to penetrate and penetration was observed most frequently after 4­5 days. 2.4 Shelf life studies of raw shell eggs Li et al (1985) studied keeping quality of eggs packaged in acrylonitrile pouches with regard to quality and compared with not-packaged, packaged in air, packaged with 15% CO2, oil coated and oiled eggs stored at refrigeration temperatures and found that there was decrease in internal quality of eggs as storage day increased but the greatest decline was observed for the notpackaged group which had the highest albumen pH and greatest weight loss. They observed that the percent weight loss of an individually packaged egg in a high barrier pouch was about 40­50% less than those not-packaged and no significant difference in Haugh unit was found between the packaged systems 32
and the oiled plus refrigeration. They concluded that controlled atmosphere was the most efficient method for preserving egg quality at room temperature for a period of 7 weeks. Clark and Bueschkens (1986) studied the effect of storage on survival of Campylobacter jejuni and found that up to 10% of the hatched birds carrying C. jejuni in the intestine when infected eggs were stored for 5.5 days before incubation and inoculation of eggs after 8 days in storage also failed to yield infected chicks. From the study it can be concluded that Campylobacter jejuni survival affected by storage period. Theron et al (2003) performed evaluation of the effects of various storage and transport conditions on the bacterial growth associated with shell eggs which were usually transported without temperature control. They subjected the shell eggs to temperature shocks to simulate various transport and storage conditions and results of their study indicated that 4-h low temperature shock was the most effective condition for limiting organism growth in or on eggs. Much higher organism counts were found in both the egg contents and on the eggshells, which had been subjected to high temperature shocks and concluded that the egg industries could incorporate a cold shock of 4 h and consequent storage at 25 °C for longer egg shelf life and enhanced egg quality. Jones et al (2004) conducted studies to determine the microbial quality of commercially processed shell eggs during extended storage. They sampled both washed and unwashed eggs and stored on pulp flats at 4°C for 10 weeks which further analysed for microbial quality namely total aerobes, yeasts and molds, Enterobacteriaceae, and Pseudomonads from external egg rinses and pooled egg contents. It was found that upon storage no differences were found between unwashed and washed eggs for Enterobacteriaceae and Pseudomonads in either shell rinses or contents as well as no differences were found between treatments for population levels of total aerobes or yeasts and molds in the egg contents throughout the storage period. Significant differences between treatments were found at each week of storage for external shell contamination by total aerobes. The highest unwashed egg contamination occurred at week 8 of storage and the lowest was at weeks 0 and 1 of storage. The highest shell contamination with aerobic bacteria on the washed eggs was found at week 0 of storage and the lowest was at week 7. Yeast and mold 33
contamination determined by shell rinses was also found to be significantly different between treatments at each week of storage. Commercialy washed eggs were significantly less contaminated than unwashed eggs. Samli et al (2005) studied the quality of eggs from old laying hens upon storage time and temperature. For experiments, shell eggs from 50 wk old Bovans-White hens subjected to various storage periods for 2, 5 and 10 days at 5, 21, and 29°C temperatures. They found that albumen height, Haugh unit, pH of albumen and yolk, specific gravity and air cell size have been found to be the most important parameters which were greatly influenced by the storage time and temperature. Extending the storage time up to 10 days and the temperature up to 29°C resulted in significant deterioration in egg quality which drastically resulted in dropped Haugh unit to 76.3, 53.7 and 40.6 at 5, 21 and 29 єC storage temperatures, respectively. It was also observed that the size of air cell has exceeded 4 mm at the storage conditions of 2 day period and over 21єC temperature, increased pH in albumen with 2 days storage time was observed regardless of storage temperature, pH in a 5 day storage period continued to raise from 7.47 to 9.2 at 5 and 21єC, respectively. Interaction effect between the storage time and temperature were also significant on the egg weight loss, specific gravity, air cell size, Haugh unit, albumen height and pH. The results of the present study suggested that Haugh unit, pH of albumen and air cell size were the most important parameters greatly influenced by the storage period and storage temperature in laying hens. Biladeau and Keener (2009) studied the effects of edible coatings on chicken egg quality under refrigerated storage and employed four food-grade coatings namely paraffin wax, mineral oil, soy protein isolate, and Whey Protein Isolate (WPI) and were applied to fresh chicken eggs. The eggs were stored for 12 wk in refrigerated storage at 7°C. Egg properties measured included Haugh units, albumen pH, yolk pH, albumen CO2 content, vitelline membrane strength, water loss, shell strength, and shell color. The results of their study found that coated eggs maintained higher Haugh units beyond 6 wk compared with the uncoated eggs. Also, coated eggs maintained a higher CO2 content and lower albumen pH than the uncoated eggs over the storage period. Vitelline membrane strength slightly decreased over time in uncoated eggs, but did not change in coated eggs. Overall, oil, wax, and WPI-coated eggs maintained higher vitelline 34
membrane strength (14%) than the uncoated eggs. Coating of chicken eggs with a food-grade film (oil, wax, WPI) extended shelf-life of eggs beyond 6 wk. Rocculi et al (2009) studied the MAP storage of shell hen eggs for 28 days of storage at 25 °C and its effect on physico-chemical characteristics of the fresh product. They observed that the Modified Atmosphere Packaging (MAP) permitted a strong reduction of the weight loss from the product while the greatest quality decline was observed for the control eggs (not packed). The eggs packed in CO2 maintained the initial values of Haugh unit during storage and the albumen pH was about 2 units lower than in the control group. Nevertheless, the light yellow colour of CO2 sample albumen deepened and the shell gradually developed a powdery surface. It was concluded that MAP with 100% N2 did not promote any additional benefits to the eggs in comparison with the product packed in air. 2.5 Pesticide and antimicrobial residues Hidalgo (1986) found organochlorines in eggs of 8 different species of aquatic birds. Between 1983 and 1984 a total of 137 eggs were collected on Pajaros Island, a nesting site located near the Nicoya Gulf (Pacific Coast of Costa Rica). Residues of p, p'DDE were found in 100% of the egg samples analyzed; the highest concentration was found in wood stork eggs (Mycteria americana) and lowest was in white ibis, Eudocimus albus. Heptachlor epoxide, HCB, p, p'DDT and endrin were present in a high percentage of the samples analyzed. A strong correlation was found between shell thickness and p,p' DDE residues in most of the bird species sampled for analysis. He also observed the cracks in some of the eggs of M. americana with the highest concentrations of DDE. Schenck et al. (1994) introduced the screening procedure for organochlorine and organophosphorus pesticide residues in eggs using a solidphase extraction cleanup and gas chromatographic detection. Eggs were extracted with acetonitrile. The extract was subjected to a cleanup on Tandem C18 and Florisil SPE columns. Organochlorine and organophosphorus pesticide 35
residues were determined by GC with electron capture and flame photometric detection, respectively They reported average recoveries of 9 spiked organochlorine pesticide residues (0.01-1.0 ppm) between 80.9 to 91.1% whereas average recoveries of 7 spiked organophosphorus pesticide residues (0.02-0.50 ppm) between 80.3 to 89.5%. They further stated that the SPE method resulted in a 90% reduction in organic solvent consumption and 85% reduction in hazardous waste production as compared to the AOAC methodology. Furusawa et al. (1998) described gel permeation and florisil chromatographic clean up and gas chromatographic determination of organochlorine pesticides in eggs. Clean up of residual organochlorine pesticides (OCPs; alpha-, beta-, gamma-, and delta-BHC; aldrin; dieldrin; p, p'DDE; o,p'-DDT; p,p'-DDD; and p,p'-DDT) in eggs by Gel Permeation Chromatography (GPC) and Florisil Mini column Chromatography were performed. Ten OCPs in purified extract were determined by gas chromatography with electron capture detection. The lipids extracted from whole egg were cleaned up first by GPC with an Envirogel column and an ethyl acetate-cychlohexane (1 + 1, v/v) mobile phase and then by Florisil mini column chromatography with 15% (v/v) diethyl ether-hexane eluant. They reported average recoveries of 10 spiked OCPs (0.0025-0.0125 ppm) between 81 to 101%, coefficients of variation between 1 and 14% with detection limit as 0.001 ppm. Donoghue and Hairston (1999) conducted study to determine whether the approved doses of Oxytetracycline (OTC) for breeder hens and meat-type poultry would produce drug residue transfer into egg components when fed to laying hens and observed that residues of OTC were not detectable during the pre dosing, dosing, or during the withdrawal period in egg yolks. They observed the Oxytetracycline residues in egg albumen on the 5th day of treatment and the 1st day of medicated feed withdrawal. They found that the concentrations were close to the limit of the assay's sensitivity (117 ppb) and concluded that the allowed doses of drugs for breeder hens or meat type poultry might not produce consistently detectable levels of residues of OTC in eggs. 36
Donoghue and Myers (2000) developed prediction models for the study of transfer or compartmentalization of drug residues into eggs as the egg yolk serves as an important storage depot for chemical residues. They employed high-resolution Magnetic Resonance Images (MRI) for visualizing compartmentalization of drug residues and the results of their study indicated that after drug dosing, images of drug residues in eggs showed that drugs could be incorporated and compartmentalized into ring structures within individual developing egg yolks. The study further suggested that even after a single dose, sequestered drug residues may be stored and later released to contaminate eggs for days to weeks after dosing. Schenck and Donoghue (2000) carried out study on determination of organochlorine and organophosphorus pesticide residues in eggs using a solid phase extraction clean up. A multiresidue solid phase extraction (SPE) method for the isolation and subsequent gas chromatographic determinations of non polar organochlorine and polar organophosphorus pesticide residues in eggs were described. The method used an acetonitrile extraction followed by SPE cleanup with graphitized carbon black and aminopropyl SPE columns. Organophosphorus pesticides were determined by gas chromatography with flame photometric detection. After further clean up of the extract using Florisil SPE columns, organochlorine pesticides were determined by gas chromatography with electron capture detection. They observed average recoveries between 86-108% for 8 fortified organochlorine pesticide residues while 61-149% for 28 fortified organophosphorus pesticide residues. Lehotay et al (2001) analyzed the samples of eggs for pesticide residues by direct sample introduction to Gas Chromatography/Tandem Mass Spectrometry for detection and confirmation. The analytical procedure developed by them had no clean up or solvent evaporation steps and the results obtained were quantitative and confirmatory with detection limit of <10 ng/g detection limits for 25 of 43 tested pesticides from several chemical classes. The procedure consisted of addition of 2 g of NaCl and 19.3 mL of acetonitrile (MeCN) to the 10 g of egg in a centrifuge tube and mixed for 1min by probe blender and centrifuged for 10min followed by injecting the 10 µL (5 mg of egg equivalent) of the extract using DSI/GC/MS-MS. The direct sample introduction (DSI) or dirty sample injection method considered as rapid, rugged, and 37
inexpensive approach to large volume injection in gas chromatography (GC) for semi volatile analytes such as pesticides. Bordet et al. (2002) conducted an inter laboratory study to validate gas chromatographic (GC) method for determination of 21 organochlorine pesticides, 6 pyrethroid pesticides, and 7 polychlorobiphenyl (PCB) congeners in milk, beef fat, fish, and eggs. The method was performed at low contamination levels, which represent relevant contents in food. It enlarged the applicable scope of the reference EN method to pyrethroid pesticides and proposed the use of SolidPhase Extraction (SPE) as a cleanup procedure. They also employed cryogenic extraction method and SPE cleanup was performed with 2 successive SPE cartridges: C18 and Florisil. Purified extract was injected on a GC column, residues were measured by electron capture detection. Food samples (liquid milk, beef fat, mixed fish, and mixed eggs) were prepared, tested for homogeneity, and sent to 17 different laboratories in France. Test portions were spiked with 27 pesticides and 7 PCBs at levels from 26 to 45, 4 to 27, 31 to 67, and 19 to127 ng/g into milk, eggs, fish, and fat, respectively. The observed the relative standard deviation of the spiked samples in the range of 1.5 to 6.8% in milk, 3 to 39% in eggs, 4.5 to 12.2% in fish, and 7 to 13% in fat. They also noticed the relative standard deviation for reproducibility were found to be ranged from 33 to 50% in milk, 29 to 59% in eggs, 31 to 57% in fish, and 30 to 62% in fat. Varga et al (2002) studied residues of Fenitrothion in chick embryos quantified in yolk and embryonic samples following exposure of fertile eggs to Fenitrothion an organophosphorus insecticide. Fertile hen's eggs were immersed in 0.33% aqueous solution of Sumithion 50 EC on day 0 or day 12 of incubation, while in the second part of the experiment eggs were injected with 0.1 ml of water/DMSO solution (9:1, v/v) containing 0.15 mg fenitrothion having the same exposure time. Following day 0 exposure, eggs were incubated until day 12, and eggs treated on day 12 were incubated until day 19. They determined the residues by gas chromatographic analysis and demonstrated that fenitrothion was able to cross the eggshell after external exposure and contaminate the developing embryo with a penetration rate that increased after the mobilization of Calcium from the eggshell. The fenitrothion residues measured in the samples indicated that the embryos were contaminated by the 38
chemical for a longer time after external exposure than after administration of the insecticide by injection. Moreover, the data suggested that fenitrothion appeared at distinct embryonic developmental stages after exposure by immersion and injection might have different toxic effects on the embryo. Donoghue and Schneider (2003) Compared between a bioassay and Liquid Chromatography-Fluorescence-Mass Spectrometry for the determination of incurred Enrofloxacin in whole eggs and results demonstrated that both methods were capable of detecting incurred fluoroquinolone residues in eggs. They found that during the three-day dosing period of hens (Day 1, 2 and 3) and following drug withdrawal (Days 5, 7 and 9) both of the methods were able to detect incurred Enrofloxacin in eggs within a 99% confidence level above the zero tolerance established by the FDA. The LC-Fluorescence-MS method had the benefit confirmation for fluoroquinolones, while the bioassay could be used as an effective, rapid screening method for detection of illegal fluoroquinolone residues in eggs. Fotina et al (2005) in their study for determination of veterinary drug elimination time from table eggs obtained from bacterial infected poultry flocks found that the residue and accumulation or their presence for drugs such as Nitrofuranes, Furazolidon Enrofloxacin, Sulfametoxin, Cefotoxin and Leveomicetin in the egg white. The highest levels of Nitrofuranes and Furazolidon were found in eggs. The concentration of antimicrobial residues on different days from 1st to 20th day of their study showed variations with a trend towards accumulation from 1st day after last treatment and Nitrofuranes concentration in egg was 315.00±1.0g/g, 3.89±1.1g/g and 2.16 ±0.8 for days 10 and 20, respectively whereas for Enrofloxacin and Sulfametoxin the concentration on 1st day was 5.12±1.8 and 5.17±1.8g/g, respectively, while for 20th day it was found to be 0.02±0.01 to 2.57±1.12 g /g. They also observed that the Cefotoxin concentration on the 1st and 20th day was 1.12±0.6 g /g. and 0.01±0.002 g /g. They concluded that the eggs were not safe for consumption even up to 20 days after the treatment. Garcia and Regueiro (2005) reported that residues of chemicals such as ions or elements including heavy metals and other trace elements, and radionuclides, mycotoxins, other organic contaminants including organochlorine compounds, dioxins, other pesticides, polychlorinated biphenyls, brominated 39
flame retardants, and mineral oil hydrocarbons plant and natural plant products and drugs determine the safety and quality of shell eggs where in the rate of transfer of residues into the egg content showed variations and newer analytical methods as a tool for determining the residues could provide better safety for consumers. Mourin (2005) collected samples of free- range chicken eggs from different sites like near waste incinerators, cement kilns, the metallurgical industry, waste dumps, and chemical production facilities and found high levels of dioxin and PCB contamination of which 70% of the samples exceeded the European Union (EU) limit for dioxins in eggs. The samples from India collected at and near by places of Hindustan Insecticides Limited contained dioxins and DDT at four and three times the European Union (E.U.) norms where as the level of Hexachlorobenzene (HCB), used as a fungicide, in the sample was found to be seven times higher than the E.U. norms. The chicken eggs served as indicators of environmental contaminants and industrial pollution. Studies by Goodman et al (2006) have shown that not all applied pesticides might actually reach the targeted pests but had the potential to get into the soil, water, and the atmosphere and acted as continuous non point sources of contamination to foods. More over the work of Daren highlighted that the pesticides act by affecting the specific body processes and not specific to targets (Daren, 2007). Marcineak et al (2006) investigated the utility of Premi®Test as screening test for detecting Sulphadimidine residues in eggs of laying hens after oral administration of Sulphadimidine (120 mg/hen/day) and compared with four-plate microbiological method, and HPLC. They confirmed positive findings of the Fourplate test (FPT) with the results of Premi®Test and found that the presence of Sulphadimidine residues was detected by Premi®Test within 8 days and by the FPT within 3 days after the last administration. The FPT test reported falsenegative results for five days (kappa < 0.6) as compared with the results of Premi®Test. The study confirmed the conformity of results obtained by both Premi®Test and HPLC (kappa = 0.6). Lohajova et al (2006) studied suitability of star and Premi®Test for the detection of amoxicillin residues in laying hens and observed that the residues were detected in eggs, muscles, kidneys, and liver with or without thermal 40
treatment. The sensitivity and correlation of results achieved by applying of STAR method and Premi®Test were compared which indicated that the Premi®Test might provide a suitable means for the detection of drug residues in poultry eggs, meat and organs. It was also showed that samples without thermal treatment produced significantly larger zones than samples thermally treated correlated with results of Premi®Test Janosova et al (2007) compared the sensitivity of antibiotic residue screening methods - the four plate test (FPT), the screening test for antibiotic residues (STAR), and the Premi®Test to 10 different standards from the Sulphonamide (SA) group. Phtalylsulphathiazole (PHT), Sulphadimidine (SD), sulphaguanidine (SG), sulphachlorpyridazine (SCHP), Sulphamerazine (SRZ), Sulphamethoxazole (SMX), Sulphanilamide (SAM), Sulphanilic acid (SAC), Sulphaquinoxaline (SQ) and Sulphathiazole (STZ) using the concentrations from 0.05g/ml to 1g/ml that represented the minimum inhibiting concentration (MIC) and the results of experiments showed that Premi®Test was found to be the most sensitive method to sulphonamides followed by STAR method and FPT. Premi®Test detected six SA at the level of the maximum residue limit (MRL) 0.1 g/ml set for SA group, STAR method detected five SA at the level of MRL, and FPT detected only one SA at the level of MRL. Study on drug residues and contaminants in poultry products in India as reported by Narahari and Amutha (2007) indicated that 79% of the egg samples had undetectable pesticide levels, and the rest had 0.026, 0.031, 0.014 and 0.013g/g egg of Chlorpyrifos, Endosulfan, Deltamethrin and Fenvalerate, respectively and in poultry meat samples, these values were 0.028, 0.022, 0.023 and 0.014g/g, respectively where as in another study about 10% of the 900 poultry liver samples were found to be positive for Chlorpyrifos residues, ranging from 0.07 to 0.35g/g, with a mean of 0.085 g /g. Among the drug residues Chloramphenicol was the most studied antibiotic and its residue in egg yolk could persist for up to 66 days, followed by Sulphadimidine and arsanilic acid residues in egg albumen. Das and Bawa (2008) studied the distribution of Oxytetracycline(OTC) residues in eggs after oral administration to hens and found that the highest OTC residue concentration in egg white 0.5944 µg/ g was significantly (p < 0.05) lower than that in yolk 0.6587 µg/ g, but the highest total OTC residue (18.8610 µg) as 41
well as total OTC residue on any of the days from 2 to 10, in the entire white portion was significantly (p < 0.01) higher than that in entire yolk. They observed that the mean cumulative residue and grand mean residue concentration were greater in egg white than those in yolk. They employed High-Performance Liquid Chromatography-Ultraviolet Visible detection (HPLC-UV) technique. Tao et al (2009) conducted a survey for Organochlorines pesticide residues in chickens and eggs at poultry farm in Beijing, China and determined the concentrations of hexachlorocyclohexane isomers (HCHs) and dichlorodiphenyltrichloroethane and metabolites (DDTs) from chicken organs, animal feed, droppings, ambient air and eggs. The results of their study revealed that mean fresh weight concentrations of HCHs and DDTs were 0.122 ± 0.061 ng/g and 0.051 ± 0.038 ng/g in the muscles and contributed to per capita daily intake of HCHs and DDTs were 487% and 88% of those of fish consumption and chicken and eggs acted as an important pathway of HCHs and DDTs exposure to human beings. 42
MATERIALS AND METHODS The present research work on quality and safety of raw shell eggs was carried out to cover a wide spectrum of analysis of raw shell eggs to know the quality (physicochemical and shelf life or keeping quality) and safety (microbiological and chemical) at the point of production (farms) and in the markets (sale and distribution points) as it destined to reach the consumers as a source of food. The study also included the effect of sanitizers on shelf life and safety aspects. Details of the study and the experimental methods adopted are described in this chapter in a brief manner. 3.1 Chemicals and reagents 3.1.1 Chemicals, culture media and reagents All the chemicals used for the study such as culture media ready to use broths and selective media plates for cultivation of bacteria, standard microbiological cultures and solutions for laboratory analysis were of analytical or laboratory grade and were procured from M/s EOS Laboratories Pvt. Ltd. Mumbai, M/s E merk (India), M/s Hi-media Laboratories Pvt. Ltd (India), M/s Qualigens (India), M/s S D Fine ­ Chem. Ltd and M/s Sigma Aldrich, USA and other reputed national and international firms. The media used for the present work included Buffered Peptone Water (BPW), Rappaport Vassiliadis broth (R.V. broth), Brilliant Green Sulfa Agar plates (BGSA), Xylose Lysine Deoxycholate agar plates (XLD), polyvalent antisera O and O1 for Salmonella, Bolton broth, modified Charcoal Cefeperazone Deoxycholate Agar plates (mCCDA), Campylobacter selective blood agar plates, Campy gas packs, Cairy Blair transport medium, Triple Sugar Iron (TSI) and urea slants, 43
hippurate and nitrate medium, Gram's stain and dilute carbol fuchsin stain for selective isolation/ detection and identification of Salmonella and Campylobacter species respectively. All the glassware/plastic ware used for isolation was of Borosil / Tarson brand and was sterilized before use. 3.1.2 Scientific instruments Gas chromatography system (Shimadzu model GC 2010) equipped with 63Ni Electron Capture Detector, and Flame Thermionic Detector fused silica capillary column (30 m x 0.25 mm i.d.), auto sampler and auto injector with GC solution software were used for detection and quantitation of pesticide residues in raw shell egg samples. 3.2 Collection of samples of raw shell eggs 3.2.1 Commercial layer farm The samples such as raw shell eggs, egg surface swabs and faecal content from cloaca of laying hens were collected from two organized commercial layer farms comprising white Leghorn breed of birds producing white shelled eggs. The birds were reared in back to back cage layer system. The strength of one layer farm was about 1.25 lakhs birds (Farm A) and the other farm was about 0.5 lakhs (Farm B). The birds were protected against major infectious diseases through recommended routine vaccinations. Per day egg production was in the range of 88 to 90%. The good quality raw shell egg samples free from dirt, visible cracks and defects were collected from commercial layer farms. The poultry farm environmental samples such as litter material, feed, water and egg tray wash samples were also collected and analyzed for Salmonella and Campylobacter species. The details of the total number of samples collected from the farms are represented in Tables 44
3.1and 3.2 and the details of the break up of the samples taken for each parameter under study are represented in Tables of respective parameter in this chapter. The swab samples collected were placed immediately into the Cairy Blair transport medium and used for further analysis. The samples of processed or finished egg break fast product from two McDonald fast food centers were also collected for analysis of Salmonella and Campylobacter species as one of the farms had integration with food processing plant as part of the egg supply chain. 3.2.2 Markets The raw shell egg samples were collected from different markets in Mumbai and the samples included white shelled eggs from Market A and C, desi eggs from Market B and brown shelled eggs from Market D. The details of the market samples collected are represented in Table 3.1. Egg rinses were used (sterile normal saline solution 10 ml /egg) for microbiological analysis of external surface and whole egg contents were used for microbiological and residue analysis. 3.3. Evaluation of physico-chemical quality of raw shell eggs 3.3.1 Sampling plan The simple random sampling method was adopted and the size of the sample is represented in the Table 3.3. The raw shell eggs were collected three times or on three occasions from the commercial layer farms and one time from four different markets A, B, C and D. 45
3.3.2 Measurements or evaluation of raw shell egg quality The physico-chemical quality of raw shell eggs were evaluated in terms of its external quality attributes such as egg weight, shape index, shell weight and shell thickness and the internal quality attributes such as height of thick albumin, Haugh Unit, yolk index and pH by egg break out method. 3.3.2.1 Measurement of external egg quality a. Egg weight (g) The eggs were weighed in an electronic balance and the weight was recorded. b. Shape index (%) The length of the eggs (mm) were measured by holding the eggs in the vertical position and measurements were taken from broad end to the tip of narrow end using a vernier dial caliper and the width/ diameter (mm) was measured by holding the eggs horizontally and then measuring the width at middle portion of the eggs. These measurements were used to calculate shape index. The shape index of each egg was calculated by dividing the egg width by egg length and multiplied by 100 as per Panda (1996). Shape index = Egg width / Egg length x 100 c. Shell weight (g) The weight of the egg shell was recorded after removing the internal contents, cleaned the inner surface with tissue paper to remove adhering albumin and later allowed to be dried for 24 hrs at room temperatures. 46
Table 3.1: Total number of samples collected from all sources for physico-chemical, microbiological, pesticide and antimicrobial residue analysis
Sr. Source No. 1. Farm A 2. Farm B 3. Market A 4. Market B 5. Market C 6. Market D Total
Physicochemical Egg Whole analysis surface Egg
90
30
30
60
30
30
20
15
15
20
15
15
20
15
15
20
15
15
230
120
120
Microbiological Analysis
Cloacal Litter Feed Water Egg Tray
swabs
washings
30
5
5
5
5
30
5
5
5
5
NA
NA NA NA
NA
NA
NA NA NA
NA
NA
NA NA NA
NA
NA
NA NA NA
NA
60
10
10
10
10
Grand Total = 644
Finished Egg Products from fast food centers 10 NA NA NA NA NA 10
Pesticide Residue 10 10 3 3 3 3 32
Antimicro bial Residue 10 10 3 3 3 3 32
47
Table 3. 2: Total number of raw shell egg samples collected for sanitation, shell penetration and recovery and shelf life studies from commercial layer farm
Egg Sanitation Pre and Post Inoculation 45 +45 = 90
Shell penetration and
recovery
Pre and post Good Vs.
Inoculation Poor shell
eggs
50 + 50 =100
36
Shelf life
Ambient Refrigeration (28 - 33 0C) (3 - 7 0C)
114
312
Total Samples 652
Table 3.3: Total number of raw shell egg samples procured from farms and markets for physico-chemical quality study.
Sr. No. Source
1
Farm A
2
Farm B
3
Market A
4
Market B
5
Market C
6
Market D
Total
NA = Not applicable
Number of collections
Lot 1
Lot 2
Lot 3
30
30
30
20
20
20
20
NA
NA
20
NA
NA
20
NA
NA
20
NA
NA
130
50
50
Total No. Samples 90 60 20 20 20 20 230
d. Shell thickness (mm) Shell thickness was measured along with its membranes with the help of disc micrometer. Three measurements were taken i.e., first at broad end of the eggs, second at the middle portion and the third at the narrow end and then average of three measurements were taken as the thickness of the shell. 3.3.2.2 Measurement of internal egg quality The internal egg quality attributes such as height of the thick albumin, Haugh unit values, albumin pH and yolk index was carried out by egg break out method.
48
The eggs were broken on to a clean flat glass surface and individual measurements were recorded. Vernier dial caliper was used for recording linear measurements and pH meter for recording pH. a. Albumin height (mm): The height of the thick albumin was recorded by depth micrometer provided in the vernier dial caliper. b. Haugh Unit: The Haugh unit which is a measure of freshness and albumin quality was calculated as per Eisen et al (1962) by employing the formula 100 log (Albumin height ­ 1.70 x Egg weight 0.37 + 7.57) and the values were recorded. c. Yolk index (%): The height of the standing yolk (mm) on the albumin and the diameter of the yolk (mm) were measured by using vernier dial caliper. The yolk index was calculated by employing the formula Yolk height / Yolk diameter x 100 as per Panda (1996). d. Albumin pH: Albumin pH was measured using digital pocket pH meter after calibration. Albumin contents were taken in a small beaker and pH meter was dipped inside and values were recorded. 3.4 Microbiological quality of eggs Microbiological quality of eggs is one of the well established methods utilized to guide, implement and improve existing practices of production, processing, preservation of the product`s safety and wholesomeness which conveys the quality of the product to consumers and quality assurance program to the industries. Total Viable Counts (TVC), detection of spoilage/ saprophytic organisms as sanitary indicators and detection of commonly and frequently associated pathogens as safety indicators are the established course of the study of microbiological quality of any food products including eggs. Therefore, in the present study TVC and 49
detection of Pseudomonas spp of fresh shell eggs as sanitary indicators and Salmonella spp and Campylobacter spp as safety indicators were used for assessing microbiological quality of eggs.
3.4.1 Total Viable Count (TVC)
The Total Viable Count (TVC) of external egg surface and whole egg contents were carried out for assessing the microbial load of the shell eggs by performing pour plate technique. The log dilutions of the sample in sterile normal saline solutions were made up to 10-5 for external egg rinse and 10 -3 for internal whole egg contents of which 0.1 ml of the inoculum was put into Petri plates in duplicates for each dilution of 10-4 and 10-5 and 10-2 and 10-3 respectively for external egg rinse and internal whole egg contents. The molten nutrient agar cooled to 450C was poured on to the inoculum and rotated in both clock wise and anti clock wise directions for thorough mixing and allowed for solidification of media in the plates. After solidification, the plates were incubated at 370 C for 24 to 48 hrs. The counts of viable Colony Forming Units (CFU) were made after the incubation period was over and the Total Viable Count (TVC) was determined.
TVC was calculated by using the following formula:
C
CFU/ml = ---------------------------
Where:
[n1 + (0.1 x n2)] x d
C = Total no. of colonies counted from all plates
n1 = No. of plates of lower dilution n2 = No. of plates of higher dilution d = Dilution factor
50
3.4.2 Detection of Pseudomonas spp. The detection and enumeration of the Pseudomonas spp was done by serial log dilutions in sterile normal saline solution up to 102 followed by selective plating of the pooled samples of external egg rinses and internal whole egg contents on to the ready made cetrimide plates procured from EOS Laboratory Pvt. Ltd. 3.4.3 Isolation and identification of Salmonella and Campylobacter species 3.4.3.1 Sampling plan For the collection of samples of raw shell eggs, surface swabs and cloacal swabs simple random sampling (probability sampling) was adopted with the estimated prevalence for the organism at 10% level so that positive samples could be obtained if they were present. Accordingly the sample size was determined and presented in Table3. 4. 3.4.3.2 Isolation of Salmonella species a. Raw shell egg surface, cloacal swabs and whole egg internal contents The procedure for isolation and identification of the Salmonella species from the samples was carried out as per US-FDA Bacteriological Analytical Manual (BAM) (2008) with necessary modifications. All procedures were carried out in an aseptic manner and sterilized glass wares and media were used for the purpose. The swab samples from the Cairy Blair transport media were put into 0.1% peptone water, mixed well and 1 ml was transferred into 10 ml buffered peptone water and incubated at 370C for 18 to 24 hrs which served as non selective enrichment. After non selective enrichment, I ml was transferred to 10 ml Rappaport Vassiliadis ( R.V.) broth and incubated at 420C for 24 to 48 hrs which served as selective enrichment for Salmonella species. Plating was carried out on selective media Brilliant Green Sulpha Agar (BGSA) and incubated at 370C for 24 to 48 hrs. Purification of the suspected colonies was done by plating on to MacConkey's plates and used for biochemical and serological confirmation of the isolates. The 51
same protocol was adopted for analysis of surface egg rinses where eggs were washed with 10 ml sterile normal saline solution per egg and 1 ml rinsate was transferred to 10 ml buffered peptone water and other steps followed in similar way as above. b. Whole egg internal contents Raw shell eggs were surface disinfected with 70% ethanol and then two eggs per sample unit were aseptically broken to obtain the 100 g (100 ml) of whole egg internal contents and were collected in plastic sample bags and homogenized in stomacher for 60 sec. 10 ml of the homogenized sample was transferred to 90 ml of buffered peptone water and incubated at 370C for 18 to 24 hrs as non selective enrichment and rest of the steps for isolation carried out as described for egg surface and cloacal swabs. c. Finished egg products Ten gram of the egg product was thoroughly minced and was transferred to 90 ml of buffered peptone water and incubated at 370C for 18 to 24 hrs as non selective enrichment. Then 1ml of the pre enriched sample was transferred to 9 ml of Rappaport-Vassiliadis selective enrichment broth and incubated for 24-48 hrs. After incubation, selective plating was done on Brilliant Green Sulpha Agar plates for isolation of the Salmonella spp. d. Feed, litter and water samples Twenty five gram of feed sample was taken in 225 ml of buffered peptone water,10 gram dry litter in 90 ml buffered peptone water and 10 ml water sample in 90 ml buffered peptone water and homogenized/ mixed thoroughly and incubated for non selective enrichment at 370C for 24 hrs. Further steps of enrichment and selective plating were carried out in the same manner as done for other samples analyzed in the present study for Salmonella spp. 52
The identification of the organism was done as per US-FDA protocol (2008) and Barrow and Feltham (2003). The suspected colonies on Brilliant Green Sulpha Agar plates were taken and purified by streaking on to MacConkey's agar plates and incubated at 370C for 24 to 48 hrs. Further confirmation of organisms was done by performing various biochemical, sugar fermentation tests and by slide agglutination test with polyvalent O and O1 antisera for Salmonella species. e. Slide agglutination test One to three loopful of cultures of presumptive positive Salmonella from Triple Sugar Iron (TSI) agar slants was suspended in 0.5 ml of sterile normal saline solution and was used as antigen. Three portions/sections were made on two clean microscopic slides, and one drop of normal saline was placed on left and right sides of both the slides and in the middle portion/section one drop of antigen suspension was taken. To this, one drop of polyvalent O" antisera was added to one of the slides and to other O1" antisera was added separately and mixed well with the help of a sterile loop. Agglutination reaction in the form of clumps or flocs in 60 sec. with antigen suspension was considered as Salmonella positive. 3.4.3.3 Isolation of Campylobacter species a. Raw shell egg surface, cloacal swabs and whole egg internal contents The procedure for isolation and identification of the Campylobacter species from the samples was carried out as per US-FDA Bacteriological Analytical Manual (BAM) (2008) with necessary modifications. The swab samples from the Cairy Blair transport media were put into 0.1% peptone water, mixed well and 1 ml was transferred into 10 ml Bolton broth and placed inside the anaerobic jar under microaerophilic conditions created by gas generating Campy gas packs. The jar was incubated at 370C for 4 to 5 hrs which served as selective pre enrichment. After selective pre enrichment, jar was transferred to the incubator maintained at 420C for 48 hrs which served as selective enrichment for Campylobacter species. Plating 53
was carried out on selective media modified Charcoal Cefeperazone Deoxycholate Agar (mCCDA) and incubated at 420 C for 48 hrs. Purification of the suspected colonies was done by plating on to mCCDA plates and used for biochemical and morphological confirmation of the isolates. The same protocol was adopted for analysis of surface egg rinses where eggs were washed with 10 ml sterile normal saline solution per egg and 1 ml of egg rinsate was transferred to 10 ml Bolton broth and other steps followed in similar way as above. b. Whole egg internal contents 10 ml of the homogenized sample was transferred to 90 ml of Bolton broth and placed inside the anaerobic jar under micro-aerophilic conditions created by gas generating Campy gas packs. The jar was incubated at 370C for 4 to 5 hrs as selective pre enrichment and rest of the steps for isolation carried out as described for egg surface and cloacal swabs. c. Finished egg products Ten gram of the egg product was thoroughly minced and was transferred to 90 ml of Bolton broth and incubated at 37 0C for 4 to 5 hrs as selective pre enrichment under micro-aerophilic conditions in an anaerobic jar. Then pre enriched sample was transferred to the incubator set at 42 0C for selective enrichment and kept for 48 hrs. After incubation, selective plating was done on modified Cefeperazone Charcoal Deoxycholate Agar plates for isolation of the Campylobacter spp. d. Feed, litter and water samples Ten gram of feed, 10 gram dry litter and 10 ml water samples were taken in 90 ml of Bolton broth separately for each of the samples, homogenized/ mixed thoroughly and incubated for selective enrichment at 37 0C for 4 -5 hrs under micro- 54
aerophilic conditions in an anaerobic jar. Further steps of selective enrichment and selective plating were carried out in the same manner as done for other samples analyzed in the present study for Campylobacters. The identification of the organism was done as per US-FDA Bacteriological Analytical Manual (BAM) protocol (2008), OIE terrestrial manual (2008) and Barrow and Feltham (2003). The suspected cultures were tested for oxidase and catalase reaction, reaction in Triple Sugar Iron Agar and microscopic appearance of organism. Only oxidase positive, catalase positive, alkaline reaction in slant and butt with H2S negative reaction in TSI agar together with characteristic microscopic appearance of curved or sea gull shaped organism with dilute carbol fuchsin stain was considered as positive for Campylobacter species. 55
Table 3.4: Number of samples collected from commercial layer farms and markets for detection of Salmonella and Campylobacter species
Source
Egg surface
Whole egg content
Farm A
30
30
Farm B
30
30
Market A
15
15
Market B
15
15
Market C
15
15
Market D
15
15
Lower
NA
NA
Parel
Kalamboli NA
NA
Total
120
120
NA ­ Not Applicable
Cloacal swabs 30 30 NA NA NA NA NA NA 60
Type of sample
Litter Feed
Water
5
5
5
5
5
5
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Egg tray washings 5 5 NA NA NA NA NA
NA
NA
NA
NA
10
10
10
10
Finished egg product
Total
NA
110
NA
110
NA
30
NA
30
NA
30
NA
30
5
5
5
5
10
350
56
3.5 Effect of various sanitizers on surface inoculation of Salmonella Typhimurium on raw shell eggs 3.5.1 Sanitary trial design It was envisaged to study the effect(s) of sanitizers' when challenged with surface inoculation of Salmonella Typhimurium on to the eggs and its recovery in the internal contents at different time intervals. The details of the sample requirements and the trial design are described in Table 3. 5. The trial consisted of three sanitizers viz, 70% alcohol, 65 ppm Polyhexamethylene Guanidine (PHMG) and 200 ppm of chlorine with the inclusion of the appropriate control samples. The sanitizers were selected on the basis such as, the 70% alcohol is US FDA recommended chemical for surface raw shell egg disinfection before microbiological analysis of liquid egg contents, Polyhexamethylene Guanidine is a new chemical with promising sanitizing abilities and chlorine is an established sanitizer used for decontamination purposes routinely in food processing establishments and commonly used sanitizer for shell egg surface disinfection in industries and egg processing plants. The desired concentration of sanitizers were prepared from respective stock concentration of the sanitizers/ chemicals or as per label instructions of the manufacturer and utilized for the trial. The chosen concentration is in the range of their germicidal concentrations. The surface inoculation or challenge studies in raw shell eggs were carried out as per FDA (USDA) (2004) protocol with little modifications. a. Raw shell eggs used for the trial The apparently clean and good quality raw shell eggs collected from the commercial layer farm were used for the experiment. Egg candling was done to remove the cracked and poor shelled eggs as per the plan. 57
Table 3. 5: Effect of different sanitizers on surface inoculation of Salmonella Typhimurium on raw shell eggs
Pre Inoculation
Treatment
Sample size X
Replicates
Salmonella free eggs
3X3=9
Inoculation control
3X3=9
70% Alcohol
3X3=9
65 ppm PHMG
3X3=9
200 ppm Chlorine
3X3=9
Total
45
Post Inoculation
Sample size X
Total eggs
Replicates
3X3=9
18
3X3=9
18
3X3=9
18
3X3=9
18
3X3=9
18
45
90
b. Preparation of Salmonella Typhimurium culture for surface inoculation
The standard culture of Salmonella Typhimurium grown on Brilliant Green Sulpha Agar from which 2-3 colonies were picked up and inoculated into 10 ml peptone water and incubated at 37 0 C for 24 hrs. The 24 hour culture was further diluted to required quantity in the ratio of 1:10 and used for surface inoculation. The dose of the inoculum was approximately 10 8 cfu/ml as enumerated by log dilution and plating on to Xylose Lysine Deoxycholate (XLD) agar.
c. Surface inoculation to raw shell eggs
The trial was carried out with two different experimental patterns i.e., first one, the eggs were first inoculated with the organism and sanitizers were applied later, termed as pre inoculation and the second one, the eggs were sanitized first with the sanitizers and organism was inoculated later, termed as post inoculation.
i. Pre inoculation
A 500 ml sterile beaker was taken to which 30 ml of fresh culture of Salmonella Typhimurium (i.e. age of the culture was 24 hrs) grown in peptone broth
58
was poured and 270 ml of sterile peptone water was added in order to get a log dilution of 1:10 and thus 300 ml of total volume of the culture was prepared. The raw shell eggs were immersed in the culture for about 10 sec., drained, placed back into the plastic pouches and allowed to be dried for 30 min. ii. Post inoculation The inoculation culture was taken in four 500 ml beakers that contained 300 ml peptone culture to which pre sanitized eggs i.e., the eggs treated with known selected concentration of sanitizers viz., 70% alcohol, 65 ppm Polyhexamethylene Guanidine and 200 ppm of chlorine were immersed separately to avoid carry over effect of sanitizers and to ensure the appropriate dose of the inoculum to each eggs in the treatments. Then eggs were immersed in the culture for about 10 sec., drained, placed back into the plastic pouches and allowed to be dried for 30 min. d. Application of different sanitizers to eggs Raw shell eggs were sprayed with 70% alcohol, 65 ppm Polyhexamethylene Guanidine and 200 ppm of chlorine with the help of sprayers to all eggs in the respective treatment groups of pre and post surface inoculated eggs with Salmonella Typhimurium. The approximate quantity or volume of sanitizers delivered to each egg was about 8 -10 ml and allowed an effective contact period of about 15 to 20 minutes. e. Assessment of efficacy (effect) of sanitizers The effectiveness of the above said sanitizer was assessed in terms of Total Viable Count (TVC) and recovery of Salmonella Typhimurium from the surface of raw shell eggs. The raw shell eggs were rinsed with sterile normal saline solution after 15-20 minutes of contact period with the sanitizers. 59
i. Total viable count (TVC) Spread plate technique was employed. Serial 10 fold dilutions up to 10-4 was prepared from 1 ml of the raw shell egg rinsate and 0.1 ml of rinsate was taken from dilution 3 and 4 and was spread on to the previously prepared plate count agar plates (in duplicates) with the help of sterile plastic spreader. Plates were incubated at 37 0C for 24-48 hrs and Total Viable Counts (CFU/ml) were made after the completion of incubation period. ii. Recovery of Salmonella Typhimurium The recovery of viable Salmonella Typhimurium was carried out as per the US FDA protocol for Salmonella isolation (2008). One ml of the egg rinsate from each egg belonged to different sanitizer treatment groups were taken in to 10 ml of non selective (pre) enrichment buffered peptone water, incubated at 370C for 24-48 hours followed by selective enrichment in 10 ml Rappaport Vassiliadis (R.V.) broth i.e., 1 ml quantity of culture from buffered peptone water transferred to enrichment medium, incubated at 42 0C for 24-48 hours and selective plating of the inoculum from R.V. broth on to Xylose Lysine Deoxycholate ( XLD) agar plates kept for incubation at 37 0C for 24-48 hrs. Plates were observed for growth of Salmonella Typhimurium and counts of the organism were made (enumerated) wherever growth was observed and the log reduction values were derived. 3.6 Shelf life study of raw shell eggs 3.6.1 Sampling plan The egg samples were collected randomly from an organized commercial layer farm and the defective eggs were replaced with good quality eggs by candling. The details of the sample requirement along with the frequency or the time interval when required numbers of samples were drawn for break out/quality study are 60
presented in the Table3.6. Four sanitizers were selected for disinfection or decontamination of the egg shell surfaces viz., 70% alcohol, 65 ppm Polyhexamethylene Guanidine, and 200 ppm chlorine, 100 ppm of hydrogen peroxide with colloidal silver and paraffin oil for coating after sanitation / disinfection. Thus the shelf life study comprised of six groups, i.e. combination of four different sanitizers and paraffin oil, one coated with paraffin oil and one as control. Control group had only the fresh raw shell eggs. The eggs were withdrawn at 4 intervals for ambient temperatures and 6 intervals for refrigeration storage. 3.6.2 Protocol / procedure for shelf life study a. Weighing, candling and allotment of eggs to treatment group The eggs procured from the farm were weighed, candled and allotted to different treatment groups viz., 70% alcohol, 65 ppm Polyhexamethylene Guanidine, 200 ppm of chlorine, sanitizer with hydrogen peroxide 100 ppm, white mineral oil and fresh raw eggs without treatment as control for both ambient storage (28-330 C) and refrigerated storage ( 3-7 0C) conditions. The cracked eggs and eggs with other shell defects were not included for shelf life study. b. Cleaning and surface disinfection of raw shell eggs with sanitizers The raw shell eggs were first cleaned for the purpose of treatment with sanitizers by dry cleaning the eggs with sand papers for sanitizing egg with alcohol, Polyhexamethylene Guanidine and hydrogen peroxide stabilized with colloidal silver and wet cleaning the eggs with hot water (temp. 39-45 0C, pH 8-9) for chlorine as both the methods are accepted method of cleaning of shell eggs in different countries of the world. The process adopted in the wet cleaning of eggs before sanitation by 200 ppm chlorine included rinsing and brushing with hot water, detergent and post rinsing or final washing with hot water. The dry cleaning of shell eggs were carried out before sanitation of the eggs by 70% alcohol, 65 ppm of Polyhexamethylene Guanidine and 100 ppm of hydrogen peroxide stabilized with colloidal silver. After disinfection of shell, the eggs were allowed to dry for some time 61
in the air and then all eggs except the control group were coated with white paraffin oil and stored at ambient storage (28 -330C) and refrigerated storage (3 -7 0C) conditions for determining shelf life of eggs. c. Measurement of quality of eggs during storage The shelf life of eggs was determined by taking the samples of raw shell eggs at fixed regular intervals of time for ambient (28 -330C) and refrigerated (3 70C) storage conditions as presented in the Table 3.6. Physico-chemical and microbiological qualities of the broken out eggs were recorded. The average loss of weight for eggs during storage was also recorded. 3.7 Shell penetration and recovery study of Salmonella Typhimurium in whole egg contents from pre and post surface inoculated and sanitized raw shell eggs Shell penetration and recovery study of SalmonellaTyphimurium were under taken in eggs treated with three different sanitizers which were preserved at refrigeration temperatures. Samples of two eggs from each of the treatment and control group were drawn on 0, 4, 14, 21 and 28 days as shown in Table 3.7. The eggs were broken aseptically and the internal contents were collected in plastic bags and homogenized in stomacher for 60 sec. The rest of the procedure was carried out as mentioned in the section 3.4.3.2 except that selective plating was done on Xylose Lysine Deoxycholate (XLD) plates and observed for the growth of the organism. The growth of the organism detected on any of the selected day in the internal contents was considered as recovery of the organism through the penetration of egg shell that survived during sanitation of the egg shell surface. 62
3.8 Measurement or fixing of performance criteria for sanitary process to achieve Food Safety Objectives (FSO) or Performance Objectives (PO) Performance criteria and performance objectives are the practical industrial tools for achieving the desired food safety goals for each of the product they process, produce and market it to the consumers. In this study, an attempt was made to measure or fix the performance criteria for shell egg sanitary process and raw shell eggs as the product by considering the whole process of shell egg sanitation, penetration by the Salmonella Typhimurium inside the eggs after sanitation and its survival or detection in the egg contents based on in vitro studies. Food Safety Objectives (FSO) just do not consider the microbiological criteria/numerical reductions of the organisms but try to focus on aspects such that whether a product is safe at the time of consumption by the consumers. For this purpose, the method elucidated by Cole (2004) was used in the present study as expressed by the following equation. FSO, H0 R and I are expressed in log10 units. H0 ­ R + I FSO where H0 = initial level of the hazard R = Total (cumulative) reduction of the hazard I = Total (cumulative) increase of the hazard FSO = Food Safety Objective 63
Table 3.6: Number of raw shell egg samples analyzed for shelf life study after treatment with paraffin oil alone and in combination with different sanitizers
Interval (days) 0 7 15 30 40 30 60 90 120 150 180 Total
Control
Paraffin oil
70% alcohol+ Paraffin oil
3
3
3
3
3
3
3
3
3
4
4
4
6
6
6
8
8
8
12
12
12
12
12
12
12
12
12
4
4
4
4
4
4
71
71
71
Treatments
65 ppm PHMG +
H2O2 stabilized with Ag 100 ppm+
Paraffin oil
Paraffin oil
Ambient (28-33 0C)
3
3
3
3
3
3
4
4
6
6
Chilled ( 3- 7 0C)
8
8
12
12
12
12
12
12
4
4
4
4
71
71
200 ppm chlorine + Paraffin oil 3 3 3 4 6 8 12 12 12 4 4 71
Total No. of samples 18 18 18 24 36 48 72 72 72 24 24 426
64
Table 3.7: Number of samples analyzed for shell penetration and recovery study of SalmonellaTyphimurium in whole egg contents treated with different sanitizers
Intervals Salmonella Positive control (days) free eggs
0
2
2
4
2
2
14
2
2
21
2
2
28
2
2
0
2
2
4
2
2
14
2
2
21
2
2
28
2
2
Total
20
20
Treatments
Total Samples
70% Alcohol 65 ppm PHMG 200 ppm Chlorine
Pre Inoculation
2
2
2
2
2
2
2
2
2
2
Post Inoculation
2
2
2
2
2
2
2
2
2
2
20
20
2
10
2
10
2
10
2
10
2
10
2
10
2
10
2
10
2
10
2
10
20
100
65
3.9 Shell penetration and recovery study of Salmonella Typhimurium in whole egg contents from surface inoculated good and poor shell quality eggs
Plan of experiment and method
The raw shell eggs were candled and good shell quality and poor shell quality eggs were selected. Eggs were surface inoculated with Salmonella Typhimurium. Eggs were preserved at refrigeration temperatures. Two eggs from different groups (good vs. poor shell) and control were drawn on 0, 4, 7, 14, 40 and 80 days as shown in Table 3.8. The eggs were broken aseptically and the internal contents were collected in plastic bags and homogenized in stomacher for 60 sec. The rest of the procedure was carried out as mentioned in the section 3.4.3.2 except that selective plating was done on XLD plates and observed for the growth of the organism. The growth of the organism detected on any of the selected day in the internal contents was considered as recovery of the organism through the penetration of egg shell.
Table 3. 8: Number of samples analyzed for shell penetration and recovery of artificially inoculated SalmonellaTyphimurium in eggs good versus poor quality shells
Interval (days) 0 4 7 14 40 80 Total
Salmonella free eggs 2 2 2 2 2 2 12
Parameter Good shell 2 2 2 2 2 2 12
Poor shell 2 2 2 2 2 2 12
Total samples 6 6 6 6 6 6 36
66
3.10 Assessment of safety of raw shell eggs for organochlorines, organophosphates and synthetic pyrethroid pesticide and antimicrobial residues 3.10.1 Pesticide residues Chemicals of diverse nature appear as contaminants in the foods via the food chain where animals and birds occupy an important trophic level in the food web. Pesticides are one such group of chemicals which may appear in the shell eggs because of it's wide spread use in agriculture, public health and pest control in birds and animals. Some of the pesticides such as organochlorines and polychlorinated biphenyls show persistent properties and remain in the environment for long time where as others remain for shorter periods of time. Not only that organochlorines and polychlorinated biphenyls have bioaccumulation and biomagnification properties in the animal or bird tissues. Shell eggs from hens have higher probability of containing residues of pesticides as its feed constitutes major food grains and animal protein sources which likely to be contaminated with pesticides. Therefore, the present work on detection of pesticide residues in shell eggs was carried out. A multiresidue Gas Chromatography (GC) technique with Electron Capture Detector (GC-ECD) for organochlorines and synthetic pyrethroids and Gas Chromatography with Flame Thermionic Detector (FTD) method for organophosphates and Gas Chromatography ­ Mass Spectrometry (GC-MS) standardized and validated at Western Region Referral Laboratory (WRRL), Dept. of Veterinary Public Health, Bombay Veterinary College, for detection and confirmation of pesticide residues in meat, poultry and eggs was used for detection and confirmation of pesticide residues in shell egg samples collected from farms and markets under the present study. The selected Organochlorine (OC) pesticides included o,p'-DDE, p,p'-DDE, o,p'-DDD, p,p'-DDD, o,p'-DDT, p,p'-DDT, -HCH, HCH, -HCH/Lindane-HCH, -Endosulfan, -Endosulfan, Aldrin and Heptachlor as well as synthetic pyrethroids included -Cyhalothrin, Cypermethrin, Fenvalerate and 67
Deltamethrin in shell egg samples were subjected to GC- ECD for detection. The selected Organophosphorus (OP) pesticides included Phorate, Parathion methyl, Malathion, Chlorpyriphos ethyl, Phenthoate, Profenophos and Ethion in shell egg samples were subjected to GC­FTD for detection. Positive samples were subjected to GC­MS for confirmation.
3.10.1.1 Sampling plan
Samples of raw shell eggs were collected from commercial layer farms and markets randomly as shown in the Table 3.9 for detection and quantitation of pesticide residues.
Table 3.9: Number of samples of raw shell eggs for pesticide and antimicrobial residues
Sr. No.
Source
1.
Farm A
2.
Farm B
3
Market A
4.
Market B
5.
Market C
6.
Market D
Total
Pesticide residues 10 10 3 3 3 3 32
Antimicrobial residues 10 10 3 3 3 3 32
Total Samples 20 20 6 6 6 6 64
3.10.1.2 Method a. Sample preparation Fifteen eggs were broken and pooled which represents one laboratory sample. 20 g of homogenized analytical sample was taken for extraction and clean up.
68
b. Extraction and clean-up of homogenized egg sample Extraction and clean-up procedure for organochlorines from egg samples were carried out as per AOAC Official Method 970.52 for organochlorine, synthetic pyrethroid and organophosphate pesticide residues - General Multiresidue Method (AOAC, 2000). The details of extraction and clean-up are given in Flowcharts 1 and 2. The preparation of working standard solutions of pesticides were made from standard stock solutions of (1 ppm) for Organochlorine (OC), Synthetic Pyrethroid (SP) and Organophosphate (OP) by diluting 50, 250, 500, 750, 1000, 1250, and 1500 µl in 5 ml volume of n- hexane in 5 ml volumetric flasks which provided pesticides concentrations of 10, 50, 100, 150, 200, and 250 ng/ml of OC, SP and OP, respectively. The linearity studies with spiked egg samples with these concentrations of OC, SP and OP pesticides were carried out during standardization of the protocol by WRRL and the same was taken for the detection and quantitation of pesticide residues for surveillance work in the present study for raw shell eggs. The maximum residue limits prescribed by Prevention of Food Adulteration Act (PFA, 2004), Codex Alimentarius Commission (2006), were used as basis for selection linearity range of 10, 50, 100, 150, 200 and 250 ng/ml and two basal folds lesser than MRL values were considered as LOQ values. c. GC­ECD and GC-FTD conditions used for detection of OC, SP and OP pesticides GC conditions used in the present study for detection and quantification of OC and SP pesticide residues in shell egg samples included nitrogen as carrier and make up gas in the column with a flow rate of 2 ml/min and 30 ml/min, respectively. The injection mode used was split type with 1l injection volume and 1:10 split flow ratio. The temperature of the ECD detector was maintained at 320 0C and that of split injector was 290 0C and the total run time for GC - ECD analysis was 26.20 minutes. The GC conditions used for detection and quantification of OP pesticide 69
residues was same as described for OC and SP compounds except that the carrier gas was helium and make up gas was hydrogen and air with the flow rate of 15 ml/min and 27.50 ml/min, respectively. The split flow ratio was maintained at 1:5. Flow chart 1: Extraction of raw shell egg sample for pesticide residues 20 gm of whole egg sample + 200 ml acetonitrile +10 gm celite Mixed well with blender at high speed for 2 min Filtered through Buchner funnel with Whatman No. 1 in 500 ml suction flask. Volume of filtrate recorded Transferred filtrate to 1 liter separating funnel 100 ml petroleum ether added, mixed for 1-2 min & then 10 ml saturated sodium chloride solution & 600 ml distilled water was added to it. Contents in separating funnel mixed for 30 ­ 45 seconds and kept in stand undisturbed for some time for separation of layers. Aqueous layer discarded, organic layer in the solvent washed twice with 100 ml of distilled water. Washings were discarded and the volume of solvent measured and passed through 15 gm of anhydrous sodium sulphate and collected in evaporating flask. Evaporated in rotary evaporator and resultant concentrate of petroleum ether extract further purified in florisil column 70
Flow chart 2: Florisil Clean-up of sample extract Florisil column (18 mm i.d) prepared with 4 g of activated florisil (approx 10 cm) topped with1 cm anhydrous. Sodium sulphate Column was pre-wet with 40 ­ 50 ml petroleum ether Petroleum ether extract passed through column with a flow rate of 5 ml / min Elute was collected in volumetric flask Containers & sodium sulphate rinsed twice with 5 ml petroleum ether All rinses were poured to the column and analytes eluted @ 5 ml / min with 200 ml eluting solvent (acetonitrile + diethyl ether) The elute was concentrated in rotary evaporator up to definite volume at (60 0C) Remaining volume was dried in nitrogen drier (400C) 5 ml HPLC grade n hexane was added and final elute was made. Elute was put to GC-ECD and FTD for detection and quantification, and GC-MS for confirmation. 3.10.2 Detection of antimicrobial residues from raw shell egg samples by Premi test Antibiotic residues in raw shell eggs constitute a major concern for the public health as well as for trade. These residues appear in the eggs because either the feed contains antibiotics as additives or by giving antibiotic therapy to the birds for the control of clinical or sub clinical infections. The pattern of appearance of 71
antibiotic residues in the eggs is not homogenous and it may be released in to the eggs from hens at unpredictable intervals. Therefore, screening for antibiotic residues in eggs serve as first step in the prevention of appearance of residues in shell eggs meant for consumption purposes. Method Detection of antimicrobial residues was carried out as per Hussein et al (2005) and samples of raw shell eggs analyzed are presented in Table 9. Pooled and homogenized (as done for pesticide residue samples) 100 µl egg sample was taken and added to Premi test kit detection vials. Then allowed to stand for 20 to 30 min. for diffusion of the sample in to the test kit medium and kept at 820C for 20 min. in an oven to inactivate the natural antimicrobial substances present in the samples. The test vials were then incubated at 640C for 3 hrs and results were read. The known negative controls without the antibiotic residues in the samples were kept with test samples. Change of colour to yellow from purple was considered as negative for antimicrobial residues and no colour change and remained as purple was considered as positive for antimicrobial residues. The antimicrobials detected by this test and sensitivity or detection limit for each antimicrobial are given in Table 3.10. 3.11 Statistical analysis The data obtained in the present study was analyzed by employing Web Agri Statistical Package (WASP) software package developed by ICAR research complex, Goa. 72
Table 3.10: Name of antimicrobials and detection level in ppb by Premi test
Sr.
Group
No.
Antimicrobials
1
-lactams
Amoxicillin
2
Cephalosporins
Ampicillin Penicillin G Cloxacilline Cectiofur
3
Macrolides
Tylosin
Erythromycin
4
Tetracyclines
Chlortetracycline
Oxytetracycline
Doxycycline
5
Sulphonamides
Sulphamethazine
6
Aminoglycosides
Sulphadiazine Gentamycin
7
Other
Streptomycin Neomycine Furazolidone
Amprolium Narasin
Sensitivity or detection limit 5 5 2, 5 100 400 50 50 600 400 200 25 25 100 1000 300 2500 2500 2500
73
RESULTS AND DISCUSSION The shell eggs from domestic chicken meant for table purposes or consumption produced from organized commercial layer farms offer nutrients of highest biological value are economically affordable and have a fairly better shelf-life at ambient temperature and quite longer period of shelf-life at refrigeration temperature, as compared to other perishable foods. The starting point for the care of the shell eggs commences right from the commercial layer farms and travels through out the egg supply chain covering processing plants, store houses, market places, fast food or ready to eat centers etc. and negligence at any point is at the cost of quality and safety of shell eggs. It is a well known scientific fact that there are some indices or indicators which are used to mark the quality and safety of the raw shell eggs that fairly reflect the various practices with which the shell eggs are associated and suggest the points / processes where corrections have to be made. These indicators are selected as found relevant to the present situation of shell egg production/ processing/ storage/ marketing etc, and included in the various parameters of the present research work. The idea of this pilot project is to know the present status of the shell egg quality and safety on a broader spectrum at the point of production (commercial layer farms) and at point of distribution (markets) and suggest appropriate Performance Objectives (POs) for industry during processing and preservation of shell eggs. In the present study raw shell eggs samples were collected from commercial layers farms and markets. The analysis of the samples focused on physico-chemical, microbial quality (Total Viable Count, Pseudomonas spp.) of raw shell eggs, detection and identification of Salmonella and Campylobacter spp. as egg safety indicators; shelf life of eggs treated with paraffin oil alone and in combination with different sanitizers and detection of pesticide and antimicrobial drug residues in eggs. A brief description of the samples collected and analysed in the entire study are given here for better clarity together with results and discussion under respective parameters taken up for the study. 74
Total 644 samples collected from all the sources for physico­chemical, microbiological {both sanitary (TVC, Pseudomonas spp) and safety (Salmonella spp and Campylobacter spp)}, pesticide and antimicrobial residues are described in Table 3.1. The total number of 652 raw shell egg samples collected for sanitation, shell penetration and recovery and shelf-life study are given in Table 3.2. All together 1296 samples were collected analyzed for different parameters of the study. An overall total of 230 shell egg samples from two commercial layer farms and four different markets were collected for physico­chemical quality analysis of shell eggs. As described in Table 3.3 the shell eggs were collected on three different occasions in the form of three lots from two commercial layer farms and each lot from farm A comprised of 30 raw shell eggs and from farm B 20 raw shell eggs while from markets, raw shell eggs were procured on single occasion i.e. one lot of 20 raw shell eggs from each of four different markets A (white shell), B (desi), C (white shell) and D (brown shell). Thus, a total of 150 eggs from farms and 80 eggs from markets were individually analysed for evaluating quality of shell eggs. The total 350 samples were analysed for overall microbiological quality and safety of the shell eggs. These comprised samples from birds (layers) i.e. cloacal swabs, environmental samples and finished egg product from two fast food centers (Table 3.1 and 3.4). For all microbiological analysis a minimum of two eggs constituted one sample. As described in Table 3.4, a total of 60 samples (120 eggs) from farm A, 60 samples from farm B (120 eggs) and 60 samples (120 eggs) from markets which comprised 15 samples each from four different markets A, B, C and D were analysed for shell contamination and presence of selected enteric pathogens from external egg surface and whole egg contents. Apart from shell eggs, a total of 60 cloacal swabs, 30 from each commercial layer farms (A and B), 40 environmental samples, 20 from each of the layer farm, which included litter samples, poultry feed samples, drinking water samples and egg tray washings i.e. 5 from each category from each farm were also analysed for prevalence of pathogens namely Salmonella spp and Campylobacter spp. As commercial layer farm A, had integration with the commercial food processing plant as part of the egg supply chain, a total of 10 finished egg products (break fast item) from two fast food centers were collected 75
and analysed for microbiological safety. The samples of shell eggs and samples from the layers from each commercial layer farm were collected on three occasions and from the market on single occasion. The results of these studies are presented and discussed in this chapter. Sixty four samples, 32 each for pesticide and antimicrobial residues analysis were collected in the present study (Tables 3.1 and 3.9). The number of samples collected included 10 each from farm A and B, 12 from markets i.e. 3 samples from each of the four markets A, B, C and D for detection of both pesticide and anti microbial drug residues. For residue analysis, one sample constituted 15 shell eggs. The number of raw shell egg samples collected from the commercial layer farm (farm A) for the purpose of sanitation trials with the artificial surface inoculation to eggs with Salmonella Typhimurium included 90 eggs, 45 eggs for pre-inoculation and 45 eggs for post­inoculation study. As described in the Table 3.5, the trials were replicated 3 times with 3 shell eggs in each treatment group of sanitizers with appropriate controls both for pre and post­inoculation studies. A sample size of 136 shell eggs were collected (Table 3.2) for analysis of shell penetration and recovery both in sanitized eggs as well as good and poor quality shell eggs. As detailed in Table 3.7 a total of 100 samples, 50 for pre­ inoculation and 50 for post­inoculation. The trial had 5 different time intervals both for pre­inoculation and post-inoculation with 10 samples of eggs (i.e. 2 each from 5 groups) per time interval were analysed. The findings of the study are described in the later part of this chapter. Similarly, as presented in Table 3.8, a total of 36 eggs, 12 samples of good quality shell eggs, 12 samples of poor quality shell eggs and 12 surface inoculated eggs as control were analysed for shell penetration and recovery of Salmonella Typhimurium at 6 different time intervals. The sample size of 426 shell eggs were collected from a commercial layer farm for shelf-life study that included 114 shell eggs for shelf life study at ambient temperature and 312 shell eggs for refrigeration temperature of storage. The shell eggs were treated with paraffin oil alone and in combination with various sanitizers and analysed at different time intervals (Table 3.2 and 3.6). The number of samples analysed for all time intervals at both ambient and 76
refrigeration temperature in the entire study comprised of 71 shell eggs under each treatment group including control. 4.1 Physico-chemical and microbiological qualities of raw shell eggs from commercial layer farms and market samples The raw shell egg samples which were collected from two commercial layer farms and different markets of Mumbai were analyzed for physico-chemical and microbiological qualities. A total of 90 egg samples from farm A, 60 from farm B, 80 eggs from markets (20 eggs from each of market A, B, C and D) were collected on different occasions (referred herein as lots) were analyzed for individual physico-chemical parameters and pooled samples for microbiological analysis. A. External quality of raw shell eggs The external egg qualities such as egg weight, egg length and width, shape index, shell weight, shell thickness and shell per cent were measured and the results of the same are presented in Table 4.1 and Figure 1. The average weights of the eggs collected from the commercial layer farms were found to be 56.69 ± 0.43 and 54.65 ± 0.38 g for farm A and B, respectively. The average weight of eggs collected from markets was found to be 57.91 ± 1.00 and 56.49 ± 0.49 g for markets A and C (white shelled eggs) where as it was 36.47 ± 0.83 and 45.51 ± 0.62 g for market B( Desi eggs) and Market D ( Brown shell) eggs. From the results it is seen that the desi eggs recorded lower average egg weights followed by brown shell eggs and white shell eggs. The results of the shell egg weights were subjected to statistical analysis between commercial layer farms and market eggs and the results of analysis are described in Tables 4.2 and 4.2 (a). From the results it is observed that even though, apparent differences were noticed in the shell egg weights between commercial layer farms and markets they were found to be non significant. 77
The weight of the shell eggs is an important quality criterion for the purpose of grading and marketing of the shell eggs. In some countries egg grading is a form of quality control used to divide variable commodity or product in to a number of quality and weight classes as per the established standards. Shell eggs are sorted, graded and assigned to respective weight classes on a lot basis such as Jumbo or XL (70 g), Extra large or Class 1(65­70 g, Large or Class 2(56­65 g),, Medium or Class 3(49­56 g, Small or Class 4(42­49 g) and Peewee (35-42g) as per USDA egg size standards in USA (USDA, egg grading manual 2000). Similarly, egg weights are classified according to Canadian egg size standards such as small (42.0­48.9 g), medium (49.0­55.9 g), large (56.0­ 63.9 g), extra-large (64.0­69.9 g) and jumbo (70.0 g or higher). The grade designations and quality of table eggs in India as stipulated by AGMARK (1968) is Extra large (60g or higher), Large (53-59g), Medium (45-52g) and Small (3844g). Though the shell eggs in India are not sold by weight basis and the consumer choices with regard to egg size are not known, the average egg weight observed in the present study for white shell eggs collected from commercial layer farms and from market A and C fall under large weight class where as brown shell eggs (from market D) fall under small and desi eggs (from market B) do not fit into any of the weight grades mentioned by AGMARK as the average weight of the eggs was 36.47 ± 0.83. Egg weight is directly proportional to the weight of albumin, yolk and shell and is chiefly controlled by breeds and selection parameters. The eggs of all breeds and localities vary (Hasting 2003) due to several reasons such as genetic, feed, climate, hen age, ovarian sequence, molting etc (Bell and Weaver 2002, Sheikh and Younis (2005). Chatterjee et al (2007) studied the comparative traits of different crosses of brown Nicobari with White Leghorn under intensive and extensive management systems in Andaman, and observed that the average egg weight was 51g and recorded non significant difference between the studied genetic groups. The findings of the present work are similar to the observations of above workers with the difference that, Chatterjee et al (2007) had recorded the medium weight grade or category for shell eggs in their work when compared to the present work. Holik (2009) also recorded the weight of 78
shell eggs of scavenging chickens from 30-50g which may well correlate with the findings of the present study with respect to desi eggs that recorded 36.47 ± 0.83. The egg length and egg width of shell eggs from all the sources were recorded and were used to determine the shape index of the eggs (Table 4.1). The egg length values found to be ranged between 51.18 ± 0.39 and 56.95 ± 0.34 mm and width was found to be 38.20 ± 0.33 to 43.11 ± 0.28 mm. The least or minimum egg length and egg width was observed in desi eggs and maximum in white shell eggs. The shape index percent calculated from values of egg length and width recorded for all the eggs were found to be between 73.79 ± 0.71 and 76.84 ± 0.34 regardless of the different categories of the shell eggs. Parmar et al (2006) evaluated the egg quality traits in indigenous Kadaknath breed of poultry from two field survey centers Jhabua-I (Meghnagar) and Jhabua-II (Jobat) of District Jhabua (Madhya Pradesh) and found that the mean shape index, pooled for both the centers was 73.93%. Shi et al (2009) recorded shape index percent of 76.77, 76.87 and 79.51 for small, medium and large brown shell eggs of Nike breed, respectively and observed significant positive correlation between egg weight and shape index. The typical shape index values range from 70 to 74 (Bell and Weaver, 2002). The shape index percent obtained in the present study is in overall agreement with these workers and showed medium sized egg recording marginally higher shape index percent as compared to that of smaller sized desi and brown shell eggs. Gawande et al (2007) who studied indigenous chicken farming in rural conditions of Assam reported similar values for egg weight and shape index percent for desi or indigenous shell eggs. The weight of the shell recorded for all categories (Table 4.1) showed that the desi (market B) eggs had least weight i.e. 3.87 ± 0.01g followed by brown shell eggs (market D) 4.26 ± 0.11g. The weights of white shell eggs from commercial layer farms and markets were found to be in the range of 5.27 ± 0.06 to 5.68 ± 0.07g. The shell percent in the eggs was highest in desi eggs that recorded 10.61% whereas the white shell eggs from farm and market showed the percentages that ranged between 9.33 and 9.84%. The brown shell eggs (market D) recorded shell percentage of 9.57%. Like egg weight which is directly proportional to that of albumin, yolk and shell and influenced by factors such as 79
genetic, feed, climate, hen age, ovarian sequence, molting, health etc, the shell weight is also influenced by such factors. Percent shell weight observed in the present study for different categories of shell eggs is within the accepted range of values where shell constitutes 11-12 % of the egg and is in consistent with the quality standards (Silversides 1994 and 2004, Akram et al 1999, USDA 2000, Bell and Weaver 2002, and Kadim et al 2008). Thickness of the shell is an important shell quality attribute. The results in the present study indicate that the average shell thickness ranged between 0.40 ± 0.00 to 0.42 ± 0.00 mm for white shell eggs from both farms (A and B) and markets (A and C) while the brown shell eggs (from market D) recorded least shell thickness of 0.37 ± 0.01mm and desi eggs (market B) recorded the maximum shell thickness of 0.43 ± 0.01mm (Table 4.1). The results were subjected to statistical analysis and it was found that the mean thickness of the shell was similar and did not differ significantly between farm shell eggs and market samples. The results of the statistical analysis are presented in Table 4.2 and 4.2 (b). Parmar et al (2006) evaluated egg quality traits in indigenous Kadaknath breed of poultry from two field survey centers [Jhabua-I (Meghnagar) and Jhabua-II (Jobat)] of District Jhabua (Madhya Pradesh) and found that the mean shell thickness pooled for both the centers was 0.31mm while the work of Gawande et al (2007) for indigenous shell eggs recorded 0.29 mm for Kamrup, Nagaon and Sibsagar breeds. The mean shell thickness reported by Baishya et al (2008) for different categories of shell eggs from different sources (farm and market) was found to range between 0.29 to 0.31mm. However, in the present study, the mean shell thickness was higher which ranged between 0.37 to 0.43 mm between different categories of shell eggs. This is in agreement with the observations of Bell and Weaver (2002) for white shell eggs. As per Stadelman and Cotterill (2002) the minimum shell thickness that is required to pass through egg supply chain with less than 50% chance of breakages during handling is 0.33 mm and the quality of shell as indicated by the shell thickness in the present study was found to be superior. 80
B. Internal quality of raw shell eggs The quality or freshness of raw shell eggs are measured by certain physico-chemical indices of the albumin and yolk where in height of the thick albumin, Haugh unit values of albumin, pH of the albumin and yolk index serve as useful quality indicators and the results of analysis are presented in Table 4.1 and Figure 2. The average height (mm) of the thick albumin from commercial layer farms was found to be 6.12 ± 0.18 (Farm A) and 6.28 ± 0.09 (Farm B) while that of the shell eggs from markets had an average albumin height of 3.63 ± 0.40 (market A), 4.58 ± 0.17 (market B), 4.02 ± 0.23 (market C) and 2.78 ± 0.43(market D). It was apparent from the results that the average height of the albumin of the eggs procured from the markets was lower or decreased height as compared to that of fresh farm eggs. The results were further subjected to statistical analysis (ANOVA) to determine whether the heights of the thick albumin showed significant variation between commercial layer farms and markets. The details of the analysis are given in Table 4.3 and 4.3(a). The statistical analysis revealed that the differences in mean albumin heights between the two commercial layer farms were non significant and indicated that the quality of the thick albumin was similar in both the farms. Similarly, the comparison of average albumin heights between commercial layer farms and market eggs showed significant (P0.01) difference which indicated that there was reduction in the thick albumin height (thinning of albumin) of the market eggs due to loss of moisture and CO2 from albumin during transport and storage and hence, losses of quality or freshness. The average Haugh unit values were calculated for shell eggs collected from layer farms and market samples (Table 4.1) and the average values were found to be 77.26 ± 1.25 and 79.91± 0.64 for farm A and farm B, respectively, whereas for market eggs from A, B, C, and D the values were found to be 52.10 ± 5.36, 77.78 ± 1.66, 58.90 ± 2.57 and 46.18 ± 6.42, respectively. It is evident from the results that, in general, all market eggs showed lower Haugh unit values except for desi eggs from market B, the values of which were near to farm eggs. The average Haugh unit values were subjected to statistical analysis between commercial layer farms and markets for comparison and variability, the 81
results of statistical analysis are depicted in Tables 4.3 and 4.3 (b). The statistical analysis revealed that the difference in Haugh unit values of farm eggs was non significant which indicated that the quality of the albumin from both the farms was similar. But significant (P0.05) differences in Haugh unit value was noticed between commercial layer farms and market eggs which suggested that quality of albumin is bound to change when it comes to market for sale. The quality of the egg yolk as percent yolk index was found to be lower for market procured eggs and values ranged between 14.54 ± 3.02 to 30.30 ± 1.23 whereas for farm eggs the indices were higher and ranged between 36.13± 0.39 and 38.70 ± 0.29. In addition to thick albumin height, Haugh unit and yolk index, the pH values of albumin are also used as indicator of freshness of eggs or egg quality. The pH of the egg albumin becomes more and more alkaline as the age of the egg increases. The results of the present work (Table 4.1) indicated that the average values of pH ranged between 8.45 ± 0.04 and 9.44 ± 0.09. The market eggs showed higher range of pH values as compared to the farm eggs. The results of pH values of albumin were subjected to statistical analysis and are presented in Tables 4.3 and 4.3 (c). The results suggest that the pH variation was found to be non significant between commercial layer farms and significant (P0.05) between commercial layer farms and market eggs. This indicated that the eggs from commercial layer possessed similar albumin quality but the loss of quality of albumin was observed in the market eggs was evident from higher pH values. The overall results of internal physico-chemical quality of the shell eggs collected from the commercial layer farms and the different markets, suggested that the egg samples collected from layer farm were of superior quality and freshness as against the market eggs which had a conspicuous loss of freshness and quality. The results of the present study on internal qualities of shell eggs from commercial layer farm and market can be compared with work of following researchers who studied the internal qualities of the shell eggs. Stadelman and Cotterill (2002) observed that the internal quality attributes namely thick albumin and yolk are lost as the age of egg increases and the time, temperature, humidity and handling are the usual factors associated with the loss of quality 82
and the rate of change in the albumin and yolk is a function of temperature and movement of carbon dioxide through the shell. Baishya et al (2008) who studied shell eggs from different sources observed that the indigenous chicken eggs were smaller in size with better internal and external qualities with appealing yolk quality where as the market chicken eggs were comparatively poor in respect of internal qualities. The findings of the present study are similar to the observations of Abo Omar and Aref (2000) who in their surveillance study of market shell eggs in Palestine observed that the source of eggs had significant effect on egg weight, Haugh unit and price per kg egg mass. Eggs obtained from super markets had significantly (P < 0.05) lower weight, the highest (P < 0.01) percentage of weight class 'small' and 'pewee1, and stained, checked eggs. Similarly, eggs from super markets had the lowest interior quality. However, these eggs were of more shell thickness and carried higher prices per egg mass unit. Most of the eggs tested were of grade (AA) with regard to the interior quality. Similar observations were reported by several research workers on internal qualities of shell eggs. Silversides and Budgell (2004), Sheikh and Younis (2005). Strahle et al (2005) observed decreased albumin height and increased albumin pH with increased egg age of both white and brown shell eggs. The pH of the newly laid egg ranges between 7.6 to 8.5 and further change in the pH of shell eggs dependent on the temperature in which eggs are held and can reach up to 9.7 within short periods as observed by Stadelman and Cotterill (2002). The Haugh unit is an expression of albumin quality which takes into consideration both egg weight and height of thick albumin. Higher Haugh values is generally accepted to indicate better albumin quality and inspite of its constraints it has been widely used as commercial and research standard as noted by Silversides (1994). Bell and Weaver, (2002),.reported that, when shell eggs are fresh the Haugh unit value of more than 90 and height of thick albumin between 8 and 10 mm were suggestive of very high quality. Roberts (2005) noted that the reduction in Haugh units was mainly affected by temperature of handling and storage. The high pH values of 9.44, reduction in albumin height and decreased Haugh units in the market egg samples observed in the present study can be corroborated with these observations as the eggs in India are 83
brought to market places in an open system and at prevailing ambient temperatures depending upon the season and geographical area. Gerber (2006) suggested that a minimum measurement in Haugh unit for eggs that goes for consumption should be 60 and the shell eggs leaving the farm should be between 75 and 85 Haugh units. The Haugh units obtained for shell eggs collected from the farm is in accordance with the Gerber's criteria. However, the USDA, has recommended four consumer grade qualities based on Haugh unit such as, more than 72 HU (AA), 60-72 HU (A), 31-60 HU (B) and less than 31HU (C). In the present study, considering this, the farm eggs were found to be of AA quality and market eggs were of B quality with the exception of desi eggs from market B that were also found to possess AA quality. C. Microbiological qualities of raw shell eggs Microbiological status is one of the key factors that determines the quality of raw shell eggs apart from its physico-chemical indices and reflects fairly on practices that are being followed in primary production at farm level, egg processing, transport and storage conditions. Moreover, the build up of the organisms on or in the eggs makes it vulnerable for rejections in the trade or reduced shelf life or increased incidence of spoilages. On these occasions, the surface contamination of the eggs and the extent of contamination determine the microbiological quality of inside of the eggs. The microbiological work on Total Viable Count (TVC) and spoilage organism Pseudomonas spp. on the external surface of eggs and in the internal contents is presented in Table 4.1 and Figure 3. The average TVC on the surface of eggs were found to be 4.5 ± 0.08 and 5.00 ± 0.03 log CFU/ml for farm A and farm B, respectively while the TVC of 5.81± 0.05, 5.44±0.05, 5.52±0.06 and 5.61±0.05 log CFU/ml was noticed for markets A, B, C and D, respectively. A glance at the results would show that the surface TVC of market shell eggs were higher than farm eggs and suggested that the market eggs were more contaminated than the eggs collected from the farms A and B. Similarly, the internal contents of the eggs recorded the average TVC of 3.09 ± 0.19 and 3.24± 0.20 log CFU/ml for farm A and farm B, respectively, whereas the TVC of 4.81± 0.06, 4.86± 0.02, 4.72± 0.08 and 3.76±0.38 log 84
CFU/ml was observed for markets A, B, C and D respectively. The results demonstrated that the market eggs showed higher bacterial load as compared to farm egg samples. The presence of Pseudomonas spp. (1.09± 0.14) was observed only in the eggs collected from market C (white shell) and it was absent from all other eggs on its external surface and was also absent in the internal contents of eggs from all the sources of samples analysed in the present study. The data pertaining to the TVC of eggs both from external and internal level of contaminations were later subjected to statistical analysis and the results of the analysis is depicted in Table 4.4 and 4.4 (a). The results of the analysis revealed that the level of contamination of the eggs on its external surface when compared between two commercial farms was found to differ significantly (P0.01) and indicated that farm A and farm B differ with respect to egg production practices. Similarly, the results of analysis between farms and market eggs suggested that the degree of contamination was found to differ significantly (P0.01) between farms and markets which further indicated that, when eggs moved from farm to market they do acquired the microorganisms. However, statistical analysis of the TVC of internal contents of eggs from the commercial layer farms showed no significance difference in microbiological quality whereas between the eggs of commercial layer farms and markets, the microbiological quality of the internal contents showed significance (P0.01) difference which suggested that the microbiological quality of market eggs was inferior and prone for earlier spoilage. The results of the present study with respect to shell egg contamination observed by estimating Total Viable Counts and Pseudomonas spp. both in commercial layer farms and market eggs can be very well compared with the work and observations of several workers. Knape et al (1999) found that the shell surface of unwashed eggs could contain more than 105 log CFU/egg and observed that off-line eggs were more contaminated than in-line eggs. They suggested that microbial load of less than 100 log CFU/egg should be considered an excellent commercial standard, whereas viable counts of more than 105 log CFU/egg could be considered unacceptable. Similarly, Musgrove et al (2008) observed that the average aerobic population levels (log10 cfu/ml) were 1.4, 0.7, 0.9, and 0.8 for washed eggs and 4.2, 2.9, 2.5, and 2.5 for 85
unwashed eggs. Similar types of total viable counts were observed in the present study on the surface of shell eggs with significantly higher counts in market egg samples. Edema and Atayese (2006) isolated Aeromonas, Alcaligenes, Pseudomonas, Escherichia and Staphylococcus from shell eggs sold for consumption in Nigerian markets. Baishya et al (2008) also found higher microbial counts in shell eggs from the markets. However, De Reu et al (2005), who employed total aerobic counts and Gram-negative flora as a tool for quality assurance in the production chain of consumption eggs observed a positive correlation between the initial bacterial eggshell contamination and the concentration of bacteria in the air of the poultry houses. They opined that the total count of aerobic bacteria and the total count of Gram-negative bacteria on the eggshell could be used as a tool to detect the critical contamination points in the egg production chain. The findings of the present study also emphasize the same observation. The Total Viable Counts observed for internal contents of shell eggs in the present study were well within the range of recommended standards or generally accepted limits by USDA, (1991) for farm eggs i.e. less than 1000 log CFU/ml at production stage and less than 10,000 log CFU/ml by expiry date. Whereas higher counts than the acceptable limit were recorded in all market egg samples except brown shell eggs from market D. However, in the present study, market eggs regardless of the source or category showed consistently higher total viable counts both on external surface and internal contents as compared to farm eggs. Similarly, the presence of Pseudomonas spp. in shell egg samples from one of the market suggested the build up of the organisms on the shell eggs as they move through the egg supply chain i.e. from farm to market. There are several factors which influence the internal microbial load of the shell eggs which is explained by several workers. Sparks and Board (1985) observed that the structures of the shell eggs though act as a significant physical and chemical barrier to pathogen invasion and growth, the drying period of the cuticle that occurs within 3 min. of lay is most vulnerable for contamination from faecal microorganisms. Similarly, Stadelman and Cotterill (2002) reported that factors, such as initial level of contamination of the egg shell and egg contents, the integrity of the shell, the storage conditions and the age of the egg were critical for the microbial 86
or pathogen contamination of inside egg contents. CAC (2005) guidelines suggested that contaminations of eggs are influenced by the method of collection, cleaning, storage and processing. However, Nys et al (2001) who studied quality and safety of hen eggs in different production systems found that the bacterial counts from furnished cages as well as controlled cages were low but hens kept at higher temperature (32°C) in the hen house laid eggs that were more prone to contamination by eggshell penetration as frequently as nonstressed hens. In the present study, the Pseudomonas spp. was detected only in one of the market shell egg samples i.e. market C (white shell eggs). The presence of Pseudomonas spp. along with other organisms like Aeromonas, Alcaligenes, Enterobacter, Flavobacterium, Micrococcus, Proteus, Pseudomonas and Serratia spp. including contamination by pathogens such as Campylobacter, Salmonella and Staphylococcus spp. etc has been reported by several workers in the literature like (Baker, 1990; Jay, 1996; and ICMSF 1998). Similarly, Bell and Weaver (2002) observed that the microflora of commercial shell eggs is large and diverse and it has been found that 46% of it was Gram­negative while 54% was Gram-positive. The predominant Gram-positive isolates included Staphylococcus, Bacillus and Lactobacillus spp. and Gram-negative isolates included Salmonella, Escherichia coli Pseudomonas spp and Proteus vulgaris. 87
Table 4.1: Physico-chemical and microbiological analysis of raw shell eggs from commercial layer farms and market samples.
Sr. No Parameter
A. External quality:
1
Egg weight (g)
2
Egg length (mm)
3
Egg width (mm)
4
Shape Index (%)
5
Shell weight (g)
6
Shell thickness (mm)
7
Shell %
B. Internal quality:
1
Albumin height (mm)
2
Haugh unit
3
Yolk Index (%)
4
Albumin pH
C. Microbiological quality:
a. External egg surface:
1
Total Viable Count
(log CFU/ml)
2
Pseudomonas spp
(log CFU/ml)
b. Internal egg contents:
1
Total Viable Count
(log CFU/ml)
2
Pseudomonas spp.
(log CFU/ml)
Layer farm A N = 90
Average values observed for different parameters
Layer farm B
Market A
Market B
Market C
N = 60
N = 20
N = 20
N = 20
Market D N = 20
56.69 ± 0.43 55.62 ± 0.24 42.72 ± 0.11 76.84 ± 0.34 5.41 ± 0.04 0.40 ± 0.00 9.64
54.65 ± 0.38 55.47 ± 0.24 42.15 ± 0.13 76.06 ± 0.42 5.38 ± 0.05 0.42 ± 0.00 9.84
57.91 ± 1.0 56.78 ± 0.45 43.11 ± 0.28 75.98 ± 0.53 5.68 ± 0.07 0.41 ± 0.01 9.81
36.47 ± 0.83 51.18 ± 0.39 38.20 ± 0.33 74.67 ± 0.76 3.87 ± 0.01 0.43 ± 0.01 10.61
56.49 ± 0.45 56.95 ± 0.34 42.77 ± 0.17 75.15 ± 0.52 5.27 ± 0.06 0.41 ± 0.00 9.33
45.51 ± 0.62 54.20 ± 0.49 40.06 ± 0.20 73.79 ± 0.71 4.26 ± 0.11 0.37 ± 0.01 9.57
6.12 ± 0.18 77.26 ± 1.25 36.13 ± 0.39 8.67 ± 0.01
6.28 ± 0.09 79.91 ± 0.64 38.70 ± 0.29 8.45 ± 0.04
3.63 ± 0.40 52.10 ± 5.36 30.30 ± 1.23 8.89 ± 0.01
4.58 ± 0.17 77.78 ± 1.66 26.36 ± 1.28 8.69 ± 0.06
4.02 ± 0.23 58.90 ± 2.57 14.54 ± 3.02 9.01 ± 0.02
2.78 ± 0.43 46.18 ± 6.42 21.31 ± 2.25 9.44 ± 0.09
4.50 ± 0.08 Absent
5.00 ± 0.03 Absent
5.81 ± 0.05 Absent
5.44 ± 0.05 Absent
5.52 ± 0.06 5.61 ± 0.05 1.09 ± 0.14 Absent
3.09 ± 0.19 Absent
3.24 ± 0.20 Absent
4.81 ± 0.06 Absent
4.86 ± 0.02 Absent
4.72 ± 0.08 3.76 ± 0.38
Absent
Absent
88
Table 4.2: Comparison of weight of raw shell eggs and shell thickness between commercial layer farms and market
Sr. Source Lot 1 /
Lot 2 / Lot 3 /
No.
Market A Market Market
B
C
A. Average weight of eggs from different lots (g)
1
Farm A
57.73
55.83 56.51
2
Farm B
53.05
54.95 55.96
3
Market
57.91
36.47 56.49
B. Average thickness of shell from different lots (mm)
1
Farm A
0.43
0.39
0.39
2
Farm B
0.42
0.43
0.42
3
Market
0.41
0.43
0.41
NA = Not applicable.
Lot 4 / Market D NA NA 45.51 NA NA 0.37
Mean 56.69 54.65 49.09 0.40 0.42 0.41
Table 4.2 (a): Analysis of variance for egg weights between commercial layer farms and market
Source of variation Treatments Error Total
Degrees of freedom 2 7 9
Sum of squares 110.028 310.853 -
Mean sum of squares 55.014 44.408 -
F cal 1.239NS 0.346 -
Table 4.2 (b): Analysis of variance for shell thickness between commercial layer farms and market
Source of variation Treatments Error Total NS = Non significant
Degrees of freedom 2 7 9
Sum of squares 0.001 0.003 -
Mean sum of squares 0.000 0.000 -
F cal 0.885NS -
89
Table 4.3: Comparison of internal physico-chemical qualities of shell eggs between commercial layer farms and market
Sr.
Source Lot 1 / Lot 2 / Lot 3 / Lot 4 /
No.
Market Market Market Market D
A
B
C
A. Average albumin height (mm) observed for different lots
1 Farm A 5.72
5.36
7.27
NA
Treatmen t means 6.12a
2 Farm B 5.75
6.27
6.81
NA
6.28a
3 Market 3.63
4.58
4.02
2.78
3.75b
B. Average Haugh units observed for different lots
1 Farm A 74.82 72.34 84.49
NA
77.22a
2 Farm B 76.69 79.78 83.26
NA
79.91a
3 Market 52.10 77.78 58.90
46.19
58.74b
C. Average pH values of albumin observed for different lots
1 Farm A 8.86
8.64
8.52
NA
8.67ab
2 Farm B 8.55
8.45
8.44
NA
8.48b
3 Market 8.89
8.69
9.01
9.44
9.01a
NA = Not applicable. Treatment means with different superscript differ significantly
Table 4.3 (a): Analysis of variance for average albumin heights of eggs between commercial layer farms and market
Source of variation Treatments
Degrees of freedom 2
Sum of squares 14.376
Mean sum of squares 7.188
F cal 11.596**
Error
7
4.339
0.620
-
Total
9
-
-
-
** = Significant at 1 and 5% level of significance CD (0.05) = 1.317 CD (0.01) = 1.948
Table 4.3 (b): Analysis of variance for average Haugh units of eggs between commercial layer farms and market
Source of variation Treatments
Degrees of freedom 2
Sum of squares 953.996
Mean sum of squares 476.998
Error
7
668.425
95.489
Total
9
-
-
* = Significant at 5% level of significance CD (0.05) = 16.342
F cal 4.995* -
90
Table 4.3 (c): Analysis of variance for average albumin pH values of eggs between commercial layer farms and market
Source of variation Treatments
Degrees of freedom 2
Sum of squares 0.502
Mean sum of squares 0.251
Error
7
0.369
0.053
Total
9
-
-
* = Significant at 5% level of significance CD (0.05) = 0.384
F cal 4.763* -
Table 4.4: Comparison of Total Viable Count (log CFU/ml) of raw shell eggs between commercial layer farms and market
Sr. No Source Lot 1 /
Lot 2 /
Lot 3 /
Lot 4 / Average
Market A Market B Market C Market D count(log
CFU/ml)
A. Average Total Viable Count (log CFU/ml)from external surface of eggs from different
1 Farm A 4.69
lots
4.45
4.37
NA
4.50c
2 Farm B 5.04
4.93
5.04
NA
5.00b
3 Market 5.81
5.44
5.52
5.61
5.96a
B. Average Total Viable Count (log CFU/ml)from internal contents of eggs from different
lots
1 Farm A 3.30
2.63
3.34
NA
3.09b
2 Farm B 3.39
3.57
2.75
NA
3.24b
3 Market 4.81
4.86
4.72
3.76
4.54a
NA = Not applicable. Treatment means with different superscript differ significantly.
Table 4.4(a): Analysis of variance for Total Viable Count from external surface of eggs between commercial layer farms and markets
Source of variation Treatments
Degrees of freedom 2
Sum of squares 2.075
Mean sum of squares 1.038
F cal 52.015**
Error
7
0.140
0.020
-
Total
9
-
-
-
** = Significant at 1% and 5% level of significance CD (0.01) = 0.349 CD (0.05) = 0.236
91
Parameter
Fig.1: Comparison of external quality parameters of shell eggs from commercial layer farms and markets
90 80 70 60 50 40 30 20 10 0 Farm A
Egg weight(g) Shape index% Shell weight(g) Shell %
Farm B
Market A
Market B
Sources
Market C
Market D
92
Parameters
Fig.2:Comparisonofinternalqualityparametersofshelleggsfromcommerciallayer farms and markets
80 70 60 50 40 30 20 10 0 FarmA
FarmB
MarketA MarketB MarketC MarketD Sources
Albumin height(mm) Haugh Unit Yolk index% Albumin pH
93
Log CFU/ml
Fig.3:Comparisonofmicrobiologicalqualityofrawshelleggsfromcommerciallayer farms and markets 6
5
4
3
ExternalTVC Internal TVC
Ext.Pseudomonas spp
2
Int.Pseudomonas spp
1
0 FarmA FarmB MarketA MarketB MarketC MarketD Sources
94
Table 4.4 (b): Analysis of variance for Total Viable Count from internal contents of eggs between commercial layer farms and markets
Source of variation Treatments
Degrees of freedom 2
Sum of squares 4.564
Mean sum of squares 2.282
F cal 10.609**
Error
7
1.506
0.215
-
Total
9
-
-
-
** = Significant at 1% and 5% level of significance CD (0.01) = 1.148 CD (0.05) = 0.776
4.2 Microbiological safety of shell eggs
The shell eggs produced and marketed as seen earlier might vary in their qualities from very good to very poor quality attributes but these quality attributes do not convey anything about egg safety aspects where consumers are at risk of contracting infection after consumption of eggs contaminated with pathogens. The contamination of shell eggs with pathogens (safety aspects) does not follow the order of standard grades which are used for merchandising egg trades and there is every possibility that an AA or A or B grade eggs can carry the pathogen and result in illness. There are a few well known pathogens, which are associated with the egg borne illness in human beings and where layers can serve as reservoirs. In this context, in the present study, two well known pathogens were considered or selected as safety indicators viz., Salmonella enterica serotypes especially Enteritidis and Typhimurium and thermophilic Campylobacter spp. especially Campylobacter jejuni and Campylobacter coli, all of them are food borne pathogens and layers may harbour these infections. A total of 350 samples which included cloacal swabs from layers, surface swabs from raw shell eggs, raw shell eggs, environment samples like feed, water, litter etc from commercial layer farms, finished egg products from consumer out let like egg rings and raw shell eggs from markets A, B, C and D were collected and analyzed for the presence of Salmonella spp and Campylobacter spp by cultural methods and the results of the analysis are presented in Table 4.5 and Figure 4. 95
The results in the present study revealed the prevalence of Salmonella enterica serotypes in layers isolated from cloacal swab samples collected from both the commercial layer farms. The percent prevalence of Salmonella spp. as obtained from the analysis of 30 cloacal swabs from each of the layer farm was found to be at the level of 6.67 for farm A and 10 for farm B. The percent prevalence of Salmonella spp. in shell eggs as indicated by the analysis of internal contents of 30 shell egg samples from each of the layer farm was found to be 3.33 and 10 in farm A and farm B, respectively where as the analysis of 60 samples of the shell eggs from external surface of the egg from both the layer farms showed no presence of Salmonella spp. Similarly, the results of the surveillance for Salmonella spp. from four different markets which comprised analysis of 120 market egg samples (Table 4.5) from external and internal contents, indicated presence of Salmonella spp. on external surface of the market A egg samples while none of the sample from the markets B, C and D was found positive for Salmonella spp. either from external or internal contents. The percent prevalence of Salmonella spp was at 13.33 in samples collected from market A and nil for eggs from markets B, C and D. Thus, in the present study, the Salmonella spp. was isolated from internal contents of the eggs and cloacal swabs of the layers from commercial layer farms and not from external surface of the eggs. Interestingly, all the isolates obtained in the present study from the shell eggs either from external surface or internal contents recovered from white shell eggs and not from desi or brown shell eggs. The rate of prevalence of Salmonella spp. is not uniform in shell eggs wherever studies have been conducted and different rate of prevalence of Salmonella underlines the basic fact that the shell eggs as a source of food and as a source of Salmonella ought to be under continuous surveillance system. The results of the present study on safety of shell eggs with respect to Salmonella point to the same basic consideration and is supported by the research of several workers. Adesiyun et al (2005) who analysed samples of swabs of egg shells and egg content observed an overall prevalence of 13·0% for Salmonella spp. in shell eggs, where 3.8% of the egg shell and 7.6% of the egg contents showed the presence of Salmonella spp. McCluskey et al (2008) analysed a total of 360 egg pools and observed that 4 were positive for Salmonella spp. out of which 3 positives were associated with the interior 96
components of the shell eggs, whereas one positive was associated with the exterior of the shell component. Jones and Musgrove (2007) in their study on prevalence of pathogenic organism in restricted shell eggs found that the prevalence of Salmonella spp. was 1.1% in the shell samples analysed. Mas and Davies (2008) also observed low prevalence of Salmonella serotype Enteritidis in shell eggs. Laura et al (2007) in a major survey of 30,000 grade A shell eggs for prevalence of Salmonella spp. in Ireland, found that the prevalence was very low as they could get only two isolates from egg shells and none from the contents. Little et al (2006) reported 3.40 percent prevalence of Salmonella spp. on surface of shell eggs and an equal prevalence rate in egg contents imported to England from out side, mainly from European Union countries. Otomo et al (2007) observed the prevalence of Salmonella spp. in layers, immature eggs, and the yolk of mature eggs in oviducts and reported that prevalence rate of 14%, 7.2%, and 6.8%, in layers, immature eggs and yolk of mature eggs, respectively. Ghosh et al (2002) found 4% positive samples out of 50 shell eggs from the surface of the shells in India. In the present study, out of 40 environmental samples analysed for the presence of Salmonella spp. from both the commercial layer farms which included litter, feed, water and wash water from egg trays, none of the samples recorded presence of Salmonella spp. Similarly, 10 samples of finished egg products collected from two fast food centers also did not show the presence of Salmonella spp. However, in contrast to the results of the present study, several workers have reported prevalence of Salmonella spp. from environmental samples. Nabbut et al (1982) isolated Salmonella serotypes from farm animals, animal feed, sewage and sludge in Saudi Arabia and found that out of 65 different serotypes, six serotypes most frequently recovered or isolated were Livingstone, Concord, Schottmuelleri, Lille, Typhimurium and Cerro. Similarly, Davies and Wray (1997) studied the distribution of salmonella contamination in ten animal feed mills and found that the Salmonella isolation rate ranged from 1.1% to 41.7% and the most contaminated mills were those where the inside of the cooling systems for pellet or mash had been colonized by Salmonella spp. The results of the epidemiological surveillance on environmental contaminants in poultry farms by Soliman et al (2009) indicated that the prevalence of Citrobacter sp (8.3%), Proteus vulgaris (8.3%), Streptococcus fecalis (5.3%) and Pseudomonas aureuginosa (16.7%) predominated in litter samples from closed house system. Klebsiella oxytoca 97
(10.0%) predominated in water of open house system and none of the samples tested positive for Salmonella spp. Similar results were recorded in the present study where Salmonella spp. was found to be absent in all the environmental (feed, litter, water etc) samples collected and analyzed from both the commercial layer farms and the detection of other organisms was not focused in this study. Since, Campylobacter spp. are considered to be of equal importance as that of Salmonella spp. same number of samples were also analysed for isolation of Campylobacters and the results of its prevalence is given in Table 4.5. It is evident from the results that there was no prevalence of Campylobacter spp. in shell eggs and layers as evidenced by absence of Campylobacter spp. from samples of shell eggs (both external surface and internal contents), cloacal swab samples and environmental samples (feed, litter, water, wash water from egg trays) collected from commercial layer farms. Similarly, there was no prevalence of Campylobacter spp in shell eggs of market samples and finished egg products like egg rings from consumer out lets. The findings of the present study are in agreement with the work carried out by several researchers who studied the profiles of Campylobacters in relation to shell eggs, layers and farm environments as potential risk factors for transmission via the shell eggs. Clark and Bueschkens (1985) in two separate experiments to study the profile of Campylobacters, infected the chicken eggs with Campylobacter jejuni and recovered the organisms in hatched chicks up to 11% but recovery from infertile eggs was found to be poor. They also observed that the storage of eggs for 8 days prior to incubation for hatching failed to yield infected chicks. Shane et al (1986) observed in a field survey of three commercial laying farms and their associated egg-packing plants that the hens demonstrated to be fecal shedders of C. jejuni with 12% to 62% incidence but did not produce infected or contaminated eggs and the organism was not found be prevalent in the environment of the packing plant, including grading machinery and effluent. On the contrary, Allen and Griffiths (2001) observed colonization and penetration in fresh and retail eggs by C. jejuni with fresh eggs being heavily colonized than retail eggs. Newell and Fearnley (2003) studied the sources of Campylobacter colonization in chickens and reported that the dry conditions of feed and fresh litter were found to be lethal to C. jejuni as the organisms were not isolated 98
from clean dry litter. Further, the feed, feed additives, and fresh litter were not found to be the potential sources of infection. Several epidemiological studies where the relationships between water sources (well or mains water) and broiler flock Campylobacter positivity were considered have shown that the water source was found to be low-risk factor. Sahin et al (2003) examined freshly laid eggs from Campylobacterinoculated specific pathogen-free (SPF) layers. They revealed that C. jejunicontamination was detected in three of 65 pooled whole eggs (5­10 eggs in each pool) via culture and PCR. The organism was not detected from any of the 800 eggs (80 pools) collected from the same SPF flock stored at 18oC for 7 days. They also reported that Campylobacter was not recovered from any of the 500 fresh eggs from commercial broiler-breeder flocks which were actively shedding Campylobacter in faeces and none from 1000 eggs of broiler breeders from a commercial hatchery. They observed that unlike Salmonella spp. the vertical transmission of C. jejuni through the egg probably is a rare event and did not play a major role in the introduction of Campylobacter to chicken flocks. In the present study, the overall observations with regard to safety of shell eggs did suggest that Salmonella spp was an issue of concern to raw shell egg safety albeit at low level for consumers and traders and an issue of biosecurity concern for commercial layer farms and producers. Drociuk et al (2003) observed that 78.0% of the out-breaks with Salmonella Enteritidis in USA were associated with consumption of shell eggs and the decrease in their incidence was largely attributed to farm based control measures and cautioned that Salmonella Enteritidis could survive boiling water temperature if the yolk was not completely solidified. Braden (2006) observed that the internal contamination of shell eggs by transovarian route was the major source of contamination for shell eggs. Both the researchers recommended that, one of the important control measures was microbiological testing of hen houses for the presence of Salmonella Enteritidis, farm based control programs and surveillance of Salmonella Enteritidis outbreaks necessary to detect changes in trends of Salmonella Enteritidis infection in the region. The points which are well observed by them are again strengthened by the present research work on safety of raw shell eggs and prevalence of Salmonella spp. The other important issue that the present work suggested was that the Campylobacters do not constitute an egg safety and biosecurity concern at present for consumers, producers and traders. 99
Table 4.5: Percent prevalence of Salmonella spp. and Campylobacter spp. in commercial layer farms and market samples
Safety Indicators/Source Farm A Salmonella spp. Campylobacter spp. Farm B Salmonella spp. Campylobacter spp. Market A (N = 15) Salmonella spp. Campylobacter spp. Market B (N = 15) Salmonella spp. Campylobacter spp. Market C (N = 15) Salmonella spp. Campylobacter spp. Market D (N = 15) Salmonella spp. Campylobacter spp. NA= Not Applicable
Samples from layers (N = 30)
Cloacal samples Layers
External surface (eggs)
Internal contents (eggs)
6.67
Nil
3.33
Nil
Nil
Nil
10
Nil
10
Nil
Nil
Nil
NA
13.33
Nil
NA
Nil
Nil
NA
Nil
Nil
NA
Nil
Nil
NA
Nil
Nil
NA
Nil
Nil
NA
Nil
Nil
NA
Nil
Nil
Litter Nil Nil Nil Nil NA NA NA NA NA NA NA NA
Environmental samples (N = 5)
Feed
Water
Wash water egg trays
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Finished egg product
from fast food centers
(N = 5)
Kalamboli
Lower
Parel
Nil
Nil
Nil
Nil
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
100
Prevalence rates
Fig.4:PercentprevalenceofSalmonella sppandCampylobacter sppfromdifferentsources
9 8 7 6 5 4 3 2 1 0 Shell eggs
Layers
%prevalenceSalmonellaspp. %Prevalence Campylobacter spp
Litter
Feed
Water
Eggtray Finishedegg
washings product
Sources
101
4.3 Effect of various sanitizers on artificially inoculated Salmonella Typhimurium on surface of shell eggs and fixing of performance criteria for sanitizers Sanitation of table eggs is an important processing step either by physical means or through chemicals now has become an integral part of shell egg processing in order to ensure safety to the consumers especially after rise in the incidence of Salmonellosis through eggs. Egg sanitation is principally done to reduce the surface microbial load and to eliminate the pathogens present on the surface of the eggs. The use of sanitizers can be reduced if there is a corresponding improvement in biosecurity of layer farms, egg production, processing and preservation practices rather it has become a necessity to over come the inadequacies in hygienic operations. In India shell eggs are sold as fresh eggs irrespective of its hygienic status or safety concerns. In the present study, trials were conducted to determine efficacy of sanitizers' viz., 70% alcohol, 65 ppm Polyhexamethylene guanidine and 200 ppm of chlorine by artificially inoculating the Salmonella Typhimurium on the surface of shell eggs and to fix the performance criteria for sanitizers by isolations or recovery of Salmonella Typhimurium in internal egg contents at regular time intervals from the eggs stored at refrigeration temperatures (370C). A total of 90 shell eggs were used for pre and post inoculation studies with sanitizers that comprised 3 experimental replicates and appropriate controls. The results of the study are given in Tables 4.6. The average initial inoculation control count of the Salmonella Typhimurium on the surface of raw shell eggs in pre inoculation studies were found to be 4.24 log CFU/ml and after treatment (sanitation) with 70% alcohol, 65 ppm Polyhexamethylene guanidine (PHMG) and 200 ppm of chlorine the average count of Salmonella Typhimurium was found to be 0.54, 0, and 0.60, log CFU/ml, respectively. The average log reductions of the organism for each treatment was found to be 3.70, 4.24, and 3.64 log CFU/ml, respectively and the percent log reductions were found to be 87.26, 100, and 85.85 for 70% alcohol, 65 ppm PHMG and 200 ppm of chlorine, respectively (Table 4.6). Like wise, the effect of sanitizers on Total Viable Count (TVC) was assessed and it was observed that after treatment with 70% alcohol, 65 ppm PHMG and 200 ppm of chlorine, the average counts were reduced to 4.47, 102
4.45 and 4.62 log CFU/ml from an initial count of 5.75 log CFU/ml. The average log reductions for each treatment were recorded as 1.28 (22.26%), 1.30 (22.61%) and 1.13 (19.65 %), log CFU/ml, respectively. The results suggested that the average log reductions brought about by each treatment were less and only ranged between 1.13 to 1.30 log CFU/ml. However, among the sanitizers, percentage of log reductions was found to be better for 65 ppm PHMG i.e. 22.61 and poor for 200 ppm chlorine i.e.19.65 (Table 4.6). The data of sanitary trials were subjected to statistical analysis and it revealed that the sanitizers were found to be effective in reducing the initial artificially inoculated load of Salmonella Typhimurium and initial TVC at 1 and 5% level of significance (P0.01) as given in Tables 4.7, 4.7(a) and 4.7(b). However, when the results of different sanitizers on log reductions of Salmonella Typhimurium and of TVC were subjected to statistical analysis, the difference in effectiveness was found to be non significant. The results of the statistical analysis are presented in Tables 4.8 and 4.8 (a) and 4.8 (b). Several researchers have carried out experiments to determine efficacy of disinfection of shell eggs employing different marker organisms and application of physical or chemical means of disinfection. Knape et al (2001) employed Salmonella Enteritidis and SalmonellaTyphimurium as marker strains for egg shell surface inoculations and subsequent disinfection by disinfectants like distilled deionised water, Iodine based disinfectant and chlorine 200 ppm. It was observed that all the treatments significantly (p < 0.05) decreased Salmonella spp. populations on the surface of the shell compared to dry (no spray) egg controls. The results of the present study are in accordance with these observations where 70% alcohol, 65 ppm PHMG and 200 ppm of chlorine were used. The disinfectants were found to be significantly effective in reducing populations of organism in both pre and post-inoculated eggs. Similarly, the findings of the present study with Salmonella Typhimurium as a marker organism to determine the efficacy of 70% alcohol, 65 ppm PHMG and 200 ppm chlorine on the surface of raw shell eggs are similar to the observations of Himathongkham et al (1999), Favier et al (2000) and Favier et al (2001) who demonstrated that sanitizers differed in their ability to reduce or eliminate the organisms on the surface of the raw shell eggs and complete elimination of the organism was not achieved with most of the sanitizers tried in the experiments. Himathongkham et al (1999) carried out experiments on efficacy of disinfection of shell eggs by Lugol's solution, chlorhexidine, ethanol, 103
quaternary ammonium solutions, ethanol followed by flaming and boiling water treatment on shell eggs externally contaminated with Salmonella Enteritidis and observed that the disinfection of shell eggs could not be achieved completely except in the boiling water treatment. The work of Favier et al (2000) on the disinfection of shell eggs with different sanitizers (Extran, Tergitol type 08, sodium hypochlorite solution containing 100 ppm of free chlorine and a combination of Tergitol and sodium hypochlorite) and Yersinia enterocolitica as marker organism revealed different degrees of log reductions of the organism and mesophilic aerobic bacteria and none of the chemicals achieved complete elimination of the organism. Tergitol/100 ppm was highest followed by chlorine in achieving the log reductions. In a similar experiment with different sanitizers, Favier et al (2001) tested efficacy of 100 ppm of chlorine, 3% sodium chloride, 1, 5, and 12% Tri Sodium Phosphate (TSP), and 4,573 W/cm2 ultraviolet radiations. They found that 100 ppm chlorine was highly effective and comparable with UV exposure for more than 25 min. with 1.28 and 1.60 log reductions of mesophilic aerobic bacteria as against control eggs with 4.55 log CFU/egg. It was also observed that the reductions achieved with 12% TSP (3.74-log reduction) was significantly higher (P < 0.05) than those obtained with the remaining treatments. Similar observations were reported by Davies and Breslin (2003) by physical methods of decontamination of egg shell surface and by Russell (2003) who employed different commercial sanitizers for surface decontamination of shell eggs. In the present research work, though the log reductions of the organism between different sanitizers were found to be non significant, PHMG as a sanitizer was found to be effective in elimination of the Salmonella Typhimurium completely from the surface of the shell eggs comparable to the boiling water treatment by Himathongkham et al (1999). Similarly, the work of Soljour et al (2004) with sodium carbonate, sodium hypochlorite, and potassium hydroxide as sanitizers for Salmonella Enteritidis inoculated onto the eggshell surface at concentrations of 104 or 106 CFU/ml indicated that none of the chemicals applied at the recommended manufacturer's concentrations (sodium carbonate 36 ppm, other treatments 200 ppm) could eliminate Salmonella Enteritidis from eggshell surface, which further substantiates the findings of the present work. 104
The results of effect of sanitizers in post inoculation studies are presented in Table 4.6. The initial average count of Salmonella Typhimurium on the surface of the raw shell eggs kept as inoculation control was found to be 4.36 log CFU/ml. The results showed that the average count of Salmonella Typhimurium was found to be 0.53, 0 and 0 log CFU/ml for 70% alcohol, 65 ppm PHMG and 200 ppm of chlorine, respectively. The average log reductions for each treatment were found to be 3.83, 4.36 and 4.36 log CFU/ml, respectively. The percent log reduction of the organism was found to be 100 for 65 ppm PHMG and 200 ppm chlorine and 87.84 for 70% alcohol. Similarly, the results of TVC showed average counts of 2.21, 3.89 and 2.40 log CFU/ml for 70% alcohol, 65 ppm PHMG and 200 ppm chlorine, in post inoculated eggs, as against the initial TVC of 6.37 log CFU/ml, respectively. The average log reduction values were found to be 4.16, 2.48, and 3.97 for respective treatments by 70% alcohol, 65 ppm PHMG and 200 ppm chlorine. The percent log reduction was found to be 65.31(highest) for 70% alcohol, 62.32 for chlorine and 38.93 (least) for PHMG. The microbiological data of the post inoculation trials were subjected to statistical analysis and the results of the analysis are described in Table 4.7 and 4.7(c) and 4.7(d). The results showed that all the sanitizers were found to be effective in reducing inoculated load of the Salmonella Typhimurium and of Total viable Counts and the reduction was found to be significant at 1 and 5% levels (P0.01). The recorded CD (0.01) values were found to be 2.100 and 1.896 for Salmonella Typhimurium and TVC, respectively. However, the statistical analysis of different sanitizers on log reductions of Salmonella Typhimurium and TVC were compared between treatments, analysis showed non significant difference in their effectiveness by all the three sanitation methods on both TVC and on Salmonella Typhimurium. The results of analysis are presented in Table 4.8 and 4.8(c) and 4.8(d). The literature available for post inoculation studies on the surface of the shell eggs for determining the efficacy of sanitizers in terms of log reductions of the organism are scanty, the research by Hong and Slavik (1998) on penetration of post inoculated Salmonella Enteritidis in raw shell eggs illustrates indirectly the effectiveness of sanitizers. Hong and Slavik (1998) washed the raw shell eggs with quaternary ammonium compound (QAC, pH 7.5), sodium carbonate (Na2CO3, pH 12) and sodium hypochlorite (NaOCl, 100 ppm, pH 7.5) solutions and later inoculated the cultures of Salmonella Enteritidis at 104 CFU/ml and 105
dried for 30 min. The eggs were analysed for recovery of the organism from day 1 to day 21 and the results indicated that the organism was recovered from all the treatments from day 1 to 21. It can be inferred that none of the sanitizers was able to eliminate the organism completely from the egg shell surface. However, in the present study, PHMG and chlorine were able to reduce the organism very effectively up to 100% level excepting 70% alcohol which showed 87.84% reduction, which suggested the results are not totally in accordance with the study of Hong and Slavik (1998) but agreed partially to the fact that complete elimination may not be achieved with all the sanitizers. It appears to be very clear from the results of the present study that the Salmonella Typhimurium responded differently to different type of sanitizers used to inactivate or kill the organism in both pre and post-inoculation studies. This seems to be agreeable with the science of antiseptics/disinfectants and their antimicrobial properties as different types of microorganisms vary in their response to antiseptics and disinfectants in view of their different cellular structure, composition, and physiology and organisms' innate resistance to antiseptics or disinfectants (McDonnell and Russell 1999). Tolerance to heat, ethanol, and hypochlorous acid has also been reported in the literature. The same analogy can be applied to the varied responses obtained to the different sanitizers with regard to the reductions of the Total Viable Counts achieved in the present study. Another significant feature of the study to be mentioned is that the present study incorporated a new sanitizer i.e. Polyhexamethylene guanidine (PHMG) along with the other two established sanitizers namely chlorine and alcohol which are widely used as either disinfectants or antiseptics. PHMG belongs to a class of polymeric biguanides with known potential biocidal activity and difficult for microorganism to acquire resistance against it. PHMG is a food grade polymer approved by Commission Regulation (EC) No. 2032/2003. Barge (2009) who conducted experiments on efficacy and validation of PHMG as sanitizer observed that 50 to 65 ppm of PHMG was found to be highly effective against artificially inoculated organisms which included Staphylococcus aureus, Salmonella Typhimurium, Escherichia coli, Pseudomonas aeruginosa and Aspergillus niger in buffalo and chicken meat samples and the findings of the present work on artificial inoculation on to surface of egg shells and effects by 65 ppm of PHMG are in total agreement with his observations. 106
Table 4.6: Effect of various sanitizers on artificially inoculated SalmonellaTyphimurium and Total Viable Count on surface of shell eggs.
Parameter
Inoculation control count
A. Pre-inoculation
Salmonella
Typhimurium 4.24
(log CFU/ml)
TVC (log CFU/ml)
5.75
B. Post-inoculation
Salmonella
Typhimurium 4.36
(log CFU/ml)
TVC (log CFU/ml)
6.37
70% Alcohol
Count
Log
Percent
after reduction
log
sanitation in count reduction
0.54
3.70
87.26
4.47
1.28
22.26
0.53
3.83
87.84
2.21
4.16
65.31
65 ppm PHMG
Count
Log
Percent
after reduction
log
sanitation in count reduction
0
4.24
100
4.45
1.30
22.61
0
4.36
100
3.89
2.48
38.93
200 ppm Chlorine
Count
Log
Percent
after reduction
log
sanitation in count reduction
0.60
3.64
85.85
4.62
1.13
19.65
0
4.36
100
2.40
3.97
62.32
107
Table 4.7: Efficacy of various sanitizers on Salmonella Typhimurium and Total Viable Count on the surface of shell eggs
Parameter
Trial 1
Trial 2
Trial 3
Average
I. Pre inoculation
A. Average log CFU/ml of Salmonella Typhimurium from different trials
Initial count/ level
4.01
70% Alcohol
0.97
65 ppm PHMG
0
4.28 0.67 0
4.57 0 0
4.24a 0.546b 0b
200 ppm Chlorine
1.14
0
0.67
0.603b
B. Average log CFU/ml of Total Viable Count
Initial count/ level
5.35
70% Alcohol
4.83
65 ppm PHMG
4.52
6.15 3.99 4.37
5.76 4.59 4.45
5.75a 4.47b 4.45b
200 ppm Chlorine
4.86
4.31
4.70
4.62b
II. Post inoculation
A. Average log CFU/ml of Salmonella Typhimurium from different trials
Initial count/ level
4.55
70% Alcohol
0
65 ppm PHMG
0
3.76 2.0 0
4.80 2.78 0
4.370a 1.59b 0.00c
200 ppm Chlorine
0
0
0
0.00c
B. Average log CFU/ml of Total Viable Count
Initial count/ level
6.54
70% Alcohol
2.49
65 ppm PHMG
2.82
6.02 1.43 4.31
6.54 2.72 4.56
6.37a 2.213c 2.407c
200 ppm Chlorine
2.67
2.92
1.63
3.897b
Treatment means with different superscripts differ significantly
Table 4.7(a): Analysis of variance for effect of sanitizers on pre inoculated Salmonella Typhimurium on surface of shell eggs
Source of variation Treatments
Degrees of freedom 3
Sum of squares
Mean sum of squares
34.132
11.377**
F cal 73.383**
Error
8
1.240
0.155
-
Total
11
-
-
-
**= Significant at 1% and 5% level of significance CD (0.01) = 1.079 CD (0.05) = 0.741
108
Table 4.7(b): Analysis of variance for effect of sanitizers on Total Viable Count on surface of shell eggs (Pre-inoculated)
Source of variation Treatments
Degrees of freedom 3
Sum of squares 3.515
Mean sum of squares 1.172**
F cal 10.826**
Error
8
0.866
0.108
-
Total
11
-
-
-
**=Significant at 1% and 5% level of significance CD (0.01) = 0.901CD (0.05) = 0.619
Table 4.7(c): Analysis of variance for effect of sanitizers on postinoculated Salmonella Typhimurium on surface of shell eggs
Source of variation Treatments
Degrees of freedom 3
Sum of squares 38.236
Mean sum of squares 12.745
F cal 21.686**
Error
8
4.702
0.588
-
Total
11
-
-
-
**= Significant at 1% and 5% level of significance CD (0.01) = 2.100 CD (0.05) = 1.443
Table 4.7(d): Analysis of variance for effect of sanitizers on Total Viable Count on surface of shell eggs (post-inoculated)
Source of variation Treatments Error Total
Degrees of freedom 3 8 11
Sum of squares 33.093 3.833 -
Mean sum of squares 11.031 0.479 -
F cal 23.021** -
**= Significant at 1% and 5% level of significance CD (0.01) = 1.896 CD (0.05) = 1.303
109
Table 4.8: Comparison of effect of different sanitizers on log reductions of Salmonella Typhimurium and Total Viable Count on surface of shell eggs
Parameter
Trial 1
Trial 2
Trial 3
Average
I. Pre-inoculation
A. Average log CFU/ml of Salmonella Typhimurium from different trials
70% Alcohol
3.27
3.57
4.24
3.69
65 ppm PHMG
4.24
4.24
4.24
4.24
200 ppm Chlorine
3.10
4.24
3.57
3.64
B. Average log CFU/ml of Total Viable Count
70% Alcohol
0.92
1.76
1.16
1.28
65 ppm PHMG
1.23
1.38
1.30
1.30
200 ppm Chlorine
0.89
1.44
1.05
1.13
II. Post-inoculation
A. Average log CFU/ml of Salmonella Typhimurium from different trials
70% Alcohol
4.36
0.66
0.93
1.98
65 ppm PHMG
4.36
4.36
4.36
4.36
200 ppm Chlorine
4.36
4.36
4.36
4.36
B. Average log CFU/ml of Total Viable Count
70% Alcohol
3.88
4.94
3.65
4.16
65 ppm PHMG
3.55
2.06
1.81
2.47
200 ppm Chlorine
3.70
3.45
4.74
3.96
Treatment means without any superscripts means non significant.
Table 4.8(a): Analysis of variance for effect of sanitizers on log reductions of pre inoculated SalmonellaTyphimurium on the surface of eggs
Source of variation Replications
Degrees of freedom 2
Treatments
2
Error
4
Total
8
NS = Non significant
Sum of squares 0.461 0.666 0.689 -
Mean sum of squares 0.230 0.333 0.172 -
F cal 1.338 0.259NS -
110
Table 4.8(b): Analysis of variance for effect of sanitizers on log reductions of Total Viable Count (pre-inoculated) on the surface of eggs
Source of variation Replications
Degrees of freedom 2
Treatments
2
Error
4
Total
8
NS = Non significant
Sum of squares 0.415 0.055 0.130 -
Mean sum of squares 0.208 0.028 0.033 -
F cal 6.366 0.847NS -
Table 4.8(c): Analysis of variance for effect of sanitizers on log reductions of post-inoculated Salmonella Typhimurium on the surface of eggs
Source of variation Replications
Degrees of freedom 2
Treatments
2
Error
4
Total
8
NS = Non significant
Sum of squares 2.836 11.297 5.673 -
Mean sum of squares 1.418 5.649 1.418 -
F cal 1.000 3.983NS -
Table 4.8(d): Analysis of variance for effect of sanitizers on log reductions of Total Viable Count (post-inoculated) on the surface of eggs
Source of variation Replications
Degrees of freedom 2
Treatments
2
Error
4
Total
8
NS = Non significant
Sum of squares 0.154 5.091 3.499 -
Mean sum of squares 0.077 2.546 0.875 -
F cal 0.088 2.910NS -
111
4.4 Shell penetration and recovery studies of Salmonella Typhimurium in whole egg contents of raw shell eggs treated with different sanitizers stored at refrigeration temperature The raw shell eggs, when contaminated with microorganisms, under favourable conditions such as moist shell surfaces, minor cracks, thin shells etc penetrate through the shell (though the cuticle and shell act as a barrier) and enter inside and show growth, multiplication and survival. The time taken for penetration through the shell and further growth depends on the temperature of storage and humidity, either ambient or refrigeration and the status of the raw shell eggs. The egg sanitation study in the present research work included two experiments. First experiment was designed as pre inoculation study where surfaces of raw shell eggs were artificially contaminated with Salmonella Typhimurium and then sanitized by different sanitizers. In second experiment, designed as post inoculation study, eggs were sanitized first and then inoculated with the Salmonella Typhimurium cultures. The results of the studies showed different degrees of log reductions of cells of Salmonella Typhimurium both in pre and post inoculation experiments and showed percent reductions even to an extent of 100%. The question that arose was whether the organism that survived sanitation penetrated the shell and could these be recovered from internal contents? Secondly, wherever disinfectant showed 100% reductions resulted in irreversible killing or they were only damaged to non culturable state? Thirdly, how many days would be required to penetrate the shell both in sanitized as well as surface inoculated eggs? Finally based on in vitro sanitation and recovery studies would it be possible for fixing performance criteria for sanitizers, so that the process could ensure better safety of eggs? The answers to these questions are presented in the form of results in Tables 4.9. It was evident from the results of pre inoculation studies that the Salmonella Typhimurium was recovered from the whole egg contents on 14th, 21st and 28th day from surface inoculated eggs without any treatment. The organisms started appearing from 14th day onwards and survived till the end of the study period from surface inoculated eggs. In 70% alcohol treated eggs, the organism was recovered on 28th day while it was recovered on 21st day from 200 ppm chlorine treated eggs. No Salmonella Typhimurium was recovered from eggs treated with 65 ppm PHMG in the entire period of the study and 112
suggested that it was able to kill all the Salmonella Typhimurium inoculated on to the surface of eggs. Similarly, the results of the post inoculation studies showed that the Salmonella Typhimurium was recovered from the whole egg contents on 14th, 21st and 28th day from surface inoculated eggs without any treatment and indicated that the organism started appearing from 14th day onwards and survived till the end of the study period from surface inoculated eggs as well as eggs treated with 70% alcohol with almost similar log CFU/ml. In 200 ppm chlorine treated eggs, the organism was recovered from internal contents on 28th day and no recovery of the organism was obtained in 65 ppm PHMG treated eggs until the end of experimental period of 28 days. It might be of value to note that the 200 ppm of chlorine that showed 100% log reductions in post inoculation studies and thus giving an impression that the organisms were effectively eliminated but the results of the recovery study proved otherwise where Salmonella Typhimurium was recovered from internal contents. The overall observations of the present study suggested that the Salmonella Typhimurium could be recovered from surface inoculated (untreated) eggs as well as from eggs treated with 70% alcohol and 200 ppm chlorine. The Salmonella Typhimurium was not recovered from the surface inoculated eggs treated with the 65 ppm PHMG in both pre and post inoculation studies. The minimum period for penetration of the organism stored at refrigeration temperature was found to be 14 days and by 28th day the organism could be recovered from sanitized/treated eggs except PHMG treated group irrespective of method of inoculations of Salmonella Typhimurium either before or after sanitizer's application. This clearly suggested that the organism was not only able to survive on the surface of the shell eggs at the recommended doses of sanitizer's but was also able to penetrate the shell, survive inside the eggs and was recoverable from whole egg contents. Many research workers have conducted several experiments to study the penetration of bacteria/microorganisms through the egg shells both hatching and table eggs and the survival of organism in hatched chick or egg contents in case of table eggs. Relatively less research work has been undertaken on shell penetration and recovery of organism from the shell eggs treated with sanitizers or disinfectants. Padron (1995) who studied the effect of dipping of shell eggs in 6% hydrogen peroxide solution to eliminate Salmonella Typhimurium from egg shell membranes found that it only reduced the average 113
number of organisms in eggshell membranes by 95% and number of SalmonellaTyphimurium-positive eggs by 55% compared to infected untreated group which suggested that the organisms could be recovered after disinfection of the shell eggs. The work by Hong and Slavik (1998) who sanitized the table eggs with quaternary ammonium compound, sodium carbonate, and sodium hypochlorite and inoculated the eggs with Salmonella Enteritidis reported more or less similar observations. Reduced penetration by Salmonella Enteritidis was seen in all time intervals of 1, 7, 14 and 21 days at 4 and 23 0C of storage in eggs treated with Quaternary Ammonium Compound (QAC) and sodium hypochlorite while increased penetration rate was observed in sodium carbonate treatments. Prolonged storage period further increased the rate of penetration. The results or observations of the present study on shell penetration and recovery of Salmonella Typhimurium after sanitation with 70% alcohol and 200 ppm of chlorine stored at refrigeration temperature at 14 days and onwards further confirms the observations of Pardon (1995) and Hong and Slavik (1998) that the routinely used sanitizers do not eliminate the pathogens completely and the organisms can be recovered from the whole egg contents. Similar findings were reported by Jones et al. (2005) who recovered reduced levels of Salmonella Enteritidis in shell membrane emulsion and whole egg contents sampled at regular intervals from stored shell eggs for a period of nine weeks after treatment with 200 ppm of chlorine as per typical U.S. wash water configuration. However, no recovery of Salmonella Typhimurium in PHMG treated eggs suggested complete elimination of the organism and found to be an effective chemical sanitizer which can be compared to the effectiveness of Pasteurization and dry heat treatment of shell eggs as demonstrated by the work of Barbour et al (2001) with Salmonella Enteritidis as a marker organism. The observation by Miyamoto et al (1998) that there is no significant difference between the penetration abilities of Salmonella Enteritidis and Salmonella Typhimurium in fresh and cooled (refrigeration) shell eggs but refrigeration prior to inoculation significantly reduced the penetration should be worth considering since Salmonella Typhimurium is used as a marker organism in the present study. 114
Berrang et al (1999) and Board and Tranter (2002) reported that the penetration of the egg shell by different microorganisms primarily occurs through trans-shell transmission of apart from transovarian and oviducal transmissions. The natural defenses of the egg are not entirely adequate to prevent penetration and survival of Salmonellae in shell eggs and observed that the outer structures of the shell eggs are not well adapted to resist bacterial penetration. The initial recovery of Salmonella Typhimurium observed in the present study was on 14th day at refrigeration temperature which is in accordance with the observations of Howard et al (2006). They conducted experiments to identify the proximate timelines for bacterial invasion of the egg by observing growth and survival of Salmonella Typhimurium which were inoculated on to shell membrane stored under refrigerated conditions for 8 weeks. They reported that the growth in albumin occurred by 2nd week. A lag period of 1 to 6 weeks is reported to be required for the appearance of the organism in the albumin especially in the absence of iron and at different temperature gradients. A similar study of Okamura et al (2008) further correlates with the findings of the present study with regard to time of appearance of Salmonella Typhimurium in whole egg contents. Similar observations were reported by Board and Tranter (2002) that the time required for the appearance of the organism in egg contents was10 to 20 days. The proof for survival capacity of Salmonella Enteritidis and Typhimurium to egg albumen has been provided by the work of Lu et al (2003) who identified Yaf D gene and the over expression of this gene conferred enhanced resistance to Salmonella serovars. Still there are several angles of looking in to finer aspects of shell penetration and recovery of the organism from inside contents of the eggs, first, as put forth by Board and Tranter (2002) that the presence of bactericides in wash water does not ensure that the viable organisms will not be lodged or enter within the pores, second, as reported by McDonnell and Russell (1999) that the capacity of organisms to acquire tolerance to chemicals or physical stresses as it has been observed with heat, ethanol, and hypochlorous acid, and thirdly, as demonstrated by Gupte et al (2009) and Gougeon et al (2006) that Salmonella Typhimurium which is capable of modifying its physiological characteristics, including entry into and recovery from the viable but non culturable state, suggests the overall possibility that Salmonella Typhimurium 115
might be able to respond uniquely to various adverse environmental conditions. These possibilities assume lot of significance to food safety in particular to egg safety or consumer concerns. The results further emphasized the need for establishment of performance criteria for sanitation of raw shell eggs to fulfill the Food Safety Objectives (FSOs) or Performance Objectives (POs) at processing plant or industry level. The performance criterion was derived in the present study by considering the initial hazard level and increase in hazard levels from in vitro surface inoculation and recovery studies and substituting these values in the following equation. H0 - I + R FSO Where - H0 = Initial level of hazard I = Cumulative increase in the hazard levels R = Cumulative decrease in hazard levels FSO = Established Food safety Objectives or criteria for Salmonella spp. in foods. The FSO is the maximum frequency and /or concentration of a (microbial) hazard in a food at the time of consumption that still provides the Appropriate Level Of Protection (ALOP) and Performance Objectives (POs) is the maximum frequency and /or concentration of a (microbial) hazard in a food at specified step in the food chain before time of consumption that still provides or contributes to an achievement of FSO or ALOP as applicable (Gorris, 2004 and Buchanan, 2004). The results of the calculations are depicted in Table 4.10. The average H0 value for surface inoculated eggs without any treatment by Salmonella Typhimurium which served as control for treatment with various sanitizers was found to be 4.30 log CFU/ml and cumulative increase in hazard levels i.e. I of internal contents of the eggs as obtained in the in vitro penetration and recovery study was found to be 2.13 log CFU/ml. Therefore, to meet the FSO of zero Salmonella Typhimurium in raw shell eggs, the expected cumulative decrease in hazard levels i.e. R should be 6.43 log CFU/ml as 116
per the equation. Likewise, the average I and R log CFU/ml values obtained in the present study were found to be 1.45, 0, 1.37 and 5.75, 4.30, and 5.67 for 70% alcohol, 65 ppm PHMG and 200 ppm of chlorine, respectively. The average I values (log CFU/ml) irrespective of treatment by different sanitizers was found to be 1.24 and the average expected cumulative decrease in hazard levels i.e. R values ((log CFU/ml) was found to be 5.54 to 6.00 approximately. It may be appropriate here to make a distinction between FSO and routinely used microbiological criteria concerning food safety. The FSO and Performance Objectives (POs) take in to consideration all the aspects like initial hazards, increase in hazards and expected log reductions to be achieved before a product is consumed laying emphasis on all points in the supply chain like sanitation, preservation, transport, distribution/sale to consumers etc. Moreover, FSO/PO is valid for all supply chains coming out with a particular product and suitable for the assessment of food safety system of a particular manufacturer or industry whereas microbiological criteria go by standards, guidelines and specifications for acceptability of a product based on the presence or absence or number of microorganisms per unit of mass, volume or lot and may not be suitable for assessment of food safety system of a manufacturer or industry and provides only information on a present lot, but not the previous or the future ones (Cordier and Nestec, 2004). Therefore, the overall results suggest that the raw shell eggs in order to be safe or to meet FSO would require cumulative log reductions (CFU/ml) of 5.54 to 6.00 by all commercially recommended and routinely employed sanitizers for complete and effective egg sanitation process. Since, there is every chance that the sanitizers may fail to achieve the complete elimination of the organism from the surface of the egg shell. Based on the results of the present study the performance criterion for sanitizers was fixed at minimum of 6 log reductions as it can be believed to contribute to an achievable ALOP at specified process step of sanitation and probably at consumer level. The performance criterion was worked out as per Cole (2004) with a worked out example for Listeria monocytogenes in whole lettuce for hydrogen peroxide and peroxy acetic acid mixture using the same equation. In addition, the prevalence rate of less than 1% for Salmonella spp. is mentioned as performance criteria for commercial layer farms as it can effectively contribute to achieve the FSOs (Cole 2004). Soljour et al (2004) also suggested that higher concentrations of sodium carbonate and sodium hypochlorite applied at 117
commercially recommended doses of 36 ppm and 200 ppm, respectively needed at least 5 to 20 times greater than recommended doses, in order to destroy Salmonella Enteritidis/ bacteria on the surface of shell eggs. This corresponds more or less to the observation of present study where 200 ppm chlorine and 70% alcohol failed to eliminate the organism.
Table 4.9: Shell penetration and recovery of Salmonella Typhimurium in whole egg contents after surface inoculation of raw shell eggs and treated with different sanitizers stored at refrigeration temperature
Average count of Salmonella Typhimurium ( log CFU/ml)
recovered after treatments with different sanitizers at different
Days
Salmonella free eggs
Surface Inoculated eggs
intervals 70% Alcohol
65 ppm PHMG
200 ppm Chlorine
A. Pre-inoculation
0
Absent
Absent
Absent
Absent
Absent
4
Absent
Absent
Absent
Absent
Absent
14
Absent
1.67
Absent
Absent
Absent
21
Absent
1.66
Absent
Absent
0.79
28
Absent
2.76
1.09
Absent
2.26
B. Post-inoculation
0
Absent
Absent
Absent
Absent
Absent
4
Absent
Absent
Absent
Absent
Absent
14
Absent
1.60
1.71
Absent
Absent
21
Absent
2.38
1.30
Absent
Absent
28
Absent
2.72
1.69
Absent
1.07
118
Table 4.10: Fixing of performance criteria for raw shell eggs to meet Food Safety Objectives based on in vitro studies of sanitation and penetration of Salmonella Typhimurium (log CFU/ml) in shell eggs
Parameter Surface inoculated untreated eggs 70% Alcohol 65 ppm PHMG 200 ppm Chlorine Average
H0 Values 4.30 4.30 4.30 4.30 4.30
I Values 2.13 1.45 0 1.37 1.24
Established FSO for Salmonella Absent/ zero Absent/ zero Absent/ zero Absent/ zero 0
Calculated R values 6.43 5.75 4.30 5.67 5.54 6
H0 = Initial level of hazard I =Cumulative increase in hazard R = Cumulative decrease in hazard
4.5 Shell penetration and recovery studies of Salmonella Typhimurium in whole egg contents of good and poor quality raw shell eggs stored at refrigeration temperature The quality of the shell of the eggs matters most in preventing the entry of organism inside the shell although does not offer full protection and is influenced by variety of external factors. The results of shell penetration and recovery of Salmonella Typhimurium in poor and good quality shells are presented in Table 4.11. The results indicated that the organism was recovered from the internal contents of the poor shell eggs on the same day of surface inoculation and continued to show recovery up to 40 days where as from good shell eggs the organism was recovered on 14th day and continued to show recovery up to 40 days as that of poor shell eggs. However, no Salmonella Typhimurium was recovered from both categories of shell eggs after 80 days of storage. The results suggested that the organism penetrated more readily in poor quality shell eggs than in intact shell eggs and poor quality shell eggs might increase the risk of Salmonella infections. Moreover, the organism was capable of survival up to 6 weeks at refrigeration temperatures and if not handled properly might become a source of contamination and infection though with low risk. The findings of the present study are supported by the research work carried 119
out by De Reu et al (2006), Bell and Weaver (2002), Schoeni et al (1995) and Shane et al (1986). De Reu et al (2006) studied the egg shell factors and shell penetration by bacteria using seven bacterial strains which included Staphylococcus warneri, Acinetobacter baumannii, Alcaligenes sp., Serratia marcescens, Carnobacterium spp., Pseudomonas spp. and Salmonella Enteritidis. They proposed that the mean cuticle deposition on the shell surface significantly influenced the bacterial penetration and organisms were recovered in egg contents after 4 to 5 days. In addition, they observed that the Gram-negative, motile and non-clustering bacteria penetrated the egg shell most frequently and the rate of penetration of Pseudomonas spp. (60%) and Alcaligenes spp. (58%) was relatively greater than Salmonella Enteritidis (43%). Bell and Weaver (2002) reported that the cracks in the eggs increase the likelihood of contamination of sweated eggs ten times more than the sound shell eggs. Schoeni et al (1995) observed that the penetration of shell by Salmonella Enteritidis, Salmonella Typhimurium or Salmonella Heidelberg occurred between one to 3 days when eggs were exposed to 104 cfu Salmonella/g feces and noticed that increased inoculum dose to 106cfu/g feces resulted in 50­75% of the contents getting contaminated by day one during storage. Similarly the work of Shane et al (1986) showed contamination of commercial table eggs with a fecal suspension containing 4.4 x 106 CFU/g Campylobacter jejuni which resulted in shell penetration in 3/70 eggs and recovery of the organism in homogenized egg contents was found in 1/70 eggs. The viability of C. jejuni on the shell surface was retained for 16 hrs. and this further suggested that the penetration had occurred before 16 hrs. The growth and survival of the Salmonella Typhimurium inside the egg contents for longer time up to 6 weeks in the present study are supported by the research work of Guan et al (2006), Howard et al (2006) and Lu et al (2003). Howard et al (2006) reported that SalmonellaTyphimurium can survive up to 8 weeks under refrigeration temperature and the proof for survival capacity of Salmonella Enteritidis and Typhimurium to egg albumen provided by the work of Lu et al (2003) because of Yaf D gene. 120
Qualitative risk analysis The penetration of shell by Salmonella Typhimurium (both poor and good shell quality eggs) and its survival in eggs for long period assumes lot of food safety significance in India's context as there are no shell egg quality monitoring programmes concerning to microbiological safety. The shell eggs are produced, stored, transported and marketed under unhygienic conditions including all eggs in one simple category as shell eggs without any distinction between their quality aspects/Grades such as clean, dirty, large, small etc. The risk to consumers from such shell eggs is a cause for real concern. The present work on shell penetration and recovery /survival, inclusive of eggs of good quality shell, poor quality shell and eggs treated with different sanitizers under refrigeration storage, pointed to one common observation that the average growth of the organism (Salmonella Typhimurium) in the whole egg contents was found to vary between 1.60 and 2.72 log CFU/ml. This when categorized for qualitative risk profiling/ rating considering the growth in the commercial shell egg product indicated that the risk posed by such eggs constituted low to medium risk category to the consumers. The qualitative risk rating such as low, medium and high is as per the Daughty et al (2005) who prepared a National Food Safety Risk Profile of eggs and egg products for Australian Egg Corporation limited. An increase of 5 log CFU/g in commercial non cracked egg constitute high risk rating at consumer points. However, when the work of Schoeni et al (1995) is taken in to consideration who observed the growth of Salmonella Enteritidis, Salmonella Heidelberg and Salmonella Typhimurium inoculated in to yolk and albumin stored at ambient temperature of 25 0C which resulted in the growth of 108 to 1010 CFU/g, a net increase of more than 3 log CFU/g points to the potential high risk profile under different storage conditions. 121
Table 4.11: Shell penetration and recovery of Salmonella Typhimurium in whole egg contents after surface inoculation of good and poor quality shell eggs stored at refrigeration temperature
Days 0 4 9 14 40 80
Salmonella free eggs
Poor shell
Good shell
Salmonella Typhimurium Counts CFU / ml
Absent
1.70
Absent
Absent
1.87
Absent
Absent
1.79
Absent
Absent
1.88
1.66
Absent
0.88
1.74
Absent
Absent
Absent
4.6 Shelf life of raw shell eggs stored at ambient (28-330C) and refrigeration (3-70C) storage temperatures treated with paraffin oil alone and in combination with various sanitizers
Shell eggs for table purposes are produced from genetically well evolved breed/ strains of poultry and generally the eggs are of high initial quality from disease free flocks and the quality of eggs there after solely depends upon the post harvest practices such as frequency of egg collection, cleanliness of the cages and egg collection trays, storage temperature, humidity, handling practices and various biosecurity measures. These factors determine the rate or rapidity of the decline in the quality of eggs. Shell eggs retain their freshness and quality when they are properly collected or stored with no abuse of temperature and humidity. The well accepted unit of physical measurement for shell egg freshness and internal quality of albumin is Haugh unit. Haugh unit is accepted by both industries as well as research institutions as a physical parameter for defining the egg quality attributes whereas for safety point microbiological criteria is used as guideline to determine the quality. Therefore, in the present study, the shelf life of raw shell eggs was determined by albumin quality at two storage conditions viz., ambient (28 ­ 33 0C) and refrigeration (3 -7 0C) treated with different sanitizers (as safety measure) and paraffin oil and paraffin oil alone. For assessing the shelf life at ambient and refrigeration storage temperatures, quality indices selected included Haugh unit, albumin height, albumin pH and yolk index. The microbiological quality assessment such as Total Viable Count (TVC), detection of Pseudomonas spp. was carried out till the end of the study period 122
whereas for safety assessment, detection of Salmonella spp and Campylobacter spp. was carried out up to first 7(ambient) and 30 (refrigeration) days of the storage. The 7 and 30 days period is considered as sufficient time for detection of Salmonella spp. and Campylobacter spp. in the internal contents of eggs as they appear before that period in case of contaminations and inadequate sanitations. 4.6.1 Shelf life studies of raw shell eggs stored at ambient temperature In the present study, The results of the internal quality assessment for freshness and retention of albumin and yolk quality at ambient storage temperature (28-330C) conditions at 0 day, 7 days, 15 days, 30 days and 40 days are given in Table 4.12 and Figure 5 & 6. The results of the individual internal quality attributes such as thick albumin height, Haugh unit, albumin pH and yolk index percentage are described in brief here. At `0' day, the average height of the albumin recorded were found to be 6.36 ± 0.49, 7.33 ± 1.13, 5.79 ± 0.28, 6.76 ± 0.66, 6.18 ± 0.85 and 8.06 ± 1.13 mm for (untreated) control eggs, paraffin oil coated, 70% alcohol plus paraffin oil, 65 ppm PHMG plus paraffin oil, 100 ppm hydrogen peroxide with colloidal silver (H2O2 with Ag) plus paraffin oil, and 200 ppm chlorine plus paraffin oil, respectively. The initial thick albumin height recorded for different treatment categories were found to be above 5.79 mm (Table No.12). By 7th day, the albumin height were found to be reduced up to 3.93 ± 0.31, 3.76 ± 0.09, 3.73 ± 0.24, 4.28± 0.39, 3.49 ± 0.45 and 3.99 ± 0.36 for (untreated) control eggs, paraffin oil coated, 70% alcohol plus paraffin oil, 65 ppm PHMG plus oil, 100 ppm hydrogen peroxide with colloidal silver (H2O2 with Ag) plus paraffin oil, and 200 ppm chlorine plus paraffin oil, respectively. The results at this stage showed considerable decline in the heights of thick albumin had occurred with minimum decline in height of about 2.06 to 2.69 units for all categories except for chlorine which recorded maximum decline in height of about 4.07 units. By 15th day, control eggs (untreated) showed complete thinning of thick albumin where as other groups showed more or less similar albumin heights with a minimum decline in height of 0.3 units and maximum of 0.46 units for chlorine treated eggs. The height of the thick albumin further decreased by 0.17 to 0.72 units among treated groups and complete thinning was observed by 40th day in chlorine treated group where as 123
other groups recorded albumin height below 2mm indicating complete loss of freshness and albumin quality (Table No.12). The overall trend with regard to albumin height clearly demonstrated that the fresh untreated eggs ( control group) retained the quality below 15 days and treated eggs lasted up to 40 days except chlorine treated samples that lasted for 30 days. The overall reduction in albumin heights compared to its initial heights were found to be 6.02, 4.20, 4.82, 4.76 and 8.06 units for groups treated with paraffin oil, 70% alcohol plus paraffin oil, 65 ppm PHMG plus oil, 100 ppm hydrogen peroxide with colloidal silver(H2O2 with Ag) plus paraffin oil, 200 ppm chlorine plus paraffin oil, respectively. Silversides and Budgell (2004) who conducted experiments to study the relationship between egg albumin height, pH and whipping volume of shell eggs from Brown Leghorn hens (commercial brown egg layer) and Babcock hens (commercial white egg layer) observed reduction in albumin height between fresh and 5 and 10 days stored eggs. Similar findings were obtained in the present study. Sheikh and Younis (2005) evaluated the influence of hen layer age and storage periods at room temperature (33.2°C) on quality of eggs of white and brown hyline strains during summer conditions. The albumin height of eggs decreased as the hen age increased and with increasing storage time or period. Longer periods of storage resulted in significantly (P<0.05) lower albumin height. They further observed that, the shelf life of eggs was between 21 to 28 days at room temperatures which is higher as compared to the findings of present study with regard to fresh untreated eggs. Samli et al (2005) studied the quality of eggs subjected to storage time or periods of for 2, 5 and 10 days and temperatures of 5, 21, and 29°C observed that the albumin height, Haugh unit, pH of albumin and yolk, specific gravity and air cell size were the most important parameters which were greatly deteriorated by the storage time and temperature. Similar observations were made in the present study that the quality of deterioration of eggs depended upon storage time and temperature at which the eggs were stored. The Haugh unit values, a marker of freshness and albumin quality, recorded for ambient stored eggs (Table No.12) were found to be initially as 80.39± 3.22, 85.35 ± 6.19, 76.64 ± 2.37, 81.96± 3.77, 76.45 ± 6.16 and 88.07 ± 1.13 for (untreated) control eggs, paraffin oil coated, 70% alcohol plus paraffin oil, 65 ppm PHMG plus paraffin oil, 100 ppm hydrogen peroxide with colloidal silver (H2O2 with Ag) plus paraffin oil, and 200 ppm chlorine plus paraffin oil, respectively. The fresh (control group) eggs without any treatment had no 124
Haugh unit value by day 15th and other treatment groups had 53.24 ± 2.38, 60.55 ± 4.15, 58.00 ± 5.61, 47.05 ± 1.08 and 56.37 ± 1.86, respectively. This suggested a considerable decline in the Haugh unit values and hence decline in quality. However, by 30th day at ambient storage, all the treated groups showed just acceptable Haugh unit values ( i.e.40 HU to 54.30) while storage for 40 days showed Haugh unit values below 30 in all except chlorine treated group which recorded `0' HU suggesting that the eggs were below acceptable Haugh unit values. The reduction in Haugh unit values at the end of first week of storage was found to be in the range of 17.17 to 31.67 units, (all groups considered) and in subsequent days of storage the reduction was in the range of 0.03 to 5.33 (15days) and 2.47 to 16.07 (30 days) units. This showed that the maximum reductions occurred in the initial first 7 days and then the intensity of Haugh unit reductions showed a declining trend. Large variations were noticed in Haugh units, between days of storage and between groups, though the constant phenomenon was a loss of Haugh units. Samli et al (2005) observed that extending the storage time up to 10 days and the temperature up to 29°C resulted in significant deterioration in egg quality which drastically resulted in dropped Haugh unit values to 76.3, 53.7 and 40.6 at 5, 21 and 29 єC storage temperatures, respectively. However, in the present study, the acceptable Haugh unit values up to 30 days of storage were obtained in paraffin oil and sanitizers plus paraffin oil treated groups. Their observations coincide well with the observations of the present study where complete thinning of albumin was noticed in fresh eggs by 15 days at ambient storage temperature. Considering the USDA criteria for consumer grade qualities based on Haugh unit, eggs in all the treated categories declined in its initial quality of AA to B by 15th day and remained in B grade up to 30 days and passed on to C grade after 30 days of storage. But as per Gerber (2006) who demonstrated that the eggs that go for consumption should have Haugh unit value of 60, none of the eggs in all the categories met this requirement by 15th day except the eggs just coated with the paraffin oil. The findings of the present study concerning albumin height and Haugh unit values as quality markers indicated that at ambient storage, the shelf life of shell eggs without any treatment was less than 15 days and for treatment groups it was about 30 days with acceptable Haugh unit values according to USDA consumer grade qualities for 125
shell eggs but with a lesser grade. Sanitizers as such did not play any role with regard to albumin quality and the extension of shelf life/quality was due to paraffin oil applications. Similar to albumin, the yolk quality also showed considerable decline at ambient storage conditions (Table 4.12). The initial percent yolk index values were between 35.48 and 39.67 which declined to 13.56 in control eggs and 22.87 to 24.89 in treated groups by 15th day and by the end of 30 days it was found to be in the range of 10.26 to 12.24 among the treated group and 4.15 for control eggs. There was almost complete loss of quality and eggs were unsuitable as raw shell eggs by 40 days of storage at ambient temperature. Sheikh and Younis (2005) and Samli et al (2005) observed the loss in quality of yolk with increasing storage period at ambient temperatures which correlates well with the results of the present study. It is quite obvious that the quality of yolk changed with the quality of albumin since they are inter related. The pH of the albumin was found to be alkaline and ranged between 8 and 9 from the beginning of the study and remained in the same range with marginal variations in between the period (Table 4.12). Silversides and Budgell (2004) and Sheikh and Younis (2005) observed that the pH of the albumin was affected by several factors such as strain and hen age, but it increased with the storage period at ambient temperatures. Longer periods of storage resulted in significantly (P<0.05) higher albumin pH regardless of hen age and strain. Samli et al (2005) also observed that over 21єC temperature, increased pH in albumin was noticed in 2 days of storage whereas a rise in pH from 7.47 to 9.2 was noticed after 5 days of storage regardless of storage temperature, i.e. 5 and 21єC. A similar trend was observed in the present study with regard to rise of pH at ambient storage. The application of oil on surface of shell eggs is one of the recommended and economical way of preserving the egg quality which greatly reduces losses by way of evaporation and where eggs must withstand high temperatures (of more than 210C), they should be coated with oil within four to six hours after lay (FAO 2003) for better results. Otherwise eggs are prone to the loss of quality as and when there is abuse of temperature and storage conditions. This could probably, be one of the reasons where even oil treated eggs also showed faster decline in quality at ambient temperatures of 28 -33 0C. (Bell and Weaver, 2002 and Stadelman and Cotterill, 2002) 126
The microbiological quality of eggs was assessed in terms of Total Viable Count (TVC), Pseudomonas spp, and presence or absence of Salmonella up to 7 days of storage (Table 4.12). It was observed that the Total Viable Count was in the range of 3.30 ± 0.08 log CFU/ml to 3.90 ± 0.16 log CFU/ml at the beginning of the study and remained almost in the similar range with little variations in between and showed a marginal decline among the treatment groups at 15th day. Pseudomonas spp were not found through out the period and both Salmonella and Campylobacters were absent in the eggs. The findings of the present study with regard to internal microbiological quality are well within the accepted microbiological limits as recommended by USDA (1991) for liquid eggs before expiration date. Theron et al (2003) who performed evaluation of the effects of various storage and transport conditions on the bacterial growth associated with shell eggs transported without temperature control observed 4 hrs low temperature shock (cold shock) was the most effective condition for limiting organism growth in or on eggs whereas at high temperature shocks, much higher counts of organism were found in both the egg contents and on the eggshells. However, in the present study, an increase in the counts of organisms were not noticed which may be because the shell eggs were prior treated with sanitizers followed by application of oils which can be noticed with the result that untreated eggs showed a marginal increase in counts. 4.6.2 Shelf life of raw shell eggs stored at refrigeration (3 ­ 70C) temperature The shelf life of raw shell eggs was analyzed at intervals of 30 days (1month), up to 180 days (6 months) by assessing the internal qualities in terms of albumen heights, Haugh unit values, yolk index %, pH, TVC and presence of Pseudomonas, Salmonella and Campylobacter species during storage for up to 30 days. The details of the results are presented in the Table 4.13. The average height of the albumin recorded for control and treated groups of eggs was 5.79 ± 0.28 to 7.33 ± 1.10 at `0' day or beginning of the study which remained steady albumin heights up to 150 days with a variations of 0.20 units to 1.95 units at 150th day. The decline in height was observed markedly after 180 days of storage by a margin of 0.97 to 2.71 units. However, the decrease in heights of the albumin was observed more in untreated (control eggs) i.e.2.7 units as compared to treated groups of eggs. But still, after 6 127
months, the heights of albumin were superior to eggs stored at ambient temperature for 7 days (Table 4.13). The Haugh units values observed (Table 4.13) for the control and treated eggs were found to be in the range of 76.45 to 85.35 at the start of the shelf life study. The Haugh units showed visible decline after 180 days of storage by units that ranged from 7.19 to 18.29 and the highest loss of units was found to be in untreated (control) eggs i.e. 18.29 and least loss of units was noticed in 70% alcohol plus paraffin oil applied eggs. However, the loss of units between sanitizers plus paraffin oil applied and only paraffin oil applied appeared to be marginal, which ranged between 7.19 and 10.44. Discounting the sanitizers' application to the eggs and looking at the results, the maximum loss of HU values was just 10.44 for paraffin oil applied eggs. When glanced at the HU values until 150 days of storage at refrigeration, eggs possessed good HU values and approached towards fair HU values by 180 days and possessed HU values far above the acceptable value. A point worth mentioning here is, under commercial/ industrial conditions, such prolonged storage may not be needed as eggs' demand and supply may not see such deficits. As the quality of albumin and yolk are inter related, the yolk showed a good quality throughout the storage period from 0 to 180 days and the percent yolk index values recorded were 39.89 to 32.30, 36.27 to 34.00, 35.48 to 34.50, 37.44 to 35.40, 37.52 to 34.80 and 35.94 to 34.90 for untreated (control) eggs, paraffin oil coated, 70% alcohol plus paraffin oil, 65 ppm PHMG plus paraffin oil, 100 ppm hydrogen peroxide with colloidal silver (H2O2 with Ag) plus paraffin oil and 200 ppm chlorine plus paraffin oil, respectively (Table 4.13). The average values of the egg albumin pH showed increase from that of the initial values of 8.57 - 8.67 at `0' days of storage to 8.93-9.40 at the end of 180 days of storage period. The eggs showed higher pH values towards alkaline side. The retention of high internal quality of shell eggs stored at refrigeration temperature is evidenced by work of several researchers such as Samli et al (2005) observed it for 2 weeks, Biladeau and Keener (2009) who observed for 12 weeks and FAO (2003) states eggs stored at cold storage at a temperature of - 1° C maintains quality for several months are well correlated with the findings of the present study where shell eggs were observed for 6 months for its internal quality. Moreover, the shell eggs with respect to consumer grade 128
qualities fulfilled the requirements of Gerber (2006) who observed that the eggs meant for consumption should have Haugh unit of 60 and above, as well as USDA consumer grade qualities. The shell eggs in the present study were of A quality in untreated eggs, oiled eggs and chlorine plus oiled eggs where as other groups (alcohol plus oil, PHMG plus oil and hydrogen peroxide with colloidal Ag plus oil) had AA quality. The USDA consumer grade shell egg qualities based on Haugh unit is more than 72 HU (AA), 60-72(A), 31-60 (B) and less than 31 HU (C). The microbiological qualities were assessed in terms of Total Viable Count, Pseudomonas spp, Salmonella and Campylobacter spp. Pseudomonas spp was not recovered through out the storage period and eggs were found to be free from Salmonella and Campylobacter spp. The results of the Total Viable Count (TVC) were found to be in the range of 3.30 to 3.90 log CFU/ml at the start of the study and the counts remained more or less within the same range till the end of 180 days of storage i.e. 3.71 to 3.83 log CFU/ml. The raw shell eggs remained acceptable in terms of microbiological quality and by physico-chemical quality with a bit of loss of freshness as compared to `0' day. The results of the present study are in accordance with recommended microbiological limits of USDA (1991) for liquid eggs which recommend the acceptable Total Viable Counts of less than 10,000 log CFU/ml before expiration date and free from Salmonella spp. in 100 g of eggs. When ambient and refrigeration storage of shell eggs were compared, the refrigeration storage extended or even preserved the original internal quality attributes of freshness, albumin and yolk quality fairly enough for the observed period of 6 months as against 1 month (30 days) shelf life of treated eggs at ambient storage. Just refrigeration storage without any type of treatment was able to preserve the internal quality attributes for 5 to 6 months while for ambient storage it was less than 15 days. As observed by Williams (1992) the oiling of eggs within 24 h of lay though very effective in retarding the deterioration of the albumin quality does not replace the need for cool storage. Similar, observations were made by FAO (2003), Stadelman and Cotterill (2002) and Bell and Weaver (2002) and the work of several researchers as quoted in the discussion above, that the factors such as temperature, humidity, air movement and storage time can all have adverse effects on the internal quality of shell eggs which if not controlled, can lead to loss of moisture and CO2 in eggs and hence, quality of eggs. 129
Table 4.12: Shelf-life of raw shell eggs treated with paraffin oil alone and in combination with various sanitizers stored at ambient (28 - 330C) temperature
Parameter 0 days Albumin Height (mm) Haugh unit Yolk Index % Albumin pH TVC (log CFU/ml) Pseudomonas spp. Salmonella spp. Campylobacter spp. 7 days Albumin Height (mm) Haugh unit Yolk Index % Albumin pH Pseudomonas spp. Salmonella spp. Campylobacter spp.
Untreated control 6.36 ± 0.49 80.39 ± 3.22 35.48 ± 2.73 8.53 ± 0.04 3.30 ± 0.08 Absent Absent Absent 3.93 ± 0.31 60.07 ± 6.26 24.64 ± 1.82 8.90 ± 0.0 Absent Absent Absent
Paraffin oil 7.33 ± 1.10 85.35 ± 6.19 37.44 ± 1.35 8.67 ± 0.07 3.90 ± 0.16 Absent Absent Absent 3.76 ± 0.09 58.30 ± 1.15 25.49 ± 2.27 8.70 ± 0.00 Absent Absent Absent
70%Alcohol + Paraffin oil 5.79 ± 0.28 76.64 ± 2.37 35.94 ± 0.73 8.67 ± 0.07 3.90 ± 0.16 Absent Absent Absent 3.73 ± 0.24 59.47 ± 2.87 25.49 ± 1.73 8.63 ± 1.0 Absent Absent Absent
65 ppm PHMG + Paraffin oil
100 ppm H2O2 with colloidal Ag + Paraffin oil
200 ppm chlorine + Paraffin oil
6.76 ± 0.66 81.96 ± 3.77 37.52 ± 2.55 8.70 ± 0.09 3.83 ± 0.20 Absent Absent Absent
6.18 ± 0.85 76.45 ± 6.16 36.27 ± 2.25 8.70 ± 0.05 3.76 ± 0.05 Absent Absent Absent
8.06 ± 1.13 88.07 ± 6.33 39.67 ± 1.43 8.60 ± 0.08 3.34 ± 0.07 Absent Absent Absent
4.28 ± 0.39 63.33 ± 4.41 24.90 ± 0.44 8.67 ± 0.03 Absent Absent Absent
3.49 ± 0.45 51.65 ± 6.13 25.88 ± 1.45 8.80 ± 0.0 Absent Absent Absent
3.99 ± 0.36 56.40 ± 3.95 27.74 ± 0.36 8.90 ± 0.0 Absent Absent Absent Contd..
130
Parameter 15 days Albumin Height (mm) Haugh unit Yolk Index % Albumin pH TVC(log CFU/ml) Pseudomonas spp. Salmonella spp. Campylobacter spp. 30 days Albumin Height (mm) Haugh unit Yolk Index % Albumin pH Pseudomonas spp. Salmonella spp. Campylobacter spp. 40 days Albumin Height (mm) Haugh unit Yolk Index % Albumin pH TVC (log CFU/ml) Pseudomonas spp. Salmonella spp. Campylobacter spp.
Untreated control
Paraffin oil
70%Alcohol + Paraffin oil
0 0 13.56±1.90 8.77±0.03 3.85 ± 0.16 Absent ND ND
3.46 ± 0.15 53.24 ± 2.38 23.13 ± 1.14 8.63 ± 0.02 3.03 ± 0.01 Absent ND ND
3.93 ± 0.39 60.55 ± 4.15 24.89 ± 0.81 8.83 ± 0.07 2.98 ± 0.09 Absent ND ND
0 0 4.15 ± 2.08 8.63 ± 0.07 Absent ND ND
3.17 ± 0.39 46.64 ± 6.90 12.24 ± 4.13 8.45 ± 0.11 Absent ND ND
3.57 ± 0.15 54.30 ± 6.89 11.94 ± 5.99 8.0 ± 0.04 Absent ND ND
0 0 5.35 ± 1.64 8.90 ± 0.0 3.92 ± 0.01 Absent ND ND
1.36 ± 0.43 22.40 ± 7.50 6.54 ± 2.77 8.58 ± 0.05 3.48 ± 0.12 Absent ND ND
1.79 ± 0.73 27.57 ± 11.28 19.00 ± 1.58 7.92 ± 0.13 3.36 ± 0.05 Absent ND ND
65 ppm PHMG + Paraffin oil 3.95 ± 0.44 58.00 ± 5.61 23.25 ± 0.40 8.77 ± 0.03 3.34 ± 0.24 Absent ND ND 3.23 ± 0.40 51.32 ± 5.51 15.22 ± 4.41 8.00 ± 0.03 Absent ND ND 1.94 ± 1.37 26.53 ± 10.20 9.98 ± 4.09 8.03 ± 0.16 3.37 ± 0.02 Absent ND ND
100 ppm H2O2 with colloidal Ag + Paraffin oil
200 ppm chlorine + Paraffin oil
3.09 ± 0.05 47.05 ± 1.88 22.21 ± 0.62 8.67 ± 0.07 2.87 ± 0.05 Absent ND ND
3.53 ± 0.07 56.37 ± 1.86 22.87 ± 0.66 8.70 ± 0.0 3.12 ± 0.06 Absent ND ND
2.92 ± 0.54 44.58 ± 7.02 20.80 ± 0.12 7.85 ± 0.03 Absent ND ND
2.96 ± 0.66 40.30 ± 11.28 10.26 ± 5.14 7.85 ± 0.03 Absent ND ND
1.42 ± 0.45 22.02 ± 7.64 11.73 ± 3.41 7.92 ± 0.05 3.48 ± 0.13 Absent ND ND
0 0 1.72 ± 1.57 8.70 ± 0.07 .3.36 ± 0.05 Absent ND ND
131
Table 4.13: Shelf-life of raw shell eggs treated with paraffin oil alone and in combination with various sanitizers at refrigeration storage (3-7 0C) temperature
Parameter 0 days Albumin Height (mm) Haugh unit Yolk Index % Albumin pH TVC (log CFU/ml) Pseudomonas spp. Salmonella spp. Campylobacter spp 30 days Albumin Height (mm) Haugh unit Yolk Index % Albumin pH TVC (log CFU/ml) Pseudomonas spp. Salmonella spp. Campylobacter spp
Untreated control 7.09 ± 0.35 84.29 ± 1.76 39.89 ± 1.69 8.57 ± 0.15 3.30 ± 0.08 Absent Absent Absent 5.87 ± 0.41 76.73 ± 3.94 38.49 ± 1.08 8.61 ± 0.05 3.27 ± 0.01 Absent Absent Absent
Paraffin oil 6.18 ± 0.85 76.45 ± 6.16 36.27 ± 2.25 8.70 ± 0.05 3.76 ± 0.05 Absent Absent Absent 5.22 ± 0.37 70.31 ± 3.25 39.49 ± 0.87 8.75 ± 0.03 3.60 ± 0.04 Absent Absent Absent
70%Alcohol + Paraffin oil
65 ppm PHMG + Paraffin oil
100 ppm H2O2 with colloidal Ag + Paraffin oil
200 ppm chlorine + Paraffin oil
6.36 ± 0.49 80.39 ± 3.22 35.48 ± 2.73 8.53 ± 0.04 3.26 ± 0.09 Absent Absent Absent
7.33 ± 1.10 85.35 ± 6.19 37.44 ± 1.35 8.67 ± 0.07 3.90 ± 0.16 Absent Absent Absent
6.76 ± 0.66 81.96 ± 3.77 37.52 ± 2.55 8.70 ± 0.09 3.83 ± 0.20 Absent Absent Absent
5.79 ± 0.28 76.64 ± 2.37 35.94 ± 0.73 8.67 ± 0.07 3.90 ± 0.16 Absent Absent Absent
5.9 0± 0.30 78.35 ± 1.02 34.39 ± 3.34 8.76 ± 0.05 3.28 ± 0.14 Absent Absent Absent
6.22 ± 0.35 79.67 ± 2.45 40.34 ± 0.90 8.68 ± 0.03 3.68 ± 0.04 Absent Absent Absent
6.00± 0.14 77.30 ± 1.96 40.63 ± 1.76 8.70 ± 0.02 3.58 ± 0.00 Absent Absent Absent
5.76 ± 0.67 76.27 ± 2.58 39.33 ± 1.19 8.69 ± 0.03 3.35 ± 0.09 Absent Absent Absent
Contd....
132
Parameter 60 days Albumin Height (mm) Haugh unit Yolk Index % Albumin pH TVC (log CFU/ml) Pseudomonas spp. Salmonella spp. Campylobacter spp. 90 days Albumin Height (mm) Haugh unit Yolk Index % Albumin pH TVC (log CFU/ml) Pseudomonas spp. Salmonella spp. Campylobacter spp. 120 days Albumin Height (mm) Haugh unit Yolk Index % Albumin pH TVC (log CFU/ml) Pseudomonas spp. Salmonella spp. Campylobacter spp
Untreated control 4.91 ± 0.20 70.39 ± 1.80 33.94 ± 1.42 8.55 ± 0.02 3.44 ± 0.08 Absent ND ND 5.30 ± 0.21 73.12 ± 1.88 33.73 ± 1.47 8.5 ± 0.02 3.34 ± 0.16 Absent ND ND 4.40 ± 0.31 65.65 ± 3.22 33.14 ± 0.92 8.58 ± 0.02 3.26 ± 0.32 Absent ND ND
Paraffin oil 5.21 ± 0.29 69.91 ± 2.54 36.60 ± 0.87 8.26 ± 0.02 3.35 ± 0.16 Absent ND ND 5.53 ± 0.40 72.21 ± 3.98 36.08 ± 0.72 8.20 ± 0.05 3.07 ± 0.19 Absent ND ND 5.21 ± 0.18 70.82 ± 1.51 34.77 ± 0.95 8.78 ± 0.02 2.81 ± 0.10 Absent ND ND
70%Alcohol + Paraffin oil
65 ppm PHMG + Paraffin oil
100 ppm H2O2 with colloidal Ag +Paraffin oil
5.43 ± 0.25 71.55 ± 2.56 39.04 ± 1.12 8.40 ± 0.06 3.53 ± 0.13 Absent ND ND
5.43 ± 0.39 73.49 ± 3.06 39.94 ± 1.45 8.20 ± 0.05 3.29 ± 0.11 Absent ND ND
5.47 ± 0.30 72.72 ± 2.12 37.55 ± 1.52 8.24 ± 0.02 3.26 ± 0.11 Absent ND ND
5.36 ± 0.27 72.71 ± 2.49 39.26 ± 0.97 8.05 ± 0.04 3.40 ± 0.13 Absent ND ND
5.41 ± 0.28 73.65 ± 1.91 38.14 ± 0.75 8.17 ± 0.03 3.56 ± 0.26 Absent ND ND
5.51 ± 0.36 74.43 ± 2.59 36.22 ± 0.88 8.09 ± 0.04 3.12 ± 0.30 Absent ND ND
5.21 ± 0.17 71.85 ± 1.35 35.53 ± 1.41 8.31 ± 0.05 2.77 ± 0.13 Absent ND ND
5.26 ± 0.35 72.18 ± 3.36 37.28 ± 0.91 8.46 ± 0.02 2.65 ± 0.26 Absent ND ND
5.25 ± 0.35 71.25 ± 3.13 36.04 ± 0.91 8.50 ± 0.06 2.88 ± 0.18 Absent ND ND
200 ppm chlorine + Paraffin oil 5.09 ± 0.35 70.90 ± 2.45 35.80 ± 0.82 8.35 ± 0.03 3.51 ± 0.13 Absent ND ND 5.40 ± 0.22 73.76 ± 1.81 34.78 ± 0.52 8.32 ± 0.03 3.39 ± 0.10 Absent ND ND 4.47 ± 0.26 67.74 ± 2.48 36.04 ± 1.10 8.63 ± 0.43 2.58 ± 0.28 Absent ND ND Contd....
133
Parameter 150 days Albumin Height (mm) Haugh unit Yolk Index % Albumin pH TVC (log CFU/ml) Pseudomonas spp. Salmonella spp. Campylobacter spp. 180 days Albumin Height (mm) Haugh unit Yolk Index % Albumin pH TVC (log CFU/ml) Pseudomonas spp. Salmonella spp. Campylobacter spp.
Untreated control 5.14 ± 0.16 72.50 ± 0.85 31.60 ± 0.89 9.68 ± 0.02 3.8 ± 0.08 Absent ND ND 4.38 ± 0.29 66.00 ± 3.28 32.30 ± 1.99 9.40 ± 0.00 3.71 ± 0.09 Absent ND ND
Paraffin oil 5.54 ± 0.19 76.20 ± 1.78 34.60 ± 0.40 9.20 ± 0.04 3.77 ± 0.08 Absent ND ND 4.77 ± 0.27 67.10 ± 2.94 34.00 ± 0.88 9.40 ± 0.0 3.83 ± 0.06 Absent ND ND
70%Alcohol + Paraffin oil 6.16 ± 0.19 78.40 ± 1.60 37.90 ± 1.58 8.93 ± 0.04 3.64 ± 0.13 Absent ND ND 5.39 ± 0.09 73.20 ± 1.00 34.50 ± 1.05 8.93 ± 0.07 3.68 ± 0.10 Absent ND ND
65 ppm PHMG + Paraffin oil
100 ppm H2O2 with colloidal Ag + Paraffin oil
200 ppm chlorine + Paraffin oil
5.31 ± 0.16 71.20 ± 1.64 36.70 ± 0.61 9.20 ± 0.06 3.48 ± 0.21 Absent ND ND
6.04 ± 0.20 78.00 ± 1.83 37.00 ± 0.88 9.13 ± 0.07 3.23 ± 0.06 Absent ND ND
4.80 ± 0.22 68.90 ± 2.16 34.90 ± 1.0 9.55 ± 0.06 3.46 ± 0.15 Absent ND ND
5.15 ± 0.18 72.10 ± 1.19 35.40 ± 0.70 9.30 ± 0.0 3.61 ± 0.14 Absent ND ND
5.35 ± 0.19 72.11 ± 2.11 34.80 ± 1.07 9.10 ± 0.04 3.68 ± 0.02 Absent ND ND
4.47 ± 0.23 66.20 ± 1.84 34.90±0.99 9.38±0.02 3.83±0.23 Absent ND ND
134
Fig. 5: Albumin height (mm) of raw shell eggs stored at ambient temperatures treated with paraffin oil alone and in combination with various sanitizers
Albumin height (mm)
9 8 7 6 5 4 3 2 1 0 Untreated
Paraffin oil
70% alcohol + 65ppm PHMG + 100 ppm (Ag 200 ppm
oil
oil
stabilized) Chlorine + oil
hydrogen
peroxide + oil
Treatments
0 day
7 days
15 days
30 days
40 days
135
Fig.6: Haugh unit of raw shell eggs stored at ambient temperatures treated with paraffin oil alone and in combination with various sanitizers
Haugh unit
90 80 70 60 50 40 30 20 10 0 Untreated
Paraffin oil
70% alcohol + oil
65 ppm PHMG +oil
100 ppm (Ag 200 ppm stabilized) chlorine + oil hydrogen peroxide + oil
Treatments
0 day
7 days
15 days
30 days
40 days
136
4.7 Shell egg weight loss stored at ambient and refrigeration temperatures The shell eggs go through loss of weight when stored for some period at different storage conditions. The results of the weight loss of the fresh shell eggs are given in Table 4.14 and Figure 7. The results demonstrated that the average weight loss for fresh raw eggs were in the range of 0.50 ± 0.15 to 5.90 ± 0.88g at ambient and 0.70 ± 0.15 to 1.29 ± 0.13g at refrigeration storage observed for a period of 6 and 8 weeks, respectively. The respective percentage weight loss observed for shell eggs stored at ambient and refrigeration temperatures were found to range from 0.88 to 10.33 and 1.24 to 2.28. At 15 days (2 weeks) the weight loss observed was found to be 2.10 %, which was found to be shelf life of fresh raw shell eggs at ambient temperature in the present study. The results indicated that the loss of weight of shell eggs was reduced to an extent of 2-6 times at 2 weeks and 6 weeks, (normally the maximum holding period of eggs in the market channel at ambient and refrigeration temperature), respectively. Bell and Weaver (2002) observed that as a general rule, the eggs stored at adequate storage conditions of 70C would lose about 1 percent of their initial weight per week and 2 percent at ambient temperatures with maximum loss of weight in the initial periods. The eggs may loose weight as high as 2-3% at ambient conditions (FAO, 2003). Jones and Musgrove (2005) reported the loss of egg weight as high as 5-6g in cold stored eggs for an extended period of 5 weeks. The results of the present study suggested more or less similar observations to that of Bell and Weaver and FAO for ambient stored eggs. The less weight loss at refrigeration temperature observed in the present study as compared to that of 1% per week as reported by Bell and Weaver may be due to the fact that eggs were placed inside the cartons and sealed properly before storage which restricted the air movement. Similar observations were recorded by Silversides and Budgell (2004) while Rocculi et al (2009) observed that the Modified Atmosphere Packaging (MAP) of shell eggs strongly reduced the weight loss from the product stored for 28 days at 25 °C. 137
Fig. 7: Percent weight loss of shell eggs stored at ambient and refrigeration temperatures 12
10
8
% weight loss
6 4
2
0
1
2
3
4
5
6
7
W e e ks
Am bi e nt
Re frige ra tion
138
Table 4.14: Average loss of egg weight (g) of fresh raw shell eggs stored at ambient (28-33 0C) and refrigeration (3-7 0C) temperatures
I. Ambient storage
Weeks
Average weight (g)
0
57.10±0.66
1
56.60±0.53
2
55.10±0.71
3
53.85±0.28
4
53.46±0.29
6
51.20±0.25
II. Refrigeration storage
0
56.64±0.30
2
55.39±0.61
4
55.18±0.63
6
55.05±0.58
8
54.79±0.49
Average weight loss (g) 0 0.50±0.15 2.00±0.39 3.25±0.68 3.64±0.82 5.90±0.88 0 0.70±0.15 0.90±0.11 1.04±0.11 1.29±0.13
% weight loss 0 0.88 2.10 5.69 6.37 10.33 0 1.24 1.59 1.84 2.28
4.8 Safety of shell eggs from pesticide and antimicrobial residues 4.8.1 Pesticides residues Residues of chemicals especially agro-chemicals like pesticides because of their extensive use in control of pests in agriculture and as a vector control measure in public health are encountered in food/feed at varying concentrations and sometimes at violative concentrations prescribed by National/ International agencies. The changing proportion or concentration of residues in animal food products meant for human consumption is related to various factors like movement or flow of contaminants in food chain, sources of contamination, farming practices and disposal of pesticides in the environment and its capacity to persist in the environment for shorter or longer periods. Shell eggs being an important food commodity for human consumption, the assessment of shell eggs for freedom from residues of pesticides is essential. The shell eggs from commercial layer farms and random market samples were analyzed using Gas Chromatography in the present study for detection of Organochlorine compounds like HCH (alpha, beta and delta HCH together), HCH/Lindane, Heptachlor, Aldrin, Endosulphan (alpha and beta 139
Endosulphan together) and DDT( o,p'-DDE, p,p'-DDE, o,p'-DDD, p,p'-DDD, o,p'-DDT and p,p'-DDT together), Synthetic pyrethroids like lambda Cyhalothrin, Cypermethrin, Fenvalerate and Deltamethrin and Organophosphate compounds like Phorate, Methyl Parathion, Malathion, Ethyl Chlorpyriphos, Phenthoate, Profenophos and Ethion. The results of the study indicated that the shell egg samples from farm and market sources analysed for selected pesticides from Organochlorines, Organophosphates and synthetic pyrethroids were found negative for presence of residues. Though the study revealed zero prevalence status in shell eggs, to state the prevalence in conclusive manner larger samples size and more extensive surveillance of farms and market may be required. Kan (2002) observed reduction in levels of organochlorines pesticide residues in food of animal origin by controlling contamination of animal or poultry feeds which restricted the entrance of pesticides via the feed chain. The registration and use of less persistent pesticides in the environment both for direct treatment of animals and of plants and plant products might have resulted in the further reduction of levels of pesticide residues in animal origin foods. Similarly, the ban on the use of persistent pesticides in the environment for crop protection and vector control further controlled the contamination of foods of animal origin. The results of the present study can be attributed to these factors, but the observations by Mourin (2005) who collected samples of free- range chicken eggs from different sites like near waste incinerators, cement kilns, the metallurgical industry, waste dumps, and chemical production facilities and found violative levels of pesticide residues. The observations of Narahari and Amutha (2007) that 79% of the egg samples had undetectable pesticide levels and rest detected at low levels needs to be viewed in the light of location/ spatial variations where contaminations from industries could be more. The studies by Khwaja (2004),Overmeire et al (2006 and 2009) indicated that the prevalence of pesticide residues is more in free range chicken eggs and home produced eggs than in commercial layer farms. These facts or observations may strengthen the findings of the present study and suggests that shell eggs produced from organized commercial layer farms do not pose risk to the consumers by way of pesticide residues. 140
4.8.2 Antimicrobial residues Antibiotic residues in raw shell eggs appear because they are added to poultry feed as feed additives or birds are treated with antibiotics for the control of clinical or sub clinical infections. The pattern of appearance of antibiotic residues in the eggs is not homogenous and it may be released in to the eggs from hens at unpredictable intervals. Therefore, screening for antibiotic residues in eggs serve as first step in the prevention of appearance of residues in shell eggs meant for consumption purposes. In the present study Premi®Test was employed for qualitative screening of shell eggs for detection of antimicrobial residues such as Amoxicillin, Ampicillin, Penicillin G, Cloxacillin, Cectiofur, Tylosin, Erythromycin, Chlortetracycline, Oxytetracycline, Doxycycline, Sulphamethazine, Sulphadiazine, Gentamicin, Streptomycin, Neomycin and others such as Furazolidone, Amprolium and Narasin with detection limit of 5, 5, 5, and 100, 400, 50, 50, 600, 400, 200, 25, 25, 100, 1000, 300, and 2500 ppb, respectively. The results suggested that except farm B, the residues of antibiotics were seen in all the shell egg samples procured from farm A and markets A, B, C, and D with different prevalence rates. The percent prevalence for farm A was found to be 60 and for farm B it was found to be zero whereas 100 percent prevalence for all of the markets A, B, C, and D. The overall indication of the present surveillance is that the use of antibiotics/antimicrobials in commercial layer farms may be the norm of the poultry practice for various reasons, may be with the exception of few commercial layer farms as seen in farm B who may use pro or pre biotics instead of antibiotics. The number of samples screened for the detection of the antimicrobial residues in the present study was less and hence to make inference on the actual prevalence of antibiotic residues in shell eggs on population basis needs larger surveillance studies and inference from small sample size may be inappropriate. Several research workers detected the presence of antimicrobial residues in shell eggs which supports the findings of the present study. Narahari and Amutha (2007) reported the residues of Chloramphenicol in eggs that persisted in egg yolk up to 66 days and Sulphadimidine and arsanilic acid residues in egg albumen. Quon(2000) examined both domestic and imported eggs in Canada for veterinary drugs like Chloramphenicol, -Lactams, Fluoroquinolones, Halofuginone, Macrolides, Sulfonamides, Tetracyclines, 141
Decoquinate, and Coccidiostats over a two-year period. They observed that more than 99% of the samples screened were found to be free of any veterinary drug residue but 1% were found to contain Tetracyclines, sulfonamides, ciprofloxacin, Macrolides, Streptomycin, Clopidol, Ethopabate, and Nitromide antimicrobial residues. Adesiyun et al (2005) conducted surveillance of table eggs from market and commercial layer farms for antimicrobial residues and observed that the farms had prevalence rate of 6.5% whereas mall and supermarket eggs showed prevalence between 16.1% and 15.0%, respectively. Marcinибk et al (2006) observed the presence of Sulphadimidine residues in shell eggs by Premi®Test up to 8 days after oral administration of the drug to the layers. Fotina et al (2005) and Das and Bawa (2008) observed that residues of drugs get accumulated in the albumin and yolk at different proportions and remained unsafe for consumption even up to 20 days of therapy to infected birds. Similar observations were obtained by Donoghue and Myers (2000) and Donkova (2005) who suggested that even after a single dose, sequestered drug residues may be stored and later released to contaminate eggs for days to weeks after dosing. The use of antibiotics in the feed for chemoprophylaxis in poultry could be the reason for the high rate of prevalence noticed in the present study. 142
SUMMARY AND CONCLUSIONS The research work on comprehensive study on quality and safety of raw shell eggs was planned to ascertain the existing conditions of table eggs meant for human consumption through random collection of samples from commercial layer farms and different markets. Two organized commercial layer farms and four markets, i.e. A (white shell eggs), B (desi eggs), C (white shell eggs) and D (brown shell eggs) were part of the present study. The total number of samples collected from different sources and analysed in the entire study for different parameters was about 1296. The analysis of the samples focused on physico-chemical quality (230 samples), microbiological quality (350 samples) both sanitary (TVC, Pseudomonas spp in shell eggs) and safety (Salmonella spp. and Campylobacter spp. in shell eggs, layers, environment and finished egg product), egg sanitation trials with 70% alcohol, 65 ppm Polyhexamethylene guanidine and 200 ppm of chlorine against SalmonellaTyphimurium spiked eggs (90 samples), shell penetration and recovery of SalmonellaTyphimurium from shell eggs (136 samples),shelf life of eggs treated with paraffin oil alone and in combination with different sanitizers at ambient and refrigeration storage temperatures (426 samples) and detection of pesticide (32 samples) and antimicrobial drug residues (32 samples) in raw shell eggs. The results of the study suggested that the physical attributes that contributed to the external quality of shell eggs such as egg weight, shell thickness and shape index of white shell eggs procured from commercial layer farms and markets showed similar quality and in line with the prescribed quality guidelines. The eggs weighed more than 56 g on an average and belonged to large weight class as per AGMARK, had thickness of more than 0.40 mm and percent shape index between 75 and 76.84. The brown shell eggs procured from the market was under small weight class and had an average egg weight of more than 45 g, with shell thickness of 0.37 mm (lowest thickness recorded in the study) and percent shape index of more than 73. Brown shell eggs were also in line with the prescribed quality guidelines. The desi eggs procured from the market did not fit in to the any of the weight grades mentioned by AGMARK as the average weight of the eggs was below the specified grade (36.47g) but had 143
highest shell thickness (0.43 mm) and had normal shape index value of 74.67 %. All shell eggs regardless of categories had recorded extremely good shell thickness which exceeded the minimum benchmark value of 0.33 mm. Shell weight and shell percent was also found to be in similar lines irrespective of categories. Microbiological quality as indicated from Total Viable Counts suggested that the market eggs were more contaminated than the farm eggs, however, all the sources (farm and market) showed significant shell surface contamination. This signaled the scope for implementation of farm level Good Egg Production Practices (GEP) and improvements required in egg supply chain. The internal quality of shell eggs were evaluated in terms of albumin height, Haugh unit, albumin pH and percent yolk index. The results suggested that market eggs recorded considerable decline in its physico-chemical attributes than farm eggs and the quality differences were significant (P0.01) for albumin height and (P0.05) for Haugh unit and pH. The farm eggs were of AA quality and market eggs were of B quality except desi eggs which had AA quality as per Haugh unit standards of consumer grade egg quality criteria. Microbiological quality of internal contents of eggs (both farm and markets) were within the USDA acceptable limits and recorded the TVC between 3.09 and 4.86 log CFU/ml, however, market eggs showed higher bacterial load and significant (P0.01) difference in count as compared to farm egg samples. The Pseudomonas spp. in shell eggs appeared to be infrequent and uncommon. The overall trend on the quality aspects of shell eggs thus can be summarized as, they were all in the acceptable consumer grade qualities but with wide and significant differences in quality. The shell eggs were also assessed for safety criteria using Salmonella spp. and Campylobacter spp. as safety indicators by cultural methods. Study included representative samples of all categories which could act as a source of contamination to shell eggs and possible carry over to finished egg products. The results of the study suggested that the prevalence of Salmonella enterica serotypes was observed only in white shell eggs procured from layer farms as well as markets and not in brown shell or desi eggs. The overall prevalence of Salmonella spp. for white shell eggs was found to be 8.88 % and in layers it was found to be 8.33 %. The litter, feed, water and wash water from egg trays and 144
finished egg products did not show the prevalence of Salmonella spp. The Campylobacter spp. was not found in shell eggs, layers and environment of commercial layer farms as well as in shell eggs of market samples and finished egg products. Results thus suggested that Salmonella spp. appeared to be major biosecurity concern and required sound farm based control measures to ensure safety of raw shell eggs. Also results suggested that though the eggs were found to possess good consumer grade qualities, it was failed to pass the safety criteria as a whole for Salmonella spp. Shell egg sanitation studies (both pre and post inoculation) demonstrated that after treatment (sanitation) with 70% alcohol, 65 ppm Polyhexamethylene guanidine (PHMG) and 200 ppm of chlorine all the sanitizers were found to be effective in reducing the initial inoculated load of Salmonella Typhimurium and Total Viable Count at 1 and 5% level of significance (P0.01). However, shell penetration and recovery studies demonstrated that the Salmonella Typhimurium could be recovered from surface inoculated untreated eggs as well as from eggs treated with 70% alcohol and 200 ppm chlorine with the exception of 65 ppm PHMG between 14 and 28 days of storage at refrigeration temperature. Results thus questioned the ability of sanitizers to ensure safety. Further these studies (in vitro penetration and recovery study after sanitation) proved valuable to fix the performance criterion for sanitizers at minimum of 6 log cumulative reductions of organisms during process of sanitation, as it can be believed to contribute to an achievable Appropriate Level Of Protection (ALOP) at specified process step in the industry. Hence, it can be summarized that the shell egg sanitation too required modification to ensure better safety of eggs. Apart from these observations, the study on quality of shells in relation to recovery of Salmonella Typhimurium suggested that the shell quality influenced the penetration and recovery of Salmonella Typhimurium. It was observed that the organism penetrated more readily in poor shell quality eggs than in shell intact eggs and survived for 6 weeks at refrigeration temperature. The shelf life study of the raw shell eggs treated with paraffin oil alone and in combination with various sanitizers stored at ambient temperature revealed that there was substantial reductions in albumin height (4.20 to 8.06 units) and Haugh unit value (30 to 40 units) by 30 days. Considering the USDA 145
criteria for consumer grade qualities based on Haugh unit, eggs in all the treated categories declined in their initial quality of AA to B by 15th day and remained in B grade up to 30 days and passed to C grade after 30 days of storage. Untreated shell eggs retained its internal quality below 15 days. Similar decline was observed in yolk quality followed by increased albumin pH. The internal microbiological quality was well with in the accepted microbiological limits recommended by USDA (1991) for liquid eggs before date of expiry and eggs were free from Salmonella spp. Campylobacter spp. and Pseudomonas spp. The shelf life study of raw shell eggs treated with paraffin oil alone and in combination with various sanitizers stored at refrigeration temperature revealed that internal quality in terms of albumin height, Haugh unit, yolk quality, albumin pH and microbiological quality were found to be good and fulfilled the requirements of consumer grade qualities of USDA i.e. they were of A quality in untreated eggs, paraffin oil applied eggs and chlorine plus paraffin oil coated eggs where as other groups (alcohol plus oil, PHMG plus oil and hydrogen peroxide with colloidal Ag plus oil) had AA quality by 180 days of storage. The eggs were free from Salmonella spp. Campylobacter spp. and Pseudomonas spp. The results of extended storage on weight loss of eggs revealed that the percentage weight loss of shell eggs was from 0.88 (0.50g) to 10.33 (5.90g) at ambient and 1.24 (0.70g) to 2.28 (1.29g) at refrigeration temperature stored eggs for a period of 6 to 8 weeks. The results indicated that the loss of weight of shell eggs was reduced to an extent of 2-6 times at 2 weeks and 6 weeks (the normal holding period of eggs in the market channel at ambient and refrigeration temperature), respectively. Shell eggs were also assessed for safety from chemical and drug residues in addition to the microbiological safety. The assessment of shell eggs for freedom from residues of pesticides indicated that the shell egg samples analysed were free from residues of major pesticides of Organochlorines, Organophosphates and Synthetic pyrethroids. The shell eggs produced from organized commercial layer farms thus do not pose risk to the consumers by way of pesticide residues whereas the residues of antibiotics were seen in all the shell egg samples procured from commercial layer farm and markets with the exception of layer farm B. The percent prevalence was found to range from 60 to 100 for farm and market samples, respectively. 146
Based on the findings of the present study following broad conclusions can be made. It can be concluded from the results of the present study on external quality of shell eggs such as egg weight, shell thickness and shape index that shell eggs were in line with the prescribed quality guidelines and accepted quality standards except the weight of desi eggs. However, microbiological quality (TVC) of shell egg surfaces led to the conclusion that egg supply chain practices were unhygienic and poor. The overall conclusion based on internal physico-chemical quality that shell eggs from layer farms were found to be of superior quality and freshness than market eggs. As per USDA grading based on Haugh unit, the farm eggs were of AA quality and market eggs were of B quality with the exception of desi eggs from market B that possessed AA quality. However, internal contents of the eggs from market had significantly higher bacterial load than farm egg samples. It was also concluded that the occurrence of Pseudomonas spp. in shell eggs was infrequent or low. It was concluded that Salmonella spp. from layers appeared to be major biosecurity concern and required sound farm based control measures to ensure safety of raw shell eggs while farm environment did not appear to be a potential source of contamination. It was also concluded that the finished egg products do not constituted risk for Salmonella spp. The absence of Campylobacter spp. in all the samples tested suggests that this organism does not constitute a safety concern for consumers, producers and traders. The results of shell penetration and recovery of Salmonella Typhimurium from surface inoculated shell eggs treated with different sanitizers led to the conclusion that the minimum period for penetration of the Salmonella Typhimurium in shell eggs stored at refrigeration temperature was 14 days. By 28th day, the organism could be recovered from 70% alcohol and 200 ppm treated eggs but not from the eggs treated with PHMG. Treatment with 70% alcohol and 200 ppm of chlorine were found to be 147
weaker than 65 ppm of PHMG with respect to penetration of Salmonella Typhimurium in egg shells at 28 days of storage at refrigeration. The Salmonella Typhimurium was able to survive on the surface of the shell eggs treated with recommended doses of sanitizer's and penetrate the shell, survive inside the eggs and could be recovered from whole egg contents. PHMG acted as an effective chemical sanitizer and completely eliminated the Salmonella Typhimurium from surface of shell eggs. The raw shell eggs in order to be safe and meet the Food Safety Objective (FSO) the performance criterion for sanitizers should be minimum 6 log cumulative reductions (CFU/ml) which would offer an achievable Appropriate Level Of Protection (ALOP) at specified process step (sanitation) and probably at consumer level at appropriate storage conditions. The Salmonella Typhimurium penetrated more readily in poor shell quality eggs than in shell intact eggs suggesting increased risk of Salmonella infection associated with poor shell quality eggs. Qualitative risk profiling/ rating considering the growth in the commercial shell eggs observed in three situations namely sanitation, storage at refrigeration temperature and shell quality suggest that shell eggs constitute low to medium risk category to the consumers when handled under said situations. The shelf life of shell eggs at ambient storage without any treatment was less than 15 days and for treatment groups it was about 30 days with acceptable Haugh unit values as per USDA consumer grade qualities. It was also concluded from the study that the shelf life of shell eggs at refrigeration was good even after 180 days and fulfilled the requirements of Gerber (i.e. Haugh unit of 60 and above) as well as USDA consumer grade qualities. Untreated shell eggs, paraffin oil applied eggs and chlorine plus paraffin oil coated eggs had `A' quality where as other 148
groups (alcohol plus oil, PHMG plus oil and hydrogen peroxide with colloidal Ag plus oil) had AA quality. The internal microbiological quality was found well with in the acceptable limits as recommended by USDA (1991) for liquid eggs for both ambient and refrigeration stored eggs. However, no such standards exist in India to grade the qualities of eggs as per Haugh units with respect to consumption of eggs. The refrigeration storage extended or even preserved the original internal quality attributes of freshness, albumen and yolk quality plus the microbiological quality fairly enough for the observed period of 6 months as against 1 month (30 days) shelf life of treated eggs at ambient storage. The percent weight loss in shell eggs stored at ambient temperature was comparatively much higher (2 to 6 times) than stored at refrigeration temperature. The shell eggs produced from organized commercial layer farms does not pose risk to the consumers by way of pesticide residues. The high rate of prevalence of antimicrobial residues could be a safety concern for consumers and traders. 149
PROPOSED AREA OF FUTURE RESEARCH The present study on raw shell egg quality and safety highlighted the scope for further studies in the areas mentioned below. 1. The study revealed the presence of Salmonella enterica serotypes in layers and shell eggs at high prevalence rates. There is scope for molecular characterization of the isolates and comparative molecular epidemiological studies on circulating Salmonella spp. among layers and shell eggs by including more number of commercial layer farms from different geographical regions. Such study will help in reducing the burden of Salmonella in commercial layer farms. 2. There is scope for identifying and quantifying antimicrobial residues from shell eggs and will help in promoting the judicious use of antimicrobials among producers. 3. Research on industrial application of PHMG as a sanitizer in shell egg processing and its efficacy on Listeria monocytogenes and other important pathogens in shell eggs can be under taken. 4. Scope for research on chemical indicators which identifies the safety and quality of shell eggs simultaneously and serve as an industrial standard (like that of furosine, uracil or other Amadori compounds) can be under taken. 5. Study on industrial and commercial applications of electronic egg tester can be under taken for rapid identification of the quality and quality assurance programs. 150
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xx US FDA (2008) Campylobacter. In Bacteriological analytical manual: Chapter 7: 1-24. US FDA(2008) Salmonella. In Bacteriological analytical manual: Chapter 5: 1-24. Varga, T., J.P. Cravedi, I. Fuzesi and L. Varnagy (2002) Residues of Fenitrothion in chick embryos following exposure of fertile eggs to this organophosphorus insecticide. Revue Mйd. Vйt. 153 (4): 275-278. Wagenaar, J.A., D.J. Mevius and A.H. Havelaar (2006) Campylobacter in primary animal production and control strategies to reduce the burden of human campylobacteriosis. Rev. science. tech. Off. int. Epiz. 25 (2): 581-594. Wales, A., M. Breslin and R. Davies (2006) Semiquantitative assessment of the distribution of Salmonella in the environment of caged layer flocks. J of Appl. Microbiology.101 (2): 309 -318. Williams, K.C (1992) .Some factors affecting albumen quality with particular reference to Haugh unit score. World's Poult. Science J. 48:5-16. Windhorst, H.W. (2008) Asia's changing role in the global egg industry- an analysis of past, present and foreseeable future dynamics, World's Poult. Science. J. 64: 533-552. WHO (2002) Methods for Foodborne Disease Surveillance in Selected Sites. Report of a WHO consultation Leipzig, Germany: 1-27.
xxi VITA Madhavaprasad C. B. was born on 13th March 1964 in Chakkodabylu village of Theerthahalli Tahasil of Shivamogga District of Karnataka State. He belongs to the family of Agriculturist. He completed his primary education in his village and S.S.L.C from Government High School, Paivalike under Kerala State Board of Education, Kerala State. Author completed his PUC II from Marimallappa's College, Mysore in the year 1980, under Pre University Education Board, Karnataka. He completed his B.V.Sc. degree from Veterinary College, Bangalore in 1986 under UAS, Bangalore and M.V.Sc. (VPH) from IVRI - Izatnagar in 1993. He obtained first class throughout his educational career. He joined the University services as Instructor in the year 1987, promoted to the Asst. Professor in 1993 and Assistant Professor (SG) in 2000 and continues to serve in the same cadre till date. All his service was put in Veterinary College, Bidar (KVAFSU- Bidar) as teacher in Veterinary Public Health with exception of 1 or 2 years of service in other colleges/ other subjects. Author has worked for several research projects as co-Principal Investigator funded by out side Agencies such as NATP, KSCST and ICAR/DBT.

CB Madhavprasad

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Title: DETECTION OF PESTICIDE RESIDUES IN MEAT
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