AN ABSTRACT OF THE THESIS OF Dunyu Xi for the degree of Master of Science in Food Science and Technology presented on May 26, 2011. Title: Application of Probiotics and Green Tea Extract in Post-harvest Processes of Pacific Oysters (Crassostrea gigas) for Reducing Vibrio parahaemolyticus and Extending Shelf Life Abstract approved: ________________________________________________________________________ Yi-Cheng Su Vibrio parahaemolyticus is a human pathogen which is prevalent in marine environment. Consumption of raw or undercooked seafood contaminated with V. parahaemolyticus can cause foodborne illness. This study investigated the application of probiotics in depuration for reducing V. parahaemolyticus in raw Pacific oysters (Crassostrea gigas) and the utilization of green tea extract on inactivating V. parahaemolyticus and extending shelf life of oyster meats during refrigeration storage. Lactobacillus plantarum ATCC 8014, which exhibited strong bactericidal effects against mixture of five clinical V. parahaemolyticus strains mainly due to the production of organic acids, was utilized in the study. Populations of lactic acid bacteria in oysters increased from 1.83 to 4.66 log CFU/g after exposing oysters to artificial seawater containing L. plantarum at a level of 6.41 log CFU/ml after 20 h accumulation at room temperature. Levels of L. plantarum colonized in oysters decreased gradually when oysters were depurated at 20°C, but remained moderately colonized in oysters (3.10 log CFU/g) after 4 days of depuration. Adding cells of L. plantarum (107 CFU/ml) to seawater for depuration of raw oysters inoculated with V. parahaemolyticus BE 98-2029 (O3:K6) at levels of 104 MPN/g did not enhance V. parahaemolyticus reductions in oysters depurated at 15±1ºC but significantly (p<0.05) decreased levels of V. parahaemolyticus in oysters depurated at 10±1°C after 5 days without mortality compared with controls. Lactic acid bacteria other than L. plantarum ATCC 8014 can be explored for potential application in post-harvest treatments of raw oysters for reducing V. parahaemolyticus contamination. Longjing Tea extract containing 4.594 g/l total phenolic contents (TPC) as gallic acid equivalents (GAE) or higher determined by Folin-Ciocalteau reduced a mixture of five clinical V. parahaemolyticus strains in tryptic soy broth plus 1.5% NaCl from around 4.5 log CFU/ml to non-detectable level (<10 CFU/ml) within 8 h. Shucked oysters treated with tea extract containing 9.054 g/l TPC as GAE for 2 h at 23±1ºC at oyster/tea extract ratio of approximate 0.9 g/ml resulted in greater (p<0.05) V. parahaemolyticus reductions (0.75 log MPN/g) compared to controls (0.15 log MPN/g). Storing oysters treated with tea extract at 5±1°C in containers filled with tea extract at oyster/tea extract ratio of approximate 0.7 g/ml enhanced reductions of V. parahaemolyticus and extended the shelf life of oyster meats from 8 days for controls to 18 days for oysters stored in tea extract. Further studies need to investigate the effects of green tea extract treatments on the sensory attributes of oyster meats. ©Copyright by Dunyu Xi May 26, 2011 All Rights Reserved Application of Probiotics and Green Tea Extract in Post-harvest Processes of Pacific Oysters (Crassostrea gigas) for Reducing Vibrio parahaemolyticus and Extending Shelf Life by Dunyu Xi A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented May 26, 2011 Commencement June 2012 Master of Science thesis of Dunyu Xi presented on May 26, 2011 APPROVED: ________________________________________________________________________ Major Professor, representing Food Science and Technology ________________________________________________________________________ Head of the Department of Food Science and Technology ________________________________________________________________________ Dean of the Graduate School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. ________________________________________________________________________ Dunyu Xi, Author ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my major professor, Dr. Yi-Cheng Su, for his patient guidance and ongoing encouragement throughout my whole master program. His sound knowledge and inspirational ideas in the field of food microbiology proved invaluable to my experience. Thank you to my thesis committee members, Dr. Goddik, Dr. Morrissey, and Dr. Gamroth for taking time to review my thesis and make the comments. Many thanks to the faculty, staff and students I know for their instruction, help and encouragement, especially the people in the OSU Seafood Laboratory. I cherish every moment I spent with them during my master program. Special thanks to my labmate, Note, for her help in my research. Sincere thanks also go to Dr. Chengchu Liu from Shanghai Ocean University for her strong recommendation and ongoing encouragement. Last but not the least, I greatly appreciate my parents for their unconditional love and support all through my life. TABLE OF CONTENTS Page Chapter 1 General Introduction .......................................................................................... 1 1.1 Overview of V. parahaemolyticus ............................................................................. 2 1.1.1 Classification of V. parahaemolyticus ................................................................ 2 1.1.2 Ecology of V. parahaemolyticus ........................................................................ 3 1.1.3 V. parahaemolyticus infections and related outbreaks ....................................... 4 1.1.4 Regulation for preventing V. parahaemolyticus infection.................................. 6 1.1.5 Post harvest treatments to reduce V. parahaemolyticus ..................................... 7 1.1.5.1 Non-thermal treatments ............................................................................... 7 1.1.5.2 Thermal treatments ...................................................................................... 8 1.1.5.3 Depuration.................................................................................................... 9 1.1.5.4 Addition of natural antibacterial compounds ............................................. 11 1.1.5.5 Biological treatments ................................................................................. 12 1.2 Overview of probiotics for investigating the potential on reducing V. parahaemolyticus in raw oysters ................................................................................... 12 1.2.1 Definition of probiotics .................................................................................... 12 1.2.2 General sources and selections of probiotics .................................................... 13 1.2.3 Mode of action .................................................................................................. 14 1.2.3.1 Competition for adhesion sites................................................................... 15 1.2.3.2 Production of inhibitory compounds ......................................................... 16 TABLE OF CONTENTS (Continued) Page 1.2.3.2.1 Organic acids ....................................................................................... 16 1.2.3.2.2 Hydrogen peroxide .............................................................................. 17 1.2.3.2.3 Bacteriocins ......................................................................................... 17 1.2.3.2.4 Other inhibitory compounds................................................................ 19 1.2.4 Application of probiotics in aquaculture .......................................................... 19 1.2.5 Application of probiotics in food...................................................................... 21 1.3 Overview of tea for investigating the potential on reducing V. parahaemolyticus in oyster meats ................................................................................................................... 22 1.3.1 Classification of tea .......................................................................................... 23 1.3.2 Major chemical compositions ........................................................................... 23 1.3.3 Factors affecting tea compositions and stability............................................... 25 1.3.3.1 Intrinsic factors .......................................................................................... 25 1.3.3.2 Manufacturing processes ........................................................................... 25 1.3.3.3 Extraction solvents ..................................................................................... 26 1.3.3.4 Brewing conditions .................................................................................... 27 1.3.3.5 Stability of chemical composition in tea .................................................... 27 1.3.4 Antibacterial activities of tea ............................................................................ 28 1.3.4.1 Mode of action ........................................................................................... 28 1.3.4.2 In vitro inhibition tests ............................................................................... 29 TABLE OF CONTENTS (Continued) Page 1.3.4.3 Factors affecting antibacterial activity of tea ............................................. 31 1.3.4.4 Food application......................................................................................... 31 Chapter 2 Impacts of Probiotics in Post-harvest Treatments for Reducing Vibrio parahaemolyticus in Pacific Oysters (Crassostrea gigas) ................................................ 33 2.1 Abstract ................................................................................................................... 34 2.2 Introduction ............................................................................................................. 35 2.3 Materials and Methods ............................................................................................ 37 2.4 Results ..................................................................................................................... 43 2.5 Discussion ............................................................................................................... 49 Chapter 3 Effects of Green Tea Extract on Reducing Vibrio parahaemolyticus and Increasing Shelf Life of Oyster Meats .............................................................................. 53 3.1 Abstract ................................................................................................................... 54 3.2 Introduction ............................................................................................................. 55 3.3 Materials and methods ............................................................................................ 56 3.4 Results and discussion............................................................................................. 62 3.5 Conclusion............................................................................................................... 72 Chapter 4 General Conclusion .......................................................................................... 73 Bibliography ..................................................................................................................... 77 LIST OF FIGURES Figure Page 3.1. Vibrio parahaemolyticus levels in shucked oysters stored at 5ºC ............................. 68 3.2 Total bacterial count in shucked oysters stored at 5ºC ............................................... 70 3.3 Psychrotrophic bacterial count in shucked oysters stored at 5ºC ............................... 71 LIST OF TABLES Table Page 2.1 Titratable acidity and pH of cell-free supernatant from growth of three lactic acid bacteria at 37ºC ................................................................................................................. 44 2.2 Inhibitory effects of cell-free supernatant from growth of Lactobacillus plantarum ATCC 8014 and lactic acid on growth of a mixture of five Vibrio parahaemolyticus strains (105 log CFU/ml) in tryptic soy agar plus 1.5% NaCl at 37ºC for 20 h ................ 45 2.3 Changes of lactic acid bacteria populations in oysters and artificial seawater (ASW) during depuration at 20±1ºC. ............................................................................................ 46 2.4 Reductions of Vibrio parahaemolyticus BE 98-2029 in laboratory inoculated oysters during depuration at 15±1 and 10±1ºC ............................................................................. 47 2.5 Mortality of oysters during depuration at 15±1 and 10±1ºC ...................................... 48 3.1 Inhibitory effects of tea extract on growth of Vibrio parahaemolyticus (106 CFU/ml) in tryptic soy agar plus 1.5% NaCl incubated at 37ºC for 20 h ........................................ 63 3.2 Effects of brewing methods on yields of total phenolic contents in extract of Longjing Tea .................................................................................................................................... 64 3.3 Antibacterial activity of Longjing tea extract against Vibrio parahaemolyticus (104 CFU/ml) in tryptic soy broth plus 1.5% NaCl incubated at 37ºC. .................................... 66 Application of Probiotics and Green Tea Extract in Post-harvest Processes of Pacific Oysters (Crassostrea gigas) for Reducing Vibrio parahaemolyticus and Extending Shelf Life Chapter 1 General Introduction The genus Vibrio belongs to family Vibrionaceae, it contains 30 species and Vibrio parahaemolyticus is one of the 13 Vibrio species that are pathogenic to humans. V. parahaemolyticus is Gram-negative halophilic, non-spore forming bacterium with the appearance of straight or curved rods (Drake and others 2007). Though only small percentage of overall V. parahaemolyticus are pathogenic and can cause illness in human (Nishibuchi and Kaper 1995), V. parahaemolyticus infections largely associated with seafood consumption are commonly reported worldwide. The first report of V. parahaemolyticus poisoning outbreak can be dated back to 1950 in Japan (FDA 2005). Since then, V. parahaemolyticus outbreaks have been reported in Asia (Wong and others 2000), Africa (Ansaruzzaman and others 2005), Europe (Lozano-León and others 2003), North America (CDC 1998, 1999, 2005, 2006) and South America (Harth and others 2009). Therefore, establishment of effective post-harvest treatments to reduce V. parahaemolyticus in seafood is crucial for reducing the risks of the infections. To investigate the potential application of probiotics and tea extract on reducing V. parahaemolyticus in Pacific oysters (Crassostrea gigas) during post-harvest treatments, 2 basic information of V. parahaemolyticus, probiotics and tea will be reviewed in this chapter. 1.1 Overview of V. parahaemolyticus 1.1.1 Classification of V. parahaemolyticus The classification of V. parahaemolyticus is historically based on their serotypes, but the presence of pathogenic genes is now also used for classification (Drake and others 2007). For the serotype, all strains have the same H (flagellar) antigen, but may have different K (capsular) antigens and O (somatic) antigens that can be used for classification (FDA 2004). In addition, pathogenic strains of V. parahaemolyticus can be identified by some virulent factors (Drake and others 2007). The thermostable direct hemolysin (TDH) can be produced by strains of V. parahaemolyticus possessing tdh gene and produce β-hemolysis on Wagatsuma agar. Such an activity is called Kanagawa phenomenon (KP) and can be used to identify virulent strains of V. parahaemolyticus (Nishibuchi and Kaper 1995). In addition to KP-positive strains, KP-negative V. parahaemolyticus has also been isolated from the gastroenteritis outbreak occurred in Republic of Maldives with ability to produce a hemolysin named TDH-related hemolysin (TRH). The TRH is encoded by the trh gene and has similar biological activities of TDH (Honda and others 1988). Other than TDH and TRH, production of urease by a V. parahaemolyticus strain was also reported could be a virulent factor (Eko 1992). It was reported that most of the trh+ strains could produce urease and presence of trh gene or ure gene in addition to the tdh gene could also increase the virulence of the tdh+ strains (DePaola and others 2003). Recently, a serine protease has been isolated from a clinical 3 strain without carrying tdh or trh genes and regarded as the potential virulent factor (Lee and others 2002). 1.1.2 Ecology of V. parahaemolyticus V. parahaemolyticus is ubiquitous in ocean and coastal waters. The range of salt concentration, temperature and pH for V. parahaemolyticus growth is 1-8% NaCl, 5-44ºC and 4.8-11.0, respectively, with the optimal condition being 2-4% NaCl, 30-35ºC and 7.6-8.6, respectively. It also tends to enter a viable but non-culturable state in some extreme conditions like sharp temperature shifts (Jay and others 2005). Many studies have focused on the factors influencing the distribution and densities of V. parahaemolyticus in marine environment. The densities of total and pathogenic V. parahaemolyticus in two Oregon oyster harvest bays (Yaquina and Tillamook) from November 2002 to October 2003 were highest during July and August, indicating the densities correlated well with the temperature of seawater while no correlation with seawater salinity was observed (Duan and Su 2005). Later, one study indicated an outbreak related to the consumption of Alaskan oysters in 2004 might result from the fact that the temperature of the seawater in harvest farm had increased gradually since 1997 (McLaughlin and others 2005). The densities of V. parahaemolyticus in oysters from three sites of Chesapeake Bay in Maryland from November 2004 to October 2005 were also correlated well with the water temperature, turbidity and dissolved oxygen (Parveen and others 2008). However, one study conducted in Spain from January 2002 to December 2004 showed that the seawater salinity instead of the temperature was the primary factor contributing to the spatial and temporal distribution of V. 4 parahaemolyticus (Martinez-Urtaza and others 2008). Another temperature-controlled study conducted during 2004 summer at two sites of the northern Gulf of Mexico confirmed that the total densities of V. parahaemolyticus in water and oysters were affected by both seawater salinity and turbidity (Zimmerman and others 2007). In addition to those factors, one study performed at Northwest Pacific area indicated that the levels of both total and pathogenic V. parahaemolyticus were lower in the oysters harvested before the exposure of oysters in the intertidal area to sun light and suggested intertidal harvest practices might also affect the bacterial load of oysters (Nordstrom and others 2004). 1.1.3 V. parahaemolyticus infections and related outbreaks Seafood can be a vehicle to transfer pathogens to humans from marine environment. V. parahaemolyticus levels in oyster can be more than 100 times greater than those in water (DePaola and others 1990). Moreover, Vibriosis including V. parahaemolyticus infection has become nationally notifiable disease since January 2007 (CDC 2010). Vibrio infection has three major clinical syndromes: gastroenteritis, primary septicemia and wound infection. Generally, the outbreaks caused by V. parahaemolyticus are related to the consumption of raw or undercooked seafood and the major symptom is gastroenteritis accompany with chills, cramps, diarrhea, headache and vomiting (Butt and others 2004). Daniels and others summarized that total V. parahaemolyticus infections occurred in the United Sates between 1988 and 1997 included 59% gastroenteritis, 34% wound infection and 5% septicemia (Daniels and others 2000). Hydration and antibiotics 5 are usually used treating gastroenteritis and severe diseases such as wound infections and septicemia, respectively (Butt and others 2004). The first confirmed foodborne outbreak caused by V. parahaemolyticus in the United States occurred in Maryland during August 1971, which was related to the consumption of crabs (Molenda and others 1972). Later, an outbreak linked to the consumption of raw oysters occurred in the Pacific Northwest region in 1981 (Nolan and others 1984). In 1997, total 209 cases of V. parahaemolyticus infections were related to the consumption of raw oysters harvested in west coast (CDC 1998). In 1998, the largest V. parahaemolyticus outbreak (416 cases) related to the consumption of raw oysters occurred in the United Sates were reported in Texas and the serotype of all clinical isolates was confirmed as O3:K6 (DePaola and others 2000). Additional outbreaks due to eating raw oysters and clams harvested in Long Island Sound also occurred in the same year, and several clinical isolated strains were also determined as O3:K6 serotype (CDC 1999). In 2004, 14 persons suffered from gastroenteritis due to the consumption of Alaskan oysters contaminated with V. parahaemolyticus (McLaughlin and others 2005). In 2006, a total of 177 cases of V. parahaemolyticus infections (72 cases were confirmed) happened in New York City, New York State, Oregon and Washington (CDC 2006). The recent market survey of the oysters harvested in North America in 2007 showed that the pathogenic V. parahaemolyticus was generally lower than total V. parahaemolyticus by several logs in oysters (DePaola and others 2010). However, proper post-harvest treatments of oysters must be taken into account to decrease the potential risks caused by V. parahaemolyticus contamination. 6 1.1.4 Regulation for preventing V. parahaemolyticus infection The U.S. Food and Drug Administration (FDA) initiated a “Quantitative Risk Assessment on the Public Health Impact of Pathogenic Vibrio parahaemolyticus In Raw Oysters” in response to the severe outbreaks occurred between 1997 and 1998 to better determine the factors resulting in infections and evaluate the public health impact of various control methods. Four components have been established for risk assessment: (1) hazard identification, (2) hazard characterization, (3) exposure assessment and (4) risk characterization with four seasons (spring, summer, autumn and winter) and six different oyster harvest areas (Gulf Coast-Louisiana, Gulf Coast-non Louisiana, Mid-Atlantic, Northeast Atlantic, Pacific Northwest-Dredged and Pacific Northwest-Intertidal). The FDA also recommended a limit of 10,000 cells/gram of V. parahaemolyticus in oysters (FDA 2005). The U.S. National Shellfish Sanitation Program (NSSP) also suggested several measures to effectively control V. parahaemolyticus in seafood before consumption. The measures include: (1) post-harvest processing (PHP) which can demonstrate that V. parahaemolyticus levels are lower than the average levels of the year resulting in V. parahaemolyticus illness (100/g and 1000/g for Pacific and Atlantic/Gulf oysters); (2) closing oyster harvest area; (3) properly labeling oysters “For Cooking Only” or “For PHP Only”, and (4) limiting the time between harvest and refrigeration (storage condition with the ambient air temperature of 45ºF/7.5ºC) to less than 5 hours or other proven times while guaranteeing the increase of V. parahaemolyticus levels is less than 0.75 log. For the validation of PHP, ten processed samples containing 10-12 oysters for each sample with adjusted geometric mean (AGM) of 104 MPN/g or greater initial levels 7 need to be obtained from three different processing days and less than 30 MPN/g with a minimum 3.52 log reduction should be observed after treatments. The initial V. parahaemolyticus levels of samples and the levels of treated samples should be detected by three-tube most probable number (MPN) and 5-tube MPN method respectively (FDA 2007). 1.1.5 Post-harvest treatments to reduce V. parahaemolyticus 1.1.5.1 Non-thermal treatments High pressure processing is a non-thermal process can be used to reduce the pathogenic bacteria without compromising the sensory features and original nutrients (Su and Liu 2007). Several studies confirmed that high pressure treatment could significantly reduce V. parahaemolyticus in oysters or shucked oyster meat to non-detectable levels (Calik and others 2002; Cook 2003; Kural and others 2008), including the serotype O3:K6 strain related to the severe outbreak occurred in Texas (Koo and others 2006). The optimal condition for the high pressure processing varied in different studies, which is mainly due the pressure resistance of different species. And the temperature of processing was suggested to be lower than 52ºC for the maintenance of meat quality (Andrews and others 2003b). More recently, high-pressure processing treatment of 293 MPa for 120 s at groundwater temperature (8±1ºC) was reported capable of reducing V. parahaemolyticus in Pacific oysters by greater than 3.52 log, which also satisfied the requirement of post harvest processing validation by FDA (Ma and Su 2011). Another non-thermal treatment is irradiation. It was reported that treatments of 1.0-1.5 kGy of gamma radiation (60Co) could reduce V. parahaemolyticus, including the 8 O3:K6 strain, in oysters to non-detectable level without changing their sensory attributes (Jakabi and others 2003; Andrews and others 2003a). X-ray treatment with 0.75, 2.0 and 5.0 kGy also achieved more than 6.0 log MPN/g reductions of V. parahaemolyticus in pure culture, half shell and whole shell oysters, respectively. In addition, X-ray treatment with 1.0 kGy could also reduce inherent microorganisms in whole shell oysters from 4.7 log CFU/g to non detectable level (Mahmoud and Burrage 2009). 1.1.5.2 Thermal treatments Icing treatment alone right after harvest did not reduce V. parahaemolyticus in oysters and even increased the mortality of the oysters in the latter cold storage (Melody and others 2008). However, levels of V. parahaemolyticus inoculated Zhe oysters (shell stock or shucked oysters) were reduced by about 2.00 log MPN/g after storage at 0ºC or 5ºC for 96 h, and the following frozen storage at -30ºC for 75 days led to around 3.80 and 5.08 log MPN/g reductions for shell and shucked oysters, respectively (Shen and others 2009). Another recent study demonstrated that the process of flash freezing (-95.5ºC for 12min) followed by 5 months storage at -21±2ºC could reach more than 3.52 log MPN/g reductions of V. parahaemolyticus in half-shell Pacific oysters, which met the requirement of post harvest treatment validation by FDA (Liu and others 2009). V. parahaemolyticus is sensitive to heat treatment. Low temperature pasteurization treatment (50ºC for 10 min) was effective to reduce V. parahaemolyticus (around 105 MPN/g) artificially inoculated in oysters to non-detectable level (Andrews and others 2000). Another study showed that it required treatments at temperatures between 50-52ºC for up to 22 min to reduce V. parahaemolyticus O3:K6 strain to non- 9 detectable level in oysters (Andrews and others 2003b). These results indicate that heat resistance of V. parahaemolyticus is strain dependent. 1.1.5.3 Depuration Depuration is “a dynamic process whereby shellfish are allowed to purge themselves of contaminants either in a natural setting or in land-based facilities”. Flowthrough and recirculating systems are two major types of depuration processes with the former one requires continuous supply of seawater at all time while the later one requires only limited amount of seawater (Richards 1988). To avoid re-contamination of water in recirculating system, water disinfection processes such as chlorination (Wells 1929), ozone (Croci and others 2002) and ultraviolet light (Kelly 1961) treatments are commonly applied separately or synergistically (de Abreu Corrêa and others 2007). Studies of oyster depuration have indicated many factors including species of oysters (Olympia, Estern, Pacific, Sydney oysters), temperature, salinity, dissolved oxygen and turbidity of seawater, harvest time and location, and species of targeted bacteria or virus could affect the efficacy of depuration (Richards 1988). In addition, initial levels of bacterial contamination also affected the depuration efficacy (Govorin 2000). Therefore, there have been controversial results observed for the efficacies of depuration processes in reducing V. parahaemolyticus obtained from different studies (Vasconcelos and Lee 1972; Son and Fleet 1980; Croci and others 2002). Optimal conditions for depuration of shellfish with appropriate parameters need to be determined to enhance the efficacy of depuration. 10 The use of a safe disinfectant instead of regular seawater could enhance the reductions of V. parahaemolyticus in oysters. Electrolyzed oxidizing (EO) water treatment (30 ppm chlorine, pH 2.82; oxidation-reduction potential, 1131mV) for 4-6 h resulted in 1.13 log MPN/g reductions of V. parahaemolyticus in raw oysters. The low pH and chlorine were believed to be the main factors contributing to the effect. However, long exposure (>12h) of oysters to EO water (>30ppm chlorine) led to the death of oysters (Ren and Su 2006). Chloride dioxide is another disinfectant which is a small, volatile and water soluble molecule and can easily permeate the cell membrane to attack the targeted molecule with strong oxidation (Gordon and Rosenblatt 2005). One study confirmed that a treatment of Pacific oysters inoculated with V. parahaemolyticus (106 CFU/ml) with 20 mg/l chlorine dioxide eliminated V. parahaemolyticus within 6 h in all the tissues without killing oysters. Furthermore, treated oysters could survive at 4ºC storage for up to 12 days. However, the gills of treated oysters became chocolate color after treatment, indicating that chlorine dioxide affected the appearance of the oyster meat (Wang and others 2010). Water temperature is also an important factor contributing to the efficacy of depuration. A recent study showed that depuration conducted at 15ºC reduced V. parahaemolyticus in American oysters by around 2.0 log MPN/g after a 2 day depuration with UV light treatment and the reduction level was greater than those obtained from depuration at other conducted temperatures (22, 10 and 5ºC) (Chae and others 2009). In addition, the harvest time may also affect the abilities of oysters to purge the bacteria during depuration. The depuration of Pacific oysters at 5ºC with UV light treatment 11 required two more days to reach more than 3-log reductions of V. parahaemolyticus in oysters harvested in summer than those harvested in winter (Su and others 2010). 1.1.5.4 Addition of natural antibacterial compounds Many naturally occurring antibacterial compounds isolated from plants and animals have been identified (Tiwari and others 2009). Mixture of equal weight of oregano and cranberry extract (0.1 mg/ml total phenolic content) has been shown to inhibit V. parahaemolyticus in fish slices. Populations of V. parahaemolyticus in fish slices were reduced from 3 log CFU/ml to non-detectable level after a treatment with fish slices/mixture extract ratio of 0.1 g/ml followed by six days of storage at 4ºC (Lin and others 2005), indicating phenolic compounds could inhibit V. parahaemolyticus in seafood. Tea, the product processed from the leaves and buds of plant Camellia sinensis, has also been reported to exhibit strong antibacterial activity mainly due to phenolic compounds against wide range of pathogenic bacteria including V. parahaemolyticus (Tagura and others 2004) and spoilage bacteria (Yam and others 1997). However, limited information is available on the antibacterial effects of tea in food application. Tea will be reviewed later in details to explore its potential application on reducing V. parahaemolyticus and retarding spoilage bacteria in oyster meats during post-harvest treatment. Heat degraded sugar was reported able to inhibit V. parahaemolyticus in pure culture and clam extract probably due to production of furfural, acetic acid, formic acid from degraded sugar, indicating byproducts of heated sugar had the potential application in food preservatives (Yoneyama and others 2007). Both commercially purchased red and 12 white wine could reduce levels of V. parahaemolyticus in oyster meat homogenate from around 4 log MPN/g to non-detectable level after 30 min at 25ºC. The mechanisms of wine to inactivate V. parahaemolyticus were not clear but appeared to be a synergistic effects of alcohol, sulfite, polyphenol and organic acids (Liu and others 2006). A later study also indicated that antibacterial activity of the three red wines (Cabernet Sauvignon, Malbec and Merlot) was correlated with their polyphenolic compounds (Vaquero and others 2007). 1.1.5.5 Biological treatments Potential biological treatments, including probiotics treatment and phage therapy, for reducing pathogenic bacteria especially Vibrio in shellfish have been studied and results suggested that both probiotics treatment and phage therapy might enhance the efficacy of traditional post-harvest treatments of shellfish (Teplitski and others 2009). Application of probiotics to control pathogenic bacteria will be reviewed in details later for investigating the potential on reducing V. parahaemolyticus in raw oysters. 1.2 Overview of probiotics for investigating the potential on reducing V. parahaemolyticus in raw oysters 1.2.1 Definition of probiotics The meaning of “pro” and “bios” in the word “probiotics” is “favour” and “life” respectively, which is the antonym of antibiotics (Sahu and others 2008). The Food and Agriculture Organization of the United Nations (FAO) and World Health Organization (WHO) defined probiotics as “live microorganisms which, when consumed in adequate amounts as part of food, confer a health benefit on the host” (FAO/WHO 2006). In addition, one of the most widely quoted definitions of probiotics is “A live microbial feed 13 supplement which beneficially affects the host animal by improving its intestinal microbial balance” suggested by Fuller (Fuller 1989). In addition to general definition of probiotics, specific definition in aquaculture has also been proposed by many researchers. Gatesoupe defined probiotics as “microbial cells that are administrated in such a way as to enter the gastrointestinal tract and to be kept alive, with the aim of improving health” (Gatesoupe 1999). Verschuere and others modified the definition to “ a live microbial adjunct which has a beneficial effect on the host by modifying the host-associated or ambient microbial community, by ensuring improved use of the feed or enhancing its nutritional value, by enhancing the host response towards disease, or by improving the quality of its ambient environment” (Verschuere and others 2000b). Irianto and Austin suggested that probiotics defined as “an entire or components of an organism that is beneficial to the health of the host” (Irianto and Austin 2002). The latter two definitions indicate that application of probiotics in aquaculture is not limited in gastrointestinal tract, which is a general target for application in promotion of human health (FAO/WHO 2006). 1.2.2 General sources and selections of probiotics Probiotics used in aquaculture include Gram-positive bacteria, Gram-negative bacteria, yeasts and unicellular algae (Irianto and Austin 2002). Commonly used species of probiotics include lactic acid bacteria, and non-pathogenic strains of spore-forming Bacillus, Pseudomonas and Vibrio (Gomez-Gil and others 2000). Potential probiotics are usually isolated from digestive guts or other organs of aquatic animals or their living environment (Verschuere and others 2000b; Balcázar and others 2006; Prado and others 14 2009), but commercial products are also used (Moriarty 1998; Scheinbach 1998). Due to the widespread and successful use in food and pharmaceutical areas, lactic acid bacteria are also now considered as one of the promising probiotics for aquaculture application. Therefore, the review of the probiotics below mainly focused on lactic acid bacteria. Many studies summarized the steps of selecting potential probiotics used in aquaculture. Briefly, the background information of selected strains should be obtained and studied at first to guarantee the safety and freedom of virulence or antibiotics resistance genes. The in vitro antagonism tests are usually conducted in solid or liquid medium to check the activity of candidate probiotics followed by the colonization tests to determine whether the targeted strain can attach to host tissues with no adverse effect. After that, in vivo challenge tests are performed to confirm its efficacy in real application (Verschuere and others 2000b; Sahu and others 2008; Kesarcodi-Watson and others 2008). However, the results of in vitro antagonism tests should be used cautiously. Different methods including well-diffusion, disc-diffusion, co-culture and cross-streaking methods can lead to different results even when they are used to test the same target species (Hai and others 2007). Furthermore, in vitro antagonism tests cannot reflect the probiotic activity if the mode of action is not due to the production of inhibitory compounds. Therefore, positive results from in vitro antagonism tests cannot assure the same effect in challenge test (Kesarcodi-Watson and others 2008). 1.2.3 Mode of action Though mechanisms of probiotics on their beneficial effects have been reviewed (Oelschlaeger 2010; Turpin and others 2010), they are still not clearly understood. The 15 possible modes of action involved in the inhibiting pathogenic bacteria by probiotics, especially by lactic acid bacteria, will be described below for a better investigation of potential application on reducing V. parahaemolyticus in raw oysters. 1.2.3.1 Competition for adhesion sites Competition exclusion is defined as “a state in which two species competing for the same resources cannot stably coexist if other ecological factors are constant” (Turpin and others 2010). Basically, all bacteria can secret adhesions to bind to certain types of surfaces, which allows pathogens to attach to the host and cause infections (Klemm and others 2010). Therefore, probiotics which can compete with pathogens to adhere to gut or other tissues of a host might be utilized to decrease or eliminate the attachment of pathogens to the host. The adhesion of a bacterium to host tissues is generally considered the first step of bacterial colonization (Ringø and Gatesoupe 1998). The adhesion can be either specific or not. Specific adhesion is possibly due to the specific interaction between the molecules produced by bacteria and receptors of epithelial cells of the host and nonspecific adhesion is possibly based on steric hindrance (Verschuere and others 2000b). In addition, passive forces, electrostatic interactions, hydrophobic steric forces and presence of lipoteichoic acid are regarded as the adhesive factors of some lactic acid bacteria (Servin 2004). To establish an infectious animal model is the usual way to demonstrate the mechanism of competition for adhesion sites. One study indicated that preemptive colonization of non-pathogenic Aeromonas and Vibrio alginolyticus could fully or partially protect against pathogenic Vibrio proteolyticus CW8T2 in Artemia nauplii and 16 the colonization capability of selected probiotics correlated well with protective ability (Verschuere and others 2000a). In addition, pre-colonization, co-colonization or postcolonization of probiotics with pathogens might also be an important factor (Vine and others 2004). It should be noted that competition for adhesion sites is not the only mode of action contributing to the beneficial effects of probiotics in most cases. Other mechanisms may also contribute to the beneficial effects synergistically with competition for adhesion sites being hypothesized as the first probiotic effect (Verschuere and others 2000b). Lactobacillus brevis (108 bacteria/ml) treatment in the Artemia culture water could inhibit the growth of dominant strain Vibrio alginolyticus in water and Artemia nauplii, and the extracellular products of the L. brevis could also inhibit V. alginolyticus in a bactericidal assay (Villamil and others 2003). Probiotics (three lactic acid bacteria isolated from fish) treatments have been report to decrease pathogens including Vibrio anguillarum attachment in fish intestinal mucus and in vitro inhibition test also showed probiotics could produce metabolites against pathogenic bacteria, but the ability of lactic acid bacteria to reduce pathogens was species specific (Balcázar and others 2008). 1.2.3.2 Production of inhibitory compounds 1.2.3.2.1 Organic acids Both homo-fermentative and hetero-fermentative lactic acid bacteria could produce lactic acid by fermentation of hexose (Ouwehand and Besterlund 2004), which is regarded one of the important compounds contributing to inhibitory effect. In particular, un-dissociated form of lactic acid released by lactic acid producing bacteria can act as 17 permeabilizer to disrupt outer membrane and reduce intracellular pH of Gram-negative bacteria (Helander and others 1997; Alakomi and others 2000). In addition to membrane disruption, organic acids may also inhibit essential metabolism, disturb intracellular pH homeostasis or accumulate toxic anions in bacteria (Brul and Coote 1999). Furthermore, one study indicated that the mechanism of lactic acid for inhibition of growth of bacteria was pH dependent. The ionic form of lactic acid contributed to the inhibition at low pH (< 6.0) while the non-dissociated form of lactic acid did at high pH (Gonçalves and others 1997). Besides, other organic acids like acetic acid also contribute to the inhibitory effect (Helander and others 1997; Schnürer and Magnusson 2005). 1.2.3.2.2 Hydrogen peroxide Most lactic acid bacteria contain flavoprotein oxidase enzymes, which can produce hydrogen peroxide by reacting directly with oxygen in the environment. The hydrogen peroxide can accumulate in the environment due to the lack of catalase in lactic acid bacteria (Condon, 1987). The mechanism of antibacterial activity is mainly based on the strong oxidizing effect of hydrogen peroxide which can destroy the structures of cellular proteins. However, hydrogen peroxide is degraded when lactic acid bacteria are enriched in MRS broth probably as the yeast extract in MRS broth has catalase activity (Schnürer and Magnusson, 2005). In addition, the inhibitory effect of hydrogen peroxide on bacterial growth is pH and temperature dependent (Brul and Coote, 1999). 1.2.3.2.3 Bacteriocins Many lactic acid bacteria can produce bacteriocins, which are proteinaceous compounds exhibiting antibacterial activity on other bacteria, especially closely related 18 strains. Baceteriocins are usually classified into three major classes mainly according to their molecular sizes, chemical structures, physical properties and mode of action. Generally, Class I, Class II and Class III bacteriocins are small peptides (<5 kDa), small (<10kDa) heat-stable (100-120 ºC) peptides and large (>30 kDa) heat-labile proteins, respectively. Besides, Class IV bacteriocins are defined as the cyclic peptides which require lipid or carbohydrate for activity, but little is known about the properties of this class (Ouwehand and Besterlund 2004). The mechanism of actions of bacteriocins depends on classes and is still not clearly understood. Most Class I bacteriocins, which are generally cationic, may easily bind to anionic lipids on the membrane of Grampositive bacteria to form pores. The loss of the energy during membrane pore forming may contribute to the inhibitory effects. In addition, other kinds of bacteriocins may interfere with the essential enzymes activities instead of forming membrane pores to exert the effect (Servin 2004). Generally, bacteriocins cannot inhibit growth of Gram-negative bacteria because of their permeability barrier of outer membranes. However, a combination of bacteriocins with chelating agents could inhibit Gram-negative bacteria because the chelating agents could disrupt the outer membranes of Gram-negative bacteria through the release of lipopolysaccharides (Alakomi and others 2000; Gálvez and others 2007). Other physical treatments like heating, chilling, freezing, high hydrostatic pressure and pulsed electric fields could also disrupt the outer membranes of Gram-negative bacteria, which can be used together with bacteriocins and allow bacteriocins to pass through the outer membrane to take effect (Gálvez and others 2007). 19 1.2.3.2.4 Other inhibitory compounds Aromatic and heterocyclic compounds, including benzoic acid and mevalonolactone produced by Lactobacillus plantarum VTT-78076, could inhibit Gramnegative bacteria in vitro and the inhibitory effect was enhanced when combined with lactic acid (Niku-Paavola and others 1999). Reuterin (β-hydroxypropionaldehyde), which is a water-soluble substance usually produced from Lactobacillus reuteri in the presence of glycerol, has the wide range of antibacterial spectrum (Schnürer and Magnusson 2005). The antibacterial effect of reuterin is pH-dependent and species specific, and the mechanism is probably attributed to its interference with DNA synthesis (Arqués and others 2004; Bian and others 2010). Furthermore, studies showed that reuterin could work synergistically with other antibacterial compounds like nisin to enhance the antibacterial effect on Gram-positive bacteria but not Gram-negative bacteria (Arqués and others 2004). Cyclic dipeptides produced by some lactic acid bacteria might contribute to both antibacterial and antifungal activity (Strom and others 2002; Schnürer and Magnusson 2005). Other kinds of probiotics may also produce deconjugated bile acids to inhibit the growth of pathogenic bacteria (Oelschlaeger 2010). 1.2.4 Application of probiotics in aquaculture The mortality of aquatic animals is mainly caused by pathogenic bacteria like Vibrio. Probiotics can be applied to aquaculture practice to control pathogens and increase the survival of aquatic animals (Kesarcodi-Watson and others 2008; Prado and others 2010). One common treatment of probiotics is to add probiotics to the live feed like rotifers or Artemia. For instance, lactic acid bacteria such as Lactobacillus plantarum 20 or Carnobacterium spp. isolated from rotifer could enhance the survival rate of turbot larvae when they were added to the rotifer to feed the turbot larvae infected with pathogenic Vibrio. The effective dosage ranged from 107 CFU/ml to 2×107 CFU/ml (Gatesoupe 1994). Other basic diets can also be mixed with probiotics to feed the aquatic animals. Diets with lyophilized Carnobacterium divergens (about 109 CFU/g) isolated from Atlantic salmon intestines could increase the survival of Atlantic cod fry when they were challenged with Vibrio anguillarum (Gildberg and others 1997). Four strains (Vibrio alginolyticus UTM102, Bacillus subtilis UTM 126, Roseobacter gallaeciensis SLV03, and Psudomonas aestumarina SLV22) isolated from adult shrimp could increase the survival of shrimp when challenged with pathogenic Vibrio parahaemolyticus via commercial shrimp feed (106 CFU/ml cell suspension mixed with equal amount of feed) after 28 days of feeding trial (Balcázar and others 2007). Adding probiotics directly to the water is also a common method. Addition of Bacillus with a final concentration of about 104cells/ml to the pond water could increase the survival of penaeid shrimps while decreasing Vibrio (Moriarty 1998). The potential probiotics Arthrobacter nicotianae isolated from Penaeus chinensis culture water, which could inhibit three Vibrio strains (V. parahaemolyticus, V. anguillarum and V. nereis) in inhibition test, could improve the survival of Penaeus chinensis when added to the culture water with same amount of pathogenic Vibrio (106 CFU/ml) once a week in a 14 days of trial (Li and others 2006). While many studies have been conducted on application of probiotics in fish and crustacean aquaculture, application of probiotics in bivalve mollusks to reduce pathogen contamination is limited (Prado and others 2010). One study indicated that Alteromonas 21 sp. strain CA2 could improve the survival and growth of oyster larvae probably due to the nutritional supply (Douillet and Langdon 1993). The following research determined that the effect of probiotics on oyster larvae was concentration dependent, and the optimal feeding concentration was 105 cells/ml (Douillet and Langdon 1994). Though these studies had a focus on the application of probiotics in oyster larvae, the application of probiotics for oyster post-harvest treatments can also be explored. For instance, probiotics could remain in oyster larvae for 4 days, which make it possible to allow probiotics to colonize in oysters and persist for a few days during depuration (Gibson and others 1998). Such a practice might be a means for reducing pathogens such as V. parahaemolytiucus in oysters after harvest. 1.2.5 Application of probiotics in food Bio-preservation can be defined as the use of nature or controlled microflora and (or) their antibacterial products to enhance the safety while extend the shelf life of the food (Stiles 1996). Lactic acid bacteria and their extracellular products have been widely studied for bio-preservation. For instance, Nisin, a bacteriocin isolated from Lactococcus lactis subsp. lactis , was the one on the market as early as in 1953 in England and now is one of the commonly used food preservatives over 48 countries (Deegan and others 2006). It also has been approved as GRAS (Generally Recognized as Safe) compound by the U.S. Food and Drug Administration (FDA 1995). Nisin is mainly added to cheese and other fermented products to inhibit spoilage bacteria and pathogenic Gram-positive bacteria like Listeria monocytogenes and Clostridium botulinum spores, and the maximum dosage used varies in each country and different products (Vandenbergh 1993). 22 Besides, studies also showed that nisin and other kinds of bacteriaocins such as carnobacteriocins produced by Carnobacterium maltaromaticum and enterocins produced by Enterococcus faecium could be applied for inhibiting L. monocytogenes and other spoilage bacteria in seafood products (Calo-Mata and others 2008). In addition to bacteriocins, other compounds like diacetyl, acetaldehyde and hydrogen peroxide produced by lactic acid bacteria during food fermentation may also contribute to inhitory effects (Vandenbergh 1993). In addition to improving the safety of foods, probiotics may also be applied in food products as a functional ingredient for health promotion. Some studies indicated that oral consumption of probiotics had beneficial effects on human gastrointestinal health and can even been utilized to treat some gastrointestinal diseases such as diarrheal diseases, inflammatory bowel diseases, Helicobacter pylori gastroenteritis and lactose intolerance (FAO/WHO 2006). The common health claims of probiotics in food products are related to the maintenance or promotion of digestive health or immune function, and the dairy products including yogurt, dairy drink and cheese are the most common products enriched with probiotics for health promotion (Saulnier and others 2009). 1.3 Overview of tea for investigating the potential on reducing V. parahaemolyticus in oyster meats Tea is the product processed from the leaves and buds of Camellia sinensis. The optimal temperature, humidity and pH of soil for tea production are 15-25ºC, 80-90% with more than 1500 mm annual rainfall and 3.5-5.6, respectively (Wong and others 2003). The origin of tea and drinking tea habits can be traced to as early as the year 2737 B.C. in China. Nowadays, tea is a popular drink consumed by people all over the world 23 (Hara, 2001). 1.3.1 Classification of tea Tea can be classified based on genotypes: Camellia sinensis variety assamica and Camellia sinensis variety sinensis. The former one has large, broad and flat leaves with light green color while the latter one has small, narrow and serrate leaves with dark green color (Hara and others 1995a). More frequently, tea is classified based on the degree of fermentation. Green tea, oolong tea and black tea are non-fermented, semi-fermented and fully fermented, respectively (Peterson and others 2005). 1.3.2 Major chemical compositions Polyphenols are secondary metabolites of plants, which are usually classified into flavonoids, phenolic acids, lignans, and stilbenes (Wang and Ho 2009). They are commonly present in tea and account for around 30% of the dry weight in fresh tea leaf. In addition, other compounds such as methylxanthines, proteins and amino acids, organic acids, mono/poly-saccharides, pigments and volatiles in tea have also been determined. Flavonoids are generally divided into six classes including flavanols, flavonols, flavones, flavanones, isoflavones and anthocyanins, and the predominant flavonoids in tea are flavanols and flavonols (Balentine and others 1997). Catechins are monomers of flavanols. The major catechins include: (-)epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG) and (-)epigallocatechin gallate (EGCG). All of them are colorless and water-soluble compounds contributing to the bitterness with sweet aftertaste (EC and EGC) or astringency (ECG and EGCG) flavor. They exist in all kinds of tea but the amount varies (Hara and others 24 1995b; Balentine and others 1997; Peterson and others 2005). Theaflavins are dimmers of flavanols. Four major theaflavins include: theaflavin (TF), theaflavin 3-gallate (TF3G), theaflavin 3’-gallate (TF3’G) and theaflavin 3,3’-digallate (TF33’G). The first two are the oxidation products of EC and EGC, respectively, and TF3’G and TF33’G are formed from oxidation of ECG/EGC and ECG/EGCG, respectively (Hara and others 1995a). The theaflavins are main pigments in black tea with orange-red color (Balentine and others 1997; Wang and others 2000) while contributing to the sensation of richness and fullness of tea infusion (Hara and others 1995b). Flavonols, the second major class of flavonoids in tea, include quercetin, kaempferol, and myricetin. Green tea and black tea contains similar amounts of flavonols except the content of myricetin, which is significantly higher in green tea than in black tea (Peterson and others 2005). In addition, flavonols usually exist in the form of glycosides instead of aglycones in tea probably due to the poor solubility of aglycones in water (Wang and others 2000). Tea also contains small amount of flavones such as apigenin and luteolin (Peterson and others 2005). The major phenolic acid in tea is gallic acid, the amount usually increases during tea fermentation processes due to the release of catechins gallates (Lin and others 1998). Gallic acid can react with catechin quinones or gallocatechin quinones to produce bright red acidic theaflavic acids or theaflagallins, respectively, in black tea (Balentine and others 1997). 25 1.3.3 Factors affecting tea compositions and stability 1.3.3.1 Intrinsic factors Factors including species and cultivating age of tea leaves, geographic sites of growth and collection, growth season and climate can affect chemical compositions of tea. In terms of two genotypes of Camelia sinensis (variety assamica and variety sinensis), the former one contains higher level of flavanols than the latter one, and the stronger polyphenol oxidase activity for variety assamica makes it more suitable for the production of black tea while variety sinensis is better for the production of green tea (Hara and others 1995a). Tea products from different regions like Assam, Ceylon, China and Darjeeling also have various flavonoids contents (Peterson and others 2004). Usually, young green tea leaves contain higher levels of caffeine and lower levels of total catechins and EGCG compared to old leaves (Lin and others 2003). The amount of metals like aluminum, fluoride and lead usually increases with the increasing age of tea leaves (Wong and others 2003). 1.3.3.2 Manufacturing processes Different manufacturing processes of tea lead to different chemical compositions with different flavor, color and aroma features (Balentine and others 1997). In a study, same batch of fresh tea leaves were processed to make both green tea and black tea to identify the change of total polyphenols, flavanols and caffeine contents. No significant loss of those three compounds was observed during production of green tea. However, flavanols decreased sharply during manufacturing black tea, confirming that oxidation of catechins triggered by polyphenol oxidase enzyme occurred (Astill and others 2001). The 26 contents of polyphenols and catechins of tea were not influenced by different polyphenol oxidase inactivation methods (parching, steaming, microwave heating and oven heating), but the use of microwave to dry tea leaves led to higher yield of total phenols and catechins compared to sun-dried and oven-dried methods (Gulati and others 2003). In addition, total catchins of green tea varied 6-fold while total theaflavins varied 11-fold for 77 commercial tea products sold in the U.S. market (Friedman and others 2005), indicating the significant differences among tea products of various brands. 1.3.3.3 Extraction solvents Water and other organic solvents including methanol, ethanol and acetonitrile are usually used to extract active compounds from tea. One study showed that boiling water had better extraction efficacy of total catechins compared to 80% methanol or 70% ethanol (Khokhar and Magnusdottir 2002). However, another study showed that 75% ethanol was more efficient than distilled water to extract EGCG and catechins in teas (Lin and others 2003). More recently, it was found that using 80% ethanol to extract tea at 60ºC for 15 min had the best yield of biologically active compounds compared to previously published results (Friedman and others 2006b). The difference among studies might be due to different parameters used in studies, but organic solvents extraction methods should be used carefully for food application due to the possible residues. The quality of water alone also affects the tea extraction. Tap water (TW), activated carbon adsorbed water (AC), deionized water (DI), distilled water (DW), reverse osmosis water (RO) and ultra-pure water (UP) were used to extract tea components from low-grade green tea. Results showed that extraction of tea components 27 with DI yielded the highest tea polyphenol while those with TW yielded the lowest. However, no significant difference among caffeine contents were observed (Zhou and others 2009). 1.3.3.4 Brewing conditions Basically, brewing temperature, time, ratio of solvents to leaves, agitation or static condition and leaf size may all influence the final yield of compounds in tea. Brewing tea at 100ºC for 5 min led to higher yields of individual catechins, total polyphenols and caffeine compared to other temperatures conducted (80 and 60ºC) (Khokhar and Magnusdottir, 2002), which indicates that higher temperature allows tea leaves to be more penetrable to solvents (Vuong and others 2010). Longer brewing time and lower tea/water ratios also tended to have better extraction of catechins and caffeine (Astill and others 2001). In addition, the extraction efficiency of catechins in green tea was not all affected by tea/water ratios. The extraction efficiency of EC and EGC in tea prepared by boiling distilled water at 80ºC for 20 min at tea/water ratios ranging from 0.25% to 3% kept consistent but that of ECG and EGCG gradually decreased when increasing tea/water ratios (Yoshida and others 1999). 1.3.3.5 Stability of chemical composition in tea Generally, theaflavins are more sensitive to temperature and pH than catechins, and they are both more stable at lower pH. EGCG, ECG, EGC and EC tended to be epimerized to (-)-gallocatechin gallate (GCG), (-)-catechin gallate (CG), (-)gallocatechin(GC) and (-)-catechin (C), respectively, when brewing tea at the temperature higher than 70°C (Su and others 2003). Since the amounts of decreased epistructured 28 catechins were not as same as those of increased corresponding non-epistructured catechins, other reactions including oxidation might happen at the same time (Wang and Helliwell, 2000). Formation of the dimmers and epimers of EGCG was identified in a study which confirmed oxidation and epimerization were two major reactions attributing to instability of EGCG (Sang and others 2005). However, no composition changes of both catechins and theaflavins were observed when exposed to mild heat treatment (24°C) within 3 h (Wang and Helliwell, 2000; Su and others 2003). 1.3.4 Antibacterial activities of tea 1.3.4.1 Mode of action The antibacterial activity of tea was mainly contributed from the flavanoids, and the mechanisms include inhibition of nucleic acid synthesis, cytoplasmic membrane function or energy metabolism (Cushnie and Lamb 2005). Catechins are the major flavanoids in tea and their different chemical structures result in various antibacterial activities. Generally, gallates derivatives of catechins have strong activity while other compounds like EC, C and gallic acid exhibit very low activity (Hamilton-Miller 1995; Yam and others 1997; Almajano and others 2008). The higher antibacterial activity of EGCG might be due to the presence of the galloyl group (Tagura and others 2004), which has the ability to damage the cell membrane of the bacteria and led to the intracellular leakage. Such mechanism may explain the fact that most Gram-positive bacteria are more sensitive to EGCG than Gram-negative bacteria with lipopolysaccharide existed in outer membrane resulting in negative charged surface and barrier of penetration (Ikigai and others 1993). Many studies have confirmed that Gram-negative bacteria were less 29 susceptible to EGCG or other tea polyphenols compared to Gram-positive bacteria (Yoda and others 2004; von Staszewski and others 2011). However, one exception was that Vibrio strains had lower average minimum inhibition concentrations than other Grampositive bacteria when exposed to EGCG and EGC (Tagura and others 2004), indicating the mode of action should be further investigated. Recently, one transmission electron microscopy (TEM) study showed that some EGCG and ECG treated pathogenic bacteria led to morphological changes of cells, suggesting that physiological conditions instead of cell wall components of the bacteria may contribute to the various sensitivity of pathogenic bacteria towards tea polyphenols (Si and others 2006). 1.3.4.2 In vitro inhibition tests According to in vitro inhibition tests, tea extract inhibits a wide range of foodborne and other pathogenic bacteria mainly due to the flavanoids constitutes. Many studies have compared the sensitivity of various pathogenic bacteria to tea extracts. One study indicated that tea extract concentrated by rotary evaporator could inhibit Bacillus cereus in disc diffusion inhibition test but no obvious effect on intestinal bacteria (Escherichia coli and Lactoacillus acidophilus) was observed (Almajano and others 2008). Crude extract of Chinese green tea (500 µg/ml) could inhibit foodborne pathogens including Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus, Bacillus cereus and Listeria monocytogenes after 5-6 h of incubation with completed inhibition of two strains of methicilin-sensitive S. aureus. Further analysis indicated that EGCG and ECG were the most active compounds contributing to the inhibition (Si and others 2006). EGCG and EGC isolated from green tea as well as theaflavins mixture from black tea all 30 inhibited tested strains of foodborne pathogens belonging to S. aureus, Salmonella, E. coli and Vibrio, and the average minimum inhibition concentrations for those four species were consistent in three treatments. Vibrio species containing V. cholera-non O1, V. parahaemolyticus and V. vulnifucus were the most sensitive ones (Tagura and others 2004). In addition, one study showed that not all tested strains of E. coli were inhibited by green tea extract (Yam and others 1997), indicating susceptibility to the same tea extraction is strain dependent. Green tea polyphenol mixture (100 µg/ml), mainly containing EGCG, could inhibit thermophilic spore-forming Bacillus stearothermophilus in Mueller-Hinton agar medium incubated at 30ºC. The heat resistance of B. stearothermophilus and anaerobic spore-forming Clostridium thermoaceticum was also decreased when heating at 120ºC for 15 min with tea polyphenol treatment (500 µg/ml) (Sakanaka and others 2000). Biological compounds including major catechins and theaflavins had antibacterial activity against B. cereus (Friedman and others 2006a). Crude catechins, theaflavins, and four major compounds of catechins (EC, ECG, EGC, and EGCG) all could inhibit phytopathogenic bacteria including Erwinia, Pseudomonas, Clavibacter, Xanthomonas and Agrobacterium, which mainly caused diseases in vegetables. In addition, pyrogallol catechins (EGC and EGCG) had better effects than catechol catechins (EC and ECG) and crude theaflavins (Fukai and others 1991). Green tea extract also inhibited the growth of spoilage bacteria including Pseudomonas aeruginosa and Serratia marcescens (Yam and others 1997). Some volatile compounds in green tea, especially nerolidol, had the inhibitory effect against some cariogenic bacteria including Saccharomyces cerevisiae, Candida utilis, Pityrosporum 31 ovale, Trichophyton mentaprophytes and Penicillium chrysogenum (Kubo and others 1992). Two review studies showed that tea polyphenol could inhibit cariogenic bacteria including Streptococcus mutans and Streptococcus sobrinus to prevent dental caries and ocular pathogenic bacteria by inhibiting gelatinase activity (Wang and others 2000; Friedman 2007). Tea polyphenol could also inhibit many other pathogens causing human disease in gastrointestinal and respiratory systems, such as Helicobacter pylori regarded as gastric pathogens , Mycoplasma pneumonia causing atypical pneumonia and Mycobacterium tuberculosis causing tuberculosis (Friedman 2007). 1.3.4.3 Factors affecting antibacterial activity of tea Factors affecting chemical compositions of tea may also affect the antibacterial activity. In addition, antibacterial activity of tea extract decreased with the increase of fermentation degrees (Almajano and others 2008; Tiwari and others 2005; Tagura and others, 2004). Methanol extract of green tea extract showed better antibacterial activity against Shigella dysenteriae than aqueous extract (Tiwari and others 2005). Irradiation (40 kGy) treated green tea showed better antibacterial activity against some skin (E. coli, Staphylococcus aureus and S. epidermidis) and oral cavity (Streptococcus mutans) related pathogenic bacteria in disc diffusion inhibition test (An and others 2004). 1.3.4.4 Food application Application of tea extraction in food matrixes becomes more complicated due to complex chemical surroundings. Green tea extract prepared by brewing 90 ml hot water (approximate 95ºC) for 10 min could not significantly reduce total bacteria and pathogens such as L. monocytogenes and S. aureus in ground beef during the storage at 7ºC for 7 32 days (Kim and others 2004). However, 95% ethanol extract of green tea (1:4 w/v) could significantly inhibit pathogens including S. aureus and E. coli in acidulate treated mutton (pH 3.8) after one day storage at 25±2ºC without compromising the sensory attributes and meat quality (Kumudavally and others 2008). Mixing green tea extract (containing 697 mg total catechins per gram of extract) with three different meat products (beef, chicken and pork) at concentrations of 2% (wt/wt) significantly inhibited growth of Clostridium perfringens in those products when chilled from 54.5 to 7.2ºC at various chill rate (12, 15, 18 and 21 h) after cooking at 71ºC within 1 h (Juneja and others 2007). Tea polyphenol dip (0.2% w/v) could also extend the shelf life of silver carp from 28 days to 35 days at -3ºC compared to control, indicating tea polyphenol retarded growth of spoilage bacteria in food (Fan and others 2008). In addition, whey protein was used as a food model to test its influence on antibacterial activity of different Argentinean green teas. The results showed that the increase in the protein concentration correlated well with the loss of antibacterial activity, indicating the interaction between protein and polyphenol might occur (von Staszewski and others 2011). 33 Chapter 2 Impacts of Probiotics in Post-harvest Treatments for Reducing Vibrio parahaemolyticus in Pacific Oysters (Crassostrea gigas) 34 2.1 Abstract Lactobacillus plantarum ATCC 8014, which exhibited strong bactericidal effects against Vibrio parahaemolyticus in well diffusion inhibition test, was added to artificial seawater for depuration of Pacific oysters (Crassostrea gigas) inoculated with V. parahaemolyticus BE 98-2029 (O3:K6) to levels of 104 MPN/g at 15±1 and 10±1ºC. L. plantarum ATCC 8014 treatment (107 CFU/ml) did not enhance reductions of V. parahaemolyticus in oysters depurated at 15±1ºC but significantly (p<0.05) decreased levels of V. parahaemolyticus in oysters depurated at 10±1°C after 5 days when compared with controls. L. plantarum ATCC 8014 can be applied to oyster depuration at low temperature to enhance reductions of V. parahaemolyticus. 35 2.2 Introduction Probiotics are “live microorganisms which, when consumed in adequate amounts as part of food, confer a health benefit on the host” (FAO/WHO 2006). Among them, lactic acid bacteria have been widely studied for their beneficial effects in human, including the antibacterial ability against gastrointestinal and urovaginal pathogenic bacteria (Servin 2004). In addition, antibacterial activity of lactic acid bacteria has also been studied to develop bio-preservatives for application in food products. The antibacterial compounds produced by lactic acid bacteria include organic acids, diacetyl, low molecular weight compounds and bacteriocins such as nisin (Ouwehand and Besterlund 2004). Nisin is a small peptide produced by certain strains of Lactococcus lactis subsp. lactis, which has been applied as bio-preservatives over 48 countries in dairy products such as cheese, milk and desserts (Vandenbergh 1993) and considered as GRAS (Generally Recognized as Safe) by the United States Food and Drug Administration (FDA 1995). In addition to application in foods, use of probiotics in aquaculture showed that certain lactic acid bacteria could enhance the survival of fish larvae exposed to Vibrio pathogens by feeding with rotifers (Gatesoupe 1994) or commercial dry feed (Gildberg and others 1997). The survival and growth of oyster larvae increased when they were fed with algae mixed with Alteromonas spp. strain CA2 as extra nutritional supply (Douillet and Langdon 1993). A later experiment defined the optimal feeding concentration of probiotics was 105cells/ml (Douillet and Langdon 1994). However, no study has been conducted to determine the potential application of probiotics, especially lactic acid 36 bacteria, for reducing human pathogens, such as V. parahaemolyticus, in raw oysters upon harvest. V. parahaemolyticus is a human pathogen occurring naturally in the marine environment and commonly found in molluscan shellfish, particularly oysters. Consumption of raw or undercooked shellfish contaminated with V. parahaemolyticus can result in food-borne illness including gastroenteritis, wound infection and septicemia (Butt and others 2004). It is reported that more than six hundred thousand tons of Pacific oysters (Crassostrea gigas) were produced every year between 2000 and 2008 worldwide (FAO 2008). Therefore, contamination of oysters with V. parahaemolyticus is a concern for public health. Numerous outbreaks of V. parahaemolyticus infections resulted from consumption of raw oysters in the U.S. were documented over the past ten years (CDC 2005, 2006). Several post-harvest treatments, such as high pressure processing (Ma and Su 2011), irradiation (Mahmoud and Burrage 2009), low temperature pasteurization (Andrews and others 2000) and flash freezing with frozen storage (Liu and others 2009), have been developed for reducing V. parahaemolyticus in oysters upon harvest. However, these processes require either a significant amount of investment in equipment or operation costs. The processes, except for irradiation, also kill oysters during the treatments. There is a need to develop an economic post-harvest process for reducing V. parahaemolyticus contamination in oysters without adverse effects to oysters. Depuration is a process to allow shellfish to purge contaminants in clean seawater either in a natural setting or in land-based facilities (Richards 1988). Although the 37 process has a long history as a post-harvest treatment of shellfish, it is ineffective in reducing Vibrio contamination in oysters at ambient temperature (Chae and others 2009). This study was conducted to investigate the effect of probiotics on reducing V. parahaemolyticus contamination in raw oysters during post-harvest depuration. 2.3 Materials and Methods 2.3.1 Vibrio parahaemolyticus Five clinical strains of V. parahaemolyticus [10290 (O4:K12, tdh+ and trh+), 10292 (O6:K18, tdh+ and trh+), 10293 (O1:K56, tdh+ and trh+), BE 98-2029 (O3:K6, tdh+) and 027-1c1 (O5:K15, tdh+ and trh+)] obtained from the Food and Drug Administration Pacific Regional Laboratory Northwest (Bothell, WA) were used in this study. Each strain was individually grown in 9 ml tryptic soy broth (Difco, Becton Dickson, Spark, MD) supplemented with 1.5% NaCl (TSB-salt) at 37°C for 18-24 h. The enriched cultures were streaked onto tryptic soy agar (Difco) supplemented with 1.5% NaCl (TSAsalt) and incubated at 37°C for 18 h. A single colony formed on the TSA-salt plate was transferred to 9 ml TSB-salt and incubated at 37°C for 4 h. Cells of each enriched culture were harvested by centrifugation at 3000 × g (Sorvall RC-5B, Kendro Laboratory Products, Newtown, CT) at 5±1°C for 15 min and re-suspended in equal amount of sterile 2% NaCl solution to prepare a culture suspension of approximate 108 CFU/ml. 2.3.2 Lactic acid bacteria Three strains of lactic acid bacteria (Lactobacillus plantarum ATCC 8014, Lactobacillus acidophilus ATCC 314 and Lactococcus lactis subsp. lactis ATCC 11454) were individually grown in de Man, Rogosa and Sharpen (MRS) broth (Acumedia 38 Manufacturers, Inc., Lansing, MI) at 37°C for 18-24 h. The enriched cultures were streaked on MRS agar prepared by mixing MRS broth with 1.5% agar (Difco, Becton Dickson, Spark, MD) and incubated at 37°C for 72 h. Colonies taken from MRS agar of each strain were transferred to 9 ml MRS broth and incubated at 37°C for 12 or 24 h. Enriched cultures were harvested by centrifugation at 3000 × g at 5±1°C for 20 min. Cells were re-suspended in equal amount of phosphate buffered saline (PBS). 2.3.3 Preparation of lactic acid bacteria culture broth supernatant Cell-free supernatant (CFS) was prepared by filtering enriched MRS broth after centrifugation (3000 × g) at 5±1°C for 20 min through 0.2 µm sterile syringe filter (VWR International, USA) with a sterile syringe (Becton Dickinson and Company, Franklin Lakes, NJ) and kept at 4°C until usage. The pH of CFS was determined by a pH meter (Symphony Meters, Beverly, MA). Titratable acidity (TA) was determined by titration with 0.1 N NaOH standardized with potassium acid phthalate (KPH) to reach an end point of pH 8.20±0.02 in triplicate. TA was reported as equivalent to lactic acid according to the equation (Sadler and Murphy, 2003): acid (w/v)%=(N×V1×EqWt×100)/(V2×1000), where N=normality of titrant (mEq/ml); V1=volume of titrant (ml); V2=volume of sample (ml); EqWt=Equivalent weight of lactic acid (90.08 mg/ mEq); and 1000=factor converting gram to milligram (mg/g). 2.3.4 Inhibitory effect of lactic acid bacteria culture broth supernatant on growth of V. parahaemolyticus One milliliter of cell suspension from mixture of five enriched V. parahaemolyticus cultures was mixed with 100 ml of sterile TSA-salt tempered to 45ºC 39 to prepare 105 CFU/ml of V. parahaemolyticus in the medium. The inoculated TSA-salt was poured onto petri dishes (25 ml) and allowed to solidify at room temperature. Wells (9 mm in diameter) were created on the TSA-salt plates using a sterile cork borer. An aliquot (200 µl) of CFS or cell suspension (108 CFU/ml) prepared from the growth of each lactic acid bacteria was added to individual wells. The plates were incubated at 37°C for 20 h and observed for clear zone surrounding each well. The inhibitory effect (%) was calculated as: [(diameter of inhibition zone-diameter of well)/diameter of well] ×100. To determine the major parameters contributing to the antibacterial effect against V. parahaemolyticus in CFS of lactic acid bacteria, CFS prepared from 24 h growth of L. plantarum ATCC 8014 was adjusted to pH 6.5 by adding 2 N NaOH to neutralize lactic acid and other organic acids or treated with 0.1 ml/ml catalase (MP Biomedicals, LLC, Solon, OH) and 2 mg/ml pepsin (Sigma-Aldrich, St. Louis, MO) at 37°C for 1 h to eliminate hydrogen peroxide and protein compounds. The neutralized or enzyme-digested CFS was tested for the inhibitory effects on the growth of V. parahaemolyticus as described previously. In addition, lactic acid solutions (Sigma-Aldrich, St. Louis, MO) at levels of 0.5, 1.0 and 2.0% (w/v) were also tested for their effects on growth of V. parahaemolyticus. MRS broth and MRS broth treated with catalase (0.1 ml/ml) or pepsin (2 mg/ml) were used as controls. All samples were conducted in triplicate. 2.3.5 Oyster preparation Raw Pacific oysters (Crassostrea gigas) were obtained from oyster farms in Oregon and Washington. Oysters were washed under tap water to remove mud on shells and acclimated in a high-density polyethylene (HDPE) tank (18 by 12 by 12 in; Nalgene, 40 Rochester, NY) containing 20 l artificial seawater (ASW) at room temperature (23±1ºC) for 2-4 h upon the delivery to the laboratory in a cooler on the harvest day. The ASW (salinity: 30 ppt) was prepared by dissolving Instant Ocean Salts (Aquatic Eco-System Inc, Apopka, FL) in deionized water according to the manufacturer’s instructions. 2.3.6 Accumulation of L. plantarum or V. parahaemolyticus in oysters Six milliliters of L. plantarum ATCC 8014 culture enriched in MRS broth overnight at 37ºC were transferred to 400 ml of fresh MRS broth and incubated at 37ºC for 24 h. The cells were harvested by centrifugation as described earlier and re-suspended in 40 ml sterile PBS. For accumulation of L. plantarum in oysters, about 30 oysters were placed in the HDPE tank of 10 l fresh ASW containing L. plantarum ATCC 8014 at a level of approximate 106 CFU/ml with water being circulated at a rate of approximately 12 l/h at 23±1ºC for 20 h. Similarly, accumulation of V. parahaemolyticus in oysters was conducted with holding about 80 oysters in the HDPE tank of 20 l fresh ASW containing V. parahaemolyticus BE 98-2029 at a level of approximate 104 CFU/ml. 2.3.7 Oyster depuration About 25 oysters after accumulation of L. plantarum were depurated in 60 l of ASW in a laboratory-scale re-circulating (25 l/min) system at 20±1ºC for four days. Lactic acid bacteria populations in oysters and ASW were tested every day to determine the ability of L. plantarum to remain colonized in oysters during depuration process. To determine the effects of applying lactic acid bacteria in depuration on reducing V. parahaemolyticus in oysters, about 70 oysters accumulated with V. parahaemolyticus BE 98-2029 were held in 60 l of ASW in a laboratory-scale re-circulating (25 l/min) 41 system equipped with a 15-W Gamma UV sterilizer (Current-USA Inc., Vista, CA) and a temperature regulator (Delta Star, Aqua Logic, Inc., San Diego, CA) capable of regulating water temperature at 10-15°C. Cells of L. plantarum ATCC 8014 were added to the ASW to reach a level of 107 CFU/ml. Depuration was conducted at 15±1 and 10±1ºC for the first 24 h without turning on the UV light followed by additional four days of process with UV light to sterilize circulating ASW. Oysters inoculated with V. parahaemolyticus and depurated in ASW without addition of L. plantarum to ASW was conducted with the UV sterilizer turned on as a control. Survival of oysters during depuration was observed daily by knocking each oyster on its shell. Oysters which opened shells upon knocking were considered dead. The mortality of oysters was expressed as total number of dead oysters divided by total number of oysters used in the study. 2.3.8 Microbiological tests 2.3.8.1 Sample preparation Five oysters were individually analyzed. Each oyster was shucked with a sterile shucking knife in a sterile stainless steel tray and blended with equal volume of sterile PBS at low speed for 1 min using a two-speed laboratory blender (Waring Laboratory, Torrington, CT) to prepare a 1:2 dilution sample suspension. Twenty-five grams of the sample suspension were then mixed with 100 ml sterile PBS to make a 1:10 dilution. Additional 10-fold dilutions of each sample suspension were prepared by adding 1 ml of each sample suspension to 9 ml of sterile PBS. ASW sample was collected every 24 h during oyster depuration at 20±1ºC and analyzed for lactic acid bacteria. 42 2.3.8.2 Detection of lactic acid bacteria Lactic acid bacteria populations in oysters and ASW were determined by the pour plate method using MRS agar with incubation at 37°C for 72 h. Results were reported as the means of five oysters plus standard deviation. 2.3.8.3 Detection of V. parahaemolyticus Populations of V. parahaemolyticus in oysters were determined by the three-tube most probable number (MPN) method according to the U.S. Food and Drug Administration’s Bacteriological Analytical Manual (FDA 2004). Briefly, one milliliter of each of the sample suspensions was individually enriched with 9 ml sterile alkaline peptone water (APW) and incubated 16-18 h at 37ºC. One loop (1 µl) of enriched APW from each turbid tube was streaked onto individual thiosulfate-citrate-bile salts-sucrose (TCBS) agar plates and incubated at 37ºC for 16-18 h. Formation of round and green colonies on TCBS agar plates was considered positive for V. parahaemolyticus. Total populations of V. parahaemolyticus in oysters were determined as MPN per gram by converting the numbers of APW tubes that were positive for V. parahaemolyticus to MPN per gram using the MPN table. Results were reported as the means from five oysters plus standard deviation. 2.3.9 Statistical analysis Results of microbiological tests were transferred to log values for statistical analysis. Bacterial populations in oysters at different treatment times were analyzed by tTest: Paired Two Samples for Means (Excel, Microsoft, Redmond, WA). Significant differences between means of treatments were established at p<0.05. 43 2.4 Results 2.4.1 Inhibitory effects of cell-free supernatant on growth of Vibrio parahaemolyticus The pH and titratable acidity (TA) of the cell-free supernatant (CFS) of three lactic acid bacteria after 12 and 24 h enrichment are reported in Table 2.1. CFS of all three strains had lower pH and higher TA after 24 h enrichment than those in CFS collected after 12 h enrichment. The CFS obtained from 24 h of growth of Lactobacillus plantarum ATCC 8014 had the lowest pH (4.19) and highest TA (2.16%). In contrast to CFS, none of the cell suspensions prepared from growth of three lactic acid bacteria after 24 h of enrichment inhibited growth of V. parahaemolyticus (Data not shown). When the CFS from 24 h growth of L. plantarum was adjusted to pH 6.5, it lost antibacterial activity against growth of V. parahaemolyticus. The inhibitory effect of the CFS slightly reduced from 66.67% to 55.56% when it was treated with pepsin or catalase (Table 2.2). Lactic acid at 0.5, 1.0 and 2.0% (w/v) all exhibited inhibitory effects against growth of V. parahaemolyticus. 2.0% lactic acid exhibited greater inhibitory effect (85.19%) than the 24 h enriched CFS (66.67%) (Table 2.2). No inhibitory effect was observed for MRS broth either before or after enzyme (catalase or pepsin) treatments on growth of V. parahaemolyticus. 2.4.2 L. plantarum attachment to oysters Changes of lactic acid bacteria populations in oysters when held in re-circulating ASW at 20±1ºC are reported in Table 2.3. Fresh oysters contained a low level of lactic acid bacteria (1.83 log CFU/g). Exposure of oysters to L. plantarum ATCC 8014 (6.41 log CFU/ml) in artificial seawater (ASW) for 20 h allowed accumulation of the bacterium 44 in oysters and increased the total lactic acid bacteria in oysters to 4.66 log CFU/g. Populations of total lactic acid bacteria in oysters decreased gradually when the oysters were held in ASW at 20ºC but remained moderately colonized in oysters after 4 days (3.10 log CFU/g). Low levels of lactic acid bacteria were detected in ASW after one day of process and the levels increased to >3.40 log CFU/ml after four days of process. Table 2.1 Titratable acidity and pH of cell-free supernatant from growth of three lactic acid bacteria at 37ºC. Bacteria L. acidophilus ATCC 314 L. lactis subsp. lactis ATCC 11454 L. plantarum ATCC 8014 a Titratable acidityb Growth (%) Time (h) 5.29±0.01a 0.689±0.010 12 5.00±0.03 0.978±0.021 24 5.08±0.03 0.672±0.019 12 4.79±0.02 0.838±0.016 24 4.78±0.01 1.137±0.013 12 4.19±0.02 2.164±0.047 24 pH Data are means of three determinations ± standard deviation. Expressed as equivalent weight of lactic acid. b 45 Table 2.2 Inhibitory effects of cell-free supernatant from growth of Lactobacillus plantarum ATCC 8014 and lactic acid on growth of a mixture of five Vibrio parahaemolyticus strains (105 log CFU/ml) in tryptic soy agar plus 1.5% NaCl at 37ºC for 20 h. Sample Treatments Inhibitory effect (%) L. plantarum ATCC 8014 Original (pH 4.19) 66.67±0.000a Neutralization (pH 6.50) Nb Catalase (0.1 ml/ml) 55.56±0.000 Pepsin (2 mg/ml) 55.56±0.000 0.5 % (w/v) 33.33±0.000 1.0 % (w/v) 55.56±0.000 2.0 % (w/v) 85.19±6.415 Original (pH 6.50) N Catalase (0.1 ml/ml) N Pepsin (2 mg/ml) N Lactic acid MRS broth a Data are means of three determinations ± standard deviation. No inhibitory effect. b 46 Table 2.3 Changes of lactic acid bacteria populations in oysters and artificial seawater (ASW) during depuration at 20±1ºC. Time (d) Oysters (log CFU/g) ASW (log CFU/ml) 0a 4.66±0.18 Ab NDc 1 4.00±0.60 B 1.78 2 3.67±0.24 B 1.58 3 3.46±0.68 B 1.74 4 3.10±0.47 B >3.40 a After 20 h of inoculation of oysters (initial lactic acid bacteria populations: 1.83±0.44 log CFU/g) with Lactobacillus plantarum ATCC 8014 (6.41 log CFU/ml) at 23±1ºC. b Data are means of five determinations ± standard deviation. Same letter in each column indicates the means are not significantly different (p>0.05). c Not detected using MRS agar. 2.4.3 Effects of L. plantarum treatment on reducing V. parahaemolyticus in oysters during depuration The efficacies of L. plantarum treatment on reducing V. parahaemolyticus in oysters during depuration at 15±1 and 10±1ºC are summarized in Table 2.4. When oysters were depurated at 15±1ºC, V. parahaemolyticus levels in oysters decreased to <10 MPN/g (>3.06 log reductions) after 4 days of depuration with L. plantarum. At the end of 5 days of depuration, reductions of V. parahaemolyticus in oysters increased to >3.42 and >3.30 log MPN/g in oysters with and without the L. plantarum treatment, respectively. While there was no significant difference (p>0.05) between the treatments, oysters processed with L. plantarum treatment had a lower mortality of 2.9% than 8.8% observed for the control group (Table 2.5). 47 Table 2.4 Reductions of Vibrio parahaemolyticus BE 98-2029 in laboratory inoculated oysters during depuration at 15±1 and 10±1ºC. V. parahaemolyticus populations (MPN/g) in oysters Time (d) LAB Treatmenta (15±1ºC) a Control (15±1ºC) LAB Treatment* (10±1ºC) Control (10±1ºC) 0 3.91±0.45 Ab 3.91±0.45 A 4.68±0.24 A 4.68±0.24 A 1 3.00±0.37 B (0.91)c 2.76±0.44 B (1.15) 3.44±0.22 B (1.24) 3.61±0.64 B (1.07) 2 1.85±0.33 C (2.06) 2.07±0.32 C (1.84) 2.98±0.38 C (1.70) 2.86±0.39 C (1.82) 3 1.78±0.28 C (2.13) 1.42±0.12 D (2.49) 2.12±0.40 D (2.56) 2.48±0.53 CD (2.20) 4 <0.85±0.35 D (>3.06) <1.17±0.78 DE (>2.74) 2.06±0.46 DE (2.62) 1.82±0.52 D (2.86) 5 <0.49±0.04 E (>3.42) <0.61±0.20 E (>3.30) 1.28±0.53 E (3.40)d 1.93±0.53 D (2.75)d Lactobacillus plantarum ATCC 8014 treatment (7 log CFU/ml). Data are means of five determinations ± standard deviation. Same letter in each column indicates the means are not significantly different (p>0.05). c Reductions of V. parahaemolyticus population. d Significant (p<0.05) difference of V. parahaemolyticus levels was observed in two groups. b 48 When the depuration was conducted at 10±1ºC, a similar trend in V. parahaemolyticus reductions was observed in oysters with or without the L. plantarum treatment. Reductions of V. parahaemolyticus in oysters increased to 3.40 and 2.75 log MPN/g in treatment group and control group, respectively, after five days, which indicated that application of lactic acid bacteria in depuration at low temperatures could enhance reductions of V. parahaemolyticus in oysters (Table 2.4). All the oysters were able to survive during cold water (10ºC) depuration for five days (Table 2.5). Table 2.5 Mortality of oysters during depuration at 15±1 and 10±1ºC. a Time Treatmenta (%) Control (%) Treatment (%) Control (%) (d) 15±1ºC 15±1ºC 10±1ºC 10±1ºC 1 0 (0/35)b 0 (0/34) 0 0 2 2.9 (1/35) 0 (0/34) 0 0 3 2.9 (1/35) 2.9 (1/34) 0 0 4 2.9 (1/35) 8.8 (3/34) 0 0 5 2.9 (1/35) 8.8 (3/34) 0 0 Lactobacillus plantarum ATCC 8014 treatment (7 log CFU/ml). Total number of dead oysters divided by the total number of oysters. b 49 2.5 Discussion Studies of the inhibitory effects of cell-free supernatant (CFS) from Lactobacillus plantarum ATCC 8014 on growth of Vibrio parahaemolyticus indicated that low pH and high titratable acidity (TA) played the major roles in inhibiting growth of V. parahaemolyticus in tryptic soy agar plus 1.5% NaCl (TSA-salt). The CFS of L. plantarum ATCC 8014 after overnight enrichment had been reported to exhibit a wide range of antibacterial spectrum within a narrow pH range (pH 4-5) (Lash and others 2005). Such a phenomenon was also observed in this study as the CFS lost its inhibitory effect on growth of V. parahaemolyticus when the pH of CFS was adjusted from 4.19 to 6.50 (Table 2.2). The previous study also reported a protein of 122 KDa in the CFS from L. plantarum ATCC 8014 to be the major compound exhibiting antibacterial activity. However, such a compound was not observed in this study because the CFS from L. plantarum ATCC 8014 still inhibited growth of V. parahaemolyticus after being treated with pepsin (Table 2.2). This observation is consistent with previous report that L. plantarum ATCC 8014 did not produce bacteriocin (Skinner and others 1999). The possible reasons for contradictory results conducted in the studies, even using the same strain, could be the different types of inhibition tests and different CFS preparation methods. In addition, a number of low molecular mass compounds, including benzoic acid, methylhydantoin and mevalonolactone, could be produced from growth of L. plantarum and be present in CFS. All of those compounds at a level of 10 ppm have been showed to inhibit Gram-negative bacteria when they were applied with 1% lactic acid. However, the inhibitory effect of each compound was much weaker than combined use 50 (Niku-Paavola and others 1999), which indicates the strong synergic effect of those compounds. Antibacterial compounds of large molecular mass cannot penetrate into the cell due to the function of lipopolysaccharide as the permeability barrier on the outer membranes of Gram-negative bacteria. Lactic acid can act as a permeabilizer and disrupt the outer membrane of the Gram-negative bacteria so that lactic acid itself or other antibacterial compounds can then enter the cell to exert antibacterial effects (Alakomi and others 2000). In this study, acids produced by L. plantarum ATCC 8014 after 24 h enrichment appears to be the major compounds for inhibiting growth of V. parahaemolyticus in TSA-salt. However, other compound(s) with antibacterial properties might exist in the CFS. Further analysis is required to determine the structures and percentages of additional antibacterial compounds in CFS. Raw oysters may contain lactic acid bacteria and exposure of oysters to artificial seawater (ASW) containing L. plantarum ATCC 8014 allowed the organism to colonize in oysters. These results demonstrated that L. plantarum ATCC 8014 could attach to oyster tissues and remain colonized in oysters during depuration process. Therefore, the organism might compete with V. parahaemolyticus for attachment to oyster tissues and prevent the colonization of V. parahaemolyticus in oysters. However, the mechanism of colonization of L. plantarum ATCC 8014 in oysters remains to be investigated. The sudden increase of lactic acid bacteria populations in ASW after four days of holding oysters was probably because several oysters died after three days in the process (Data not shown) and provided nutrients for the multiplication of the bacteria in ASW, so that 51 depuration of oysters at 20ºC for more than 3 days should not be considered for reducing V. parahaemolyticus in oysters upon harvest. Several studies have reported that depuration at ambient temperatures are ineffective in reducing V. parahaemolyticus contamination in oysters. Ren and Su (2006) reported that holding laboratory-contaminated Pacific oysters in ASW for 24 h did not yield apparent reductions of V. parahaemolyticus or V. vulnificus in oysters. A study of depuration of laboratory-contaminated American oysters (Crassostrea virginica) in ASW at 22ºC for 48 h observed in a limited reduction (1.2 log MPN/g) of V. parahaemolyticus in oysters. However, the reduction of V. parahaemolyticus in oysters was slightly increased to 2.1 log MPN/g when the depuration was conducted at 15ºC for 48 h (Chae and others 2009). In this study, we investigated the potential application of lactic acid bacteria in depuration at 10 and 15ºC for enhancing efficacy in reducing V. parahaemolyticus contamination in oysters. When cells of L. plantarum ATCC 8014 were added to ASW for depuration of oysters inoculated with V. parahaemolyticus BE 98-2029 at 15±1ºC, slightly greater reductions of V. parahaemolyticus were observed in the process than those observed without L. plantarum treatments. Although no significant differences between reductions of V. parahaemolyticus in oysters of the treatment group and the control group were noted, addition of L. plantarum ATCC 8014 to ASW for depuration helped reduce V. parahaemolyticus cells in oysters down to <10 MPN/g in 4 days instead of five days by the process without the L. plantarum treatment (Table 2.4). In addition, application of L. plantarum in oyster depuration reduced the mortality rate of oysters depurated at 15±1ºC from 8.8% to 2.9% (Table 2.5). 52 Reducing the depuration temperature to 10±1ºC did not enhance the efficacy of depuration in reducing V. parahaemolyticus in oysters when compared with reductions observed at 15ºC (Table 2.4). However, the reduction of V. parahaemolyticus in oysters treated with L. plantarum ATCC 8014 after 5 days of depuration at 10±1ºC (3.40 log MPN/g) were significantly greater than that (2.75 log MPN/g) observed in controls (Table 2.4). No oysters were found dead after five days of depuration at 10±1ºC (Table 2.5). In conclusion, cell-free supernatant of L. plantarum ATCC 8014 for 24 h enrichment exhibited inhibitory effects against growth of V. parahaemolyticus in TSAsalt. Application of L. plantarum ATCC 8014 to depuration resulted in greater than 3.0 log MPN/g reductions of V. parahaemolyticus in raw Pacific oysters after five days of process at 10±1ºC with no mortality. The efficacy of applying lactic acid bacteria in depuration on reducing V. parahaemolyticus in oysters might be dependent on the species used and the ability of a species to compete attachment to oyster tissues with cells of V. parahaemolyticus. Further studies are needed to explore the utilization of other lactic acid bacteria in oyster depuration for decontaminating V. parahaemolyticus. 53 Chapter 3 Effects of Green Tea Extract on Reducing Vibrio parahaemolyticus and Increasing Shelf Life of Oyster Meats 54 3.1 Abstract This study investigated effects of tea extract on growth of pathogenic Vibrio parahaemolyticus and potential utilization in post-harvest treatment to extend shelf life of Pacific oysters (Crassostrea gigas). Longjing Tea, which exhibited strong bactericidal activity against V. parahaemolyticus, was selected to use in this study. Tea extract containing equal or higher than 4.594 g/l total phenolic contents (TPC) as gallic acid equivalents (GAE) determined by Folin-Ciocalteau method could reduce a mixture of five clinical V. parahaemolyticus strains in tryptic soy broth plus 1.5% NaCl from 4.5 log CFU/ml to non-detectable level (<10 CFU/ml) within 8 h. A treatment of shucked oysters with tea extract containing 9.054 g/l TPC as GAE for 2 h at 23±1ºC with oyster/tea extract ratio of approximate 0.9 g/ml resulted in greater (p<0.05) V. parahaemolyticus reductions (0.75 log MPN/g) compared to controls (0.15 log MPN/g). The following shelf life study indicated that green tea treatment at oyster/tea extract ratio of approximate 0.7 g/ml could enhance reducing V. parahaemolyticus while retarding the growth of total bacteria in oysters during 5±1ºC storage. Therefore, green tea might be used as a natural antimicrobial agent to inactivate V. parahaemolyticus in oysters and extend the shelf life during refrigeration storage. 55 3.2 Introduction Tea is rich in polyphenols, which are generally classified into flavonoids, phenolic acids, lignans and stilbenes. Major tea polyphenols (TP) are flavanols (flavan-3ols), which are a subgroup of flavonoids, and the monomers of flavanols (also called catechins) are the major TP in green tea. Predominant catechins include epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC) and epigallocatechin gallate (EGCG) (Peterson and others 2005; Wang and Ho 2009). Tea extract has been reported capable of inhibiting growth of a number of spoilage bacteria and foodborne pathogens such as Bacillus, Campylobacter, Clostridium, Escherichia coli, Listeria monocytogenes, Pseudomonas, Staphylococcus aureus, Salmonella and Vibrio. EGCG, which generally is the most abundant TP in green tea, has been reported the compound mainly contributing to the antibacterial activity (Diker and others 1991; Chou, and others 1999; Sakanaka and others 2000; Hara 2001; Tagura and others 2004; Si and others 2006; Almajano, and others 2008). Extensive studies on application of green tea extract for reducing pathogenic and spoilage bacteria in foods including beef, pork, chicken and mutton have been reported (Kim and others 2004; Juneja and others 2007; Kumudavally and others 2008). However, no study has been conducted to evaluate effects of tea extract on reducing pathogenic bacteria and retarding growth of spoilage bacteria in molluscan shellfish. Vibrio parahaemolyticus is a human pathogen occurring naturally in the coastal and estuarine environments and commonly found in oysters. Consumption of raw or undercooked oysters contaminated with V. parahaemolyticus can result in food-borne 56 illness with major symptoms of gastroenteritis (Butt and others 2004). Numerous outbreaks of V. parahaemolyticus infections resulted from consumption of raw oysters in the U.S. were documented over the past decade (CDC, 2005, 2006). Since oysters are commonly consumed raw, contamination with V. parahaemolyticus in oysters is a public health concern. In addition, growth of spoilage bacteria during storage is also a concern for the highly perishable raw molluscan shellfish. The microflora of oysters is dependent on the quality of water surroundings and the growth environment. Generally, the predominant spoilage bacteria at the beginning of storage are Pseudomonas and Acinetobacter-Moraxella spp. while enterococci, lactobacilli and yeasts became predominate at a later stage (Jay and others 2005). Oyster meats typically have a shelf life of 10-14 days when stored in sealed jars filled with water at refrigeration temperatures (47ºC). The aim of this study was to investigate the effects of green tea on reducing V. parahaemolyticus and retarding spoilage bacteria in shucked oysters stored at 5±1ºC. 3.3 Materials and methods 3.3.1 Bacterial preparation Five clinical strains of Vibrio parahaemolyticus [10290 (O4:K12, tdh+ and trh+), 10292 (O6:K18, tdh+ and trh+), 10293 (O1:K56, tdh+ and trh+), BE 98-2029 (O3:K6, tdh+) and 027-1c1 (O5:K15, tdh+ and trh+)] obtained from the Food and Drug Administration Pacific Regional Laboratory Northwest (Bothell, WA) were used in this study. Each strain was individually grown in 9 ml tryptic soy broth (Difco, Becton Dickson, Spark, MD) supplemented with 1.5% NaCl (TSB-salt) at 37°C for 18-24 h. The enriched cultures were streaked onto tryptic soy agar (Difco) supplemented with 1.5% NaCl (TSA- 57 salt) and incubated at 37°C for 18 h. A single colony formed on the TSA-salt plate was transferred to 9 ml TSB-salt and incubated at 37°C for 4 h. Cells of each enriched culture were harvested by centrifugation at 3000 × g (Sorvall RC-5B, Kendro Laboratory Products, Newtown, CT) at 5±1°C for 15 min and re-suspended in equal volume of sterile 2% NaCl solution to prepare a culture suspension of approximate 108 CFU/ml. 3.3.2 Effects of tea extract on inhibiting growth of V. parahaemolyticus in pure culture 3.3.2.1 Selection of tea extract with antibacterial activity Tea products purchased from supermarkets in the U.S. and China were tested for their bactericidal activities against growth of V. parahaemolyticus. Tea extract was prepared by steeping dry tea leaves or powder at a tea/water (g/ml) ratio of 10% for 5 min followed by filtering through a 0.2 µm filter (VWR international, USA) using sterile syringes (Becton Dickinson and Company, Franklin Lakes, NJ). One milliliter of V. parahaemolyticus culture suspension of each strain was added to 100 ml warm (45ºC) TSA-salt to reach 106 CFU/ml levels of V. parahaemolyticus in the medium. The TSAsalt containing V. parahaemolyticus was poured into petri dishes (25 ml) and allowed to solidify at room temperature. Wells (9 mm in diameter) were created on the TSA-salt plates. An aliquot (200 µl) of each tea extract solution was added to the wells in triplicate and plates were incubated at 37°C for 20 h. Inhibition zones of each tea extract solution were observed and measured. 3.3.2.2 Antibacterial activity of Longjing Tea extract against V. parahaemolyticus Extract of Longjing Tea, which exhibited the strongest inhibitory effect on growth of V. parahaemolyticus in well diffusion inhibition test, was prepared by steeping tea 58 leaves in boiling TSB-salt at various tea/water (g/ml) ratios (2, 4, 6, 8, and 10%) for 5 min. Each brewed tea was filtrated through a No.2V-folded filter paper (Whatman, Kent, England) to prepare TSB-salt containing crude tea extract. To compare the inhibitory effect of tea extract prepared by boiling with steeping method, extract of Longjing Tea prepared by boiling tea leaves with boiling DI water at tea/water (g/ml) ratios of 8% and 10% for 10 min was diluted with an equal volume of double strength TSB-salt after filtration (No.2V-folded filter paper) to prepare TSB-salt containing 4% and 5% of tea extract. One milliliter of mixed V. parahaemolyticus culture (108 CFU/ml) was added to TSB-salt with or without tea extract to reach a level of 104 CFU/ml. The mixture was incubated at 37ºC and 1 ml of the mixture was diluted with phosphate buffered saline (PBS) to determine the survival of V. parahaemolyticus at 0.5, 1, 2, 4, 6 and 8 h. Total phenolic contents (TPC) of the tea extract which reduced V. parahaemolyticus in TSBsalt to non-detectable level within 8 h were also determined. 3.3.2.3 Effects of brewing conditions on total phenolic contents of tea extract To compare brewing conditions for extracting TPC in tea extract, tea extract was prepared by boiling dry leaves in water at a tea/water (g/ml) ratio of 4%. Extraction of TPC by the boiling method was determined after 5, 10 and 15 min and compared with TPC of tea extract prepared by steeping method at a tea/water (g/ml) ratio of 8%. 3.3.2.4. Determination of total phenolic contents TPC was determined by Folin-Ciocalteau method (Waterhouse, 2001) with minor modifications. Briefly, 160 µl of tea extract sample, gallic acid (TCI America, Portland, 59 OR) calibration standard or deionized (DI) water blank were diluted with 12.64 ml DI water, mixed with 800 µl Folin-Ciocalteau Phenol reagent (MP Biomedicals, LLC, Solon, OH) and incubated for 5 min at room temperature. Each solution was then mixed thoroughly with 2.4 ml of sodium carbonate solution, incubated in dark for 2 h at room temperature, and measured at 765 nm using a UV-Vis spectrophotometer (UV-2401 PC, Shimadzu, Kyoto, Japan). A gallic acid standard curve was established according to absorbance values and the TPC (g/l) as gallic acid equivalents (GAE) of each sample were reported as means of three determinations plus standard deviation. 3.3.3 Effects of tea extract on reducing V. parahaemolyticus and retarding bacteria growth in oysters 3.3.3.1 Oyster preparation and inoculation Raw Pacific oysters (Crassostrea gigas) were delivered from oyster farms in Oregon and Washington to the laboratory in a cooler with ice on the harvest day. Twelve dozens of oysters were washed under tap water and acclimated in a high-density polyethylene (HDPE) tank (18 by 12 by 12 in; Nalgene, Rochester, NY) containing 20 l artificial seawater (ASW) at room temperature (23±1ºC) for 4 h. The ASW (salinity: 30 ppt) was prepared by dissolving Instant Ocean Salts (Aquatic Eco-System Inc, Apopka, FL) in DI water according to the instructions. The oysters were then transferred to an identical tank containing 20 l fresh ASW with five clinical V. parahaemolyticus strains at a level of 104 CFU/ml. Accumulation of V. parahaemolyticus in oysters was allowed at room temperature for 20 h with the ASW being circulated at a rate of 12 l/h. 3.3.3.2 Treatments of oysters with tea extract 60 Due to the complex food surroundings in oysters compared to pure culture, extract of Longjing Tea prepared by boiling tea leaves for 10 min at tea/water ratio of 10% was used in oyster challenge tests to determine its antibacterial activity against V. parahaemolyticus. Oysters contaminated with V. parahaemolyticus were shucked with a sterile shucking knife and immersed in tea extract or DI water at oyster/liquid ratio of approximate 0.9 g/ml for 2 h at room temperature. Populations of V. parahaemolyticus in oysters were determined before and after the treatments. 3.3.3.3 Shelf life study Oyster meats treated with tea extract for 2 h were transferred to sterile containers filled with tea extract (10%) or DI water at oyster/liquid ratio of approximate 0.7 g/ml. Oysters treated with DI water for 2 h were transferred to containers filled with DI water at the same oyster/liquid ratio as controls. All the containers were stored at 5±1ºC for up to 18 days. V. parahaemolyticus levels, total bacterial count and psychrotrophic bacterial count in oysters were analyzed every other day. 3.3.4 Microbiological tests 3.3.4.1 Detection of V. parahaemolyticus V. parahaemolyticus levels in TSB-salt were determined by pour plate method using TSA-salt and incubation at 37ºC for 24 h. Results were reported as means of three determinations plus standard deviations. For oyster challenge tests, each oyster was blended with equal volume of sterile PBS at low speed for 60 s using a two-speed laboratory blender (Waring Laboratory, Torrington, CT) to prepare a 1:2 dilution sample suspension. Twenty grams of sample 61 suspension were then mixed with 80 ml sterile PBS to make 1:10 sample dilution. Additional 10-fold dilutions were prepared by adding 1 ml of sample suspensions to 9 ml sterile PBS. V. parahaemolyticus levels in oysters were determined by three-tube most probable number (MPN) method according to the U.S. Food and Drug Administration’s Bacteriological Analytical Manual (FDA 2004). Briefly, sample suspensions were enriched in alkaline peptone water (APW) at 37ºC overnight. One loop (1 µl) of the enriched APW from each turbid tube was streaked onto thiosulfate-citrate-bile saltssucrose agar (TCBS) plates and incubated at 37ºC overnight, Formation of round greenish colonies on TCBS plates was considered positive for V. parahaemolyticus. Total V. parahaemolyticus levels were reported as MPN/g according to a MPN table. Results were reported as means of four oysters plus standard deviations. 3.3.4.2 Detection of total bacteria and psychrotrophic bacteria Total bacteria in oysters were determined by the pour plate method using tryptic soy agar (TSA) and incubation at 37ºC for 48 h. Psychrotrophic bacteria in oysters were determined by spreading 0.1 ml of sample suspension on a TSA plate followed by incubating at 7ºC for 10 days. Results were reported as means of four oysters plus standard deviations. 3.3.5 Statistical analysis Results from microbiological tests were transferred to log values and statistically analyzed. Bacterial populations in oysters in different treatments were analyzed by t-Test: Paired Samples for Means (Excel, Microsoft, Redmond, WA). Significant differences between the means of two treatments were determined at p<0.05. 62 3.4 Results and discussion 3.4.1 Comparison of antibacterial activities of tea extracts prepared from various products against Vibrio parahaemolyticus Table 3.1 shows the inhibitory effects of tea extracts prepared from various products on five individual V. parahaemolyticus strain in well diffusion inhibition test. Each strain of V. parahaemolyticus exhibited different susceptibility towards each tea extract solution, and extract of Longjing Tea exhibited the highest degree of inhibitory effect against growth of all five strains of V. parahaemolyticus by producing the biggest inhibition zone. It was used in the subsequent tests to confirm the inhibitory effect on growth of V. parahaemolyticus. 3.4.2 Effects of brewing conditions on yields of total phenolic contents in tea extract Table 3.2 shows total phenolic contents (TPC) as gallic acid equivalents (GAE) in extracts of Longjing Tea prepared by both steeping method and boiling method for 5, 10 and 15 min. TPC as GAE in tea extract prepared by boiling method at a tea/water (g/ml) ratio of 4% after brewing for 10 min was higher than those of extracts prepared by steeping method for 10 or 15 min at a ratio of 8%. Brewing tea in boiling water allowed water temperature to be kept at a consistent boiling point while the water temperature gradually decreased from boiling point during tea brewing by the steeping method. It appeared that keeping water temperature at boiling point during tea brewing enhanced the yield of TPC from tea leaves because higher temperature allowed tea leaves more penetration to the solvents (Vuong and others 2010). In addition to temperature, lower tea/water ratio and longer brewing time generally resulted in greater extraction of soluble solids (Astill and others 2001). Although we also observed an increased TPC in tea 63 Table 3.1 Inhibitory effects of tea extract on growth of Vibrio parahaemolyticus (106 CFU/ml) in tryptic soy agar plus 1.5% NaCl incubated at 37ºC for 20 h. Diameters (mm) of inhibition zones Green Teab Longjing Teac Maofengd DI water 12.0 11.0 12.0 12.0 Nf 10.0 11.0 10.0 12.0 10.0 N 10293 12.0 12.0 12.0 13.0 12.0 N BE 98-2029 11.0 11.0 10.0 12.0 10.0 N 027-1c1 12.0 12.0 10.0 12.0 10.0 N V. parahaemolyticus Premium Green Fusion Green & Decaf Teaa White Teaa 10290 11.0e 10292 a The Stash Tea Co., OR. USA. Lipton Co., NJ. USA. c Shifeng Tea Co., Ltd, Hangzhou, China. d Tianhu Tea Co., Fujian, China. e Data are means of three determination, and standard deviation is omitted due to zero value. f No inhibition zones observed. b 64 extract prepared from brewing tea in boiling water for 15 min, the longer brewing time led to a substantial water loss after 15 min as more water was either absorbed into the leaf or evaporated (Data not shown). The increase in TPC in tea extract after 15 min of brewing in boiling water was likely due to loss of water, than to increased extraction of TPC beyond 10 min of brewing. In addition, a longer time of brewing tea at 100°C could also result in higher degree of catechins epimerization in 20 min (Wang and Helliwell 2000). Therefore, brewing tea leaves in boiling deionized (DI) water for 10 min was used to prepare tea extract for oyster challenge studies. Table 3.2 Effects of brewing methods on yields of total phenolic contents in extract of Longjing Tea. Total phenolic contents (g/l) as gallic acid equivalents Steeping Method (8%)a Boiling Method (4%) 5 4.083±0.015 Ab 3.696±0.008 A 10 4.620±0.000 B 4.939±0.042 B 15 4.848±0.037 C 5.145±0.000 C Brewing Time (min) a Tea/water ratio (g/ml). Data are means of three determinations ± standard deviation. Same letter in each column indicates the means are not significantly different (p>0.05). b 65 3.4.3 Antibacterial activity of tea extract against V. parahaemolyticus Table 3.3 shows the antibacterial activity of extracts of Longjing Tea prepared by steeping method and boiling method against growth of a mixture of five clinical V. parahaemolyticus strains in tryptic soy broth plus 1.5% NaCl (TSB-salt) incubated at 37ºC. Only tea extract prepared by steeping method at tea/liquid (g/ml) ratios of 8% and 10% containing TPC as GAE were 4.594 g/l and 5.034 g/l, respectively, could reduce populations of five strains mixture of V. parahaemolyticus from 104 CFU/ml to nondetectable levels (<10 CFU/ml) in TSB-salt within 8 h. On the other hand, tea extract prepared by boiling method at tea/water (g/ml) ratios of 4% and 5%, which contained 4.648 and 5.103 g/l TPC as GAE, could also reduce populations of V. parahaemolyticus to non-detectable levels within 4 and 2 h of incubation at 37ºC, respectively. These results indicate that inhibitory effect against V. parahaemolytcius depended on TPC in Longjing Tea extract. Numerous studies have reported that epigallocatechin gallate (EGCG) was the compound mainly contributing to the antibacterial activity (Chou and others 1999; Tagura and others 2004; Si and others 2006; Almajano and others 2008). In this study, we also observed that EGCG solution (4.0 g/l) prepared by commercial powder (Cayman Chemical, Ann Arbor, MI) isolated from green tea could reduce levels of V. parahaemolytcius from 104 CFU/ml to non-detectable level in TSB-salt within 1 h (Data not shown). Nevertheless, further studies such as testing fractionated tea extracts for antimicrobial effects and structural identification of the phenolic compounds are required to determine other antibacterial compound(s) in tea extract against V. parahaemolyticus. 66 Table 3.3 Antibacterial activity of Longjing tea extract against Vibrio parahaemolyticus (104 CFU/ml) in tryptic soy broth plus 1.5% NaCl incubated at 37ºC. V. parahaemolyticus (log CFU/ml) in tryptic soy broth plus 1.5% NaCl Steeping in boiling TSB-salt for 5 min Incubation Control Time (h) Tea extract (8%)a Tea extract (10%) (4.594 g/l) b (5.034 g/l) Boiling in boiling DI water for 10 min Control Tea extract (4%) Tea extract (5%) (4.648 g/l) (5.103 g/l) 0 4.46±0.04c 4.46±0.04 4.46±0.04 4.50±0.02 4.50±0.02 4.50±0.02 0.5 4.49±0.01 2.77±0.03 2.35±0.11 4.57±0.06 2.27±0.11 1.49±0.20 1 4.54±0.06 2.65±0.04 1.57±0.23 4.63±0.04 1.49±0.20 1.46±0.15 2 5.96±0.03 2.29±0.05 ND 5.61±0.05 1.16±0.28 ND 4 8.30±0.06 1.20±0.17 ND 8.47±0.04 ND ND 6 7.81±0.04 NDd ND 8.31±0.08 ND ND 8 7.64±0.03 ND ND 7.86±0.08 ND ND a Tea/water (g/ml) ratio. Total phenolic compounds (g/l) as gallic acid equivalents. c Data are means of three determinations ± standard deviation. d Not detectable in tryptic soy broth plus 1.5% NaCl with a detection limit of <10 CFU/ml. b 67 3.4.4 Effects of green tea extract on reducing V. parahaemolyticus in oyster meats Soaking oyster meats containing V. parahaemolyticus (4.69 log MPN/g) in DI water for 2 h at room temperature slightly reduced V. parahaemolyticus levels to 4.54 log MPN/g (0.15 log reduction) (Fig. 3.1). However, immersion of oyster meats in 10% tea extract (containing 9.054 g/l TPC as GAE) significantly (p<0.05) reduced V. parahaemolyticus levels to 3.94 log MPN/g (0.75 reduction). Changes of V. parahaemolyticus levels in oyster meats with and without tea extract immersion process followed by storing in containers with DI water or tea extract at 5±1ºC are summarized in Fig. 3.1. V. parahaemolyticus levels in oysters soaked in DI water for 2 h at room temperature followed by storing in DI water decreased slightly during 5ºC storage and were reduced by 0.95 log MPN/g after 8 days of storage, which is probably due to the effects of low salt and low temperature environments. However, greater than one log reductions of V. parahaemolyticus were observed in oysters treated with tea extract for 2 h at room temperature followed by storing in 10% tea extract (containing 9.054 g/l TPC as GAE) or DI water at 5ºC after 8 days of storage. No significant difference (p>0.05) of V. parahaemolyticus levels were observed between oysters treated with tea extract at room temperature and stored in tea extract or DI water except on Day 10, which suggested a lasting bactericidal effect of tea extract treatment on inactivating V. parahaemolyticus in oysters even with the oysters being stored in DI water at 5ºC. Vibrio parahaemolyticus (log MPN/g) 68 5 0.00 0.50 0.39 0.19 T 4 0.81 0.55 3 1.07 1.29 0.39 2 D 0.95 0.43 0.00 DT 0.75 0.82 2.33 2.04 1 2.81 0 0 2 4 6 8 10 2.96 >2.95 >3.44 >3.46 12 14 18 16 Fig. 3.1 Vibrio parahaemolyticus levels in shucked oysters stored at 5ºC. The values represent the means from four oysters plus standard deviation. Numbers shown on each data line indicate V. parahaemolyticus reductions. T: oysters treated with tea extract for 2 h at room temperature and stored in tea extract. DT: oysters treated with tea extract for 2 h at room temperature and stored in deionized (DI) water. D: oysters soaked in DI water for 2 h at room temperature and stored in DI water. Tea extract used in the study contained 9.054 g/l total phenolic compounds as gallic acid equivalents. 69 3.4.5 Effects of green tea extract on retarding growth of bacteria in oyster meats Total bacterial count (TBC) and psychrotrophic bacterial count (PBC) in oyster meats stored with tea extract or DI water during 5ºC following tea extract or DI water treatment for 2 h at room temperature are summarized in Fig. 3.2 and Fig. 3.3, respectively. In general, PBC were higher than TBC in all three treatments, which indicated that most bacteria in oyster meats were psychrotrophic. TBC and PBC in oysters treated with 10% tea extract (containing 9.054 g/l TPC as GAE) for 2 h and stored in tea extract (10%) at 5ºC decreased to around 3.00 and 4.00 log CFU/g, respectively, during the first 4 days of storage and then gradually increased to around 5.00 and 7.00 log CFU/g, respectively, after 18 days of storage. Meanwhile, TBC and PBC in oysters treated with tea extract for 2 h and stored in DI water increased to 6.00 and 7.00 log CFU/g, respectively, after 12 days of storage. Compared with oysters treated with tea extract at room temperature and stored in tea extract or DI water, TBC and PBC in oysters stored in tea extract were significant lower (p<0.05) than those of oysters stored in DI water after 8 and 4 days of storage at 5ºC, respectively. However, TBC and PBC in oysters without tea extract treatment and stored in DI water increased to around 6.00 and 7.00 log CFU/g, respectively, after 8 days of storage. Oysters soaked in DI water for 2 h at room temperature and stored in DI water at 5°C had a shelf life of about 8 days while those treated with tea extract at room temperature for 2 h followed by storing in DI water or in tea extract at 5°C had a longer shelf life of 12 or 18 days. These results indicate storing oysters in tea extract retarded growth of psychrotrophic bacteria and increased shelf life of shucked oysters stored at refrigeration temperature. 70 Total bacterial count ( log CFU/g) 7 6 5 4 3 T DT D 2 0 2 4 6 8 10 12 14 16 18 Day Fig. 3.2 Total bacterial count in shucked oysters stored at 5ºC. The values represent the means from four oysters plus standard deviation. T: oysters treated with tea extract for 2 h at room temperature and stored in tea extract. DT: oysters treated with tea extract for 2 h at room temperature and stored in deionized (DI) water. D: oysters soaked in DI water for 2 h at room temperature and stored in DI water. Tea extract used in the study contained 9.054 g/l total phenolic compounds as gallic acid equivalents. Psychrotrophic bacterial count (log CFU/g) 71 8 7 6 5 T DT D 4 3 0 2 4 6 8 10 12 14 16 18 Day Fig. 3.3 Psychrotrophic bacterial count in shucked oysters stored at 5ºC. The values represent the means from four oysters plus standard deviation. T: oysters treated with tea extract for 2 h at room temperature and stored in tea extract. DT: oysters treated with tea extract for 2 h at room temperature and stored in deionized (DI) water. D: oysters soaked in DI water for 2 h at room temperature and stored in DI water. Tea extract used in the study contained 9.054 g/l total phenolic compounds as gallic acid equivalents 72 3.5 Conclusion Tea extract contains phenolic compounds with antimicrobial activities and might be utilized to retard growth of spoilage bacteria and reduce Vibrio parahaemolyticus in oysters stored at refrigeration temperature to extend shelf life of shucked oysters and reduce risks of V. parahaemolyticus infections associated with oyster consumption. Further studies need to investigate the effects of green tea extract treatments on the sensory attributes of oyster meats and identify individual compounds in the tea extract contributing to the antimicrobial properties. 73 Chapter 4 General Conclusion Vibrio parahaemolyticus is one of the foodborne pathogens that can cause human diseases including gastroenteritis, would infections and septicemia. Most reported V. parahaemolyticus infections were gastroenteritis mainly caused by consumption of raw or undercooked seafood, especially raw oysters. The U.S. Food and Drug Administration recommended a limit of 10,000 cells/g of V. parahaemolyticus in oysters, but oysters containing V. parahaemolyticus below this limit level could still cause foodborne illnesses (FDA 2005). Recently, vibiosis including V. parahaemolyticus infections has become nationally notifiable disease since January 2007, indicating an increased concern for public health (CDC 2010). Several post-harvest treatments including high pressure processing (Ma and Su 2011), irradiation (Mahmoud and Burrage 2009), low temperature pasteurization (Andrews and others 2000) and flash freezing followed by frozen storage (Liu and others 2009) have been reported to be effective in reducing V. parahaemolyticus in oysters. However, most treatments require either high initial investment or equipment operations, and the processes (except irradiation) all kill oysters during treatments. Therefore, it is needed to develop economic post-harvest treatments for effectively reducing V. parahaemolyticus in oysters without adverse effects. Our study showed that cell-free supernatant (CFS) from the growth of Lactobacillus plantarum ATCC 8014 after 24 h at 37ºC exhibited the strongest inhibitory effects against growth of five V. parahaemolyticus strains in tryptic soy agar 74 supplemented with 1.5% NaCl among three strains of lactic acid bacteria tested (Lactobacillus plantarum ATCC 8014, Lactobacillus acidophilus ATCC 314 and Lactococcus lactis subsp. lactis ATCC 11454). Organic acids in CFS of L. plantarum ATCC 8014 were the major compound(s) contributing to the antibacterial activity. Exposure of oysters to L. plantarum (6.41 log CFU/ml) in artificial seawater (ASW) for 20 h allowed accumulation of the bacterium in oysters and increased total lactic acid bacteria in oysters from initial 1.83 log CFU/g to 4.66 log CFU/g. Populations of total lactic acid bacteria in oysters decreased gradually when the oysters were held in ASW at flow rate of 20 l/h at 20ºC but remained moderately colonized in oysters after 4 days (3.10 log CFU/g). Addition of cells of L. plantarum at a level of 107 CFU/ml to ASW resulted in greater (p<0.05) V. parahaemolyticus reductions (3.40 log MPN/g reductions) in V. parahaemolyticus BE 98-2029 (O3:K6) inoculated oysters (4.68 log MPN/g) after five days of depuration at 10±1ºC with UV sterilizer turning on after 24 h of depuration compared to controls (2.75 log MPN/g reductions). In addition, no mortality of oysters was observed during five days of depuration conducted at 10ºC. These results indicated that L. plantarum ATCC 8014 could enhance reducing V. parahaemolyticus levels in raw oysters during depuration at 10±1ºC compared to regular depuration treatment probably due to the ability to compete for attachment sites to oyster tissues with cells of V. parahaemolyticus. However, the efficacy of applying lactic acid bacteria in depuration on reducing V. parahaemolyticus in oysters might be dependent on the species of lactic acid bacteria. Further studies are needed to explore the use of other lactic acid bacteria in oyster depuration for decontaminating V. parahaemolyticus. 75 In addition to the application of probiotics for reducing V. parahaemolyticus in raw oysters during depuration treatment, this study also found that extract of Longjing Tea containing equal or higher than 4.594 g/l total phenolic contents (TPC) as gallic acid equivalents (GAE) determined by Folin-Ciocalteau method also reduced a mixture of five clinical V. parahaemolyticus strains in tryptic soy broth plus 1.5% NaCl from 4.5 log CFU/ml to non-detectable level (<10 CFU/ml) within 8 h. The treatment of shucked oysters with the tea extract containing 9.054 g/l TPC as GAE for 2 h at room temperature (23±1ºC) with an oyster/tea extract ratio of approximate 0.9 g/ml resulted in greater (p<0.05) V. parahaemolyticus reductions (0.75 log MPN/g) compared to oysters treated with deionized (DI) water at the same ratio (0.15 log MPN/g). Greater than one log reduction of V. parahaemolyticus was observed in oysters treated with tea extract for 2 h at room temperature followed by storing in 10% tea extract (containing 9.054 g/l TPC as GAE) at an oyster/tea extract ratio of approximate 0.7 g/ml (2.04 log MPN/g reductions) or DI water (1.07 log MPN/g reductions) at 5ºC after 8 days of storage. However, only 0.95 log MPN/g reductions of V. parahaemolyticus levels in oysters soaked in DI water for 2 h at room temperature followed by storing in DI water were observed after 8 days of storage. Total bacterial count (TBC) and psychrotrophic bacterial count (PBC) in oysters treated with 10% tea extract for 2 h and stored in tea extract at 5ºC decreased to around 3.00 and 4.00 log CFU/g, respectively, during the first 4 days of storage and then gradually increased to around 5.00 and 7.00 log CFU/g, respectively, after 18 days of storage. Meanwhile, TBC and PBC in oysters treated with tea extract for 2 h and stored in DI water increased to 6.00 and 7.00 log CFU/g, respectively, after 12 days of storage. 76 However, TBC and PBC in oysters without tea extract treatment increased to around 6.00 and 7.00 log CFU/g, respectively, after 8 days of storage. Oysters soaked in DI water for 2 h at room temperature and stored in DI water at 5°C had a shelf life of about 8 days while those treated with tea extract at room temperature for 2 h followed by storing in DI water or in tea extract at 5°C had a longer shelf life of 12 or 18 days. 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