antimicrobial activity and preliminary characterization of peptides produced by lactic acid bacteria isolated from some vietnamese fermented foods

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE

PHAM THI DIU

ANTIMICROBIAL ACTIVITY AND PRELIMINARY
CHARACTERIZATION OF PEPTIDES PRODUCED BY
LACTIC ACID BACTERIA ISOLATED FROM SOME
VIETNAMESE FERMENTED FOODS
Program:
Code:
Supervisor:

Food technology
Master
Dr. Nguyen Hoang Anh

AGRICULTURAL UNIVERSITY PRESS - 2016


COMMITMENTS
I assure that the data and the research results in this thesis are true. They have
not been used. And, I assure that all the helps in this thesis have been acknowledged and
information used in the thesis has been cited the sources.
Ha Noi, day

month

Master candidate

Pham Thi Diu

i

year 2016


ACKNOWLEDGEMENTS
During this thesis, I learned many useful experiences in laboratory. I would
like to express my deepest appreciation to all those who help me to complete
thesis.
First of all, I would like to express the deep gratitude to Dr. Nguyen Hoang Anh
who helped and supported me to complete my thesis.
I also thank Dr. Nguyen Thi Thanh Thuy and my colleagues from faculty of
Food Science and Technology who provided insight and expertise that greatly assisted
the research.
Finally, I thank very much to my friends, my family who supported me in
this time.
Ha Noi, day

month

Master candidate

Pham Thi Diu

ii

year 2016


TABLE OF CONTENTS

Commitments ..................................................................................................................... i
Acknowledgements ........................................................................................................... ii
Table of contents .............................................................................................................. iii
List of tables ...................................................................................................................... v
List of figures ................................................................................................................... vi
List of diagram ................................................................................................................. vi
Abbreviation & acronyms ............................................................................................... vii
Chapter i. Introduction................................................................................................... 1
1.1.

Introduction ........................................................................................................... 1

1.2.

Objective of research ............................................................................................ 2

Chapter ii. Literature review ......................................................................................... 3
2.1.

Lactic acid bacteria ............................................................................................... 3

2.1.1. Lactic acid bacteria ............................................................................................... 3
2.1.2. Bacteriocins of lactic acid bacteria ....................................................................... 4
2.1.3. Application of bacteriocin .................................................................................... 7
2.2.

Popular pathogenic bacteria contaminated in food ............................................... 8

2.2.1. Listeria monocytogenes......................................................................................... 9
2.2.2. Bacillus cereus .................................................................................................... 10

2.2.3. Salmonella spp. ................................................................................................... 11
2.2.4. Escherichia coli................................................................................................... 12
2.3.

Some vietnamese traditional fermented foods .................................................... 13

2.3.1. Fermented chili sauce.......................................................................................... 13
2.3.2. Vietnamese traditional fermented meat (nem chua) ........................................... 14
2.3.3. Fermented meat ................................................................................................... 14
2.3.4. Cassava leaf and bamboo pickled ....................................................................... 15
Chapter iii. Materials and research methodology...................................................... 16
3.1.

Material ............................................................................................................... 16

3.2.

Research methodogy ........................................................................................... 17

iii


3.2.1. Collection of samples .......................................................................................... 17
3.2.2. Isolation of lactic acid bacteria ........................................................................... 17
3.2.3. Storage of isolated bacteria ................................................................................. 17
3.2.4. Screening of Lactic acid bacteria ........................................................................ 17
3.2.5. Screening of antimicrobial activity of isolated lab strains .................................. 19
3.2.6. Effect of cultivation time on peptides production ........................................ 20
3.2.7. Characterization of crude bacteriocin ............................................................ 20
Chapter iv. Results and discussion .............................................................................. 22

4.1.

Isolation and identification of lactic acid bacteria .............................................. 22

4.2.

Screening of antimicrobial activity of lactic acid bacteria .................................. 23

4.3.

Effect of cultivation time on production peptides ............................................... 24

4.4.

Characterization of concentrated cell free supernatant ....................................... 26

4.4.1. Effect of Enzymes ............................................................................................... 26
4.4.2. Characterization of concentrated cell free supernatant ....................................... 26
Chapter v. Conclusions and recommendation ........................................................... 29
5.1.

Conclusions ......................................................................................................... 29

5.2.

Recomendations .................................................................................................. 29

References ...................................................................................................................... 31
Appendix ........................................................................................................................ 40

iv


LIST OF TABLES
Table 2.1. Popular bacteriocins produced by Lactobacilli.............................................. 7
Table 3.1. Materials, tools, chemical and equipment required for the whole
experiment.................................................................................................... 16
Table 3.2. Morphological and Biochemical characteristics of selected potential
strains producing Lactic acid ....................................................................... 18
Table 4.1. Characteristics of isolated LAB strains........................................................ 23
Table 4.2. Vietnamese fermented foods that were collected to isolate LAB ................ 24
Table 4.3. Antimicrobial activity of concentrated cell free supernatant of
FME1.7 and CS3.7 ....................................................................................... 24

v


LIST OF FIGURES
Figure 2.1. Structure of Nisin ......................................................................................... 5
Figure 2.2. Structure of Sakacin P.................................................................................. 6
Figure 2.3. Morphology of L. monocytogenes cell ...................................................... 10
Figure 2.4. Morphology of B. cereus cell .................................................................... 11
Figure 2.5. Morphology of Salmonella spp. cell ......................................................... 12
Figure 2.6. Morphology of Escherichia coli cell ......................................................... 13
Figure 2.7. Rice chili and Muong Khuong chili sauce ................................................. 13
Figure 2.8. Thanh Hoa pork roll ................................................................................... 14
Figure 2.9. Thit chua Thanh Son .................................................................................. 15
Figure 2.10. Cassava leaf and bamboo pickled .............................................................. 15
Table 3.1.

Materials, tools, chemical and equipment required for the whole
experiment ................................................................................................. 16

Table 3.2.

Morphological and Biochemical characteristics of selected potential
strains producing Lactic acid ..................................................................... 18

Figure 4.1.

Culture characteristics of selected strain on MRS medium added 1%
CaCO3......................................................................................................... 22

Figure 4.2. Anti- Bacillus and Salmonella activity of concentrated cell free
supernatant of FME1.7 (a) and CS3.7 (b) .................................................. 23
Figure 4.3.

Effect of cultivation time on antimicrobial activity of FME1.7 and
CS3.7 ................................................................................................................... 26

Figure 4.4.

Effect of papain enzyme ............................................................................... 26

Figure 4.5. Antimicrobial activity of concentrated crude bacteriocin of FME1.7
and CS3.7 to pathogenic bacteria with pH range of 2-9, (Original :
Crude peptides) .......................................................................................... 28

LIST OF DIAGRAM
Diagram 3.1. Procedure of Screening of isolates for antimicrobial activity................... 19

vi


ABBREVIATION & ACRONYMS

Abbreviation

Meaning

LAB

: Lactic Acid Bacteria

MRS

: DE MAN, ROGOSA and SHARPE

LB

: Luria Broth

OD

: Optical Density

vii


CHAPTER I. INTRODUCTION

1.1. INTRODUCTION
Food is essential for human being’s to live, and as a result, food safety has
received increased attention. Consumption of food contaminated with pathogens
may cause certain disease events when it is contaminated with a very low
infective dose. In addition, foods contaminated with antibiotic resistant bacteria
could be a major threat to public health as the antibiotic resistance determinants
can be transferred to other pathogenic bacteria that later on cause compromises in
the treatment of severe infections.
Recently, food safety has not only been an intractable problem in
developing countries like Vietnam, but also in many countries around the world.
The risk of pathogenic microorganism contamination is increasing in agricultural
products and food processing products. Undoubtedly, the major threat to food
safety is the emergence of pathogens such as Escherichia coli, Salmonella spp.,
Campylobacter

spp.,

Listeria

monocytogenes,

Clostridium

botulinum,

Clostridium perfringens, or Bacillus cereus, which have been considered to be
foodborne microorganisms (Castellano et al., 2008). There are several methods
used to prevent foods from pathogenic contamination, such as freezing and
thawing or using chemical substances. However, food quality is decreased in
terms of both nutrition and food safety when using those methods (Parada et al.,

2007). So, new approaches to controlling foodborne pathogens in food
processing and food preservation have been prompted. For the past two decades,
many studies have focused on the natural compounds produced by lactic acid
bacteria (LAB) to apply in food preservation as LAB have been, so far,
considered a food grade organism (Fricourt et al., 1994; Ogunbanwo et al., 2003;
Parada et al., 2007). Moreover, LAB produce antimicrobial substances, such as
acids, peptides, and hydrogen peroxide, among others, during their growth and
development, of which, peptides have been proven to be the main group to have
antimicrobial activity and to be safely applied in food preservation (Deegan et
al., 2006; Settanni and Corsetti, 2008). A great deal of evidence has been
reported that peptides produced by LAB have broad range capabilities against

1


pathogenic bacteria activity (Nomoto, 2005). In addition, peptides are safe and
stable in food processing and preservation, and are not deleterious to food.
Therefore, up to date, many studies on antimicrobial peptides from isolated lactic
acid bacteria with expectations for food preservation have been published.
However, peptides from these studies have narrow range antimicrobial
activity, and almost all of them against only gram-positive bacteria (Ivanova et
al., 1998). Meanwhile, many bacteria contaminating food are gram-negative
bacteria, such as E.coli and Salmonella spp. That is why this study aims to isolate
lactic acid bacteria from a selection of Vietnamese fermented foods, including
fermented vegetables, fermented milks, and fermented meats, to explore new
peptides with high ranges of antimicrobial activity and characterize the peptides
for further applications.
1.2. OBJECTIVE OF RESEARCH
 General

objective

of

research:

Antimicrobial

activity

and

characterization of peptides produced by lactic acid bacteria from some
Vietnamese fermented foods

 Specific objectives of this study are:
 Isolation and identification of lactic acid bacteria from some Vietnamese
fermented foods
 Antimicrobial activity screening of isolates
 Effect of cultivation time on peptides production
 Characterization of bacteriocin: effect of enzymes (proteolytic enzymes,
amylase), heat stability, pH sensitivity.

2


CHAPTER II. LITERATURE REVIEW
2.1. LACTIC ACID BACTERIA
2.1.1. Lactic acid bacteria
Lactic acid bacteria (LAB),a diverse group of Gram-positive bacteria,

have been characterized by some common morphological, metabolic and
physiological traits. They are anaerobic bacteria, non-sporulating, acid tolerant
and produce mainly lactic acid as an end product of carbohydrate fermentation.
LAB consist of a number of diverse genera which include both homofermenters
and heterofermenters based on the end product of their fermentation.The genera
of homofermenters such as Lactococcus, Streptococcus and Pediococcus produce
lactic acid as the major product of glucose fermentation. In contrast, the
heterofermenters produce a number of products besides lactic acid, such as
carbon dioxide, acetic acid, and ethanol from the fermentation of glucose, they
are the genus Leuconostoc and a subgroup of the genus Lactobacillus, the βbacteria (Jay, 1986; Kandler et al., 1986).
Lactic acid bacteria consist of a number of bacterial genera within the
phylum Firmicutes. Recent taxonomic studies showed that the LAB group
includes the following genera; Carnobacterium, Enterococcus, Lactobacillus,
Lactococcus,

Lactosphaera,

Leuconostoc,

Melissococcus,

Oenococcus,

Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella
(Ercolini et al., 2001; Holzapfel et al., 2001; Carr et al., 2002). Species of these
genera can be found in the gastrointestinal tract of man and animal as well as in
fermented food. LAB strains used as probiotics usually belong to species of the
genera Lactobacillus, Enterococcus or Bifi dobacterium.
Gram-positive bacteria belonging to the phylum Actinobacteria, such as
the genera Aerococcus, Microbacterium, Propionibacterium (Sneath and Holt,

2001) and Bifi dobacterium (Gibson and Fuller, 2000; Holzapfel et al., 2001)
also produce lactic acid. The core LAB genera Lactobacillus, Lactococcus,
Leuconostoc, Pediococcus and Streptococcus share a long history of safe usage
in the processing of fermented foods. The antimicrobial effects and safety of

3


LAB in food preservation are widely accepted (Vuyst and Leroy, 2007; Sit and
Vederas, 2008). Their preservative effect is mainly due to the production of lactic
acid and other organic acids which result in pH reduction (Daeschel, 1987).
Preservation is enhanced by the production of other antimicrobial compounds
including hydrogen peroxide, CO2, diacetyl, acetaldehyde, and bacteriocins
(Cintas et al., 2001).
2.1.2. Bacteriocins of lactic acid bacteria
Bacteriocins, peptides produced by gram- positive bacteria have been
proved as antimicrobial agents.

Many researches have been focusing on

Bacteriocin from LAB and applying in food preservation as LAB have been
strongly considered as food grade organism. The LAB bacteriocins have many
attractive characteristics that make them suitable candidates for use as food
preservatives, such as: 1) Protein nature, inactivation by proteolytic enzymes of
gastrointestinal tract, 2) Non-toxic to laboratory animals tested and generally
non-immunogenic, 3) Inactive against eukaryotic cells, 4) Generally thermo
resistant (can maintain antimicrobial activity after pasteurization and
sterilization), 5) Broad bactericidal activity affecting most of the gram-positive
bacteria and some, damaged, gram-negative bacteria including various
pathogens but usually only when the integrity of the outer membrane has been

compromised, for example after osmotic shock or low pH treatment, in the
presence of a detergent or chelating agent, 6) Genetic determinants generally
located in plasmid, which facilitates genetic manipulation to increase the
variety of natural peptide analogues with desirable characteristics (Juodeikiene
et al. 2012).
Bacteriocin peptide is much more stable than protein because the two
factors affect protein stability do not exist for synthetic peptides. These two
factors are the tertiary folding and proteinase contamination. Due to their short
length, most peptides do not have tertiary structure. The tertiary structure is
unstable because it is held together by non-covalent bonds such as electrostatic
interaction. Therefore, peptide can not be denatured. Under this assumption, a
peptide can only be damaged by covalent modification or break of peptide bonds.
Unlike protein purified from cells that are full of various proteinase, the chance

4


of proteinase contamination is extremely small for a synthetic peptide. Reactions
could damage peptide such as oxidation require high or low pH. They are very
slow under the neutral pH condition that most biological experiments are
performed. Bacteria contamination is probably a more serious threaten than those
reactions, because peptide is a good nutrition source for bacteria. Therefore,
solvent filtration is important for peptide stability.
LAB Bacteriocins are very diverse with different sizes, structures,
physicochemical properties, and inhibitory spectrum. These bacteriocins are
classified into three major classes, namely Lantibiotics, Non-Lantibiotics and
bacteriocins (José Luis Parada, 2007).
Class I: Lantibiotics
Lantibiotics are small (<5kDa) heat stable peptides, which are extensively
modified after translation with the formation of characteristic thioether amino

acids lanthionine and β- methyllanthionine. They are further devided into two
types based on structural similarities, A and B types. Type A comprises of
relatively elongated, screw shaped, positively charged, amphipatic, flexible
molecules. Their molecular mass varies between 2 to 4 kDa. Nisin is member of
type A bacteriocins. Type B lantibiotics, are globular in structure and interfere
with cellular enzymatic reactions. Their molecular mass, is between 2 to 3 kDa
and either they have no net charge or a net negative charge.

Figure 2.1. Structure of Nisin
Class II: The Non-Lantibiotics
Class II bacteriocins are also small (<10 kDa) relatively heat stable, non-

5


lanthionine containing membrane active peptides. Class IIa bacteriocins are
Listeria-active peptides as pediocin PA-1, sakacin P, arnobacteriocin X. Class IIb
bacteriocins require two different unmodified peptides such as lactacin F, ABP118. Members of class IIc are circular peptide bacteriocins including carnocyclin
A, enterocin AS-48…. Class IId bacteriocins are linear, non-pediocinlike, singlepeptide bacteriocins, including epidermicin NI01, lactococcin A.

Figure 2.2. Structure of Sakacin P
Class III: Bacteriocins
This group consists of heat labile proteins which are in general of large
molecular weight (>30 kDa). Class III can be further subdivided into two distinct
groups (A and B). Group A are lytic- bacteriocins while group B are non- lytic
proteins
Antimicrobial mechanism of LAB bacteriocins
Most of bacteriocins are membrane active compounds that increase the
permeability of the cytoplasmic membrane. Bacteriocins generally act through
pore formation, through membrane depolarization of the cytoplasmic membrane

of the sensitive target species. Group A of Class III bacteriocins killing the
sensitive strains by lysis of the cell wall while group B bacteriocins are non-lytic
proteins.

6


Table 2.1. Popular bacteriocins produced by Lactobacilli
Bacteriocin

Bacteriocin Producing Strain

Lactacin F
Lactocin 705
Lactoccin G
Lactococcin MN
Nisin
Leucocin H
Plantaricin EF, Plantaricin W Plantaricin JK,
Plantaricin S

L. johnsonii spp.
L. casei spp.
L. lactis spp.
Lactococcus lactis var cremoris
Lactococcus lactis spp.
Leuconostoc spp.
L. plantarum spp.

2.1.3. Application of bacteriocin

Bacteriocins are now widely used in food preservation to extend selflife of
food as they inhibit pathogen infection of animal diseases and pharmaceutical
industry and medical society to treatment for malignant cancer. Bacteriocins
produced by probiotic can reduce the number of pathogens or change the
composition of intestinal microbiota in animal models, such as nisin produced by
Lactococcus lactis strains.
In food technology, bacteriocins are used for food preservation in order
to extend shelf-life of food and get rid of pathogen contaminant. They are
added into foods to inhibit microbial growth and possible corruption. Unlike
antibiotics and chemical preservative, in gastrointestinal tract, bacteriocins are
harmless for consumer as they are hydrolyzed by protease into unfunctional
peptides (Shih-Chun Yang, 2014). Previous researches indicated that nisin A
reduces undesirable bacteria in meat products (Cutter et al., 1998) and inhibits
L. monocytogenes for 8 weeks (Davies et al., 1997). Lactocin 705 inhibits
growth of L. monocytogenes in ground beef (Vignolo et al., 1996). Lacticin
3147 against different species of Enterococcus, Leuconostoc, Pediococcus,
Streptococcus, L. monocytogenes, Listeria innocua, Bacillus spp. (Ryan et al.,
1996). Sakacin A and Sakacin P against different E.coli strains producing
toxin and causing diseases in animal. Antibiotic use reduced the number of
animal death from bacterial infection, however, antibiotic resistant pathogens
become increasingly serious because of the abuse of antibiotics. Bacteriocins
prevent pathogenic bacteria from binding to receptor of bacteria and cause

7


cytotoxicity. Mixture of purified colicin E1 and colicin N has againsted
enterotoxigenic E. coli pathogens which caused post-weaning diarrhea in
piglets (Stahl et al., 2004).
In cancer therapy, some researches indicated that bacteriocins against

tumor cells. Colicin E1 inhibits the growth of several human cell-cell lines, with
fibrosarcoma HS913T being the most sensitive while the standard fibroblast
MRC5 cells show a reduced sensitivity. On the other hand, colicin A shows a
stronger inhibitory effect on this standard cell line. This bacteriocin also has
inhibitory effect on the leiomyosarcoma cells SKUT-1. (Chumchalová and
Smarda et al., 2003).
Bacteriocins from LAB are bioactive peptides or proteins with
antimicrobial activity toward Gram positive bacteria, including closely related
strains and/or spoilage and pathogenic bacteria (Tagg et al., 1976). Bacteriocins
are ribosomaly synthesized and extracellulary released bioactive peptides or
peptide complexes which have bactericidal or bacteriostatic effect (Garneau et
al., 2002). Use of either the bacteriocins or the bacteriocin-producing LAB like
starter cultures for food preservation has received a special attention (Sabia et al.,
2002). Moreover, bacteriocins are innocuous due to proteolytic degradation in
the gastrointestinal tract (Cintas et al., 1995), S. thermophilus is a lactic acid
bacterium of major importance in food industry like the manufacture of yoghourt
(Purwandari et al, 2007). Some of S. thermophilus strains produce a bacteriocin
named thermophilin which is active against several LAB and food spoilage
bacteria such as Clostridium sporogenes. In view of its technological and
biochemical properties the above bacteriocin can be considered as a potential
bioprerservative (Aktypis et al., 2007). Some of other LAB like Enterococcus,
Lactococcus, and Pediococcus are also widely used as natural preservatives, due
to the potential production of metabolites with antimicrobial activity such as
organic acids, hydrogen peroxide, antimicrobial enzymes and bacteriocins
(Mataragas et al., 2003).
2.2. POPULAR PATHOGENIC BACTERIA CONTAMINATED IN FOOD
Toxin produced by pathogenic bacteria in food during their growth cause
consumer

illness.

Of

particular

concern

8

are

Listeria

monocytogenes


(L.monocytogenes), Escherichia coli (E.coli), Salmonella spp., Bacillus cereus
(B. cereus). These pathogenic bacteria can be contaminated in to food through
raw materials or during steps of food processing. They are available from the air,
unclean hands, insanitary utensils and equipment, contaminated water, or sewage
and through cross-contamination between raw and cooked product.
2.2.1. Listeria monocytogenes
General characteristics: Listeria monocytogenes is a Gram-positive
bacterium, nonspore-forming, motile, rod-shaped bacterium. It is catalasepositive and oxidase-negative. It belongs to the genus Listeria along with L.
ivanovii, L. innocua, L. welshimeri, L. selligeri and L. grayi (Rocourt et al.,
2007). L. monocytogenes can grow under both aerobic and anaerobic conditions,
although it grows better in an anaerobic environment (Sutherland et al., 2003). In
food, the growth and survival of L. monocytogenes is influenced by many factors
including temperature, pH, water activity, salt and preservatives. The temperature
range for growth of L. monocytogenes is between -1.5 and 45°C and the optimal

temperature is 30–37°C. Temperatures above 50°C are lethal to L.
monocytogenes. Freezing can also lead to a reduction in L. monocytogenes
numbers (Lado et al., 2007). L. monocytogenes grows in a broad pH range of
4.0–9.6 (Lado et al., 2007). It becomes more sensitive to acidic conditions at
higher temperatures. Like most bacterial species, L. monocytogenes grows
optimally at a water activity (aw) of 0.97. However, L. monocytogenes also has
the ability to grow at aw of 0.91. (Farber et al., 1992) demonstrated that L.
monocytogenes is reasonably tolerant to salt. It can grow in 13–14% sodium
chloride. But, survival in the presence of salt is effected by the storage
temperature. The survival rate of L. monocytogenes is higher when the
temperature is lower (Lado et al., 2007). Some of the preservatives can inactivate
L. monocytogenes as lysozyme (100 mg/kg), 0.2% sodium benzoate at pH 5,
0.25- 0.3% sodium propionate ), and 0.2-0.3% potassium sorbate (pH 5.0).
Transmission: L. monocytogenes is widespread in the environment
including soil, vegetatable, water and sewage. The most common transmission of
L. monocytogenes to humans is via the consumption of contaminated food during
additional handling steps such as peeling, slicing and repackaging. In addition, L.

9


monocytogenes can be transmitted directly from mother to child, from contact
with animals and through hospital acquired infections (Bell et al., 2005). L.
monocytogenes causes Listeriosis with symptoms of fever, stiff neck, confusion,
weakness, vomiting, and diarrhea. It severely affects pregnant women, babies,
and people by reducing immunity. L. monocytogenes was found in milk products
(eg. cheese, butter…), egg, and seafood.

Figure 2.3. Morphology of L. monocytogenes cell
2.2.2. Bacillus cereus

General characteristics: Bacillus. cereus is a Gram-positive, motile, sporeforming, rod shaped bacterium that belongs to the Bacillus genus. B. cereusis
widespread in nature and readily found in soil, where it adopts a saprophytic life
cycle; germinating, growing and sporulation in this environment (Vilain et al.,
2006). B. cereus produces two types of toxins: emetic (in food) and diarrhoeal (in
small intestine). The temperature range for growth of B. cereus is from 40oC to
55oC and the optimal temperature is 30–40°C. The broad pH of B. cereus is
between 4.9-10 and the optimal pH is 6.0- 7.0. B. cereus can grow at a water
activity (aw) of 0.93- 0.99. The maximum salt concentration tolerated by B.
cereus for growth is 7.5% (Rajkowski et al., 2003). B. cereus growth is optimal
in the presence of oxygen, however they can grow under anaerobic conditions. B.
cereus cells grown under aerobic conditions are less resistant to heat and acid
than under anaerobic condition (Mols et al., 2009). The growth and survival of B.
cereus effected by preservatives. Nisin was used to inhibit the germination and
outgrowth of spores. In addition, Jenson et al., 2003 indicated that some

10


antimicrobials including benzoate, sorbates and ethylene diamine tetra-acetic
acid inhibited the growth of B. cereus ()
Transmission: B. cereus causes diarrhea and emetic during consumption of
contaminated foods, improper food handling/storage and improper cooling of
cooked foodstuffs (Schneider et al., 2004).

Figure 2.4. Morphology of B. cereus cell
2.2.3. Salmonella spp.
General characteristics: Salmonella spp. are Gram-negative, non-spore
forming rod-shaped bacteria and are members of the family Enterobacteriaceae
(Jay et al., 2003). It is found worldwide in both cold-blooded and warm-blooded
animals, and in the environment. Salmonella spp. have relatively simple

nutritional requirements. However, the growth and survival of Salmonella spp. is
influenced by some factors as temperature, pH, water activity, preservatives. The
temperature for growth of Salmonella spp. is 5.2–46.2° and the optimal
temperature is 35–43°C. Salmonella spp. can survive long term frozen storage at
low temperature (Jay et al., 2003). The broad pH range of Salmonella spp. is
from 3.8 to 9.5, an optimum pH range for growth of 7–7.5. The optimum aw for
growth is 0.99 and the lower limit is 0.93. Growth of Salmonella spp. can be
inhibited by benzoic acid, sorbic acid or propionic acid. Salmonella spp. are
classed as facultative anaerobic organisms as they do not require oxygen for
growth (Jay et al., 2003).
Transmission: Salmonella spp. are transmitted by the faecal-oral route by
either consumption of contaminated food or water, person-to-person contact, or

11


from direct contact with infected animals (Jay et al., 2003). The incubation
period of Salmonella spp. is 8–72 hours (usually 24–48 hours) and symptoms last
for 2–7 days (Darby and Sheorey, 2008). After infection, it causes disease in
gastrointestinal tract with symptoms as abdominal cramps, nausea, diarrhoea,
mild fever, vomiting, and dehydration.

Figure 2.5. Morphology of Salmonella spp. cell
2.2.4. Escherichia coli
General

characteristics:

Escherichia

coli

is

a gram-

negative, anaerobic, rod-shaped bacterium of the genus Escherichia. It lives in the
intestines of healthy people and animals, cattle. Most strains of this bacteria are
harmless. Escherichia coli can survive well in chilled and frozen foods, in low
pH (down to 3.6) conditions. The optimal temperature of E. coli is 37°C. The
broad pH range of is from 4.4 to 9.0 and optimal pH is 6-7. The optimum aw for
growth of E. coli is 0.995. The presence of preservatives can inhibit growth of E.
coli as sodium benzoate, potassium sorbate, and eugenol.
Transmission: Diarrhea genic pathotypes can be passed in the feces of
humans and other animals. Transmission of E. coli occurs through the fecal-oral
route, primarily via contaminated food or water. Transmission also occurs through
person-to-person contact, as well as contact with animals or their environment.
Although some animals may carry non-STEC diarrhea genic E. coli, people
constitute the main reservoir for strains causing diarrhea in humans. The intestinal
tracts of animals, especially cattle and other ruminants, are the primary reservoirs
of STEC.

12


Figure 2.6. Morphology of Escherichia coli cell
2.3. SOME VIETNAMESE TRADITIONAL FERMENTED FOODS
2.3.1. Fermented chili sauce
Muong Khuong chili sauce shaped like a thick sauce, fresh red color,
spicy tasty and aroma is made from rice chili, which are grown in the

mountainous district of Muong Khuong, Lao Cai and other special spices.

Figure 2.7. Rice chili and Muong Khuong chili sauce

13


Chili sauce is produced as follows:
Chilies are washed, drained and then put in the blender, crushed chilies
with garlic. Ingredients such as coriander seed, fennel seed, seed teams,
cardamom seeds have been roasted to make a distinct aroma. Then, individually
minced ingredients, then mix into the bucket chili was pureed with wine and
water mixed with salt.
2.3.2. Vietnamese traditional fermented meat (nem chua)
Nem chua is well known and most favorite fermented pork product. Nem
chua is made from a series of ingredients, namely ground pork thigh, minced
pork skin, chili, garlic, fish sauce, sugar, salt, those are mixed, pressed and then
naturally fermented by tender Figure or guava leaves.

Figure 2.8. Thanh Hoa pork roll
2.3.3. Fermented meat
Thit chua is the food specialty of Muong ethnic in Xuan Son, Thanh Son,
Phu Tho. Mán pig is used as raw materials for processing which is grilled by
using straw. Then, the best area of pig meat such as bacon, beef rump, lean
shoulders are brought sliced, marinated in a little seasoning salt, mix well with
flour of roast maize, beans. And the bamboo shoot is washed, dried, lined guava
leaves, leaf pulse down and let the meat was marinated with flour of roast maize;
cover guava leaf layer on the surface and tightly knot the tube. Then drape where
tall, airy.

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Figure 2.9. Thit chua Thanh Son
2.3.4. Cassava leaf and bamboo pickled
Cassava leaf and bamboo pickled is very close with Muong ethnic in Hoa
Binh. Young cassava leaves are collected, washed with mineral water and then
washed off with rain water. The leaves are dried, out of resin then put on a jar,
each turn leaves is added moderate salt (somewhere not add salt), then the jar is
poured by rain water and covered for fermentation

Figure 2.10. Cassava leaf and bamboo pickled
Bamboo shoots are peeled away the hairy coating, washed, sliced,
blanched in hot water to remove the bitter and acrid taste. Chili is washed, lightly
crushed. Garlic is removed bark and sliced. Shoots, garlic, chili are mixed in jars,
poured the vinegar and added a little salt and sugar and finally the jar is covered
for fermentation.

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CHAPTER III. MATERIALS
AND RESEARCH METHODOLOGY
3.1. MATERIAL
 Twenty-two samples of chili sauce, fermented meat, fermented
eggplant, fermented milk, fermented bamboo shoot, fermented cassava leaf
collected from different areas of the northern part of Vietnam were used to
isolate LAB.
 E. coli, B. cereus, L. monocytogenes, and Salmonella spp. supplied by
the Faculty of Veterinary Medicine, Vietnam National University of Agriculture

and Lactobacillus plantarum were chosen as pathogen indicators for
antimicrobial activity testing.
 Other materials, tools, equipment and chemicals required for the whole
study were mentioned in table 3.1.
Table 3.1. Materials, tools, chemical and equipment required for the whole
experiment
Work
Isolation of LAB
from
some
Vietnamese
fermented food

Tools and equipment
- Alcohol flame
- Autoclave
- Petri disk, needle - Fridge (40C)
- Biosafety Cabinet - Freeze (-350C)
- Incubator

Antimicrobial
activity
characterization

- Petri disk, needle - Incubator
- Alcohol flame
- Autoclave
- Biosafety Cabinet - Rotary evaporator

Effect

of
cultivation time on
peptides
production

- Petri disk, needle
- Alcohol flame
- Biosafety Cabinet
- Incubator

- Autoclave
- Rotary evaporator
Spectrophotometer

Characterization of
crude peptides

- Petri disk, needle
- Alcohol flame
- Biosafety Cabinet

- Incubator
- Autoclave
- Rotary evaporator

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Medium and Chemical
- MRS agar, broth
medium

- LB agar, broth medium
- NaOH 1N
- CaCO3
- MRS agar, broth
medium
- LB agar, broth medium
- NaOH 1N
- Indicator bacteria
MRS
agar,
broth
medium
- LB agar, broth medium
- NaOH 1N
MRS
agar,
broth
medium
- LB agar, broth medium
- NaOH 1N
-Phosphat buffer(pH 2-9)
- Enzymes: Papain, αAmylase


3.2. RESEARCH METHODOGY
3.2.1. Collection of samples
Twenty-two samples were collected as representative in each region.
After collection, samples were stored on ice for further studies (Farhana S. Diba
et al., 2013)
3.2.2. Isolation of lactic acid bacteria

Isolation of LAB was described by Yi-sheng Chen (2010). After crushing,
samples were diluted to a 10-1 - 10-6 concentration by mixing with sterilized
water. 100 µl sample of diluted solution was spread directly onto the surface of
MRS agar plates with 1% CaCO3. Dilutions of the mixed solution( 0.1ml) were
spread directly onto the surface of MRS agar plates. Samples were incubated
under anaerobic conditions at 37°C for 24 hours. Colonies of LAB appear as
large, white colonies embedded in or on MRS agar and form a clear zone around
each colony. These colonies were randomly selected from MRS-agar plates and
purified by replating on MRS-agar plates. After carrying out tests, only Grampositive, catalase negative strains were selected.
3.2.3. Storage of isolated bacteria
Bacteria were stored by two ways which were described by Lan Dung
Nguyen, 1978 as follow:
 Bacteria was cultured in MRS agar tubes. These tubes were incubated
o

at 37 C, 24hrs. Then, cultured tests were stored at 4oC
 Purified LAB was stored with glycerol. Cultured suspension was
added glycerol of 30% with ratio 1:1 and 1ml of cultured suspension with
glycerol was stored at -80oC.
3.2.4. Screening of Lactic acid bacteria
Screening of LAB was described by Barnali Ashe, 2010. The colonies
showing a transparent zone were selected, due to LAB produce lactic acid which
reduce CaCO3 in medium. Then, they were identified following morphological
and biochemical characterization as summarized in table 3.2.

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