Characterization of Pathogenic and Non-Pathogenic Vibrio

AN ABSTRACT OF THE THESIS OF

Jingyi Du for the degree of Master of Science in Food Science and Technology presented on August 31, 2017.

Title: Characterization of Pathogenic and Non-pathogenic Vibrio parahaemolyticus Strains and the Antimicrobial Activity of Fruit Juice and Fruit Extracts against Seafood Pathogens

Abstract approved: ______Christina Ann Mireles DeWitt

Vibrio parahaemolyticus is frequently isolated or detected from raw seafoods, especially shellfish. Also, Listeria monocytogenes, Salmonella spp. are pathogens that are frequently found in ready-to-eat (RTE) seafood, such as smoked fish and shellfish, seafood salad, cooked shrimp and crabmeat, and seafood consumed raw. Two studies to improve safety in RTE seafoods were conducted.

FDA’s regulatory limit, 10,000 cells/g, for V. parahaemolyticus in RTE fish products encompasses both pathogenic and non-pathogenic V. parahaemolyticus.

However, limited studies exist on the factors that influence growth and die-off rate of pathogenic and non-pathogenic V. parahaemolyticus strains. This study investigated the influence of temperature (5-30 °C) on the growth (positive µmax) and die-off rates

(negative µmax) of pathogenic and non-pathogenic V. parahaemolyticus strains. There was a significant effect of strain type (non-pathogenic vs pathogenic, P < 0.001), temperature (P < 0.001) and a strain type x temperature interaction (P = 0.026). At

5 °C, µmax was negative for both strain types indicating die-off. At 10 °C, µmax was negative for two pathogenic and one non-pathogenic strain. From 15-30 °C, µmax was positive for all strains. When evaluating differences between strain type at each temperature, significant difference (P < 0.001) between strain type only occurred at

30 °C. When evaluating the effect of temperature on µmax within either non-pathogenic or pathogenic strain type, there was no significant difference between 5-15 °C. The mean µmax for non-pathogenic and pathogenic strains was 0.448 and 0.340, respectively. The faster rate of growth of non-pathogenic strains suggests an increased likelihood of positives from environmental sampling, especially as environmental temperature increases. This study revealed differences between growth and die-off rates of pathogenic and non-pathogenic V. parahaemolyticus strains at various temperatures. This information is useful to the risk assessment of raw and RTE seafood consumption. Future studies need to be conducted to investigate the specific mechanism responsible for different responses to temperature change.

Foodborne pathogens Listeria monocytogenes, Salmonella spp. and Vibrio parahaemolyticus are especially problematic in RTE foods. Consumer demand for healthy, minimally processed RTE food suggests natural methods are needed to control these pathogens. The aim of this study was to investigate antimicrobial activity of seven fruit extracts against five L. monocytogenes, four Salmonella strains and five

V. parahaemolyticus strains from human clinical samples, seafood, raw milk, produce, and meat sources. Pomegranate peel (PPE) exhibited the highest antimicrobial activity and it was found to be similar in efficacy when compared to cranberry juice (CJ), a well-known source of antimicrobial activity. Limited inhibition was observed for blueberry, strawberry, plum meat, whole plum and pomegranate seed. The minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) of

PPE for L. monocytogenes was 22.5% and 37.5%, respectively. MBC of PPE against

Salmonella was 37.5%. MBC of PPE against V. parahaemolyticus strains was 8.1%.

The MIC of PPE against L. monocytogenes was 22.5% while the MIC of PPE against

Salmonella was >22.5% and < 37.5%. The MIC of PPE against V. parahaemolyticus strains was >4.86% and <8.1%, which was much lower that against L. monocytogenes and Salmonella. The MBC of CJ against V. parahaemolyticus strains was 12.5%. The

MBC of CJ against L. monocytogenes and Salmonella was 25%, whereas the MIC against L. monocytogenes was 12.5%, but this concentration did not fully inhibit the growth of Salmonella strains. The MIC of CJ against Salmonella was >12.5% and <

25% and that against V. parahaemolyticus was>6.25% and < 12.5%. Concentration (P

˂ 0.0001), pathogen type (P ˂ 0.0001) and treatment type (PPE vs CJ; P ˂ 0.0001) have an effect on log reductions. In addition, there was a significant three-way interaction between all main effects (P ˂ 0.0001). This study demonstrated the potential of PPE, a natural food by-product, to be used as an antimicrobial in RTE seafood. More research is needed to optimize concentrations that exhibit effective

antimicrobial activity, but have minimal impact on sensory qualities and shelf-life of seafood products.

Characterization of Pathogenic and Non-pathogenic Vibrio parahaemolyticus Strains and the Antimicrobial Activity of Fruit Juice and Fruit Extracts against Seafood Pathogens

by Jingyi Du

A THESIS

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of

Master of Science

Presented August 31, 2017 Commencement June 2018

Master of Science thesis of Jingyi Du presented on August 31, 2017

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.

Jingyi Du, Author

ACKNOWLEDGEMENTS

I would like to express my deeply appreciation to Dr. Christina Ann DeWitt, my major professor and Dr. Jovana Kovacevic, my co-major professor, for their support help and guidance to my graduation. Their encouragement and knowledge especially helped me through the hard times.

I would also like to dedicate the dissertation to my previous major professor, Dr.

Yi-Cheng Su, with all the memory we had and his support for my time as a graduate student. Miss you, Dr. Su.

I would like to thank my committee members: Dr. Jae Park and Dr. Claudia Häse for their advice and participation in my M.S. program.

Thanks to people from OSU and Food Science Department for their knowledge.

Many thanks to the Astoria Seafood Laboratory: Dr. Kwon, Dr. Guo, Sue, Craig, and my friends: Clara, Kaitline, Ziwen, Robin, Ning, Nut and many others. Thank them so much for your help and all the time that we spent together.

Finally, I am truly thankful to my parents. Without their support and understanding, I could not achieve what I have and become the person that I want to be. I cherish every moment in my Master life.

TABLE OF CONTENTS

Page Chapter 1 General Introduction ...... 1

1.1 Overview ...... 1

1.1.1 Seafood consumption ...... 1 1.1.2 Seafood hazard ...... 1

1.2 Prevalence of L. monocytogenes, Salmonella and V. parahaemolyticus in ready-to eat (RTE) seafood ...... 2

1.2.1 Taxonomy and characteristics of L. monocytogenes, Salmonella and V. parahaemolyticus ...... 2 1.2.2 Detection of L. monocytogenes, Salmonella and V. parahaemolyticus ...... 8 1.2.3 Incidence of L. monocytogenes, Salmonella and V. parahaemolyticus in ready-to-eat (RTE) seafood ...... 14 1.2.4 Outbreaks of L. monocytogenes, Salmonella and V. parahaemolyticus infection in ready-to-eat (RTE) seafood ...... 16

1.3 Regulation of L. monocytogenes, Salmonella and V. parahaemolyticus in ready-to- eat (RTE) seafood ...... 18

1.4 Control and prevention of L. monocytogenes, Salmonella and V. parahaemolyticus infection by using plant extracts ...... 20

1.4.1 Berry ...... 20 1.4.2 Pomegranate ...... 26 1.4.3 Plum ...... 29

1.5 Objective ...... 30

Chapter 2 Comparison of Growth and Die-off of Pathogenic and Non-pathogenic Vibrio parahaemolyticus at Various Temperatures ...... 37

2.1 Abstract ...... 38

2.2 Introduction ...... 39

2.3 Material and methods ...... 41

2.3.1 Bacteria culture preparation ...... 41

TABLE OF CONTENTS (Continued)

Page 2.3.2 Effect of temperature on growth of V. parahaemolyticus ...... 41 2.3.3 Statistical analysis ...... 42

2.4 Results ...... 44

2.4.1 Growth study of V. parahaemolyticus in TSB-Salt at different temperature ...... 44 2.4.2 Growth rate and die-off rate of V. parahaemolyticus in TSB-Salt at different temperatures ...... 45

2.5 Discussion ...... 48

2.6 Conclusions ...... 50

2.7 Acknowledgements ...... 51

Chapter 3 Antimicrobial Activity of Fruit Extracts and Fruit Juice against L. monocytogenes, Salmonella and V. parahaemolyticus ...... 62

3.1 Abstract ...... 63

3.2 Introduction ...... 64

3.3 Material and methods ...... 66

3.3.1 Bacteria culture preparation ...... 66 3.3.2 Preparation of fruit extract and fruit juice ...... 66 3.3.3 Determination of total phenolic content (TPC) in fruit pomaces, pomegrante peel (PPE) and cranberry juice (CJ) ...... 67 3.3.4 Determination of antimicorbial activity of fruit pomace against L. monocytogenes and Salmonella ...... 68 3.3.5 Determination of minimal inhibition concentration (MIC) and minimum bactericidal concentration (MBC) of pomegrante peel (PPE) and cranberry juice (CJ) against L. monocytogenes, Salmonella and V. parahaemolyticus ...... 68 3.3.6 Statistical analysis ...... 69

3.4 Results ...... 71

TABLE OF CONTENTS (Continued)

Page 3.4.1 Determination of total phenolic contents (TPC) ...... 71 3.4.2 Determination of antimicrobial activity, minimal inhibition concentration (MIC) and minimum bactericidal concentration (MBC) ...... 71

3.5 Discussion ...... 74

3.6 Conclusions ...... 77

3.7 Acknowledgements ...... 78

Chapter 4 General Conclusions ...... 87

Bibliography ...... 90

Appendices ...... 107

Appendix A. Inhibitory effect of fresh fruit pomaces and cranberry juice against Salmonella and L. monocytogenes after 48 h storage at 37 °C ...... 108 Appendix B. Inhibitory effects of color-reduced and non-color-reduced cranberry juice (CJ) and pomegranate peel extract (PPE) against Salmonella and L. monocytogenes after 48 h storage at 37 °C ...... 109

LIST OF FIGURES

Figure Page

1.1 Foods linked to known ounreaks (2003-2012) ...... 35

1.2 Pathogens implicated in solved outbreaks (2003-2012) ...... 36

LIST OF TABLES

Table Page

1.1 Worldwide seafood consumption (kg/captica/yr) from 1963 to 2013 (data adapted from FAOSTAT Database) ...... 32

1.2 Listeria monocytogenes outbreaks associated with consumption of ready-to-eat (RTE) seafood (data adapted from Jami et al., 2014) ...... 33

1.3 Recent Vibrio parahaemolyticus outbreaks associated with consumption of ready-to- eat (RTE) seafood in United States ...... 34

2.1 Vibrio parahaemolyticus strains ...... 43

2.2 Viable counts of Vibrio parahaemolyticus after storage at 5 °C in TSB-Salt over 96 hours...... 55

2.3 Viable counts of Vibrio parahaemolyticus after storage at 10 °C in TSB-Salt over 96 hours ...... 56

2.4 Viable counts of Vibrio parahaemolyticus after storage at 15 °C in TSB-Salt over 96 hours ...... 57

2.5 Viable counts of Vibrio parahaemolyticus after storage at 20 °C in TSB-Salt over 36 hours ...... 58

2.6 Viable counts of Vibrio parahaemolyticus after storage at 25 °C in TSB-Salt over 12 hours ...... 59

2.7 Viable counts of Vibrio parahaemolyticus after storage at 30 °C in TSB-Salt over 12 hours ...... 60

2 2 2.8 Fitted parameters of growth rates, die-off rates (µmax) and adjusted R (Adj. R ) for V. parahaemolyticus strains after storage at various temperatures ...... 61

3.1 List of foodborne pathogens information used in this study...... 70

3.2 Total phenolic content (TPC) and pH of fruit pomaces, pomegranate peel extract (PPE) and cranberry juice (CJ) for antimicrobial activity evaluation ...... 83

LIST OF TABLES (Continued)

Table Page

3.3 Effect of pomegranate peel extract (PPE) on microbial growth of L. monocytogenes, Salmonella and V. parahaemolyticus in tryptic soy broth (TSB) and tryptic soy broth supplemented with 1.5% NaCl (TSB-Salt) at 37 °C for 48 h ...... 84

3.4 Effect of lower concentration of pomegranate peel extract (PPE) and cranberry juice (CJ) on microbial growth of V. parahaemolyticus in tryptic soy broth supplemented with 1.5% NaCl (TSB-Salt) at 37 °C for 48 h ...... 85

3.5 Effect of cranberry juice (CJ) on microbial growth of L. monocytogenes and Salmonella in tryptic soy broth (TSB) and V. parahaemolyticus in tryptic soy broth (TSB) and tryptic soy broth supplemented with 1.5% NaCl (TSB-Salt) at 37 °C for 48 h ...... 86

Chapter 1 General Introduction

1.1 Overview

1.1.1 Seafood consumption

Seafood is defined as products from marine and freshwater fish and shellfish

(Hellberg et al., 2012). Different categories of food includes: mollusks (e.g., oysters, clams, and mussels, finfish such as salmon and tuna, and marine mammals (e.g., seal and whale, fish eggs, and crustaceans); (Iwamoto et al., 2010). The seafood consumption around the world shown in Table 1.1. According to the FAOSTAT datatbase (2013), the amount of seafood consumption in the United States in 2013 was 21.51 kg/capita/yr, compared to 13.04 kg/capita/yr in 1963. Because seafood is a low-fat source of protein and contains long-chain omega-3 fatty acids, it has become a popular choice in peoples’ diet (Hellberg et al., 2012). National Research Council (NRC) recommends fish as a food source to substitute high fat diets to minimize the risk of coronary heart disease (CHD;

Hellberg et al., 2012). Fish is also a great source of vitamins (B-complex vitamins, vitamin D, and vitamin A) and minerals (selenium, zinc, iodine, and iron) (Seafood

Nutrition Overview). Fishery products contribut billions of dollars, and millions of jobs to the national economy (White, 2016).

1.1.2 Seafood hazard

Even though seafood has a lot of benefits for us to eat as a nutritional food, it also naturally contains contaminants, such as chemicals, metals, marine toxins, and infectious agents (Ghanbari et al., 2013). Illnesses that are recorded by the Center for Science in the

Public Interest (CSPI)’s database from 2003 to 2012 are reported in Figure 1.1. The percentage of bacterial pathogens, viruses, chemicals and toxins, and parasites is shown

in Figure 1.2. The most frequent bacterial pathogens include Salmonella spp. (19%),

Clostridium spp. (11%), Bacillus cereus and Escherichia coli spp. (both 6%), and

Staphylococcus spp. (5%). Heavy metals are one part of the potential hazard in seafood.

The most common two heavy metals are mercury and persistent organic pollutants

(POPs) (Hellberg et al., 2012). Mercury could be released from natural and anthropogenic sources and converted into MeHg by aquatic microorganisms. Mercury concentrations could be affected by seafood species, age, and habitat of fish (Rasmussen et al., 2005). Contamination of seafood could be caused by naturally present contaminants in harvest water, human reservoir, sewage-contaminated water, handling, processing, or preparation, storage and transportation at inappropriate temperatures

(Ghanbari et al., 2013).

1.2 Prevalence of L. monocytogenes, Salmonella and V. parahaemolyticus in teady-to eat seafood

1.2.1 Taxonomy and characteristics of L. monocytogenes, Salmonella and V. parahaemolyticus

1.2.1.1 Listeria monocytogenes

Listeria monocytogenes was first found in 1926 by Murray et al. as Bacterium monocytogenes. This pathogen was isolated from laboratory rabbits and guinea pigs. The first isolation of this bacterium from human was made in 1929 from humans (Farber and

Peterkin, 1991). Even though this bacterium has been studied for many years, it wasn’t recognized as a source of food-borne human disease until the 1980s.

L. monocytogenes is a Gram-positive, nonspore-forming, facultative anaerobic bacterium (Farber and Peterkin, 1991; Gray and Killinger, 1966). L. monocytogenes has the ability to survive in a number of barriers including anaerobiososis, pH shifts and high

osmolality (Lungu et al., 2009). Because of different virulence factors in L. monocytogenes, this organism has the ability to enter the host cell, escape from phagosomes and marcrophages, multiply in cytoplasm, and spread from one host cells to another host cell (Low and Donachie, 1997). The temperature growth range for L. monocytogenes ranges between -1.5 and 45 °C. The optimum growth temperature for this bacterium is between 30 to 37°C (FDA 2011a; Lado and Yousef, 2007). The motility of this organism is temperature dependent. Tumbling mobility is observed under room temperature. However, limited motility is typically seen at 37 °C due to the reduction of flagellin expression (Peel et al., 1988). The pH range of L. monocytogenes growth is from 4.0-9.6 (Low and Donachie, 1997; FDA, 2011a; Lado and Yousef, 2007). Most bacteria grow at the water activity (aw) of 0.97. This water activity is also the optimum water activity for L. monocytogenes. However, L. monocytogenes has been reported to grow at the water activities as low as 0.90 (FDA, 2011a; Lado and Yousef, 2007). L. monocytogenes is also salt-tolerant and reported to grow in 13-14% sodium chloride

(Farber et al., 1992).

Christie et al. (1944) and Darling et a. (1975) observed that group B streptococci strains produced a β-toxin when grown on sheep or ox blood agar plates (BAP). This blood cell lytic phenomenon is referred to as the Christie–Atkins–Munch-Petersen

(CAMP) phenomenon (Darling, 1975). The CAMP phenomenon was found in L. monocytogenes (Brzin and Seeliger, 1975; Groves and Welshime, 1977). According to the somatic ‘O’ and flagellar ‘H’ antigens, L. monocytogenes could be grouped into 13 serotypes (1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, and 7). Serotypes 1/2a,

1/2b, and 4b are typically associated with food borne infections (Low and Donachie,

1997; FDA, 2012).

There are two forms of disease that caused by L. monocytogenes. The non-invasive illness presents itself as gastroenteritis, with relatively mild symptoms, such as nausea, vomiting, aches, fever, and sometimes diarrhea. For the gastroenteritis caused by L. monocytogenes, the incubation period is relatively short. It ranges from a few hours to three days (McLauchlin, 1996; FDA, 2012). The other form of disease could lead to very serious complications because of possible infection of bloodstream and nervous system.

The infection of L. monocytogenes is also host-dependent. For healthy and immune system intact people, it could cause gastroenteritis, but generally does not cause more serious disease. In contrast, for vulnerable populations, including the elderly and immuno-compromised people, sepsis and central nervous system infections could occur, and even lead to death (Doganay, 2003; FDA, 2012). For pregnant women with invasive listeriosis, the symptoms might be with fever and gastrointestinal symptoms. However, spontaneous abortion or stillbirth or neonatal infection could result in their offspring

(Painter, 2007; FDA, 2012).

1.2.1.2 Salmonella

Salmonella was first seen by Theobald Smith in pigs with hot cholera in 1855 (Eng et al., 2015). Twenty nine years later, this microorganism was named after Daniel E.

Salmon in 1884 (Kim et al., 2004; Eng et al., 2015).

Salmonella are Gram-negative, non-spore forming rods. Most of Salmonella are oxidase-negative and lactose-negative. That means most of them are not able to ferment lactose (Jay, 2012; Acheson and Hohmann, 2001).

This organism is not fastidious, so it grows and multiplies under a wide range of environmental conditions (Jay, 2012; Pui et al., 2011).These bacteria could form visible colonies on large number of different culture media within 24 hours under 37 °C (Jay,

2012). The growth temperature range for Salmonella is from 5 to 47 °C (Pui et al., 2011;

Gray and Fedorka-Cray, 2002), above 70 °C the pathogen is killed (Pui et al., 2011).

Also, Pui et al. (2011) reported pH and salt content could affect the growth of

Salmonella. The optimum pH for Salmonella to grow is around neutrality which is from

6.5 to 7.5. The minimum and maximum growth pH for Salmonella are 4 and 9, respectively. Salmonella could not grow when salt concentrations were 9%. Growth inhibition was reported at water activity of 0.94.

Based on the host preferences, Salmonella is divided into three groups: human infectious, host-adapted, and unadapted. Those serotypes that only infect humans belong to the first group. They are S. Typhi, S. Paratyphi A, and S. Paratyphi C. These bacteria cause paratyphoid fevers. The paratyphoid syndrome caused by S. Typhi is milder than that caused by S. Paratyphi. The second group is the host-adapted serovars. It includes S.

Gallinarum (poultry), S. Dublin (cattle), S. Abortus-equi (horses), S. Abortus-ovis

(sheep), and S. Choleraesuis (swine). The last group is unadapted serovars which means they are not host preference and pathogenic to neither humans nor animals. Three frequent serotypes in this group are S. Enteritidis, S. Typhimurium and S. Heidelberg

(Jay, 2012; Pui et al., 2011). Almost 99% of Salmonella strains are belong to S. enterica.

Only a few of Salmonella strains are S. bongori. The strains belonging to S. enterica can be divided into six subspecies. S. enterica subsp. enterica, S. enterica subsp. salamae, S.

enterica subsp. arizonae, S. enterica subsp. diarizonae, S. enterica subsp. houtenae and

S. enterica subsp. indica (Amagliani et al, 2012; Pui et al., 2011).

Based on the clinical patterns in human salmonellosis, there are two kinds of illness, nontyphoidal and typhoidal illness. S. Typhi and S. Paratyphi are the two serotypes that cause typhoidal fever. Both of them are only found in humans. Infection occurs when contaminated food or water is consumed (FDA, 2012; Andino and Hanning, 2015; Pui et al., 2011). The incubation period varies from one week to three weeks, and sometimes even as long as two months. The symptoms for this type of salmonellosis are high fever

(103 to 104 °F), lethargy, and gastrointestinal symptoms. The mortality is as high as 10% if people do not get treated. Nontyphoidal (NT) salmonellosis is caused by serotypes other than S. Typhi and S. Paratyphi (FDA, 2012). At least 150 Salmonella serotypes are involved in this type of salmonellosis (Jay, 2012). Unlike typhoid fever, NT salmonellosis is not human restricted. The incubation time for this type of salmonellosis varies from six hours to three days. The symptoms includes nausea, vomiting, abdominal cramps, diarrhea, fever, and headache. These symptoms might last four to seven days.

Sometimes, nontyphoidal salmonellosis could cause blood poisoning or infect the blood and internal organs (FDA, 2012). The mortality is usually lower than 1%, but more severe symptoms will be seen in infants, elderly people, young children, and immunocompromised individuals (FDA, 2012; Scallan et al., 2011).

1.2.1.3 Vibrio parahaemolyticus

Fujino et al. (1953) detected V. parahaemolyticus in 1950 in Japan. This outbreak caused 272 illness with 20 deaths (Letchumanan et al., 2014; Odeyemi, 2016). V. parahaemolyticus is a Gram-negative halophilic rod-shaped bacterium with a single polar

flagellum (Su, 2012; Su and Liu, 2007). Since it is a halophilic bacteria, salt is required for growth (Su and Liu, 2007). The optimum salt concentration ranges from 2 % to 4%

(FDA, 2012). However, growth can occur in the presence of 1-8% NaCl (Jay, 2012; Su,

2012). The growth temperature for V. parahaemolyticus is between 5 °C and 44°C. The best growth temperature occurs at 30-35°C with pH 7.6-8.6 (Su, 2012). The growth pH ranges from 4.8 to 11.0 (Jay, 2012; Su, 2012).

V. parahaemolyticus is divide into two groups: non-pathogenic and pathogenic. Non- pathogenic V. parahaemolyticus is isolated from environment and is not pathogenic to humans. Pathogenic V. parahaemolyticus represent the group of V. parahaemolyticus that are pathogenic to human and cause disease. The majority of total V. parahaemolyticus strains in the environment are non-pathogenic (Su, 2012; Letchumanan et al., 2014). The difference between pathogenic V. parahaemolyticus and non-pathogenic V. parahaemolyticus is characterizes by strain’s ability to produce a thermostable direct hemolysin (TDH). TDH is an enzyme that lyses red blood cells on Wagatsuma blood agar. This enzyme reaction is also called the Kanagawa phenomenon (KP) (Su, 2012; Su and Liu, 2007). Despite the virulence factor of TDH, some KP- negative V. parahaemolyticus strains are not able to produce TDH, but have the ability to produce a

TDH-related hemolysin (TRH) (Honda et al., 1988; Letchumanan et al., 2014). TRH were found in patients from an outbreak in the Republic of Maldives in 1985 (Honda et al., 1988). The two genes that encode TDH and TRH are tdh and trh (Nishibuchi et al.,

1986; Nelapati et al., 2012). Gene tl encodes thermolabile hemolysin (TLH) which is carried by all V. parahaemolyticus strains (McCarthy et al., 1999).

The incubation time for symptoms range from 4 hours to 90 hours. Symptoms include diarrhea, abdominal cramps, nausea, vomiting, and fever (FDA, 2012). These symptoms might last two to six days (FDA, 2012; Su, 2012). The gastroenteritis usually is mild and self-limiting. However, it could cause more serious infection for those people who have weakened immune system. Therefore for people who have diabetes, liver disease, kidney disease, cancer and other illness related to a weaken immune system (FDA, 2012), they should more careful when consume seafood.

1.2.2 Detection of L. monocytogenes, Salmonella and V. parahaemolyticus

1.2.2.1 Listeria monocytogenes

L. monocytogenes can grow at low temperatures. Early studies reported that Listeria was isolated from clinical samples by incubating them on an agar at 4 °C until there is colony form there (Gasanov et al., 2005).

Since FDA has a zero tolerance for in L. monocytogenes RTE food regulation, two culture reference methods are used widely in industries. They are FDA bacteriological and analytical method (BAM) (Hitchins et al., 2001) and the International Organization of Standards (ISO) 11290 method (Gasanov et al., 2005; Hitchins et al., 2001). PALCAM

(polymyxin acriflacin lithium-chloride ceftazidime aesculin manitol) and Oxford are the two most frequently recommended selective media by FDA, ISO, and USDA (Zunabovic et al., 2011; Gasanov et al., 2005). PALCAM agar was developed in 1989 to detect and enumerates of L. monocytogenes and other Listeria spp. in food. This agar contains

Columbia Blood Agar with protease peptones, glucose, starch, yeast extract, and sodium chloride (Van Netten et al., 1989; Magalhães et al., 2014). Since Listeria spp. can hydrolyze esculin, they could form gray-green colonies with a black sunken center and a

black halo after incubated for 24–48 h at 37°C. Sometimes, Enterococcus spp. or

Staphylococcus spp. might grow on PALCAM agar, but, the color of colonies they form are gray or red to yellow which is different from Listeria spp. Colonies of these mannitol fermenting organisms are yellow with a yellow halo or gray with a brown-green halo

(Van Netten et al., 1989; Ajay Kumar et al., 2014).

Oxford agar is another media that could be used to isolation of L. monocytogenes since 1989 (Curtis et al., 1989). Oxford contains Columbia Blood Agar with protease peptones, sodium chloride, and starch. L. monocytogenes colonies after 24 h incubation are olive-green with a black halo. However, L. monocytogenes colonies turns darker with a black sunken center and surrounded by black zones after 48 h incubation. Other Listeria spp. colonies have a similar appearance to L. monocytogenes colonies. Colonies of other

Listeria spp. are black with a black halo after 24 h of incubation and they remain the same after 48 h of incubation but with a sunken center (Curtis et al., 1989; Magalhães et al., 2014; Hitchins et al., 2001). Sometimes Staphylococcus spp. may grow on Oxford agar, but their colonies are irregular size and shape, yellow in color (Curtis et al., 1989).

Many modified Oxford Agar (MOX) has been developed to isolation and identification of L. monocytogenes in meat and poultry samples (Magalhães et al., 2014).

CHROMagarTM Listeria is one of the variations of ALOA that have been developed for the isolation and detection of L. monocytogenes. On CHROMagarTM Listeria, colonies of

L. monocytogenes are blue with a white halo and colonies of other Listeria spp. are blue without halo (Law et al., 2015).

Because the simplicity, sensitivity, and accuracy, immunoassay methods were used to test Listeria for many years. Enzyme-Linked immunosorbent assay (ELISA) and

immune-capture are two methods that used very often to detection L. monocytogenes from food. To distinguish L. monocytogenes and other Listeria species, specific antibodies on the basis of virulence factors from L. monocytogenes are used to achieve this goal (Gasanov et al., 2005).

Molecular test are other tests that used to identification L. monocytogenes form food.

This method is the most accurate method to identification L. monocytogenes (Gasanov et al., 2005). However, it requires special instrumentation and personnel skills (Law et al.,

2015; Jadhav et al., 2012). Detection of L. monocytogenes could be achieved by target specific DNA or RNA sequences of pathogen. Many methods are available to detect L. monocytogenes in foods, for instance, PCR, multiplex polymerase chain reaction

(mPCR), real-time/quantitative polymerase chain reaction (qPCR), and nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification

(LAMP), and DNA microarray (Law et al., 2015). Also, DNA hybridization is the method that used very often to detect L. monocytogenes in foods (Gasanov et al., 2005).

1.2.2.2 Salmonella

Conventional Salmonella detection methods consist of two parts. The first part is the nonselective pre-enrichment. The following step is a selective enrichment step, like plating onto selective agars, and biochemical and serological confirmation of suspect colonies (lee et al., 2015; Tietjen and Fung, 1995). Buffered peptone water (BPW) and lactose broth are two commonly media that used for pre-enrichment methods. Besides that, FDA Bacteriological Analytical Manual (BAM) and Food Emergency Response

Network (FERN) has approved Rappaport-Vassiliadis (RV) and tetrathionate (TT) broth as official Salmonella enrichment media (lee et al., 2015). The pre-enrichment of

Salmonella are streak on the solid selective media. Selective media includes Salmonella -

Shigella agar (SS), brilliant green (BGA), bismuth-sulfite agar (BSA), Hektoen enteric

(HE), and xylose-lysine-deoxycholate agar (XLD) (Tietjen and Fung, 1995; BAM, 2016;

Ruiz, 1996). Salmonella species form blue-green to blue colonies with or without black centers on Hektoen enteric (HE) agar. Some cultures of Salmonella may produce colonies may appear as almost completely black colonies. Many cultures of Salmonella produce pink colonies with or without black centers on XLD agar and brown, gray, or black colonies on BS agar (BAM, 2016). The color of colonies of Non-serovar Typhi and

Paratyphi Salmonella on SS agar, while the colonies of S. enterica serovar Typhi are transparent colonies on SS (Maddocks et al., 2002). The color of colonies of Salmonella enterica are red to pink-white colonies with red zone (Hendriksen, 2003). More media have been developed to reduce cost and labor to get faster detection of Salmonella. They are chromogenic and fluorogenic growth media. On CHROMagarTM Salmonella,

Salmonella form mauve colonies while other bacteria form blue or colorless colonies or no growth (CHORMagarTM, 2016).

Many immunology-based assay has been involved to detect Salmonella from food.

Enzyme-linked immune absorbent assay (ELISA) is the most common immunology- based assay that used to detect Salmonella spp. Specific antigen of Salmonella spp. combines with the antibody that targets this micro-organism. Chromogenic substrate is added to have color change which indicate the presence of Salmonella (lee et al., 2015;

Blivet et al., 1998). Latex agglutination assay has been apply to detect Salmonella by visually aggregation of reaction between antigens on the surface of Salmonella and latex particles coated antibodies (lee et al., 2015; Tietjen and Fung, 1995). Immunodiffusion

assay need more prepare steps than previous two methods. After enrichment on the tetrathionate brilliant green broth, the enriched sample is inoculated to a tetrathionate brilliant green broth. Then Salmonella was moved to mobility chamber that antibody has been added. Three–dimensional immunodiffusion band produced after incubation for 14 h (lee et al., 2015; Thorns et al., 1994). Besides these three immunology-based assay, immunochromatography (dipstick) assays has been developed to detect Salmonella cells.

Like other methods, pre-enriched and selectively enriched in media are necessary for this test. After enrichment, Salmonella cells would be captured by the dipstick which contains all reagents (lee et al., 2015; Brinkman et al., 1995).

Another rapid detection test is nucleic acid-based assay which target specific nucleic acid sequence in microorganism (lee et al., 2015). Polymerase chain reaction (PCR) and

DNA probe hybridization assay are two major and common assays that used by a lot researchers (lee et al., 2015). Purified nucleic acid could be achieved by lyse Salmonella cells. Then DNA probe could hybridize with target sequence. There are many methods that could use to detect the hybrid, including radioisotopes and enzymatic reactions. Last, culture methods are used to confirm the presumptive Salmonella (lee et al., 2015; Fung,

2002).

1.2.2.3 Vibrio parahaemolyticus

Thiosulfate citrate bile salts sucrose Agar (TCBS) is the most common medium that used to isolation and cultivation of Vibrio species. Because Vibrio spp. could produce oxidase and catalase, and also ferment glucose, they could form yellow or green colonies on the medium. Chromogenic medium (Bio-Chrome Vibrio medium) could be used for

distinguish Vibrio species. V. parahaemolyticus form a purple colonies on the medium

(BAM, 2004).

The most probable number (MPN) is commonly used for detection V. parahaemolyticus. The test sample were incubated in the enrichment media (alkaline salin peptone water) and following by plated on selective media (TCBS) (BAM, 2004).

However, this method is time and labor consuming and lack of accuracy since the thiosulfate-citrate-bile salts-sucrose agar (TCBS) cannot distinguish the difference between V. parahaemolyticus and other Vibrio species, like V. mimicus and V. vulnificus

(Su and Liu, 2007).

Since tdh and trh genes are encoding thermostable direct hemolysin (TDH) and TDH- related hemolysis (TRH), these two genes are used to detect pathogenic V. parahaemolyticus (Tada et al., 1992). All V. parahaemolyticus strains carry tlh gene, therefore the detection of gene tlh is used to count for total V. parahaemolyticus in food samples (Taniguchi et al., 1986). For increase the accuracy of these detection method of fecal samples, it always combine with MPN procedure (Su and Liu, 2007). Real-time

PCR developed recent for rapid and quantitative determination of V. parahaemolyticus in food samples. A real-time PCR assay was reported by detecting gene tlh in oyster samples (Kaufman et al., 2004).

Nishibuchi reported that the colony hybridization test with oligodeoxyribonucleotide probes labeled with P32 was more accurate than immunological assays for test positive reactions in Kanagawa Phenomenon test (Nishibuchi et al., 1985). In 1992, Yamanoto et al. constructed an enzyme-labeled olionucleotide that target gene tdh and trh by hybridization (Yamamoto et al., 1992). Later, to have more specific and sensitive

detection of tlh gene, McCarthy et al. developed two non-radioactive probes [alkaline phosphatase (AP)-labeled and digoxigenin (DIG) labeled probes] (McCarthy et al.,

1999).

Besides all the detection methods, there also some rapid detection method have been developed. They are API 20E and API NE, RapIDTM NF PLUS System, and Crystal

Enteric/ Non-Fermenter ID Kit. However, the prices for these tests are slightly higher than traditional methods (Su and Liu, 2007).

1.2.3 Incidence of L. monocytogenes, Salmonella and V. parahaemolyticus in ready- to-eat (RTE) seafood

1.2.3.1 Listeria monocytogenes

L. monocytogenes is a bacterium that has been isolated from a lot of RTE seafood, including crab, shrimp, prawns, oyster, clams and lobster (Jinneman and Wekell, 1999).

Contamination of RTE seafood with L. monocytogenes is typically a result of post-cook cross contamination and in linked to inadequate cleaning and sanitation. Weagant et al

(1988) tested 57 samples that included shrimp, crabmeat, lobster tail, langostinos, scallops and surimi-based imitation seafood and 15 of them were L. monocytogenes positive. Among all ready to eat products (e.g., cooked meats, cured meats, and soft cheeses), smoked salmon were reported to be the highest contamination of L. monocytogenes in Spain’s supermarket (Vitas, 2004). Loncarevic et al. (1995) reported that the highest incidence of L. monocytogenes in fish products in Sweden is gravid fish, while hot smoked rainbow trout has the highest viable cells population. In Japan,

Yamazaki et al. investigated that the highest incidence of L. monocytogenes was salmon products including processed salmon products (Yamazaki et al., 2000). Pagadala et al. surveyed 1,736 samples and found 11% percent of crab meat samples were positive for

Listeria spp., but only 0.2% of crab meat samples were positive for L. monocytogenes

(Pagadala et al., 2012). Compared the incidence of crab meat (7.9%) that surveyed by

Rawles, the contamination level of L. monocytogenes in crab meat that Pagadala investigated reduced a lot. This lower level of contamination might because good sanitation practices (Rawles et al., 1995).

1.2.3.2 Salmonella

Salmonella transmission could happened through food-borne and water-borne routes, person-to-person contact, and contact with animals, particularly reptiles (Iwamoto et al.,

2010). Fraiser et al. reported that Salmonella were detected in oysters, clams and crabs

(Fraiser and Koburger, 1984). Monfort et al. also isolated the salmonella from shellfish in

France (Monfort et al., 1994). Shellfish like oysters are often consumed as raw or slightly cooked products (Iwamoto et al., 2010. The Public Health Laboratory System in British investigated 566 raw shellfish and found 22 shellfish samples are Salmonella positive

(Public Health Laboratory System, 1993). Salmonella spp. as also detected in cooked ready-to-eat shrimp and crabmeat, dried/salted seafood, smoked fish, and other prepared ready-to-eat seafood entrees and salads (Buchanan et al., 1991; Okonko et al., 2008;

Heinitz et al., 2000). Heinitz et al. tested 2,734 ready-to-eat seafood samples. They found

10 cooked crab samples, 10 smoked fish, 25 dried and salted fish, and 31 prepared ready- to-eat (RTE) items were Salmonella positive (Heinitz et al., 2000).

1.2.3.3 Vibrio parahaemolyticus

V. parahaemolyticus can be isolated from the natural marine environment (Su, 2012;

Su and Liu, 2007). Wong tested 686 samples of seafood that were imported from Hong

Kong, Indonesia, Thailand and Vietnam and isolated V. parahaemolyticus form shrimp,

crab, snail, lobster, sand crab, fish and crawfish (Wong et al., 1999). Vanderzant et al. isolated V. parahaemolyticus from oyster samples (Vanderzant et al., 1973). Between

1988 and 1977, 345 cases of V. parahaemolyticus infections associated with oysters where reported to CDC (Daniels et al., 2000). Kirs et al. conducted a survey to test the levels of V. parahaemolyticus in Pacific oyster samples from New Zealand. Among the tested oyster samples, 94.8% of them were V. parahaemolyticus positive (Kirs et al.,

2011). The occurrence of V. parahaemolyticus in shellfish has also been reported in southern Italy (Di Pinto et al., 2008). Chitov et al. investigated potential pathogenic

Vibrio species that occurred in raw, processed and ready-to-eat seafood products. The results were presented as being present or absent in 25 grams of food. At least one pathogenic Vibrio species were found that presented in 25 grams in ready-to-eat seafood product samples (Chitov et al., 2009). Thompson et al. reported that 87 % of 153 oyster samples from Galveston Bay were V. parahaemolyticus positive (Chitov and Vanderzant,

1976).

1.2.4 Outbreaks of L. monocytogenes, Salmonella and V. parahaemolyticus infection in ready-to-eat (RTE) seafood

1.2.4.1 Listeria monocytogenes

Table 1.2 lists listeriosis outbreaks related to ready-to-eat seafood, like smoked fish and shellfish, seafood salad, cooked shrimp and crabmeat, and raw seafood (Jami et al.,

2014). Other ready-to-eat seafood recalls associated with contaminated with L. monocytogenes include cold smoked salmon and ready-to-eat shrimp. In 1989, an L. monocytogenes outbreak associated with cooked shrimp was reported in United States

(Riedo et al, 1994).

1.2.4.2 Salmonella

Seafood related outbreaks that are associated with Salmonella include fish, shrimp, oysters and clams (Iwanoto et al., 2010). From 1980 to 1994, there were four Salmonella outbreaks associated with seafood in New York (Wallace et al., 1999). On May 18, 2000, an outbreak of gastroenteritis associated with peel-and-eat shrimp and seafood stew occurred on the cruise ship Palm Beach Princess. A total of 100 passengers and 10 crew members reported to have the symptoms. Investigation of the outbreak revealed that cooked shrimp, which had been served at the lunch buffet, was contaminated with

Salmonella Newport (Hwang and Huang, 2010). Another Salmonella outbreak associated with seafood happened in Netherlands in 2012. Two hundred people were sickened after consumption of smoked salmon. After that, the U.S. Food and Drug Administration, the

Department of Agriculture and the Centers for Disease Control reported that 85 people from 27 states may have been linked to the same contaminated smoked salmon

(Bottemiller, Food Safety News, 2012). In Netherlands, among 1,149 confirmed cases between August and December 2012, four elderly (76–91 years) were reported to have died due to the infection (Friesema et al., 2014).

1.2.4.3 Vibrio parahaemolyticus

There is an estimated of 80,000 illnesses caused by Vibrio species each year in United

State (Newton et al., 2012).

The first US outbreak of V. parahaemolyticus was associated with crabs in Maryland in 1971. Four hundred and twenty-five cases of V. parahaemolyticus infections were reported. The contaminated food was cooked blue crab. Forty outbreaks of V. parahaemolyticus infections were reported to the CDC between 1973 and 1998. Most of them happened in a warmer water temperatures which provided preference growth

condition for V. parahaemolyticus and raised the risk of infection (Daniels and Shafaie,

2000).

In 1997, 209 illness associated with consumption of raw oysters were reported from

July to August in North America. Several states were involves in this large V. parahaemolyticus outbreak, including California, Oregon, and Washington (CDC, 1998).

Another V. parahaemolyticus outbreak associated with raw oysters happened in 1998 in

Washington and Texas (Depaola et al., 2000). In the same year, a small V. parahaemolyticus outbreak accociated with raw oysters and clams was reported in

Connecticut, New Jersey, and New York during summer time (CDC, 1999). In 2006, a V. parahaemolyticus outbreak associated with consumption of raw shellfish occurred from

May to July. A total of 177 illness were reported in New York City, New York, Oregon, and Washington (CDC, 2006). In 2012, 6 persons were involved in a V. parahaemolyticus outbreak in Maryland, USA. This outbreak was associated with raw and cooked seafood in a restaurant (CDC, 2012). In 2013, a large V. parahaemolyticus outbreak involving about 30,104 cases of infections occurred as a result of consuming raw oyster or clams harvested along the Atlantic Coast (CDC, 2013). The most recent V. parahaemolyticus outbreak occurred in 2015 in Massachusetts. The Massachusetts

Department of Health and the state Division of Marine Fisheries reported 6 illnesses were linked to the consumption of contaminated raw oysters (CDC, 2015). Table 1.2. shows the most recent data of V. parahaemolyticus outbreaks in United States.

1.3 Regulation of L. monocytogenes, Salmonella and V. parahaemolyticus in ready- to-eat (RTE) seafood

The United States Department of Agriculture (USDA) and the Food and Drug

Administration (FDA) regulate and inspect food safety and quality for food products in

United States. FDA is the agency that regulates seafood products and industries (Granata, et al, 2012).

1.3.1 Listeria monocytogenes

The different tolerable levels of L. monocytogenes for RTE seafood vary from country to country. In United States, Australia, Italy, and New Zealand, a zero-tolerance policy (absence of the pathogen in 25 g samples of foods) has been set up (FDA, 2011b;

FAO, 1999). This regulation level is the same for refrigerated food in China. For other

RTE food in China, the microbiological counts of L. monocytogenes should not exceed

20 cells per gram (Anonymous, 2007). While in other European countries, the regulation level of listeria in RTE foods is less than 100 cells per gram at the time of consumption.

RTE foods that exceed this level are considered unacceptable or potentially hazardous

(Lianou and Sofos, 2007; Gilbert et al., 2000). According to the Health and Consumer protection directorate-general from the European Commission, the infection dose that causes listeriosis usually is more than 1,000 cells per gram in food (Lianou and Sofos,

2007). In Canada, high priority for oversight RTE foods are those RTE foods that L. monocytogenes can occur throughout the stated shelf-life. Medium priority for oversight

RTE foods are those RTE foods that limited L. monocytogenes can potential occur throughout the stated shelf-life. Low priority for oversight RTE foods are those RTE foods that L. monocytogenes cannot occur throughout the stated shelf-life. For RTE food that need high priority for oversight, the regulation level is the same when compare to

United States. For other RTE seafood products that don’t need high priority of oversight, the microbiological level should not exceed 100 cells per gram (Lianou and Sofos, 2007;

Health Canada, 2011b).

1.3.2 Salmonella

Like L. monocytogenes, Salmonella spp. also has a zero-tolerance policy (absence of the pathogen in 25 g samples of foods) according to the FDA (FDA, 2011b). There is no international agreement on ‘acceptable levels’ of Salmonella spp. right now (FAO, 1999).

However, the same zero tolerance policy of salmonella in RTE food has been established in many other countries, like China, European, New Zealand, and Australia (Anonymous,

2007; FAO, 1999; Norhana et al., 2010).

1.3.3 V. parahaemolyticus

Post-harvest processed seafood (clams, mussels, oysters, and whole and roe-on scallops) should not exceed the V. parahaemolyticus level of 30 MPN per gram in United

States. For other RTE fishery products, the V. parahaemolyticus contamination level is required to be less 104 cells per gram (FDA, 2011b). In China, the detection of V. parahaemolyticus cells in RTE food needs to be less than 103 per gram (Anonymous,

2007).

1.4 Control and prevention of L. monocytogenes, Salmonella and V. parahaemolyticus infection by using plant extracts

Preservation of food is important because contaminated food could cause sickness and disease. Consumers concern much about the quality and hygiene of food products.

They also prefer to have healthier food product with natural additives. Natural biocides could be divided into two categories which are plant-based biocides and microbe-based biocides. Many biocides are derived from plant (Rai, 2011). Fraise separates biocides into three groups. They are disinfectants, antiseptics and preservatives (Fraise, 2002).

Preservatives could be added to products to avoid the contaminations (Rai, 2011).

1.4.1 Berry

Berries are rich in different phenolics, including flavonoids flavonoids (anthocyanins, flavonols, flavones, flavanols, flavanones, and isoflavonoids), stilbenes, stilbenes, tannins, and phenolic acids (Paredes-López et al., 2010; Chung et al., 1998). Ascorbic acid is an important water-soluble vitamins which has been found in different fruits and vegetables (Szajdek and Borowska, 2005). Many studies have indicated that a lot of berries have the antimicrobial bioactive compounds and also might could solve the problem of antibiotic resistance (Paredes-López et al., 2010; Puupponen-Pimiä et al.,

2005a).

Glycosides or complex polymerized molecules with high molecular weights are the mainly form of phenolics in plant tissues (Puupponen-Pimiä et al., 2005a). The higher concentration has been found in the epidermis tissue, following by that in the central part of the fruit (Szajdek and Borowska, 2005).Other factors might affect the concentration of phenolics content in berries, such as species, variety, geographic region, storage conditions, ripeness, climate and others (Paredes-López et al., 2010; Häkkinen et al.,

1999b).

Stilbenes are small phenolic compounds that naturally occur in various plant and foods sources, including berries (Paredes-López et al., 2010). Berries is a great resource of tannins which comprise condensed non-hydrolysable tannins and hydrolysable tannins.

Esters of gallic acid and ellagic acid are categorized as hydrolysable tannins.

Proanthocyanidins are one common condensed tannins found in berries (Puupponen-

Pimiä et al., 2005b).

Anthocyanins are really common and important phenolics in berries. Usually they are found in the peel of fruit and is the reason for the pigment for the color (Paredes-López et

al., 2010). For most berries, anthocyanins are responsible for most water-soluble pigments (Szajdek and Borowska, 2005). Many factors are involved to distinguish the different anthocyanins, including hydroxyl groups in a molecule, the degree of methylation of these groups, the type, number and place of attachment of sugar molecules, and the type and number of aliphatic or aromatic acids attached to sugars in an anthocyanin molecule (Kong et al., 2003). Cyanidin has been identified in many berry fruits (Paredes-López et al., 2010). In berry fruits, anthocynins could form mono-, di- or triglycosides by substitute at C3, C5 or C7 of glycoside residues (Shahidi and Naczk,

2004). Also, tannins could bind anthocyanins to stabilize them as copolymers (Cheynier et al., 2006).

In berry fruits, phenolic acids has been found, including cinnamic acid and benzoic acid derivatives (Kim, 2012). However, they often occurs as esters or glycosides. In berry fruits, p-hydroxybenzoic acid, salicylic acid, gallic acid and ellagic acid has been found in benzoic acid derivatives. Ellagic acid is not present in fresh berries since they are the product from ellagitanins hydrolysis (Macheix and Fleuriet, 1990). For cinnamic acid derivatives in berries, p-coumaric acid, caffeic acid and ferulic acid was identified.

Among them, caffeic and ferulic acids are two most common phenolic acids in berries

(Taruscio et al., 2004; Zadernowski et al., 2005). However, they usually esterifies with other molecules as carbohydrates and organic acids instead of free. Besides that, chlorogenic acid derivatives are the most common esters of hidroxycinnamic acids

(Paredes-López et al., 2010).

1.4.1.1 Cranberry

Large quantities of lignans have been found in cranberry. This diphenolic compounds is one part of phytoestrogens (Mazur et al., 2000). Also, cranberry fruit is also rich in ferulic acid (Häkkinen et al., 1999a). High concentration of myricetin, flavonols quercetin and kaempferol were found in cranberry (Paredes-López et al., 2010;

Puupponen-Pimiä et al., 2005a; Häkkinen et al., 1999b; Häkkinen et al., 1999a; Bhagwat et al., 2014). Only small amount of trans-resveratrol were detected in cranberry (Rimando et al., 2004).

Some studies have identified the large presence of condensed tannins. They also test the anti-adhesion of Escherichia coli in urinary tract (Paredes-López et al., 2010;

Puupponen-Pimiä et al., 2005a; Howell, 2002) and adherence of Helicobacter pylori in gastric ulcer development (Shmuely et al., 2004). Hydroxycinnamate esters in cranberry fruit were also detected (Paredes-López et al., 2010; Pappas and Schaich, 2009). Phenolic acid, like ellagic acid and gallic acid, has been found in cranberry fruit and showed highly response to the antimicrobial activity (Vattem et al., 2004a; Vattem et al., 2004b).

In summary, phenolic acids, condensed tannins, proanthocyanidins, flavonoids and flavonols have been detected in cranberry fruit (Häkkinen et al., 1999a; Leitão et al.,

2005; Puupponen-Pimiä et al., 2005a).

One study has been investigated the antimicrobial activity of nitrite and cranberry powder in cooked boneless pork. Limited growth of bacteria was observed in cranberry powder and sodium nitrite added meat samples after three and six day’s incubation at

10 °C. After incubated for nine days, at least one log CFU/g less growth of L. monocytogenes was observed when compared the population of positive control. This

results might indicate the interaction between nitrite and cranberry could cause the antimicrobial activity against L. monocytogenes (Xi et al., 2011).

Another study has been conducted to see the inhibitory effect of combination of oregano and cranberry in cooked ground beef at 4 °C for 10 days. All tested samples showed limited growth of L. monocytogenes. However, compared to the controlled sample, no significant growth reduction of L. monocytogenes were observed. When applied addition 2% sodium lactate with 750 ppm oregano-cranberry extract to ground beef, one log CFU/g less growth could be achieved compared to untreated samples. The inoculum level is also important to the inhibitory effect. The low level inoculum (3–4 log

CFU/ml) was able to have more antimicrobial activity compared to high level inoculum

(5–6 log CFU/ml) (Apostolidis et al., 2008).

In addition, one study investigate the oregano-cranberry extract with lactic acid against V. parahaemolyticus in cod fish fillet and shrimp. After 8 days incubated at 4 °C, individual cranberry and oregano extract added fish and shrimp samples showed less growth compared to the untreated samples. The population of bacteria in fish and shrimp could even reduce to less than the initial inoculum level (~3 log CFU/ml) (Lin et al.,

2005).

1.4.1.2 Blueberry

Many phenolic compounds has been found in blueberry, including resveratrol, pterostilbene, and piceatannol (Paredes-López et al., 2010a; Lyons et al., 2003). After test contents of the favonols quercetin, myricetin and kaempferol in 25 edible berries,

Häkkinen et al. found that one of the highest concentration of myricetin is in blueberry

(Häkkinen et al., 1999b).

Anthocyanins have been found in blueberries. The different composition of anthocyanins in different blueberries has also been found. In methanol pigments extracts, ten anthocyanins has been identified in American planting blueberry, while fifteen anthocyanins identified in French blueberries. Since these two blueberries are cultivated in the same mold, the different results might cause by the difference ripeness of test samples (Kader et al., 1996).

Besides these phenolic compounds, phenolic acid and flavonoids are also important phenolics to contribute as health-promoting compounds. Blueberries are found that rich in ferculic acid and chlorogenic acid (Häkkinen et al., 1999a; Može et al., 2011). In addition, caffeic, ellagic, p-coumarnnnnnnic acid, and gallic acids were identified in blueberries (Može et al., 2011).

Blueberry extract were involved in a study to extend the self-life of raw beef meet ball. Self-life of raw beef meet ball extended to 10 days by adding blueberry extract to it.

Also, the sensory test of blueberry in meat ball is the best among the test samples (Gök and Bor, 2011).

1.4.1.3 Strawberry

Strawberry is a great resource of vitamin C (Hägg et al., 1995). Large quantities of lignans have been found in strawberry. It also called diphenolic compounds and is one part of phytoestrogens (Mazur et al., 2000). Compare to lignans, trans-resveratrol only occurs as small content in strawberry (Ehala et al., 2005). Besides these phenolic compounds, kaempferol, myricetin, and quercetin have been identified in strawberry fruits (Häkkinen et al., 2000; Sultana and Anwar, 2008).

In addition, Hydrolysable tannins has been detected in strawberries, especially ellagitannins (Foo and Porter, 1981). Nohynek et al. conduct a study and found out that the presence of large amount of ellagitannins are due to the stable glucose conformation.

He also indicated that ellagitannins might be the phenolic compounds that response for the antimicrobial activity of strawberries (Nohynek et al., 2006).

Different kind of phenolic acids have been found in strawberries. Strawberry fruits are rich in ellagic acid (Häkkinen et al., 2000; Skupień and Oszmiański, 2004). Kim et al. reported that trans-cinnamic acid, p-hydroxybenzoic acid, and kaempferol were identified in thinned strawberry fruits. However, p-coumaric acid, caffeic acid, and gallic acid have not been detected in this study (Kim et al., 2012).

One study has been conducted to investigate the strawberry antimicrobial activity in inoculated chocolate samples. Population of L. monocytogenes in inoculated chocolate samples were slight reduced within 24 hours, however, the population of L. monocytogenes in chocolate samples were slight increased storage at 7 °C. On the contrast, the strawberry extracts added chocolate samples could achieve 1.5 and 1.2 log reduction in dry and humidified environment, respectively (Kotzekidou et al., 2008).

1.4.2 Pomegranate

Pomegranate is a great source of phenolic compounds. Alkaloids were found in pomegranate juice. Pomegranate juice mainly contains straight chain fatty acids (Wang et al., 2010).

Tannins are also one important phenolic compounds that have been found in pomegranate. Hydrolysable tannins have been identified to be the majority in pomegranate including ellagitannins and gallotannins. Condensed tannins are rarely

found in this fruit (Gil et al., 2000; Wang et al., 2010). In pomegranate peel, ellagitannins, like punicalin and punicalagin, is a dominant constitute (Gil et al., 2000).

Hydrolysable tannins (such as punicalin, pedunculagin, punicalagin, gallagic and ellagic acid esters of glucose) are main factors for the antimicrobial properties of peel extract

(Sadaka et al., 2013).

Besides tannins, flavonoids is another phenolic compounds that has detected in pomegranate. Flavones, flavonols, anthocyanidins and flavan-3-ols are rich in pomegranate peel and juice. Flavan-3-ols occurs in pomegranate only as unglycosylated form, including catechin, epicatechin, epigallocatechin and their derivatives (Wang et al.,

2010).

Both fresh pomegranate juice and peel contain anthocyanins (Aviram et al., 2002;

Opara et al., 2009) which are the phenolic compounds that responsible for the red color

(Gil et al., 2000; Turfan et al., 2011). Gil et al. (2000) and Turfan et al (2011) detected six anthocyanins, 3-glucosides and 3, 5-diglucoside of cyanidin, delphinidin and pelargonidin in pomegranate juice.

Pomegranate contains organic acid. In methanolic pomegranate peel extract, 3 hydroxybenzoic acids (vanillic, gallic and ellagic acids), 2 hydroxycinnamic acids

(caffeic and p-coumaric acids), and one flavonol (quercetin) have been found (Mansour et al., 2013) Moreover, tartaric acid, oxalic acid and succinic acid were detected in the juice (Poyrazoğlu et al., 2002) and caffeic acid, fumalic acid, chlorogenic acid and p- counatic acid were identified both in pomegranate juice and peel (Artik, 1998). Citric acid and malic acid are the most dominant the organic acids (Artik, 1998; Poyrazoğlu et al., 2002).

In a study which studied on the fresh chilled cold fish, after dipping the fish in WME

(water-methanol extract) (0.26% as GAE) pomegranate peel extracts, the viable counts were immediately decreases to < 3 log10 CFU/g while the Listeria-contaminated fish slices were more than 3 log10/g. After six days of storage at 4 °C, WME of pomegranate peel extracts could prevent the growth of L. monocytogenes in fish samples. The viable counts remained below the inoculated level (3 log10 CFU/g). Compare to the viable counts of Listeria-contaminated fish slices after 6 days, the pomegranate peel treated fish samples could achieve 1 log reduction (Al-Zoreky, 2009).

Pomegranate peel coating film has been involved in one study to investigate the antimicrobial activity in ground beef. Compared to the untreated ground beef samples at

4 °C, the total viable bacteria were significantly higher than that of meat samples coated with antibacterial film. After storage at 4 °C in nine days, the population of

Staphylococcus aureus for untreated ground beef sample were much higher than that of meat samples coated with antibacterial film (Emam‐Djomeh et al., 2015).

Another study conducted to test antimicrobial activity of pomegranate peel extract in ready-to-eat meat pate, showed that the inhibition of L. monocytogenes growth in meat pate of pomegranate peel depends on temperature. At 4 °C, the population of L. monocytogenes in meat pate increased 1.5 log from initial counts after 46 days, while the untreated inoculated samples were increased to 9.2 log (Hayrapetyan et al., 2012).

When the pomegranate peel extract was used to extend the self-life of chicken samples. The population of total plate counts in both treated chilled chicken sample and untreated chilled chicken samples increased. Untreated chicken samples spoiled within 7 days. However, treated chicken samples spoiled within 20 days which is much extend the

self-life of chicken samples. Also, the self-life of treated chicken lollipop could be extended to 20 days. They also did sensory test and found out that PE (pomegranate peel extract) has no effect on the sensory in chicken samples. Color, flavor, taste and texture of PE treated and control samples were similar (Kanatt et al., 2010).

1.4.3 Plum

Fresh plum and dried plum has been reported as phenolic compounds. Cevallos-

Casals et al. reported that the phenolic content in plum skin was 3- to4- fold higher than that in flesh. Similar result of anthocyanin content was found in plum samples (Yurdugul and Bozoglu, 2009; Cevallos-Casals et al., 2006).

Besides anthocyanins, other hydroxycinnamates detected in plum, including flavan 3- ols and falvonols, catechin, epicatechin, ethyl cinnamate and coumarins, and quercetin 3- rytinoside (Kim et al., 2003; Stöhr et al., 1975). Rutin was the principal flavonol in plums

(Kim et al., 2003). Also, anthocyanins were mainly found as rutinoside derivatives in fresh plums (Raynal et al., 1989).

Different types of phenolic acids have been found in plum. In dried plum products, high amount of malic acid and lower levels of citric, benzoic and chlorogenic acids has been detected (Siddiq, 2006). Malic and citric acids have been reposted as preservatives and food additives for a long history (Ricke, 2003). Fang et al. detected many other phenolic acids in plum, including cryptochlorogenic acid neochlorogenic acid, chlorogenic acid, and sorbic acid (Fang et al., 2002). Hydroxycinnamic acid derivatives were identified in fresh plum flesh and peel (Nunes et al., 2008). Among different hydroxycinnamic acid in plum, 3-caffeoylquinic acid is the main phenolic compounds in

plum flesh and peel. The other main phenolic compounds found in plum flesh is 5- caffeoylquinic acid (Nunes et al., 2008).

One study has been conducted to investigate different antimicrobial activity of plant extracts in chocolate samples. Plum extracts that added in chocolate could completely inhibited the growth of E. coli and cause a 0.4 log reduction after 9 days incubation at

7 °C. The complete inhibition growth of E. coli in plum added chocolate was also observed at 20 °C (Kotzekidou et al., 2008).

In addition, Valtierra-Rodríguez tested inhibition growth of different plant extracts that added in chicken skins. The combination of lime and plum extract could achieve more than 4 log reduction of Campylobacter jejuni and Campylobacter coli in chicken skin after storage at 4 °C for 2 days. They also did the sensory analysis using a preference ranking test. The results showed the mixture of lime and plum in chicken wings are the most popular and acceptable flavor among all tested samples (Valtierra-Rodríguez et al.,

2010).

1.5 Objective

FDA set up a regulation level that 10,000 Vibrio parahaemolyticus cells/g in ready- to-eat fishery products. This number includes pathogenic and non-pathogenic V. parahaemolyticus. However, there is limited research available about the difference between pathogenic and non-pathogenic V. parahaemolyticus in different temperature environment. The first part of this study is to investigate growth and die-off of pathogenic and non-pathogenic V. parahaemolyticus strains at different temperatures (5-30°C).

There are many ready-to-eat (RTE) seafood incidences and outbreaks of L. monocytogenes, Salmonella and V. parahaemolyticus happened in United States each

year. Also, many fruits have been reported to be rich in phenolics. Some studies showed antimicrobial activity of freeze-dried or rotary evaporated fruit extracts (Emam-Djomeh et al., 2015; Kanatt et al., 2010; Negi and Jayaprakasha, 2003; Puupponen-Pimia et al.,

2001). However, limited studies have been conducted to investigate the antimicrobial activity of fresh fruit extracts. The second part of this study is to investigate the antimicrobial activity of fresh fruit extracts against L. monocytogenes, Salmonella and V. parahaemolyticus and also there potential application on RTE seafood products.

Table 1.1 Worldwide seafood consumption (kg/captica/yr) from 1963 to 2013 (data adapted from FAOSTAT Database) Country Seafood Consumption (kg/capita/yr) 1963 1973 1983 1993 2003 2013 Americas 9.29 11.36 11.68 13.88 14.3 14.2 Africa 4.96 6.9 8.2 7.46 8.31 10.77 Asia 8.11 9.61 10.12 13.62 17.86 21.43 Europe 15.28 19.93 20.48 16.69 19.72 21.85 Australia 10.79 13.68 15.24 20.05 25 25.91 & New Zealand World 9.51 11.54 11.88 13.4 16.41 18.98

Table 1.2 Listeria monocytogenes outbreaks associated with consumption of ready-to-eat (RTE) seafood (data adapted from Jami et al., 2014) Product Country Year Number of Serotype cases Herring cutlet marinated in Germany 2010 8 cases, 1 oil deatha Smoked rainbow trout Finland 1999 5 cases 1/2a Tuna-corn salad Italy 1997 1566b 4b Imitation crab meatc Canada 1996 2 cases 1/2b Cold-smoked rainbow trout Sweden 1994 to 9 cases, 2 4b (suspected) 1995 deaths Smoked mussels New Zealand 1992 3 casesd, 1 1/2a death Smoked mussels Australia 1991 4 cases 1/2a (Tasmania) Shrimp United States 1989 2 cases 4b Smoked cod roe Denmark 1989 1 case 4b Fish or molluscan shellfish New Zealand 1980 22 cases, 7 1b-1/2af deathse a Three of eight patients do not remember about the consumption of this particular fish; 1 fatal outcome could be directly linked to fish consumption 3 d before death. b Non-invasive listeriosis. Possible cross-contamination from other untreated foods. c Artificially flavored Alaska Pollock. d DNA analysis using pulsed-field gel electrophoresis (PFGE) showed that the PFGE patterns of isolates from patients 1 and 2 were indistinguishable from the isolates from the mussels. Patient 3 had a history of consuming mussels and PFGE analysis of isolates of L. monocytogenes serogroup 1/2 revealed that the isolates were indistinguishable from the isolates of patients 1 and 2. e The link was on the basis of recall of food consumption, not microbiological testing f While in the first report, the serovar 1b indicated for isolated L. monocytogenes, mentioned the serovar 1/2a for the pathogen.

Table 1.3 Recent Vibrio parahaemolyticus outbreaks associated with consumption of ready-to-eat (RTE) seafood in United States Year Products States Numbers of illness 1997 Oysters Pacific Northwest 209 1998 Oysters Washington 43 1998 Oysters Texas 416 1998 Shellfish Connecticut, New 23 Jersey, and New York 2001 Oysters California 14 2002 Oysters California 9 2004 Oysters Alaska 14 2006 Shellfish New York City, 177 New York, Oregon, Washington 2011 Oysters Washington 22 2012 Shellfish Missouri 3

Figure 1.1 Foods linked to known outbreaks (2003-2012)

Figure 1.2 Pathogens implicated in solved outbreaks (2003-2012)

Chapter 2

Comparison of Growth and Die-off of Pathogenic and Non-pathogenic Vibrio parahaemolyticus at Different Temperatures

Jingyi Du, Yi-Cheng Su, Christina Ann DeWitt, Jovana Kovacevic

(To be Submit to Food Microbiology)

2.1 Abstract

Vibrio parahaemolyticus is a foodborne pathogen frequently isolated from seafood, especially shellfish. FDA regulations state that 10,000 Vibrio parahaemolyticus cells/g are permitted in ready-to-eat (RTE) fishery products, which includes pathogenic and non- pathogenic V. parahaemolyticus. This study investigated growth (positive µmax) and die- off rates (negative µmax) of pathogenic and non-pathogenic V. parahaemolyticus strains from 5-30°C . There was a significant effect of strain type (non-pathogenic vs pathogenic,

P < 0.001), temperature (P < 0.001) and a strain type x temperature interaction (P =

0.026). At 5 °C, µmax was negative for both strain types indicating die-off. At 10 °C, µmax was negative for 2 pathogenic and 1 non-pathogenic strain. From 15-30 °C, µmax was positive for all strains. When evaluating differences between strain type at each temperature, significance of strain type only occurred at 30 °C. When evaluating the effect of temperature on µmax within either non-pathogenic or pathogenic strain type, there was no significant difference between 5-15 °C. The mean µmax for non-pathogenic and pathogenic strains was 0.448 and 0.340, respectively. The faster rate of growth for non-pathogenic strains suggests the parameters set by FDA, 10,000 V. parahaemolyticus cells/g, are more likely to represent non-pathogenic strains, rather than pathogenic strains, especially as environmental temperature increases.

Keywords: Vibrio parahaemolyticus, growth rate, risk assessment

2.2 Introduction

Vibrio parahaemolyticus is a Gram-negative, halophilic food-born pathogen that naturally occurs in marine environments (Su and Liu, 2007). This bacterium is frequently isolated from various seafood including codfish, sardine, mackerel, flounder, clam, octopus, shrimp, crab, lobster, crawfish, scallop and oyster (Liston, 1990). Shellfish, especially oysters, are the source of contamination of V. parahaemolyticus. The occurrence of V. parahaemolyticus is more likely to happen in summer months because of warm temperature and low-salinity (Kelly and Stroh, 1988). Consuming raw or undercooked seafood contaminated with V. parahaemolyticus causes gastroenteritis.

Symptoms include diarrhea, abdominal cramps, nausea, vomiting, and fever (Daniels et al., 2000). The first V. parahaemolyticus outbreak happened in Japan in 1950 and caused

272 illness with 20 deaths (Letchumanan et al., 2014; Odeyemi, 2016). The first V. parahaemolyticus outbreak in United States happened in 1971. Sporadic V. parahaemolyticus incidences were reported after that (Dadisman et al., 1972). Between

1988 and 1997, 345 cases of V. parahaemolyticus infections associated with oysters were reported to the Centers for Disease Control and Prevention (CDC) (Daniels et al., 2000).

V. parahaemolyticus is divided into two groups: non-pathogenic and pathogenic.

Non-pathogenic V. parahaemolyticus is isolated from the environment and is not pathogenic to humans. The majority of V. parahaemolyticus strains in the environment are non-pathogenic (Letchumanan et al., 2014; Nishibuchi and Kaper, 1995). The difference between pathogenic V. parahaemolyticus and non-pathogenic V. parahaemolyticus is the ability to produce a thermostable direct hemolysin (TDH). TDH is an enzyme that lyses red blood cells on Wagatsuma blood agar. This enzyme reaction

is also called the Kanagawa phenomenon (KP) (Joseph et al., 2008; Su and Liu, 2007).

Another virulence factor was involved in 1985 in an outbreak in the Republic of

Maldives (Honda et al., 1988). TDH-related hemolysin (TRH) was found in KP- negative

V. parahaemolyticus strains (Honda et al., 1988; Letchumanan et al., 2014). The two genes that encode TDH and TRH are tdh and trh (Nelapati et al., 2012; Nishibuchi et al.,

1986). Gene tl encodes thermolabile hemolysin (TLH) which is carried by all V. parahaemolyticus strains (McCarthy et al., 1999). Therefore, TDH and TRH have been identified as major virulence factors of V. parahaemolyticus (Ceccarelli et al., 2013;

Fukui et al., 2005; Raghunath, 2015).

According to the United States Food and Drug and Administration (FDA), post- harvest processed fresh or frozen seafood (clams, mussels, oysters, and whole and roe-on scallops) should not exceed the V. parahaemolyticus level of 30 MPN per gram. For other

RTE fish products, the V. parahaemolyticus contamination level is required to be less than 104 cells per gram (FDA, 2011). The regulation level includes the pathogenic and non-pathogenic V. parahaemolyticus. However, limited studies have been conducted to investigate the different growth and die-off response of pathogenic and non-pathogenic V. parahaemolyticus in various temperature environments. In 2009, Burnam and others studied the growth of V. parahaemolyticus in broth at only low temperatures (5, 8, and

10 °C) (Burnham et al., 2009). Moreover, Yoo won Kim (2012) investigated the effect of temperature on growth of V. parahaemolyticus in broth and oyster meat. However, only three pathogenic V. parahaemolyticus strains were tested in that study and the difference in growth between pathogenic and nonpathogenic V. parahaemolyticus was not compared

(Kim et al., 2012). Therefore, this study is to investigate growth and die-off of pathogenic and non-pathogenic V. parahaemolyticus strains at different temperatures (5-30 °C).

2.3 Material and Methods

2.3.1 Bacteria Culture Preparation

Five clinical V. parahaemolyticus strains [10290 (O4:K12, tdh+ and trh+), 10292

(O6:K18, tdh+ and trh+), 10293 (O1:K56, tdh+ and trh+), BE98-2029 (O3:K6, tdh+ and trh-), and 1C1-O27 (O5:K15, tdh+ and trh+)] obtained from the culture collection of the

Food and Drug Administration Pacific Regional Laboratory Northwest (Bothell, WA) were used in the study. Also, five environmental strains of V. parahaemolyticus which were isolated from oysters were used in this study (Table 2.1). Each frozen stock culture was activated in 10 ml of tryptic soy broth supplemented (TSB; Difco, Becton, Dickson,

Spark, MD, USA) with 1.5% NaCl (TSB-Salt) and incubated overnight at 35 ± 2 °C. One loopful of each enriched culture was streaked on thiosulfate citrate bile salt sucrose

(TCBS; Difco, Becton, Dickson, Spark, MD, USA) plate and incubated overnight at 35 ±

2 °C. A single colony was picked from the TCBS plate and enriched in 10 ml of TSB-

Salt at 37 °C for 4h. Cells of V. parahaemolyticus were harvested by centrifugation

(Sorvall RC-5B, Kendri Laboratory products, Newtown, CT, USA) at 3000 × g at 5 °C for 15 min and re-suspended in equal volume of sterile 2% NaCl solution to prepare the

108-9 CFU/ml culture suspension.

2.3.2 Effect of temperature on growth of V. parahaemolyticus

To determine effects of temperature on growth of pathogenic V. parahaemolyticus and non-pathogenic V. parahaemolyticus, each strain was incubated in tryptic soy broth supplemented with 1.5% NaCl (TSB-Salt) at 5, 10 and 15°C for 96 hours, 20°C for 36

hours, and 25 and 30°C for 12 hours. For 5 and 10 °C, changes of V. parahaemolyticus populations in TSB-Salt were determined every 24 hours by the pour-plate method using

TSA-Salt with incubation at 35-37 °C for 24 hours. To determine the V. parahaemolyticus populations in TSB-Salt after incubation at 15 °C for 96 hours and

20 °C for 36 hours, changes of population were tested every four hours for the first 12 hours. After the first 12 hours, the change of population of V. parahaemolyticus was determined every 12 hours by the pour-plate method using TSA-Salt with incubation at

35-37 °C for 24 hours. For 25 and 30 °C, changes of V. parahaemolyticus populations in

TSB-Salt were determined every 2 hours by the pour-plate method using TSA-Salt with incubation at 35-37 °C for 24 hours.

2.3.3 Statistical analysis

The viable cell counts of each strain from different sampling times were analyzed using DMFit Excel Add-In software (Baranyi, J., Institute of Food Research, Norwich

2 Research Park, and Norwich, UK). The growth rates, die-off rates (µmax) and adjusted R

(Adj. R2) were obtained from this primary model and generated from analysis. The growth rates and die-off rates showed the growth response to different temperatures.

Adjusted R2 showed how good the model fit the data. Bacterial counts at different times and temperatures and growth or die-off rates were analyzed using Tukey-Kramer multiple-comparison Test in R program (R foundation, Vienna). The significant difference was set up at a level of P < 0.05. One-Way ANOVA and Two-Way ANOVA were used to analyze growth or die-off rates in SigmaPlot (Systat Software, San Jose,

CA). The significance level was P < 0.05.

Table 2.1 Vibrio parahaemolyticus Strain: Strain Virulence factora Source of Isolation Pathogenic 10290 tdh+, trh+ Clinical Sample, Washington 10292 tdh+, trh+ Clinical Sample, Washington 10293 tdh+, trh+ Clinical Sample, Washington BE 98-2029 tdh+, trh- Clinical Sample, Texas 027-1c1 tdh+, trh+ Clinical Sample, Oregon Non-pathogenic 100813W01 tdh-, trh- Oyster, Washington 102713Y01 tdh-, trh- Oyster, Oregon 090811Y02 tdh-, trh- Oyster, Oregon 100311Y04 tdh-, trh- Oyster, Oregon 100311Y11 tdh-, trh- Oyster, Oregon a Virulence factors (tdh and trh) were determined with a multiplex polymerase chain reactions (PCR) according to Bej and others (1999).

2.4 Results

2.4.1 Growth Study of V. parahaemolyticus in TSB-Salt at different temperature

The initial inoculum for ten V. parahaemolyticus strains at different temperature were at 3–4 log CFU/ml. The population of V. parahaemolyticus in broth increased when incubation temperature was increased.

At 5 °C, all V. parahaemolyticus strains start to decline. The population of non- pathogenic V. parahaemolyticus strain 100311Y11 decreased from 3.22 log CFU/ml to non-detectable level after 96 hours at 5 °C (Table 2.2). However, the population of pathogenic V. parahaemolyticus strain 10290 remained at 2.13 log CFU/ml and only decreased by 1.09 log CFU/ml (Table 2.2). Three log reduction was achieved in 3 out of

5 non-pathogenic strains and 1 of 5 pathogenic strains after 96 hours at 5 °C. Limited growth or slight decrease of V. parahaemolyticus was observed at 10 °C after 96 hours incubation (Table 2.3). Pathogenic V. parahaemolyticus strains 10292 and 10293 and non-pathogenic V. parahaemolyticus strain 090811Y02 decreased 0.24, 2.24 and 0.32 log, respectively. The remaining V. parahaemolyticus strains increased after 96 hours at

10 °C (Table 2.3).

At 15 °C, pathogenic V. parahaemolyticus strains 10290 and 10293 and non- pathogenic strain 102713Y01 reached the stationary phase after 84 hours and 96 hours, respectively, while the remaining seven V. parahaemolyticus strains reached the stationary phase after either 36 hours or 48 hours (Table 2.4). All five non-pathogenic V. parahaemolyticus strains increased by 5 log CFU/ml. However, only three pathogenic V. parahaemolyticus strains achieved the 5 log CFU/ml increase. The population of pathogenic V. parahaemolyticus strain 10293 was 6.78 log CFU/ml while that of other

strains were above 8 log CFU/ml at 20 °C (Table 2.5). This result showed that strain

10293 increase the least after 36 hours incubation. Pathogenic V. parahaemolyticus strain

10290 increased all the time after 36 hours incubation while other strains reach the stationary phase and faintly declined.

After incubation at 25 °C for 8 hours, all five non-pathogenic V. parahaemolyticus strains reached the stationary phase while pathogenic strains 10290 and 10293 where still in log phase. At 30°C, all five non-pathogenic V. parahaemolyticus strains reached the stationary phase after 6 hours storage. Pathogenic V. parahaemolyticus strain 10290 increased continuously during the 12 hours incubation.

2.4.2 Growth rate and die-off rate of V. parahaemolyticus in TSB-Salt at different temperatures

The growth rates or die-off rates (µmax) of each strain were obtained from the primary model fit generated from DMFit Excel Add-In software. The adjusted R2 (Adj. R2) represents how well the data fit in the primary model. All Adj. R2 of the ten V. parahaemolyticus strains were above 0.90.

According to the Two-Way AVOVA analysis results, a significant effect of strain type (non-pathogenic vs pathogenic, P < 0.001), temperature (P < 0.001) and a strain type x temperature interaction (P = 0.026) were observed. For both pathogenic and non- pathogenic strain types, µmax was negative which indicated die-off at 5 °C. At 10 °C, negative values of µmax were observed for 2 pathogenic and 1 non-pathogenic strains as reported in the previous section. When temperature ranged from 15 to 30 °C, µmax was positive for all type of strains. When evaluating differences between pathogenic and non- pathogenic V. parahaemolyticus strains at each temperature, significance of strain type only happened at 30 °C. One-Way ANOVA was used to evaluate the effect of

temperature on µmax within either non-pathogenic or pathogenic strain type and no significant difference was observed between 5-15 °C. The mean value of µmax for non- pathogenic and pathogenic strains was 0.448 and 0.340, respectively. Faster growth rate was observed in non-pathogenic strains, which suggests a higher possibility of false positives from environmental sampling, especially when environmental temperature goes up.

At 5 °C , µmax is negative for all strain type which suggested die-off. The die-off rates

(µmax) of V. parahaemolyticus strains were similar to each other. The µmax of pathogenic strain 10292 (0.06 log CFU/h) is significantly higher (P < 0.05) than the other nine V. parahaemolyticus strains.

At 10 °C, for the strains that decreased, there is no significant (P < 0.05) difference between the die-off rates (µmax) of pathogenic strain 10292 (0.01 log CFU/h) and 10293

(0.02 log CFU/h), and non-pathogenic strain 090811Y02 (0.003 log CFU/h). For the V. parahaemolyticus strains that grew at 10°C, non-pathogenic V. parahaemolyticus strain

100813W01 (0.08 log CFU/h) grew significantly faster (P < 0.05) than the other six V. parahaemolyticus strains.

At 15 °C, all strains started to grow and the growth rates (µmax) of ten V. parahaemolyticus strains were similar to each other at 15 °C. The range of growth rates

(µmax) of five pathogenic V. parahaemolyticus strains ranged from 0.04 log CFU/h to 0.16 log CFU/h. Three V. parahaemolyticus strains, pathogenic strain 10290 and 10293 and non-pathogenic strain 102713Y01, grew at a similar rate and significantly (P < 0.05) slower than the other seven V. parahaemolyticus strains. No significant (P < 0.05) growth rate (µmax) differences were observed between these three strains.

At 20 °C, pathogenic strain 10293 (0.23 log CFU/h) grew significantly slower (P <

0.05) than the other nine V. parahaemolyticus strains and had the lowest growth rate

(µmax (g)). However, non-pathogenic strain 100311Y11 (0.65 log CFU/h) grew significantly faster (P < 0.05) than the other nine V. parahaemolyticus strains and had the highest growth rate (µmax). In addition, the growth rates (µmax) of pathogenic 10290 (0.33 log CFU/h) and BE 98-2029 (0.36 log CFU/h) were significantly higher (P < 0.05) than pathogenic strain 10293, but significantly lower (P < 0.05) than the other seven V. parahaemolyticus strains. The range of growth rates (µmax) for pathogenic V. parahaemolyticus strains were from 0.23 log CFU/h to 0.49 log CFU/h. However, the growth rates (µmax) of non-pathogenic V. parahaemolyticus strains ranged from 0.41 log

CFU/h to 0.65 log CFU/h.

At 25 °C, pathogenic V. parahaemolyticus strain 10290 (0.43 log CFU/h) grew significantly slower (P < 0.05) than the other nine V. parahaemolyticus strains and had the lowest growth rate (µmax) among the nine V. parahaemolyticus strains. Moreover, pathogenic strain 10293 grew significantly faster (P < 0.05) than pathogenic strain 10290, but significantly slower (P < 0.05) than the other eight V. parahaemolyticus strains. The growth rates (µmax) of pathogenic V. parahaemolyticus strains ranged from 0.43 log

CFU/h to 0.84 log CFU/h. However, the growth rates (µmax) of the five non-pathogenic strains ranged from 0.73 to 0.88 log CFU/h.

At 30 °C, the growth rates (µmax) of the pathogenic V. parahaemolyticus strains were significantly lower (P < 0.05) than the other five non-pathogenic V. parahaemolyticus strains, excepting pathogenic V. parahaemolyticus strain 10292. Pathogenic strain 10292

(1.41 log CFU/h) and non-pathogenic strain 100311Y11 (1.45 log CFU/h) showed no

significant (P < 0.05) difference between their growth rates (µmax). Pathogenic strain

10290 (0.67 log CFU/h) had the lowest growth rate (µmax) among the other nine V. parahaemolyticus strains. Also, except for pathogenic strain 10292, the growth rates

(µmax) of the other four pathogenic strains were lower than 1 log CFU/h. However, the growth rates (µmax) of the five non-pathogenic V. parahaemolyticus strains were all above

1 log CFU/h.

2.5 Discussion

The results from this study show that different V. parahaemolyticus strains have different growth responses under different temperatures. All ten V. parahaemolyticus strains decreased during 5 °C storage. This finding confirmed the results from a study conducted by Burnham et al.(Burnham et al., 2009), which reported the lowest and highest viable counts after 10 days storage at 5 °C at 3.46 and 5.28 log CFU/ml, respectively. These viable counts were much higher than the results in this study. The reason may be due to the lower inoculum level (3-4 log CFU/ml) used in this study, compared to 6 log CFU/ml used by Burnham et al. (2009). It also indicated that different inoculum levels could also affect the growth or die-off response to different temperatures.

In our study, 3 of 10 V. parahaemolyticus strains slightly declined under 10 °C storage. This result indicates that strains 10292, 10293, and 090811Y02 might be more sensitive to cold temperature storage than the other seven V. parahaemolyticus strains tested. According to the study conducted by Kim et al., the minimum growth temperatures of V. parahaemolyticus in broth is 13 °C, which means 10 °C could not support all V. parahaemolyticus strains growth (Kim et al., 2012). However, early reports of growth temperature of V. parahaemolyticus were at 5 and 8.3 °C (Miles et al., 1997).

Burnham et al. also reported that all eight V. parahaemolyticus strains they tested had increased viable counts after 10 days storage under 10 °C (Burnham et al., 2009). Besides these results of V. parahaemolyticus growth at 10 °C, Yoon et al., reported the observation of growth of V. parahaemolyticus in broth (Yoon et al., 2008). Many reasons could explain the different results under 10 °C storage. Different material and methods used in different studies are likely contributing factors to different observed, for example, different media, salinities, strains, number of strains, and inoculum levels.

At 15 °C, all ten V. parahaemolyticus strains grew after four days of storage.

Pathogenic V. parahaemolyticus strains 10290 and 10293 and non-pathogenic V. parahaemolyticus strain 102713Y01 grew significantly slower (P < 0.05) than other seven V. parahaemolyticus strains that were tested in this study. The differences between pathogenic and non-pathogenic V. parahaemolyticus strains are not obvious at this temperature. However, pathogenic strain 10293 grew significantly slower (P < 0.05) than other V. parahaemolyticus strains and the growth rate of non-pathogenic 100311Y11 was significantly higher than other V. parahaemolyticus strains at 20 °C. According to Yoon et al. (2008), the difference in lag time (LT) values between pathogenic and non- pathogenic V. parahaemolyticus decreased when temperature increased. This result confirms that there is not obviously different growth response between pathogenic and non-pathogenic V. parahaemolyticus at 15 °C. Since Yoon and others only used one pathogenic and one non-pathogenic V. parahaemolyticus strain in their study, it is hard to tell whether there is difference between pathogenic and non-pathogenic strains.

Based on our results, different growth rates between strains were observed in this study. Pathogenic strain 10292 decreased significantly faster than the other nine V.

parahaemolyticus strains at 5 °C and also slightly declined at 10 °C. Besides that, this pathogenic strain grew significantly faster than the other nine V. parahaemolyticus strains at 30 °C. These results indicated that pathogenic strain 10292 is more sensitive to temperatures than other V. parahaemolyticus strains tested in this study. Also, pathogenic

V. parahaemolyticus strain 10290 grew significantly slower than other V. parahaemolyticus strains at 25 °C and increased or decreased slowly under different temperatures. It indicates that this pathogenic strain might not be sensitive to the temperature changes and had relatively stable growth rate upon different temperatures.

2.6 Conclusion

Vibrio parahaemolyticus is a leading cause of illness associated with ready-to-eat

(RTE) seafood, especially shellfish. FDA regulations state that RTE fishery products have a limit of 10,000 V. parahaemolyticus cells/g, which includes pathogenic and non- pathogenic V. parahaemolyticus.

There was a significant effect of strain type (non-pathogenic vs pathogenic, P <

0.001), temperature (P < 0.001) and a strain type x temperature interaction (P = 0.026) on

µmax. At 5 °C, µmax was negative for both pathogenic and non-pathogenic strain type, which revealed die-off. Two pathogenic and one non-pathogenic strains showed negative values of µmax at 10 °C. When temperature increased (15 - 30 °C), µmax was positive for all type of strains. When evaluating differences between pathogenic and non-pathogenic

V. parahaemolyticus strains at each temperature, the only temperature that revealed significance difference of strain type was 30 °C. No significant difference of µmax was observed between 5-15 °C when evaluating the effect of temperature on µmax within either non-pathogenic or pathogenic strain type. The mean value of µmax for non-

pathogenic and pathogenic strains was 0.448 and 0.340, respectively. The faster rate of growth for non-pathogenic strains suggests the parameters set by FDA, 10,000 V. parahaemolyticus cells/g, are more likely to represent non-pathogenic strains, rather than pathogenic strains.

2.7 Acknowledgement:

This research were supported by the Agriculture and Food Research Initiative of the

USDA National Institute of Food and Agriculture grant number #2011-68003-30005 and the Coastal Oregon Marine Experiment Station’s Seafood Research & Education Center in Astoria, OR.

Reference: Burnham, V.E., Janes, M.E., Jakus, L.A., Supan, J., DePaola, A., Bell, J., 2009. Growth and Survival Differences of? Vibrio vulnificus ?and? Vibrio parahaemolyticus ?Strains during Cold Storage. J. Food Sci. 74, M314–M318. doi:10.1111/j.1750-3841.2009.01227.x Ceccarelli, D., Hasan, N.A., Huq, A., Colwell, R.R., 2013. Distribution and dynamics of epidemic and pandemic Vibrio parahaemolyticus virulence factors. Front. Cell. Infect. Microbiol. 3. doi:10.3389/fcimb.2013.00097 Dadisman, T.A., Nelson, R., Molenda, J.R., Garber, H.J., 1972. VIBRIO PARAHAEMOLYTICUS GASTROENTERITIS IN MARYLAND I. CLINICAL AND EPIDEMIOLOGIC ASPECTS. Am. J. Epidemiol. 96, 414–426. doi:10.1093/oxfordjournals.aje.a121474 Daniels, N., MacKinnon, L., Bishop, R., Altekruse, S., Ray, B., Hammond, R., Thompson, S., Wilson, S., Bean, N., Griffin, P., Slutsker, L., 2000. Vibrio parahaemolyticus Infections in the United States, 1973?1998. J. Infect. Dis. 181, 1661–1666. doi:10.1086/315459 Fukui, T., Shiraki, K., Hamada, D., Hara, K., Miyata, T., Fujiwara, S., Mayanagi, K., Yanagihara, K., Iida, T., Fukusaki, E., Imanaka, T., Honda, T., Yanagihara, I., 2005. Thermostable Direct Hemolysin of Vibrio parahaemolyticus Is a Bacterial Reversible Amyloid Toxin ? Biochemistry (Mosc.) 44, 9825–9832. doi:10.1021/bi050311s Honda, T., Ni, Y.X., Miwatani, T., 1988. Purification and characterization of a hemolysin produced by a clinical isolate of Kanagawa phenomenon-negative Vibrio parahaemolyticus and related to the thermostable direct hemolysin. Infect. Immun. 56, 961–965. Joseph, S.W., Colwell, R.R., Kaper, J.B., 2008. Vibrio Parahaemolyticus and Related Halophilic Vibrios. CRC Crit. Rev. Microbiol. 10, 77–124. doi:10.3109/10408418209113506 Kelly, M.T., Stroh, E.M., 1988. Temporal relationship of Vibrio parahaemolyticus in patients and the environment. J. Clin. Microbiol. 26, 1754–1756. Kim, Y., Lee, S., Hwang, I., Yoon, K., 2012. Effect of Temperature on Growth of Vibrio paraphemolyticus and Vibrio vulnificus in Flounder, Salmon Sashimi and Oyster Meat. Int. J. Environ. Res. Public. Health 9, 4662–4675. doi:10.3390/ijerph9124662 Letchumanan, V., Chan, K.-G., Lee, L.-H., 2014. Vibrio parahaemolyticus: a review on the pathogenesis, prevalence, and advance molecular identification techniques. Front. Microbiol. 5. doi:10.3389/fmicb.2014.00705

Liston, J. (University of W., 1990. Microbial hazards of seafood consumption. Food Technol. USA. McCarthy, S.A., DePaola, A., Cook, D.W., Kaysner, C.A., Hill, W.E., 1999. Evaluation of alkaline phosphatase- and digoxigenin-labelled probes for detection of the thermolabile hemolysin (tlh) gene of Vibrio parahaemolyticus. Lett. Appl. Microbiol. 28, 66–70. Miles, D.W., Ross, T., Olley, J., McMeekin, T.A., 1997. Development and evaluation of a predictive model for the effect of temperature and water activity on the growth rate of Vibrio parahaemolyticus. Int. J. Food Microbiol. 38, 133–142. doi:10.1016/S0168-1605(97)00100-1 Nelapati, S., Nelapati, K., Chinnam, B., 2012. Vibrio parahaemolyticus- An emerging foodborne pathogen. Vet. World 48. doi:10.5455/vetworld.2012.48-63 Nishibuchi, M., Hill, W.E., Zon, G., Payne, W.L., Kaper, J.B., 1986. Synthetic oligodeoxyribonucleotide probes to detect Kanagawa phenomenon-positive Vibrio parahaemolyticus. J. Clin. Microbiol. 23, 1091–1095. Nishibuchi, M., Kaper, J.B., 1995. Thermostable direct hemolysin gene of Vibrio parahaemolyticus: a virulence gene acquired by a marine bacterium. Infect. Immun. 63, 2093–2099. Odeyemi, O.A., 2016. Incidence and prevalence of Vibrio parahaemolyticus in seafood: a systematic review and meta-analysis. SpringerPlus 5. doi:10.1186/s40064-016- 2115-7 Pathogenic Vibrios and Food Safety - Yi-Cheng Su - Google Books [WWW Document], n.d. URL https://books.google.com/books/about/Pathogenic_Vibrios_and_Food_Safety.htm l?id=yW7MygAACAAJ (accessed 6.13.17). Raghunath, P., 2015. Roles of thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) in Vibrio parahaemolyticus. Front. Microbiol. 5. doi:10.3389/fmicb.2014.00805 Su, Y.-C., Liu, C., 2007. Vibrio parahaemolyticus: A concern of seafood safety. Food Microbiol. 24, 549–558. doi:10.1016/j.fm.2007.01.005 US Food and Drug Administration (FDA), 2005. Risk & Safety Assessment - Quantitative Risk Assessment on the Public Health Impact of Pathogenic Vibrio parahaemolyticus in Raw Oysters [WWW Document]. URL https://www.fda.gov/food/foodscienceresearch/risksafetyassessment/ucm050421. htm (accessed 6.14.17b). US Food and Drug Administration (FDA), 2011. Seafood - Fish and Fishery Products Hazards and Controls Guidance - Fourth Edition [WWW Document]. URL

https://www.fda.gov/food/guidanceregulation/guidancedocumentsregulatoryinfor mation/seafood/ucm2018426.htm (accessed 6.14.17a). Yoon, K.S., Min, K.J., Jung, Y.J., Kwon, K.Y., Lee, J.K., Oh, S.W., 2008. A model of the effect of temperature on the growth of pathogenic and nonpathogenic Vibrio parahaemolyticus isolated from oysters in Korea. Food Microbiol. 25, 635–641. doi:10.1016/j.fm.2008.04.007

Table 2.2 Viable counts of Vibrio parahaemolyticus after storage at 5 °C in TSB-Salt over 96 hours Strain Log CFU/mL 0h 24h 48h 72h 96h Pathogenic 10290 3.22±0.01Aa 2.97±0.01B 2.73±0.02C 2.53±0.04D 2.13±0.04E 10292 3.88±0.05A 3.71±0.07B 3.00±0.03C 1.85±0.03D 0.24±0.24E 10293 3.23±0.01A 3.04±0.00B 2.41±0.01C 1.92±0.01D 1.09±0.05E BE 98-2029 3.70±0.05A 3.47±0.02B 3.19±0.02C 2.43±0.08D 1.31±0.03E 027-1c1 3.76±0.02A 3.03±0.01B 2.40±0.05C 1.78±0.02D 0.95±0.05E Non-Pathogenic 100813W01 3.72±0.01A 3.41±0.02B 2.85±0.02C 2.36±0.03D 1.96±0.03E 102713Y01 3.67±0.03A 3.37±0.04B 2.73±0.02C 2.31±0.02D 1.69±0.03E 090811Y02 3.68±0.00A 3.11±0.00B 2.46±0.05C 1.57±0.04D 0.54±0.06E 100311Y04 3.84±0.02A 3.25±0.01B 2.26±0.01C 1.19±0.01D 0.39±0.08E 100311Y11 3.84±0.00A 3.10±0.02B 2.34±0.00C 1.48±0.09D NDb E a Means for each sampling time (Horizontal columns) followed by different letters were significantly different from each other (P < 0.05). b ND = Not Detected

Table 2.3 Viable counts of Vibrio parahaemolyticus after storage at 10 °C in TSB-Salt over 96 hours Strain Log CFU/mL 0h 24h 48h 72h 96h Pathogenic 10290 3.52±0.03ABa 3.49±0.01A 3.52±0.02AB 3.56±0.07B 3.74±0.01C 10292 3.88±0.05A 3.76±0.02B 3.71±0.01C 3.59±0.04D 3.34±0.02E 10293 3.52±0.06A 3.39±0.04B 2.59±0.06C 2.00±0.01D 1.28±0.10E BE 98-2029 3.70±0.05A 3.70±0.04A 3.73±0.02A 3.90±0.02B 3.93±0.01B 027-1c1 3.74±0.01A 3.81±0.06B 4.26±0.01C 4.76±0.06D 4.82±0.01E Non-Pathogenic 100813W01 3.72±0.01A 3.72±0.04A 3.75±0.02A 4.54±0.01B 4.51±0.03B 102713Y01 3.84±0.02A 3.84±0.03A 3.84±0.03A 4.25±0.01B 4.59±0.01C 090811Y02 3.75±0.01A 3.72±0.02A 3.64±0.02B 3.59±0.02C 3.43±0.03D 100311Y04 3.81±0.02A 4.05±0.02B 4.50±0.04C 4.84±0.03D 4.80±0.01D 100311Y11 3.53±0.03A 3.78±0.02B 4.22±0.01C 4.37±0.08D 4.62±0.04E a Means for each sampling time (Horizontal columns) followed by different letters were significantly different from each other (P < 0.05).

Table 2.4 Viable counts of Vibrio parahaemolyticus after storage at 15 °C in TSB-Salt over 96 hours Strain Log CFU/mL 0h 4h 8h 12h 24h 36h 48h 60h 72h 84h 96h Pathogenic

10290 3.06±0.02A 3.46±0.01 3.66±0.01 3.82±0.04 4.51±0.02 5.17±0.00F 5.69±0.01 6.45±0.02 7.81±0.01I 8.33±0.00J a B C D E G H 10292 3.49±0.07A 3.99±0.05 4.30±0.02 5.06±0.01 5.91±0.05 7.50±0.05F 8.63±0.03 8.86±0.03I 8.78±0.01H 8.76±0.00H 8.64±0.01 B C D E G G 10293 3.25±0.03A 3.56±0.00 3.84±0.03 3.98±0.02 4.14±0.06 4.67±0.01F 4.97±0.06 5.46±0.01 5.92±0.01I 6.67±0.03J 7.78±0.01 B C D E G H K BE 98- 3.33±0.01A 3.47±0.04 3.72±0.00 4.22±0.01 5.76±0.02 7.69±0.01G 8.49±0.06J 8.87±0.04 8.03±0.01I 7.61±0.01F 7.92±0.01 2029 B C D E K H 027-1c1 3.65±0.01A 4.06±0.02 4.38±0.04 4.96±0.01 6.71±0.04 8.46±0.05F 8.91±0.02I 8.89±0.03I 8.86±0.05I 8.74±0.00H 8.66±0.08 B C D E G Non-Pathogenic 100813W0 3.56±0.06A 3.79±0.07 4.14±0.02 4.50±0.05 6.28±0.03 7.98±0.01F 8.96±0.04I 8.96±0.03I 8.91±0.04I 8.76±0.02H 8.67±0.04 1 B C D E G 102713Y01 3.72±0.02A 4.02±0.00 4.30±0.03 4.77±0.00 5.08±0.03 5.16±0.03F 5.63±0.06 6.68±0.08 8.48±0.09I 8.98±0.01J 8.97±0.04J B C D E G H 090811Y02 3.82±0.01A 4.22±0.02 4.75±0.01 5.35±0.00 7.35±0.00 8.95±0.01G 8.92±0.01 8.92±0.03 8.90±0.03F 8.85±0.01F 9.16±0.02 B C D E G G G H 100311Y04 3.79±0.00A 4.20±0.02 4.56±0.04 5.28±0.02 7.31±0.01 8.90±0.03G 8.91±0.02 8.97±0.01I 8.86±0.01F 8.88±0.03FG 8.84±0.06 B C D E H H G H F 100311Y11 3.51±0.02A 3.97±0.01 4.78±0.06 5.57±0.05 7.13±0.00 8.48±0.03F 8.88±0.05I 8.93±0.04J 8.84±0.03I 8.75±0.04H 8.60±0.07 B C D E G a Means for each sampling time (Horizontal columns) followed by different letters were significantly different from each other (P < 0.05).

Table 2.5 Viable counts of Vibrio parahaemolyticus after storage at 20 °C in TSB-Salt over 36 hours Strain Log CFU/mL 0h 4h 8h 12h 24h 36h Pathogenic 10290 3.06±0.02Aa 3.83±0.01B 4.80±0.02C 7.12±0.32D 7.83±0.00E 8.20±0.01F 10292 3.49±0.07A 4.37±0.02B 7.03±0.00C 8.88±0.05F 8.52±0.06F 8.28±0.01E 10293 3.39±0.04A 3.96±0.05B 5.06±0.03C 6.06±0.05D 6.98±0.06F 6.78±0.04E BE 98-2029 3.33±0.01A 4.55±0.01B 6.29±0.01C 7.51±0.00D 8.92±0.01F 8.48±0.02E 027-1c1 3.65±0.01A 4.57±0.05B 6.50±0.05C 8.10±0.03D 8.89±0.07E 8.87±0.05E Non-Pathogenic 100813W01 3.56±0.06A 4.49±0.06B 6.37±0.02C 7.94±0.01D 8.77±0.00F 8.66±0.08E 102713Y01 3.86±0.06A 4.74±0.01B 6.52±0.02C 8.17±0.01D 8.85±0.06F 8.73±0.07E 090811Y02 3.82±0.01A 5.29±0.01B 7.17±0.02C 8.56±0.05D 8.99±0.03E 8.98±0.00E 100311Y04 3.79±0.00A 5.24±0.02B 7.03±0.02C 8.51±0.01D 9.01±0.02F 8.95±0.03E 100311Y11 3.51±0.02A 4.65±0.02B 7.10±0.03C 8.78±0.02E 8.70±0.02D 8.70±0.01D a Means for each sampling time (Horizontal columns) followed by different letters were significantly different from each other (P < 0.05).

Table 2.6 Viable counts of Vibrio parahaemolyticus after storage at 25 °C in TSB-Salt over 12 hours Strain Log CFU/mL 0h 2h 4h 6h 8h 10h 12h Pathogenic 10290 3.50±0.01Aa 3.58±0.03B 4.39±0.04C 5.26±0.04D 6.29±0.04E 7.01±0.01F 7.70±0.04G 10292 3.66±0.04A 3.74±0.04B 4.51±0.01C 6.26±0.02D 7.74±0.02E 8.71±0.07F 8.91±0.03G 10293 3.28±0.03A 3.60±0.00B 4.29±0.01C 5.26±0.01D 6.27±0.01E 7.31±0.04F 7.95±0.05G BE 98-2029 3.51±0.03A 3.76±0.05B 4.90±0.01C 6.20±0.01D 7.64±0.01E 8.72±0.00F 8.93±0.02G 027-1c1 3.80±0.10A 4.20±0.01B 5.37±0.03C 6.74±0.03D 8.43±0.03E 8.75±0.10F 8.99±0.07G Non-Pathogenic 100813W01 3.56±0.02A 3.80±0.02B 5.07±0.05C 6.85±0.04D 8.09±0.05E 8.83±0.08F 8.98±0.02G 102713Y01 3.74±0.03A 3.84±0.07B 5.12±0.01C 6.62±0.04D 8.10±0.02E 8.88±0.03F 8.99±0.05G 090811Y02 3.62±0.00A 3.89±0.01B 5.20±0.06C 6.67±0.06D 7.88±0.07E 8.87±0.00F 8.89±0.03F 100311Y04 3.76±0.03A 4.07±0.01B 5.48±0.01C 7.13±0.01D 8.38±0.06E 8.92±0.03F 9.04±0.01G 100311Y11 3.55±0.01A 3.82±0.01B 5.29±0.03C 6.80±0.02D 8.51±0.11E 8.91±0.01F 8.95±0.02F a Means for each sampling time (Horizontal columns) followed by different letters were significantly different from each other (P < 0.05).

Table 2.7 Viable counts of Vibrio parahaemolyticus after storage at 30 °C in TSB-Salt over 12 hours Strain Log CFU/mL 0h 2h 4h 6h 8h 10h 12h Pathogenic 10290 3.73±0.01Aa 4.16±0.16B 5.38±0.03C 6.76±0.03D 7.89±0.01E 8.57±0.05F 8.70±0.04G 10292 3.35±0.01A 3.81±0.06B 5.89±0.01C 8.44±0.05D 8.89±0.07F 8.94±0.05F 8.68±0.03E 10293 3.38±0.02A 3.88±0.06B 5.66±0.01C 7.47±0.01D 8.46±0.03F 8.54±0.03G 8.26±0.02E BE 98-2029 3.56±0.07A 4.27±0.02B 5.90±0.01D 5.59±0.04C 8.82±0.05G 8.49±0.00F 7.62±0.03E 027-1c1 3.69±0.01A 4.65±0.05B 6.18±0.02C 8.77±0.03D 8.96±0.04E 8.93±0.00E 8.93±0.07E Non-Pathogenic 100813W01 3.53±0.03A 3.82±0.05B 5.87±0.02C 8.07±0.00D 8.85±0.04G 8.79±0.04F 8.39±0.04E 102713Y01 3.57±0.02A 3.75±0.02B 5.73±0.07C 8.03±0.04D 8.87±0.01E 8.97±0.01F 8.88±0.00E 090811Y02 3.71±0.01A 3.75±0.03A 5.84±0.04B 7.95±0.02C 8.81±0.02E 8.98±0.02E 8.91±0.02F 100311Y04 3.75±0.09A 4.13±0.03B 6.47±0.01C 8.44±0.05D 8.89±0.02F 8.86±0.02F 8.64±0.04E 100311Y11 3.55±0.02A 3.78±0.08B 6.36±0.04C 8.55±0.04D 8.83±0.07E 8.88±0.07F 8.82±0.02E a Means for each sampling time (Horizontal columns) followed by different letters were significantly different from each other (P < 0.05).

2 2 Table 2.8 Fitted parameters of growth rates, die-off rates (µmax) and adjusted R (Adj. R ) for V. parahaemolyticus strains after storage at various temperatures Temperature 5 10 15 20 25 30 (°C) µ (log Adj. µ (log Adj. µ (log Adj. µ (log Adj. µ (log Adj. µ (log Adj. max 2 max 2 max 2 max 2 max 2 max 2 CFU/h)a R CFU/h) R CFU/h) R CFU/h) R CFU/h) R CFU/h) R Pathogenic Strain 10290 -0.01b,cA 0.98 0.01bA 0.97 0.06A 0.99 0.33B 0.95 0.43A 0.99 0.67A 0.99 10292 -0.06B 0.99 -0.01A 0.91 0.11B 0.99 0.49E 0.94 0.84DE 0.99 1.41G 0.99 10293 -0.02A 0.95 -0.02A 0.96 0.04A 0.96 0.23A 0.99 0.53B 0.99 0.98D 0.99 BE 98-2029 -0.04A 0.98 0.01A 0.99 0.13BC 0.96 0.36B 0.99 0.73C 0.99 0.73B 0.93 027-1c1 -0.03A 0.99 0.02A 0.99 0.16C 0.99 0.47E 0.99 0.81D 0.99 0.84C 0.96 Non-pathogenic Strain 100813W01 -0.02A 0.99 0.08B 0.99 0.16C 0.99 0.46DE 0.99 0.81D 0.99 1.22F 0.99 102713Y01 -0.02A 0.98 0.02A 0.98 0.06A 0.93 0.46DE 0.99 0.80D 0.99 1.23F 0.99 090811Y02 -0.03A 0.98 -0.003A 0.90 0.18C 0.99 0.42CD 0.99 0.73C 0.99 1.16E 0.99 100311Y04 -0.04A 0.99 0.01A 0.89 0.18C 0.99 0.41C 0.99 0.80D 0.99 1.26F 0.99 100311Y11 -0.03A 0.99 0.01A 0.97 0.15BC 0.99 0.65F 0.99 0.88E 0.99 1.45G 0.99 a The growth or die-off rate (µmax) of each strain during storage was obtained from the primary model fit generated from DMFit Excel Add-In software b Negative values represent the die-off rates. Positive values represent the growth rates. c Means for each sampling time (Vertical columns) followed by different letters were significantly different from each other (P < 0.05).

Chapter 3

Antimicrobial Activity of Fruit Extracts and Fruit Juice against L. monocytogenes, Salmonella and V. parahaemolyticus

Jingyi Du, Yi-Cheng Su, Christina Ann DeWitt, Jovana Kovacevic

(To be Submit to Food Microbiology)

3.1 Abstract

Listeria monocytogenes, Salmonella spp. and Vibrio parahaemolyticus are

problematic in ready-to-eat (RTE) seafoods. To explore ways of controlling these

foodborne pathogens often contaminated in RTE foods, this study investigated

antimicrobial activity of fruit extracts against Listeria monocytogenes, Salmonella strains

and V. parahaemolyticus.

Pomegranate peel (PPE) exhibited a high antimicrobial activity similar to cranberry

juice (CJ), while limited inhibition was observed for blueberry, strawberry, plum meat,

whole plum and pomegranate seed. The minimum inhibitory concentrations (MIC) and

the Minimum bactericidal concentrations (MBC) of PPE for L. monocytogenes were

22.5% and 37.5%, respectively. The MBC of PPE against Salmonella was 37.5%. The

MBC of PPE against V. parahaemolyticus strains was 8.1%. The MIC of PPE against

Salmonella was >22.5% and < 37.5%. The MIC of PPE against V. parahaemolyticus

strains was >4.86% and <8.1%, which was much lower that against L. monocytogenes

and Salmonella. The MBC of CJ against L. monocytogenes and Salmonella was 25% and

that against V. parahaemolyticus strains was 12.5%. For MIC of CJ against L.

monocytogenes was 12.5%, but that against Salmonella was >12.5% and <25%. The MIC

of CJ against V. parahaemolyticus strains was >6.25% and <12.5%. This study

demonstrates potential of PPE to be used as natural antimicrobials in RTE seafood.

Keywords: Listeria monocytogenes, Salmonella spp., Vibrio parahaemolyticus, cranberry

juice, pomegranate peel

3.2 Introduction

Seafood is a low-fat source of protein and contains long-chain omega-3 fatty acids, which makes it a popular food to include in the diet (Hellberg et al., 2012). There are many different kinds of seafood products that are at higher risk for contamination by pathogens. These include fresh products that are eaten raw or minimally processed and ready-to-eat (RTE) products. Food-borne pathogens of concern for RTE seafood products, include L. monocytogenes, Salmonella and V. parahaemolyticus (Amagliani et al., 2012; Eklund et al., 2007; Iwamoto et al., 2010; Jami et al., 2014; Lianou and Sofos,

2007; Su and Liu, 2007). Consumption of L. monocytogenes, Salmonella, and V. parahaemolyticus-contaminated RTE seafood causes mild gastroenteritis to severe infections of the blood stream or the central nervous system (Carpentier and Cerf, 2011).

(Carpentier and Cerf, 2011). Many outbreaks and recalls of RTE seafood related to L. monocytogenes, Salmonella, and V. parahaemolyticus were reported to the United States

Center for Disease Control and Prevention (Lee et al., 2015; Lianou and Sofos, 2007;

Newton et al., 2012; Su and Liu, 2007). There is an estimate of 80,000 illnesses caused by Vibrio species each year in United State (Newton et al., 2012). The different tolerable levels of L. monocytogenes for RTE seafood vary from country to country. In United

States, Australia, Italy, and New Zealand, a zero-tolerance policy (absence of the pathogen in 25 g samples of foods) has been established (FAO, 1999; FDA, 2011). There is also a zero-tolerance policy (absence of the pathogen in 25 g samples of foods) for

Salmonella spp. according to the US Food and Drug Administration (FDA, 2011). Post- harvest processed seafood (clams, mussels, oysters, and whole and roe-on scallops) should not exceed a level of 30 MPN of V. parahaemolyticus per gram in United States.

For other RTE fishery products, the V. parahaemolyticus contamination level is required to be less than 104 cells per gram (FDA, 2011; Su and Liu, 2007).

Consumers are very concerned about the quality and hygiene of food products. They also prefer to have healthier food products with no additives or natural additives (Rai and

Chikindas, 2011). Fruits, especially berries, are rich in different phenolic compounds, including flavonoids (anthocyanins, flavonols, flavones, flavanols, flavanones, and isoflavonoids), stilbenes, tannins, and phenolic acids (Chung et al., 1998; Paredes-López et al., 2010). Studies have indicated that many berries have antimicrobial bioactive compounds and also might be able to control the wild scope of pathogens (Paredes-López et al., 2010; Puupponen-Pimiä et al., 2005). Studies have investigated the antimicrobial activity of cranberry in cooked boneless pork, cooked ground beef, and cod fish fillet and shrimp (Apostolidis et al., 2008; Lin et al., 2005; Xi et al., 2011). Also, the antimicrobial activity of pomegranate extracts have been evaluated in fresh chilled cold fish, ground beef, chicken, and RTE meat pate (Al-Zoreky, 2009; Emam-Djomeh et al., 2015;

Hayrapetyan et al., 2012; Kanatt et al., 2010). Valtierra-Rodríguez et al. (2010) tested inhibition growth of plum and lime extracts added to chicken skins. Many studies have also demonstrated the antimicrobial activity of freeze-dried or rotary evaporated fruit extracts (Emam-Djomeh et al., 2015; Kanatt et al., 2010; Negi and Jayaprakasha, 2003;

Puupponen-Pimia et al., 2001). However, these studies were not conducted to investigate the antimicrobial activity by using fresh fruit extracts. This study will investigate the antimicrobial activity of fresh fruit extracts against L. monocytogenes, Salmonella and V. parahaemolyticus, major pathogens of concern for many RTE seafood products.

3.3 Material and Methods

3.3.1 Bacteria and culture preparation

Five strains of Listeria monocytogenes, four strains of Salmonella and five clinical V. parahaemolyticus strains were used in this experiment (Table 3.1). V. parahaemolyticus frozen stock culture was activated in 10 ml of tryptic soy broth supplemented with 1.5%

NaCl (TSB-Salt). Frozen stock culture of other pathogens were activated in 10 ml of tryptic soy broth (TSB; Difco, Becton, Dickson, Spark, MD, USA) and incubated at 35 ±

2 °C overnight. One loopful of enriched V. parahaemolyticus culture was streaked on thiosulfate citrate bile salt sucrose (TCBS; Difco, Becton, Dickson, Spark, MD, USA) plate and incubated overnight at 35 ± 2 °C. A single colony was picked from the TCBS plate and enriched in 10 ml of TSB-Salt at 37 °C for 4h. For enriched Listeria monocytogenes and Salmonella cultures, one loopful of each enrichment was transferred to 10 ml of TSB and incubated at 35 ± 2 °C for 12h. The enriched cells were harvested by centrifugation (Sorvall RC-5B, Kendri Laboratory products, Newtown, CT, USA) at

3000 × g at 5 °C for 15min to get the 108- 109 CFU/ml culture suspension.

3.3.2 Preparation of fruit extract and fruit juice

3.3.2.1 Fruit Pomace and juice for antimicrobial activity determination

Fresh plums, pomegranates, blueberries, and strawberries were obtained from local supermarket to create fresh fruit extracts. One commercial cranberry juice (CJ; Simple truth®) was utilized as a reference for effectiveness of fresh fruit extract on antimicrobial activity.

The following fresh fruit pomace preparations were made: Whole Plum Pomace

(WPLP), Plum Meat Pomace (PMP), Pomegranate Seed Pomace (PSP), Whole Blueberry

Pomace (WBP) and Whole Strawberry Pomace (WSP). Pomaces were prepared by blending (Waring commericial, Torrington, CT, USA) for 1 min at room temperature. In addition, to get 25% plum peel pomace (PLPP), whole pomegranate pomace (WPOP) and pomegranate peel pomace (POPP), 5 grams of fresh plum peel, whole pomegranate, or pomegranate peel were blended with 15 ml of sterile distilled water for 1 min at room temperature. Each sample preparation was measured with a pH meter (VWR® symp

Hony™ benchtop meters, Radnor, PA, USA).

3.3.2.2 PPE20% and PPE33% for antimicrobial studies

Ten grams of pomegranate peels were blended with 20 and 40 ml of sterile deionized water (DI) for 1 min at room temperature to prepare PPE33% and PPE20%, respectively.

The PPE was centrifuged at 5000 × g for 20 min at room temperature.

3.3.2.3 Color reduced treatment of extracts, PPE20%, PPE33% and CJ

In addition, for color-reduced studies the extracts/juice were passed through

Discovery DPA-6S Solid Phase Extraction tubes (Sigma-Aldrich, St. Louis, Missouri,

USA) to reduce the color.

3.3.2.4 Pomegranate peel extract (PPE50%) for Minimal Inhibition Concentration (MIC) and Minimum Bactericidal Concentration (MBC) determination

Forty grams of pomegranate peels were blended with 40 ml of sterile deionized water

(DI) for two min at room temperature to get 50% w/v PPE. The PPE50% was centrifuged at 8,000 × g for 30 min at room temperature. This PPE50% was used to determine the MIC and MBC against L. monocytogenes, Salmonella and V. parahaemolyticus by using TSB or TSB-Salt to obtain final concentrations of 37.5%, 22.5%, and 13.5% PPE.

3.3.3 Determination of TPC in fruit pomaces, PPE and CJ

The TPC in fruit pomaces, PPE and CJ was determined by the Folin-Ciocalteau method (Waterhouse, 2001). Two hundred microliters of each fruit pomaces, PPE and CJ were added to 15.8 ml of deionized water (DI). One milliliter of Folin-Ciocalteau Phenol reagent (MP Biomedicals, Solon, OH, USA) was added and mixed by vortexing. After five minutes, three milliliters of sodium carbonate solution were added, mixed, and held at room temperature for 2 h. TPC in fruit pomaces, PPE and CJ were determined by UV-

Vis Spectrophotometer (UV-2401 PC, Shimadzu, Kyoto, Japan) with absorbance measured at 765 nm. Results are reported at Gallic Acid Equivalent of each sample according to the standard curve prepared from the absorbance values.

3.3.4 Determination of antimicrobial activity of fruit pomaces against L. monocytogenes and Salmonella

One milliliter of Listeria or Salmonella culture suspension of each strain was added to

100 ml warm (45 °C) tryptic soy agar (TSA; Difco, Becton, Dickson, Spark, MD, USA) to reach 106-7 CFU/ml in the medium. The TSA containing either L. monocytogenes or

Salmonella was poured into petri dishes and allowed to solidify at room temperature.

Five wells (9 mm in diameter) were created on the TSA plates by using a sterile cork borer. Each plant extract solution was added to the wells and plates were incubated at

37 °C for 48 h. Inhibition zones of each plant extract solution were measured.

3.3.5 Determination of MIC and MBC of PPE and CJ against L. monocytogenes, Salmonella and V. parahaemolyticus

The L. monocytogenes and Salmonella culture suspension of each strain was diluted to reach a level of 105-6 CFU/ml in TSB. The V. parahaemolyticus culture suspension was diluted to reach the same level in TSB-Salt. All dilutions of CJ or PPE were inoculated with a L. monocytogenes, Salmonella or V. parahaemolyticus culture suspension in sterile

transparent glass tubes to reach the final inoculation level. A positive control (bacteria without the antimicrobial substrates) and a negative control (medium only) were also prepared. All tubes were incubated at 35 ± 2 °C for 48 h. Results were reported as means of triplicate tests. Bacterial growth reductions were determined by the pour plate method using TSA for L. monocytogenes and Salmonella and tryptic soy agar supplemented with

1.5% NaCl (TSA-Salt) for V. parahaemolyticus. The MBC was the lowest concentration at which no growth on the pour plate occurred, which means this concentration could not only inhibit the growth of pathogen, but also kill the pathogen and have zero pathogen cell on the plate. The MIC values was the lowest concentration at which a two log reduction occurred, which means this concentration could inhibit the growth of pathogen, but still have growth of pathogen on plate.

3.3.6 Statistical analysis

Bacterial counts of each strain at different time were analyzed using Tukey-Kramer multiple-comparison Test in R program (R foundation, Vienna). Three-Way ANOVA were used in SigmaPlot (Systat Software, San Jose, CA). The significance level was P <

0.05.

Table 3.1 List of foodborne pathogens information used in this study Species Strain No. Source of Isolation Listeria monocytogenes Scott A Clinical sample Listeria monocytogenes M0507 Retail frozen shrimp Listeria monocytogenes SFL0404 Retail frozen shrimp Listeria monocytogenes F5027 Raw milk Listeria monocytogenes H222 Potatoes Salmonella Weltevreden SFL0319 Shrimp Salmonella Newport ATCC 6962 Meat Salmonella Newport H1275 Sprout Salmonella Typhimurium ATCC 14028 Chicken Vibrio parahaemolyticus 10290 Clinical Sample, Washington Vibrio parahaemolyticus 10292 Clinical Sample, Washington Vibrio parahaemolyticus 10293 Clinical Sample, Washington Vibrio parahaemolyticus BE 98-2029 Clinical Sample, Texas Vibrio parahaemolyticus 027-1c1 Clinical Sample, Oregon

3.4 Result

3.4.1 Determination of TPC

Among all tested treatments, CJ had the lowest pH value (2.91; Table 3.2) and a relatively low TPC (2.37 mg/ml). The highest TPC (44.36 mg/ml) was in POPP, which also had a relatively high pH value (3.81). In general, pomegranate peel has higher TPC than plums, strawberry, blueberry and pomegranate seed. Previous research has demonstrated peels are a source of phenolic compounds such as tannins (Gil et al., 2000).

3.4.2 Determination of antimicrobial activity, MIC and MBC

The antimicrobial activities of all samples in Table 3.2 were preliminarily screened using well diffusion inhibition studies. After 48 hours incubation at 35 ± 2 °C, the inhibition zones of WPLP, PMP, PSP, WBP, WSP and PLPP against L. monocytogenes and Salmonella were smaller than those of CJ, WPOP and POPP (data not shown). There was some difficulty in determining the zone of inhibition for POPP due to color from the peel. As a result, extracts were prepared from the peel of pomegranate (PPE20%, PPE33%) in order to reduce color interference. In addition, pomegranate sample extracts were passed through a solid phase extraction column containing a reverse phase polyamide resin in order to further reduce color intensity. TPC of color-reduced CJ and PPE were lower than those of non-color-reduced CJ and PPE, however, pH was not affected.

Comparing the inhibition zones of color-reduced and non-color-reduced samples, color- reduced samples had smaller inhibition zones (data not shown). Based on this preliminary inhibition study, PPE was selected to test MIC and MBC against L. monocytogenes,

Salmonella and V. parahaemolyticus. CJ was used as a positive control.

To get the lower concentration of PPE in MIC and MBC test, PPE50% was used.

Concentration (P ˂ 0.0001), pathogen type (P ˂ 0.0001) and treatment type (PPE vs CJ;

P ˂ 0.0001) have an effect on log reductions. In addition, there was a significant three- way interaction between all main effects (P ˂ 0.0001). At the lowest concentrations of each treatment, there was not a significant pathogen type by treatment type interaction.

There was a significant interaction at the higher concentrations of each treatments.

Significant differences between pathogen types were observed for all concentrations of treatment, except high concentration of PPE. At 37.5% PPE, log reduction of Salmonella and V. parahaemolyticus was not significantly different. The least square means of PPE was 3.688 log reduction and for CJ was 4.177.

No growth of L. monocytogenes and Salmonella was observed when strains were exposed to 37.5% PPE (Table 3.3). At 22.5% PPE, the log reductions of Salmonella were >1 log CFU/ml, while those of L. monocytogenes strains were >2 log CFU/ml. The log reduction of 22.5% PPE against Salmonella strains ranged from 1.66 log CFU/ml to

1.97 log CFU/ml, which was significantly (P < 0.05) lower than those of 22.5% PPE against L. monocytogenes (2.32 log CFU/ml – 3.22 log CFU/ml). For 13.5% PPE, the growth of L. monocytogenes was inhibited after 48 hours incubation. However, this concentration of PPE could not inhibit the growth of all four Salmonella strains. After 48 hours, the viable counts of Salmonella strains increased to 6-7 log CFU/ml.

For V. parahaemolyticus, all concentrations of PPE showed no growth (Table 3.3). As a result, lower concentrations were evaluated. No growth was observed at 4.86% PPE for

V. parahaemolyticus strains 10290 and 10293 (Table 3.4). However, for 4.86% PPE, the log reduction of V. parahaemolyticus strains 10292, BE 98-2029, and 027-1c1 were 0.51,

2.1, and 3.03 log CFU/ml, respectively. For 2.92% PPE, the population of all five V. parahaemolyticus strains was over 7 log CFU/ml, which is 1-2 log CFU/ml higher than the initial inoculum. Compared to the concentration of PPE needed to completely inhibit the growth of L. monocytogenes and Salmonella, a considerably lower concentration of

PPE was able to inhibit growth of V. parahaemolyticus.

No growth of L. monocytogenes and Salmonella was observed when strains were incubated in the mix of 25% CJ (Table 3.5). At 12.5% CJ, inhibition of L. monocytogenes strains was achieved the log reduction ranged from 0.16 log CFU/ml to 1.63 log CFU/ml after 48h. Conversely, Salmonella strains were not inhibited and increased to 8 log

CFU/ml over the same time period.

For 1.56% CJ, complete inhibition of V. parahaemolyticus strain BE 98-2029 was observed after 48 hours (Table 3.4). However, the population of this strain increased by

3.47 log CFU/ml when only exposed to 0.78% CJ. No growth was observed at 3.13% CJ for V. parahaemolyticus strains 10292 and 027-1c1. Besides that, the viable counts of V. parahaemolyticus strain 10292 and 027-1c1 were increased to 8.87 and 8.71 log CFU/ml, respectively, in 1.56% CJ after incubation for 48 hours. No growth was observed in

12.5% CJ for V. parahaemolyticus strain 10290 and 10293. Also, the viable counts of V. parahaemolyticus strain 10290 and 10293 decreased by 1.78 and 4.67 log CFU/ml in

6.25%CJ, respectively. In 3.13% CJ, the log reduction of V. parahaemolyticus strain

10290 and 10293 was 2.33 and 4 log CFU/ml, respectively. For 1.56% CJ, the population of V. parahaemolyticus strain 10290 increased to 8.41 log CFU/ml while those of V. parahaemolyticus strain 10293 decreased to 1.55 log CFU/ml after 48 hours. Moreover,

the total counts of V. parahaemolyticus strain 10293 increased by 3.15 log CFU/ml in

0.78% CJ.

Results demonstrate that the MBC for L. monocytogenes and Salmonella was 37.5%

PPE and 25% for CJ. MBC for was 8.1%PPE and 12.5%CJ. The MIC for L. monocytogenes was 22.5% PPE and >12.5% and <25% for CJ. For Salmonella, the MIC was >22.5% and <37.5% PPE and for CJ was >12.5% and <25%. For V. parahaemolyticus, the MIC was <8.1% and >4.86% PPE and for CJ was >6.25% and

<12.5%.

3.5 Discussion

In this study, CJ and PPE showed strong antimicrobial activity against L. monocytogenes, Salmonella and V. parahaemolyticus. A lower antimicrobial activity, lower TPC and lower pH value was found in the color-reduced CJ and PPE compared to unaltered extracts. This implies that phenolic compounds from the color of fruit played an important role in antimicrobial activity. It also indicates that low pH might be another reason for antimicrobial activity of CJ. This hypothesis also been discussed by Wu et al. in 2008. They tested same pH level CJ and acidic solutions and found that CJ showed stronger antimicrobial effects (Wu et al., 2008). Because of the loss of H+-ATPase, low pH could cause sublethal injury to cell membranes. This damage also enhances the ability of phenolic compounds in CJ as antimicrobial compounds (Lin et al., 2004). Other studies also indicated that phenolic compounds are key to inhibiting the growth of pathogens. Puupponen-Pimia et al. indicated that the inhibitory effects of berry extracts might be caused by complex phenolic polymers instead of simple phenolic compounds

(Puupponen-Pimia et al., 2001). Caillet et al. tested fractions of phenolic acids,

anthocyanins, flavonols or proanthocyanidins derived from cranberries and found that all of them had antimicrobial activity against many foodborne pathogens (Caillet et al.,

2012). Therefore, both low pH and phenolic compounds contribute to the antimicrobial activity of CJ.

Like CJ, PPE is another great source of phenolic compounds, such as ellagic acid, catechins, and chlorogenic, caffeic and ferulic acids, and other flavonoids (Negi and

Jayaprakasha, 2003). A possible reason for inhibition of pathogen growth by phenolics might be from damage to cell walls and membranes. Wu et al. (2008) did a TEM study and found out that CJ has a strong, damaging effect on the cell wall, cell membrane and also induced cell lysis. Because of cell wall differences, damage might occurs differently in Gram-positive and Gram-negative bacteria (Puupponen-Pimia et al., 2001). Compared to the Gram-negative bacteria, Gram-positive bacteria have simpler membrane structure.

Therefore, hyperacidification of membrane and disruption of H+-ATPase of bacteria cell wall might be easier for Gram-positive bacteria than Gram-negative bacteria (Vattem et al., 2004). That is likely why antimicrobial activity against L. monocytogenes was stronger than that of Salmonella at 12.5% CJ.

Phenolic acids and flavonoids contribute to membrane perforation and disruption of membrane fluidity (Gullon et al., 2016). Additionally, pomegranate peel is rich in tannins. Cowan hypothesized that the antimicrobial mechanism of PPE might inactivate microbial adhesions, enzymes, and cell envelope transport proteins, and modify the morphology of microorganisms (Cowan, 1999; Puupponen-Pimia et al., 2005). In addition, tannins may combine with metal ions (e.g. Fe++ and Cu++) to affect the metalloenzymes of bacteria (Puupponen-Pimia et al., 2005). Some studies showed that

pomegranate extract had limited antimicrobial activity against E. coli (Kanatt et al., 2010;

Negi and Jayaprakasha, 2003). Other studies also showed that plant extracts had less effective antimicrobial activity against Gram-negative bacteria (Oliveira et al., 2008). In our study, the antimicrobial activity of PPE against L. monocytogenes is stronger compared to Salmonella strains. These results in hazelnut are similar to the observations found in other studies(Oliveira et al., 2008).

Ellagic acid has been found in cranberry and pomegranate peel and its partial hydrophobicity could act at the membrane-water interface of the bacteria (Vattem et al.,

2004). Since ellagic acid stacks on the membrane of bacteria, it could cause a membrane disruption or destabilization. This action of ellagic acid is required for Gram-negative bacteria because the second layer present is more hydrophobic (Vattem et al., 2004).

More peptidoglycan has been found in the cell wall of Gram-positive bacteria which causes cell wall rigidity (Seltmann and Holst, 2002). This could explain why lower concentration of CJ and PPE against V. parahaemolyticus was needed to achieve the same log reduction when compared to L. monocytogenes. Vattem et al. hypothesized that

E. coli O157:H7 might manage acidic stress better than V. parahaemolyticus. Ellagic acid might disrupt the membrane of V. parahaemolyticus by key ion channels which make it sensitive to phenolic compounds (Vattem et al., 2004). That might be the reason that the antimicrobial activity of CJ and PPE against V. parahaemolyticus was stronger than that against Salmonella strains test in this study.

For L. monocytogenes and Salmonella, the lower concentration of CJ has higher viable counts. However, for pathogenic V. parahaemolyticus strain 10290, the population of V. parahaemolyticus in 3.13% CJ is lower than that in 6.25% CJ. Similar results were

reported demonstrating higher populations at lower concentrations were reported by (Wu et al., 2008). They found lower populations of L. monocytogenes in 25 when compared to 50 µl/ml cranberry-BHI after 3 and 5 days storage. They suggest these results are the result of nutrient availability. At the higher concentration, more nutrients are available than at the lower concentration, thus counteracting antimicrobial effects.

3.6 Conclusion

Vibrio parahaemolyticus, Listeria monocytogenes, Salmonella spp. are pathogens that reading contaminate in ready-to-eat (RTE) seafood, such as smoked fish and shellfish, seafood salad, cooked shrimp and crabmeat, and raw seafood. This study was conducted to explore natural ways of controlling these pathogens in seafood by using natural fruit extracts and juice.

When plum, plum meat, plum peel, pomegranate seed, blueberry, strawberry and plum peel were tested for their antimicrobial activity, limited inhibition was observed.

Pomegranate peel (PPE) exhibited a high antimicrobial activity similar to cranberry juice

(CJ). There was a significant effect of concentration (P ˂ 0.0001), pathogen type (P ˂

0.0001) and treatment type (PPE vs CJ; P ˂ 0.0001) on log reductions. Also, there was a significant three-way interaction between all main effects (P ˂ 0.0001). The MBC of PPE against L. monocytogenes and Salmonella was 37.5% and for V. parahaemolyticus was

8.1%. The MBC of CJ against L. monocytogenes and Salmonella was 25%, against V. parahaemolyticus was 12.5%.

The MIC of PPE against L. monocytogenes was 22.5% while the MIC of PPE against

Salmonella was >22.5% and < 37.5%. The MIC of PPE against V. parahaemolyticus strains was >4.86% and <8.1%, which was much lower that against L. monocytogenes

and Salmonella. The MIC of CJ against L. monocytogenes was 12.5%, but this concentration did not fully inhibit the growth of Salmonella strains. The MIC of CJ against Salmonella was >12.5% and < 25% and that against V. parahaemolyticus was>6.25% and < 12.5%.

3.7 Acknowledgement

This research were supported by the Agriculture and Food Research Initiative of the

USDA National Institute of Food and Agriculture grant number #2011-68003-30005 and the Coastal Oregon Marine Experiment Station’s Seafood Research & Education Center in Astoria, OR.

Reference: Al-Zoreky, N.S., 2009. Antimicrobial activity of pomegranate (Punica granatum L.) fruit peels. Int. J. Food Microbiol. 134, 244–248. doi:10.1016/j.ijfoodmicro.2009.07.002 Amagliani, G., Brandi, G., Schiavano, G.F., 2012. Incidence and role of Salmonella in seafood safety. Food Res. Int. 45, 780–788. doi:10.1016/j.foodres.2011.06.022 Apostolidis, E., Kwon, Y.-I., Shetty, K., 2008. Inhibition of Listeria monocytogenes by oregano, cranberry and sodium lactate combination in broth and cooked ground beef systems and likely mode of action through proline metabolism. Int. J. Food Microbiol. 128, 317–324. doi:10.1016/j.ijfoodmicro.2008.09.012 Caillet, S., Côté, J., Sylvain, J.-F., Lacroix, M., 2012. Antimicrobial effects of fractions from cranberry products on the growth of seven pathogenic bacteria. Food Control 23, 419–428. doi:10.1016/j.foodcont.2011.08.010 Carpentier, B., Cerf, O., 2011. Review — Persistence of Listeria monocytogenes in food industry equipment and premises. Int. J. Food Microbiol. 145, 1–8. doi:10.1016/j.ijfoodmicro.2011.01.005 Chung, K.-T., Wei, C.-I., Johnson, M.G., 1998. Are tannins a double-edged sword in biology and health? Trends Food Sci. Technol. 9, 168–175. doi:10.1016/S0924- 2244(98)00028-4 Cowan, M.M., 1999. Plant Products as Antimicrobial Agents. Clin. Microbiol. Rev. 12, 564–582. Eklund, M., Jinneman, K., Wekell, M., 2007. Incidence and Behavior of Listeria monocytogenes in Fish and Seafood, in: Ryser, E., Marth, E. (Eds.), Listeria, Listeriosis, and Food Safety, Third Edition. CRC Press, pp. 617–653. doi:10.1201/9781420015188.ch15 Emam-Djomeh, Z., Moghaddam, A., Yasini Ardakani, S.A., 2015. Antimicrobial Activity of Pomegranate ( Punica granatum L.) Peel Extract, Physical, Mechanical, Barrier and Antimicrobial Properties of Pomegranate Peel Extract-incorporated Sodium Caseinate Film and Application in Packaging for Ground Beef: Properties of Sodium Caseinate Film. Packag. Technol. Sci. 28, 869–881. doi:10.1002/pts.2145 FAO Expert consultation on the trade impact of Listeria in fish products - Table of Contents [WWW Document], n.d. URL http://www.fao.org/docrep/003/x3018e/x3018e00.htm (accessed 7.5.17). Folin-Ciocalteau Micro Method for Total Phenol in Wine — Waterhouse Lab [WWW Document], n.d. URL http://waterhouse.ucdavis.edu/faqs/folin-ciocalteau-micro-method- for-total-phenol-in-wine (accessed 8.2.17). Gil, M.I., Tomás-Barberán, F.A., Hess-Pierce, B., Holcroft, D.M., Kader, A.A., 2000. Antioxidant Activity of Pomegranate Juice and Its Relationship with Phenolic

Composition and Processing. J. Agric. Food Chem. 48, 4581–4589. doi:10.1021/jf000404a Gullon, B., Pintado, M.E., Pérez-Álvarez, J.A., Viuda-Martos, M., 2016. Assessment of polyphenolic profile and antibacterial activity of pomegranate peel (Punica granatum) flour obtained from co-product of juice extraction. Food Control 59, 94–98. doi:10.1016/j.foodcont.2015.05.025 Hayrapetyan, H., Hazeleger, W.C., Beumer, R.R., 2012. Inhibition of Listeria monocytogenes by pomegranate (Punica granatum) peel extract in meat paté at different temperatures. Food Control 23, 66–72. doi:10.1016/j.foodcont.2011.06.012 Hellberg, R.S., DeWitt, C.A.M., Morrissey, M.T., 2012. Risk-Benefit Analysis of Seafood Consumption: A Review. Compr. Rev. Food Sci. Food Saf. 11, 490–517. doi:10.1111/j.1541-4337.2012.00200.x Iwamoto, M., Ayers, T., Mahon, B.E., Swerdlow, D.L., 2010. Epidemiology of Seafood- Associated Infections in the United States. Clin. Microbiol. Rev. 23, 399–411. doi:10.1128/CMR.00059-09 Jami, M., Ghanbari, M., Zunabovic, M., Domig, K.J., Kneifel, W., 2014. Listeria monocytogenes in Aquatic Food Products-A Review: L. monocytogenes in aquatic food products…. Compr. Rev. Food Sci. Food Saf. 13, 798–813. doi:10.1111/1541- 4337.12092 Kanatt, S.R., Chander, R., Sharma, A., 2010. Antioxidant and antimicrobial activity of pomegranate peel extract improves the shelf life of chicken products. Int. J. Food Sci. Technol. 45, 216–222. doi:10.1111/j.1365-2621.2009.02124.x Lee, K.-M., Runyon, M., Herrman, T.J., Phillips, R., Hsieh, J., 2015. Review of Salmonella detection and identification methods: Aspects of rapid emergency response and food safety. Food Control 47, 264–276. doi:10.1016/j.foodcont.2014.07.011 Lianou, A., Sofos, J.N., 2007. A Review of the Incidence and Transmission of Listeria monocytogenes in Ready-to-Eat Products in Retail and Food Service Environments. J. Food Prot. 70, 2172–2198. doi:10.4315/0362-028X-70.9.2172 Lin, Y.T., Labbe, R.G., Shetty, K., 2005. Inhibition of Vibrio parahaemolyticus in seafood systems using oregano and cranberry phytochemical synergies and lactic acid. Innov. Food Sci. Emerg. Technol. 6, 453–458. doi:10.1016/j.ifset.2005.04.002 Lin, Y.T., Labbe, R.G., Shetty, K., 2004. Inhibition of Listeria monocytogenes in Fish and Meat Systems by Use of Oregano and Cranberry Phytochemical Synergies. Appl. Environ. Microbiol. 70, 5672–5678. doi:10.1128/AEM.70.9.5672-5678.2004 Negi, P.S., Jayaprakasha, G.K., 2003. Antioxidant and Antibacterial Activities of Punica granatum Peel Extracts. J. Food Sci. 68, 1473–1477. doi:10.1111/j.1365- 2621.2003.tb09669.x

Newton, A., Kendall, M., Vugia, D.J., Henao, O.L., Mahon, B.E., 2012. Increasing Rates of Vibriosis in the United States, 1996-2010: Review of Surveillance Data From 2 Systems. Clin. Infect. Dis. 54, S391–S395. doi:10.1093/cid/cis243 Oliveira, I., Sousa, A., Morais, J.S., Ferreira, I.C.F.R., Bento, A., Estevinho, L., Pereira, J.A., 2008. Chemical composition, and antioxidant and antimicrobial activities of three hazelnut (Corylus avellana L.) cultivars. Food Chem. Toxicol. 46, 1801–1807. doi:10.1016/j.fct.2008.01.026 Paredes-López, O., Cervantes-Ceja, M.L., Vigna-Pérez, M., Hernández-Pérez, T., 2010. Berries: Improving Human Health and Healthy Aging, and Promoting Quality Life—A Review. Plant Foods Hum. Nutr. 65, 299–308. doi:10.1007/s11130-010-0177-1 Puupponen-Pimiä, R., Nohynek, L., Alakomi, H.-L., Oksman-Caldentey, K.-M., 2005. The action of berry phenolics against human intestinal pathogens. BioFactors 23, 243– 251. doi:10.1002/biof.5520230410 Puupponen-Pimia, R., Nohynek, L., Hartmann-Schmidlin, S., Kahkonen, M., Heinonen, M., Maatta-Riihinen, K., Oksman-Caldentey, K.-M., 2005. Berry phenolics selectively inhibit the growth of intestinal pathogens. J. Appl. Microbiol. 98, 991–1000. doi:10.1111/j.1365-2672.2005.02547.x Puupponen-Pimia, R., Nohynek, L., Meier, C., Kahkonen, M., Heinonen, M., Hopia, A., Oksman-Caldentey, K.-M., 2001. Antimicrobial properties of phenolic compounds from berries. J. Appl. Microbiol. 90, 494–507. doi:10.1046/j.1365-2672.2001.01271.x Rai, M., Chikindas, M. (Eds.), 2011. Natural antimicrobials in food safety and quality. CABI, Wallingford. doi:10.1079/9781845937690.0000 Seltmann, G., Holst, O., 2002. The Bacterial Cell Wall. Springer Berlin Heidelberg, Berlin, Heidelberg. doi:10.1007/978-3-662-04878-8 Su, Y.-C., Liu, C., 2007. Vibrio parahaemolyticus: A concern of seafood safety. Food Microbiol. 24, 549–558. doi:10.1016/j.fm.2007.01.005 US Food and Drug Administration (FDA), 2011. Seafood - Fish and Fishery Products Hazards and Controls Guidance - Fourth Edition [WWW Document]. URL https://www.fda.gov/food/guidanceregulation/guidancedocumentsregulatoryinformation/s eafood/ucm2018426.htm (accessed 6.14.17). Valtierra-RodríGuez, D., Heredia, N.L., GarcíA, S., SáNchez, E., 2010. Reduction of Campylobacter jejuni and Campylobacter coli in Poultry Skin by Fruit Extracts. J. Food Prot. 73, 477–482. doi:10.4315/0362-028X-73.3.477 Vattem, D.A., Lin, Y.-T., Labbe, R.G., Shetty, K., 2004. Antimicrobial activity against select food-borne pathogens by phenolic antioxidants enriched in cranberry pomace by solid-state bioprocessing using the food grade fungus Rhizopus oligosporus. Process Biochem. 39, 1939–1946. doi:10.1016/j.procbio.2003.09.032

Wu, V.C.-H., Qiu, X., Bushway, A., Harper, L., 2008. Antibacterial effects of American cranberry (Vaccinium macrocarpon) concentrate on foodborne pathogens. LWT - Food Sci. Technol. 41, 1834–1841. doi:10.1016/j.lwt.2008.01.001 Xi, Y., Sullivan, G.A., Jackson, A.L., Zhou, G.H., Sebranek, J.G., 2011. Use of natural antimicrobials to improve the control of Listeria monocytogenes in a cured cooked meat model system. Meat Sci. 88, 503–511. doi:10.1016/j.meatsci.2011.01.036

Table 3.2 Total phenolic content (TPC) and pH of fruit pomaces, pomegranate peel extract (PPE) and cranberry juice (CJ) for antimicrobial activity evaluation Treatment Total phenolic contents pH (mg/mL) Cranberry Juice (CJ) 2.37 2.91 Color-reduced Cranberry Juice (RCJ) 1.60 2.91 Whole Plum Pomace (WPLP) 3.80 3.56 Plum Meat Pomace (PMP) 3.64 3.61 Pomegranate Seed Pomace (PSP) 2.13 4.54 Whole Blueberry Pomace (WBP) 2.32 2.93 Whole Strawberry Pomace (WSP) 2.90 3.53 Plum Peel Pomace (PLPP; 25%) 2.45 3.35 Whole Pomegranate Pomace (WPOP; 25%) 24.54 4.01 Pomegranate Peel Pomace (POPP; 25%) 44.36 3.81 Pomegranate Peel Extract (PPE; 20%) 13.34 3.73 Color-reduced Pomegranate Peel Extract (RPPE; 20%) 12.68 3.79 Pomegranate Peel Extract (PPE; 33%) 21.81 3.66 Color-reduced Pomegranate Peel Extract (RPPE; 33%) 18.74 3.77

Table 3.3 Effect of pomegranate peel extract (PPE) on microbial growth of L. monocytogenes, Salmonella and V. parahaemolyticus in tryptic soy broth (TSB) and tryptic soy broth supplemented with 1.5% NaCl (TSB-Salt) at 37 °C for 48 h Target species Strains Day 37.5% 22.5% 13.5% L. monocytogenes Scott A 0 h 6.57±0.03a 6.57±0.03a 6.57±0.03a 48 h -b 3.35±0.02b 4.53±0.63b L. monocytogenes M0507 0 h 6.43±0.01a 6.43±0.01a 6.43±0.01a 48 h -b 3.74±0.49b 4.37±0.42b L. monocytogenes SFL0404 0 h 6.32±0.05a 6.32±0.05a 6.32±0.05a 48 h -b 3.89±0.50b 4.12±0.53b L. monocytogenes F5027 0 h 5.48±0.02a 5.48±0.02a 5.48±0.02a 48 h -b 3.16±0.15b 3.65±0.03b L. monocytogenes H222 0 h 6.06±0.09a 6.06±0.09a 6.06±0.09a 48 h -b 3.33±0.63b 4.45±0.42b S. Weltevreden SFL 0319 0 h 5.52±0.10a 5.52±0.10a 5.52±0.10a 48 h -b 3.86±0.40b 7.48±0.33b S. Newport ATCC 6962 0 h 5.60±0.07a 5.60±0.07a 5.60±0.07a 48 h -b 3.86±0.13b 6.76±0.44b S. Newport H1275 0 h 5.76±0.02a 5.76±0.02a 5.76±0.02a 48 h -b 3.98±0.07b 7.23±0.73b S. Typhimurium ATCC 14028 0 h 5.48±0.01a 5.48±0.01a 5.48±0.01a 48 h -b 3.51±0.02b 6.54±0.42b V. parahaemolyticus 10290 0 h 5.09±0.06a 5.09±0.06a 5.09±0.06a 48 h -b -b -b V. parahaemolyticus 10292 0 h 5.72±0.04a 5.72±0.04a 5.72±0.04a 48 h -b -b -b V. parahaemolyticus 10293 0 h 5.35±0.01a 5.35±0.01a 5.35±0.01a 48 h -b -b -b V. parahaemolyticus BE 98-2029 0 h 5.36±0.03a 5.36±0.03 5.36±0.03a 48 h -b -b -b V. parahaemolyticus 027-1c1 0 h 5.85±0.06a 5.85±0.06a 5.85±0.06a 48 h -b -b -b a, b Means for each concentration (Vertical columns) within in each strain followed by different letters were significantly different from each other (P < 0.05).

Table 3.4 Effect of lower concentration of pomegranate peel extract (PPE) and cranberry juice (CJ) on microbial growth of V. parahaemolyticus in tryptic soy broth supplemented with 1.5% NaCl (TSB-Salt) at 37 °C for 48 h Strains Day 8.1% (PPE) 4.86% (PPE) 2.92% (PPE) 6.25% (CJ) 3.13% (CJ) 1.56% (CJ) 0.78% (CJ) 10290 0 h 5.09±0.06a 5.09±0.06a 5.09±0.06a 4.42±0.05a 4.42±0.05a 4.42±0.05a 4.42±0.05a 48 h -b -b 7.11±0.11b 2.64±0.13b 2.09±0.25b 8.41±0.06b NTb 10292 0 h 5.72±0.04a 5.72±0.04a 5.72±0.04a 5.49±0.21a 5.49±0.21a 5.49±0.21a 5.49±0.21a 48 h -b 5.21±0.68b 7.48±0.07b -b -b 8.87±0.04b NTb 10293 0 h 5.35±0.01a 5.35±0.01a 5.35±0.01a 5.35±0.05a 5.35±0.05a 5.35±0.05a 5.35±0.05a 48 h -b -b 7.44±0.10b 0.68±1.18b 1.35±1.18b 1.55±1.35b 8.50±0.04b BE 98-2029 0 h 5.36±0.03a 5.36±0.03a 5.36±0.03a 5.54±0.09a 5.54±0.09a 5.54±0.09a 5.54±0.09a 48 h -b 3.26±0.04b 7.18±0.05b -b -b -b 9.01±0.09b 027-1c1 0 h 5.85±0.06a 5.85±0.06a 5.85±0.06a 5.64±0.27a 5.64±0.27a 5.64±0.27a 5.64±0.27a 48 h -b 2.82±0.27b 7.79±0.01b -b -b 8.71±0.13b NTb a,b Means for each concentration (Vertical columns) followed by different letters were significantly different from each other (P < 0.05). c NT Means not tested.

Table 3.5 Effect of cranberry juice (CJ) on microbial growth of L. monocytogenes and Salmonella in tryptic soy broth (TSB) and V. parahaemolyticus in tryptic soy broth (TSB) and tryptic soy broth supplemented with 1.5% NaCl (TSB-Salt) at 37 °C for 48 h Target species Strains Day 50% 25% 12.5% L. monocytogenes Scott A 0 h 6.17±0.12a 6.17±0.12a 6.17±0.12a 48 h -b -b 5.14±0.14b L. monocytogenes M0507 0 h 5.86±0.06a 5.86±0.06a 5.86±0.06a 48 h -b -b 5.70±0.12b L. monocytogenes SFL0404 0 h 6.07±0.23a 6.07±0.23a 6.07±0.23a 48 h -b -b 4.44±0.84b L. monocytogenes F5027 0 h 5.57±0.02a 5.57±0.02a 5.57±0.02a 48 h -b -b 5.22±0.02b L. monocytogenes H222 0 h 6.16±0.09a 6.16±0.09a 6.16±0.09a 48 h -b -b 5.43±0.03b S. Weltevreden SFL 0319 0 h 5.62±0.10a 5.62±0.10a 5.62±0.10a 48 h -b -b 7.94±0.03b S. Newport ATCC 6962 0 h 5.70±0.07a 5.70±0.07a 5.70±0.07a 48 h -b -b 8.06±0.01b S. Newport H1275 0 h 5.86±0.02a 5.86±0.02a 5.86±0.02a 48 h -b -b 8.06±0.12b S. Typhimurium ATCC 14028 0 h 5.58±0.01a 5.58±0.01a 5.58±0.01a 48 h -b -b 8.15±0.03b V. parahaemolyticus 10290 0 h 4.42±0.05a 4.42±0.05a 4.42±0.05a 48 h -b -b -b V. parahaemolyticus 10292 0 h 5.49±0.21a 5.49±0.21a 5.49±0.21a 48 h -b -b -b V. parahaemolyticus 10293 0 h 5.35±0.05a 5.35±0.05a 5.35±0.05a 48 h -b -b -b V. parahaemolyticus BE 98-2029 0 h 5.54±0.09a 5.54±0.09a 5.54±0.09a 48 h -b -b -b V. parahaemolyticus 027-1c1 0 h 5.64±0.27a 5.64±0.27a 5.64±0.27a 48 h -b -b -b a,b Means for each concentration (Vertical columns) followed by different letters were significantly different from each other (P < 0.05).

Chapter 4. General Conclusion

Vibrio parahaemolyticus is frequently isolated or detected from raw seafoods, especially shellfish. Also, Listeria monocytogenes, Salmonella spp. are pathogens that easily contaminate ready-to-eat (RTE) seafood, like smoked fish and shellfish, seafood salad, cooked shrimp and crabmeat, and raw seafood. Two studies to improve safety in

RTE seafoods were conducted.

FDA regulations state that RTE fishery products have a limit of 10,000 V. parahaemolyticus cells/g, which includes pathogenic and non-pathogenic V. parahaemolyticus. However, limited studies have been conducted to investigate the difference in growth and die-off between pathogenic and non-pathogenic V. parahaemolyticus in various environments. The first study investigated growth rates

(positive µmax) and die-off rates (negative µmax) of five pathogenic and five non- pathogenic V. parahaemolyticus strains at different temperatures (5-30°C). V. parahaemolyticus strains exhibited different growth or die-off rate under different temperatures. Both strain type (non-pathogenic vs pathogenic) and temperature had significant effect on growth or die-off rates (µmax). No significant difference between 5-

15 °C when evaluating the effect of temperature on µmax within either non-pathogenic or pathogenic strain type. However, µmax within either non-pathogenic or pathogenic was significant difference when temperature goes higher (20-30 °C). Significance of strain type only occurred at 30 °C when evaluating differences between strain type at each temperature. The mean µmax for non-pathogenic and pathogenic strains was 0.448 and

0.340, respectively. The faster rate of growth for non-pathogenic strains suggests the parameters set by FDA, 10,000 V. parahaemolyticus cells/g, are more likely to represent

non-pathogenic strains, rather than pathogenic strains. This information is useful to the risk assessment of RTE seafood consumption. Further study are needed to identify the mechanism of temperature response of different V. parahaemolyticus strains.

Foodborne pathogens Listeria monocytogenes, Salmonella spp. and Vibrio parahaemolyticus are especially problematic in RTE foods. Also, fruits, especially berries, are rich in different phenolic compounds. The aim of the second study was to investigate antimicrobial activity of fruit extracts and juices against five L. monocytogenes, four Salmonella strains and five V. parahaemolyticus strains from human clinical samples, seafood, raw milk, produce, and meat sources. Limited inhibition was observed for plum, plum meat, plum peel, pomegranate seed, blueberry, strawberry and plum peel. Pomegranate peel (PPE) exhibited a high antimicrobial activity similar to cranberry juice (CJ). The MBC of PPE against L. monocytogenes and Salmonella was

37.5% and for Vibrio was 8.1%. The MBC of CJ against L. monocytogenes and

Salmonella was 25%, against V. parahaemolyticus was 12.5%.

For inhibition (MIC) of V. parahaemolyticus much lower concentrations of PPE and

CJ were required than for L. monocytogenes and Salmonella. Salmonella inhibition required the highest concentrations of PPE and CJ. The MIC of PPE against L. monocytogenes was 22.5% while against Salmonella was >22.5% and <37.5%. The MIC of PPE against V. parahaemolyticus strains was >4.86% and <8.1%. The MIC of CJ against L. monocytogenes was 12.5%, but this concentration did not fully inhibit the growth of Salmonella strains as the MIC was >12.5% and <25%.

Pomegranate peel is a by-product of fruit juice. This study demonstrates potential of

PPE to be used as natural antimicrobials against L. monocytogenes, V. parahaemolyticus,

and Salmonella in RTE seafood. However, more research is needed to understand the impact on how these antimicrobial applications impact both sensory and shelf-life of ready-to-eat seafood.

Bibliography Acheson, D., & Hohmann, E. L. (2001). Nontyphoidal salmonellosis. Clinical Infectious Diseases, 32(2), 263-269.

Ajay Kumar V. J., Latha C., Sunil B., Dhanya V. R. (2014).Comparison of different cultural techniques in isolation of Listeria monocytogenes from various samples. J. Foodborne Zoonotic Dis.2 10–14. 1.

Al-Zoreky, N. S. (2009). Antimicrobial activity of pomegranate (Punica granatum L.) fruit peels. International journal of food microbiology, 134(3), 244-248.

Amagliani, G., Brandi, G., & Schiavano, G. F. (2012). Incidence and role of Salmonella in seafood safety. Food Research International, 45(2), 780-788.

Andino, A., & Hanning, I. (2015). Salmonella enterica: survival, colonization, and virulence differences among serovars. The Scientific World Journal, 2015.

Anonymous. (2007). Microbiological guidelines for ready to eat food. Centre for Food Safety, Food and Environmental Hygiene Department, Hong Kong, China. Retrieved from: http://www.cfs.gov.hk/english/food_leg/food_leg_mgref.html

Apostolidis, E., Kwon, Y. I., & Shetty, K. (2008). Inhibition of Listeria monocytogenes by oregano, cranberry and sodium lactate combination in broth and cooked ground beef systems and likely mode of action through proline metabolism. International journal of food microbiology, 128(2), 317-324.

Artik N. (1998). Determination of phenolic compounds in pomegranate juice by using HPLC. Fruit Processing, 8:492-499.

Aviram, M., Dornfeld, L., Rosenblat, M., Volkova, N., Kaplan, M., Coleman, R., ... & Fuhrman, B. (2000). Pomegranate juice consumption reduces oxidative stress, atherogenic modifications to LDL, and platelet aggregation: studies in humans and in atherosclerotic apolipoprotein E–deficient mice. The American journal of clinical nutrition, 71(5), 1062-1076.

Bacteriological Analytical Manual (BAM). 2004. Chapter 9: Vibrio, Bacteriological Analytical Manual. Retrieved from: https://www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm070830.htm

Bacteriological Analytical Manual (BAM). 2016. Chapter 5: Salmonella US Food and Drug Administration's Bacteriological Analytical Manual. Retrieved from: https://www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm070149.htm

Barfblogm, safe food from farm to fork. (2015). 6 sick from Vibrio in oysters in Mass. Retrieved from: http://barfblog.com/2015/09/6-sick-from-vibrio-in-oysters-in-mass/

Bhagwat, S., Haytowitz, D.B. Holden, J.M. (Ret.). (2014). USDA Database for the Flavonoid Content of Selected Foods, Release 3.1. U.S. Department of Agriculture, Agricultural Research Service. Nutrient Data Laboratory. Retrieved from: http://www.ars.usda.gov/nutrientdata/flav

Blivet, D., Soumet, C., Ermel, G., & Colin, P. (1998). Rapid detection methods for pathogens. In 3rd Karlsruhe Nutrition Symposium European Research towards Safer and Better Food (p. 3).

Brinkman, E., van Beurden, R., Mackintosh, R., & Beumer, R. (1995). Evaluation of a new Dip-Stick test for the rapid detection of Salmonella in food. Journal of Food Protection, 58, 1023e1027.

Brzin, B., and H. P. R. Seeliger. A brief note on the CAMP phenomenon in Listeria. In M. Woodbine (ed.), Problems of listeriosis. Leicester University Press, Leicester, England. 1975. p. 34-37.

Buchanan, R. L. (1991). Microbiological criteria for cooked, ready-to-eat shrimp and crabmeat. Food Technology, 45(4), 157-160.

Burnham, V. E., Janes, M. E., Jakus, L. A., Supan, J., DePaola, A., & Bell, J. (2009). Growth and survival differences of Vibrio vulnificus and Vibrio parahaemolyticus strains during cold storage. Journal of food science, 74(6).

Caillet, S., Côté, J., Sylvain, J. F., & Lacroix, M. (2012). Antimicrobial effects of fractions from cranberry products on the growth of seven pathogenic bacteria. Food Control, 23(2), 419-428.

Carpentier, B., & Cerf, O. (2011). Persistence of Listeria monocytogenes in food industry equipment and premises. International journal of food microbiology, 145(1), 1-8.

Ceccarelli, D., Hasan, N. A., Huq, A., & Colwell, R. R. (2013). Distribution and dynamics of epidemic and pandemic Vibrio parahaemolyticus virulence factors. Frontiers in cellular and infection microbiology, 3.

Centers for Disease Control and Prevention [CDC]. (1998). Outbreak of Vibrio parahaemolyticus Infections Associated with Eating Raw Oysters -- Pacific Northwest, 1997. Retrieved from https://www.cdc.gov/mmwr/preview/mmwrhtml/00053377.htm

Centers for Disease Control and Prevention [CDC]. (1999). Outbreak of Vibrio parahaemolyticus infection associated with eating raw oysters, and clams harvested from Long Island Sound-Connecticut. New Jersey and New York, 1998. Morb. Mortal. Wkly. Rep. 48 48–51.

Centers for Disease Control and Prevention [CDC]. (2006). Vibrio parahaemolyticus Infections Associated with Consumption of Raw Shellfish --- Three States, 2006. Retrieved from https://www.cdc.gov/mmwr/preview/mmwrhtml/mm55d807a1.htm

Centers for Disease Control and Prevention [CDC]. (2012). Pandemic Vibrio parahaemolyticus, Maryland, USA, 2012. Retrieved from https://wwwnc.cdc.gov/eid/article/20/4/13-0818_article

Centers for Disease Control and Prevention [CDC]. (2013). Increase in Vibrio parahaemolyticus illnesses associated with consumption of shellfish from several Atlantic coast harvest areas, United States, 2013 Retrieved from https://www.cdc.gov/vibrio/investigations/vibriop-09-13/index.html

Cevallos-Casals, B. A., Byrne, D., Okie, W. R., & Cisneros-Zevallos, L. (2006). Selecting new peach and plum genotypes rich in phenolic compounds and enhanced functional properties. Food Chemistry, 96(2), 273-280.

Cheynier, V., Dueñas-Paton, M., Salas, E., Maury, C., Souquet, J. M., Sarni-Manchado, P., & Fulcrand, H. (2006). Structure and properties of wine pigments and tannins. American Journal of Enology and Viticulture, 57(3), 298-305.

Chitov, T., Wongdao, S., Thatum, W., Puprae, T., & Sisuwan, P. (2009). Occurrence of potentially pathogenic Vibrio species in raw, processed, and ready-to-eat seafood and seafood products. Maejo International Journal of Science and Technology, 3(1), 88-98.

Christie, R., Atkins, N. E., & Munch-Petersen, E. (1944). A note on a lytic phenomenon shown by group B streptococci. Australian Journal of Experimental Biology & Medical Science, 22(3).

CHORMagarTM Salmonella. 2016. CHROMagar Microbiology. Retrieved from: http://www.chromagar.com/clinical-microbiology-chromagar-salmonella-focus-on- salmonella-species-27.html#.WSdrH7V6zIU

Chung, K. T., Wei, C. I., & Johnson, M. G. (1998). Are tannins a double-edged sword in biology and health?. Trends in Food Science & Technology, 9(4), 168-175.

Cowan, M. M. (1999). Plant products as antimicrobial agents. Clinical microbiology reviews, 12(4), 564-582.

Curtis, G. D. W., Mitchell, R. G., King, A. F., & Griffin, E. J. (1989). A selective differential medium for the isolation of Listeria monocytogenes. Letters in applied microbiology, 8(3), 95-98.

DADISMAN Jr, T. A., NELSON, R., MOLENDA, J. R., & GARBER, H. J. (1972). Vibrio parahaemolyticus gastroenteritis in Maryland I. Clinical and epidemiologic aspects. American journal of epidemiology, 96(6), 414-426.

Daniels, N. A., & Shafaie, A. (2000). A review of pathogenic Vibrio infections for clinicians. Infections in medicine, 17(10), 665-685.

Daniels, N. A., MacKinnon, L., Bishop, R., Altekruse, S., Ray, B., Hammond, R. M., ... & Slutsker, L. (2000). Vibrio parahaemolyticus infections in the United States, 1973– 1998. Journal of Infectious Diseases, 181(5), 1661-1666.

Darling, C. L. (1975). Standardization and evaluation of the CAMP reaction for the prompt, presumptive identification of Streptococcus agalactiae (Lancefield group B) in clinical material. Journal of clinical microbiology, 1(2), 171-174.

DePaola, A., Kaysner, C. A., Bowers, J., & Cook, D. W. (2000). Environmental investigations of Vibrio parahaemolyticus in oysters after outbreaks in Washington, Texas, and New York (1997 and 1998). Applied and environmental microbiology, 66(11), 4649-4654.

Di Pinto, A., Ciccarese, G., De Corato, R., Novello, L., & Terio, V. (2008). Detection of pathogenic Vibrio parahaemolyticus in southern Italian shellfish. Food Control, 19(11), 1037-1041.

Doganay, M. (2003). Listeriosis: clinical presentation. FEMS Immunology & Medical Microbiology, 35(3), 173-175.

Eklund, M., Jinneman, K., Wekell, M., (2007). Incidence and Behavior of Listeria monocytogenes in Fish and Seafood, in: Ryser, E., Marth, E. (Eds.), Listeria, Listeriosis, and Food Safety, Third Edition. CRC Press, pp. 617-653.

Ehala, S., Vaher, M., & Kaljurand, M. (2005). Characterization of phenolic profiles of Northern European berries by capillary electrophoresis and determination of their antioxidant activity. Journal of Agricultural and Food Chemistry, 53(16), 6484-6490.

Emam‐Djomeh, Z., Moghaddam, A., & Yasini Ardakani, S. A. (2015). Antimicrobial Activity of Pomegranate (Punica granatum L.) Peel Extract, Physical, Mechanical, Barrier and Antimicrobial Properties of Pomegranate Peel Extract‐incorporated Sodium Caseinate Film and Application in Packaging for Ground Beef. Packaging Technology and Science, 28(10), 869-881.

Eng, S. K., Pusparajah, P., Ab Mutalib, N. S., Ser, H. L., Chan, K. G., & Lee, L. H. (2015). Salmonella: A review on pathogenesis, epidemiology and antibiotic resistance. Frontiers in Life Science, 8(3), 284-293.

Fang, N., Yu, S., & Prior, R. L. (2002). LC/MS/MS characterization of phenolic constituents in dried plums. Journal of Agricultural and Food Chemistry, 50(12), 3579- 3585.

Farber, J. M., & Peterkin, P. I. (1991). Listeria monocytogenes, a food-borne pathogen. Microbiological reviews, 55(3), 476-511.

Farber, J. M., Coates, F., & Daley, E. (1992). Minimum water activity requirements for the growth of Listeria monocytogenes. Letters in Applied Microbiology, 15(3), 103-105.

Folin-Ciocalteau Micro Method for Total Phenol in Wine – Waterhouse lab. Retrieved from: http://waterhouse.ucdavis.edu/faqs/folin-ciocalteau-micro-method-for-total-phenol- in-wine

Foo, L. Y., & Porter, L. J. (1981). The structure of tannins of some edible fruits. Journal of the Science of Food and Agriculture, 32(7), 711-716.

Food and Agriculture Organization Corporate Statistical Database (FAOSTAT Database) (2017). Retrieved from http://www.fao.org/faostat/en/#data/CL

Food and Agriculture Organization (FAO) (1999). – Report of the FAO expert consultation on the trade impact of Listeria in fish products, 17-20 May, Amherst, Massachusetts. FAO Fisheries Report no. 604.

Food Drug and Administration (FDA) (2011a) Retrieved from https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact =8&ved=0ahUKEwjn5ZXU28_TAhVE6mMKHUotCW4QFggoMAA&url=https%3A% 2F%2Fwww.foodstandards.gov.au%2Fpublications%2FDocuments%2FListeria%2520m onocytogenes.pdf&usg=AFQjCNGo0DPronEY2H3RRUrUP98ojnrauQ&sig2=c00T_Jgj CXPWtKlfjFJoJw

Food and Drug Administration. (2011b). Fish and fishery products hazards and controls guidance. US Department of Health and Human Services Food and Drug Administration Center for Food Safety and Applied Nutrition.

Food and Drug Administration. Bad Bug Book, Food borne Pathogenic microorganisms and natural toxins. Second Edition.2012. Retrieved from https://www.fda.gov/downloads/food/foodsafety/foodborneillness/foodborneillnessfoodb ornepathogensnaturaltoxins/badbugbook/ucm297627.pdf

Food Safety News: Has Imported Dutch Smoked Salmon Sickened 100 Americans? Retrieved from http://www.foodsafetynews.com/2012/10/has-dutch-smoked-salmon-sickened-100- americans/#.Vfh8YLEmguo

Fraise, A. P. (2002). Biocide abuse and antimicrobial resistance—a cause for concern?. Journal of antimicrobial chemotherapy, 49(1), 11-12.

Fraiser, M. B., & Koburger, J. A. (1984). Incidence of Salmonellae in Clams, Oysters, Crabs and Mullet 1. Journal of food protection, 47(5), 343-345.

Friesema, I., de Jong, A., Hofhuis, A., Heck, M., van den Kerkhof, H., de Jonge, R., ... & Notermans, D. (2014). Large outbreak of Salmonella Thompson related to smoked salmon in the Netherlands, August to December 2012. Euro Surveill, 19, 39. Retrieved from http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20918

Fukui, T., Shiraki, K., Hamada, D., Hara, K., Miyata, T., Fujiwara, S., ... & Imanaka, T. (2005). Thermostable direct hemolysin of Vibrio parahaemolyticus is a bacterial reversible amyloid toxin. Biochemistry, 44(29), 9825-9832.

Fung, D. Y. (2002). Rapid methods and automation in microbiology. Comprehensive reviews in food science and food safety, 1(1), 3-22..

Gaillard, J. L., Berche, P., Frehel, C., Gouln, E., & Cossart, P. (1991). Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from gram-positive cocci. Cell, 65(7), 1127-1141.

Gasanov, U., Hughes, D., & Hansbro, P. M. (2005). Methods for the isolation and identification of Listeria spp. and Listeria monocytogenes: a review. FEMS microbiology reviews, 29(5), 851-875.

Gil, M. I., Tomás-Barberán, F. A., Hess-Pierce, B., Holcroft, D. M., & Kader, A. A. (2000). Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. Journal of Agricultural and Food chemistry, 48(10), 4581- 4589.

Gilbert, R. J., De Louvois, J., Donovan, T., Little, C., Nye, K., Ribeiro, C. D & Bolton, F. J. (2000). Guidelines for the microbiological quality of some ready-to-eat foods sampled at the point of sale. PHLS Advisory Committee for Food and Dairy Products. Communicable disease and public health/PHLS, 3(3), 163-167.

Gök, V., & Bor, Y. (2012). Effect of olive leaf, blueberry and Zizyphus jujuba extracts on the quality and shelf life of meatball during storage. Journal of Food, Agriculture & Environment, 10(2), 190-195.

Granata, L. A., Flick Jr, G. J., & Martin, R. E. (2012). The seafood industry: species, products, processing, and safety. John Wiley & Sons.

Gray, M. L., & Killinger, A. H. (1966). Listeria monocytogenes and listeric infections. Bacteriological reviews, 30(2), 309.

Gray, J.T., and Fedorka-Cray, P.J. (2002). Salmonella, pp. 55-68. In Foodborne Diseases. 2nd Ed. Cliver, D.O. and Riemann, H. (Eds.) Academic Press, New York.

Groves, R. D., & Welshimer, H. J. (1977). Separation of pathogenic from apathogenic Listeria monocytogenes by three in vitro reactions. Journal of clinical microbiology, 5(6), 559-563.

Gullon, B., Pintado, M. E., Pérez-Álvarez, J. A., & Viuda-Martos, M. (2016). Assessment of polyphenolic profile and antibacterial activity of pomegranate peel (Punica granatum) flour obtained from co-product of juice extraction. Food Control, 59, 94-98.

Hägg, M., Ylikoski, S., & Kumpulainen, J. (1995). Vitamin C content in fruits and berries consumed in Finland. Journal of Food Composition and Analysis, 8(1), 12-20.

Häkkinen, S., Heinonen, M., Kärenlampi, S., Mykkänen, H., Ruuskanen, J., & Törrönen, R. (1999a). Screening of selected flavonoids and phenolic acids in 19 berries. Food Research International, 32(5), 345-353.

Häkkinen, S. H., Kärenlampi, S. O., Heinonen, I. M., Mykkänen, H. M., & Törrönen, A. R. (1999b). Content of the flavonols quercetin, myricetin, and kaempferol in 25 edible berries. Journal of Agricultural and Food Chemistry, 47(6), 2274-2279.

Häkkinen, S. H., & Törrönen, A. R. (2000). Content of flavonols and selected phenolic acids in strawberries and Vaccinium species: influence of cultivar, cultivation site and technique. Food research international, 33(6), 517-524.

Hayrapetyan, H., Hazeleger, W. C., & Beumer, R. R. (2012). Inhibition of Listeria monocytogenes by pomegranate (Punica granatum) peel extract in meat paté at different temperatures. Food Control, 23(1), 66-72.

Health Canada. (2011). Policy on Listeria monocytogenes in ready-to-eat foods. Bureau of Microbial Hazards, Food Directorate, Health Products and Food Branch, Health Canada. Retrieved from: http://www.hc-sc.gc.ca/fn-an/legislation/pol/policy_listeria_monocytogenes_2011- eng.php

Heinitz, M. L., Ruble, R. D., Wagner, D. E., & Tatini, S. R. (2000). Incidence of Salmonella in fish and seafood. Journal of food protection, 63(5), 579-592.

Hendriksen, R. S., (2003). Laboratory Protocols Level 1 Training Course Isolation of Salmonella 4th Ed. Contents 1.Isolation, identification and serotyping of Salmonella – from faeces and food. Retrieved from: http://www.antimicrobialresistance.dk/data/images/salmonella1_pdf.pdf

Hellberg, R. S., DeWitt, C. A. M., & Morrissey, M. T. (2012). Risk‐benefit analysis of seafood consumption: A review. Comprehensive reviews in food science and food safety, 11(5), 490-517.

Hitchins A. D., Jinneman K. (2013). Bacteriological Analytical Manual (BAM) Chapter 10: Detection and Enumeration of Listeria monocytogenes in Foods. Retrieved from:https://www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm2006949.ht m

Honda, T. A. K. E. S. H. I., Ni, Y. X., & Miwatani, T. O. S. H. I. O. (1988). Purification and characterization of a hemolysin produced by a clinical isolate of Kanagawa phenomenon-negative Vibrio parahaemolyticus and related to the thermostable direct hemolysin. Infection and Immunity, 56(4), 961-965.

Howell, A. B. (2002). Cranberry proanthocyanidins and the maintenance of urinary tract health. Critical reviews in food science and nutrition, 42(S3), 273-278.

Hwang, A., & Huang, L. (Eds.). (2010). Ready-to-eat foods: microbial concerns and control measures. CRC Press. 132. Retrieved from https://books.google.com.hk/books?id=OQPLBQAAQBAJ&pg=PA132&lpg=PA132&d q=outbreak+ready+to+eat+shrimp&source=bl&ots=uZcA4sOiXK&sig=nNCR1HF0V_f pcQyc8exIpWUHUk8&hl=zh-CN&sa=X&ved=0CDIQ6AEwAmoVChMIpa- p9czvxwIV0aOICh223AJ7#v=onepage&q=outbreak%20ready%20to%20eat%20shrimp &f=false

Iwamoto, M., Ayers, T., Mahon, B. E., & Swerdlow, D. L. (2010). Epidemiology of seafood-associated infections in the United States. Clinical microbiology reviews, 23(2), 399-411.

Jadhav, S., Bhave, M., & Palombo, E. A. (2012). Methods used for the detection and subtyping of Listeria monocytogenes. Journal of microbiological methods, 88(3), 327- 341.

Jay J M. Modern food microbiology [M]. Springer Science & Business Media, (2012). Chapter 26. Foodborne Gastroenteritis Caused by Salmonella and Shigella. Chapter 28. Foodborne Gastroenteritis Caused by Vibrio, Yersinia, and Campylobacter Species

Jami, M., Ghanbari, M., Zunabovic, M., Domig, K. J., & Kneifel, W. (2014). Listeria monocytogenes in aquatic food products—a review. Comprehensive Reviews in Food Science and Food Safety, 13(5), 798-813.

Jinneman, K.C. and Wekell, M.M. (1999) Incidence and behaviour of Listeria monocytogenes in fish and seafood. In: Ryser, E.T. & Marth, E.H. (eds.), Listeria, Listeriosis and Food Safety. Marcel Dekker, Inc., New York, United States. pp. 601-630.

Joseph, S. W., Colwell, R. R., & Kaper, J. B. (1982). Vibrio parahaemolyticus and related halophilic vibrios. CRC Critical Reviews in Microbiology, 10(1), 77-124.

Kader, F., Rovel, B., Girardin, M., & Metche, M. (1996). Fractionation and identification of the phenolic compounds of high bush blueberries (Vaccinium corymbosum, L.). Food Chemistry, 55(1), 35-40.

Kanatt, S. R., Chander, R., & Sharma, A. (2010). Antioxidant and antimicrobial activity of pomegranate peel extract improves the shelf life of chicken products. International journal of food science & technology, 45(2), 216-222.

Kaysner, C. A., & DePaola Jr, A. (2000). Outbreaks of Vibrio parahaemolyticus gastroenteritis from raw oyster consumption: assessing the risk of consumption and genetic methods for detection of pathogenic strains.

Kaufman, G. E., Blackstone, G. M., Vickery, M. C. L., Bej, A. K., Bowers, J., Bowen, M. D., ... & DePaola, A. (2004). Real-time PCR quantification of Vibrio parahaemolyticus in oysters using an alternative matrix. Journal of food protection, 67(11), 2424-2429.

Kelly, M. T., & Stroh, E. M. (1988). Temporal relationship of Vibrio parahaemolyticus in patients and the environment. Journal of Clinical Microbiology, 26(9), 1754-1756.

Kim, D. O., Chun, O. K., Kim, Y. J., Moon, H. Y., & Lee, C. Y. (2003). Quantification of polyphenolics and their antioxidant capacity in fresh plums. Journal of Agricultural and Food Chemistry, 51(22), 6509-6515.

Kim, D. S., Na, H., Song, J. H., Kwack, Y., Kim, S. K., & Chun, C. (2012). Antimicrobial activity of thinned strawberry fruits at different maturation stages. Korean Journal of Horticultural Science and Technology, 30(6), 769-775.

Kirs, M., Depaola, A., Fyfe, R., Jones, J. L., Krantz, J., Van Laanen, A., & Castle, M. (2011). A survey of oysters (Crassostrea gigas) in New Zealand for Vibrio parahaemolyticus and Vibrio vulnificus. International journal of food microbiology, 147(2), 149-153.

Kong, J. M., Chia, L. S., Goh, N. K., Chia, T. F., & Brouillard, R. (2003). Analysis and biological activities of anthocyanins. Phytochemistry, 64(5), 923-933.

Kotzekidou, P., Giannakidis, P., & Boulamatsis, A. (2008). Antimicrobial activity of some plant extracts and essential oils against foodborne pathogens in vitro and on the fate of inoculated pathogens in chocolate. LWT-Food Science and Technology, 41(1), 119- 127.

Lado, B. H. (2003). Characteristics of Listeria Monocytogenes Important for Pulsed Electric Field Process Optimization (Doctoral dissertation, The Ohio State University).

Law, J. W. F., Ab Mutalib, N. S., Chan, K. G., & Lee, L. H. (2015). An insight into the isolation, enumeration, and molecular detection of Listeria monocytogenes in food. Frontiers in microbiology, 6.

Lee, K. M., Runyon, M., Herrman, T. J., Phillips, R., & Hsieh, J. (2015). Review of Salmonella detection and identification methods: aspects of rapid emergency response and food safety. Food Control, 47, 264-276.

Leitão, D. P., Polizello, A. C. M., Ito, I. Y., & Spadaro, A. C. C. (2005). Antibacterial screening of anthocyanic and proanthocyanic fractions from cranberry juice. Journal of medicinal food, 8(1), 36-40.

Letchumanan, V., Chan, K. G., & Lee, L. H. (2014). Vibrio parahaemolyticus: a review on the pathogenesis, prevalence, and advance molecular identification techniques. Frontiers in microbiology, 5, 705.

Lianou, A., & Sofos, J. N. (2007). A review of the incidence and transmission of Listeria monocytogenes in ready-to-eat products in retail and food service environments. Journal of Food Protection, 70(9), 2172-2198.

Lin, Y. T., Labbe, R. G., & Shetty, K. (2004). Inhibition of Listeria monocytogenes in fish and meat systems by use of oregano and cranberry phytochemical synergies. Applied and environmental microbiology, 70(9), 5672-5678.

Lin, Y. T., Labbe, R. G., & Shetty, K. (2005). Inhibition of Vibrio parahaemolyticus in seafood systems using oregano and cranberry phytochemical synergies and lactic acid. Innovative Food Science & Emerging Technologies, 6(4), 453-458.

Liston, J. (1990). Microbial hazards of seafood consumption. Food Technol, 44.

Loncarevic, S., Tham, W., & Danielsson-Tham, M. L. (1995). Prevalence of Listeria monocytogenes and other Listeria spp. in smoked and 'gravad'fish. Acta Veterinaria Scandinavica, 37(1), 13-18.

Low, J. C., & Donachie, W. (1997). A review of Listeria monocytogenes and listeriosis. The Veterinary Journal, 153(1), 9-29.

Lungu, B., Ricke, S. C., & Johnson, M. G. (2009). Growth, survival, proliferation and pathogenesis of Listeria monocytogenes under low oxygen or anaerobic conditions: a review. Anaerobe, 15(1), 7-17.

Lyons, M. M., Yu, C., Toma, R. B., Cho, S. Y., Reiboldt, W., Lee, J., & Van Breemen, R. B. (2003). Resveratrol in raw and baked blueberries and bilberries. Journal of Agricultural and Food Chemistry, 51(20), 5867-5870.

Macheix, J. J., & Fleuriet, A. (1990). Fruit phenolics. CRC press.

Maddocks, S., Olma, T., & Chen, S. (2002). Comparison of CHROMagar Salmonella medium and xylose-lysine-desoxycholate and Salmonella-Shigella agars for isolation of Salmonella strains from stool samples. Journal of clinical microbiology, 40(8), 2999- 3003.

Magalhães R., Mena C., Ferreira V., Almeida G., Silva J., Teixeira P. (2014). “Traditional methods for isolation of Listeria monocytogenes,” in Listeria monocytogenes: Methods and Protocols, eds Jordan K., Fox E. M., Wagner M., editors. (New York, NY: Springer Science;), 15–30.

100

Mansour, E., Ben Khaled, A., Lachiheb, B., Abid, M., Bachar, K., & Ferchichi, A. (2013). Phenolic compounds, antioxidant, and antibacterial activities of peel extract from Tunisian pomegranate. Journal of Agricultural Science and Technology, 15, 1393-1403

Mazur, W. M., Uehara, M., Wähälä, K., & Adlercreutz, H. (2000). Phyto-oestrogen content of berries, and plasma concentrations and urinary excretion of enterolactone after a single strawberry-meal in human subjects. British journal of nutrition, 83(04), 381-387.

McCarthy, S. A., DePaola, A., Cook, D. W., Kaysner, C. A., & Hill, W. E. (1999). Evaluation of alkaline phosphatase‐and digoxigenin‐labelled probes for detection of the thermolabile hemolysin (tlh) gene of Vibrio parahaemolyticus. Letters in applied microbiology, 28(1), 66-70.

Miles, D. W., Ross, T., Olley, J., & McMeekin, T. A. (1997). Development and evaluation of a predictive model for the effect of temperature and water activity on the growth rate of Vibrio parahaemolyticus. International journal of food microbiology, 38(2), 133-142.

Monfort, P., Le Gal, D., Le Saux, J. C., Piclet, G., Raguenes, P., Boulben, S., & Plusquellec, A. (1994). Improved rapid method for isolation and enumeration of Salmonella from bivalves using Rambach agar. Journal of microbiological methods, 19(1), 67-79.

Može, S., Polak, T., Gašperlin, L., Koron, D., Vanzo, A., Poklar Ulrih, N., & Abram, V. (2011). Phenolics in Slovenian bilberries (Vaccinium myrtillus L.) and blueberries (Vaccinium corymbosum L.). Journal of agricultural and food chemistry, 59(13), 6998- 7004.

Negi, P. S., & Jayaprakasha, G. K. (2003). Antioxidant and antibacterial activities of Punica granatum peel extracts. Journal of food science, 68(4), 1473-1477.

Nelapati, S., Nelapati, K., & Chinnam, B. K. (2012). Vibrio parahaemolyticus-An emerging foodborne pathogen. Veterinary World, 5(1), 48-63.

Newton, A., Kendall, M., Vugia, D. J., Henao, O. L., & Mahon, B. E. (2012). Increasing rates of vibriosis in the United States, 1996–2010: review of surveillance data from 2 systems. Clinical Infectious Diseases, 54(suppl 5), S391-S395.

Nishibuchi, M. I. T. S. U. A. K. I., & Kaper, J. B. (1985). Nucleotide sequence of the thermostable direct hemolysin gene of Vibrio parahaemolyticus. Journal of Bacteriology, 162(2), 558-564.

Nishibuchi, M. I. T. S. U. A. K. I., Hill, W. E., Zon, G. E. R. A. L. D., Payne, W. L., & Kaper, J. B. (1986). Synthetic oligodeoxyribonucleotide probes to detect Kanagawa phenomenon-positive Vibrio parahaemolyticus. Journal of clinical microbiology, 23(6), 1091-1095.

101

Nishibuchi, M., & Kaper, J. B. (1995). Thermostable direct hemolysin gene of Vibrio parahaemolyticus: a virulence gene acquired by a marine bacterium. Infection and Immunity, 63(6), 2093.

Nohynek, L. J., Alakomi, H. L., Kähkönen, M. P., Heinonen, M., Helander, I. M., Oksman-Caldentey, K. M., & Puupponen-Pimiä, R. H. (2006). Berry phenolics: antimicrobial properties and mechanisms of action against severe human pathogens. Nutrition and cancer, 54(1), 18-32.

Norhana, M. W., Poole, S. E., Deeth, H. C., & Dykes, G. A. (2010). Prevalence, persistence and control of Salmonella and Listeria in shrimp and shrimp products: A review. Food Control, 21(4), 343-361.

Nunes, C., Guyot, S., Marnet, N., Barros, A. S., Saraiva, J. A., Renard, C. M., & Coimbra, M. A. (2008). Characterization of plum procyanidins by thiolytic depolymerization. Journal of agricultural and food chemistry, 56(13), 5188-5196.

Odeyemi, O. A. (2016). Incidence and prevalence of Vibrio parahaemolyticus in seafood: a systematic review and meta-analysis. SpringerPlus, 5(1), 464.

Okonko, I. O., Ogunjobi, A. A., Fajobi, E. A., Onoja, B. A., Babalola, E. T., & Adedeji, A. O. (2008). Comparative studies and microbial risk assessment of different Ready-to- Eat (RTE) frozen sea-foods processed in Ijora-olopa, Lagos State, Nigeria. African Journal of Biotechnology, 7(16).

Oliveira, I., Sousa, A., Morais, J. S., Ferreira, I. C., Bento, A., Estevinho, L., & Pereira, J. A. (2008). Chemical composition, and antioxidant and antimicrobial activities of three hazelnut (Corylus avellana L.) cultivars. Food and Chemical Toxicology, 46(5), 1801- 1807.

Opara, L. U., Al-Ani, M. R., & Al-Shuaibi, Y. S. (2009). Physico-chemical properties, vitamin C content, and antimicrobial properties of pomegranate fruit (Punica granatum L.). Food and Bioprocess Technology, 2(3), 315-321.

Painter, J., & Slutsker, L. (2007). Listeriosis in humans. FOOD SCIENCE AND TECHNOLOGY-NEW YORK-MARCEL DEKKER-, 161, 85.

Pagadala, S., Parveen, S., Rippen, T., Luchansky, J. B., Call, J. E., Tamplin, M. L., & Porto-Fett, A. C. (2012). Prevalence, characterization and sources of Listeria monocytogenes in blue crab (Callinectus sapidus) meat and blue crab processing plants. Food microbiology, 31(2), 263-270.

Pappas, E., & Schaich, K. M. (2009). Phytochemicals of cranberries and cranberry products: characterization, potential health effects, and processing stability. Critical reviews in food science and nutrition, 49(9), 741-781.

102

Paredes-López, O., Cervantes-Ceja, M. L., Vigna-Pérez, M., & Hernández-Pérez, T. (2010). Berries: improving human health and healthy aging, and promoting quality life— a review. Plant foods for human nutrition, 65(3), 299-308.

Peel, M., Donachie, W., & Shaw, A. (1988). Temperature-dependent expression of flagella of Listeria manocytogenes studied by electron microscopy, SDS-PAGE and Western blotting. Microbiology, 134(8), 2171-2178.

Poyrazoğlu, E., Gökmen, V., & Artιk, N. (2002). Organic acids and phenolic compounds in pomegranates (Punica granatum L.) grown in Turkey. Journal of food composition and analysis, 15(5), 567-575.

Pui, C. F., Wong, W. C., Chai, L. C., Robin, T., Ponniah, J., Sahroni, M. & Radu, S. (2011). Salmonella: A foodborne pathogen. International Food Research Journal, 18(2), 465-473.

Public Health Laboratory System. (1993). Surveillance group update: salmonella contamination of foods. PHLS Microbiol. Dig. 10:105

Puupponen‐Pimiä, R., Nohynek, L., Meier, C., Kähkönen, M., Heinonen, M., Hopia, A., & Oksman‐Caldentey, K. M. (2001). Antimicrobial properties of phenolic compounds from berries. Journal of applied microbiology, 90(4), 494-507.

Puupponen-Pimiä, R., Nohynek, L., Alakomi, H. L., & Oksman-Caldentey, K. M. (2005a). The action of berry phenolics against human intestinal pathogens. Biofactors, 23(4), 243-251.

Puupponen-Pimiä, R., Nohynek, L., Alakomi, H. L., & Oksman-Caldentey, K. M. (2005b). Bioactive berry compounds—novel tools against human pathogens. Applied Microbiology and Biotechnology, 67(1), 8-18.

Rai, M. (2011). Natural antimicrobials in food safety and quality. CABI.

Raghunath, P. (2014). Roles of thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) in Vibrio parahaemolyticus. Frontiers in microbiology, 5.

Rasmussen, R. S., Nettleton, J., & Morrissey, M. T. (2005). A review of mercury in seafood: Special focus on tuna. Journal of Aquatic Food Product Technology, 14(4), 71- 100.

Rawles, D., Flick, G., Pierson, M., DIALLO, A., WITTMAN, R., & CROONENBERGHS, R. (1995). Listeria monocytogenes occurrence and growth at refrigeration temperatures in fresh blue crab (Callinectes sapidus) meat. Journal of food protection, 58(11), 1219-1221.

103

Raynal, J., Moutounet, M., & Souquet, J. M. (1989). Intervention of phenolic compounds in plum technology. 1. Changes during drying. Journal of Agricultural and Food Chemistry, 37(4), 1046-1050.

Ricke, S. C. (2003). Perspectives on the use of organic acids and short chain fatty acids as antimicrobials. Poultry science, 82(4), 632-639.

Riedo, F. X., Pinner, R. W., de Lourdes Tosca, M., Cartter, M. L., Graves, L. M., Reeves, M. W., ... & Broome, C. (1994). A point-source foodborne listeriosis outbreak: documented incubation period and possible mild illness. The Journal of Infectious diseases, 170(3), 693-696.

Rimando, A. M., Kalt, W., Magee, J. B., Dewey, J., & Ballington, J. R. (2004). Resveratrol, pterostilbene, and piceatannol in vaccinium berries. Journal of agricultural and food chemistry, 52(15), 4713-4719.

Ruiz, J., Nunez, M. L., Diaz, J., Sempere, M. A., Gömez, J., & Usera, M. A. (1996). Note: Comparison of media for the isolation of lactose‐positive Salmonella. Journal of Applied Microbiology, 81(5), 571-574.

Sadaka, F., Nguimjeu, C., Brachais, C. H., Vroman, I., Tighzert, L., & Couvercelle, J. P. (2013). WITHDRAWN: Review on antimicrobial packaging containing essential oils and their active biomolecules. Innovative Food Science & Emerging Technologies, 20, 350.

Scallan, E., Hoekstra, R. M., Angulo, F. J., Tauxe, R. V., Widdowson, M. A., Roy, S. L., ... & Griffin, P. M. (2011). Foodborne illness acquired in the United States—major pathogens. Emerg Infect Dis, 17(1).

Seltmann, G., Holst, O., (2002). The Bacterial Cell Wall.

Seafood Nutrition Overview. Seafood Health Facts. Retrieved from: http://www.seafoodhealthfacts.org/seafood-nutrition/healthcare-professionals/seafood- nutrition-overview

Shahidi F, Naczk M (2004) Phenolic compounds in fruits and vegetables. In: Phenolics in food and nutraceutical, CRC LLC, pp 131–156. 13.

Shmuely, H., Burger, O., Neeman, I., Yahav, J., Samra, Z., Niv, Y., ... & Ofek, I. (2004). Susceptibility of Helicobacter pylori isolates to the antiadhesion activity of a high- molecular-weight constituent of cranberry. Diagnostic microbiology and infectious disease, 50(4), 231-235.

Siddiq, M. (2006). Plums and prunes. Handbook of Fruits and Fruit Processing, Blackwell Publishing Professional, Lowa, 553-564.

104

Skupień, K., & Oszmiański, J. (2004). Comparison of six cultivars of strawberries (Fragaria x ananassa Duch.) grown in northwest Poland. European Food Research and Technology, 219(1), 66-70.

Stöhr, H., Mosel, H. D., & Herrmann, K. (1975). The phenolics of fruits. VII. The phenolics of cherries and plums and the changes in catechins and hydroxycinnamic acid derivatives during the development of fruits. Zeitschrift für Lebensmittel-Untersuchung und-Forschung, 159(2), 85-91.

Su, Y. C. (2012). Pathogenic Vibrios and Food Safety. Phuvasate, S. & Su, Y. C. Chapter 3. Vibrio parahaemolyticus

Su, Y. C., & Liu, C. (2007). Vibrio parahaemolyticus: a concern of seafood safety. Food microbiology, 24(6), 549-558.

Sultana, B., & Anwar, F. (2008). Flavonols (kaempeferol, quercetin, myricetin) contents of selected fruits, vegetables and medicinal plants. Food Chemistry, 108(3), 879-884.

Szajdek, A., & Borowska, E. J. (2008). Bioactive compounds and health-promoting properties of berry fruits: a review. Plant Foods for Human Nutrition, 63(4), 147-156.

Tada, J., Ohashi, T., Nishimura, N., Shirasaki, Y., Ozaki, H., Fukushima, S., ... & Takeda, Y. (1992). Detection of the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction. Molecular and cellular probes, 6(6), 477-487.

Taniguchi, H., Hirano, H., Kubomura, S., Higashi, K., & Mizuguchi, Y. (1986). Comparison of the nucleotide sequences of the genes for the thermostable direct hemolysin and the thermolabile hemolysin from Vibrio parahaemolyticus. Microbial pathogenesis, 1(5), 425-432.

Taruscio, T. G., Barney, D. L., & Exon, J. (2004). Content and profile of flavanoid and phenolic acid compounds in conjunction with the antioxidant capacity for a variety of northwest Vaccinium berries. Journal of agricultural and food chemistry, 52(10), 3169- 3176.

Thompson, C. A., & Vanderzant, C. (1976). Relationship of Vibrio parahaemolyticus in oysters, water and sediment, and bacteriological and environmental indices. Journal of Food Science, 41(1), 117-122.

Thorns, C. J., McLaren, I. M., & Sojka, M. G. (1994). The use of latex particle agglutination to specifically detect Salmonella enteritidis. International journal of food microbiology, 21(1-2), 47-53.

Tietjen, M., & Fung, D. Y. (1995). Salmonellae and food safety. Critical reviews in microbiology, 21(1), 53-83.

105

Turfan, Ö., Türkyılmaz, M., Yemiş, O., & Özkan, M. (2011). Anthocyanin and colour changes during processing of pomegranate (Punica granatum L., cv. Hicaznar) juice from sacs and whole fruit. Food Chemistry, 129(4), 1644-1651.

Valtierra-Rodríguez, D., Heredia, N. L., García, S., & Sánchez, E. (2010). Reduction of Campylobacter jejuni and Campylobacter coli in poultry skin by fruit extracts. Journal of food protection, 73(3), 477-482.

Van Netten, P., Perales, I., Van de Moosdijk, A., Curtis, G. D. W., & Mossel, D. A. A. (1989). Liquid and solid selective differential media for the detection and enumeration of L. monocytogenes and other Listeria spp. International journal of food microbiology, 8(4), 299-316.

Vanderzant, C., Thompson Jr, C. A., & Ray, S. M. (1973). Microbial flora and level of Vibrio parahaemolyticus of oysters (Crassostrea virginica), water and sediment from Galveston bay. a. Journal of Milk and Food Technology, 36(9), 447-452.

Vattem, D. A., Lin, Y. T., Labbe, R. G., & Shetty, K. (2004a). Phenolic antioxidant mobilization in cranberry pomace by solid-state bioprocessing using food grade fungus Lentinus edodes and effect on antimicrobial activity against select food borne pathogens. Innovative food science & emerging technologies, 5(1), 81-91.

Vattem, D. A., Lin, Y. T., Labbe, R. G., & Shetty, K. (2004b). Antimicrobial activity against select food-borne pathogens by phenolic antioxidants enriched in cranberry pomace by solid-state bioprocessing using the food grade fungus Rhizopus oligosporus. Process Biochemistry, 39(12), 1939-1946.

Vitas, A. I. (2004). Occurrence of Listeria monocytogenes in fresh and processed foods in Navarra (Spain). International Journal of Food Microbiology, 90(3), 349-356.

Wallace, B. J., Guzewich, J. J., Cambridge, M., Altekruse, S., & Morse, D. L. (1999). Seafood-associated disease outbreaks in New York, 1980–1994. American journal of preventive medicine, 17(1), 48-54.

Wang, R., Ding, Y., Liu, R., Xiang, L., & Du, L. (2010). Pomegranate: constituents, bioactivities and pharmacokinetics. Fruit, Vegetable and Cereal Science and Biotechnology, 4(2), 77-87.

Weagant, S. D., Sado, P. N., Colburn, K. G., Torkelson, J. D., Stanley, F. A., Krane, M. H., ... & Thayer, C. F. (1988). The incidence of Listeria species in frozen seafood products. Journal of Food Protection, 51(8), 655-657.

White, C. (2016). American seafood consumption up in 2015, landing volumes even. Retrieved from: https://www.seafoodsource.com/news/supply-trade/american-seafood-consumption-up- in-2015-landing-volumes-even

106

Wong, H. C., Chen, M. C., Liu, S. H., & Liu, D. P. (1999). Incidence of highly genetically diversified Vibrio parahaemolyticus in seafood imported from Asian countries. International journal of food microbiology, 52(3), 181-188.

Wu, V. C. H., Qiu, X., Bushway, A., & Harper, L. (2008). Antibacterial effects of American cranberry (Vaccinium macrocarpon) concentrate on foodborne pathogens. LWT-Food Science and Technology, 41(10), 1834-1841.

Xi, Y., Sullivan, G. A., Jackson, A. L., Zhou, G. H., & Sebranek, J. G. (2011). Use of natural antimicrobials to improve the control of Listeria monocytogenes in a cured cooked meat model system. Meat science, 88(3), 503-511.

Yamamoto, K., Honda, T., Miwatani, T., Tamatsukuri, S., & Shibata, S. (1992). Enzyme- labeled oligonucleotide probes for detection of the genes for theromostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus. Canadian journal of microbiology, 38(5), 410-416.

Yamazaki, K., Tateyama, T., Kawai, Y., & Inoue, N. (2000). Occurrence of Listeria monocytogenes in retail fish and processed seafood products in Japan. Fisheries science, 66(6), 1191-1193.

Yoon, K. S., Min, K. J., Jung, Y. J., Kwon, K. Y., Lee, J. K., & Oh, S. W. (2008). A model of the effect of temperature on the growth of pathogenic and nonpathogenic Vibrio parahaemolyticus isolated from oysters in Korea. Food microbiology, 25(5), 635-641.

Yurdugul, S., & Bozoglu, F. (2009). Studies on antimicrobial activity and certain chemical parameters of freeze-dried wild plums (Prunus spp.). Pakistan Journal of Nutrition, 8(9), 1434-1441.

Zadernowski, R., Naczk, M., & Nesterowicz, J. (2005). Phenolic acid profiles in some small berries. Journal of Agricultural and Food Chemistry, 53(6), 2118-2124.

Zunabovic M., Domig K. J., Kneifel W. (2011). Practical relevance of methodologies for detecting and tracing of Listeria monocytogenes in ready-to-eat foods and manufacture environments – a review. LWT-Food Sci. Technol. 44 351–362.

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Appendices

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Appendix A. Inhibitory effect of fresh fruit pomaces and cranberry juice against Salmonella and L. monocytogenes after 48 h storage at 37 °C . Strain Diameter of inhibition zone (mm)a WPLPc PMP PSP WBP WSP PLPP WPOP POPP CJ (25%) (25%) (25%) S. Weltevreden NDb ND ND ND 10.5 ND 16.5 16.2 13.0 SFL0319 S. Newport ATCC ND ND ND ND 11.0 ND 18.0 17.0 12.8 6962 S. Newport H1275 ND ND ND ND 11.0 ND 18.0 16.5 12.9 S. Typhimurium ND ND ND ND 11.0 ND 17.8 16.7 12.5 ATCC 14028 L. monocytogenes 12.5 12.0 12.0 15.3 12.0 ND 17.5 23.3 20.7 Scott A L. monocytogenes 10.5 9.2 ND 17.0 9.2 ND 21.2 26.5 19.8 M0507 L. monocytogenes 13.0 11.0 11.2 14.1 11.0 ND 20.0 28.2 20.5 SFL0404 L. monocytogenes 12.0 12.2 11.2 16.2 12.2 ND 19.8 25.5 21.4 F5027 L. monocytogenes 12.5 12.0 11.0 16.5 12.0 ND 20.5 30.8 21.2 H0222 a Diameter of the zone was measured with the diameter of the wells (9 mm). b ND, not detected. c WPLP: Whole Plum Pomace, PMP: Plum Meat Pomace, PSP: Pomegranate Seed Pomace, WBP: Whole Blueberry Pomace, WSP: Whole Strawberry Pomace, PLPP: Plum Peel Pomace, WPOP: Whole Pomegranate Pomace, POPP: Pomegranate Peel Pomace, CJ: Cranberry Juice, RCJ: Color-reduced Cranberry Juice, PPE: Pomegranate Peel Extract and RPPE: Color-reduced Pomegranate Peel Extract.

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Appendix B. Inhibitory effects of color-reduced and non-color-reduced cranberry juice (CJ) and pomegranate peel extract (PPE) against Salmonella and L. monocytogenes after 48 h storage at 37 °C . Strain Diameter of inhibition zone(mm)a PPE (20%) PPE (33%) CJ (100%) Beforeb Afterb Before After Before After S. Weltevreden SFL0319 19.3 18.3 20.6 20.0 12.8 12.0 S. Newport ATCC 6962 18.8 18.0 21.0 20.0 12.5 12.0 S. Newport H1275 18.5 18.1 20.2 19.5 12.0 11.3 S. Senfterberg 18.0 17.8 20.0 19.0 12.0 11.2 S. Typhimurium ATCC 14028 18.0 17.5 19.8 19.0 11.7 11.0 L. monocytogenes Scott A 18.5 18.5 20.0 19.6 23.0 22.8 L. monocytogenes M0507 20.2 20.2 23.8 22.0 19.5 19.2 L. monocytogenes SFL0404 20.0 19.8 21.0 20.8 20.3 20.3 L. monocytogenes F5027 19.0 18.3 20.2 19.8 21.0 20.5 L. monocytogenes H0222 20.0 19.8 21.0 20.8 20.3 20.0 a Diameter of the zone was measured with the diameter of the wells (9mm) b Before means the fruit extracts we measured is the supernatants after centrifuge. After means the fruit extracts after centrifuge went thought the Discovery DPA-6S Solid Phase Extraction Products.