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 VIETNAME

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: Food technology Code: Master

Supervisor: 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

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 year 2016

Master candidate

Pham Thi Diu

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

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

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

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

ABBREVIATION & ACRONYMS

Abbreviation Meaning

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

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

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 β-bacteria (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

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

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-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, ABP-

118 Members of class IIc are circular peptide bacteriocins including carnocyclin

A, enterocin AS-48… Class IId bacteriocins are linear, non-pediocinlike, peptide bacteriocins, including epidermicin NI01, lactococcin A

single-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

Table 2.1 Popular bacteriocins produced by Lactobacilli

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

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 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 catalase-positive 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

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, forming, 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

spore-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

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

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 anegative,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

gram-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

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

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 2.9 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

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

- LB agar, broth medium

- LB agar, broth medium

- Enzymes: Papain,

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 Gram-positive, 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

at 37oC, 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

Table 3.2 Morphological and Biochemical characteristics of selected

potential strains producing Lactic acid

Morphological

+ve: positive ; -ve: negative

Morphology colony

Through observation, these colonies having morphological colony as mentioned table 3.2 were selected for further studies

Gram staining

Protocol used to identify gram of bacteria is described as below

Step 1: Prepare a slide with the culture by transferring the specimen to be examined onto a drop of distilled water Spread the specimen on the slide

Step 2: Fix the culture by heating the slide over an alcohol burner to evaporate the water

Step 3: Drop a few drops of crystal violet strain onto the fixed culture and let it stand for 20 seconds Then, pour off the crystal violet strain and gently rinse the excess strain with distilled water

Step 4: Drop a few drops ofiodinesolution on the smear and let it stand for 20 seconds Then pour off the iodine solution and rinse the slide with distilled water

Step 5: Decolorize using 95% ethyl alcohol Then, rinse the slide off with distilled water after 5 seconds

Step 6: Drop a few drops of fuchsine on the slide and let it sit for 20 seconds, then wash off the solution with distilled water

Step 7: View the smear using a light-microscope

Motility

LAB is no- moving bacteria Motility of LAB was checked by streaking bacteria in surface of suitable agar medium dish and then incubate for 24hrs, at

370C Finally, determination of results Only colonies grown on streak are selected

Catalase test

Reaction between catalase enzyme and H2O2 create O2 (oxygen) and effervesce However, LAB don’t produce catalase enzyme So, only the isolated colonies with negative catalase are accepted Protocol was showed as below:

Step1: Transferring colony into a slide

Step 2: Drop a few 3% H2O2 on colony and stir

Step 3: Observe results Negative catalase results were accepted

3.2.5 Screening of antimicrobial activity of isolated lab strains

The antibacterial activity of isolated LAB was performed by diffusion method which described by Al-Allaf (2009) It was summarized in below diagram

well-Diagram 3.1 Procedure of Screening of isolates for antimicrobial activity

3.2.6 Effect of cultivation time on peptides production

Isolated LAB were cultured in 1000 ml of MRS broth at 37oC Every

2 hours, 100 ml of culture medium was taken out to centrifuge at 4oC, 6000 rpm for 15 min Cell free supernatant was concentrated 3 times by rotary evaporator at 35oC, 70 rpm for 45 min before testing antimicrobial activity

by the well diffusion method

3.2.7 Characterization of crude bacteriocin

Characterization of crude bacteriocin was carried out according to method of Vinod Kumar Joshi (2006) after concentrating bacteriocin using rotary evaporator

Six hundred milliliter of cell free supernatant in container of rotary evaporator was concentrated 3 times at 35oC, 70 rpm for 45 min 200ml of concentrated cell free supernatant was used for characterization

3.2.7.1 Sensitivity of cell free supernatant to enzymes

The effect of proteolytic enzymes on the concentrated cell free supernatant as described by Joshi et al., (2006) was applied to show that the peptides of LAB are agents of antimicrobial activity First, the concentrated cell free supernatant was adjusted to pH 6.5 Second, 5ml of concentrated cell free supernatant was taken in test tubes and treated with papain at a final concentration of 1mg/ml, pH= 7

The test tubes with and without the enzymes (control) were incubated for 2 hours at 37 oC and then heated for 3 min at 100oC to inactivate the enzymes Both the control and the samples were assayed for antimicrobial activity using the well diffusion method

3.2.7.2 Heat stability of bacteriocin

A volume of 5 ml of concentrated crude peptides in different test tubes were overlaid with paraffin oil to prevent evaporation and then heated

at 68oC and 100oC for 10 and 20 min each, and at 121oC for 15 min under pressure The heat-treated samples were then assayed for antimicrobial activity as described previously