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1-1-2013
Antibacterial efects of proteases on diferent strains
of Escherichia coli and Listeria monocytogenes
Hanan Eshamah
Clemson University, heshama@g.clemson.edu
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Eshamah, Hanan, "Antibacterial efects of proteases on diferent strains of Escherichia coli and Listeria monocytogenes" (2013). All
Dissertations. Paper 1177.









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
ii
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 consumers
demand for natural ingredients has made the investigations of effectiveness of natural
antimicrobials necessary. In this study, in vitro antimicrobial activity of bromelain and
papain 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 beef 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 enzyme 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
iii
controlled nature of the study and that meat broth media exposes enzymes 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
iv
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.

v
DEDICATION

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




vi
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.
vii
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.






































viii
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








ix
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





x
LIST OF TABLES


Table Page

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

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

2.3 Cysteine proteinases (bromelain) from pineapples (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





xi
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




xii
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
xiii
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
1
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
2
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 protease 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 papaya fruit (Carica papaya, Caricaceae). It is
characterized by its ability to hydrolyze large proteins into smaller peptides 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 cysteine protease derived from
pineapple fruit (Ananas comosus) which is a member of Bromeliaceae family (Hale et al.,
3
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 (Tart, 2009). Moreover, it does not affect
sensory attributes of meat (flavor and odor) compared to the other thiol 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 seconddegree burns 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
4
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).
5
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),
6
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
7
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
peoples 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
8
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 milk, 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
9
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)






10









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









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
substrate 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.
?
HUS
Late complications
3% - 5%
death
~5% chronic
renal failure,
stroke, and
other major
sequelae
~30%
proteinuria and
other minor
sequelae
~60%
resolution
5%
1 2 days
3 4 days
5 -7 days
Abdominal cramps, non-bloody diarrhea
Bloody diarrhea
Resolution HUS
E.coli O157:H7 ingested
95%
11
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. Meat
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).
12
Brooks (2007) concluded that enzymes marination 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 product.
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 USDAs 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 ginger (zingibain)
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, medicalpharmaceutical,
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 peptide 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 endopeptidases (Gonzlez-Rbade et al., 2011).
13
Exopeptidases are divided into aminopeptidases which are able to hydrolyze peptide bond
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 active site. 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 industrial
enzymes 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
Serine/ Threonine
Trypsin, chymotrypsin, subtilisin,
elastase
Aspartate Pepsin
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 cell wall structure and to
14
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 (Gonzlez-Rbade et al., 2011).




15
Table 2. 2
Plant cysteine proteinases and their sources (Stepek. 2004)
Plant species
(common name)
Enzyme
pH
optimum
Stability
to acid
Carica
papaya (papaya)
Papain 410 To pH4

Chymopapain 310
To
<pH1.2

Caricain 310 To pH4

Glycyl
endopeptidase
310 To pH3
Ficus
carica (Mediterranean
fig)
Ficin 48.5 To pH4
Ficus glabrata Ficain 48.5
To
pH3.3
Ananas
comosus (pineapple)
Stem
bromelain
5.58 NA

Fruit
bromelain
5.58 NA

Ananain 5.58 NA

Comosain 5.58 NA
Actinidia
chinensis (kiwi fruit)
Actinidin 410 NA
Calotropis
gigantea (madar
plant)
Calotropin 48 NA
Asclepias spp.
(milkweed)
Asclepain 610 NA

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
16
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 disulfide bridges (cys22-cys63, cys56-
cys95 and cys153-cys200) and a free cysteine cys25 which takes part in the catalysis.
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 catalytic triad which is made up of 3 amino
acids: the chemically sensitive cysteine-25, histidine-159 and asparagine-158.
17
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
2
O) 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 stem bromelain (80%), fruit bromelain
(10%), and ananain (5%), whereas non-protease components include phosphatases,
glucosidases, peroxidases, cellulases, glycoproteins and carbohydrates (Chobotova et al.,
2010; Maurer, 2001).






18
Table 2. 3
Cysteine proteinases (bromelain) from pineapples (Ananas comosus) (Maurer, 2001)
Name (EC
number)
Abbreviation
Molecular
mass
(Dalton)
Isoelectr
ic point
Sequences Glycosylation
From pineapple
stems:

Stem bromelain
(EC 3.4.22.32)
F4 & F5
23,800
(sequence
+ sugar)
> 10
Completely
sequenced
(212 amino
acid)
glycosylated
Ananain
(EC 3.4.22.31)
F9
23,464
sequence
> 10
Completely
sequenced
(216 amino
acid)
Non
glycosylated
Comosain
SBA/a &
SBA/b
F9/b
23,550 &
23,560
24,509 &
23,569
4.8 and
4.9
> 10
N-term.
sequence
N-term.
sequence
highly
glycosylated
glycosylated
From pineapple
fruits:

highly
glycosylat
ed
4,6
N-term.
sequence
not
glycosylated Fruit bromelain
(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).

19

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
20
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
21
cuts than injection. 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, seeds, latex and extracts have been used traditionally to treat
various human ailments. Papaya seed 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, glycyl
endopeptidase and caricain 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
22
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)
23
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 lysine 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 collagenases and cathepsins 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 (Tart, 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).
24


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 burn 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 -glucuronidase activity and promoting the activity of -
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).


25
References
Amid, A., Ismail, N. A., Yusof, F., & Salleh, H. M. (2011). Expression, purification, and
characterization of a recombinant stem bromelain from Ananas Comosus. Process
Biochemistry, 46 (12), 2232-2239

Amri, E., & Mamboya, F. (2012). Papain, a plant enzyme of biological importance: A
review. American Journal of Biochemistry and Biotechnology, 8 (2), 99-104

Anibijuwon, I. I., & Udeze, A. O. (2009). Antimicrobial Activity of Carica Papaya
(Pawpaw Leaf) on Some Pathogenic Organisms of Clinical Origin from South-
Western Nigeria. Ethno botanical leaftels, 13, 850-64

Anuar, N. S., Zahari, S. S., Taib, I. A., & Rahman, M. T. (2008). Effect of green and ripe
carica papaya epicarp extracts on wound healing and during pregnancy. Food and
Chemical Toxicology, 46, 2384-2389.

Baker, E. N. (1980). Structure of actinidin after refinement at 1.7-A resolution. Journal
of Molecular Biology, 141, 441-484.

Basile, A., Vuotto, M., Violante, U., Sorbo, S., Martone, G., & Castaldo-Cobianchi, R.
(1997). Antibacterial activity in actinidia chinensis, feijoa sellowiana and aberia
caffra. International Journal of Antimicrobial Agents, 8, 199-203.

Bavaro, M. F. (2009). Escherichia coli O157: what every internist and gastroenterologist
should know. Current gastroenterology reports, 11(4), 301-306

Bekhit, A. E. D., Han, J., Morton, J., & Sedcole, R. (2007). Effect of kiwifruit juice and
water pre-rigor infusion on lamb quality. In Proceedings of the 53rd International
Conference of Meat Science and Technology, (pp. 377-378), Beijing, China.

Benkerroum, N., Daoudi, A., & Kamal, M. (2003). Behavious of L. monocytogenes in
raw sausages in presence of a bacteriocins producing lactococcal strain as a
protective culture, Meat Science, 63, 479-484

26
Borrelli, F., Capasso, R., Severino, B., Fiorino, F., Aviello, G., De Rosa, G., Mazzella,
M., Romano, B., Capasso, F., Fasolino, I., & Izzo, A.A. (2011). Inhibitory effects
of bromelain, a cysteine protease derived from pineapple stem (Ananas comosus),
on intestinal motility in mice. Neurogastroenterology and Motility 23, 745-e331

Bromelain Monograph (2010). Alternative medicine review, 15(4), 361-368

Brooks, C. (2007). Marinating of Beef for Enhancement. Beef Facts: Product
Enhancement. National Cattlemans Beef Association. <www.beefresearch.org>

Calkins, C.R. & Sullivan, G. (2007). Adding Enzymes to Improve Beef Tenderness. Beef
facts product enhancement. National Cattlemans Beef association. Centennial
Colorado: Cattlemen's Beef Board 2007.

Cartwright, E.J., Jackson, K.A., Johnson, S.D., Graves, L.M., Silk, B.J., & Mahon, B.E.
(2013). Listeriosis outbreaks and associated food vehicles, United States, 1998
2008. Emerging Infectious Diseases, 19, 1-9.

Center of Disease Control and Prevention. (2013). Listeriosis. Available at <
www.cdc.gov/listeria/index.html>

Center of Disease Control and Prevention. (2011). Estimates of foodborne illness in the
United States available at <http://www.cdc.gov/foodborneburden/2011-foodborne-
estimates.htm>

Chobotova, K., Vernallis, A. B., & Majid, F. A. A. (2010). Bromelains activity and
potential as an anti-cancer agent: Current evidence and perspectives. Cancer
letters, 290(2), 148-156.

Christensen, M., Torngren, M. A., Gunvig, A., Rozlosnik, N., Lemetsch, R., Karlsson, A.
H. & Ertbjerg, P. (2009). Injection of marinade with actinidin increases tenderness
of procine M. biceps femoris and affects myofibrils and connective tissue. Journal
of Science Food Agriculture, 89, 1607-1614

27
Conner, D.E. (1993). Naturally occurring compounds. In: Davidson, P.M., Branen, A.L.
(Eds.), Antimicrobials in Foods. Marcel Dekker, New York, pp. 441- 468.

Corzo, C. A., Waliszewski, K. N., & Welti-Chanes, J. (2011). Pineapple fruit bromelain
affinity to different protein substrates. Food Chemistry, 133(3), 5-9.

Dorman, H. J.D., & Dean, S.G. (2000). Antimicrobial agents from plants; antimicrobial
activity of plant volatile oils. Journal of Applied Microbiology, 88: 308-316.

Drenth, J., Jansonius, J. M., Koekoek, R. & Wolthers B. G. (1971). The structure of
papain. Advanced Protein Chemistry, 25, 79-115

Elder, R. O., Keen, J. E., Siragusa, G. R., Barkocy-Gallagher, G. A., Koohmaraie, M., &
Laegreid, W. W. (2000). Correlation of Enterohemorrhagic Escherichia coli O157
prevalence in feces, hides, and carcasses of beef cattle during processing.
Proceedings of the National Academy of Sciences, 97(7), 2999-3003.

Emeruwa, A. C. (1982) Antibacterial substance from Carica papaya fruit extract. Journal
of Natural Products, 45(2), 123-127

Farouk, A. (1982). Antibacterial activity of proteolytic enzymes. International Journal of
Pharmaceutics, 12, 295-298

FDA. (2012). Bad Bug Book-Foodborne Pathogenic Microorganisms and Natural Toxins,
(2
nd
Ed.). www.cfsan.fda.gov.

Frazier, W. C. & Westhoff, D. C. (1988). In Food Microbiology, (4
th
Ed). McGraw-Hill,
New Delhi, pp. 412-419.

FSIS Salmonella Compliance Guidelines for Small and Very Small Meat and Poultry
Establishments that Produce Ready-to-Eat (RTE) Products. Available at: http://
www.fsis.usda.gov/PDF/Salmonella_Comp_Guide_091912.pdf.

28
Glazer, A. N., & Smith, E. L. (1971). Papain and Other Plant Sulfhydryl Proteolytic
Enzymes. The enzymes, 3, 501-546.

Gonzlez-Rbade, N., Badillo-Corona, J. A., Aranda-Barradas, J. S., & Oliver-Salvador,
M. D. C. (2011). Production of plant proteases in vivo and in vitro A review.
Biotechnology Advances, 29, 983-996.

Gould, G. W., & Russell, N. J. (2003). Major, new, and emerging food-poisoning and
food-spoilage microorganisms. In: Russell, N. J., Gould, G. W., editors. Food
preservatives. 2nd Ed. New York: Kluwer Academic/Plenum Publishers. pp. 1-13.

Grover, R. K. et al. (2005). Standardization of chicken gizzard pickle using sodium tri
polyphosphate and papain. Indian Journal of Poultry Science, 40(2), 202-205.

Ha, M., Bekhit, A. E. A., Carne, A., & Hopkins, D. L. (2012). Characterization of
commercial papain, bromelain, actinidin and zingibain protease preparations and
their activities toward meat proteins. Food Chemistry, 134, 95-105.

Hafezi, F., Rad, H. E., Naghibzadeh, B., Nouhi, A. H., & Naghibzadeh, G. (2010).
Actinidia deliciosa (kiwifruit), a new drug for enzymatic debridement of acute burn
wounds. Burns, 36, 352-355.

Hale, L. P., Greer, P. K., Trinh, C. T., & James, C. L. (2005). Proteinase activity and
stability of natural bromelain preparations. International Immunopharmacology,
5(4), 783-793.

Han, J., Morton, J. D., Bekhit, A. E. D., & Sedcole, J. R. (2009). Pre-rigor infusion with
kiwifruit juice improves lamb tenderness. Meat science, 82(3), 324-330.

Jakubowski, H. (2010). Biochemistry online: an approach based on chemical logic.
Available at:
http://employees.csbsju.edu/hjakubowski/classes/ch331/catalysis/olcatenzmech.htl

Jay, J. M. (1994). Modern food microbiology. 3
rd
edition, Wayne State University. S.A.
Zaragoza: Editorial acribia, 84,200-0746-3.
29

Junttila, R., Niemala, I., & Hirn, J. (1988). Minimum growth temperature of Listeria
monocytogenes and non-hemolytic Listeria. Journal of Applied Bacteriology, 65,
321-327.

Kamphius, I. G., Drenth, J., & Baker, E. N. (1985). Thiol protease. Journal of Molecular
Biology, 182, 317-329

Katsaros, G., Katapodis, P., & Taoukis, P. (2009). Modeling the effect of temperature and
high hydrostatic pressure on the proteolytic activity of kiwi fruit juice. Journal of
Food Engineering, 94, 40-45.

Kentnawa, S., & Rawdkuen, S. (2011). Application of bromelain extract for muscle foods
tenderization. Food and Nutrition Sciences, 2, 393-401

Ketnawa, S., Rawdkuen, S., & Chaiwut, P. (2010). Two phase partitioning and collagen
hydrolysis of bromelain from pineapple peel nang lae cultivar. Biochemical
Engineering Journal, 52, 205-211.

Koak, J. H., Kim, H. S., Choi, Y. J., Baik, M. Y., & Kim, B. Y. (2011). Characterization
of a protease from over-matured fruits and development of a tenderizer using an
optimization technique. Food Science and Biotechnology, 20, 485-490.

Lawrie, R. A. (1998). Lawrie's meat science .6
th
Ed., In: Wood head publishing Ltd.:
England

Leary, D., Vierros, M., Hamon, G., Arico, S., & Monagle, C. (2009). Marine genetic
resources: A review of scientific and commercial interest. Marine Policy, 33, 183-
94.

Lewis, D. A. & Luh, B. S. (1988). Application of actinidin from kiwifruit to meat
tenderization and characterization of beef muscle protein hydrolysis. Journal of
Food Biochemistry, 12(3), 147-158.

30
Llerena-Suster, C. R., Priolo, N. S., & Morcelle, S. R. (2011). Sodium Tetrathionate
effect on papain purification from different Carica papaya latex crude extracts.
Preparative Biochemistry and Biotechnology, 41, 107-121.

Ma, Q. (2011). Evaluation of pre-rigor proteases injections on cooked beef volatiles at 1
day and 21 days post-mortem (Doctoral dissertation, AUT University).

Maiti, A. K., Ahlawat, S.S., D.P. Sharma, D. P., & Khanna, N. (2008). Application of
natural tenderizers in meat - A review. Agricultural Review, 29 (3), 226 - 230.

Marsh, B. B., & Leet, N. G. (1966). Studies in meat tenderness III. The effects of cold
shortening on meat tenderness. Journal of Food Science, 31, 450-459.

Martin, E., Griffis, C., Vaughn, K., O'Bryan, C., Friedly, E., Marcy, J., . . . & Lary Jr, R.
(2009). Control of listeria monocytogenes by lauric arginate on frankfurters
formulated with or without lactate/diacetate. Journal of Food Science, 74(6),
M237-M241.

Maurer, H.R. (2001) Bromelain: biochemistry, pharmacology and medical use. Cell.
Molecular Life Science, 58, 1234-1245

McKeith, F.K., Brewer, M. S., & K.A. Bruggen, K. A., (1994). Effects on enzyme
application on sensory, chemical, and processing characteristics of beef steaks and
roasts. Journal of Muscle Foods, 5,149-164.

Mead, P. & Griffin, P. (1998). Escherichia coli O157:H7. The Lancet 352:1207-1212.

Mead, P. S., Slutsker, L., Dietz, V., McCaig, L.F., Bresee ,J. S., Shapiro, C., Griffin, P.
M., & Tauxe, R. V.(1999). Food-related Illness and Death in the United States,
Emerging Infectious Diseases, 5, 607-625. Available at
http://www.cdc.gov/ncidod/eid/vol5no5/mead.htm

Ming, C. C., Bono A., Krishnaiah, D., & Han, T. S. (2002). Effects of Ionic and non-
Ionic surfactants on papain activity. Borneo Science, 12, 71-77.

31
Mohajeri, G., Masoudpour, H., Heidarpour, M., Khademi, E. F., Ghafghazi, S., Adibi, S.,
& Akbari, M. (2010). The effect of dressing with fresh kiwifruit on burn wound
healing. Surgery, 148, 963-968.

Molan, A. L., Kruger, M. C., & Drummond, L. N. (2008). Kiwifruit: the ability to
positively modulate key markers of gastrointestinal function in: Proceeding of
nutrition society of New Zealand, 2008 vol32, pp. 66-71. Forty second annual
conference held in combination with the Australian Nutrition Society at Massey
University, Albany campus Auckland, Dec 2007.

Nakamura, Y., Yoshimoto, M., Murata, Y., Shimoishi, Y., Asai, Y., Park, E.Y., & Sato,
K. (2007). Papaya seed represents a rich source of biologically active
isothiocyanate. Journal of Agriculture and Food Chemistry, 55, 4407-4413.

Names, V. (2012). Actinidia Chinensis. Edible Medicinal and Non-Medicinal Plants:
Volume 1, Fruits, 1, 12.

Nataro, J. P. & Kaper, J. B. (1998). Diarrheagenic Escherichia coli. Clinical
Microbiology Reviews, 11(1), 142 - e201.

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 Of-cinale Roscoe (Ginger Rhizome). Meat Science, 68, 363-369.

Noveir, M. R., Dogan, H. B., & Kadir Halkman, A. (2000). A note on Escherichia coli
O157: H7 serotype in Turkish meat products. Meat science, 56(4), 331-335

Nishimura, T., Hattori, A., & Takahashi, K. (1995). Structural weakening of
intramuscular connective tissue during conditioning of beef. Meat Science, 39, 127-
133.

Nunnelly, M. C. (2012). Survival and persistence of foodborne pathogens on packaging
materials and reduction of Escherichia coli O157:H7 and Salmonella in beef
trimmings (Doctoral dissertation, Colorado State University).

32
Oloyede, O. I. (2005). Chemical profile of unripe pulp of Carica papaya. Pakistan
Journal of Nutrition, 4, 379-381.

Osato, J. A., Santiago, L. A., Remo, G. M., Cuadra, M. S., & Mori, A. (1993).
Antimicrobial and antioxidant activities of unripe papaya. Life Sciences, 53, 1383-
1389.

Park, S., Worobo, R.W. & Durst, R.A. (1999) Escherichia coli O157:H7 as an emerging
foodborne pathogen: A literature review. Critical Reviews in Food Science and
Nutrition 39, 481-502.

Payne, C.T. (2009). Enzymes. In: Tarte R, editor. Ingredients in meat products:
properties, functionality and applications. New York: Springer Science + Business
Media, LLC. Pp. 173-98.

Rangel, J. M., Sparling, P. H., Crowe, C., Griffin, P. M., & Swerdlow, D. L. (2005).
Epidemiology of Escherichia coli O157: H7 outbreaks, United States, 1982
2002. Emerging infectious diseases, 11(4), 603.

Rawdkuen, S., Jaimakreu, M., & Benjakul, S. (2012). Physicochemical properties and
tenderness of meat samples using proteolytic extract from Calotropis procera
latex. Food Chemistry, 136(2), 909-916

Rawling, N. D., Barrett, A. J., & Bateman, A. (2010). MEROPS. The peptidase database.
Nucleic Acids Research, 38, D227-33.

Rowan, A., & Buttle, D. J. (1994). Pineapple cysteine endopeptidases. Methods in
Enzymology, 244, 555-568.

Roxas, M. (2008). The role of enzyme supplementation in digestive disorders. Alternative
medicine review, 13(4), 307-14.

Salas, C. E., Gomes, M. T. R., Hernandez, M., & Lopes, M. T. P. (2008). Plant cysteine
proteinases: Evaluation of the pharmacological activity. Phytochemistry, 69, 2263-
2269.
33

Scallan, E., Hoekstra, R.M., Angulo, F.J., Tauxe, R.V., Widdowson, M. A., & Roy, R. L.
(2011). Foodborne illness acquired in the United Statesmajor pathogens.
Emerging Infectious Diseases, 17, 7-15.

Scharff, R.L. (2012). The economic burden from health losses due to foodborne illness in
the United States. Journal of Food Protection, 75, 123-131.

Schneider, G., & Wolfing, J. (2004). Synthetic cardenolides and related compounds.
Current Organic Chemistry. 8, 1381-1403.

Shrinithivihahshini, D. N., Sheelamary, M., Mahamuni, D., & Chithradevi, R.
(2011).Occurrence of Listeria monocytogenes in food and ready to eat food
products available in tiruchirappalli, Tamil nadu, India.

Sofos, J. N., & Geornaras, I. (2010). Overview of current meat hygiene and safety risks
and summary of recent studies on biofilms, and control of Escherichia coli
O157:H7 in nonintact, and Listeria monocytogenes in ready-to-eat, meat products.
Meat Science, 86, 2-14.

Stepek, G., Behnke, J. M., Buttle, D. J., & Duce, I. R. (2004). Natural plant cysteine
proteinases as anthelmintic. Trends in parasitology, 20(7), 322-327.

Sullivan, G. A., Calkins, C. R. (2010). Application of exogenous enzymes to beef muscle
of high and low-connective tissue. Meat Science, 85, 730-734.

Tart, R. (2009). Ingredients in meat products: Properties, functionality and applications.
Springer, New York, pp. 174-179.

Todd, E.C.D. (1989). Preliminary estimates of costs of foodborne disease in Canada and
costs to reduce Salmonellosis. Journal of Food Protection, 52, 586-594

United States Department of Agriculture. (2006). Economic research service. Economics
of foodborne disease. Available at:
<http://www.ers.usda.gov/Briefing/FoodborneDisease.>
34
United States Department of Agriculture. (2008). Additives in Meat and Poultry Products
available at <http:// www.fsis.usda.gov/factsheets/Additives>

United States Department of Agriculture. (2012). FSIS Compliance Guideline:
Controlling Listeria monocytogenes in Post-lethality Exposed Ready-to-Eat Meat
and Poultry Products. Available at
http://www.fsis.usda.gov/.../Controlling_LM_RTE_guideline

United States Department of Agriculture (2008). Additive in meat and poultry products.
Food safety Information. Available at < http://www.fsis.usda.gov/pdf/ Additive in
meat and poultry products.pdf>

Wada, M., Hosaka, M., Nakazawa, R., Kobayashi, Y. & Hasegawa, T. (2004). The
solubilization of unheated cattle Achilles tendon with actinidin under neutral and
acidic conditions. Food Science and Technology Research, 10(1), 35-37.

Walker, S. J., Archer, P., & Banks, J. G. (1990). Growth of Listeria monocytogenes at
refrigeration temperatures. Journal of Applied Microbiology, 68(2), 157-162.

Walsh, G. P. (2002). Biochemistry and Biotechnology. West Sussex, England: John
Wiley and Sons Ltd., pp. 420.

Warriss, P. D. (2010). Meat science: an introductory text. Cab International.

Zamyatnin, A. A. & Brochikov, A. S. (2007). Enzymatic fragments as possible regulatory
oligopeptides. Neurochemical Journal, 1(3), 188-195





35
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
36

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
37
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
38
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
39
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
40
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 m syringe filter membrane (0.45 m 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
41
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 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
42
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).


43
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
44
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
45
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
46
cysteine, which is located at the active site of the enzyme (Mamboya, 2012). Bromelain
preferentially cleaving at amino acid sites involving lysine, alanine, tyrosine and glycine.
Whereas, papain prefers hydrophobic sites that include Valine 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
47
enzymes used as meat-tenderizing agents reported here and in other sources may enhance
the safety and shelf life of marinated meat products.

References
Amid, A., Ismail, N. A., Yusof, F., & Salleh, H. M. (2011). Expression, purification, and
characterization of a recombinant stem bromelain from ananas comosus. Process
Biochemistry, 46(12), 2232-2239. http://dx.doi.org/10.1016/j.procbio.2011.08.018

Anibijuwon, I. I., & Udeze, A. O. (2009). Antimicrobial Activity of Carica Papaya
(Pawpaw Leaf) on Some Pathogenic Organisms of Clinical Origin from South-
Western Nigeria. Ethnobotanical leaftels, 13, 850-64.

Bhardwaj A., Ballal S., & Velmurugan, N. (2012). Comparative evaluation of the
antimicrobial activity of natural extracts of Morinda citrifolia, papain and aloe vera
(all in gel formulation), 2% chlorhexidine gel and calcium hydroxide, against
Enterococcus faecalis: An in vitro study. Journal of Conservative Dentistry, 15,
293-297. http://dx.doi.org/10.4103/0972-0707.97964

Borrelli, F., Capasso, R., Severino, B., Fiorino, F., Aviello, G., De Rosa, G., Izzo, A. A.
(2011). Inhibitory effects of bromelain, a cysteine protease derived from pineapple
stem (Ananas comosus), on intestinal motility in mice. Neurogastronterology and
Motility, 23, 745-751. http://dx.doi.org/10.1111/j.1365-2982.2011.01735.x

Bromelain Monograph. (2010). Alternative medicine review, 15(4), 361-368. Retrieved
from
http://www.chiro.org/alt_med_abstracts/ABSTRACTS/Bromelain_Monograph.pdf

Corzo, C. A., Waliszewski, K. N., & Welti-Chanes, J. (2012). Pineapple fruit bromelain
affinity to different protein substrates. Food Chemistry, 133(3), 5-9.
http://dx.doi.org/10.1016/j.foodchem.2011.05.119

Emeruwa, A. C. (1982). Antibacterial substance from Carica papaya fruit extract. Journal
of Natural Products, 45(2), 123-127.http://dx.doi.org/10.1021/np50020a002
48
Gudbjomsdottir, B., Suihko, M. L., Gustavsson, G., Thorkelsson, G., Salo, S., Sjoberg, A.
M., Bredholt, S. (2004). The incidence of Listeria monocytogenes in meat, poultry
and seafood plants in the Nordic countries. Food Microbiology, 21(2), 217-225.
http://dx.doi.org/10.1016/S0740-0020(03)00012-1

Hale, L. P., Greer, P. K., Trinh, C. T., & James, C. L. (2005). Proteinase activity and
stability of natural bromelain preparations. International Immunopharmacology,
5(4), 783-793. http://dx.doi.org/10.1016/j.intimp.2004.12.007

Ketnawa, S., & Rawdkuen, S. (2011). Application of bromelain extract for muscle foods
tenderization. Food and Nutrition Sciences, 2, 393 401.
http://dx.doi.org/10.4236/fns.2011.25055

Ketnawa, S., Rawdkuen, S., & Chaiwut, P. (2010). Two phase partitioning and collagen
hydrolysis of bromelain from pineapple peel Nang laecultivar. Biochemical
Engineering Journal, 52(2-3), 205-211. http://dx.doi.org/10.1016/j.bej.2010.08.012

Lee, G. Y., Jang, H. I., Hwang, I. G., & Rhee, M. S. (2009). Prevalence and classification
of pathogenic Escherichia coli isolated form fresh beef, poultry, and pork in Korea.
International Journal of Food Microbiology, 134(3), 196-200.
http://dx.doi.org/10.1016/j.ijfoodmicro.2009.06.013

Llerena-Suster, C. R., Priolo, N. S., & Morcelle, S. R. (2011). Sodium Tetrathionate
effect on papain purification from different Carica papaya latex crude extracts.
Preparative Biochemistry and Biotechnology, 41(2), 107-121.
http://dx.doi.org/10.1080/10826068.2011.544230

Lopez-Garcia, B., Hernandez, M., & Segundao, B. S. (2006). Bromelain, a cysteine
protease from pineapple (Ananas comosus), is an inhibitor of fungal plant
pathogens. Letters in Aplied Microbiology, 55(1), 62-67.
http://dx.doi.org/10.1111/j.1472-765X.2012.03258.x

Marsh, B. B., Woodham, P. R., & Leet, N. G. (1966). Studies in meat tenderness III. The
effects of cold shortening on meat tenderness. Journal of Food Science, 31, 450-
459. http://dx.doi.org/10.1111/j.1365-2621.1966.tb00520.x
49
Mamboya, E. A. F. (2012). Papain, a plant enzyme of biological importance: A Review.
American Journal of Biochemistry and Biotechnology, 8(2), 99-104.

Ming, C. C., Awang, B., Duduku, K., & Sing, H. (2002). Effects of Ionic and non-Ionic
surfactants on papain activity. Borneo Science, 12, 71-77.

Nishimura, T., Hattori, A., & Takahashi, K. (1995). Structural weakening of
intramuscular connective tissue during conditioning of beef. Meat Science, 39, 127-
133. http://dx.doi.org/10.1016/0309-1740(95)80014-X

Osato, J. A., Santiago, L. A., Remo, G. M., Cuadra, M. S., & Mori, A. (1993).
Antimicrobial and antioxidant activities of unripe papaya. Life Sciences, 53, 1383-
1389. http://dx.doi.org/10.1016/0024-3205(93)90599-X

Prescott, L. M., Harley, J. P., & Klein, D. A. (1990). Procaryotic cell structure and
function. In K. Kane (Ed), Microbiology (pp. 48-51). Dubuque, IA: W. C. Brown
Publishers.

Salampessy, J., Phillips, M, Senewera, S., & Kailasapathy, K. (2006). Release of
antimicrobial peptides through bromelain hydrolysis of leatherjacket (Meuchenia
sp.) insoluble proteins. Food Chemistry, 120(2), 556-560.
http://dx.doi.org/10.1016/j.foodchem.2009.10.054

Schirmer, T. (1998). General and specific porins from bacterial outer membranes.
Journal of Structural Biology, 121(2), 101-109.
http://dx.doi.org/10.1006/jsbi.1997.3946

Sofos, J. N., & Geornaras, I. (2010). Overview of current meat hygiene and safety risks
and summary of recent studies on biofilms, and control of Escherichia coli
O157:H7 in non-intact, and Listeria monocytogenes in ready-to-eat, meat products.
Meat Science, 86(1), 2-14. http://dx.doi.org/10.1016/j.meatsci.2010.04.015


50
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.

USDA-FSIS. (2005). US Department of Agriculture, Food Safety and Inspection Service,
FSIS food recalls. Retrieved from
http://www.fsis.usda.gov/Fsis_Recalls/Quantity_Recovered_by_Case_2005/index.asp



































51
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)
52

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)
53


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)
54
5.a. Papain against E. coli JM 109 at 5 C.
5.b. Papain against E. coli JM 109 at 25 C.
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)


0
1
2
3
4
5
0 4 8 12 16 20 24 28 32 36 40 44 48
L
o
g

C
F
U
/
m
l
Time (hours)
0 mg/ml
0.0625 mg/ml
0.125 mg/ml
0.25 mg/ml
0.5 mg/ml
a
b
a
b
0
1
2
3
4
5
6
0 4 8 12 16 20 24 28 32 36 40 44 48
L
o
g

C
F
U
/
m
l
Time (hours)
0 mg/ml
0.0625 mg/ml
0.125 mg/ml
0.25 mg/ml
0.5 mg/ml
a
b
a
b
a
b
0
1
2
3
4
5
0 4 8 12 16 20 24 28 32 36 40 44 48
L
o
g

C
F
U
/
m
l
Time (hours)
0 mg/ml
0.0625 mg/ml
0.125 mg/ml
0.25 mg/ml
0.5 mg/ml
a
b
a
b
bc
c
a
b
55
6.a. Papain against L. monocytognes at 5 C.
6.b. Papain against L. monocytogenes at 25C.
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)

0
1
2
3
4
5
6
0 4 8 12 16 20 24 28 32 36 40 44 48
L
o
g

C
F
U
/
m
l
Time (hours)
0 mg/ml
0.0625 mg/ml
0.125 mg/ml
0.25 mg/ml
0.5 mg/ml
a
b
0
1
2
3
4
5
6
0 4 8 12 16 20 24 28 32 36 40 44 48
L
o
g

C
F
U
/
m
l
Time (hours)
0 mg/ml
0.0625 mg/ml
0.125 mg/ml
0.25 mg/ml
0.5 mg/ml
a
b
a
b
a
b
0
1
2
3
4
5
6
0 4 8 12 16 20 24 28 32 36 40 44 48
L
o
g

C
F
U
/
m
l
Time (hours)
0 mg/ml
0.0625 mg/ml
0.125 mg/ml
0.25 mg/ml
0.5 mg/ml
a
b
a
b
a
b
56
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)


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)







0
1
2
3
4
5
6
0 0.125 0.25 0.375 0.5
L
o
g

C
F
U
/
m
l
papain concentration (mg/ml)
5C
25C
35C
a
b
c
a
b
c
a
b
c
0
1
2
3
4
5
6
0 0.125 0.25 0.375 0.5
L
o
g

C
F
U
/
m
l
papain concentration (mg/ml)
5C
25C
35C
a
b
a
b
a
b
a
b
57
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.
58
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 Bacus, 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 for safe, convenient and wholesome food (Nychas et al., 2003; Smid
and Gorris, 1999) although, their potential as food preservatives has not been fully
explained.
59
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
60
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 m
syringe filter membrane (0.45 m Supor

Membrane, Pall Corporation, Ann Arbor, MI).

Enzyme concentrations were prepared by adding appropriate amounts of cooked meat
61
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 C. All reaction mixtures were incubated
62
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 m syringe filter (0.45 m 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
mol 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
63
techniques and linear contrasts. Mean were separated using Fishers 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 35C from initial populations. However, at 3 h, there was an
64
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 25C
(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.
65
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).
66
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
67
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.

References
Bourn, D., & Prescott, J. (2002). A comparison of the nutritional value, sensory qualities,
and food safety of organically and conventionally produced foods. Critical Reviews
in Food Science and Nutrition, 42(1), 134.

Burt, S. (2004). Essential oils: their antibacterial properties and potential applications in
Foods - A review. International Journal of Food Microbiology, 94 (3), 223253.

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

Campos, C. A., Castro, M. P., Gliemmo, M. F., & Schelegueda, L. I. (2011). Use of
natural antimicrobials for the control of Listeria monocytogenes in foods. Science
against microbial pathogens: communicating current research and technological
advances

68
Davidson, P. M., Sofos, J. N., & Branen, A. L. (Eds.) (2010). Antimicrobials in food
(Vol. 145). CRC Press.

Eshamah, H., Han, I., Naas, H., Rieck, J., & Dawson, P. (2013). Bactericidal Effects of
Natural Tenderizing Enzymes on Escherichia coli and Listeria monocytogenes.
Journal of Food Research, 2(1), p 8.

Gutierrez, J., Barry-Ryan, C., & Bourke, P. (2008). The antimicrobial efficacy of plant
essential oil combinations and interactions with food ingredients. International
Journal of Food Microbiology, 124(1), 91-97.

Gutierrez, J., Barry-Ryan, C., & Bourke, P. (2009). Antimicrobial activity of plant
essential oils using food model media: efficacy, synergistic potential and
interactions with food components. Food Microbiology, 26(2), 142-150.

Hao, Y.Y, Brackett, R. E., & Doyle, M. P. (1998). Efficacy of plant extracts in inhibiting
Aeromonas hydrophila and Listeria monocytogenes in refrigerated, cooked poultry.
Food Microbiology, 15, 367-378.

Hsieh, P. C., Mau, J. L., & Huang, S. H. (2001). Antimicrobial effect of various
combinations of plant extracts. Food Microbiology, 18, 3543.

Hudson, J. A., Mott, S. J., & Penney, N. (1994). Growth of Listeria monocytogenes,
Aeromonas hydrophila, Yersinia enterocolitica on vacuum and saturated carbon
dioxide controlled atmosphere-packaged sliced roast beef. Journal of Food
Protection, 57(3), 204-208.

Hughner, R. S., McDonagh, P., Prothero, A., Shultz, C. J., & Stanton, J. (2007). Who are
organic food consumers? A compilation and review of why people purchase
organic food. Journal of consumer behavior, 6(23), 94-110

Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein
measurement with the Folin phenol reagent. Journal of biochemistry, 193(1), 265-
275

69
Murray P. R., Baron J. H., Pfaller M. A., Jorgensen J. H. & Yolken R. H., (Eds.) (2003).
Manual of Clinical Microbiology, 8th Ed., American Society for Microbiology,
Washington, D.C.

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.

Nychas, G. J. E., Skandamis, p., & Tassou, C. C. (2003). Antimicrobials from Herbs and
Spices. In: Natural Antimicrobials for the Minimal Processing of Foods, S. Roller
(Ed.), CRC Press, Woodhead Publishing Limited, Cambridge, UK pp. 176 200.

Olsen, S. J., MacKinnon, L. C., Goulding, J. S., Bean, N. H., & Slutsker, L., (2000)
MMWR CDC Surveillance Summary 2000; 49,1.

Raghubeer, E. V., & Matches, J. R. (1990). Temperature range for growth of Escherichia
coli serotype O157: H7 and selected coliforms in E. coli medium. Journal of
clinical microbiology, 28(4), 803-805.

Robinson, G. W. (1975). Isolation and characterization of papaya peptidase A from
commercial chymopapain. Biochemistry, 14(16), 3695-3700.

Sebranek, J. G., & Bacus, J. N. (2007). Cured meat products without direct addition of
nitrate or nitrite; what are the issues? Meat Science. 77,136-147.

Smid, E. J., & Gorris, L. G. M. (1999). Natural Antimicrobials for Food Preservation. In:
Handbook of Food Preservation, M.S. Rahman (Ed.), Marcel Dekker, New York,
NY, USA pp. 285308.

Ultee, A., & Smid, E. J. (2001). Influence of carvacrol on growth and toxin production
by Bacillus cereus. International Journal of Food Microbiology, 64, 373378.

Valero, M., & Salmeron, M. C. (2003). Antibacterial activity of 11 essential oils against
Bacillus cereus in tyndallized carrot broth. International Journal of Food
Microbiology, 85, 73-8
70

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)








71
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)










72
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)









73
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)












74
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.
75
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.
76
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
77
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 Agricultures 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 doesnt 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
78
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 seconddegree 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
79
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
80
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
81
were sterilized using 0.45 m syringe filter membrane (0.45 m 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 (04 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.
82
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 C. 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 m syringe filter (0.45 m 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 mol 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.
83
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 Fishers 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).
84
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 ( 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
85
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
86
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
87
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 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.

88
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
89
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 Arginine, Lysine and Phenylalanine (Menard et al., 1990).
Actinidin has a similar pattern of hydrolysis to that of papain. Actinidin also prefers
hydrophobic sites including Leucine, 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
90
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.

References
Aminlari, M., Shekarforoush, S. S., Gherisari, H. R., & Golestan, L. (2009). Effect of
actinidin on the protein solubility, water holding capacity, texture, electrophoretic
pattern of beef, and on the quality attributes of a sausage product. Journal of Food
Science, 74 (3), C221-6.

Banatvala, N., Griffin, M., & Greene, D. (2001). The United States national prospective
hemolytic uremic syndrome study: microbiologic, serologic, clinical, and
epidemiologic findings. Journal of Infectious Diseases, 183, 1063-70.

Basile, A., Vuotto, M., Violante, U., Sorbo, S., Martone, G., & Castaldo-Cobianchi, R.
(1997). Antibacterial activity in actinidia chinensis, feijoa sellowiana and aberia
caffra. International Journal of Antimicrobial Agents, 8, 199-203.

Benkerroum, N., Daoudi, A., & Kamal, M. (2003). Behaviour of Listeria monocytogenes
in raw sausages (merguez) in presence of a bacteriocin-producing lactococcal strain
as a protective culture. Meat Science, 63(4), 479-484.

Bloomfield, S. F. (1991). Methods for Assessing Antimicrobial Activity. In: Mechanisms
of Action of Chemical Biocides, Denyer, S. P., Hugo, W. B. (Eds.), Blackwell
Scientific Publications, Oxford, UK, pp. 1-22.

Boland, M., & Moughan, P. J. (2013). Nutritional Benefits of Kiwifruit. In: Kiwifruit
proteins and enzymes, Mike Boland (Ed). Academic Press. MA, USA. pp. 59 -74.
ISBN: 9780123942944

91
Brocklehurst, T. F., Mitchell, G.A., & Smith A.C. (1997) A model experimental gel-
surface for the growth of bacteria on foods. Food Microbiology, 14, 303-311.

Centers for Disease Control and Prevention (CDC) (2011). Estimates of Foodborne
Illness in the United States. Available at
http://www.cdc.gov/foodborneburden/index.html
Conner, D. E., & Beuchat, L. R. (1984). Effects of essential oils from plants on growth of
food spoilage yeasts. Journal of Food Science, 49, 429 434.
Cutter, C. N. (2000). Antimicrobial effect of herb extracts against Escherichia coli
O157:H7, Listeria monocytogenes, and Salmonella Typhimurium associated with
beef. Journal of Food Protection, 63, 601607.

Emeruwa, A. C. (1982) Antibacterial substance from Carica papaya fruit extract. Journal
of Natural Products. 45(2), 123-127.

Ensign, J. C., & Wolfe, R. S. (1966). Characterization of a small proteolytic enzyme
which lyses bacterial cell walls. Journal of bacteriology, 91(2), 524-534.

Eshamah, H., Han, I., Naas, H., Rieck, J. & Dawson, P. (2013). Bactericidal Effects of
Natural Tenderizing Enzymes on Escherichia coli and Listeria monocytogenes.
Journal of Food Research, 2(1), p 8.

Eyob, S., Martinsen, B. K., Tsegaye, A., Appelgren, M., & Skrede, G. (2008).
Antioxidant and Antimicrobial activities of Extract and Essential Oil of Korarima
(Aframomum corrorima (Braun) P. C. M. Jansen). African Journal of Biotechnology,
7, 2585-2592

92
Farbood, M. I., MacNei, l. J. H., & Ostovar, K. (1976). Effect of rosemary spice
extractive on growth of microorganisms in meats. Journal of Milk Food Technology,
39(10), 675-79.
FDA. (2012). Bad Bug Book. www.cfsan.fda.gov.

Foegeding, E. A., & Larick, D. K. (1986). Tenderization of beef with bacterial
collagenase. Meat Science, 19, 201214.

FSIS Salmonella Compliance Guidelines for Small and Very Small Meat and Poultry
Establishments that Produce Ready-to-Eat (RTE) Products. (2012). Available at:
http:// www.fsis.usda.gov/PDF/Salmonella_Comp_Guide_091912.pdf.

Ha, M., Bekhit, A. E-D., Carne, A., & Hopkins, D. L. (2012). Characterization of
commercial papain, bromelain, actinidin and zingibain protease preparations and
their activities toward meat proteins. Food Chemistry, 134, 95-105.

Ha, M., Bekhit, A. E-D., Carne, A., & Hopkins, D.L. (2012). Characterization of
kiwifruit and asparagus enzyme extracts, and their activities toward meat proteins,
Food Chemistry. doi: http://dx.doi.org/ 10.1016/j.foodchem.2012.09.034

Hafezi, F., Rad, H. E., Naghibzadeh, B., Nouhi, A. H., & Naghibzadeh, G. (2010).
Actinidia deliciosa (kiwifruit), a new drug for enzymatic debridement of acute burn
wounds. Burns, 36, 352-355.

Hao, Y. Y., Brackett, R. E., & Doyle, M. P. (1998). Efficiency of plant extracts in
inhibiting Aeromonas hydrophila and Listeria monocytogenes in refrigerated, cooked
poultry. Food Microbiology. 15, 367- 378.
Hsieh, P. C., Mau J. L., & Huang S. H. (2001). Antimicrobial effect of various
combinations of plant extracts. Food Microbiology. 18, 35-43.
93

Jay, J. M. (1994). Microbiologa moderna de los alimentos. 3rd edicin Wayne State
University. S.A. Zaragoza: Editorial acribia, ISBN 84-200-0746-3.

Junttila, R., Niemala, I., & Hirn, J. (1988). Minimum growth temperature of Listeria
monocytogenes and non-haemolytic Listeria. Journal of Applied Bacteriology, 65,
321-327.

Katsaros, G., Katapodis, P., & Taoukis, P. (2009). Modeling the effect of temperature and
high hydrostatic pressure on the proteolytic activity of kiwi fruit juice. Journal of
Food Engineering, 94, 40-45.

Kaur, L., Rutherfurd, S. M., & Moughan, P. J., Drummond, L., & Boland, M. J. (2010).
Actinidin enhances protein digestion in the small intestine as assessed using an in
vitro digestion model. Journal of agricultural and food chemistry, 58(8), 5074-5080.

Ketnawa, S., & Rawdkuen, S. (2011). Application of bromelain extract for foods
tenderization. Food and Nutrition Sciences, 2, 393-401.

Koak, J. H., Kim, H. S., Choi, Y. J., Baik, M. Y., & Kim, B. Y. (2011). Characterization
of a protease from over-matured fruits and development of a tenderizer using an
optimization technique. Food Science and Biotechnology, 20, 485-490.

Kudva, I. T., Davis, M. A., Griffin, R. W., Garren, J., Murray, M., John, M., &
Calderwood, S. B. (2012). Polymorphic Amplified Typing Sequences and Pulsed-
Field Gel Electrophoresis Yield Comparable Results in the Strain Typing of a
Diverse Set of Bovine Escherichia coli O157: H7 Isolates. International journal of
microbiology, 2012.
94
Llerena-Suster, C. R., Priolo, N. S., & Morcelle, S. R. (2011). Sodium Tetrathionate
Effect on Papain Purification from Different Carica papaya Latex Crude
extracts. Preparative Biochemistry and Biotechnology, 41, 107-121

Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, J. R. (1951). Protein
measurement with the folin phenol reagent. Journal of Biological Chemistry, 193,
265-275

Mamboya, E. A. F. (2012). Papain, a plant enzyme of biological importance: A Review.
American Journal of Biochemistry and Biotechnology, 8(2), 99-104.

Martin, E., Griffis, C., Vaughn, K., O'Bryan, C., Friedly, E., Marcy, J., & Lary Jr. R.
(2009). Control of Listeria monocytogenes by lauric arginate on frankfurters
formulated with or without lactate/diacetate. Journal of Food Science. 74 (6), M237-
M241.

Menard, R., Khouri, H. E., Plouffe, C., Dupras, R., & Ripoll, D. (1990). A protein
engineering study of the role of aspartate 158 in the catalytic mechanism of papain.
Biochemistry, 29: 6706-6713. DOI: 10.1021/bi00480a021

Ming, C. C., Awang, B., Duduku, K., & Sing, H. (2002). Effects of Ionic and non-Ionic
surfactants on papain activity. Borneo Science, 12, 71-77.

Mohajeri, G., Masoudpour, H., Heidarpour, M., Khademi, E. F., Ghafghazi, S., Adibi, S.,
& Akbari, M. (2010). The effect of dressing with fresh kiwifruit on burn wound
healing. Surgery, 148, 963-968.

Naveena, B. M., Mendiratta, S. K., & Anjaneyulu, A. S. R. (2004). Tenderization of
muscle buffalo meat using plant proteases from Cucumis Trigonus Roxb (Kachri) and
Zingiber Of-cinale Roscoe (Ginger Rhizome). Meat Science, 68, 363369
95
Osato, J. A., Santiago, L. A., Remo, G. M., Cuadra, M. S., & Mori, A. (1993).
Antimicrobial and antioxidant activities of unripe papaya. Life Sciences, 53,1383-
1389

Pathania, A., McKee, S., Bilgili, S., & Singh, M. (2010). Antimicrobial activity of
commercial marinades against multiple strains of Salmonella spp. International
Journal of Food Microbiology, 139(3), 214-217.

Rangel, J., Sparling, P., Crowe, C., Griffin, M., & Swerdlow, L. (2005). Epidemiology of
Escherichia coli O157:H7 outbreaks, United States, 19822002. Emerging Infectious
Diseases, 11, 603-9

Robins, M. M., & Wilson, P. D. G. (1994) Food structure and microbial growth, Trends
Food Science and Technology, 5, 289-293.

Robinson, G. W. (1975). Isolation and characterization of papaya peptidase A from
commercial chymopapain. Biochemistry, 14(16), 3695-3700.

Sabah, R., Juneja, K., & Fung, C. (2004). Effect of spices and organic acids on the
growth of Clostridium perfringens during cooling of cooked ground beef. Journal of
Food Protection 67(9), 1840-1847.

Scallan, E., Hoekstra, R. M., Angulo, F.J., Tauxe, R.V., Widdowson, M. A., & Roy, R.L.
(2011). Foodborne illness acquired in the United Statesmajor pathogens. Emerging
Infectious Diseases, 17, 7-15.

Shelef, A. (1984). Antimicrobial effects of spices. Journal of Food Safety, 6, 29-44.
96
Shelef, L. A., Jyothi, E. K., & Bulgarellii, M. A. (2006). Growth of Enteropathogenic and
Spoilage Bacteria in Sage Containing Broth and Foods. Journal of Food Science,
49(3), 737-740.

Shrinithivihahshini, D. N., Sheelamary, M., Mahamuni, D., & Chithradevi, R. (2011).
Occurrence of Listeria monocytogenes in food and ready to eat food products
available in Tiruchirappalli, Tamil nadu, India.

Stecchini, M. L., Sarais, I., & Giavedoni, P. (1993). Effect of essential oils in a culture
medium and in cooked pork. Journal of Food Protection, 56, 406409.

Sullivan, G. A., & Calkins, C. R. (2010). Application of exogenous enzymes to beef
muscle of high and low-connective tissue. Meat Science, 85, 730734.

Tart, R. (2009). Plant proteins. In Ingredients in meat products: Properties, functionality
and applications. New York: Springer, pp. 111-129.

Uhart, M., Marks, N., & Ravishankar, S. (2006). Effect of spices on growth and survival
of Salmonella Typhimurium DT 104 in ground beef stored at 4 and 8
o
C. Journal of
Food Safety, 26(2), 115-25.

United Sates Department of Agriculture (USDA) (2006). Economic research service.
Economics of foodborne disease. Available at:
http://www.ers.usda.gov/Briefing/FoodborneDisease.

United States Department of Agriculture-Food Safety and Inspection Service (USDA-
FSIS) (2012). FSIS Salmonella Compliance Guidelines for Small and Very Small
Meat and Poultry Establishments that Produce Ready-to-Eat (RTE) Products.
Available at: http://www.fsis.usda.gov/PDF/Salmonella_Comp_Guide_091912.pdf

97
Volk WA, & Wheeler MF (1988). The Basic Microbiology. Erlangga. Jakarta, 1: 218.

Walker, J., & Stringer, f. (1987). Growth of Listeria monocytogenes and Aeromonas
hydrophila at chill temperatures. Journal of Applied Bacteriology, 63:R20.

Yousef, E., & Courtney, D. (2003). Basics of stress adaptation and implications in new
generation foods. In: Yousef, A. E., Juneja, V. K. (Eds.), Microbial Stress Adaptation
and Food Safety. CRC press, Boca Raton, FL, pp. 1-30






























98
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
(mg/ml)
6 h 24 h 6 h 24 h
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)







99






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
(mg/ml)

1 h 3 h 1 h 3 h
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)













100










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
(mg/ml)

6 h 24 h 6 h 24 h
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)













101










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
(mg/ml)
1 h 3 h 1 h 3 h
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)







102





Table 5.5
Total and specific activity of actinidin and papain measured by spectrophotometer
Concentration
(mg solid/ml)
Protein
a
(mg / ml) Activity (units / ml)
b
Unit activity / mg
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 1mol of
tyrosine per minute at 660 nm/min at pH 7.5 at 37 C for 10 minutes, (n=6).








103
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)

104
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)



105
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
106
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.
107
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.
















108
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 100C. 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.

109
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 m 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 121C for 15 minutes

110
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 = 11,500 g/ml)
Vial
Volume of Diluent
(D.W)
Volume and Source of BSA Final BSA conc.
1 150 l 750 l of Stock 1,500 g/ml
2 625 l 625 l of Stock 1,000 g/ml
3 310 l 310 l of vial 1 dilution 750 g/ml
4 625 l 625 l of vial 2 dilution 500 g/ml
5 625 l 625 l of vial 4 dilution 250 g/ml
6 625 l 625 l of vial 5 dilution 125 g/ml
7 800 l 200 l of vial 6 dilution 25 g/ml
8 800 l 200 l of vial 7 dilution 5 g/ml
9 800 l 200 l of vial 8 dilution 1 g /ml
10 1000 l 0 0 g/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.
111
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 tubes 10-minute incubation period, add 100 l 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 g/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

112

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






113

Enzyme activity assay
Reagents preparation:
1. 50 mM Potassium Phosphate buffer, pH 7.5 at 37C (Buffer)
Prepare 11.4 mg/mL in purified water using Potassium Phosphate, Dibasic,
Trihydrate. Adjust to pH 7.5 at 37C 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 80-85C for approximately 10 minutes until a homogeneous
dispersion is achieved. Do not boil.
Adjust the pH to 7.5 at 37C, 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 & Ciocalteus Phenol Reagent (F-C)
Prepare a 1:4 dilution of 2 M Folin & Ciocalteus 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 37C
(Enzyme Diluent)
114
Prepare 1.4 mg/mL Sodium Acetate, Trihydrate, and 0.8 mg/mL Calcium Acetate
in purified water. Adjust the pHto 7.5 at 37C 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 m 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
2
CO
3
.

115



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.
solution
0.05 0.1 0.2 0.4 0.5 0
Purified
water
1.95 1.9 1.8 1.6 1.5 2
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 37C for 30 minutes.
Remove the vials and allow it cool to room temperature.
6. Filter each Blank, Standard, and Test using a 0.45 m syringe filter into suitable
cuvettes.
7. Record the A660nm of each Test, Standard, and Blank solution.







116
Results & calculations


Figure A.2 Enzymes activity standard curve



Sample Determination
For protease:
A660nm (Test) = A660nm (Test) - A660nm (Test Blank)
= 1.667- 0.201 = 1.466
Units/mL enzyme = (mole 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
y = 2.404x - 0.0112
R = 0.9995
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 0.1 0.2 0.3 0.4 0.5 0.6

A

6
6
0
n
m
umoles of Tyrosine
Standard curve
117
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 OD600 (or optical density at
600 nm). This value can then be converted to a useful concentration value using the standard
conversion factor where 1 OD600 = 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
118
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





119
Figure A.3: Growth curve of E. coli O157:H7 380-94








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







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