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8-2013 Antibacterial effects of on different strains of Escherichia coli and Listeria monocytogenes Hanan Eshamah Clemson University, [email protected]

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ANTIBACTERIAL EFFECTS OF PROTEASES ON DIFFERENT STRAINS OF ESCHERICHIA COLI AND LISTERIA MONOCYTOGENS

A Dissertation Presented to the Graduate School of Clemson University

In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Food Technology

by Hanan Lotfi Eshamah August 2013

Accepted by Dr. Paul L. Dawson, Committee Chair Dr. Anthony Pometto III Dr. James Rieck Dr. Xiuping Jiang

ABSTRACT

Escherichia coli O157:H7 and Listeria monocytogenes are pathogens that have received special attention by federal agencies, food safety researchers and food industries due to their economic and human health impact. To reduce the presence of these pathogens, alternative interventions have been studied. However, increasing consumer’s demand for natural ingredients has made the investigations of effectiveness of natural antimicrobials necessary. In this study, in vitro antimicrobial activity of and against E. coli JM109 and L. monocytogenes was investigated . Furthermore, actinidin and papain were evaluated to reduce populations of L. monocytogenes strain and three mixed strains of E. coli O157:H7 in cooked meat media and on when held at three different temperatures.

In vitro , bromelain levels of 4 mg/ml and 1 mg/ml were the most effective concentrations tested against E. coli JM 109 and L. monocytogenes , respectively, at 25 and 35 °C , reducing the populations by (3.37, 5.02) and (5.7, 4.1 ) log CFU/ml after 48 h, respectively. While papain levels of (0.0625 mg/ml) and (0.5 mg/ml) were the most effective concentration tested at 25 and 35 °C against E. coli and L. monocytogenes, respectively, reducing populations by (4.94, 5.64) and (6.58, 5.78) log CFU/ml after 48h, respectively.

While significant effects on bacterial populations in cooked meat media were found in this study, significant differences (P ≤ 0.05) were sometimes ≤ 1-log unit, which are not typically considered of practical significance. However, due to the highly

ii controlled nature of the study and that meat broth media exposes and bacteria to concentrated amounts of meat protein, the results may indicate positive results are possible when enzymes are applied to foods surfaces.

In cooked meat media, at 25 and 35 °C, for all actinidin and papain concentrations there were not significant reductions found in E. coli O157:H7 and L. monocytogenes populations for any time-temperature combination. Moreover, there was bacterial growth from 1 to 3 h for 25 and 35 °C. The bacterial growth at 35 °C was significantly higher than that at 25 °C. At 5 °C, actinidin and papain did not significantly reduce the populations of E. coli O157:H7 and L. monocytogenes except for actinidin at 50 mg/ml on L. monocytogenes at 24 h. Also, no difference was found between bacterial populations at 6 and 24 h for both pathogens except for L. monocytogenes at 24 h where there was bacterial growth for both papain levels tested.

On beef, the average reduction of E. coli O157:H7 was greater than that of L. monocytogenes and higher concentrations of either proteases yielded greater reduction in bacterial populations . For instance, actinidin at 700 mg/ml significantly (p ≤ 0.05) reduced the population of L. monocytogenes by 1.49 log cfu/ml after 3 h at 25 & 35 °C, and by 1.45 log cfu/ml after 24 h at 5 °C. Also, the same actinidin concentration significantly (p ≤ 0.05) reduced the populations of three mixed strains of E. coli O157:H7 by (1.81 log cfu/ml) after 3 h at 25 & 35 °C, and by (1.94 log cfu/ml) after 24 h at 5 °C.

While papain at 10 mg/ml reduced the population of L. monocytogenes by 0.56 log cfu/ml after 3 h at 25 & 35 °C and by (0.46 log cfu/ml) after 24 h at 5 °C. Also, the same

iii papain concentration significantly (p ≤ 0.05) reduced the populations of three mixed strains of E. coli O157:H7 by 1.48 log cfu/ml after 3 h at 25 & 35 °C, and by 1 .57 log cfu/ml after 24 h at 5 °C. These findings suggest that, in addition to improving the sensory attributes of beef, proteases can enhance meat safety and shelf life when stored at suitable temperatures. The findings also propose a promising approach in developing antimicrobial systems for beef products . If these enzymes are combined with current antimicrobial technologies, higher pathogen reductions may be achieved if present.

iv DEDICATION

I would like to dedicate this work to my beloved parents and family, for their endless love, support and encouragement.

v ACKNOWLEDGMENTS

The Prophet Muhammad (peace and blessings be upon him) said: “He who does not thank people, does not thank God” and for this, I would like to express my deepest appreciation to all those who provided me the possibility to complete this dissertation.

First and foremost, my utmost gratitude to my advisor, Dr. Paul Dawson, for his valuable guidance, continuous support and encouragement throughout the duration of my study. I would never be able to finish this dissertation without his immense patience and encouragements. Appreciation is also expressed to my graduate committee members,

Drs. Anthony Pometto III, James Rieck, and Xiuping Jiang, for their kindness in guiding and advising. My deepest gratitude to Dr. Han for her guidance and persistent help. Her ideas and tremendous support had a major influence on this dissertation.

I would also like to acknowledge Libyan Ministry of Higher Education for their financial and moral support. This project would not have been accomplished without their support.

I must never forget my parents, sisters and brothers; they are always my reminders of how important family is. I feel motivated and encouraged every time I call them. Thank you all. My deepest appreciations to my beloved husband Dr. Hesham Naas for his love, support, strong encouragement and sacrifices to have me finish this study.

Also, my love and deep thanks to my sweethearts Raoum, Mohamed and Rawan Hesham

Naas who I wish to have a good education and a better future.

vi Last but not the least, I would like to thank the one above all of us, God ”Allah”, for answering my prayers, giving me the strength, power and patience to be able to continue in this study. Without Him, I would be lost. Thank you so much.

vii TABLE OF CONTENTS

Page

TITLE PAGE ...... i

ABSTRACT ...... ii

DEDICATION ...... v

ACKNOWLEDGMENTS ...... vi

LIST OF TABLES ...... x

LIST OF FIGURES ...... xii

CHAPTER

1. INTRODUCTION ...... 1

2. LITERATURE REVIEWS ...... 5

Food borne pathogens ...... 5 Listeria monocytogenes ...... 6 Escherichia coli O157:H7 ...... 8 Natural food additives for meat and poultry products ...... 10 Meat tenderizers ...... 11 Proteolytic enzymes ...... 12 Plant proteolytic enzymes ...... 14 Role of proteolytic enzymes on protein hydrolysis ...... 16 Bromelain ...... 17 Papain ...... 20 Actinidin ...... 23 References ...... 25

viii 3. BACTERICIDAL EFFECTS OF NATURAL TENDERIZING ENZYMES ON ESCHERICHIA COLI AND LISTERIA MONOCYTOGENES ...... 35

Abstract ...... 35 3.1. Introduction ...... 36 3.2. Materials and Methods ...... 39 3.3. Results ...... 41 3.4. Discussion ...... 45 References ...... 47

4. ANTIBACTERIAL EFFECTS OF PROTEASES ON DIFFERENT STRAINS OF ESCHERICIA COIL O157:H7 AND LISTERIA MONOCYTOGENES IN COOKED MEAT MEDIA ...... 57

Abstract ...... 57 4.1. Introduction ...... 58 4.2. Materials and methods ...... 59 4.3. Results ...... 63 4.4. Discussion ...... 65 References ...... 67

5. ANTIBACTERIAL EFFECTS OF PROTEASES ACTINIDIN AND PAPAIN ON DIFFERENT STRAINS OF ESCHERICIA COIL O157:H7 AND LISTERIA MONOCYTOGENES ON BEEF ...... 74

Abstract ...... 74 5.1. Introduction ...... 75 5.2. Materials and methods ...... 79 5.3. Results ...... 83 5.4. Discussion ...... 85 5.5. Conclusion ...... 89 References ...... 90

6. CONCLUSIONS ...... 105

APPENDIX………………...... 108 Laboratory media preparation, protein concentration and enzyme activity assays and growth curve of E. coli O157:H7 strains ...... 108

ix LIST OF TABLES

Table Page

2.1 Classification of proteases by characterizing active sites ...... 13

2.2 Plant proteinases and their sources ...... 15

2.3 Cysteine proteinases (bromelain) from ( Ananas Comosus ) ...... 18

4.1 Differences in E. coli O157:H7 populations exposed to various concentrations of actinidin in cooked meat media compare to time 0 ...... 70

4.1a. at 5 °C ...... 70

4.1b. at 25 and 35 °C ...... 70

4.2 Differences in L. monocytogenes populations exposed to various concentrations of actinidin in cooked meat media compare to time 0 ... 71

4.2a. at 5 °C ...... 71

4.2b. at 25 and 35 °C ...... 71

4.3 Differences in E. coli O157:H7 populations exposed to various concentrations of papain in cooked meat media compare to time 0 ...... 72

4.3a. at 5 °C ...... 72

4.3b. at 25 and 35 °C ...... 72

4.4 Differences in L. monocytogenes populations exposed to various concentrations of papain in cooked meat media compare to time 0 ...... 73

4.4a. at 5 °C ...... 73

4.4b. at 25 and 35 °C ...... 73

x Table Page

5.1 Reduction in E. coli O157:H7 and L. monocytogenes populations at 5 °C exposed to various concentrations of actinidin on beef……………….98

5.2 Reduction in E. coli O157:H7 and L. monocytogenes populations at 25 and 35 °C exposed to various concentrations of actinidin on beef……..99

5.3 Reduction in E. coli O157:H7 and L. monocytogenes populations at 5 °C exposed to various concentrations of papain on beef ...... 100

5.4 Reduction in E. coli O157:H7 and L. monocytogenes populations at 25 and 35 °C exposed to various concentrations of papain on beef ...... 101

5.5 Total and specific activity of actinidin and papain measured by spectrophotometer…………………………………………..…..……..102

A.1. Preparation of diluted albumin (BSA) standard…………………..……110

A.2. Result of diluted albumin (BSA) standard ……………………...……...111

A.3. Actinidin absorptions and concentrations……………………….….…..112

A. 4. Papain absorptions and concentrations ………………...…………….….112

A.5. Specific activity of different actinidin concentrations………..……….....117

A.6. Specific activity of different papain concentrations ………………...…..117

xi LIST OF FIGURES

Figure Page

2.1 Natural progression infection with E. coli O157:H7…………………...…10

2.2 Natural progression of post-diarrheal HUS…………………………….....10

2.3 Optimum temperature and pH of bromelain………………………….…...19

2.4 Optimum temperature and pH of papain……………………………….....22

2.5 Optimum temperature and pH of actinidin……………………………...... 24

3.1 Effect of bromelain on E. coli E. coli JM 109 at 5, 25 and 35 °C over 48 hours…………………………………………………………….....51 . 3.1. a. Bromelain against E. coli JM 109 at 5 °C……………………...... 51 3.1. b. Bromelain against E. coli JM 109 at 25 °C…………………...... 51 3.1. c. Bromelain against E. coli JM 109 at 35 °C …………...……...... 51

3.2 Effect of bromelain on L. monocytogenes at 5, 25 and 35 °C over 48 h. ….52

3.2. a. Bromelain against L. monocytogenes at 5 °C…………………....52 3.2. b. Bromelain against L. monocytogenes at 25 °C…………………..52 3.2. c. Bromelain against L. monocytogenes at 35 °C…………………..52

3.3 Effect of temperature on bromelain efficiency against on E. coli JM 109 after 48 h………………………………………………………….…...53

3.4 Effect of temperature on bromelain efficiency against L. monocytogenes after 48 h ………………………………………………………….…..53

3.5 Effect of papain on E. coli JM 109 at 5, 25 and 35 °C over 48 hours… ... ..54

3.5. a. Papain against E. coli JM 109 at 5 °C………………………....…54 3.5. b. Papain against E. coli E. coli JM 109 at 25 °C...... 54 3.5. c. Papain against E. coli E. coli JM 109 at 35 °C……...... 54

3.6 Effect of papain on L. monocytogenes at 5, 25 and 35 °C over 48 h……. .. 55

3.6. a. Papain against L. monocytogenes at 5 °C………………..………..55

xii 3.6. b. Papain against L. monocytogenes at 25 °C………………… .…55 3.6. c. Papain against L. monocytogenes at 35 °C……………….….…..55

3.7 Effect of temperature on papain efficiency against on E. coli JM 109 after 48 h ..... ………………………………………………………….56

3.8 Effect of temperature on papain efficiency against L. monocytogenes after 48 h ...... 56

5.1 Effect of different concentrations of actinidin on L. monocytogenes and E. coli O157:H7 at 5 °C. The standard error for the means was 0.18. .. 103

5.2 Effect of different concentrations of actinidin on L. monocytogenes and E. coli O157:H7 at 25 and 35 °C. The standard error for the means was 0.11……………………………………………………………….…103 5.3 Effect of different concentrations of papain on L. monocytogenes and E. coli O157:H7 at 5 °C. The standard error for the means was 0.17…………………………………………………………………..…104 5.4 Effect of different concentrations on L. monocytogenes and E. coli O157:H7 at 25 and 35 °C. The standard error for the means was 0.14…...104

A.1. Diluted Albumin (BSA) Standards curve…………………………...... 112

A.2. Enzyme activity standards curve…………………………………....…116

A. 3. Growth curve of E. coli O157:H7 38094……………………………....119

A. 4. Growth curve of E. coli O157: H7 E-0654…………………….……....119

A. 5. Growth curve of E. coli O157: H7 C7929………….………….….…...120

xiii CHAPTER ONE

INTRODUCTION

Increasing in world population and changing in lifestyles have resulted in major concerns about food quality of animal origin. The meat from a healthy animal is initially sterile, but may become contaminated by hide, skin, hooves, hair, gastrointestinal tract contents, knives, cutting tools, infected staff, polluted water, air, improper slaughtering technique, post slaughter handling or during storage (Frazier and Westhoff, 1988).

Different types of pathogenic and spoilage organisms may be introduced into meat during slaughtering and processing, which causes rapid spoilage, great loss of valuable protein and affects human health. Therefore, it is very important to reduce the initial microbial load to increase shelf-life of meat .

It has been reported that 90% of the estimated food-related deaths involve the pathogens Salmonella (28%), Toxoplasma (24%), Listeria monocytogenes (19%),

Norwalk-like viruses (11%), Campylobacter (6% ) (CDC, 2011), and Escherichia coli

O157:H7 (3%) (Mead et al.,1999).

Escherichia coli O157:H7 and Listeria monocytogenes are pathogens that have received special attention by federal agencies and food safety researchers due to their economic and human health impact. These two pathogens are responsible for 3 billion dollars in economic losses each year (USDA, 2006).

The worldwide awareness of health risks associated with non-natural additives added to control some pathogen of concerns have prompted investigations into the use of

1 natural products as antimicrobials obtained from various sources including plants and spices.

Using exogenous proteases to tenderize meat has been of considerable interest with focus on some members of the plant cysteine family such as papain, bromelain, ficin and actinidin (Ha et al., 2012; Ketnawa and Rawdkuen, 2011; Koak et al., 2011; Naveena et al., 2004; Sullivan and Calkins 2010). Plant proteolytic enzymes are superior to bacterially derived enzymes as meat tenderizers because of safety concerns such as pathogenicity. Plant proteolytic enzymes can digest muscle proteins including collagen and elastin, which lessens the toughness of meat. However, the proper quantity of enzymes must be used because excessive amounts would result in meat decomposition (Rawdkuen et al ., 2012).

Papain is an important plant peptidase due to its powerful proteolytic activity. It derived from the latex of unripe fruit ( Carica papaya , Caricaceae ). It is characterized by its ability to hydrolyze large proteins into smaller and amino acids. Its ability to break down fibers has been used for many years in food industry

(Llerena-Suster et al., 2011). Studies found that papain and other papaya extracts possess antimicrobial activities against Bacillus subtilis , Enterobacter cloacae , E. coli ,

Salmonella typhi , Staphylococcus aureus , and Proteus vulgaris (Osato et al., 1993;

Emeruwa, 1982).

Bromelain is also a proteolytic enzyme which is a derived from fruit (Ananas comosus) which is a member of family (Hale et al.,

2 2005). Many studies revealed that bromelain has antimicrobial effect such as antihelminthic activity against gastrointestinal nematodes and anti-candida effects.

Bromelain can also cause complete resolution of infectious skin diseases like pityriasis lichenoides chronica (Alternative Medicine Review, 2010)

Actinidin is another member of cysteine protease family present in kiwi fruit and it belongs to the same class of enzymes as ficin, papain and bromelain. It has many applications in the food industry replacing other plant proteases like papain and ficin because of its mild tenderizing reaction even at high concentrations preventing surface mushiness; it has a relatively low inactivation temperature (60 °C) which makes it easier to control tenderization without overcooking (Tarté, 2009). Moreover, it does not affect sensory attributes of meat (flavor and odor) compared to the other proteases

(Christensen et al., 2009). Actinidin also has beneficial effects on lipid oxidation and color stability of lamb meat (Bekhit et al ., 2007; Ha et al., 2012).

Actinidin has potential pharmaceutical usages, for example Mohajeri et al., (2010) concluded that kiwi fruit extract has dramatic antibacterial and debridement effects when used as a dressing on deep second–degree due to its strong proteolytic effects

(Hafezi et al., 2010). Moreover, Basile (1997) found that Actinidia chinensis extract has significant antibacterial activity against various Gram-positive and Gram-negative strains.

Meat consumption is increasing around the world, prompting concerns related to the meat quality (tenderness), hygiene and safety. Meat toughness can be addressed in different ways while meat hygiene concerns are mostly of a biological nature and

3 includes bacterial pathogens, such as Escherichia coli O157:H7, Salmonella and

Campylobacter in raw meat and poultry, and Listeria monocytogenes in ready-to-eat processed products (Sofos and Geornaras, 2010). Proteolytic enzymes are used in meat marinades and meat tenderizers and these natural enzymes may also reduce risk of meat pathogens. This study examined three proteolytic enzymes (actinidin, papain and bromelain) for antimicrobial activity against pathogenic bacteria. Three bacteria such as three mixed strains of E. coli O157:H7, E. coli JM109 and L. monocytogenes were used to determine the effect of these proteolytic enzymes on the population (log CFU/ml) when held at different temperatures (5, 25 and 35 °C).

4 CHAPTER TWO

LITRATURE REVIEW

This review will cover foodborne pathogens ( L. monocytogenes and E. coli

O157:H7), plant proteolytic enzymes (bromelain, papain and actinidin), their meat tenderizing and antimicrobial effects.

Foodborne pathogens

Foodborne pathogens are considered a major cause of human suffering through illnesses, deaths and massive economic losses in both under developed and developed countries. Gould and Russell, (2003) have estimated that there are more than 1 billion cases of gastroenteritis and up to 5 million deaths annually in under developed countries.

According to the US Centers for Disease Control and Prevention (CDC), foodborne diseases cause 48 million illnesses, more than 128,000 hospitalizations and 3000 deaths annually in the United States (CDC, 2011). Scharff (2012) announced that the annual foodborne illnesses cost about $ 77 billion and total annual health related cost of food safety is around

$103 billion in US, whereas economic Research Service of the United States Department of

Agriculture (USDA, 2006) reported that the economic losses caused by E.coli O157:H7 and

L.monocytogenes are more than 3 billion dollars each year. Todd (1989) reported that cost to treat the foodborne disease due to meat and meat products contamination is estimated to $500 million per year.

The increase in foodborne microbial hazards caused by some pathogens such as L. monocytogenes and E. coli O157:H7 have received great attention and concern by regulatory agencies, food industries and the food safety researchers. According to the CDC (2011),

5 foodborne outbreaks still occur frequently suggesting that new alternatives to reduce health hazards and economic losses due to foodborne microorganisms are needed. The use of natural products as antibacterial compounds (Conner, 1993; Dorman and Dean, 2000) appear to be an interesting way to control the presence of pathogenic bacteria and to extend the shelf life of fresh and processed food.

Listeria monocytogenes

L. monocytogenes is a Gram positive, motile, nonsporeforming rod that grows at wide temperature 1.7 °C – 50 °C and pH ranges 4.5 to 7.0 (Junttila et al ., 1988; Walker et al., 1990). L. monocytogenes is widely distributed in the nature with some studies indicating that 1 - 10% of humans are intestinal carriers of L. monocytogenes (FDA Bad

Bug Book, 2012). Its association with meat and slaughter environments is well established (Benkerroum et al., 2003). Consumption of raw and partially cooked contaminated meat can result in Listeriosis, especially among the immune-compromised populations, elderly and pregnant (Shrinithivihahshini et al., 2011). According to the

CDC (2011) the rate of listeriosis has decline by 38 % from 1996-2010. Yet, L. monocytogenes causes an estimated 1600 cases of listeriosis, 1450 hospitalizations and

255 deaths annually in the United States (Scallan et al., 2011; CDC, 2011). Moreover, 24 confirmed listeriosis outbreaks were reported during 1998-2008, resulting in 359 illnesses, 215 hospitalization and 38 deaths (Cartwright et al., 2013). As L. monocytogenes is a ubiquitous organism able to multiply at refrigeration temperatures and under anaerobic conditions, it is of major concern especially in RTE meat and poultry

6 products (Martin et al., 2009). The minimum infective dose of L. monocytogenes is unknown but is thought to vary with the strain and individual susceptibility (FDA Bad

Bug Book, 2012). However, indications are that the intake of up to 100 cells does not affect the health of healthy consumers (Jay, 1994).

The United States Department of Agriculture- -Food Safety and Inspection

Survives recommended that food industry re-evaluate HACCP plans with specific regard to the threat of L. monocytogenes with a goal of 0.24 /100,000 people in 2010, targeting the reduction in overall incidence of listeriosis by 25% to 0.2 / 100,000 in 2020 (USDA-

FSIS, 2012; Cartwright et al., 2013). USDA-FSIS currently enforces a zero-tolerance policy on foods labeled as ready-to-eat (USDA-FSIS, 2012). The viability of zero- tolerance policy is always a subject of dispute between industry and academia, however, the push for such an extreme measure is highly indicative of the problem the pathogen causes for consumers.

L. monocytogenes is quite hardy and resists the danger effects of freezing, drying, and heating treatments. According to the CDC, general recommendations to reduce people’s risk of listeriosis include: thoroughly cooking raw food from animal sources such as beef, pork and poultry, washing raw vegetables thoroughly before eating, and keeping uncooked meats separated from vegetables and cooked ready-to-eat foods, consuming perishable and ready-to-eat foods as soon as possible (CDC, 2013).

Escherichia coli

E. coli is a ubiquitous Gram-negative bacterium commonly found within the colonic flora of humans and warm blooded animals. Some strains of E. coli can cause

7 adverse effects to the gastrointestinal system, and are classified according to their virulence properties into enteropathogenic E. coli (EPEC), enterotoxinogenic E. coli

(ETEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), enteroaggregative E. coli ( EAEC), diffusely adherent E. coli (DAEC), and necrotoxinogenic E. coli (NTEC). Of these, the first four groups are known to be transmitted via contaminated food or water; EHEC, mainly E. coli O157:H7, are often implicated in major foodborne outbreaks worldwide (Nataro and Kaper, 1998; Mead and

Griffin, 1998).

Escherichia coli O157:H7

E. coli O157:H7 is an emerging pathogen responsible for about 63,000 illnesses,

2000 hospitalizations and 20 deaths each year in the United States (Scallan et al., 2011).

Most of these illnesses are associated with eating undercooked contaminated ground beef.

Foods usually get contaminated through improper slaughtering processes, shedding of pathogens from colonized cattle into , use of contaminated soil or contaminated irrigation water in produce production, or cross-contamination. Harvesting procedures often applied in food processing such as fruit, vegetable or meat are considered to be at lower risk of contamination (Elder et al., 2000).

In addition to its traditional association with ground beef, E. coli O157:H7 has also been found in nonmeat foods such as radish sprouts in Japan in 1996 (Park et al.,

1999) and hazelnuts in the Great Lakes region in 2011 (Nunnelly, 2012).

E. coli O157:H7 was recognized as a significant foodborne pathogen in the early

1980s and continues to be a major cause of diarrheal illness in North America. Human

8 infection with E. coli O157:H7 is associated with asymptomatic shedding, non-bloody diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (HUS) , and thrombotic thrombocytopenic purpura (TTP), which may lead to kidney failure in children and elderly (Bavaro, 2009) . E. coli O157:H7 may be shed in the stool for several weeks following the resolution of symptoms. The average interval between exposure to the organism and illness is 3 days with incubation periods as short as 1 day and as long as 8 days being reported (Mead and Griffin, 1998) . E. coli O157:H7 is thought to account for over 90% of all HUS cases, however, only 5% of E. coli O157:H7 infections result in

Hemolytic Uremic syndrome development in the patient.

Figures 2.1 and 2.2 from Mead and Griffin (1998) explain the natural progression of infection with E.coli O157:H7. The infective dose of E. coli O157:H7 is estimated to be very low, in the range of 10 to 100 cells (FDA Bad Bug Book, 2012). According to the CDC, 350 outbreaks were reported from 1982 to 2002 (Rangel et al., 2005).

E. coli O157:H7 is resistant to acidic conditions, low temperatures, freezing and competitive flora. Due to its pathogenicity and ability to survive under a wide range of environmental conditions, its presence in foods and clinical specimens has been the focus of many studies (Noveir et al., 2000)

9

E.coli O157:H7 ingested

3 – 4 days

Abdominal cramps, non-bloody diarrhea

1 – 2 days

Bloody diarrhea

95% 5 -7 days 5%

Resolution HUS

Figure 2. 1. Natural progression of infection with E coli O157:H7 (Mead and Griffin, 1998)

HUS

3% - 5% death ~60% resolution

~5% chronic ~30% renal failure, proteinuria and stroke, and other minor ? other major sequelae sequelae

Late complications

Figure 2. 2. Natural progression of post-diarrheal HUS (Mead and Griffin, 1998)

Natural food additives for meat and poultry products

Foods are very susceptible to different biological deterioration and are a suitable for pathogen growth. Heating, cooling, drying or fermenting have been popular methods to achieve quality and safety goals since prehistoric times. However, it has been only during the last century that the use of chemicals to control spoilage and pathogenic microorganisms in food became extensive.

10 Recently, the use of food additives has become more popular due to the increased production of prepared, processed and convenience foods (USDA, 2008). Additives are used for technological purposes in the manufacturing, processing, preparation, treatment, packaging, transportation or storage of certain foods. Thus, food additives are widely used and are essential in food manufacturing industries.

Food additives have been used in meat products for various reasons such as changing and/or stabilizing of pH values, increasing water holding capacity in order to attain higher yields, decreasing cooking losses, improving texture and sensory properties

(tenderness, juiciness, color and flavor), and extending shelf-life.

Important food additives to modify texture include proteolytic enzymes such as bromelain, ficin and papain (USDA, 2008) that can dissolve or degrade the proteins collagen and elastin to soften meat and poultry tissue.

Meat tenderizers

Tenderness is one of the most important quality attributes of meat. The consumer acceptance or rejection for cut or processed meat depends on its tenderness. is basically related to structural integrity of myofibrillar and connective tissues proteins (Marsh and Leet, 1966; Nishimura et al ., 1995). Many studies have investigated methods to improve tenderness and overall meat quality. These studies attempted to reduce the toughening effect of connective tissues using different tenderizing methods including chemical tenderization of meat with enzymes, salts, and physical tenderization by pressure treatments, blade tenderization or electrical stimulation (Ketnawa et al.,

2011).

11 Brooks (2007) concluded that enzymes not only improves tenderness but can also be added to enhance juiciness, flavor, yields, and water holding capacity, shelf-life, and anti-microbial attributes thus providing a more valuable .

Plant proteolytic enzymes have gained special attentions in the field of medicine and biotechnology due to their proteolytic properties. Five exogenous proteases that have been classified as “Generally Recognized as Safe” (GRAS) by USDA’s Food Safety

Inspection Service (FSIS) (Payne, 2009) are papain, bromelain, ficin, and microbial enzymes sourced from Bacillus and Aspergillus spp. These enzymes are shown to have varying degrees of activity against myofibrillar and collagenous proteins. In addition to these GRAS enzymes, enzymes isolated from kiwi fruit (actinidin) and () showed potential for future inclusion in meat systems for tenderization (Han et al., 2009;

Naveena et al., 2004; Ma, 2011; Wada et al., 2004 & Ketnawa et al ., 2010).

Proteolytic enzymes have been widely used in food, medical–pharmaceutical, cosmetic and other industries. In the food industry, the primary application has been for meat tenderization. Many studies have investigated meat tenderness using different proteases. However, to date there has been few studies investigating antimicrobial effects of proteolytic enzymes.

Proteolytic Enzymes

The term of proteolytic enzymes also refer to proteases or proteinases which are able to hydrolyze bonds of protein. Proteolytic enzymes that can act near the termini of polypeptides chains are called exopeptidases, while proteolytic enzymes that can act away from termini are called (González-Rábade et al., 2011).

12 Exopeptidases are divided into which are able to hydrolyze at the N-terminus, and carboxypeptidases which hydrolyze peptide bond at C-terminus.

Endopeptidases are classified on basis of their mechanism of action at the . In plants, there are five types of endopeptidases which include Cysteine, Serine, Aspartic,

Metallo and Threonine (Rawlings et al., 2010; Jakubowski, 2010 ) examples for them are shown in Table 2.1. Proteolytic enzymes form the most important group of currently in use due to their important roles in the food and detergent industries, and also in leather processing and as therapeutic agents (Walsh, 2002).

Table 2.1 Classification of proteases by amino acids characterizing actives sites (Jakubowski, 2010)

Class (active site) Example , , subtilisin, Serine/ Threonine elastase Aspartate Metallo Thermolysin Cysteine Papain family

Farouk (1982) investigated the antibacterial activity of proteolytic enzymes against different types of bacteria and found that the tested proteolytic enzymes showed higher killing effect against the Gram-negative bacteria ( Escherichia coli , Proteus vulgaris , and Pseudomonas aeruginosa ) than the gram-positive bacteria ( Staphylococcus aureus and Streptococcus pyogenes ). Farouk theorized these effect differences were due to the differences of Gram negative and Gram positive bacterial structure and to

13 high amount of lipoproteins in Gram negative than Gram positive bacteria. He also concluded the lethal activity of proteolytic enzymes was dependent on enzyme concentrations.

Leary et al. (2009) reported that proteases are the most common type in the $3 billion world market of enzymes. Also, Rowan and Buttle (1994) estimated that sales of proteolytic enzymes account for over 60% of total market share of these types of biochemical products indicating the great importance of proteases as a group of industrial enzymes.

Plant Proteolytic Enzymes

Proteolytic enzymes have been studied from the latex of several plant families such as Caricaceae, Asteraceae, Asclepiadaceae, Moraceae, Apocynaceae and

Euphorbiaceae . Most plant proteolytic enzymes are cysteine proteases with few Aspartic proteases (Rawling et al., 2010). Plant proteolytic enzymes such as papain, bromelain, actinidin and ficin have been used frequently in several industrial applications because of their ability to act over a wide temperature and pH range (Table 2.2). These industrial applications include the food industry, e.g. brewing, meat tenderization, and beverage industry (González-Rábade et al., 2011).

14 Table 2. 2 Plant cysteine proteinases and their sources (Stepek. 2004)

Plant species pH Stability Enzyme (common name) optimum to acid Carica Papain 4–10 To pH4 papaya (papaya) To 3–10

Glycyl 3–10 To pH3 carica (Mediterranean Ficin 4–8.5 To pH4 fig) To Ficus glabrata 4–8.5 pH3.3 Ananas Stem 5.5–8 NA comosus (pineapple) bromelain Fruit 5.5–8 NA bromelain 5.5–8 NA

Comosain 5.5–8 NA

Actinidia Actinidin 4–10 NA chinensis (kiwi fruit) Calotropis gigantea (madar Calotropin 4–8 NA plant) Asclepias spp. 6–10 NA (milkweed)

Treatment by plant proteolytic enzymes is popular method for meat tenderization.

Papain, bromelain and ficin have been widely used as meat tenderizers in most parts of the world (Ketnawa and Rawdkuen, 2011). About 95% of the meats tenderizing enzymes used in the United States are from plant proteases papain and bromelain. This marked

15 tenderizing effect is due to the strong proteolytic activity of these enzymes (Amid et al.,

2011).

Plant proteases also have important applications in the pharmaceutical industry such as a debrider as an alternative to mechanical cleansing for rapid removal of dead tissue. Salas et al. (2008) established that there was a clear association between plant cysteine proteases (bromelain and papain) and therapeutic treatment of digestive disorders, dermal and gastric ulcers of different origins, immunological modulation, and tumoral/metastatic disorders.

Role of proteolytic enzymes on protein hydrolysis

Papain, bromelain, and actinidin belong to the cysteine protease family. These enzymes and others from figs are part of the papain family. This group of enzymes shows only few variations in primary structure, however, they are not identical.

Collectively, they are characterized by having a chemically sensitive sulfhydryl group at their active site (Glazer and Smith, 1971).

Papain consists of 212 amino acids with 3 bridges (cys22-cys63, cys56- cys95 and cys153-cys200) and a free cysteine cys25 which takes part in the .

Catalytic activity is proportional to the thiol content of the enzyme. Papain tertiary structure consists of 2 distinct structural domains with a cleft between them. This cleft contains the active site, which contains a which is made up of 3 amino acids: the chemically sensitive cysteine-25, -159 and -158.

16 The mechanism by which it breaks peptide bonds involves deprotonating of Cys-

25 by His-159. Cys-25 then performs a nucleophilic attack on the carbonyl carbon of a peptide backbone. This frees the amino terminal of the peptide and forms a covalent acyl-enzyme intermediate. The enzyme is then deacylated by a water molecule (H 2O) and releases the carboxyl terminal portion of the peptide (Amri and Mamboya, 2012).

As expected for papain family enzymes, actinidin has a titratable free sulfhydryl group that is essential for activity (Baker, 1980). The 3-D structure of actinidin was determined by X-ray crystallographic analysis, which showed that the polypeptide chain conformation was essentially identical to that of papain (Drenth et al., 1971). Therefore, actinidin is likely to perform in a similar way to papain on protein hydrolysis.

Bromelain

Bromelain is a complex mixture of proteolytic enzymes which are mainly cysteine proteases. It is derived from pineapple plant (Ananas comosus) which is a member of

Bromeliaceae family (Hale et al., 2005). Bromelain contains not only protease components but also contains non-protease components. Proteases constitute the major components of bromelain (Table 2.3) including (80%),

(10%), and ananain (5%), whereas non-protease components include phosphatases, glucosidases, peroxidases, cellulases, glycoproteins and carbohydrates (Chobotova et al.,

2010 ; Maurer, 2001) .

17 Table 2. 3 Cysteine proteinases (bromelain) from pineapples ( Ananas comosus ) (Maurer, 2001) Molecular Name (EC Isoelectr Abbreviation mass Sequences Glycosylation number ) ic point (Dalton) From pineapple

stems: Completely 23,800 Stem bromelain sequenced F4 & F5 (sequence > 10 glycosylated (EC 3.4.22.32) (212 amino + sugar) acid) Completely Ananain 23,464 sequenced Non F9 > 10 (EC 3.4.22.31) sequence (216 amino glycosylated acid) 23,550 & N-term. SBA/a & 4.8 and highly 23,560 sequence Comosain SBA/b 4.9 glycosylated 24,509 & N-term. F9/b > 10 glycosylated 23,569 sequence From pineapple highly fruits: N-term. not glycosylat 4,6 Fruit bromelain sequence glycosylated ed (EC 3.4.22.33)

Corzo et al., (2011) reported that optimum pH and temperature conditions for proteolytic activity of bromelain are in range of 6.5-7.5 and 50-60 °C, respectively (Fig.

2.3). In the food industry, bromelain has been used widely in meat tenderization processes because of its ideal temperature range of 50-70 °C which is appropriate for a food processing applications (Amid et al., 2011; Calkins & Sullivan, 2007).

18

Figure 2.3. Optimum temperature and pH of bromelain (Corzo et al ., 2011)

Ketnawa and Rawdkuen (2011) concluded that the technology of using bromelain as a meat tenderizer is easily and cheaply available and can be accomplished at the household or industrial level. They also suggested that bromelain can be applied as a better alternative to other tenderizers such as chemical or other plant proteases. They also observed the difference between muscle fibers treated with or without bromelain using scanning electron micrographs. The non- treated muscle fibers were closely bound to each other while muscle fibers treated with bromelain were less attached, and there was a loss of muscle fiber interaction. Moreover, there was a disintegration of myofibrillar structure with an excess of exudates. In addition, Calkins and Sullivan (2007) found that bromelain tenderizing action related to damaging of both myofibrillar and collagen components of the muscle ultrastructure, and it is more effective when bromelain solution is injected into muscle compared to dipping or tumbling in brine (McKeith et al., 1994).

In the United States, bromelain is sold in health food stores as a nutritional supplement to promote digestive health and is used as anti-inflammatory drug (Borrelli et al., 2011). A study by Zamyatnina and Brochikov (2007) revealed that tetra-, penta-, and hexa-peptide fragments of the bromelain molecules are involved in amino acid sequences

19 of many natural oligopeptides including antimicrobial oligopeptides, toxins, neuropeptides, and hormones. Therefore, these fragments with antimicrobial effects can be considered as natural preservatives of food products that will increase shelf life without dangerous side effects. Bromelain also has antimicrobial effects such as antihelminthic activity against gastrointestinal nematodes, anti-candida effects, and bromelain can cause complete resolution in case of infectious skin disease like pityriasis lichenoides chronica (Alternative Medicine Review, 2010; Roxas, 2008; Maurer, 2001).

Papain

Papain is another important plant peptidase due to its powerful proteolytic activity derived from the latex of unripe papaya ( Carica papaya , Caricaceae ). Papain is characterized by its ability to hydrolyze large proteins into small peptides and amino acids. Its ability to break down tough fibers was used for many years in the USA and is now included as a component in powdered meat tenderizers (Llerena-Suster et al., 2011).

Papain has a highly aggressive tenderizing action on myofibrillar and collagen proteins yielding protein fragments of several sizes. Moreover, it shows massive disruption of the

Z disc thus, it was found to be unsatisfactory for use at a commercial level because it over-tenderizes the surface of the meat producing a mushy texture and unusual bitter flavor (Lawrie, 1998).

Papain is more effective when injected into the product due to its poor ability to penetrate surfaces (Brooks, 2007). However, another study by Maiti et al. (2008) showed that papain infusion with forking technology was more effective for tenderizing hen meat

20 cuts than . While Grover et al. (2005) concluded that sodium tri polyphosphate has a synergistic effect on papain in increasing the tenderness of chicken gizzard.

Papain has many other applications in different industries, especially in personal care products such as in shower gels and soaps, and in the food industry including the preparation of chewing gum, brewing to remove cloudiness in beer, and in dairy products for cheese manufacture. It is also used in the pharmaceutical industry, textile industry and in the tanning industry (Ming et al., 2002).

Papaya fruits, , latex and extracts have been used traditionally to treat various human ailments. Papaya is found to be a rich source of biologically active isothiocyanate (Nakamura et al., 2007). Unripe pulp of Carica papaya is rich in carbohydrate and starch (Oloyede, 2005) and also contains cardenolides and saponins that have medicinal value for use in the treatment of congestive heart failure (Schneider and

Wolfing, 2004).

The papaya-latex is well known for being a rich source of the four cysteine endopeptidases namely papain, a well-known proteolytic enzyme, chymopapain, and that may contribute to latex`s antimicrobial properties (Anuar et al., 2008). Osato et al. (1993) revealed that the papaya latex possess bacteriostatic properties against Bacillus subtilis , Enterobacter cloacae , E. coli , Salmonella typhi ,

Staphylococcus aureus , and Proteus vulgaris by inhibiting of either bacterial cell wall synthesis or protein synthesis.

Anibijuwon and Udeze (2009) reported that Carica papaya may be used for treatment of gastroenteritis, urethritis, otitis media and wound infections. They also

21 concluded that the antimicrobial activity against both Gram-negative and Gram-positive bacteria is an indication that the Carica papaya is a potential source for production of

medicine with broad-spectrum bactericidal activity. Moreover, Emeruwa (1982) indicated that Carica papaya fruit extracts contain an antibacterial substance which is bactericidal on several species of Gram-positive and -negative bacteria. However, the bacteria varied widely in the degree of their susceptibility, reporting that small amounts

0.03 % (W/V) of the extract inhibited growth of Gram-positive bacteria such as S. aureus

and B. cereus. On the other hand, a wider range and higher concentrations 0.4 % (W/V)

were required for the inhibition of the Gram-negative bacteria such as E.coli

Emeruwa (1982) also suggested that the site of action of the antibacterial was at

the cell wall because the cell morphology appeared changed after exposure to the extract.

Ming et al. (2002) and Calkins & Sullivan (2007) reported that optimum pH and

temperature conditions for proteolytic activity of papain are in range of 6.0-7.0 and 65-80

°C, respectively (Fig. 2.4). While Anibijuwon and Udeze (2009) found that the increase

in temperature enhances the activity, whereas alkaline pH decreases the activity of

papain.

Figure 2.4. Optimum temperature and pH of papain (Ming et al., 2002)

22 Actinidin

Actinidin [derived from kiwi fruit ( Actinidia chinensis )] is one of the plant thiol proteinases which contain a free sulfhydryl group, similar to papain, bromelain and ficin

(Kamphuis et al., 1985). Actinidin has wide substrate specificity, hydrolyzes most amide and ester bonds at the carboxyl side of a residue and is active at wide pH range 4-

10. The amino acid sequence of actinidin shows about 52 % homology with papain

(Katsaros et al., 2009). However, it has advantages over other plant proteases because of its mild tenderizing action (Lewis and Luh, 1988). It is very active against both globular proteins such as myosin and fibrous proteins such as collagen of muscle tissue (Wada et al., 2004; Lewis and Luh, 1988). Furthermore, it is able to hydrolyze the myofibrillar structure by enhancing the action of and which are active at low pH (Warriss, 2000). It has a lower inactivation temperature (60 °C) compared to that of papain and bromelain (80 °C), which makes it easier to control the tenderizing action without overcooking (Tarté, 2009). Moreover, it does not affect sensory attributes of meat (flavor and odor) (Christensen et al., 2009), and has beneficial effects on lipid oxidation and color stability of lamb meat (Bekhit et al., 2007). Therefore, it would appear that the meat tenderizing ability of actinidin could be a practical option for the commercial meat industry that would benefit consumers (Lewis and Luh, 1988). Besides meat tenderizing, actinidin has other food applications such as beer chill haze removers, cereals quality improvers, and plant milk clotting enzymes for novel dairy products

(Katsaros et al., 2009). Optimum pH and temperature of actinidin are of 8.5-9 and 30-

50° C, respectively (Fig. 2.5).

23

Figure 2.5. Optimum temperature and pH of actinidin (Katsaros et al., 2009)

Actinidin has potential pharmaceutical usages. Mohajeri et al. (2010) and Hafezi

et al. (2010) concluded that kiwi fruit extract were used as dressing on deep second – degree because of its dramatic antibacterial and debridement effects which was thought to be due to its potent proteolytic effects. Moreover, Basile (1997) found that

Actinidia chinensis extract has significant bacteriostatic activity against both Gram - positive (Staphylococcus aureus and Streptococcus mutans ) and Gram -negative

(Salmonella Typhimurium and Escherichia coli ) pathogenic bacteria. Molan et al. (2008)

reported that water gold kiwifruit possess the ability to positively influence intestinal

bacteria enzymes by inhibiting β-glucuronidase activity and promoting the activity of β- glucosidase. Moreover, extracts prepared from gold kiwifruit and green kiwifruit are able to promote the growth of intestinal lactic acid bacteria especially at high concentrations and reduce the growth of Escherichia coli (Names, 2012) .

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34 CHAPTER THREE

BACTERICIDAL EFFECTS OF NATURAL TENDERIZING ENZYMES ON

ESCHERCIA COLI AND LISTERIA MONOCYTOGENES

Abstract

The objective of this study was to determine the antimicrobial activity of proteolytic enzymes (papain and bromelain), meat-tenderizing agents, against

Escherichia coli and Listeria monocytogenes at three different temperatures (5, 25 and

35 °C). Two overnight cultures of E. coli JM109 and L. monocytogenes were separately suspended in 0.1% (w/v) peptone water and exposed to the proteolytic enzyme (papain and bromelain) at three different temperatures. Bromelain concentrations (4 mg/ml) and

(1 mg/ml) tested at 25 °C against E. coli and L. monocytogenes, respectively, were the most effective concentrations tested reducing mean log CFU/ml populations by 3.37 and

5.7 after 48 h, respectively. Papain levels of (0.0625 mg/ml) and (0.5 mg/ml) were the most effective concentration tested at 25 °C against E. coli and L. monocytogenes, respectively, reducing mean log CFU/ml populations by 4.94 and 6.58 after 48 h, respectively. Interestingly, the lower papain concentration tested (0.0625 mg/ml) was more effective than the higher concentration (0.5 mg/ml) against E. coli at all three temperatures. As expected, the temperature was directly related to enzyme efficacy against both E. coli and L. monocytogenes.

Keywords: proteolytic enzymes, bromelain, papain, meat tenderizing, Escherichia coli,

Listeria monocytogenes

35

3.1. Introduction

Consumer acceptance or rejection for cut or processed meat after initial purchase is strongly influenced by tenderness. Meat tenderness is related to structural integrity of myofibrillar and connective tissues proteins (Marsh et al., 1991 & Nishimura et al.,

1995). Many studies have investigated methods to improve tenderness and overall meat quality using different tenderizing methods including: chemical tenderization of meat with enzymes, salts, or calcium chloride, and physical tenderization by pressure treatments, blade tenderization or electrical stimulation (Ketnawa et al ., 2011).

Pathogenic bacteria are also a serious concern for consumers in further processed meat products. Gudbjomsdottir et al. (2004) reported the incidence of Listeria monocytogenes in meat processing plants was between 0 and 15% and in poultry plants was 20.6 to

24.1%. A majority of food product recalls associated with L. monocytogenes contamination involve ready - to - eat meat and poultry products (USDA-FSIS, 2005).

Lee et al. (2009) reported 9.1% of beef, poultry and pork raw samples contained

Escherichia coli with 39 pathogenic isolates found among these isolates.

Plant proteolytic enzymes have also received attention in the field of medicine and biotechnology due to their proteolytic properties including papain from papaya

(Carica papaya) , bromelain from pineapple (Ananas comosus) and ficin from figs ( Ficus spp.) (Ketnawa et al., 2010). These enzymes have been widely used in the food, medical– pharmaceutical, cosmetic and other industries. In the food industry, the primary application has been for meat tenderization. About 95% of tenderizing enzymes used for

36 meat in the United States are from plant proteases. This marked tenderizing effect is due to the strong proteolytic activity of these enzymes (Amid et al., 2011).

Bromelain is a mixture of proteolytic enzymes, many of which are cysteine proteases derived from the pineapple plant (Ananas comosus) , which is a member of

Bromeliaceae family (Hale et al., 2005). In the United States, bromelain is sold in health food stores as a nutritional supplement to promote digestive health and as an anti- inflammatory drug (Borrelli et al., 2011). Bromelain also has demonstrated antimicrobial effects including antihelminthic activity against gastrointestinal nematodes, anti-candida effects, and can resolve infectious skin diseases such as pityriasis lichenoides chronica

(Alternative medicine review, 2010). Corzo et al. (2012) reported that optimum pH and temperature conditions for proteolytic activity of bromelain are in range of pH 6.5-7.5 and 50-60 °C, respectively. Lopez-Garcia et al., (2006) reported that bromelain could be used as an alternative to chemical fungicides against Fusarium spp . plant pathogens.

Salampessy et al. (2006) isolated antimicrobial peptides produced through bromelain hydrolysis of raw food.

Papain is another important plant peptidase derived from the latex of unripe papaya fruit ( Carica papaya , Caricaceae ) useful as a meat tenderizer due to its powerful proteolytic activity. Papain is characterized by its ability to hydrolyze large proteins into smaller peptides and amino acids. Its ability to break down tough fibers has been used for many years in the US as a natural tenderizing agent and is included as a component in meat tenderizers (Llerena-Suster et al., 2011).

Anibijuwon & Udeze (2009) concluded that Carica papaya maybe used for

37 treatment of gastroenteritis, urethritis, and otitis media and wound infections. They also concluded that antimicrobial activity against both Gram-negative and Gram-positive bacteria is an indication that the Carica papaya is a potential source for production of a broad-spectrum bactericide. Moreover, Emeruwa (1982) supported that Carica papaya fruit extract had antibacterial activity against both Gram-positive and Gram-negative bacteria like Staphylococcus aureus and Escherichia coli . Emeruwa (1982) also suggested that the site of action of the antibacterial was at cell wall since the cell morphology appeared to be changed. Raw papaya extract was mixed with hydroxy methyl cellulose at a 1:2 ratio and tested against Enterococcus faecalis as a debriding gel for dentistry and showed 68% inhibition (Bhardwaj et al ., 2012)

Ming et al. (2002) reported that optimum pH and temperature conditions for proteolytic activity of papain is in range of pH 6.0-7.0 and 65-80 °C respectively. While

Anibijuwon & Udeze, (2009) said that the increase in temperature enhances the activity, whereas alkaline pH decreases the activity of papain. Meat consumption is increasing around the world, there are some concerns related to the meat quality (tenderness) and meat hygiene and safety.

Meat tenderness can be addressed in different ways and meat hygiene concerns are mostly of a biological nature and include bacterial pathogens, such as Escherichia coli O157:H7, Salmonella and Campylobacter in raw meat and poultry, and Listeria monocytogenes in ready- to -eat processed products (Sofos et al., 2010). Since proteolytic enzymes are used in meat marinades as meat tenderizers and also have displayed antimicrobial activity, they may have used in reducing pathogen risk in meat. Tests

38 against common meat pathogens at temperatures used to hold and store meat seem appropriate. Therefore, the objective of this study was to examine two proteolytic enzymes (bromelain and papain) for antimicrobial activity against E. coli and L. monocytogenes when held at different temperatures (5, 25 and 35 °C).

3.2. Materials and Methods

3. 2.1. Inoculum preparation

Ampicillin-resistant E. coli JM 109 was preserved by freezing at -70 °C in vials containing tryptic soy broth (Becto™ Tryptic Soy Broth, Becton Dickinson and company

Sparks, MD 21152 USA) supplemented with 20% (v/v) glycerol (Sigma, St. Louis, MO).

To propagate the culture, a frozen vial was thawed at room temperature, and 0.1 ml of the thawed culture was transferred to 10 ml of Enrichment TSB with 0.5% (W/V) ampicillin

(DIFCO, Detroit, MI) in screw-capped tubes and incubated aerobically for 16-18 h at

37 °C with shaking (Thermolyne Maxi-Mix III type 65800, Barnstead/ Thermolyne,

Dubuque, IA). The inoculum was prepared from a second transfer of this culture (0.1 ml) to another 10 ml tube of Enrichment TSB (DIFCO, Detroit, MI), and incubated aerobically for 16-18 h at 37 °C with shaking. After overnight incubation, washed cells were harvested by centrifugation for 10 min at 1107 ×g (IEC HN-SII Centrifuge,

International Equipment CO., Inc., Needham Heights, MA), the pellet resuspended in sterile peptone water 0.1% (w/v) (Bacto peptone, Becton Dickinson) to obtain a population of approximately 8-9 log CFU/ml. One ml of the suspension was transferred into 99 ml of sterile 0.1% (w/v) peptone water to obtain a final population of approximately 5-6 log CFU/ml. Initial cell populations were verified by enumeration of

39 the cells following surface-plating in TSA with 0.5% (W/V) ampicillin (DIFCO, Detroit,

MI) and incubating at 37 °C for 24 h. The same procedure was followed with Listeria monocytogenes (ATCC 15313) grown in Listeria broth (DIFCO™ Listeria Enrichment broth, Becton Dickinson and Company Sparks, MD 21152 USA).

3.2.2. Preparation of enzyme concentrations

The concentrations of bromelain (B4882-25G, sigma Chemicals, St Louis, MO) used with L. monocytogenes were 0, 0.25, 0.375 and 1 mg/ml while for E. coli , 0, 1, 2 and

4 mg/ml were used based on preliminary experiments. Both enzymes were sterilized using 0.45 µm syringe filter membrane (0.45 µm Supor ® membrane, Pall Corporation,

Ann Arbor, MI). These concentrations were prepared by mixing appropriate amount of

0.1% (W/V) peptone water, enzyme stock solution and bacterial solution. The same procedure was followed with papain (P4762-500MG, sigma Chemicals, St Louis, MO) using different concentrations. For example, concentrations of papain with E.coli and L. monocytogenes were 0, 0.0625, 0.125, 0.25, and 0.5 mg/ml.

Enzyme and peptone water of the different concentrations were mixed for 30 sec. until a homogenized solution was achieved. At t = 0 h the bacteria were added to the different mixtures and finally transferred to sterile petri dishes and placed on an orbit shaker at 40 rpm (Model 3520 Orbit shaker, Lab-Line Instruments, Melrose park, IL) at different temperatures 5, 25, 35 °C.

3.2.3. Sampling time:

At t = 0, 2, 4, 8, 24, and 48 h, 0.1 ml of each enzyme concentration was serially diluted and appropriate serial dilutions were surface plated on enrichment agar, Listeria

40 agar (DIFCO Detroit, MI, ) for L. monocytogenes and TSA (DIFCO, Detroit, MI ) for E. coli , in duplicate. The inoculated plates were incubated (Model 2300 incubator, VWR

Scientific Products, West Chester, PA) at 37 °C for 48 h for L. monocytogenes and 24 h for E. coli and dilution plates with 25-250 colonies were counted (LEICA, QUEBEC

DARK FIELD colony counter, Buffalo, NY 14240 USA model 3325) and populations were reported a CFU/ml and log CFU/ml. All experiments were repeated three times.

3.2.4. Statistical analysis

The experiment was conducted as a repeated measures split-plot experimental design. The response variable was logarithmic function of the colony forming units (log

CFU) per ml. The whole-plot treatment factor was enzyme concentration and sub-plot treatment factor was temperature. Measurements were repeated over time (0, 2, 4, 8, 24 and 48 h) the covariance matrix was modeled using spatial power law that is a generalization of the first-order autoregressive covariance structure. The PROC MIXED procedure from SAS ® was used to analyze the data and the Tukey multiple comparison procedure was for mean separation. All comparisons were made using α ≤ 0.05.

3.3. Results

3.3.1. Bromelain

3.3.1.1. Effect of bromelain on E. coli

Bromelain was tested at concentrations from 1 to 4 mg/ml at 5, 25, and 35 °C and was effective at all concentrations in reducing bacterial populations after 24 and 48 h compared to no added bromelain (P ≤ 0.0001) (Figure 3. 1). However, there was not a significant difference (P > 0.05) in E. coli populations among samples exposed to

41 bromelain concentrations of 1, 2 or 4 mg/ml at 5, 25 and 35 °C. At 48 h, a bromelain concentration of 4 mg/ml was the most efficient on E. coli reducing the log CFU/ml population by 5.5 at 35 °C (P < 0.0001). Similar results were observed by Sparso &

Moller (2002) who added bromelain to soy protein films to inhibit E. coli . The exact mechanism by which bromelain inhibits the growth of E. coli is not completely understood but could be related to compromise of the Gram-negative outer membrane which also contains proteins. These surface proteins may be digestible by bromelain, weakening the cell wall to allow leakage, swelling of the cell and finally cell fracture.

3. 3.1.2 Effect of bromelain on L.monocytogenes

Bromelain reduced L. monocytogenes populations after 4 h for all 3 temperatures tested (P < 0.0001) (Figure 3. 2) however, there was no a significant difference in populations at concentrations of 0.25 and 0.375 mg/ml after 4 h. After 2 and 4 h at 35 °C, the 1 mg/ml bromelain level reduced L. monocytogenes by 3 log cycles which was significantly greater than the other concentrations tested. After 8 h, L. monocytogenes population exposed to 1 mg/ml was significantly low compared to bacteria exposed to concentrations of 0.25 and 0.375 mg/ml at all temperatures tested. This finding contradicts Sparso & Moller (2002) who concluded that bromelain is more efficient against Gram-negative than Gram-positive bacteria. Overall, bromelain at the 1 mg/ml level was more effective in reducing L. monocytogenes populations than all other levels tested (P < 0.0001).

42 3. 3.1.3 Effect of temperature on bromelain efficiency

The 48-hour exposure time was used as a comparison point for the temperature effects of the enzymes on bacteria. As temperature increased from 5 to 35 °C, the efficacy of bromelain to reduce both E. coli and L .monocytogenes increased (Figures 3.3 and 3.4). The optimum temperature conditions for proteolytic activity of bromelain are in range of 50-60 °C (Corzo et al., 2012) thus as this optimal temperature was approached the greater activity yielded greater cell destruction. At 5 °C, bromelain had no significant effect on E. coli populations after 24 hours at all concentrations tested. However at

25 °C, E. coli populations were reduced at least 3 log cycles after 24 h at all concentrations. At 35 °C, E. coli populations were reduced by 5 log cycles using 1 mg/ml and by 5.5 logs (below detection) at 4 mg/ml. The effect of increasing temperature and increasing activity against E. coli by bromelain was not observed against

L. monocytogenes (Figure 3. 4). Bromelain reduced L. monocytogenes by 1 log cycle at

0.25 and 0.375 mg/ml and by 3 logs at 1 mg/ml after 24 hours at 5 °C. These reductions were only 1 - 1.5 logs greater at 25 and 35 °C, respectively after 24 hours for bromelain against L. monocytogenes .

3.3.2. Papain

3.3.2.1 Effect of papain on E. coli

Papain was tested in concentrations from 0.0625 to 0.5 mg/ml at 5, 25 and 35 °C with all concentration reducing E. coli populations significantly compared to the control

(no papain). At room temperature (25 °C), the lower papain concentration of 0.0625 mg/ml was the most efficient on E. coli (Figure 3.5b). It reduced the log of CFU/ml by

43 4.94 (P < 0.0001) at 48 h, while the higher papain concentration of 0.5 mg/ml was the least effective. At 5 °C the activity of papain against E. coli was reduced only yielding a

1-log reduction compared to the control except for the lowest concentration (0.0625 mg/ml) that showed nearly a 2.5 log reduction after 48 h. The most significant reduction in E. coli population with papain was at 35 °C where all concentration reduced populations below detection after 48 h while the 0.0625 mg/ml level achieved this in 24 hours while other concentrations lagged behind this rate. Papain did not have a significant effect on E. coli at 35 °C (Figure 3.5c) until 4 h and the lower papain concentration (0.0625 mg/ml) was most effective. The antibacterial effect of papain found in this study is similar to those of Sparso & Moller (2002) where Lactobacillus plantarum was significantly reduced by lower concentrations of papain. They explained that the relatively high concentration of protein in enzymes might inhibit or even destroy the enzyme because of the proteolytic properties.

3.3.2.2 Effects of papain on L. monocytogenes

At 5 °C papain slowly reduced L. monocytogenes populations at all concentrations tested yielding about a 2-log cycle reduction after 48 h compared to controls (Figure

3.6a). Papain concentrations of 0.0625 to 0.5 mg/ml at room temperature (25 °C) significantly reduced L. monocytogenes population by 2 log cycles after 8 h and 4 to 6 log after 24 and 48 h, respectively, (Figure 3.6b) compared to the control 0 mg/ml, which increased in population (log 0.44 CFU/ml) after 48 h. At 35 °C, the rate of L. monocytogenes inactivation was greatest reaching 4.5 to 5 log reductions in 8 h and complete elimination of detectable cells by 24 h. All papain concentrations were equally

44 effective in reducing L. monocytogenes with the 0.5 mg/ml level significantly reduced bacterial numbers after 24 h at 5 °C and 8 h at 35 °C compared to the 0.0625 mg/ml concentration. The antibacterial effect of papain would be similar to that of bromelain and was theorized to inhibit either bacterial cell wall synthesis or general protein synthesis (Osato et al., 1993).

3.3.2.3 Effect of temperature on papain efficiency

There was no significant difference in the effect of papain concentrations (0.0625,

0.125, 0.25 and 0.5 mg/ml) on E. coli population after 24 h at 5 °C (Figure 3. 7).

However, all papain concentrations reduced L.monocytogenes population after 24 h at

5 °C compared to controls. At 25 °C, there was at least a 3-log reduction of E. coli and a

4-log reduction of L. monocytogenes at all concentrations tested compared to controls

(Figure 3. 8). The 24-hour reduction in L. monocytogenes held at 25 °C and 35 °C did not differ among the concentrations tested. However for E. coli , the 25 °C was about 1- log higher in populations after 24 hours than the 35 °C treatment at each concentration.

3.4. Discussion

In general, 35 °C was the most effective temperature for all enzyme concentrations tested. However, 25 and 35 °C did not affect papain activity but did affect bromelain activity. Temperature effects on enzyme activity was demonstrated by Ming et al. (2002) as the optimum temperature conditions for proteolytic activity of bromelain and papain are in range of 50-60 °C and 65-80 °C, respectively. Moreover, Anibijuwon &

Udeze, 2009 reported that the increase in temperature enhances papain activity. Both bromelain and papain belong to a family of cysteine proteases that are activated by

45 cysteine, which is located at the active site of the enzyme (Mamboya, 2012). Bromelain preferentially cleaving at amino acid sites involving lysine, , and glycine.

Whereas, papain prefers hydrophobic sites that include and also Lysine. The enzyme breaks bonds at selected locations dividing the protein chain into fragments.

Gram-negative bacterial cell walls are more complex than Gram-positive cell walls containing an outer membrane comprised of protein, lipoprotein and lipopolysaccharides, a peptidoglycan layer then a plasma membrane that also contains proteins. Gram-positive bacteria have a thick peptidoglycan layer and an inner plasma membrane. The surface layer of both Gram-positive and Gram-negative bacteria contains protein components that can be targeted by proteases to compromise cell wall structure to varying degrees. For example, the peptidoglycan layer (outer layer of Gram-positive bacteria) consists of subunits that are joined by crosslinks between the amino group of one amino acid and the carboxyl group of alanine (Prescott et al., 1990), a preferred scission site for bromelain. In contrast, Gram-negative bacteria have an outer lipopolysaccharide layer that contains porin proteins that a lined with exclusively charged amino acids to facilitate passage of molecules through the membrane (Schirmer, 1998); papain prefers uncharged (hydrophobic) amino acid sites. The different responses observed to bromelain and papain for E. coli and L. monocytogenes are likely to be due, in part to the differences in cell wall/membrane structure amino acid presence between

Gram-negative ( E. coli ) and Gram-positive ( L. monocytogenes ). The presence and availability of amino acids in bacterial cell wall proteins that are enzyme targets will enhance or inhibit protease antibacterial activity. The antimicrobial activity of proteolytic

46 enzymes used as meat-tenderizing agents reported here and in other sources may enhance the safety and shelf life of marinated meat products.

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49 Sparso, H. M., & Moller, S. M. (2002). Proteolytic enzymes as antimicrobial agents and incorporation of hydrophobic additives into thermally compacted soy protein-based films . Research Thesis at Clemson University exchange with The Technical University of Denmark.

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50 Figures

1. a. Bromelain against E. coli JM 109 at 5 °C.

1. b. Bromelain against E. coli JM 109 at 25 °C.

1. c. Bromelain against E. coli at 35 °C. Figure 3. 1: Effect of bromelain on E. coli JM 109 at 5, 25 and 35 °C over 48 hours. a, b, c data points or groups of points with the same superscripts are not significantly different (p> 0.05). Standard error of the mean = 0.18. (n=6)

51

2.a. Bromelain against L. monocytogenes at 5 °C.

2.b. Bromelain against L. monocytogenes at 25 °C.

2.c. Bromelain against L. monocytogenes at 35 °C. Figure 3. 2: Effect of bromelain on L.monocytogenes at 5, 25 and 35 °C over 48 hours. a, b, c data points or groups of points with the same superscripts are not significantly different (p> 0.05). Standard error of the mean = 0.24. (n=6)

52

Figure 3. 3: Effect of temperature on bromelain efficiency against on E. coli JM 109 after 48 hour. a, b, c data points or groups of points with the same superscripts are not significantly different (p> 0.05). Standard error of the mean = 0.25. (n=6)

Figure 3. 4: Effect of temperature on bromelain efficiency against L. monocytogenes after 48h. a, b, c data points or groups of points with the same superscripts are not significantly different (p> 0.05). Standard error of the mean = 0.24. (n=6)

53 5

4 a a

3 0 mg/ml 2 b 0.0625 mg/ml b 0.125 mg/ml 1 Log Log CFU/ml 0.25 mg/ml 0 0.5 mg/ml 0 4 8 12 16 20 24 28 32 36 40 44 48 Time (hours) 5.a. Papain against E. coli JM 109 at 5 °C.

6 a a 0 mg/ml a 5 0.0625 mg/ml 4 0.125 mg/ml 3 0.25 mg/ml 0.5 mg/ml b 2 b

Log Log CFU/ml 1 b 0 0 4 8 12 16 20 24 28 32 36 40 44 48 Time (hours) 5.b. Papain against E. coli JM 109 at 25 °C. 5

4 a a a 0 mg/ml 3 0.0625 mg/ml 0.125 mg/ml 2 b 0.25 mg/ml b 0.5 mg/ml 1 Log Log CFU/ml bc b c 0 0 4 8 12 16 20 24 28 32 36 40 44 48 Time (hours) 5.c. Papain against E. coli at 35 °C. Figure 3. 5: Effect of papain on E. coli JM 109 at 5, 25 and 35 °C. a, b, c data points or groups of points with the same superscripts are not significantly different (p> 0.05). Standard error of the mean = 0.46. (n=6)

54 6 a 5

4 0 mg/ml b 3 0.0625 mg/ml 2 0.125 mg/ml 0.25 mg/ml

Log Log CFU/ml 1 0.5 mg/ml 0 0 4 8 12 16 20 24 28 32 36 40 44 48 Time (hours) 6.a. Papain against L. monocytognes at 5 °C. a 6 a a 0 mg/ml 5 0.0625 mg/ml 4 0.125 mg/ml 3 b 0.25 mg/ml 2 0.5 mg/ml

Log Log CFU/ml 1 b b 0 0 4 8 12 16 20 24 28 32 36 40 44 48 Time (hours) 6.b. Papain against L. monocytogenes at 25°C.

6 a a a 5 0 mg/ml 4 0.0625 mg/ml 3 0.125 mg/ml 0.25 mg/ml 2 0.5 mg/ml

Log Log CFU/ml b 1 b b 0 0 4 8 12 16 20 24 28 32 36 40 44 48 Time (hours) 6.c. Papain against L. monocytogenes at 35 °C. Figure 3. 6: Effect of papain on L.monocytogenes at 5, 25, and 35 °C. a, b, c data points or groups of points with the same superscripts are not significantly different (p> 0.05). Standard error of the mean = 0.26. (n=6)

55 6 5C 5 25C a 4 a a 35C 3 b 2 b b

Log Log CFU/ml 1 c c c 0 0 0.125 0.25 0.375 0.5 papain concentration (mg/ml) Figure 3. 7: Temperature effect of papain on E. coli JM 109 after 48 h. a, b, c data points or groups of points with the same superscripts are not significantly different (p> 0.05). Standard error of the mean = 0.23. (n=6)

5C 6 25C 5 a a a 35C a 4 3 2

Log Log CFU/ml 1 b b b b 0 0 0.125 0.25 0.375 0.5

papain concentration (mg/ml) Figure 3. 8: Effect of temperature of papain on L. monocytogenes after 48 h. a, b, c data points or groups of points with the same superscripts are not significantly different (p> 0.05). Standard error of the mean = 0.35. (n=6)

56 CHAPTER FOUR

ANTIBACTERIAL EFFECTS OF PROTEASES ON DIFFERENT STRAINS OF ESCHERICHIA COLI O157:H7 AND LISTERIA MONOCYTOGENES IN COOKED MEAT MEDIA

Abstract

The objective of this study was to determine the efficacy of actinidin and papain on reducing L. monocytogenes and three mixed strains of E. coli O157:H7 populations in a cooked meat medium . Overnight cultures of L. monocytogenes and a three-strain mix of E. coli O157:H7 were separately suspended in 0.1 % (w/v) peptone water and were individually exposed to a proteolytic enzyme (actinidin or papain) in cooked meat media at three different temperatures (5, 25 and 35 °C). At 25 and 35 °C, for all actinidin and papain concentrations there were no significant reductions in E. coli O157:H7 and L. monocytogenes populations for any time-temperature combination. Moreover, there was bacterial growth from 1 h to 3 h for 25 and 35 °C. The bacterial growth at 35 °C was significantly higher than that at 25 °C. At 5 °C, actinidin and papain did not significantly reduce the populations of E. coli O157:H7 and L. monocytogenes except for actinidin at

50 mg/ml on L. monocytogenes at 24 h. Also, no significant difference was found between bacterial populations at 6 and 24 h for both pathogens except for L. monocytogenes at 24 h where there was bacterial growth for both papain levels tested.

These findings indicate that proteolytic enzymes have a little antibacterial effect when added to cooked meat media.

57 Keywords: Proteolytic enzymes, actinidin, papain, meat tenderizing, Escherichia coli

O157:H7, Listeria monocytogenes, cooked meat media.

4.1. Introduction

Using natural antimicrobial compounds to enhance food safety, quality and shelf- life have received special attention by food safety researchers. This is likely due to increased consumer demands for foods with fewer additives perceived as being un- natural. Consumers tend to believe that natural or organic foods are more nutritious or safer to eat ( Hughner et al., 2007). Therefore, there has been market growth in the United

States in the natural and organic food sector (Sebranek and B acus, 2007; Bourn and

Prescott, 2002).

Bacteria are considered the main food safety hazard and spoilage source of animal origin foods. Enteric pathogens like E. coli spp. and psychrotrophic bacteria such as

Listeria monocytogenes are one of the potential microbial hazards in meat and poultry products (Olsen et al., 2000). Inhibition and/or inactivation of these bacteria may improve the safety and extend the shelf-life of meat products.

Throughout history, plants have provided natural antimicrobials and aromatic compounds (Cowan, 1999). Using agricultural products, wastes and by- products as a source of natural antimicrobials has been investigated and antimicrobials from plants have been found to be an alternative to chemical preservatives in order to satisfy consumers’ demand f or safe, convenient and wholesome food (Nychas et al., 2003; Smid and Gorris, 1999) although, their potential as food preservatives has not been fully explained.

58 Proteolytic enzymes derived from plants, such as papain, bromelain, ficin and most recently actinidin have been widely used as meat tenderizers in most parts of the world (Naveena et al., 2004). However, little attention to their antibacterial effects on pathogenic bacteria related to meat has been received.

A previous in vitro study concluded that plant proteolytic enzymes (papain and bromelain) had antibacterial affects against Gram-negative E. coli JM 109 and Gram- positive L. monocytogenes (Eshamah et al., 2013). However, there is little information about the inhibitory effects of these proteolytic enzymes on foodborne pathogens in meat systems, which contain significant amounts of protein. Therefore, this research was conducted to determine the antibacterial activities of papain and actinidin against three mixed strains of Escherichia coli O157:H7 and Listeria monocytogenes in a cooked meat medium.

4.2. Materials and methods

4.2.1. Inoculum preparation

Three strains of E. coli O157:H7 ( E. coli O157:H7 E-380-94: CDC, E. coli

O157:H7 C-7929 and E. coli O157: H7- 0654 supplied by Dr. Mike Doyle, UGA) were preserved by freezing at -70 °C in vials containing trypticase soy broth (TSB) (DIFCO,

Detroit, MI) supplemented with 20% (v/v) glycerol (Sigma, St. Louis, MO). To propagate the culture, a frozen vial was thawed at room temperature, and 0.1 ml of the thawed culture was transferred to 10 ml of enrichment TSB in screw capped tubes and incubated aerobically for 16-18 h at 37 °C with shaking (Thermolyne Maxi-Mix III type

65800, Barnstead/ Thermolyne, Dubuque, IA). The inoculum was prepared from a

59 second transfer of this culture (0.1 ml) to another 10 ml tube of enrichment TSB, and incubated aerobically for 16-18 h at 37 °C with shaking. After incubating overnight, the washed cell suspension of the organism was harvested by centrifugation for 10 min at

1107 ×g (IEC HN-SII Centrifuge, International Equipment CO., Inc., Needham Heights,

MA), then the pellet resuspended in sterile 0.1% (w/v) peptone water (Bacto peptone,

Becton Dickinson, Sparks, MD) to obtain a population of approximately 8-9 log CFU/ml.

Three ml of each strain were mixed together then 1 ml of the suspension transferred into

99 ml of sterile 0.1% (w/v) peptone water to obtain population of approximately 5-6 log

CFU/ml. Initial cell populations were verified by enumeration of the cells following surface-plating in TSA (DIFCO, Detroit, MI) and incubating at 37 °C for 24 h. The same procedure was followed for L. monocytogenes (ATCC 15313) which was surface-plated in Listeria Enrichment Agar (DIFCO, Detroit, MI) and incubated at 37 °C for 48 h.

4.2.2. Preparation of enzyme concentrations

The concentrations of actinidin (KFPE OT1005X, Ingredient Resources Pty Ltd,

Australia) with L. monocytogenes were 0, 25 and 50 mg/ml, while with E. coli O157:H7 the concentrations were 0, 50 and 100 mg/ml. The concentrations of papain (P4762-

500MG, Sigma Chemicals, St Louis, MO) used with L. monocytogenes were 0, 1 and 2 mg/ml while with E. coli O157:H7 the concentrations were 0, 2 and 4 mg/ml. These concentrations were chosen based on preliminary experiments using standard media

(Tryptic Soy and Listeria Enrichment). Both enzymes were sterilized using 0.45 µm syringe filter membrane (0.45 µm Supor ® Membrane, Pall Corporation, Ann Arbor, MI).

Enzyme concentrations were prepared by adding appropriate amounts of cooked meat

60 broth, enzyme stock solution and bacterial solution. Enzymes and cooked meat broth of the different concentrations were mixed for 30 sec. until a homogenized solution was achieved. At 0 h, bacteria were added to the different mixtures and then held at different temperatures 5, 25 and 35 °C.

4.2.3. Enumeration of surviving bacteria and sampling time

Cooked meat media-enzyme mixtures held at 25 and 35 °C were sampled at 0, 1, and 3 h while those held at 5 °C were sampled at 0, 6 and 24 h. Approximately 0.1 ml of each mixture were serially diluted and appropriate serial dilutions were surface plated on enrichment agar, Listeria agar (DIFCO Detroit, MI) for L. monocytogenes and TSA for E. coli O157:H7, in duplicate. The inoculated plates were incubated (Model 2300 incubator,

VWR Scientific Products, West Chester, PA) at 37 °C for 48 h for L. monocytogenes and

24 h for E. coli O157:H7 and dilute on plates with 25-250 colonies were counted

(LEICA, QUEBEC DARK FIELD colony counter, Buffalo, NY 14240 USA model 3325) and populations were reported a CFU/ml and log CFU/ml. All experiments were repeated three times.

4.2.4. Determination of Enzymatic Activity and Protein Content

The activity of actinidin and papain was measured spectrophotometrically according to the modified method of Robinson (1975). The assay mixture contained 1 ml of actinidin (KFEP OT1005X) or papain of these concentrations (2.5, 5 and 10 mg/ml) and 5 ml of 0.65% (w/v) of substrate, casein (Sigma, St. Louis, MO), dissolved in 50 mM potassium phosphate buffer, pH 7.5 at 37 ºC. All reaction mixtures were incubated

61 at 37 °C for 10 minutes. The reaction was terminated by adding 5 ml of 110 mM trichloroacetic acid (TCA) and precipitated protein was removed by filtration through a

0.45 µm syringe filter (0.45 µm Supor ® membrane, Pall Corporation, Ann Arbor, MI).

The absorbance of filtrate was measured at 660 nm (Spectrophotometer GENESYS 20

4001-4, 100-240V 50/60 Hz, Madison, WI). Blank samples were prepared by adding the enzyme at the end of the incubation time, just before TCA addition and precipitation.

One unit of the enzyme activity was defined as the amount of enzyme that released 1

µmol of tyrosine per minute under the assay conditions. Specific activity was expressed as enzyme units per mg protein. Protein content of actinidin (KFEP 0T1005X) and papain were measured using a modified Lowry protein assay method (Lowry et al., 1951) with bovine serum albumin (Pierce Biotechnology, modified Lowry protein assay kit,

Rockford, IL) as the protein standard.

4.2.5. Statistical analysis

A split-split plot design was used for the study. The factor at the highest level was actinidin and papain concentrations (actinidin concentrations used with E. coli

O157:H7 were 0, 50 and 100 mg/ml while with L. monocytogenes were 0, 25 and 50 mg/ml, and papain concentrations used with E. coli O157:H7 were 0, 2 and 4 mg/ml while with L. monocytogenes were 0, 1 and 2 mg/ml). The second level was temperature

(5, 25 and 35 °C) and the third level was time (6 h and 24 h for 5 °C and 1 h and 3 h for

25 and 35 °C). The response variable was the log increase in the bacterial count compared to time 0. Treatment effects were evaluated using analysis of variance

62 techniques and linear contrasts. Mean were separated using Fisher’s protected least signification difference (LSD) test with alpha ≤ 0.05.

4.3. Results

While significant enzyme effects on bacterial populations in cooked meat media were found in this study, significant differences were sometimes less than 1-log unit, which are not typically considered of practical significance. However, due to the highly controlled nature of the study and that cooked meat media exposes enzymes and bacteria to concentrated amounts of meat protein, the results may indicate positive results are possible when enzymes are applied to meat surfaces and not mixed with high levels of protein.

4.3.1. Actinidin

4.3.1.1. Effect of actinidin on E. coli O157:H7

For E. coli O157:H7 held at 5 °C, there were no significant reductions found due to the presence of actinidin at 6 h, however, at 24 h there was a significant reduction in the bacterial population due to only time effect for 0 and 50 mg/ml (Table 4.1a). At 25 and 35 °C, both actinidin concentrations used in this study did not have any effect on the reduction of E. coli O157:H7 populations for any time-temperature combination (Table

4.1b). There was bacterial growth between 1 h and 3 h for both temperatures and at 1 h, there was no significant reduction in E. coli O157:H7 populations for any of the test conditions for either 25 or 35°C from initial populations. However, at 3 h, there was an

63 increase in E. coli O157:H7 populations for 25 and 35 °C and E. coli O157:H7 growth at

35 °C was significantly higher than that at 25 °C (Table 4. 1b).

4.3.1.2 Effect of actinidin on L. monocytogenes

There were no significant reductions due to presence of actinidin on L. monocytogenes populations held at 5 °C after 6 h. While at 24 h, the bacterial population was lower for populations exposed to 50 mg/ml actinidin compared to 0 mg/ml. (Table

4.2a).

At 25 and 35 °C, there was no significant reduction in L. monocytogenes population due to actinidin at any level for any time-temperature combination (Table

4.2b). There was actually L. monocytogenes growth between 1 h and 3 h for both 25 and

35 °C. However, at 3 h, there was greater L. monocytogenes growth at 35 °C than at 25°C

(Table 4.2b).

4.3.2 Papain

4.3.2.1 Effect of papain on E. coli O157:H7.

At 5 °C, there was no significant reduction in E. coli O157:H7 for any of the test conditions. In addition, for papain levels 2 and 4 mg/ml, there was no difference in the bacterial populations between 6 h and 24 h (Table 4.3a). At 25 and 35 °C, neither papain level had an effect on E. coli O157:H7 populations for any time-temperature combination

(Table 4.3b). There was bacterial growth between 1 h and 3 h for both 25 and 35 °C.

64 However, at 3 h, E. coli O157:H7 growth at 35 °C was significantly greater than that at

25 °C (Table 4.3b).

4.3.2.2. Effect of papain on L. monocytogenes

At 5 °C , there were reductions in L. monocytogenes populations for both papain concentrations after 6 h, whereas at 24 h there was bacterial growth for both papain levels tested (Table 4.4a). At 25 and 35 °C, neither papain level reduced L. monocytogenes populations for any time-temperature combination (Table 4.4b) and L. monocytogenes populations actually increased between 1 h and 3 h at 25 and 35 °C. However, at 3 h, the growth in L. monocytogenes population at 35 °C was significantly greater than that at 25

°C (Table 4.4b).

4.4. Discussion

Many studies have been conducted using agar-based growth media in order to evaluate the antimicrobial activity of plant extracts against spoilage and foodborne pathogens, however, results obtained from media modeling food components (such as cooked meat media) may be useful in predicting antimicrobial efficacy in food, since the model media more closely reflects nutrient availability and composition of food

(Gutierrez et al., 2009). In this aspect, some researchers have already used fruits and vegetables model media to investigate antimicrobial efficacy of several plant extracts

(Hsieh et al., 2001; Ultee and Smid, 2001; Valero and Salmeron, 2003). In most of these cases the plant extracts’ efficacy decreased in the food model media by comparison with the in vitro control media and a higher concentration is needed to achieve the same effect as in vitro (Burt, 2004).

65 In this study, the antimicrobial efficacy of actinidin and papain was evaluated in cooked meat media. Significant differences were observed between antibacterial activity of papain in 0.1% (w/v) peptone water (Eshamah et al., 2013) and cooked meat media.

Often, an antimicrobial that performed well in microbiological media is shown to have little or no effect in food ( Gutierrez et al., 2008 ). This is due to many factors that include the presence of proteins and lipids, binding to food components, inactivation by other additives, pH effects on antimicrobial stability and activity, uneven distribution in the food matrix and poor solubility (Davidson et al., 2010).

In addition, the physical structure of the food may also have a role in the antimicrobial action of plant extracts. Bacteria may enter in the pores of the food surface such as beef reducing the probability and duration of direct contact with the antimicrobial

(Hao et al., 1998)

Cooked meat medium used in this study had the ability to promote growth of bacteria from low inoculum and to maintain the viability of cultures over time. This could be one reason that leads to increase bacterial populations observed. Moreover, cooked meat medium contains beef heart which provides amino acids and other nutrients to the inoculated bacteria (Murray et al., 2003). Furthermore, the presence of meat proteins and the mixture of enzymes in this media may have accelerated the inactivation of the proteases by interacting with the meat proteins, ultimately reducing the direct inactivation of bacteria by the enzymes.

There was bacterial growth for both E. coli O157:H7 and L. monocytogenes at all temperatures tested. This bacterial growth increased with increasing temperatures from 5

66 to 35 °C and time from 6 to 24 h at 5 °C and 1 to 3 h at 25 and 35 °C as would be expected since E. coli O157:H7 has an optimum temperature range between 25 to 45 °C

(Raghubeer & Matches, 1990). Similarly, the temperature range for L. monocytogenes growth is -1.5 to 44 °C (Hudson et al., 1994).

If these enzymes are combined with other antimicrobial technologies or if less interaction with food proteins were possible, higher pathogen reductions may be achieved. Campos et al. (2011) concluded that natural antimicrobials in combination with other stress factors are a valuable tool to control the growth of L. monocytogenes in foods. The use of multiple stress factors may increase the effectiveness of plant extracts, thereby achieving the dual goal of reducing any undesirable organoleptic impact and controlling foodborne pathogens and spoilage bacteria.

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Naveena, B. M., Mendiratta, S. K., & Anjaneyulu, A. S. R. (2004). Tenderization of buffalo meat using plant proteases from Cucumis trigonus Roxb (Kachri) and Zingiber officinale roscoe (Ginger rhizome). Meat Science , 68(3), 363-369.

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69

TABLES Table 4.1 . Differences in E. coli O157:H7 populations exposed to various concentrations of actinidin in cooked meat media compared to time 0. a. 5 °C

Time Concentration (mg/ml) 6 h 24 h 0 -0.26 aA -0.20 aB 50 -0.32 aA -0.26 bB 100 -0.32 aA -0.32 cA Standard error of the mean = 0.11. b. 25 and 35 °C

Temperature 25 °C 35 °C Concentration (mg/ml) Time Time 1 h 3 h 1 h 3 h 0 -0.09 aA +0.56 aB -0.07 aA +1.40 aB 50 -0.17 aA +0.54 aB -0.12 aA +1.44 aB 100 -0.21 aA +0.35 bB -0.16 aA +1.26 aB Standard error of the mean = 0.13. Bold value indicates the change in the mean log count was significantly different than 0. Means with the same superscripts are not significantly different within the same temperature and time (column). Level of significance used was 0.05. Lower case superscripts are for the comparison of enzyme concentration and upper case superscripts are for time comparisons. (n=6)

70 Table 4.2 . Differences in L. monocytogenes populations at 5 °C exposed to various concentrations of actinidin in cooked meat media compared to time 0. a. 5 °C

Time Concentration (mg/ml) 6 h 24 h 0 -0.05 aA +0.18 aB 25 -0.08 aA +0.12 abB 50 -0.12 aA -0.03 bA Standard error of the mean = 0.06. (n=6) b. 25 and 35 °C

Temperature 25 °C 35 °C Concentration (mg/ml) Time Time 1 h 3 h 1 h 3 h 0 +0.05 aA +0.52 aB +0.08 aA +0.86 aB 25 +0.01 aA +0.39 aB +0.07 aA +0.70 aB 50 -0.03 aA +0.25 aB +0.03 aA +0.60 aB Standard error of the mean = 0.12. Bold value indicates the change in the mean log count was significantly different than 0. Means with the same superscripts are not significantly different within the same temperature and time (column). Level of significance used was 0.05. Lower case superscripts are for the comparison of enzyme concentration and upper case superscripts are for time comparisons. (n=6)

71 Table 4.3. Differences in E. coli O157:H7 populations exposed to various concentrations of papain in cooked meat media compared to time 0. a. 5 °C Time Concentration (mg/ml) 6 h 24 h 0 -0.24 aA -0.14 aB 2 -0.29 aA -0.30 bA 4 -0.35 aA -0.35 bA Standard error of the mean = 0.14. (n=6) b. 25 and 35 °C

Temperature 25 °C 35 °C Concentration (mg/ml) Time Time 1 h 3 h 1 h 3 h 0 -0.10 aA +0.56 aB -0.06 aA +1.40 aB 2 -0.10 aA +0.56 aB -0.06 aA +1.54 aB 4 -0.12 aA +0.58 aB -0.16 aA +1.55 aB Standard error of the mean = 0.17. Bold value indicates the change in the mean log count was significantly different than 0. Means with the same superscripts are not significantly different within the same temperature and time (column). Level of significance used was 0.05. Lower case superscripts are for the comparison of enzyme concentration and upper case superscripts are for time comparisons. (n=6)

72 Table 4.4. Differences in L. monocytogenes populations exposed to various concentrations of papain in cooked meat media compared to time 0. a. 5 °C

Time Concentration (mg/ml) 6 h 24 h 0 0.00 aA +0.08 aB 1 -0.07 bA +0.03 abA 2 -0.14 cA +0.02 bA Standard error of the mean = 0.02. (n=6) b. 25 and 35 °C Temperature 25 °C 35 °C Concentration (mg/ml) Time Time 1 h 3 h 1 h 3 h 0 +0.02 aA +0.51 aB +0.17 aA +0.89 aB 1 -0.01 aA +0.42 aB +0.02 aA +0.84 aB 2 -0.02 aA +0.41 aB -0.16 aA +0.76 aB Standard error of the mean = 0.11. Bold value indicates the change in the mean log count was significantly different than 0. Means with the same superscripts are not significantly different within the same temperature and time (column). Level of significance used was 0.05. Lower case superscripts are for the comparison of enzyme concentration and upper case superscripts are for time comparisons. (n=6)

73 CHAPTER FIVE

ANTIBACTERIAL EFFECTS OF PROTEASES ACTINIDIN AND PAPAIN ON

DIFFERENT STRAINS OF ESCHERICHIA COLI O157:H7 AND LISTERIA

MONOCYTOGENES ON BEEF

Abstract

Tenderization of beef is used to increase consumer acceptance. This study determined the efficacy of actinidin and papain on reducing Listeria monocytogenes and three mixed strains of Escherichia coli O157:H7 populations on beef . Overnight cultures of L. monocytogenes and a three-strain mix of E. coli O157:H7 were separately suspended in 0.1 % (w/v) peptone water and were individually inoculated onto beef surface (ca. 10 6 CFU/cm 2). After a 5-minute attachment time different enzyme concentrations were added. Treated samples were then held for either 3 h at 25 and 35 °C or 24 h at 5 °C. The average reduction of E. coli O157:H7 was greater than that of L. monocytogenes and higher concentrations of either protease yielded greater reduction in bacterial populations . For instance, actinidin at 700 mg/ml significantly (p ≤ 0.05) reduced the population of L. monocytogenes by 1.49 log cfu/ml after 3 h at 25 & 35 °C, and by 1.45 log cfu/ml after 24 h at 5 °C. Also, the same actinidin concentration significantly (p ≤ 0.05) reduced the populations of three mixed strains of E. coli

O157:H7 by 1.81 log cfu/ml after 3 h at 25 & 35 °C, and 1.94 log cfu/ml after 24 h at

5 °C.

74 While papain at 10 mg/ml significantly reduced the population of L. monocytogenes by

0.56 log cfu/ml after 3 h at 25 & 35 °C and by 0.46 log cfu/ml after 24 h at 5 °C. Also, the same papain concentration significantly (p ≤ 0.05) reduced the populations of three mixed strains of E. coli O157:H7 by 1.48 log cfu/ml after 3 h at 25 & 35 °C, and by 1.57 log cfu/ml after 24 h at 5 °C. These findings suggest that, in addition to improving the sensory attributes of beef, proteolytic enzymes can enhance meat safety and shelf life when stored at suitable temperatures. The findings also propose a promising approach in developing antimicrobial systems for beef products. If these enzymes are combined with current antimicrobial technologies, higher pathogen reductions may be achieved.

Keywords: Proteolytic enzymes, actinidin, papain, meat tenderizing, Escherichia coli

O157:H7, Listeria monocytogenes, meat surface.

5.1 Introduction

Food borne diseases have a major impact in the United States with estimated 48 million illnesses, 128,000 hospitalizations and up to 3000 deaths occurring each year from bacteria, viruses, parasites and fungi (CDC, 2011). E. coli O157:H7 and L. monocytogenes are pathogens that have received special attention by federal agencies, industries, and food safety researchers due to their economic and human health impact.

These pathogens are responsible for 3 billion dollars in economic losses each year

(USDA, 2006), therefore, alternative interventions are being studied to control these microorganisms.

75 L. monocytogenes is a Gram positive, motile, microaerophilic and nonsporeforming rod that grows at a wide temperature (1.7 °C – 50 °C) and pH range

(4.5 to 7.0) (Junttila et al ., 1988; Walker et al., 1987). L. monocytogenes is widely distributed in the nature with some studies indicating that 1- 10 % of humans are intestinal carriers of L. monocytogenes (FDA Bad Bug Book, 2012). Its association with meat and slaughter environments is well established (Benkerroum et al., 2003).

Consumption of raw and partially cooked contaminated meat can result in Listeriosis, especially among the immune-compromised populations, elderly and pregnant

(Shrinithivihahshini et al., 2011). According to the US Centers for Disease Control and

Prevention (CDC, 2011), the rate of listeriosis has fallen by 38 % from 1996-2010. Yet,

L. monocytogenes causes an estimated 1600 cases of listeriosis, 1450 hospitalizations and

255 deaths annually in the United States (Scallan et al., 2011; CDC, 2011). As L. monocytogenes is a ubiquitous organism able to multiply at refrigeration temperatures and under anaerobic conditions, they are of major concern especially in RTE meat and poultry products (Martin et al., 2009). The minimum infective dose of L. monocytogenes is unknown but thought to vary with strain and individual susceptibility (FDA Bad Bug

Book, 2012). However, indications are that intake of up to 100 cells does not affect a healthy consumer (Jay, 1994).

Escherichia coli O157:H7 is an emerging pathogen responsible for about 63,000 illnesses, 2,000 hospitalizations, and 20 deaths each year in the United States (Kudva, et al., 2012; CDC, 2011). Some of these illnesses are associated with eating undercooked, contaminated ground beef. E. coli O157:H7 was recognized as a significant foodborne

76 pathogen in the early 1980s and continues to be a major cause of diarrheal illness in

North America. E. coli O157:H7 infections are the primary cause of hemolytic uremic syndrome (HUS) in children (Banatvala et al., 2001). According to the CDC, 350 outbreaks were reported from 1982 to 2002 (Rangel et al., 2005).

The U.S. Department of Agriculture’s Food Safety and Inspection Service

(USDA-FSIS, 2012) has updated the lethality regulations for meat and poultry products.

A 5-log lethality of E. coli O157:H7 for RTE products containing beef and a 3-log reduction of L. monocytogenes should be achieved, although a 5-log reduction or greater is desirable for providing an even greater safety margin for ensuring that L. monocytogenes doesn’t grow during cold storage to detectable levels.

In spite the fact that foodborne pathogens are subjected to physical, chemical, and nutritional stresses during processing (Yousef and Courtney, 2003), their elimination and/ or inhibition remains a big hurdle to processors. Remarkable advances have been made in developing thermal and non-thermal intervention technologies to eliminate food borne pathogens from meat and poultry product, yet their ability to grow and proliferate at a wide range of temperatures and pH are major concerns during food preparation, storage or distribution (Pathania et al., 2010). Consumers’ increasingly demand for convenience foods of the highest quality have triggered the use of marinades to enhance food safety

(Shelef, 1984; Sabah et al., 2004)

Using exogenous proteases to tenderize meat has been of considerable interest, with focus on some plant cysteine proteases such as papain, bromelain and actinidin (Ha et al.,

2012; Ketnawa and Rawdkuen , 2011; Koak et al., 2011; Naveena et al., 2004; Sullivan

77 and Calkins, 2010). Papain is an important plant peptidase due to its powerful proteolytic activity, derived from the latex of unripe papaya fruit ( Carica papaya , Caricaceae ).

Papain is characterized by its ability to hydrolyze large proteins into smaller peptides and amino acids. Its ability to break down tough fibers has been used for many years

(Llerena-Suster et al., 2011). Studies found that papain and other papaya extracts possess antimicrobial activities against Bacillus subtilis , Enterobacter cloacae , E. coli ,

Salmonella typhi , Staphylococcus aureus , and Proteus vulgaris (Osato et al., 1993;

Emeruwa, 1982).

Actinidin is another member of cysteine protease family present in kiwi fruit and belongs to the same class of enzymes as ficin, papain and bromelain. The important features of actinidin include a wide pH range for catalytic activity and stability at moderate temperatures, but the enzyme is susceptible to oxidation, a feature in common with other plant thiol proteases (Kaur et al., 2010). Actinidin has many applications in the food industry replacing other plant proteases like papain and ficin because of its mild tenderizing reaction even at high concentrations preventing surface mushiness; It has a relatively low inactivation temperature (60 °C) which makes it easier to control tenderization without overcooking (Tarte´, 2009). Actinidin has potential pharmaceutical usage. Mohajeri et al. (2010) concluded that kiwi fruit extract has dramatic antibacterial and debridement effects when it is used as a dressing on deep second–degree burns, due to its potent proteolytic effects (Hafezi et al., 2010). Moreover, Basile (1997) found that kiwi fruit ( Actinidia chinensis ) extract has significant antibacterial activity against various Gram-positive and Gram-negative strains. Many studies have addressed the roles

78 of these plant proteases as meat tenderizers (Ha et al., 2012; Ketnawa and Rawdkuen ,

2011; Koak et al., 2011; Naveena et al., 2004; Sullivan and Calkins, 2010). However, few of these studied their roles as antifungal, antioxidant and antibacterial properties on meat and poultry products. Antibacterial activity depends on many factors as pH, temperature and level of target microbial population (Bloomfield, 1991). Nevertheless, the most important factor that affects the fate of microorganisms in foods is the structure of the food matrix. Immobilized bacterial cells on solid surfaces behave differently in terms of growth rate and survival; thus liquid laboratory media are not suitable to simulate real food conditions ( Brocklehurst et al., 1997; Robins et al., 1994 ). In the present study, L. monocytogenes and three mixed strains of E. coli O157:H7 were used to determine the effect of these proteolytic enzymes on bacteria on beef at different temperatures (5, 25 and 35 °C).

5.1 Materials and methods

5. 2.1. Inoculum preparation

Three strains of E. coli O157:H7 (E. coli O157:H7 E-380-94 by CDC, E. coli

O157:H7 C-7929 and E . coli O157: H7- 0654 supplied by Dr. Mike Doyle at UGA) were preserved by freezing at -70 °C in vials containing trypticase soy broth (TSB) (DIFCO,

Detroit, MI) supplemented with 20% (v/v) glycerol (Sigma, St. Louis, MO). To propagate the culture, a frozen vial was thawed at room temperature, and 0.1 ml of the thawed culture was transferred to 10 ml of enrichment TSB in screw capped tubes and incubated aerobically for 16-18 h at 37 °C with shaking (Thermolyne Maxi-Mix III type

79 65800, Barnstead/ Thermolyne, Dubuque, IA). The inoculum was prepared from a second transfer of this culture (0.1 ml) to another 10 ml tube of enrichment TSB, and incubated aerobically for 16-18 h at 37 °C with shaking. After incubating overnight, the washed cell suspension of the organism was harvested by centrifugation for 10 min at

1107 ×g (IEC HN-SII Centrifuge, International Equipment CO., Inc., Needham Heights,

MA), then the pellet resuspended in sterile 0.1% (w/v) peptone water (Bacto peptone,

Becton Dickinson, Sparks, MD) to obtain a population of approximately 8-9 log CFU/ml.

Three ml of each strain were mixed together then 1 ml of the suspension transferred into

99 ml of sterile 0.1% (w/v) peptone water to obtain population of approximately 5-6 log

CFU/ml. Initial cell populations were verified by enumeration of the cells following surface-plating in TSA and incubating at 37 °C for 24 h. The same procedure was followed for L. monocytogenes (ATCC 15313) which was surface-plated in Listeria

Enrichment Agar (DIFCO, Detroit, MI) and incubated at 37 °C for 48 h.

5.2.2. Preparation of enzyme concentrations

The concentrations of actinidin (KFPE OT1005X, Ingredient Resources Pty Ltd,

Australia) used with L. monocytogenes and three mixed strains of E. coli O157:H7 were

0, 175, 350, and 700 mg/ml, while the concentrations of papain (P4762-500MG, Sigma

Chemicals, St Louis, MO) used with L. monocytogenes and three mixed strains of E. coli

O157:H7 were 0, 5, 8 and 10 mg/ml. These concentrations were chosen based on preliminary experiments using a greater range of enzyme concentrations. Both enzymes

80 were sterilized using 0.45 µm syringe filter membrane (0.45 µm Supor ® Membrane, Pall

Corporation, Ann Arbor, MI).

5.2.3. Meat sample preparation and inoculation

Chunk beef was purchased from a local store, transported to the laboratory under refrigerated conditions (0–4 °C) and stored at 4 °C until use. The beef meat was cut using a sterile sharp knife and a stainless steel square template into approximately 3 x 3 cm 2 and thickness 1.5 - 2 cm. Meat samples were transferred into individual sterile plastic bags (WHIRL-PAK®, Nasco, CA, USA). A 0.5 ml aliquot of the inoculum was pipetted on meat surface in the bags, giving a surface inoculum of 5-6 log CFU/cm 2 and allowed to remain undisturbed for 5 minutes at room temperature to permit bacterial cell attachment before subjecting enzyme treatments. After inoculation, 1 ml of each enzyme concentrations was pipetted on the meat surface. The samples were then held at three different temperatures 5, 25, and 35 °C.

5.2.4. Enumeration of surviving bacteria and sampling time

Meat samples held at 25 and 35 °C were sampled at 0, 1, and 3 h while those at

5 °C were sampled at 0, 6, and 24 h. Twenty milliliters of 0.1% (w/v) peptone water was added to each bag and hand massaged for 1 minute then 0.1ml were aseptically removed, serially diluted and appropriate serial dilutions were surface plated on enrichment agar,

Listeria agar (DIFCO, Detroit, MI) for L. monocytogenes and TSA (DIFCO, Detroit, MI) for E. coli O157:H7, in duplicate. Inoculated plates were incubated (Model 2300 incubator, VWR Scientific Products, West Chester, PA) at 37 °C for 48 h for L.

81 monocytogenes and 24 h for E. coli O157:H7. Dilution plates with 25-250 colonies were counted (LEICA, QUEBEC DARK FIELD colony counter, Buffalo, NY 14240 USA model 3325) and populations were reported as a CFU/ml and log CFU/ml. All experiments were repeated three times.

5.2.5. Determination of Enzymatic Activity and Protein Content

The activity of actinidin and papain was measured spectrophotometrically according to the modified method of Robinson (1975). The assay mixture contained 1 ml of actinidin (KFEP OT1005X) or papain dilutions of these concentrations (2.5, 5 and 10 mg/ml) and 5 ml of 0.65% (w/v) of substrate, casein solution, (Sigma, St. Louis, MO) dissolved in 50 mM potassium phosphate buffer, pH 7.5 at 37 ºC. All reaction mixtures were incubated at 37 °C for 10 minutes. The reaction was stopped by adding 5 ml of 110 mM trichloroacetic acid (TCA) and precipitated protein was removed by filtration through a 0.45 µm syringe filter (0.45 µm Supor ® membrane, Pall Corporation, Ann

Arbor, MI). The absorbance of filtrate was measured at 660 nm (Spectrophotometer

GENESYS 20 4001-4, 100-240V 50/60 Hz, Madison, WI). Blank samples were prepared by adding the enzyme at the end of the incubation time, just before TCA addition and precipitation. One unit of the enzyme activity was defined as the amount of enzyme which releases 1 µmol of tyrosine per minute under the assay conditions. Specific activity was expressed as enzyme units / mg protein. Protein content of actinidin (KFEP

0T1005X) and papain were measured using a modified Lowry protein assay method

(Lowry et al., 1951) with bovine serum albumin (Pierce Biotechnology, modified Lowry protein assay kit, Rockford, IL) as the protein standard.

82 5.2.6. Statistical analysis

A split-split-split plot design was used for the study. The factor at the highest level was the bacterium type ( L. monocytogenes and E. coli O157:H7). The next level was the concentration level for papain (0, 5, 8 and 10 mg/ml) and for actinidin (0, 175,

350 and 700 mg/ml) and the third level was temperature (5, 25 and 35 °C). The fourth level was time (6 h and 24 h for 5 °C and 1 h and 3 h for 25 and 35 °C). The response variable was the log increase in the bacterium count compared to time 0. Treatment effects were evaluated using analysis of variance techniques and linear contrasts. Mean were separated using Fisher’s protected least signification difference (LSD) test with alpha ≤ 0.05.

5.3. Results

5.3.1. Effect of actinidin on L. monocytogenes and E. coli O157:H7 at 5 °C

The average reduction of E. coli O157:H7 was greater than that of L. monocytogenes and the higher concentrations of actinidin yielded greater reduction in bacterial populations.

The population of E. coli O157:H7 was significantly reduced at actinidin concentrations ≥ 175 mg/ml from 6 h to 24 h (Table 5. 1).

L. monocytogenes populations were significantly reduced at actinidin concentrations ≥ 350 mg/ml after 6 and 24 h compared to starting populations. Whereas, for E. coli O157:H7 there was no significant difference in log reductions for concentrations greater than or equal to 175 mg/ml. The reductions when averaged for all concentrations were 1.21 log cfu/ml after 6 h and 1.74 log cfu/ml after 24 h (Table 5. 1).

83 5.3.2. Effect of actinidin on L. monocytogenes and E. coli O157:H7 at 25 °C and

35 °C

There was no significant difference in the log reduction for L. monocytogenes or

E. coli O157:H7 at 25 and 35 °C, therefore, the data were pooled for these two temperatures (Table 5. 2). This is in agreement with Aminlari et al. (2009) who found that actinidin increased protein solubility by 20% at 37 °C for 2 hours, indicating that the optimum temperature of actinidin ranges between 30-50 °C. Moreover, Katsros et al.

(2009) found actinidin did not show any activity at temperatures higher than 55 °C.

There was no significant difference in L. monocytogenes population between 1 and 3 h at actinidin concentrations of 0 and 175 mg/m, however, the population of L. monocytogenes at actinidin concentrations greater than or equal to 350 mg/ml decreased between 1 and 3 h. Actinidin concentrations ≥ 350 mg/ml after 1 h and actinidin concentrations ≥ 175 mg/ml after 3 h significantly reduced L. monocytogenes compared to the initial population (Table 5. 2).

On the other hand, there were differences in the population of E. coli O157:H7 between 1 h and 3 h with all actinidin levels tested. Also, the average population of E. coli O157:H7 was reduced significantly for actinidin concentrations ≥ 175 mg/ml by

(1.15 log cfu/ml average) after 1 h and by (1.56 log cfu/ml average) after 3 h (Table 5. 2).

5.3.3. Effect of papain on L. monocytogenes and E. coli O157:H7 at 5 °C

As found in actinidin, higher concentrations of papain resulted greater population reductions ( α ≤ 0.05) for L. monocytogenes (Table 5. 3). For papain concentrations tested with L. monocytogenes , 10 mg/ml was the most effective concentration in reducing

84 bacterial counts after 6 h and 24 h, whereas E. coli O157:H7 population was reduced significantly with papain levels ≥ 5 mg/ml by (1.13 log cfu/ml) after 6 h and by (1.4 log cfu/ml) after 24 h (Table 5. 3).

5.3.4. Effect of papain on L. monocytogenes and E. coli O157:H7 at 25 °C and 35 °C

There was no significant difference in the population reduction for L. monocytogenes or E. coli O157:H7 at 25 and 35 °C, therefore, the data for these two temperatures were pooled (Figure 5. 4). Bacterial reductions for both 25 and 35 °C were significantly different from 5 °C (Figure 5. 3). This could be due to the optimum temperature range for papain being between 65 - 80 °C (Ming et al., 2002).

At papain concentrations greater than or equal to 5 mg/ml, there was a significant decrease in the population of L. monocytogenes and E. coli O157:H7 between 1 h and 3 h

(Table 5. 4).

As with actinidin, where the higher concentrations were more effective in reducing bacterial populations, papain at 10 mg/ml reduced the average population of L. monocytogenes after 1 h and 3 h, while for E. coli O157:H7, there was no difference in the reductions for concentrations ≥ 5 mg/ml. The average reductions at these concentrations were 1.06 log cfu/ml after 1 h and 1.38 log cfu/ml after 3 h (Table 5. 4).

5.4. Discussion

5.4.1. Effects of proteolytic enzymes concentrations

As was found in the present study, Eyob et al. (2008) reported that increasing concentrations of the active antimicrobial substance from plant extracts yielded greater antimicrobial activity. In the current study, antibacterial activities of papain and kiwifruit

85 extract (actinidin) were dependent on concentration. For instance, actinidin at 700 mg/ml reduced E. coli O157:H7 population by 1.81 log cfu/ml after 3 h at 25 and 35 °C and by

1.94 log cfu/ml after 24 h at 5 °C, while the same concentration reduced L. monocytogenes population by 1.49 log cfu/ml after 3 h at 25 and 35 °C, and by 1.45 log cfu/ml after 24 h at 5 °C (Figures 5. 1 & 2). On the other hand, papain at 10 mg/ml reduced E. coli O157:H7 population by 1.48 log cfu/ml after 3 h at 25 and 35 °C, and by

1.57 log cfu/ml after 24 h at 5 °C, while the same concentration reduced L. monocytogenes population by 0.56 log cfu/ml after 3 h at 25 and 35 °C, and by 0.46 log cfu/ml after 24 h 5 °C (Figures 5. 3 & 4).

Interestingly, L. monocytogenes showed a nearly linear response in log reduction to actinidin concentration at 6 h (R 2=0.97) and 24 h (R 2=0.94) for 5 °C while E. coli

O157:H7 log reduction response to actinidin concentration was not linear at the concentrations tested (R 2= 0.64 and 0.61 for 6 and 24 h, respectively) (Figure 5. 1). This trend was also evident in the pooled 25 and 35 °C temperatures with L. monocytogenes having log reduction/actinidin concentration R 2 values of 0.98 (1 h) and 0.99 (3 h) while

E. coli O157:H7 displayed R 2 values of 0.58 (1 h) and 0.64 (3 h) (Figure 5. 2). This trend does not hold for papain (linear response to concentration by L. monocytogenes ).

Consequently, it is important to determine enzyme activity for enzyme preparations. Papain concentration of 10 mg solid/ml had 0.3 activity units/ml while actinidin at the same concentration had 0.01activity units/ml (Table 5. 5). The specific activity of the two protease preparations, expressed as enzyme activity unit per mg protein, was 0.1 and 0.02 for papain and actinidin, respectively, showing that the papain

86 protease preparation displayed the highest specific activity towards the bovine serum albumin substrate. This is with agreement with Foegeding and Larick (1986) and Ha et al. (2012) who demonstrated that actinidin possessed the minimal activity compared with other plant cysteine proteases such as papain, bromelain and zingibain.

5.4.2. Effects of bacterial species

The present study also reported that the average log reduction of E. coli O157:H7 was greater than that of L. monocytogenes for both enzymes and at all temperatures used in this study (Figures 5. 1-4). This could be due to the structural differences of bacterial cell wall between Gram negative and Gram positive bacteria. According to Volk and

Wheeler (1988), bacteria have a three-layer of cell wall structure. This structure is composed of: (1) cytoplasmic membrane, (2) thicker peptidoglycan membrane, and (3) varied outer membrane that is mainly composed of proteins and lipids that are susceptible to proteolytic nature of those enzymes. Volk and Wheeler (1988) also explained the bacterial destruction by papain and actinidin is due to proteolytic enzyme precipitation of the outer membrane proteins, rupture of the cell wall and coagulation resulting in loss of cell contents and energy through cell wall leakages. In addition, Conner & Beuchat, 1984 concluded that antimicrobial compounds might change the functions of the microbial and sensitivity of the cell to various antimicrobial compounds to increase the inactivation of membrane-bound enzymes. Therefore, effective antimicrobial preservatives might act on more than one target site on the bacterial membrane, resulting in leakage or autolysis and inhibition of growth or even death of the cell.

87 5.4.3. Effects of media

The inhibitory effect of papain against foodborne pathogens used in this study was lower on beef than that in laboratory buffer (0.1% w/ v peptone water) (Eshamah et al., 2013). This is in agreement with the results of Shelef et al. (2006) and Stecchini et al.

(1993) who found that the potency of natural antimicrobial extracts decreases in complex food systems.

Moreover, Hoa et al. (1998) suggested that the differences in results could be due to the complexity of the food system tested and/ or the specific characteristic of the natural antibacterial used. Shelef et al. (2006) also concluded that the antimicrobial activity of plant extracts increases by increasing its solubility in meat systems. Cutter

(2000) and Hsieh et al. (2001) both reported that the activity also increases under acidic conditions, high water contents, high salt and low fat contents of meat products.

However, Robins and Wilson (1994) concluded that the growth of foodborne pathogens in liquid media provides a baseline for their behavior in complex structures.

Uhart et al. (2006) also observed the differences in the efficacy of natural antimicrobials when studied in vitro versus when added to a food matrix. They concluded that fat, oil droplets, and/or protein interaction are responsible for this reduction in activity.

Furthermore, Farbood et al. (1976) explained that a high lipid fraction in meat may absorb rosemary extract and decrease its concentration in the aqueous phase and consequently its antibacterial effect. They also mentioned that a potential decrease in the penetration of the spice into the microbial cell could be due to the formation of a fat coat around the cell. Regardless of the low antimicrobial achieved, if these natural

88 antimicrobials are used as a part of a hurdle system, higher pathogen reductions may be attained.

5.4.4. Effects of proteolytic enzymes

Bacterial cell walls contain peptides with some amino acids in the L configuration, therefore, proteolytic enzyme are able to hydrolyze these peptides. Most of the cell-wall lytic enzymes are characterized by ability to hydrolyze bonds between the amino sugars of the glycosaminopeptide (Ensign and Wolfe, 1966). Both papain and actinidin belong to a family of cysteine proteases that are activated by cysteine, which is located at the active site of the enzyme. Cysteine-25 attacks the carbonyl carbon in the backbone of the peptide chain freeing the amino terminal portion, breaking the protein chain (Mamboya, 2012)

Papain shows extensive proteolytic activity towards proteins, short chain peptides, amino acid esters and amide links. It preferentially cleaves peptide bonds involving basic amino acids, particularly , Lysine and (Menard et al., 1990).

Actinidin has a similar pattern of hydrolysis to that of papain. Actinidin also prefers hydrophobic sites including , Valine, or Phenylalanine but not Tyrosine (Boland

& Moughan, 2013).

5.5. Conclusion

The average reduction of E. coli O157:H7 was greater than that of L. monocytogenes and higher concentrations of actinidin and papain yielded greater reduction in bacterial populations at all temperatures tested. These findings suggest that, in addition to improve the sensory attributes of beef, tenderization with proteolytic

89 enzymes may enhance the safety and shelf life when stored at suitable temperatures. The findings also propose a promising approach in developing antimicrobial systems for beef.

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97 TABLES AND FIGURES

Table 5.1 Reduction in E. coli O157:H7 and L. monocytogenes populations at 5 °C exposed to various concentrations of actinidin on beef

E. coli O157:H7 L. monocytogenes Time Time Concentration 6 h 24 h 6 h 24 h (mg/ml) Log cfu/ml rinse Log cfu/ml rinse 0 0.02 b -0.019 b 0.05 b 0.55 cb 175 1.08 a 1.52 a 0.32 ab 0.53 c 350 1.19 a 1.77 a 0.43 ab 1.03 ab 700 1.34 a 1.94 a 0.77 a 1.45 a Bold value indicates the reduction in the mean log bacterium count was significantly greater than 0. Means with the same superscripts are not significantly different within the same bacterial type and time (column). Level of significance used was 0.05. The standard error of the mean was 0.18. (n=6)

98

Table 5.2 Reduction in E. coli O175:H7 and L. monocytogenes populations at 25 and 35 °C exposed to various concentrations of actinidin on beef E. coli O157:H7 L. monocytogenes Time Time Concentration 1 h 3 h 1 h 3 h (mg/ml) Log cfu/ml rinse Log cfu/ml rinse 0 -0.18 b -0.36 c 0.08 c 0.07 c 175 1.06 a 1.35 b 0.20 bc 0.34 c 350 1.10 a 1.51 ab 0.49 b 0.80 b 700 1.28 a 1.81 a 1.11 a 1.49 a Bold value indicates the reduction in the mean log bacterium count was significantly greater than 0. Means with the same superscripts are not significantly different within the same bacterial type and time (column). Level of significance used was 0.05. The standard error of the mean was 0.11. (n=6)

99

Table 5.3 Reduction in E. coli O175:H7 and L. monocytogenes populations at 5 °C exposed to various concentrations of papain on beef E. coli O157:H7 L. monocytogenes Time Time Concentration 6 h 24 h 6 h 24 h (mg/ml) Log cfu/ml rinse Log cfu/ml rinse 0 -0.02 b 0.28 b -0.04 b -0.21 b 5 1.08 a 1.29 a -0.07 b -0.01 b 8 1.12 a 1.40 a 0.04 ab 0.15 ab 10 1.18 a 1.57 a 0.38 a 0.46 a Bold value indicates the reduction in the mean log bacterium count was significantly greater than 0. Means with the same superscripts are not significantly different within the same bacterial type and time (column). Level of significance used was 0.05. The standard error of the mean was 0.17. (n=6)

100

Table 5.4 Reduction in E. coli O175:H7 and L. monocytogenes populations at 25 and 35 °C exposed to various concentrations of papain on beef E. coli O157:H7 L. monocytogenes Time Time Concentration 1 h 3 h 1 h 3 h (mg/ml) Log cfu/ml rinse Log cfu/ml rinse 0 0.07 b -0.38 b 0.03 bc -0.2 c 5 0.98 a 1.29 a -0.15 c -0.01 bc 8 1.02 a 1.37 a 0.12 ab 0.14 b 10 1.19 a 1.48 a 0.33 a 0.56 a Bold value indicates the reduction in the mean log bacterium count was significantly greater than 0. Means with the same superscripts are not significantly different within the same bacterial type and time (column). Level of significance was 0.05. The standard error of the mean was 0.14. (n=6)

101

Table 5.5 Total and specific activity of actinidin and papain measured by spectrophotometer Concentration Protein a (mg / ml) Activity (units / ml) b Unit activity / mg (mg solid/ml) protein Actinidin 10 0.58 0.01 0.02 Papain 3 0.3 0.1 a measured by recording absorbance at 750 nm b one unit actinidin or papain activity is the amount of enzyme that release 1µmol of tyrosine per minute at 660 nm/min at pH 7.5 at 37 °C for 10 minutes, (n=6).

102 Figure 5.1. Effect of different concentrations of actinidin on L. monocytogenes and E. coli O157:H7 at 5 °C. The standard error of the mean was 0.18. (n=6)

Figure 5.2. Effect of different concentrations of actinidin on L. monocytogenes and E. coli O157:H7 at 25 and 35 °C. The standard error of the mean was 0.11. (n=6)

103 Figure 5.3. Effect of different concentrations of papain on L. monocytogenes and E. coli O157:H7 at 5 °C. The standard error of the mean was 0.17. (n=6)

Figure 5.4. Effect of different concentrations on L. monocytogenes and E. coli O157:H7 at 25 and 35 °C. The standard error of the mean was 0.14. (n=6)

104 CHAPTER SIX

CONCLUSIONS

Antibacterial effect of proteases, bromelain and papain, tested in vitro was found to be concentration, temperature, bacterial type and time dependent. Bromelain and papain were found to be effective antimicrobials in reducing the populations of E. coli and L. monocytogenes . Reductions of more than 4.0 log cycles were achieved when E. coli and L. monocytogenes were in contact with bromelain level of 4 mg/ml and 1 mg/ml, respectively, at both 25 and 35 °C for 48 h. While the reductions of these bacterial populations when subjected to papain levels of 0.0625 and 0.5 mg/ml, respectively, were below the detection level at 35 °C for 48 h.

When actinidin and papain were applied on beef, the highest reduction in E. coli

O157:H7 populations were 1.94 log cycles at 5 °C after 24 h for actinidin concentration

700 mg/ml. While for L. monocytogenes, the highest reduction was 1.49 at 25 and 35 °C after 3 h for the same actinidin concentration.

In addition, the highest reduction in E. coli O157:H7 populations were 1.57 at

5 °C after 24 h for papain concentration at 10 mg/ml. While for L. monocytogenes, the highest reduction was 0.56 at 25 and 35 °C after 3 h for the same papain concentration.

Fat and protein content of meat, binding to food components, inactivation by other additives, pH effects on antimicrobial stability and activity, uneven distribution in the food matrix and poor solubility among the others, were believed to be factors affecting the efficacy of natural antimicrobials. In addition, the physical structure of beef also has

105 a role in the antimicrobial action of protease. Probably, bacteria may enter in the pores of the surface of certain foods such as beef reducing the chance and time of direct contact with the antimicrobial

The average reduction of E. coli O157:H7 was greater than that of L. monocytogenes. This could be due to the structural differences of bacterial cell wall between Gram negative and Gram positive bacteria. According to Volk and Wheeler

(1988), bacteria have a three-layer of cell wall structure. This structure is composed of

(1) cytoplasmic membrane, (2) thicker peptidoglycan membrane, and (3) varied outer membrane that is mainly composed of proteins and lipids that are susceptible to proteolytic nature of those enzymes. Volk and Wheeler (1988) also explained the bacterial inhibition by papain and actinidin is due to proteolytic enzyme precipitation of the outer membrane proteins, rupture of the cell wall and coagulation resulting in loss of cell contents and energy through cell wall leakages. In addition, Conner & Beuchat, 1984 concluded that antimicrobial compounds might change the functions of the microbial cell membrane and sensitivity of the cell to various antimicrobial compounds to increase the inactivation of membrane-bound enzymes. Therefore, effective antimicrobial preservatives might act on more than one target site on the bacterial membrane, resulting in leakage or autolysis and inhibition of growth or even death of the cell.

The higher the concentrations of actinidin and papain the greater the reduction in bacterial populations at all temperatures tested on beef. This is in agreement with many researchers who reported that increasing concentrations of the active antimicrobial substance from plant extracts yielded greater antimicrobial activity.

106 These findings suggest that, in addition to improving the sensory attributes of beef, tenderization with proteolytic enzymes may enhance the safety and shelf life when stored at suitable temperatures. The findings also propose a promising approach in developing antimicrobial systems for beef. Regardless of the low antimicrobial achieved, if these natural antimicrobials are used as a part of a hurdle system, higher pathogen reductions may be attained. However, this research needs further investigation.

107 APPENDIX

Laboratory media preparation

Preparation of Listeria Agar

• 36.1g of Listeria Enrichment broth

• 1 liter of DW

• 15 g of granulated agar

The products were mixed and heated to a temperature of 100°C. And it

separated into tubes.

Preparation of Listeria Broth

• 36.1g of Listeria Enrichment broth

• 1 liter of DW

The products were mixed to a homogenized solution. And it separated into

tubes of 10 ml each.

Preparation of TSB solution

• 30g of Tryptic Soy Broth

• 1 liter of DW

The products were mixed to a homogenized solution. And it separated into tubes of 10 ml each.

108 Preparation of TSA

• 40g of Tryptic Soy Agar

• 1 liter of DW

The products were mixed and heated to temperature of 100 °C and separated into

tubes of 200 ml.

Before pouring the TSA into plates, 1ml Ampicillin solution was added to 200ml

media.

Preparation of Ampicillin solution (Ampicillin Sodium Salt Sigma A 9518)

• 0.2g of Ampicillin sodium salt

• 10ml of sterile water

This solution is sterilized by using 0.45 µm syringe filter

Preparation of Peptone water

• 1g of Bacto™ Peptone

• 1 liter of DW

The products were mixed to a homogenized solution. And it separated into tubes

of 9.0, 9.9 and 99.0 ml.

The tubes of agars, solutions and peptone were autoclaved for 20 min. at 121 °C

Preparation of cooked meat broth

• Place 1.25 g of meat granules into a test tube and add 10 mL of purified water.

• Autoclave at 121°C for 15 minutes

109 Protein concentration assay (Modified Lowry protein assay method) Preparation of Standards and Folin-Ciocalteu Reagent

1. Preparation of Diluted Albumin (BSA) Standards

Table A.1: Preparation of Diluted Albumin (BSA) Standards

Dilution Scheme for Test Tube Procedure (Working Range = 1–1,500 µg/ml) Volume of Diluent Vial Volume and Source of BSA Final BSA conc. (D.W) 1 150 µl 750 µl of Stock 1,500 µg/ml 2 625 µl 625 µl of Stock 1,000 µg/ml 3 310 µl 310 µl of vial 1 dilution 750 µg/ml 4 625 µl 625 µl of vial 2 dilution 500 µg/ml 5 625 µl 625 µl of vial 4 dilution 250 µg/ml 6 625 µl 625 µl of vial 5 dilution 125 µg/ml 7 800 µl 200 µl of vial 6 dilution 25 µg/ml 8 800 µl 200 µl of vial 7 dilution 5 µg/ml 9 800 µl 200 µl of vial 8 dilution 1 µg /ml 10 1000 µl 0 0 µg/ml = Blank 11 (Sample) ?

1. Preparation of 1X Folin-Ciocalteu Reagent

Prepare 1X (1 N) Folin-Ciocalteu Reagent by diluting the supplied 2X (2 N)

reagent 1:1 with ultrapure water. Because the diluted reagent is unstable, prepare

only as much 1X Folin-Ciocalteu Reagent as will be used in one day.

Procedure

1. Pipette 0.2 ml of each standard and unknown sample (Actinidin) replicate into an appropriately labeled test tube.

110 2. At 15-second intervals, add 1.0 ml of Modified Lowry Reagent to each test tube. Mix well and incubate each tube at room temperature (RT) for exactly 10 minutes.

3. Exactly at the end of each tube’s 10-minute incubation period, add 100 µl of prepared

1X Folin-Ciocalteu Reagent, immediately vortex to mix the contents. Maintain the 15- second interval between tubes established in Step 2.

4. Cover and incubate all tubes at RT for 30 minutes.

5. With the spectrophotometer set to 750 nm, zero the instrument on a cuvette filled only with water. Subsequently, measure the absorbance of all the samples.

6. Subtract the average 750 nm absorbance values of the Blank standard replicates from the 750 nm absorbance values of all other individual standard and unknown sample replicates.

7. Prepare a standard curve by plotting the average Blank-corrected 750 nm value for each BSA standard vs. its concentration in µg/ml. Use the standard curve to determine the protein concentration of each unknown sample.

Results: Table A.2: Standard curve:

Conc.(mg/ml) Abs(nm) 1.5 2.29 1 1.99 0.75 1.68 0.5 1.19 0.25 0.72 0.125 0.45 0.025 0.24 0.005 0.085 0.001 0.06 0 0.04

111

Figurer A.1: Standard curve of absorption of Diluted Albumin (BSA)

Table A.3: Actinidin absorption and concentration

A1(10mg/ml) A2(5mg/ml) A3(2.5mg/ml) Abs. 1.15 0.538 0.458 Conc. 0.58 0.22 0.17

Table A.4: Papain absorption and concentration

P1(10mg/ml) P2(5mg/ml) P3(2.5mg/ml) Abs. 3.39 1.8 1.13 Conc. 3 0.9 0.4

112

Enzyme activity assay

Reagent’s preparation:

1. 50 mM Potassium Phosphate buffer, pH 7.5 at 37ºC (Buffer)

Prepare 11.4 mg/mL in purified water using Potassium Phosphate, Dibasic,

Trihydrate. Adjust to pH 7.5 at 37ºC with 1 N HCl.

2. 0.65% (w/v) Casein Solution (Casein)

* Prepare 6.5 mg/mL of casein in 50 mM potassium phosphate,

* Heat gently with stirring to 80-85ºC for approximately 10 minutes until a homogeneous dispersion is achieved. Do not boil.

Adjust the pH to 7.5 at 37ºC, if necessary, with 0.1 N NaOH or 0.1 N HCl.

3. 110 mM Trichloroacetic Acid Reagent (TCA)

Prepare 1:55 dilution of Trichloroacetic Acid, 6.1N, approximately 100% (w/v) with purified water.

4. 0.5 M Folin & Ciocalteu’s Phenol Reagent (F-C)

Prepare a 1:4 dilution of 2 M Folin & Ciocalteu’s Phenol Reagent with purified water.

6. 500 mM Sodium Carbonate Solution (Na2CO3)

Prepare 53 mg/mL in purified water using Sodium Carbonate, Anhydrous

7. 10 mM Sodium Acetate Buffer with 5 mM Calcium Acetate, pH 7.5 at 37ºC

(Enzyme Diluent)

113 Prepare 1.4 mg/mL Sodium Acetate, Trihydrate, and 0.8 mg/mL Calcium Acetate in purified water. Adjust the pHto 7.5 at 37ºC with 0.1 M Acetic Acid or 0.1 N NaOH.

8. 1.1 mM L-Tyrosine Standard (Std. solution)

Prepare 0.2 mg/mL L-Tyrosine, Free Base in purified water. Heat gently (do not boil) until tyrosine dissolves Cool to room temperature.

9. Protease Enzyme Solution

Immediately before use, prepare a solution containing 0.1 – 0.2 units/mL of Protease in cold Reagent (Enzyme Diluent). Prepare sample at 10 mg solid/mL in cold Reagent

(Enzyme Diluent ).

Procedure:

Setting up the protease assay and standard curve

1. Pipette the following into suitable vials (in milliliters):

Protease T1 Protease T2 Protease T3 blank Casein 5 5 5 5 Water bath at 37C for 5min. Enzyme sol. 0.5 0.7 1.0 - Incubate at 37C for 10min. TCA 5 5 5 5

Enzyme sol. 0.5 0.3 - 1 Incubate at 37C for 30 min. 2. Filter each solution using a 0.45 µm syringe membrane.

3. Pipette the following reagent into 4 dram vials (in milliliters): For more consistent

results, add F-C immediately following the addition of Na 2CO 3.

114

T1 T2 T3 blank

Test filtrate 2 2 2 -----

Blank filtrate ------2

Na2CO3 5 5 5 5

F-C 1 1 1 1

4. Prepare a standard curve by pipetting the following reagents into suitable vials (in milliliters). Std.1 Std.2 Std.3 Std.4 Std.5 Std. blank Std. 0.05 0.1 0.2 0.4 0.5 0 solution Purified 1.95 1.9 1.8 1.6 1.5 2 water Na2CO3 5 5 5 5 5 5

F-C 1 1 1 1 1 1

5. Mix by swirling and incubate Blanks, Standards, and Tests at 37ºC for 30 minutes.

Remove the vials and allow it cool to room temperature.

6. Filter each Blank, Standard, and Test using a 0.45 µm syringe filter into suitable cuvettes.

7. Record the A660nm of each Test, Standard, and Blank solution.

115 Results & calculations

Figure A.2 Enzymes’ activity standard curve Standard curve

1.4 1.2 y = 2.404x - 0.0112 R² = 0.9995 1 0.8 0.6

A 660nm A 0.4 ∆ 0.2 0 0 0.1 0.2 0.3 0.4 0.5 0.6 -0.2 umoles of Tyrosine

Sample Determination

For protease:

∆A660nm (Test) = ∆A660nm (Test) - ∆A660nm (Test Blank)

= 1.667- 0.201 = 1.466

Units/mL enzyme = ( µmole Tyrosine equivalents released) (11)/ (1) (10) (2)

Where

11 = Total volume of assay in milliliters

2 = Volume (in milliliters) used in Colorimetric Determination

1 = volume of enzyme used for assay

10 = time (in minutes) of assay

Units/mL enzyme = 0.61 X 11 / 20 = 0.33 unit/ml enzyme

Units/mg solid = Units/mL enzyme mg solid/ml enzyme

116 Units/mg protein = Units/mL enzyme mg protein/ml enzyme

Table A.5: Specific activity of different concentrations of actinidin

Mg solid / ml Unites activity / mg protein 10 0.02 700 1.4 350 0.7 175 0.35

Table A.6: Specific activity of different concentration of papain

Mg solid / ml Unites activity / mg protein 10 0.1 8 0.08 5 0.05

Measuring optical density and growth curve of E. coli O157:H7 strains

The bacterial population in the culture will be estimated by measuring its turbidity with a spectrophotometer. Traditionally, turbidity is defined as the absorbance of the culture at a wavelength of 600 nanometers, commonly referred to as the OD 600 (or optical density at

600 nm). This value can then be converted to a useful concentration value using the standard conversion factor where 1 OD 600 = 1 x 10 9 cells/mL

Equipment/Reagents

250 mL Erlenmeyer flasks containing 100 mL of growth medium (TSB)

37 °C incubator shaker

Timer

Sterile pipettes (5 & 1 mL)

Spectrophotometer

117 Plastic cuvettes

Overnight of Escherichia coli cultures (10 mL per culture)

Blank samples of un-inoculated media (TSB)

Procedure

1. Set the wavelength to 600 nm.

2. Blank the spectrophotometer with a cuvette containing 1 mL of un-inoculated TSB broth

3. Read the absorbance of the culture.

4. Place the culture flask in the shaker incubator set at 120 rpm at 37° C, and time for the required 30- minute intervals.

5. Every 20 minutes, aseptically transfer 1 mL of the culture to a cuvette and determine its absorbance. Try to remove your 1 mL aliquots as quickly as possible to avoid cooling the culture.

6. Using the computer program Excel, plot a bacterial growth curve with the absorbance on the y-axis and the incubation time on the x-axis. Draw the best line connecting the plotted points.

Results

118 Figure A.3: Growth curve of E. coli O157:H7 380-94

Figure A.4: Growth curve of E. coli o157: H7 E-0654

119 Figure A.5: Growth curve of E. coli O157: H7 C7929

120