Protection of Washed and Pasteurized Shell Eggs against Fungal Growth by Application of Natamycin-Containing Shellac Coating THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Yang Song Graduate Program in Food Science and Technology The Ohio State University 2016 Master's Examination Committee: Dr. Ahmed Yousef, Advisor Dr. Dennis R. Heldman Dr. Luis Rodriguez-Saona Copyrighted by Yang Song 2016 Abstract Mold contamination of commercial shell eggs can potentially cause significant economic loss to the egg industry during storage. Studies indicated that molds from varies sources can propagate on commercial eggs when storage condition is less ideal. The current egg processing procedures such as commercial washing and pasteurization can weaken the egg shell, which is the primary defense of egg content, and expose processed eggs to contaminations. Generally, processed eggs are coated with mineral oil to overcome this problem. However, oil application is not very effective when used to protect eggs against mold contamination during storage. The food grade anti-fungal agent natamycin can be used to improve egg defense against mold contamination; however, direct application on egg surface will cause it to lose activity rapidly. Therefore, incorporation of natamycin and a food-grade coating is necessary to extend its anti-fungal effectiveness. As a food-grade coating, shellac can retain egg quality better compare to other coating materials; moreover, it can also serve as a matrix for natamycin to treat egg surface. Research is needed to investigate whether natamycin can remain effective in shellac coating; determine the minimum inhibitory concentration (MIC) of natamycin in shellac coating against typical mold contaminants, and whether the natamycin-shellac coating is effective when used on commercial washed eggs and pasteurized eggs. ii The main objective of this research were (i) to assess natamycin efficacy on molds isolated from contaminated eggs using agar plates; and (ii) to evaluate the anti- fungal effectiveness of natamycin-shellac coating when applied on commercially washed eggs and mild heat/ ozone pasteurized eggs. Natural mold species were isolated from contaminated eggs, and identified using polymerase chain reaction (PCR) method with ITS primers. Identification results revealed three mold contaminants on shell eggs, these are: Cladosporium romotenellum, Penicillium commune, and Mucor hiemalis. Identified mold species were used to inoculate potato dextrose agar (PDA) plates, and the MIC of natamycin-shellac coating were tested with prepared test disks. Test disks were soaked in natamycin-shellac coating mixture, which were prepared by mixing natamycin suspension (1mg per 1 ml methanol) and shellac coating (1:4 weight/weight ratio of shellac in ethyl alcohol) at different concentrations (12.5, 25, 50, 100, 200, 400, and 800 µg natamycin/ml (mixture suspension). The efficacy of natamycin-shellac coating at different concentrations was measured by inhibition zone on PDA plates after 5 days of incubation. Identification results revealed three mold contaminants on shell eggs, these are: Cladosporium romotenellum, Penicillium commune, and Mucor hiemalis. Results suggest that natamycin-shellac coating inhibits the tested molds at these threshold concentrations of natamycin: Mucor, 97.7 µg/ml; Cladosporium, 51.6 µg/ml; and Penicillium, 44.9 µg/ml. iii Shellac-containing natamycin was tested on proceed shell eggs. Inhibitory concentration was adjusted based on preliminary tests and related published data. A formulation of natamycin-shellac coating was finalized to contain natamycin concentration at 400 µg/ml. Studied eggs were prepared into three groups: eggs that received no coating (control), eggs that were coated with only shellac (treatment one), and eggs that were coated with shellac containing 400 µg/ml of natamycin (treatment two). Each group of eggs were inoculated with mold spores and stored at 25°C for 18 days, and mold populations were enumerated on different days by plating on PDA plates. The same experimental set up was repeated in triplicates on eggs inoculated with Mucor, Cladosporium and Penicillium. Growth of molds on shell eggs were fitted with the logistic model, and kinetic parameters were estimated and compared. Statistical analysis showed that natamycin-coated eggs had significant antifungal effects compare to other treatments. Under the influence of natamycin, estimated overall mold growth was significantly reduced (Tukey test, p<0.05), maximum specific growth rates were significantly lower (Tukey test, p<0.05), and mold growth lag time was significantly increased (Tukey test, p<0.05) on both washed and pasteurized egg. This study illustrated that shellac coating can be used as a carrier for natamycin to create an effective protection against mold contaminations on washed and pasteurized egg surface, hence extend their shelf-life and minimize economic loss associated with moldy eggs. iv Acknowledgments I would like to offer my sincerest thanks to my advisor Dr. Ahmed Yousef for believing in me, offering me a second chance to pursue my goals. His guidance, discipline, and encouragements are the most valuable instructions that helped me become a better scientist. I am very blessed and very thankful to have Dr. Yousef as my advisor. To my committee member: Dr. Dennis Heldman, thank you for your teaching in food engineering class. You spiked my interest in modeling and provided invaluable advises to me. To my other committee member: Dr. Luis Rodriguez-Saona, thank you for your suggestions and lectures. You once told me to pursue the career that I truly love, and you helped me fall in love with food science. I would also like to thank the past and current members of the Yousef lab members: Dr. Jin-Gab Kim, Dr. En Huang, Dr. Baosheng Liu, Dr. Ismet Ozturk, Dr. Rui Li, David Kasler, Xu yang, Mustafa Yesil, Greg Culbertson, Nathan Morrison, Michelle Gerst, Ebrahim El-Khtab, Emily Holman, and Walaa Hussein for their precious friendship and constructive criticism. Special thanks go to Dr. En Huang, Xu Yang, David Kasler and Mustufa Yesil for their encouragements and helps during my experiment. None of this research would have been possible if they didn’t help me. To my parents, thank you so much for your understanding and support to my education, it’s the best experience and wealth that you showed me. v Vita September 25, 1983 .......................................Born, Guangzhou, Guangdong, China 2002................................................................New Mexico Military Institute 2006................................................................B.S. Biological Science, The Ohio State University Fields of Study Major Field: Food Science and Technology vi Table of Contents Abstract…………………………………………………………………………………..ii Acknowledgements………………………………………………………………………v Vita………………………………………………………………………………………vi List of figures…………………………………………………………………………....xi List of tables…………………………………………………………………………….xiv Chapter 1 : Literature Review ...................................................................................... 1 1.1 Mold ..................................................................................................................... 1 1.1.1 Introduction ................................................................................................... 1 1.1.2 Fungi taxonomy and nomenclature ............................................................... 2 1.1.3 Ecology of fungi contamination.................................................................... 4 1.1.4 Mold isolation, enumeration and identification ............................................ 7 1.2 Egg ..................................................................................................................... 13 1.2.1 Introduction ................................................................................................. 13 1.2.2 Structure ...................................................................................................... 15 1.2.3 Egg processing ............................................................................................ 17 1.3 Edible coating ..................................................................................................... 22 vii 1.3.1 Introduction ................................................................................................. 22 1.3.2 Types of edible coating ............................................................................... 23 1.3.3 Related regulations...................................................................................... 25 1.3.4 Edible film antifungal efficacy ................................................................... 25 1.3.5 Advantage and disadvantage of edible coating ........................................... 29 1.4 Natamycin .......................................................................................................... 29 1.4.1 Introduction ................................................................................................. 29 1.4.2 Natamycin application history and regulation ............................................ 31 1.4.3 Natamycin toxicity ...................................................................................... 32 1.4.4 Natamycin application in edible films ........................................................ 33 1.5 Modeling ...........................................................................................................