Flavor, Enzymatic and Microbiological Profiles of Pressure-Assisted Thermal Processed (PATP) Milk Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Francisco Parada-Rabell Graduate Program in Food Science and Technology The Ohio State University 2009 Dissertation Committee: Valente B. Alvarez, Advisor David B. Min Ahmed E. Yousef V. M. Balasubramaniam Copyright by Francisco Parada-Rabell 2009 Abstract The main objective of this study was to evaluate the application of high pressure processing (HPP) and pressure-assisted thermal processing (PATP) as an alternative technology to process high quality fluid milk. The specific objectives of this work were to compare the microbial load, chemical stability and flavor profiles of HPP and PATP milk to that of HTST pasteurized and ultra high temperature (UHT) processed milk. Milk (2% milkfat) was subjected to combined pressure-heat treatment using a factorial 3x1x3 model at temperature (32, 72 and 105 oC), pressure (650 MPa), and time (0, 1, and 5 min). Milk samples were processed within 72 hr, packed in light – protected polyethylene teraphtalane (PET) bottles without head space, and stored at either room temperature (25±1 oC) or refrigeration (4±1 oC) conditions depending on the treatment applied. The shelf life of pressure treated milk samples was examined over a period of 20, 45 and 90 d. Additionally, pasteurized (78±0.8 oC for 18 sec) and UHT processed (138±1 oC for 2 sec) milk samples obtained from commercial source were analyzed along with pressure treated milk. The quality of milk samples was analyzed by the following tests: (1) Microbiological tests: Total plate count (TPC) and spore-forming bacteria survival analyses ( Bacillus stearothermophilus and B. amyloliquefaciens) . (2) Residual Plasmin activity in milk samples was measured using a BODIPY FL-Casein ii spectrofluorometric assay. (3) The chemical stability of milk samples was assessed by measuring the extent of proteolysis and lipolysis. Proteolysis was evaluated by SDS- PAGE analysis and lipolysis was measured by a modified copper soap method. (4) The flavor profile of milk samples was evaluated using selected ion flow tube mass spectrometry (SIFT-MS) to identify and quantify volatile organics compounds in milk on real time basis. Preliminary data showed that the application of pressure treatment is capable of rendering milk with longer shelf life than HTST pasteurized and UHT milk. 6 Up to 6-log reduction was obtained when a suspension (N 0 = 7x10 spore/mL) of B. stearothermophilus was inoculated in UHT milk treated at 700 MPa for 3 min at 105 oC. 7 Similarly, a 7-log reduction was achieved when a suspension (N 0 = 1x10 spore/mL) of B. amyloliquefaciens was inoculated in UHT milk treated at the conditions described above. Pressure treatments at room temperature (32±1 oC) delivered milk with similar microbial load than pasteurized milk. The microbial population of milk further decreased o 4 with increasing temperature (72 and 105 C). Up to 4 log reduction (N 0 = 2x10 CFU/mL) was obtained in PATP milk samples processed at 650 MPa and 105 oC for 0, 1 and 5 min, respectively. Storage temperature had an effect on the microbial growth rate in PATP milk. Microorganisms recovered in pressure treated milk processed at 650 MPa and 72 oC for 0, 1 and 5 min stored at refrigeration conditions showed slower growth rates than pressure-treated samples stored at room temperature conditions. No additional efforts were made to characterize the microorganisms recovered during storage. No significant proteolysis was observed in pressure-treated samples at the end of their shelf life. Plasmin inactivation rates increased with increasing temperature. Although the enzyme was not iii completely inactivated, combinations of mild temperature with ultra high pressure were sufficient to inactivate the enzyme to levels similar to UHT processes. However, lipolysis was enhanced by pressure, holding time during pressurization and storage temperature. The formation of volatile aroma compounds in HPP and PATP milk was different than in milk processed with traditional heat treatments. Pressure treatments enhanced only the formation of straight-chain aldehydes and sulfur compounds; whereas higher concentrations of methyl ketones and straight-chain aldehydes were observed in pasteurized and UHT milk samples. These results indicate that pressure treatment is capable of rendering milk with quality characteristics close to pasteurized and UHT milk. However, processing temperature and storage conditions have a significant role on the quality of milk. iv Dedication Dedicated to Toñe Thank you for your unconditional love and always believe in me. v Acknowledgements a) The author particularly acknowledges the support given by CONACyT – México for scholarship # 196181. b) I would like to show my most sincere gratitude to my advisor Dr. Valente B. Alvarez for his unconditional support during my graduate studies. Thank you for sharing with me your advice, your friendship and the work ethics that changed my life completely. c) I would like to particularly thank my committee members Dr. David Min, Dr. Ahmed Yousef and Dr. V. M. Balasubramaniam. This thesis would not have been possible unless your valuable suggestions and guidance. d) I would also like to thank Dr. Luis Rodriguez-Saona, Dr. James Harper and Dr. Sheryl Barringer for their always available and helpful advice. e) I owe my deepest gratitude to my parents (Dr. Francisco Parada Aguirre and Dr. Ana T. Rabell Urbiola) and my siblings (Rodolfo, Andres, Ana Giselle and Pablo Alonso) for their unconditional love and guide me to make these lines possible today. f) It is an honor for me to particularly acknowledge Bob, Paula and Meghan Quinn. Thank you for your selfless support. g) I am indebted to Mr. Gary Wenneker. Most of the research data presented in this work would not have been possible without your help. h) Lastly, I would like to kindly acknowledge the assistance provided by Dr. Balasubramaniam’s PATP laboratory staff with the processing of pressure treated milk samples vi Vita 2004 .......................................................... B.S. Food Engineering, National Autonomous University (Mexico City, Mexico) (Best Student of the 2000 – 2004 Food Engineering Class) 2005 to Present ......................................... Graduate Research Associate, Department of Food Science and Technology, The Ohio State University Publications Parada-Rabell, F. and V. B. Alvarez. 2007. Food allergens: Status, risks and implications in the food industry. Industria Alimentaria 29 (2): 28 – 33. Fields of Study Major Field: Food Science and Technology vii Table of Contents Abstract.......................................................................................................................... ii Dedication.......................................................................................................................v Acknowledgments ..........................................................................................................vi Vita .............................................................................................................................. vii List of Tables................................................................................................................ xii List of Figures ............................................................................................................. xiii Chapter 1: Literature Review ...........................................................................................1 1.1 Introduction ...............................................................................................................1 1.2 Pasteurized Milk........................................................................................................4 1.2.1 Characteristics of fresh milk ...................................................................................4 1.2.2 Chemistry of milk ....................................................................................................6 1.2.3 Milk Production ......................................................................................................9 1.2.4 Microbiology of pasteurized milk ..........................................................................11 1.2.5 Chemical characteristics of pasteurized milk ........................................................13 1.3 UHT Milk................................................................................................................15 1.3.1 Milk processing .....................................................................................................15 1.3.2 Microbiology of UHT milk ....................................................................................16 viii 1.3.3 Chemical characteristics of UHT milk ...................................................................19 1.4 High Pressure Processed (HPP) Milk .......................................................................22 1.4.1 High pressure processing ......................................................................................23 1.4.2 Microbiology of high pressure processed milk ......................................................24 1.4.3 Effects of high pressure on milk constituents .........................................................26 1.4.3.1 Effects on milk proteins..............................................................................27