i

ANTIMICROBIALS FROM Lactobacillus plantarum ISOLATED

FROM TURMERIC (Curcuma longa linn.) AND THEIR

APPLICATIONS AS BIOPRESERVATIVE AND IN EDIBLE FILM

by

Melada Supakijnoraset

A thesis submitted in partial fulfillment of the requirements for the

degree of Master of Engineering

Examination Committee : Dr. Anil K. Anal (Chairperson)

Prof. Athapol Noomhorm

Prof. Sudip K. Rakshit

Nationality : Thai

Previous Degree : Bachelor of Science in Biotechnology

King Mongkut’s Institute of Technology Ladkrabang

Bangkok, Thailand

Scholarship Donor : RTG Fellowship

Asian Institute of Technology

School of Environment, Resources and Development

Thailand

May 2011

ii

ACKNOWLEDGEMENT

The author would like to hearty thank to her advisor, Dr.Anil Kumar Anil for his

very enthusiastic guidance, timely consulting feedback and encouragement for completion

of this thesis with great success. Hearty appreciation and thankful to the thesis committee

members, Prof. Athapol Noomhorm and Prof. Sudip Kumar Rakshit for their valuable

guidance, suggestions and advice regarding to the author’s thesis.

The author would like to sincere thanks to Mr. Krirkpong, Ms. Plangpin and Ms.

Deepika for their generous support and providing the microorganism and raw materials and

sincere thanks to Mr.Ong-Ard for providing the chemical preservative.

Sincere thanks to the FEBT laboratory supervisor, Mr. Imran Ahmad for his kind

help, keen support and guidance about the instrumentations. Furthermore, the author would

also like to thank to Mr. Songkla, the FEBT laboratory technician who facilitate and keep

the knowledge and generous advice on the use of various instruments during operation and

to Ms.Tasana, FEBT senior Administrative officer for her support.

Most importantly, heartfelt thanks to my family who always encourage and support

in all every aspects and made this project successfully complete very well.

The author would like to thank sincerely to Royal Thai Government (RTG) and the

Asian Institute of Technology (AIT) for the fellowship to pursue the Master degree.

Finally, the author would also like to express her gratitude to all the friends in those

helped and encourages her to complete this thesis work.

iii

ABSTRACT

The antimicrobial is a substance that kills or inhibits the growth of microorganism.

The antimicrobial substance can be organic acid, hydrogen peroxide, carbon dioxide,

diacetyl and bacteriocin.

Lactobacillus plantarum (L. plantarum) was isolated from fresh turmeric rhizome

(Curcuma longa Linn.). The antimicrobial substances from Lactobacillus plantarum and

Lactobacillus casei TISTR 1463 (L. casei) showed significant antibacterial activities

against Escherichia coli, Salmonella typhimurium and Staphylococcus aureus. The

bacteriocin of L. plantarum was purified by ammonium sulphate precipitation followed by dialysis

through 1000 molecular weight-cut-off-dialysed membrane. The activity of bacteriocin from L.

plantarum is 5,120 AU/ml, and was not affected when treated at 95°C for 10 min, and remained

stable at pH 5 and pH 7. The molecular weight of bacteriocin from L. plantarum is approximately

1-2 kDa and 12-14 kDa. The bacteriocin from both strains was able to inhibit the growth of

indicator strains. L. plantarum and L. casei produced the maximum bacteriocin at 37 °C

and 30 °C, respectively. The bacteriocin activity from L. plantarum was found efficient

than from L. casei TISTR 1463.

The unpurified extracts and partially purified bacteriocin from L. plantarum were

incorporated with cassava starch film in the form of edible packaging. The surface of all

types of edible films based on the cassava starch was rough and with small ridges all over

the film. Addition of extracts from L. plantarum into the film significantly (p˂0.05)

reduced tensile strength and increased elongation at break. On the other hand, addition of

partially purified bacteriocin had not shown any changes in tensile strength and elongation

at break as like of cassava starch film. In case of water vapor transmission (WVT) and

water vapor transmission rate (WVTR), the addition of antimicrobial substance and

bacteriocin into the cassava starch film increase significantly (p<0.05) WVT and WVTR.

The various types of cassava starch based film were applied on the fresh fermented pork

product. The result shows that the cassava starch film incorporated with the whole extracts

could inhibit the growth of microorganism. However the cassava starch based film can be

applied on the products for the short period because of the barrier properties problem. For

the application, the bacteriocin from L. plantarum has ability to prolong the shelf-life of

product. In case of pasteurized orange juice and pasteurized milk, there were not observed

the growth of microorganism during period of storage on both products when incorporated

with bacteriocin. On the other hand, for the control of both pasteurized orange juice and

pasteurized milk, there were observed the growth of microorganism during period of

storage.

iv

Table of Contents

CHAPTER TITLE PAGE

Title Page i

Acknowledgements ii

Abstract iii

Table of Contents iv

List of Tables vi

List of Figures vii

List of Abbreviations viii

1 Introduction 1

1.1 General background 1

1.2 Statement of problem 2

1.3 Scope of study 2

1.4 Objective 2

2 Literature Review 3

2.1 Probiotics 3

2.2 Turmeric 3

2.3 Identification of probiotics 3

2.4 Antimicrobial components from lactic acid bacteria 5

2.5 Classification of bacteriocins 7

2.6 Edible packaging 12

2.7 Film composition 13

2.8 Antimicrobial packaging 15

3 Materials and Methods 21

3.1 Materials 21

3.2 Chemicals 21

3.3 Equipments 22

3.4 Methodology 24

4 Result and Discussion 36

4.1 Isolation and identification of Lactic acid bacteria (LAB) 36

From turmeric rhizome

4.2 Growth of L. plantarum and L. casei TISTR 1463 37

4.3 Antimicrobial and bacteriocin activities 38

4.4 Purification of bacteriocin 39

4.5 Effect of temperature and pH on bacteriocin activity 40

4.6 Molecular mass determination of bacteriocins 42

4.7 The physical properties of edible film based on cassava starch 43

v

4 4.8 Tensile strength and Elongation at break of edible film 46

based on cassava starch

4.9 Water vapor transmission and water vapor transmission rate 49

of edible film based on cassava starch

4.10 Effect of antimicrobial of edible film based on cassava starch 51

on bacteria strains

4.11 Effect of antimicrobial packaging in fermented pork 52

4.12 Effect of bacteriocin in orange juice 54

4.13 Effect of bacteriocin in pasteurized milk 56

5 Conclusions and Recommendations 58

5.1 Conclusions 58

5.2 Recommendation for further studies 59

References 60

Appendices 68

vi

List of Tables

TABLE TITLE PAGE

2.1 Classification of bacteriocins for Lactic acid bacteria 11

2.2 Summarization of bacteriocins, purification methods 12

and molecular weight of bacteriocin

2.3 Application of bactericidal food packaging systems 19

4.1 Inhibition zones by Agar well diffusion test 38

4.2 The bacteriocin activity of L. plantarum 41

at 16, 18, 20, 22, 24 and 26 h against L. casei TISTR 1463

4.3 The thickness of edible film based on cassava starch 44

4.4 The tensile strength and elongation at break of various types of 47

edible film based on cassava starch

4.5 Water vapor transmission (WVT) and water vapor transmission rate 49

(WVTR) of various types of edible film based on cassava starch

4.6 Antimicrobial activity of edible film based on cassava starch 52

on the indicator strains

4.7 The number of microorganism in the fermented pork which 53

were wrapped with plastic bag and three type of edible films

based on cassava starch

4.8 The pH value of various types of orange juice during storage period 55

4.9 The number of microorganism in orange juice 56

4.10 The pH value of various types of pasteurize milk during storage period 57

4.11 The number of microorganism in various types of pasteurize milk 57

vii

List of Figures

FIGURE TITLE PAGE

2.1 Life cycle of biodegradable packaging 12

3.1 Flow-chart of overall experiment 23

3.2 Show the purification step; the bacteriocin was purified 26

by using dialysis membrane (1,000 MWCO)

3.3 Flow-chart of purification of the bacteriocin 27

3.4 Flow-chart of sensitivity of bacteriocin to temperature and pH 28

3.5 Flow-chart of cassava starch based films formation 30

3.6 Flow-chart of orange juice experiment 34

3.7 Flow-chart of pasteurized milk experiment 35

4.1 Cell morphology of lactic acid bacteria isolated from fresh turmeric 36

rhizomes

4.2 Batch culture profile of Lactobacillus plantarum and 37

Lactobacillus casei in MRS medium at 37°C

4.3 Bacteriocin activity of L. plantarum against L. casei TISTR 1463 40

at 18 h of culture

4.4 The effect of temperature on bacteriocin activity 41

4.5 The effect of pH on bacteriocin activity 41

4.6 Molecular mass determination by SDS-PAGE 43

4.7 Appearance of edible films based on cassava starch 44

4.8 Scanning electron micrograph of the surface of cassava starch films 45

4.9 Scanning electron micrograph of the surface of cassava starch films 46

incorporated with partially purified bacteriocin

4.10 Scanning electron micrograph of the surface of cassava starch film 46

incorporated with the whole extracts

4.11 Tensile strength of cassava starch films, cassava starch film 48

incorporated with the whole extracts and cassava starch

films incorporated with partially purified bacteriocin

4.12 Elongation at break of cassava starch films cassava starch film 48

incorporated with the whole extracts and cassava starch

films incorporated with partially purified bacteriocin

4.13 Water vapor transmission (WVT) of edible film based on cassava starch 50

4.14 Water vapor transmission rate (WVTR) of edible film based 50

on cassava starch

4.15 The number of microorganism versus time of plastic bag and 53

three types of edible films based on cassava starch

viii

List of Abbreviations

C Cytosine

cfu Colony Forming Unit

Da Dalton

EB Elongation at break

FAO Food and Agriculture Organization

G Guanine

g Gram

h Hour

kDa Kilodalton

LAB Lactic Acid Bacteria

log Logarithm

min Minute

ml Milli liter

mM Milli molar

mm Milli meter

MRS Man Rogosa Sarpe media

NB Nutrient Broth

nm Nama Meter

OD Optical density

PCA Plate Count Agar

SD Standard deviation

TS Tensile strength

V Voltage

v/v Volume by volume

w/v Weight by volume

WVT Water Vapor Transmission

WVTR Water Vapor Transmission Rate

°C Degree Celsius

μl Micro liter

μm Micro meter

WHO World Health Organization

1

CHAPTER 1

INTRODUCTION

1.1 General background

Recently, the quality of foods has been defined as their degree of excellence, and

includes factors of taste, appearance and nutritional content. Quality of food is the

composite of characteristics that have significance and make for acceptability. However,

acceptability can be highly subjective. Based on deterioration factors and determination

procedures, quality may include various aspects such as sensory quality, microbial quality,

and toxicological quality. These aspects are not separated from one another – for example,

microbial contamination damages sensory quality and safety. Microbial food quality

relates to all three groups of factors, since the growth of bacteria generates undesirable

odors and life-threatening toxins, changes the color, taste and texture of food, and also

reduces the shelf life of the product (Han, 2005). Microbial growth on foods significantly

decreases the safety of food and the security of public health.

The microbial contamination is the main cause of food spoilage. Bacteria, moulds

and yeasts are the major cause of microbial contamination of food. The main function of

the food additive is to reduce spoilage of foods. The food preservatives used in food

industries today is largely a chemical preservative. There are many kinds of chemical

preservative available. The chemical food preservatives are designed either to inhibit or

kill the growth of pathogens. Moreover, the chemical food preservatives are also used to

retard or prevent the chemical reactions. To avoid the chemical food preservative,

biopreservatives could be alternative. Bacteriocins are considered as natural

biopreservatives. Nisin is a type of bacteriocin which is commonly used as food

biopreservative (Yin et al., 2007).

There is hence abundance scope for the research to find such natural substances,

which could enhance the shelf life of food products. Probiotics are live microorganisms

that generally found in human and animal body. Probiotics have a lot of benefits. They

have ability to produce some substances which prevent other microorganism to grow.

These substances are called antimicrobial.

The antimicrobial substance is a substance that inhibits or kills the growth of

microorganism. The antimicrobial substance is composing of organic acid, diacetyl, carbon

dioxide, hydrogen peroxide, low molecular weight antimicrobial substances and

bacteriocin. Bacteriocins are defined as antimicrobial compounds mostly extracted from

probiotic bacteria and having the ability to kill other related and unrelated micro-

organisms. Bacteriocins are non-toxic and do not alter the nutritional properties. They are

effective at low concentrations and remain active even under refrigerated

2

temperature. Edible food packaging technologies that incorporate with bacteriocins can

extend the shelf-life of products and reduce the risk from pathogens.

Many researchers have studied the application of bacteriocins from probiotic

bacteria. Some studies incorporate bacteriocin with edible film. And other studies apply

directly bacteriocin to foods. The main objective of this thesis was to find new bacteriocin

from probiotic bacteria and study on their application.

1.2 Statement of problem

The greatest threat to the safety and quality of food products come from microbial

spoilage. Foods are valuable sources of nutrients for newest of the microbes. As they grow

on the food, they cause many problems such as unpleasant smell, bad taste, food discolors

and poor appearance. More importantly, the growth of microbes leads to danger levels of

toxins in the food. This makes the food becomes dangerous to eat. The preservative food

additive has an important role to solve the microorganism contamination problem.

Biopreservative is one of the best options that can protect consumers from harmful

preservative. The bacteriocin from probiotic bacteria has study about its efficiency of

inhibits the foodborne spoilage. Therefore, this research focuses on a new type of

bacteriocin which will extend the shelf life of product. The bacteriocin producer stain in

this thesis was classified from turmeric rhizome. Turmeric is an herb plant that commonly

found in Thailand.

1.3 Scope of study

Bacteriocin from probiotic bacteria has been used as biopreservative in food

product to prolong the shelf life, and incorporated into cassava starch based edible films.

1.4 Objective

1. Compare the efficiency of bacteriocin production between Lactobacillus

plantarum and Lactobacillus casei.

2. Extraction and purification of the bacteriocin from probiotic bacteria from

turmeric rhizome and study the effect of temperature and pH to activity of

bacteriocin and determination of molecular mass of bacteriocin.

3. To study the application of bacteriocin as a food biopreservative.

4. Preparation, characterization and application of bacteriocin-cassava starch film.

3

CHAPTER 2

LITERATURE REVIEW

2.1 Probiotics

The probiotics are defined as the live microbial feed supplements that beneficially

affect the host by improving its intestinal microbial balance. Probiotics are also defined as

“Live microorganisms (bacteria or yeasts), which when ingested or locally applied in

sufficient numbers confer one or more specified demonstrated health benefits for the host”

(FAO/WHO, 2002). In human, lactobacilli are commonly used as probiotics, either as

single species or in mixed culture with other bacteria. The largest group of probiotic

bacteria in the intestine is lactic acid bacteria. Currently, most probiotic bacteria are

cultivated from the genera Lactobacillus and Bifidobacterium, but Enterococcus spp.,

Bacillus spp., and Streptococcus spp. also have great potential.

Probiotic bacteria are generally found in many healthy foods such as dairy product

and non dairy product. The examples of dairy food products which contain probiotic

bacteria are yogurts, milk, kefir and etc. However, some probiotic strains are found in non-

dairy food such as meat, fruits, vegetables and herbs.

2.2 Turmeric

Turmeric (Curcuma longa Linn.) is a rhizomatous medicinal herbaceous plant of

the ginger family. Turmeric is generally found in tropical South Asia and also Thailand.

Curcuma spp. contains essential oils, turmerin (water-soluble peptide) and curcuminoids

including curcumin (Sharma et al., 2005).

Turmeric has ability to inhibit the growth of microbes. Turmeric has antimicrobial

in itself. The traditional medicine had been used turmeric as an anti-inflammatory, tumors

and skin wounds. Many researchers have confirmed that the compounds from turmeric

have effect as anti-oxidant, antimicrobial, anticancer agents, and anti-inflammatory.

Sindhu et al., (2011) studied on the essential oil from turmeric leaves to inhibit

Aspergillus flavus and aflatoxin. They found that the extent of inhibition of fungal growth

and aflatoxin depend on the concentration of essential oil used.

2.3 Identification of probiotics

Although the first recordings on the potential health-promoting effects if lactic acid

bacteria date from the beginning of the 20th

century. The most common used probiotics

among all other microorganism are genera Lactobacillus and Bifidobacterium, stains

belonging to other gram-positive genera such as Enterococcus, Lactococcus, Pediococcus,

4

and Bacillus have also appeared on the market as well as yeasts such as Saccharomyces

boulardii.

2.3.1. Taxonomy

2.3.1.1. Genus Lactobacillus

Lactobacilli are generally characterised as gram-positive, non-sporeforming, non-

motile rods or coccobacilli. Lactobacilli are mainly group of lactic acid bacteria.

Lactobacillus has ability to produce lactic acid. Lactic acid is by-product of glucose

metabolism. They are aero-tolerant or strict anaerobic. Glucose may be fermented by

homofermentation or heterofermentation (Charteris et al., 1997).

The Lactobacillus genus currently contains approximately 125 species, including

such organisms as L.acidophilus, L. plantarumm, L. curvatus, L. bulgaricus, and L.

rhamnosus. Many Lactobacillus species used to be classified simply as L. acidopholus,

resulting in the term “acidophilus” being used almost synonymously with probiotic

organisms.

2.3.1.2. Genus Bifidobacterium

Bifidobacteria are generally characterised as gram-positive, non-sporeforming,

non-motile, strictly anaerobic, catalase-negative anaerobes. The suitable temperature for

bifidobacteria is 37°C to 41°C (least temperature growth for bifidobacteria: 25-28°C;

highest temperature for bifidobacteria: 42-45°C) and optimum pH is 6.5 to 7.0 (no growth

at 4.5-5.0 or 8.0-8.5). The morphology of bifidobacteria is rods of various shapes that are

often in a “Y” shaped or “bifid” form (Goktepe, 2006).

Of the more than 500 bacteria that inhabit the human body, bifidobacteria are the

most abundant microorganisms in the human body. In infants, Bifidobacterium longum,

Bifidobacterium infantis and Bifidobacterium breve predominate. In the adult colon,

however, Bifidobacterium adolescentis and Bifidobacterium longum are isolated more

frequently with Bifidobacterium longum being the more dominant species in human (Vuyst

et al., 2004).

2.3.2. Differential and Selective plating methodologies

The cultivation methods were the mainstay for identifying microbial content. These

relied on physiological and/or phenotypic characteristics of the microorganism. Selective

media and/or incubation condition are important to isolate the interesting bacterial group.

The different microorganisms require different nutritions and conditions for growth.

The classic differential plating strategies are applicable for probiotic bacteria.

Generally, differential or selective media are used to monitor the specific microorganism.

Differential media allows the certain types of microorganism to grow. On the other hand,

5

differential media shows a color change when microorganisms with certain metabolic

capabilities are present.

Bujalance et al. (2006) reveals that the LPSM (Lb. plantarum selective medium)

was developed to isolate and enumerate L. plantarum from faecal samples. LPSM have

ability to inhibit the growth of L. casei and L. fermentum.

A differential medium distinguishes between different types of bacteria based on

some characteristic of the bacteria that is growing on it. In some case, differential media

may even allow the tentative classification of organisms. Typically there is a color change

that results from certain bacterial metabolic products reacting with substances or chemicals

that have been added to the media.

2.4 Antimicrobial components from lactic acid bacteria

The procedure for food preserving by using the ability of lactic acid bacteria (LAB)

which prevent microorganism to grow has been found for long time back. Preservation of

milk by fermentation has been used early in history; Sumerian writings about dairying

about 6000 B.C ago (Arthur and Satu, 2004).

Fermentation reduces the amount of available carbohydrates and results in a range

of small, molecular mass organic molecules that exhibit antimicrobial activity, the most

common being lactic, acetic, and propionic acids. Moreover the production of these

inhibitory primary metabolites, many other antimicrobial components can be formed by

different LAB. The biological significance is thought to be that of amensalism, a means of

one bacterium gaining advantage over another competing microbe. This can be achieved

by changing the environment, e.g., acidification, or production of toxins against

competitors (Arthur and Satu, 2004).

2.4.1 Organic acids

In the fermentation of hexoses process, lactic acid may be produced by

homofermentation or heterofermentation.

It has long been observed that weaker acids are more powerful antimicrobial

substances at low pH than at neutral pH. Of the two acids, acetic acid is the strongest

inhibitor and has a wide range of inhibitory activity, inhibiting yeasts, molds, and bacteria,

while propionic acid has been observed to exert a strong antimicrobial effect, in particular

towards yeasts and molds. This stronger antimicrobial activity of acetic and propionic acid

can be explained in part by their higher pKa of as compared to lactic acid. At, for example,

pH4, only 11% of lactic acid is undissociated, whereas 85% of aceticacid and 92% of

propionnic acid is undissociated (Eklund, 1983). When a mixture of acids is present, it is

likely that lactic acid contributes mainly to the reduction in pH, while propionic and acetic,

which become undissociated, are the antimicrobial agents (Arthur and Satu 2004).

6

2.4.2 Hydrogen peroxide

In the presence of oxygen, lactic acid bacteria are able to generate hydrogen

peroxide (H2O2) through the action of flavoprotein-containing oxidases, NADH oxidases,

and superoxide dismutase. In the absence of source of heme, lactic acid bacteria will not

produce catalase for the removal of hydrogen peroxide. Other systems that eliminate

hydrogen peroxide are less active than the ones producing it. This allows for the

accumulation of hydrogen peroxide (Codon, 1987). The bactericidal effect of hydrogen

peroxide has been attributed to its strong oxidizing effect on the bacterial cell; sulfhydryl

groups of cell proteins and membrane lipids can be oxidized (Arthur and Satu, 2004).

Under natural conditions, the antimicrobial effects of hydrogen peroxide may be

enhanced because of the presence of lactoperoxidase and thiocyanate (SCN¯). The

glycoprotein lactoperoxidase is found in saliva, tears, and milk. It catalyzes the oxidation

of thiocyanate by hydrogen peroxide, generating hypothyanite (OSCN¯) (Arthur and Satu,

2004):

SCN¯ + H2O2 OSCN¯ + H2O

Structural damage and changes in bacterial membranes due to exposure to OSCN¯

have been reported (Kamau et al., 1990). However, the main antimicrobial effect is

contributed to blocking of the glycolysis. The activity toward gram-positive bacteria,

including lactic acid bacteria, is generally bacteriostatic, whereas many gram-negative

bacteria are rapidly killed (Arthur and Satu 2004).

2.4.3 Carbon dioxide

Carbon dioxide (CO2) is mainly formed during heterofermentation of hexoses, but

also many other metabolic pathways generate carbon dioxide during fermentation. Its

formation creates an anaerobic environment and carbon dioxide in itself has an

antimicrobial activity. The mechanism of this activity is unknown, but it has been

suggested that enzymatic decarboxylations are inhibited (King and Nagel, 1975) and that

accumulation of carbon dioxide in the lipid bilayer causes dysfunction in membrane

permeability (Lindgren and Dobrogosz, 1990). At low concentrations carbon dioxide can

stimulate the growth of some organisms, whereas at higher concentrations it can prevent

growth (Arthur and Satu, 2004). Gram-negative bacteria have been reported that they have

more sensitive to carbon dioxide than gram-positive bacteria (Arthur and Satu, 2004).

2.4.4 Diacetyl

Diacetyl (2,3-butanedione) is the aroma and flavor component in butter. LAB have

ability to produce diacetyl. When hexoses are metabolized, the formation of diacetyl will

lactoperoxidase

7

be repressed. However, diacetyl can be overproduced if citrate is metabolized. Citrate is

converted via pyruvate into diacetyl. Diacetyl was found to be more active against gram-

negative bacteria, yeast, and molds than against gram-positive bacteria; lactic acid bacteria

were the least sensitive (Arthur and Satu, 2004).

2.4.5 Low molecular weight antimicrobial substances

Lactic acid bacteria have ability to produce low molecular weight components with

antimicrobial activity. The properties of low molecular weight antimicrobial substances are

active at low pH, thermostable, broad spectrum of activity, and soluble in acetone.

Reuterin, reutericyclin, and 2-pyrrolidone-5-carboxylic acid are examples of low molecular

weight antimicrobial substances (Arthur and Satu 2004).

2.4.6 Bacteriocins

Bacteriocins are defined as antimicrobial peptides which are produced by different

groups of bacteria and kill other related and unrelated microorganisms. Bacteriocins from

lactic acid bacteria have proven non-toxic to humans, do not alter the nutritional properties,

effective at low concentration and active under refrigerated storage and can be used as food

biopreservation by the use of bacteriocin produced by LAB (Kumari and Garg, 2007). The

bacteriocin was first found in Gram-negative bacteria. LAB are Gram-positive bacteria

which can produce bacteriocins.

The antimicrobial proteins or peptides produced by bacteria are termed

bacteriocins. They are ribosomally synthesized and kill closely related bacteria by various

mechanisms such as inhibiting cell wall synthesis, permeabilizing the target cell

membrane, or by inhibiting RNase or DNase activity (Cleveland et al., 2001). Bacteriocin

from lactic acid bacteria have been shown to be safe to human, and have potential to

preserve food. Nowadays, bacteriocins have many applications in food industry to prolong

the shelf-life of perishable food products.

2.5 Classification of bacteriocins

Generally, bacteriocins are ribosomally synthesized polypeptides, and are produced

by microorganisms that are immune to their own action. They are normally modified post-

translationally to some degree, with the secreted mature peptides size usually between 20

to 60 amino acids, and possessing bactericidal activity (O’Connor et al., 2005; Joerger &

Klaenhammer, 1986). The number of bacteriocins continues to grow, significant diversity

in their structure and activity is evident, and this has meant. Therefore, bacteriocins

classification continues to be updated. The current classification divides bacteriocins into

three main classes (O’Connor et al, 2005).

8

Class I bacteriocins, termed the lantibiotics, were initially broadly grouped according to

structure; with type A being elongated amphiphilic peptides and type B more compact and

globular. Bacteriocins class I are small peptides which less than 5 kDa. Lantibiotics

contain unusual amino acids not normally found in nature (e.g., lanthionine and β-methyl-

lanthionine), in addition to a number of dehydrated amino acids. More recently, lantibiotics

subdivided into six subgroups based on primary sequence comparisons.

Class II bacteriocins are known to be small (<10kDa), non-modified, heat-stable

bacteriocins, non-lanthionine-containing, and membrane-active peptides. The inhibition

spectrum of class II bacteriocins is mostly narrow. Class II bacteriocins can be divided into

three subclasses.

Subclass IIa bacteriocins, the largest subgroup, are highly effective in killing

Listeria (Muriana, 1996).

Subclass IIb bacteriocins, the activities depend upon the complementary action of

two peptides such as plantaricin A from Lactobacillus plantarum (Nissen-Meyer et al.,

1993).

Subclass IIc bacteriocins, this subclass is a diverse set of bacteriocins containing all

non-lantibiotics that do not belong to classes IIa or IIb, including sec-dependent secreted

bacteriocins.

Class III bacteriocins are large, heat-labile protein bacteriocins of which very few have

been described from Lactobacillus and bifidobacteria. They are less well-characterized.

The properties of bacteriocins that produced by lactic acid bacteria are suitable for

food preservation because of the following reasons (Gálvez A. et al., 2007):

1. They are recognized as safe substances.

2. For eukaryotic cells, they will become inactive and non-toxic.

3. The digestive proteases can inhibit activity of bacteriocin and having little

influence on the gut microbiota.

4. They are generally tolerance with wide range of pH and temperature.

5. They have a relatively broad antimicrobial spectrum. The antimicrobial affect many

food-borne pathogenic and spoilage bacteria.

6. They show a bactericidal mode of action, usually acting on the bacterial

cytoplasmic membrane and no cross resistance with antibiotics.

7. Their genetic determinants are usually plasmid-encoded, facilitating genetic

manipulation.

9

The benefits of using bacteriocins as biopreservation in food (Thomas et al., 2000):

1. The shelf-life of food can be extended.

2. Provide extra protection for food during in non suitable condition such as

temperature.

3. Decrease the risk form foodborne pathogens.

4. It can protect the food from food spoilage that can improve the economic losses.

5. Using of bacteriocins can reduce the amount of chemical preservatives in food

products.

6. Permit the application of less severe heat treatments without compromising food

safety: better preservation of food nutrients and vitamins, as well as organoleptic

properties of foods.

7. Permit the marketing of “novel” foods (less acidic, with a lower salt content, and

with higher water content).

8. They are safe for using in food industries and consumers.

10

Table 2.1 Classification of bacteriocins for Lactic acid bacteria

Class Subgroup/subclass Bacteriocin Microorganism Reference(s)

Class I: Lantibiotics

Class II: Bacteriocins

Class III: Bacteriocins

Subgroup I – Nisin

Subgroup II – Lacticin 481

Subgroup III – Mersacidin

Subgroup IV – LtnA2

Subgroup V – Cytolysis group

Subgroup VI – Lactocin S

Subclass IIa – Pediocin-like

Subclass IIb – Two peptides

Subclass IIc – Sec-dependent

Class III

Nisin

Plantaricin C

Plantaricin plwa

Plantaricin plwb

Cytolysin CyILL&CyILS

Lactosin S

Bifidocin 1454

Lactocin 705

plantaricin A

Acidocin B

Lactacin B

Lac. Lactis subsp. Lactis

L. plantarum

L. plantarum

L. plantarum

E. faecalis

L. sake

B. bifidum

L. casei CRL 705

L. plantarum

L. acidophilus M 46

L. acidophilus N 2

Roger and Whiter, (1928)

Gonzalez et al., (1994)

Holo et al., (2001)

Holo et al., (2001)

Gilmore et al., (1994)

Skaugen et al., (1994)

Yildirim et al., (1998)

Cuozzo et al., (2000)

Nissen-Meyer et al., (1993)

Leer et al., (1995)

Barefoot and Klaenhammer, (1984)

10

11

Table 2.2 Summarization of bacteriocins, purification methods and molecular weight of bacteriocins

Bacteriocin Strain Medium Purification Molecular weight References

A5-11A

A5-11B

Enterococcus durans M17 3 steps

- Cation-exchange chromatography

- Reverse phase chromatography

- RP-HPLC

5206 Da

5218 Da

Batdorj et al., 2006

162W

AM09

L. curvatus

L. Plantarum

MRS Compare between 2 methods

- Amberlite resin

- 55% Ammonium Sulphate

55% Ammonium Sulphate give a higher

antimicrobial activity (AU/ml)

8.1 kDa

15.5 kDa

-

Acidocin

D20079

L. acidophilus DSM

20079

MRS Ammonium sulphate precipitation

(400g/l) followed by sequential cation

exchange and hydrophobic interaction

chromatography.

6.6 kDa Deraz et al., 2005

Enterococcin

EFS2

Enterococcus faecalis APT 60% Ammonium Sulphate precipitation

followed by RP-HPLC

6756 Da Maisnier-Patin et al.,

1996

11

12

2.6 Edible packaging

Food packaging plays an important role in the Food Industry, where plastic has

been one of the major component in food packaging since last 30 years. The consumption

of plastics in the world with the annual grow is of approximately 5%. Plastics that are

contaminated by foodstuffs and biological substances cannot recycle.

An edible packaging can be defined as a material which can be consumed and

preserve the food from spoilage. Most of edible packagings in nowadays are in form of

edible films and coatings. Edible films are used to apply on the food products after being

formed separately, whereas edible coatings are used to apply and form directly on the food

products. Edible film is a thin layer of material which can be consumed and provides a

barrier to moisture, carbon dioxide, oxygen, aroma, lipid and solute movement for the

food. Moreover, edible films can be used in form of food coatings. Edible coatings may

contribute to extend the shelf-life of product such as fresh-cut fruits. Edible packaging is a

part of biodegradable packaging.

The traditional food packagings based on plastics such as polyolefins, polyester,

etc. They are totally non-biodegradable and lead to environmental pollution. The

biodegradable packaging could be derived from plant-based materials, marine food

processing industry wastes or other renewable natural sources. Food packaging

applications can make from these materials. Biodegradable packagings are considered as

user-friendly and eco-friendly. Therefore, biodegradable packagings offer a possible

alternative to solve the environmental pollution and create the new markets for agricultural

products. Biodegradable packaging can be degraded by the enzymatic action of

microorganisms and the optimum condition. The composting biopackagings are converted

into carbon dioxide, water and biomass within 6-12 weeks.

Figure 2.1 Life cycle of biodegradable packaging

13

The advantages of edible films over traditional synthetic polymeric packaging

materials have been listed as follows (Robertson, 2006):

1. The films can be consumed with the packaged product, leading no residual

packaging to be disposed of.

2. The reduction of environmental pollution can still contribute even if the films are

not consumed. Films are likely to degrade more completely than synthetic

polymeric materials, and are produced exclusively from renewable, edible

ingredients.

3. The films can enhance the organoleptic properties of packaged foods provided that

various components such as flavorings, colorings and sweeteners are incorporated

into them.

4. The films can supplement the nutritional value of foods (this is particularly true for

films made from proteins).

5. A film can be used for a small portion of food individual packaging. Peas, beans,

nuts and strawberries, etc. are not currently individually packaged.

6. The films can be applied inside heterogeneous foods at the interfaces between

different layers of components. They can be tailored to prevent deteriorative

intercomponent moisture and solute migration in foods such as pizzas, pies and

candies.

7. The films can function as carriers for antimicrobial and antioxidant agents. In a

similar application, they can also be used at the surface of foods to control the

diffusion rate of preservative substances from the surface to the interior of the food.

8. The films can be very conveniently used for microencapsulation of food flavoring

and leavening agents to efficiently control their addition and release into the

interior of foods.

9. The films could be used in multilayer food packaging materials together with

nonedible films, in which case the edible films would be the internal layers in direct

contact with food materials.

2.7 Film composition

2.7.1 Film-forming materials

The main film-forming materials are biopolymers, such as polysaccharides, protein,

lipids and resins (Han & Gennadios, 2005). They can be used alone or in combinations.

Edible and biodegradable films must meet a number of specific functional requirements

such as moisture barrier, solute and/or gas barrier, water or lipid solubility, colour and

appearance, mechanical and rheological characteristics, non-toxicity, etc. These properties

14

are dependent on the type of material used, its formation and application (Guilbert et al.,

1996). Film-forming materials can be hydrophilic or hydrophobic. However, in order to

maintain edibility, solvents used are restricted to water and ethanol (Han and Gennadios,

2005; Peyron, 1991).

Biopolymer composites can modify film properties and create desirable film

structures for specific applications (Han & Gennadios, 2005). Similar to multi-layered

composite plastic films, biopolymer films can be produces as multiple composite layers,

such as protein coatings (or film layers) on polysaccharide films, or lipid layers on

protein/polysaccharide films (Han & Gennadios, 2005). This multi-layered film structure

optimizes the characteristics of the final film. Composite films can also be created by

mixing two or more biopolymers, yielding one homogeneous film layer (Han and

Gennadios, 2005; Yildirim and Hettiarachchy, 1997; Debeaufort et al., 1998; Were et al.,

1999). Various biopolymers can be mixed together to form a film with unique properties

that combine the most desirable attributes of each component (Han and Gennadios, 2005;

Wu et al., 2002)

2.7.1.1 Cassava starch

Cassava is one of the most economic crops in Thailand. The starch from cassava

has is important in many industry such as paper industry, food industry. Moreover cassava

starch has ability to produce the edible packaging. The edible packaging from cassava

starch has excellent properties such as their transparency, grease and oil resistance and heat

sealability. (Zhong and Xia, 2008).

Cassava starch granules are mostly round with a flat surface on one side. The

granules exhibit wide variation in size range (5-40 m). The starch has very little lipid and

phosphorus content. The amylase content in the starch is in the range of 20-27% similar to

most other starches. The viscosity of cassava starch is high level compared with most other

tuber starches and the cereal starches. Setback is one of factors which relate to viscosity

and is attributed to the retrogradation of starch during cooling. Cassava starch has

relatively low setback and this may be due to lower content and also the structure of the

amylopectin (Moorthy, 2004).

2.7.2 Plasticizers

Plasticizers are required for edible films and coatings, especially for

polysaccharides and proteins (Han & Gennadios, 2005). These film structures are often

brittle and stiff due to extensive interactions between polymer molecules (Han and

Gennadios, 2005; Krochta, 2002). Generally, Plasticizers are required for polysaccharides

(or proteins) based edible films. Their amount added into hydrocolloid film-forming

preparations varies between 10% and 60% by weight of the hydrocolloids. Plasticizers are

small size, high polarity, low molecular weight agents incorporated into the polymeric

film-forming materials, which decrease the glass transition temperature of the polymer.

15

Plasticizers, such as glycerol, propylene, polyethylene glycol, acetylated monoglyceride,

sugar alcohol and glucose are often used to modify the mechanical properties of a film.

Plasticizers are low molecular weight agents incorporated into the polymeric film-forming

materials, which decrease the glass transition temperature of the polymers. They are able to

position themselves between polymer molecules and to interfere with the polymer-polymer

interaction to increase flexibility and processability (Han and Gennadios, 2005; Krochta,

2002; Guilbert and Gontard, 1995). Plasticizers increase the free volume of polymer

structures or the molecular mobility of polymer molecules (Han and Gennadios, 2005;

Sothornvit and Krochta, 2000). The addition of plasticizers can decrease the glass

transition temperature. Moreover, plasticizers affect not only the elastic modulus and other

mechanical properties, but also the resistance of edible films and coatings to permeation of

vapors and gases (Han and Gennadios, 2005; Sothornvit and Krochta, 2000, 2001). Most

plasticizers are very hydrophilic and hygroscopic so that they can attract water molecules

and form a large hydrodynamic plasticizer-water complex (Han and Gennadios, 2005).

Water molecules in the films function as plasticizers. It has a very good plasticizer, but it

can easily be lost by dehydration at a low relative humidity (Han and Gennadios, 2005;

Guilbert and Gontard, 1995). Therefore, the addition of hydrophilic chemical plasticizers

to films can reduce water lose through dehydration, increase the amount of bound water,

and maintain a high water activity.

2.7.3 Additives

The new generation of edible films and coatings is being especially designed to

increase their functionalities. Edible films and coatings can incorporated with various

active agents such as emulsifiers, antioxidants, antimicrobials, nutraceuticals, enzymes,

flavors, and colorants, thus enhancing food quality and safety, up to the level where the

additives interfere with physical and mechanical properties of the films (Han and

Gennadios, 2005; Han, 2002, 2003; Howard and Gonalea, 2001; Guilbert et al., 1996;

Baldwin et al., 1995; Kaster and Fennema, 1986). Although many biopolymers possess

certain levels of emulsifying capacity, it is necessary to incorporated into film-forming

solutions to produce lipid-emulsion films (Han and Gennadios, 2005). In the case of

protein films, some film-forming proteins have sufficient emulsifying capacity due to their

amphiphilic structure (Han and Gennadios, 2005). Antimicrobial agents can be

incorporated into edible films and coatings to inhibit the growth of microorganisms. The

most frequently used biopreservatives for antimicrobial are lysozyme and nisin. Lysozyme

shows antimicrobial activity mainly on gram-positive and does not show antibacterial

activity against gram-negative bacteria. Common other biopreservatives that may be used

in edible films and coatings are bacteriocin. Most antimicrobial compounds have

antioxidant properties.

2.8 Antimicrobial packaging

Antimicrobial packaging is a system that can kill or inhibit the growth of target

microorganisms and thus extend the shelf-life of perishable products and enhance the

16

safety of packaged products (Han, 2005; Han, 2000). There are many applications of

antimicrobial packaging such as oxygen-scavenging packaging and moisture-control

packaging. Antimicrobial packaging is one of the most promising innovations of active

packaging technologies. It can be constructed by using antimicrobial packaging materials

and/or antimicrobial agents inside the package space or inside foods. Most food packaging

systems consist of the food products, the headspace atmosphere and the packaging

materials (Han, 2005). Any one of these three components of food packaging systems

could possess an antimicrobial element to increase antimicrobial efficiency (Han, 2005).

Edible antimicrobial agents can be incorporated into food ingredients, while

antimicrobial resources can be incorporated into food ingredients, while antimicrobial

resources can be interleaved in the in-package headspace in the form of sachets, films,

sheets or any in-package supplements, to generate antimicrobial atmospheres (Han, 2005).

Besides the use of antimicrobial packaging materials or antimicrobial inserts in the

package headspace, gaseous agents have been used to inhibit the growth of

microorganisms (Han, 2005). Common gases are carbon dioxide for modified atmosphere

packaging, sulfur dioxide for berries, and ethanol vapor for confections (Han, 2005). These

gases are injected into the package headspace or into palletized cases after shrink-wrapping

of a unit load on a pallet (Han, 2005). Vacuum, nitrogen-flushing and oxygen-scavenging

packaging, which were originally designed for preventing the oxidation of packaged foods,

also possess antifungal and antimicrobial properties against aerobic bacteria as a secondary

function, since these microorganisms are restrictively aerobic (Han, 2005). However, these

technologies, which control the low oxygen concentration to inhibit the growth of aerobic

microorganisms, could cause the onset of anaerobic microbial growth (Han, 2005).

Controlling anaerobic bacteria in modified atmosphere packaging is very important issue

in maintaining the quality and safety of the products (Han, 2005).

2.8.1 Type of Antimicrobial agents in food packaging

Various antimicrobial agents could be incorporated into conventional food

packaging systems and materials to create new antimicrobial packaging systems. The

antimicrobial agents can generally be classified into three groups; Synthetic agents, natural

agents, and probiotics.

2.8.1.1 Synthetic antimicrobial agents

For the purpose of food preservation, all packaging ingredients should be food-

grade additives. The synthetic agents can be mixed with food ingredients, incorporated into

packaging additives or inserted into the headspace atmosphere. The antimicrobial agents

are in contact with and consumed with the food products in these applications. Therefore,

the synthetic antimicrobial agents should be controlled as food ingredients regardless of

where the synthetic antimicrobial agents were positioned initially – in the food products, in

the packaging materials, or in the package headspace atmosphere (Han, 2005). In case of

17

non-food-grade synthetics, the only way to incorporate the synthetic into the food

packaging system is through the synthetic binding of the antimicrobial agents to packaging

material polymers (immobilization) (Han, 2005). In this case, the migration or residual

amount of the non-food-grade synthetic in the food products is prohibited by regulation

(Han, 2005). Therefore, it is necessary to verify that there is no migration of the synthetic

from packaging materials to foods, and there is no residual free synthetic after the

immobilization reaction (Han, 2005).

Organic acids are widely used as synthetic antimicrobial agents because their

efficacy is generally well understood and cost effective. Many organic acids, including

fatty acids, are naturally existing synthetics and have been used historically. Currently,

most of them are produced by chemical synthesis or chemically modified from natural

acids. Organic acids have characteristic sensitivities to microorganisms. For example,

sorbic acid and sorbates are very strong antifungal agents, while their antibacterial

activities are not effective – they have various antimicrobial mechanisms. Therefore, the

correct selection of organic acids is essential to have effective antimicrobial agents.

Mixtures of organic acids have a wider antimicrobial spectrum and stronger activity than a

single organic acid (Han, 2005).

Fungicides are also common antimicrobial agents. Imazalil has been incorporated

into the wax coating of oranges and other citrus fruits (Han, 2005). Since fungicides are

not permitted as a direct food preservative, they cannot be mixed into food ingredients or

added to food-contact packaging materials as food-contact substances (Han, 2005).

Therefore, it is necessary to design antimicrobial food packaging systems when non-food-

grade antimicrobial agents, such as fungicides, are used (Han, 2005).

2.8.1.2 Natural antimicrobial agents

The natural antimicrobial agents include herb extracts, spices, enzymes, and

bacteriocins as naturally occurring antimicrobial agents. Herb and spice extracts contain

multiple natural compounds, and are known to have a wide antimicrobial spectrum against

various microorganisms (Han, 2005). Apart from antimicrobial activity, other advantages

they offer include antioxidative activity and their effect as alternative medicines (Han,

2005). However, their mode of action and kinetics are generally unknown, and their

chemical stability is also of concern. In addition, they create some problems with respect to

flavors (Han, 2005).

Specificity of enzymes should be considered carefully, since antimicrobial activity

is very sensitive to the environments and substrates. For an example, the activity of

lysozyme can be significantly affected by temperature and pH. In most cases, lysozyme is

not effective against gram-negative bacteria because gram-negative bacteria have the

complex structure of cell wall and the specificity of lysozyme for peptidoglycan (Han,

2005). However, Güçbilmez et al. (2007) studies the edible zein films incorporated with

lysozyme, albumin proteins and disodium EDTA. They found that zein films effective on

18

Escherichia coli and Bacillus subtilis. Therefore, lysozyme and other ingredients in zein

films can improve the antimicrobial and antioxidant activity of the films.

Various bacteriocins, such as nisin, pediocin, lacticin, propionicin etc., can be

incorporated into foods and/or food packaging systems to inhibit the growth of spoilage

and pathogenic microorganisms (Han, 2005; Daeschul, 1989). Sanjurjo et al. (2006)

studies the performance of nisin supported in edible tapioca films formulated with tapioca

starch and glycerol reduced L. innocus growth, producing count decrease and acting as a

barrier to contamination after processing. In the case of fermented food products, live

bacteria which produce bacteriocins can be intentionally added as probiotics in the

packaged food system to obtain antimicrobial effectiveness.

2.8.1.3 Probiotics

Various microorganisms, e.g. lactic acid bacteria, produce bacteriocins and non-

peptide growth-inhibiting chemicals such as reuterin. These naturally produced

antimicrobials can inhibit the growth of other bacteria. Use of probiotics can therefore

effectively control the competitive undesirable microorganisms (Han, 2005). Many

traditional fermented food products contain antimicrobial probiotics. There has been much

research and development regarding the function of antimicrobial probiotics for the

preservation of fermented foods (Han, 2005). Currently there is only limited research into

the use of probiotics for the purpose of antimicrobial packaging design. With the new

technology development for the delivery of love probiotics, the use of probiotics as an

antimicrobial source for antimicrobial food packaging will be more popular in future due

to its safety and effectiveness (Han, 2005).

Rößle et al. (2010) indicated that dipping fresh-cut apple slices in a solution

containing probiotic bacteria (Lactobacillus rhamnosus GG) and measure entrapment and

stability of the microorganism.

19

Table 2.3 Application of bactericidal food packaging systems

Bacteriocins Packaging materials Foods Microorganisms References

Nisin HPMC Culture media L. monocytogenes,

S. aureus

Coma et al., 2001

Nisin HPMC, stearic acid Culture media L. monocytogenes,

S. aureus

Sebti et al.,2002

Nisin Corn zein Shredded cheese Total aerobes Cooksey et al., 2000

Nisin Corn zein, wheat gluten Culture media Lactobacillus plantarum Dawson et al., 2003

Nisin Agar coating Fresh poultry S. typhimurium Natrajan and Sheldon, 1995

Nisin Alginate Beef S. aureus Millette et al., 2007

Nisin Tapioca starch Culture media L. innocua Sanjurjo et al., 2006

Nisin, lacticins LDPE, Poly-amide Culture media M. flacus,

L. monocytogenes

An et al., 2000

Nisin, lacticins LDPE, Poly-amide Oyster, beef Total aerobes, coli-form Kim et al., 2002

Nisin, EDTA PE, PE-PE oxide Beef B. thermosphacta Cutter et al., 2001

Nisin, lauric acid Zein Simulants Migration test Hoffman et al., 2001

19

20

Table 2.3 Application of bactericidal food packaging systems (Cont.)

Bacteriocins Packaging materials Foods Microorganisms References

Nisin, lauric acid Soy protein Turkey bologna L. monocytogenes Dawson et al., 2002

Nisin, pediocin Cellulose casing Turkey breast, ham, beef L. monocytogenes Ming et al.,1997

Nisin, grape seed

extract, EDTA

Soy protein Drop culture on film and

enumerate after 1 h

(25°C)

L. monocytogenes,

E. coli O157:H7,

Salmonella typhimurium

Sivarooban et al., 2008

Enterocins Alginate, zein, polyvinyl

alcohol (biodegradable

film)

Ham L. monocytogenes Marcos et al., 2007

Enterocins 416K1 Low-density polyethylene Culture media,

frankfurters and fresh

cheeses

L. monocytogenes Iseppi et al., 2008

20

21

CHAPTER 3

MATERIAL AND METHODS

3.1 Materials

3.1.1 Lactobacillus plantarum isolated from Turmeric (Curcuma longa Linn.)

3.1.2 Lactobacillus casei TISTR 1463

3.1.3 Escherichia coli TISTR 780

3.1.4 Salmonella typhimurium TISTR 292

3.1.5 Staphylococcus aureus TISTR 029

3.1.6 Listeria monocytogenes

3.2 Chemicals

3.2.1 MRS agar (HiMedia, India)

3.2.2 MRS broth (HiMedia, India)

3.2.3 Nutrient broth (HiMedia, India)

3.2.4 Nutrient agar (HiMedia, India)

3.2.5 PCA agar (HiMedia, India)

3.2.6 Yeast extract (HiMedia. India)

3.2.7 Peptone

3.2.8 Catalase enzyme

3.2.9 Glycerine (Union Chemical 1986 Co., Ltd., Thailand)

3.2.10 Glycerol (Ajax)

3.2.11 Ammonium sulphate (Qrec)

3.2.12 Dipotassium hydrogen phosphate (Ajax)

3.2.13 Potassium dihydrogen phosphate (Ajax)

3.2.14 Sodium hydroxide (Merck)

3.2.15 Hydrogen chloride

3.2.16 Ethyl alcohol

3.2.17 40% Acrylamide/Bis Solution (BIO-RAD, USA)

3.2.18 10X TRis/Glycine/SDS (BIO-RAD, USA)

3.2.19 Coomassie Blue G-250

3.2.20 Sodium Dodecly Sulfate (SDS)

3.2.21 Tris Hydrochloride

22

3.2.22 Ammonium Persulfate

3.2.23 Tetramethylethylenediamine (TEMED)

3.2.24 Tricine Sample Buffer (BIO-RAD, USA)

3.2.25 Polypeptide SDS-PAGE Molecular Weight Standards (BIO-RAD, USA)

3.3 Equipments

3.3.1 Plastic plates (Hycon)

3.3.2 Glasswares

3.3.3 Anaerobic jar and GasPak (BBL Gas Pak System, USA)

3.3.4 Microscope (Olympus, Japan)

3.3.5 Magnetic stirrer and magnetic bar

3.3.6 Autoclave (ALP Co., Ltd., Japan)

3.3.7 Vortex mixer (Maxi mix II,Thermolyte, USA)

3.3.8 Laminar flow

3.3.9 Centrifuge

3.3.10 Incubator (Orbital SI 50)

3.3.11 Hot air oven

3.3.12 Balance

3.3.13 pH meter (Portamess, Germany)

3.3.14 Dialysis membrane 1 kD MWCO (Spectro/por 7)

3.3.15 UV Spectrophotometer (Unicam, UK)

3.3.16 Colony counter (Stuart Scientific, UK)

3.3.17 Water bath (Memert, Germany)

3.3.18 Hand micrometer,0-25 mm, 0.01 mm (Miyuyoyo, Japan)

3.3.19 Desiccator (Nikko, Japan)

3.3.20 Texture analyzer (Model TA.XT2 Texture Technologies Corp., NY. USA)

3.3.21 Stomacher (BagMixer®400, Interscience, France)

3.3.22 Micropipette

3.3.23 Cork borer

3.3.24 Mini-PROTEAN® II Electrophoresis Cell (BIO-RAD, USA)

3.3.25 Cellulose nitrate membrane, 0.45μm (Whatman, Japan)

23

Figure 3.1 Flow-chart of overall experiment

Bacteria culture

Screen for bacteriocin production

Agar spot test

Well diffusion agar test

Inhibition test of bacteriocins

Purification of the bacteriocins by

ammonium sulphate and then dialysis bag

Cassava starch – partially purified

bacteriocin based edible film

Effect of temperature and pH

on bacteriocin activity

Assay for bacteriocin activity

Effect of temperature control

on bacteriocin production

Partially purified bacteriocin

Physical properties, mechanical

properties and antibacterial activities

of edible film

Biopreservative test

Edible packaging for fermented pork

Milk Orange juice

Detection of molecular weight

24

3.4 Methodology

3.4.1 Isolation of Lactobacillus plantarum from turmeric rhizome

10 g of the fresh turmeric rhizomes were chopped and mixed with 90 ml of 0.85%

NaCl. The sample was homogenizing for 2 min by stomacher. Appropriate dilutions were

spread on MRS agar containing 0.5% w/v CaCo3 and incubated anaerobically at 37°C for

48 h. Well-developed individual colonies were restreaked on MRS agar containing 0.006

% w/v bromocresol purple.

Isolates were screened for morphology by Gram-stain technique and biochemical

identification test by catalase activity. The catalase activity was performed by isolates were

smeared on a clean slide glass and drop 3% hydrogen peroxide (H2O2) over the isolates.

Finally, isolates were identified the genus and species by using API kits (bioMérieux SA,

Marcy l'Etoile, France).

3.4.2 Bacterial culture and media

The bacteriocin producer Lactobacillus plantarum was isolated from turmeric

rhizome and Lactobacillus casei TISTR 1463 was obtained from TISTR (Thailand

Institute of Scientific and Technological Research). Pathogen strains which are

Escherichia coli TISTR 780, Salmonella typhimurium TISTR 292 and Staphylococcus

aureus TISTR 029 were obtained from TISTR (Thailand Institute of Scientific and

Technological Research) and Listeria monocytogenes was available in the Bioprocess

Technology Laboratory (BPT) at AIT. All of pathogen strains used as indicator strains to

testing the efficiency of bacteriocin producers. Lactobacillus strains were grown in MRS

medium (HiMedia, India) and Pathogen strains were grown in Nutrient broth medium

(HiMedia, India). Before use, all strains were subcultured three successive times in 10 ml

of medium. The transfer volume was 1% (v/v). The incubation for Lactobacillus plantarum

was at 37°C for 18 h., Lactobacillus casei was at 37°C for 18 h. and pathogen strains were

at 37°C at 18 h.

3.4.3 Screen for antimicrobial production by agar spot test

The bacteriocin producer strains were cultured in 5 ml of MRS broth for 18 h.

Aliquots 10 μl of each culture was spot onto 20 ml MRS agar plates (1.5% w/v agar) and

incubated for 24 h at 37°C until the colonies developed. After that, the plates were overlaid

with 5 ml of the Nutrient soft agar (0.75% agar) incubated with the indicator strains cell

suspension at a final concentration of 105 cfu/ml. The plates were incubated at 37°C for 24

h and the appearance of inhibitory zones was observed and measured.

25

3.4.4 Bacteriocin activity assay by agar well diffusion test

The indicator strains were swabbed on the Nutrient soft agar plate (0.75% w/v

agar). Well of 5 mm. diameter was made with a sterile cork borer. Bacteriocin producing

strains were grown in 10 ml of MRS broth and were centrifuged at 12,000g at 4°C for 15

min. The pH of the supernatants were adjusted to 6.5 either with 1N NaOH or 1N HCl to

reduce the effects of acid such as lactic acid, acetic acid, etc. followed by addition of

catalase enzyme at the final concentration 1 mg/ml to reduce the effect of hydrogen

peroxide. The mixtures were then incubated at 37°C for 1 h. The aliquots of supernatant

(50 μl) were placed into each well of the seed plates. After pre-diffusion at 25°C for 30

min, the plates were incubated at 37°C. The antimicrobial activity, expressed in centimeter,

was determined by measuring the diameter of the inhibition zone around the wells.

3.4.5 Effect of temperature control on bacteriocin production

The producer strains were propagated in MRS broth with 1% inocula. The

cultivation temperature was varying at 30 and 37°C without the pH control. The samples of

1 ml were removed for activity assays at 18 h and 24 h. The samples were centrifuge at

12,000g at 4°C for 15 min. The supernatants were adjusted to pH 6.5 and added catalase

enzyme (final concentration of 1 mg/ml) and incubated for 1 h. The supernatants were

determined by an agar well diffusion test.

3.4.6 Bacteriocin production by L. plantarum in MRS broth at 37°C

Five hundred milliliter of MRS broth was inoculated with L. plantarum with 1%

inocula and incubated at 37°C. The 50 ml of samples were withdrawn at regular intervals

every 2 h form 16 h to 26 h. The samples were centrifuged at 12,000 g for 15 min at 4°C,

and ammonium sulphate (ANALAR B.D.H. grade, England) (65% of saturation, at 25°C)

was added to the supernatants to precipitate proteins. The mixtures had been stirred for 2 h

at 4°C, the protein precipitates were collected by centrifugation at 12,000 g for 45 min at

4°C. The pellets were solubilised in 2 ml of 50 mM potassium phosphate buffer pH 7 and

then exhaustively dialysed overnight through 1000 molecular weight-cut-off-dialysed

membrane against the same buffer opposite membrane. The dialysed was further filtered

through cellulose nitrate membrane 0.45 μM (Whatman, Japan) and stored at -20°C until

further use.

The inhibitory activity in the MRS supernatants after ammonium sulphate

precipitation and dialysis membrane steps were determined by an agar well diffusion test.

The inhibitory activities of each interval time were tested to compare the highest

bacteriocin activity. For a semiquantitative assay of the bacteriocin, two-fold serial

dilutions were used (Barefoot and Klaenhammer, 1994) with L.casei as the indicator strain.

26

3.4.7 Purification of the bacteriocin produced by Lactobacillus plantarum in MRS broth

Each strain producing bacteriocin-like substance was propagated in 1 L of MRS

broth and after 18 h of incubation, the culture was centrifuged at 19000g for 10 min. The

cell free solution was precipitated with ammonium sulphate (65 % saturation). The mixture

will be stirred for 10 h at 4°C and latter centrifuged at 12000g for 45 min at 4°C. The

precipitate was resuspended in 20 ml of potassium phosphate buffer (50 mmol/l, pH 7.0)

and exhaustively dialyzed overnight through 1000 molecular weight-cut-off-dialysed

membrane against the same blank buffer inside membrane. The dialyzed was further

filtered through cellulose nitrate membrane 0.45 μm (Whatman, Japan) and stored at -20°C

until further use.

Figure 3.2 Show the partially purification step; the bacteriocin was purified by using

dialysis membrane (1,000 MWCO)

27

Figure 3.3 Flow-chart of partially purification of the bacteriocin

3.4.8 Assays for bacteriocin activity

The antimicrobial activity of partial purification of bacteriocin solution was expressed

in arbitrary units per ml (AU/ml) and it was determined by an agar well diffusion assay.

This method was modified from Vallani et al. (1993). Briefly, a serial two-fold dilution in

phosphate buffer solution 50 mmol/l, pH 7.0 (Appendix A) of bacteriocin was prepared,

and 50 μl were placed into each well of the seed plates (MRS soft plate (0.75% agar) seed

with about 105 CFU/ml of indicator strain). After pre-diffusion at 25°C for 30 min, the

plates were incubated at 37°C. The antimicrobial activity, expressed in centimeter, was

determined by measuring the diameter of the inhibition zone around the wells.

The AU/ml was calculated as:

AU/ml = (1000/A)D

Where:

A is the volume of bacteriocin aliquot placed into each well of agar plate

D is the reciprocal of the highest dilution showing a clear inhibition of the indicator

strain.

Purification of the bacteriocin

Bacteriocin precipitation by ammonium

sulphate 65% (NH4)2SO4

Dialyze overnight with 1 kDa MWCO

Resuspend in potassium phosphate

buffer

Filter with 0.45 μM and store at -20°C

28

3.4.9 Sensitivity of bacteriocin to heat

Bacteriocin was treated at the various temperatures (40, 60, 80, 95°C) and each

various temperatures do with the various times (0, 30, 60, 90 min). After treat the

bacteriocin, samples were taken and determined the remaining activity by agar well

diffusion assay and compare with the sample that did not pass the heat processing.

3.4.10 Sensitivity of bacteriocin to pH

The pH stability was determined by measuring the activity of the partial purified

bacteriocin with 5M NaOH or 5M HCl at different pH values ranging (pH 3, 5, 7 and 9)

followed by incubation for 2 hours at 25°C. Activities were assayed by the agar well

diffusion method as mentioned previously in section 3.4.7.

Figure 3.4 Flow-chart of sensitivity of bacteriocin to temperature and pH

3.4.11 Determination of molecular mass of bacteriocin

The molecular mass of bacteriocins was estimated in a SDS-PAGE system as

described by Schägger H. & Von Jagow G. (1987), using 16.5% v/v, 10% v/v and 4% v/v

acrylamide in the separation, spacer and stacking gel respectively (Appendix B1).

Electrophoresis was performed in vertical gels in a Mini-Protean II cell (Bio-Rad

Laboratories, Richmond, CA, USA) at 200V for 45 min. After electrophoresis, the gel was

cut in two vertical parts. One part was fixed and stained with Coomassie brilliant blue R-

250 (1 g/L) in 50% v/v methanol and 10% v/v acetic acid (Appendix B2) for 20 min and

then destain with 10% glacial acetic acid and 5-10% v/v methanol (Appendix B2). The

other part was assayed for antimicrobial activity according to Bhunia et al. (1987). Briefly,

0 min 30 min 60 min 90 min

Agar well diffusion

test

pH 3 pH 5 pH 7 pH 9

Agar well diffusion

test

Sensitivity of bacteriocin to heat and pH

pH Heat

40°C 60°C 80°C 95°C Adjust pH with 5M NaOH and 5M

HCl

29

the gel was fixed for 30 min (25% v/v isopropanol, 10% v/v acetic acid and 65% v/v

distilled water), rinsed with distilled water (1 h initial rinse followed by two washes of 5

min), and overlaid with 25 ml of MRS (0.75% w/v agar) seed with 105 CFU/ml of L. casei

TISTR 1463. After incubation at 30°C for 24 h the gel was examined for the presence of an

inhibitory zone. Molecular Mass Markers for Peptides (Bio-Rad) (Appendix B3) were used

for mass standards.

3.4.12 Preparation of cassava starch based films

For the control film, the mixture of film forming solution which composed of 5%

w/w cassava starch, 2% w/w glycerine and 93% w/w water was prepared. For the whole

extracts film, the mixture of film forming solution which composed of 5% w/w cassava

starch, 2% w/w glycerine, 46.5% w/w water and 46.5% w/w supernatant of L. plantarum

culture at 18 h was prepared. For the partially purified bacteriocin film, the mixture of film

forming solution which composed of 5% w/w cassava starch, 2% w/w glycerine, 1% w/w

partially purified bacteriocin and 92% w/w water was prepared. After that the prepared

film forming solution was heated on a hot plate with a magnetic stirrer until the mixture

entered in the gelatinization step which was 70oC for cassava starch (Flores et al., 2007).

Gelatinization of cassava starch solution was completed after 20 min by using water bath

for controlling the temperature at 70oC (Cereda, 2000). The film forming solutions were

kept at ambient temperature (25oC) for at least 3 h to allow bubbles to dissipate. The film

forming solution was then casted over the tray that covers with plastic bag. Those trays

were dried at 50oC for 20-24 hours by using the hot air oven and the films were then peeled

off. The dried films were kept in the plastic bags and stored in the dessicators at 30-40%

RH for further measurement.

30

Figure 3.5 Flow-chart of cassava starch based films formation

Control film

Mixture of cassava starch, glycerine

and water at ratio of 5.0: 2.0: 93.0 to

obtain film forming solution

Film forming solution was heated on a magnetic

stirrer with hot plate until the system entered in the

gelatinization step (≈70°C) for 20 minutes

The whole extracted film

Mixture of cassava starch, glycerine, supernatant of

L. plantarum culure at 18 h and water at ratio of 5.0:

2.0: 46.5: 46.5 to obtain film forming solution

The partially purified bacteriocin film

Mixture of cassava starch, glycerine,

bacteriocin and water at ratio of 5.0: 2.0: 1.0:

92.0 to obtain film forming solution

The film forming solutions were kept at ambient

temperature for at least 3 h to allow bubbles to dissipate

Film forming solution was casted over

the tray that covers with plastic bag

Drying at 50°C for 20-24

h

Film was peeled off

The dried films were kept in the plastic bags

and stored in the dessicators at 30-40% RH for

further measurement

31

3.4.13 Physical properties, mechanical properties and antibacterial activities of cassava

starch based film

3.4.13.1 Film thickness measurement

Hand micrometer (0-25 mm, 0.01 mm, Miyuyoyo, Japan) was used to determine

the thickness of the film. The method was followed as described by Rodríguez et al.

(2006). The thickness of films was calculated from the mean of five random positions of

all over the films.

3.4.13.2 Microstructure of edible film

Microstructure of film samples was determined by scanning electron microscopy

(SEM) by following the method of Anal et al. (2006). Briefly, the fragments of the film

were mounted on copper stubs, fixed with double-sided tape, and coated with copper. The

sample was then observed with a scanning electron microscope.

3.4.13.3 Tensile strength and elongation at break analysis (Pranoto, 2004)

The tensile strength (TS) and elongation at break (EB) properties of film were

measured by using Lloyd Instrument Testing Machine type LRX 5K. The method was

followed as described by Pranoto et al. (2004). Tested samples were cut into 1×8 cm

strips. The films were held parallel with an initial grip separation of 3.5 cm. The film

sample was pulled apart at 50 mm/min head speed. TS is force per a unit of cross section

area of film which is calculated by using the following formula:

TS =

Where; Fmax = maximum force at break, N

x = wide of film, m

d = thickness of film, m

EB is calculated based on the length extended compared to the original length of

films. The following formula is used to calculate EB of film.

EB =

Where; H = head speed, 50 mm/min

t = time for film extension until break, min

l = initial length of film, m

32

3.4.13.4 Water vapor transmission

The water vapor transmission (WVT) of film was measured based on ASTM as

described by Pranoto (2004) with some modifications. A cup containing 20 ml. of distilled

water (70% RH was covered with a test film membranes and then placed in a desiccators

filled with silica gel (0% RH). The room temperature was maintained at 25oC. The

temperature and relative humidity values were used to calculate the partial pressure by

using saturated steam tables. The moisture loss inside the cup was weighed every 2 hours

until R2 of the graph between cumulative weight loss (g) and time (day) was more than

0.99. The constant rate of weight increased was obtained by linear regression. The WVT

was calculated from the following formula:

WVT = (g H2O mm/cm2)

Where; w = the weight of water absorbed in the cup (g)

x = the average thickness of the film (mm)

A = the permeation area (cm2)

WVTR = (g H2O mm/h cm2)

Where WVTR = Water Vapor Transmission Rate

x/t = linear regression from the points of weight gain and time

during constant rate period

3.4.13.5 Antimicrobial activities of cassava starch based films

Antimicrobial activity of cassava starch based films was determined by agar

diffusion method. The cultures of target microorganisms (Escherichia coli TISTR 780,

Salmonella typhimurium TISTR 292, Staphylococcus aureus TISTR 029, Listeria

monocytogenes) were obtained by incubation in nutrient broth at 37oC for 24 hours. The

indicator strain was swabbed on the Nutrient soft agar plate (0.75% agar). Then the square

cut out of the dried films (10 mm), which were placed under the UV light for 1 hour, were

placed onto the agar plate. The plates were incubated at 37oC for 24-48 hours or until

growth is visible. Observations of the diameter of the inhibitory zone surrounding film and

contact area of edible film with agar surface were made. Experiments were done in

triplicate.

33

3.4.14 Shelf-life of fermented pork which was wrapped with bacteriocin-coated cassava

starch film

Lean pork (500 g) was brought from supermarket. The lean pork was washed in

water and then dried with paper towel. The pork was minced and then chilled in the freezer

(-18°C). The cooked pork rind (250 g) was cleaned and cut into small and long strips. The

Nam Powder Seasoning Mix together with Nam salt into the chopped pork. The mixture

was kneaded quickly together while the pork was still cold. Pork rind, garlic and bird chili

were kneaded together. The mixture was called the fermented pork. Fermented pork was

weighted 5 g per piece. The fermented pork was then wrapped with plastic bag as a control

and wrapped with three types of edible film which are cassava starch film, cassava starch

film incorporated with the whole extracts and cassava starch film incorporated with

partially purified bacteriocin. The samples were kept at 4°C in refrigerator. The samples

were taken at sampling time. Experiments were done in triplicate.

For sampling, the sample was taken and removed all cover with aseptic technique

and then put into 45 ml of 0.1% sterile peptone water in plastic bag. The sample was mixed

together by stomacher for 1 min. Samples (100 μl) were taken and spread on plate count

agar (PCA). The plates were incubated at 37°C for 48 hours.

3.4.15 Shelf-life of orange juice which was added partially purified bacteriocin as

biopreservative and compared with orange juice which was added Sodium

benzoate as chemical preservative

The orange juice was made from fresh orange which bought from market. The

orange juice was separated into five study treatments.

First treatment : without partially purified bacteriocin (Control)

Second treatment : 0.1% Sodium benzoate

Third treatment : 0.5% partially purified bacteriocin

Forth treatment : 1% partially purified bacteriocin

Fifth treatment : 2.5% partially purified bacteriocin

The 10 ml of orange juice was dispensed into the sterile glass tube. The tubes were

sealed using polypropylene screw cap. The samples were pasteurized at 90°C for 15

second and then cooled down immediately by put the samples into ice water bath. The

samples were stored at room temperature (25°C). The samples were taken at sampling time

for pH measurement and microbiological testing. Experiments were done in triplicate.

For pH measurement, the samples were taken at sampling time. The pH of the

sample was measured by pH meter (Portamess, Germany).

34

For microbiological testing, the samples were taken at sampling time. 100 μl of

sample were taken and spread on Plate Count Agar (PCA). The plates were incubated at

37°C for 48 hours.

Figure 3.6 Flow-chart of orange juice experiment

3.4.16 Shelf-life of pasteurized milk which was added partially purified bacteriocin as

biopreservative

The pasteurized milk (CP-Meiji) was bought from the convenience store (108 shop

in AIT, Thailand). The pasteurized milk was separated into four study treatments to study

the effect on milk quality during refrigerated storage.

First treatment : without partially purified bacteriocin (Control)

Orange

juice

Orange juice without

bacteriocin (Control)

Orange juice with

0.5% bacteriocin

Orange juice with 0.1%

Sodium benzoate

Orange juice with

2.5% bacteriocin

Orange juice with

1% bacteriocin

Pasteurization at

90°C for 15 seconds

Store at room temperature

25°C

Take the sample

Cool down

pH measurement Microbiological testing

35

Second treatment : 0.5% partially purified bacteriocin

Third treatment : 1% partially purified bacteriocin

Forth treatment : 2.5% partially purified bacteriocin

The pasteurized milk was aseptically dispensed into the sterile glass tube about 10

ml/tube. The tubes were sealed using polypropylene screw cap. The samples were stored at

4°C in the refrigerator. The samples were taken at sampling time for pH measurement and

microbiological testing. Experiments were done in triplicate.

For pH measurement, the samples were taken at sampling time. The pH of sample

was measured by pH meter (Portamess, Germany).

For microbiological testing, the samples were taken at sampling time. 100 μl of

samples were taken and spread on Plate Count Agar (PCA). The plates were incubated at

37°C for 24-48 hours.

Figure 3.7 Flow-chart of pasteurize milk experiment

Pasteurize milk

Pasteurize milk

without bacteriocin

(Control)

Pasteurize milk

0.5% bacteriocin

Pasteurize milk

2.5% bacteriocin

Pasteurize milk

1% bacteriocin

Store at 4°C

Take the sample

pH measurement Microbiological testing

36

CHAPTER 4

RESULT AND DISCUSSION

4.1 Isolation and identification of Lactic acid bacteria (LAB) from turmeric rhizome

The MRS medium was used to isolate the lactic acid bacteria (LAB) from fresh

turmeric rhizome. All of the LAB were selected from the colonies that developed on MRS

plus bromocresal purple agar of which media color changed to yellow (representative to

acid production). The pure colonies were primarily identified according to their Gram-

staining reaction and catalase test. Further identification was carried out by using API kits

(bioMérieux SA, Marcy l'Etoile, France) to identify the genera and species. API 50 CHL

intended for the identification of the genus Lactobacillus and related species by

examination of carbohydrates fermentation. API 20 Strep was demonstrated of

Streptococcus of Enterococcus by enzymatic activity or fermentation of sugars. There were

three strains of LAB originate from turmeric: Lactobacillus plantarum, Enterococcus

faecium and Lactococcus lactis subsp. lactis as shown in figure 4.1 (i), (ii), (iii),

respectively.

(i) Lactobacillus plantarum morphology: (ii) Enterococcus faecium morphology:

Long-rods shape Cocci shape

(iii) Lactococcus lactis subsp. lactis morphology:

Short-rods shape

Figure 4.1 Cell morphology of lactic acid bacteria isolated from fresh turmeric rhizomes in

phase contrast microscopy, under 1000 magnification

37

4.2 Growth of L. plantarum and L. casei TISTR 1463

The L. plantarum and L. casei TISTR 1463 used in this study were able to grown in

MRS medium without pH control. The L. plantarum and L. casei TISTR 1463 were

inoculated from stock culture into fresh MRS broth and statically subcultured more than

three times before experiments. Growth in terms of changes in optical density of cell was

observed at regular time intervals. The data of optical density and viable cell count is

shown in Appendix C1. The growth curve of L. plantarum and L. casei TISTR 1463 is

shown in figure 4.2. The growth of both strains was observed that they were very short lag

phage at the initial time indicating that the growth conditions were optimum for the

microorganisms. After 2 h of lag phase period, the cells initially adjusted to the new

environment in the batch culture. The optical density intensively increased during the

exponential phase or log phase. Both of L. plantarum and L. casei TISTR 1463 reached the

end of log-phase at 16 h after inoculation. The cell concentration in terms of CFU/ml of L.

plantarum and L. casei TISTR 1463 at the end of log phase were 2.23 × 1011

and 1.97 ×

1011

cfu/ml, respectively. Form figure 4.2, the optical density data shows the similar trends

for both L. plantarum and L. casei TISTR 1463 as with the colony counting as shown in

fugure 4.2.

Figure 4.2 Batch culture profile of Lactobacillus plantarum and Lactobacillus casei in

MRS medium at 37°C. : growth curve of L. plantarum at absorbance 600 nm;

. : growth cure of L. casei at absorbance 600 nm; : viable cell count of L.

plantarum; : viable cell count of L.casei.

38

4.3 Antimicrobial and bacteriocin activities

The Lactic acid bacteria (LAB) can produce antimicrobial substances with the

capacity to inhibit the growth of pathogenic and spoilage microorganisms. Organic acids,

hydrogen peroxide, diacetyl and bacteriocins are included among these compounds

(Daeschel, 1989).

Antimicrobial activity of L. plantarum and L. casei TISTR 1463 were tested against

indicator strains (Escherichia coli TISTR 780, Salmonella typhimurium TISTR 292 and

Staphylococcus aureus TISTR 029).The agar spot test was used to screen the antimicrobial

activity of both strains. The diameters of inhibition were included between 16 to 20 mm.

Bacteriocin activities of both strains were tested against the same indicator strains.

The agar well diffusion method was used to screen the bacteriocin activity. The

supernatants of both stains at vary temperatures and times were taken and adjusted to pH

6.5 and added catalase enzyme. The bacteriocin activity was determined by checking the

inhibition zone produced by both of Lactobacillus strains in a plate of indicator strains. In

this experiment the sample was taken every two hours from 16 hours after inoculation to

26 hours after inoculation and the result found that L. plantarum produced the highest

bacteriocin at 18 h and L. casei produced the highest bacteriocin at 24 h. The temperature

of culture also affects the bacteriocin production. It was found that L. plantarum produced

the highest bacteriocin at 37°C, L. casei TISTR 1463 produced showed the highest

bacteriocin at 30°C. The best result of both strains is shown in table 4.1 and their data is

included in Appendix C2. As shown in table 4.1, the diameter of inhibition zone of both

strains for sample with and without pH adjustment and catalase enzyme were different

because for sample without pH adjustment and without catalase enzyme has an effect of

acid condition and hydrogen peroxide.

Table 4.1 Inhibition zones by Agar well diffusion test

strain

Diameter of inhibition zone of

L. plantarum (mm) (18 h, 37°C)

Diameter of inhibition zone of

L. casei (mm) (24 h, 30°C)

No pH

adjustment and

no catalase

enzyme

With pH

adjustment and

catalase

enzyme

No pH

adjustment and

no catalase

enzyme

With pH

adjustment and

catalase

enzyme

E. coli TISTR 780 18.0 ± 0.00 10.5 ± 0.50 18.0 ± 1.00 10.0 ± 0.87

S. typhimurium

TISTR 292 18.0 ± 0.00 10.0 ± 0.00 17.5 ± 1.04 -

S. aureus TISTR

029 18.0 ± 0.00 10.0 ± 1.00 18.0 ± 0.00 10.0 ± 1.00

39

4.4 Purification of bacteriocin

From the part of bacteriocin activity, L. plantarum were chosen to purify the

bacteriocin. To confirm the time that L. plantarum produced the highest bacteriocin, 50 ml

of sample were took every two hour from 16 h to 26 h. The sample was take the two major

steps for purification of bacteriocin were used in this study. The first step was ammonium

sulphate precipitation and follow by dialysis through 1000 molecular weight-cut-off-

dialysed membrane. Then bacteriocin activity test were used to test. The result was shown

in table 4.2. After purification step, the bacteriocin from L. plantarum and L. casei TISTR

1463 did not have activity against indicator strains (Escherichia coli TISTR 780,

Salmonella typhimurium TISTR 292, Staphylococcus aureus TISTR 029 and Listeria

monocytogenes). However, the bacteriocin of L. plantarum showed the activity against L.

casei TISTR 1463.

According to the result of another study (Leal et al., 1998) on the ability of the

Lactobacillus plantarum LPCO10 strain to produce bacteriocin, the activity of bacteriocin

was not detected either in the culture supernatants or in the ammonium sulphate

precipitated and 40-fold concentrated samples. However, it was detected after passing the

concentrated samples through HIC columns.

From table 4.2, the result was shown that L. plantarum produce the highest

bacteriocin at 18 h. Figure 4.3 is shown the two-fold serial dilution of partially purified

bacteriocin from L. plantarum at 18 h. The partially purified bacteriocin inhibited the

indicator strain up to ten-time of two-flow serial solution. The partially purified bacteriocin

activity is 5,120 AU/ml, when tested with L. casei TISTR 1463.

Table 4.2 The bacteriocin activity of L. plantarum at 16, 18, 20, 22, 24 and 26 h against

L. casei TISTR 1463

Time (hour) Bacteriocin activity (AU/ml)

16 2,560

18 5,120

20 2,560

22 2,560

24 2,560

26 2,560

40

Figure 4.3 Bacteriocin activity of L. plantarum against L. casei TISTR 1463 from 18 h of

culture

4.5 Effect of temperature and pH on bacteriocin activity

The partially purified bacteriocin from L. plantarum which was purified by

ammonium sulphate precipitation and followed by dialysis through 1000 molecular

weight-cut-off-dialysed membrane was used in this experiment. The partially purified

bacteriocin was treated with various temperatures and pH. The effect of temperature and

pH on bacteriocin activity is necessary to study because in some food processing is needed

heat treatment or low pH. The heat stable and the pH stable of bacteriocin was very useful

characteristic in case of using bacteriocin as a food preservative. Therefore it is necessary

to study both effects on bacteriocin activity.

The effects of temperature and pH on the partially purified bacteriocin from L.

plantarum for its activity are presented in Appendix C3 and C4, respectively. Figure 4.4

shows the effect of temperature on bacteriocin activity. The bacteriocin activity of L.

plantarum was not altered by the heat treatment after 60 min at 40°C, 60°C and 80°C, and

after 10 min at 100°C. The bacteriocin activity was found decreasing at 100°C only after

10 min of incubation. The pasteurization temperature has not shown the effect on the

bacteriocin activity. Figure 4.5 shows the effect of pH on bacteriocin activity. The

bacteriocin produced by L. plantarum was completely stable at pH 5 and pH 7, and 50 %

of bacteriocin activity remained after subjection to the pH 3 and pH 9. The experiment was

done in triplicate and found that no significant difference (p˂0.05).

41

Figure 4.4 Effect of temperature on partially purified bacteriocin activity, extracted from

L. plantarum isolated from turmeric rhizome

Figure 4.5 Effect of pH on partially purified bacteriocin activity, extracted from L.

plantarum from turmeric rhizome

42

4.6 Determination of molecular mass of bacteriocins

Electrophoretic analysis was performed with partially purified bacteriocins

extracted by ammonium sulphate. In this experiment, SDS-polyacrylamide gel

electrophoresis was carried out with a discontinuous buffer system. The most cases, SDS-

polyacrylamide gel electrophoresis is carried out with a discontinuous buffer system in

which the buffer in the reservoirs is of a pH and ionic strength different from that of the

buffer used to cast the gel. The SDS- polypeptide complexes in the sample that is applied

to the gel are swept along by a moving boundary created when an electric current is passed

between the electrodes (Sambrook and Russell, 2001). The sample and the stacking gel

contain Tris-Cl (pH 6.8), the upper and lower buffer reservoirs contain Tris-glycine (pH

8.3), and the resolving gel contains Tris-Cl (pH 8.8).

The result of the one part of SDS-polyacrylamide gel electrophoresis did not give

definite bands when stained the gel in Coomassie blue G-250. However another part of

SDS-polyacrylamide gel electrophoresis was done in the post-electrophoretic detection

analysis and found that the partially purified bacteriocin was active against the indicator

strain L. casei TISTR 1463. The result shows the two clear inhibition bands which

corresponded to a molecular mass of approximately 1-2 kDa and 12-14 kDa. The

photograph of the result is shown in figure 4.6.

According to the result of another study (Hata et al., 2010) on a new bacteriocin

produced by L. plantarum A-1, the molecular mass of plantaricin ASM1 which was

analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

analysis showed a mass of 5045.7 Da.

43

Figure 4.6 Molecular mass determination by SDS-PAGE

(A) Polypeptide SDS-PAGE Molecular Weight Standards of one part of SDS-

polyacrylamide gel electrophoresis extracts peptide from the L. plantarum

(B) The clear inhibition bands of the protein in another part of SDS-

polyacrylamide gel electrophoresis which overlaid with L. casei TISTR

1463

4.7 The physical properties of edible film based on cassava starch

The cassava starch films were prepared by casting method. The sample of the dried

cassava starch films is shown in figure 4.7.

Molecular weight

(Daltons)

14,437

6,512

3,496

1,423

M 1

A

B

Inhibition zone

12-14 kDa

1-2 kDa

44

Figure 4.7 Appearance of edible films based on cassava starch

(A) Cassava starch

(B) Cassava starch film incorporated with the whole extracts

(C) Cassava starch film incorporated with partially purified bacteriocin

4.7.1 Film thickness of edible film based on cassava starch

The thickness of edible films is an important parameter since it directly affects the

physical properties and biological properties of the coated food. The data of film thickness

is shown in table 4.3 (Appendix C5). The cassava starch film incorporated with the whole

extracts has the most thickness. The supernatant of L. plantarum which was added into the

cassava starch film affects on the thickness of the film. Moreover, the addition of partially

purified bacteriocin also increases the thickness of the film. Iamareeray (2010) made an

edible film from cassava starch. The thickness of cassava starch film which was made by

Iamareeray is 0.29 ± 0.02 mm.

Table 4.3 The thickness of edible film based on cassava starch

Type of edible film Thickness (mm)

Cassava starch film 0.100 ± 0.005

Cassava starch film incorporated with the whole extracts 0.152 ± 0.006

Cassava starch film incorporated with partially purified 0.115 ± 0.004

4.7.2 Scanning Electron Microscope analysis (SEM)

The surface microstructure of three types of cassava starch film was studied by

Scanning electron microscope analysis (SEM). The samples were prepared on copper stubs

and fixed with double-sided tape. The samples were coated with copper, and then the

samples were observed with Scanning electron microscope.

Figure 4.8 illustrates small ridges all over the cassava starch film. The cracks and

pores did not appear on the cassava starch film. The surface of film was uniform. The

A B C

45

result of the surface of cassava starch film in this research is similar to the finding of

Iamareeray, 2010.

Figure 4.9 illustrates a lot of small ridges all over the cassava starch film

incorporated with partially purified bacteriocin. The pores and cracks were also not

observed with this film. The surface of cassava starch was rougher than the surface of

cassava starch film incorporated with partially purified bacteriocin.

In case of the cassava starch film incorporated with the whole extracts from L.

plantarum, the machine could not zoom more than 60 magnification because the effect of

component in the whole extracts. In the supernatant of L. plantarum has a lot of remaining

nutrients and various substances which are synthesized by L. plantarum. Figure 4.10 shows

that the surface of cassava starch film incorporated with the whole extracts from L.

plantarum had rough surface with bulges. The bulges may occur due to the presence of

various polymers and their interaction in the film, and also the effect of electron wave from

electron gun. However, at the right corner of figure 4.10 shows the surface of the edible

film which has a little effect of electron wave.

Figure 4.8 Scanning electron micrograph of the surface of cassava starch films (A) cassava

starch film at 2000 magnification (B) cassava starch film at 5000 magnification

A B

46

Figure 4.9 Scanning electron micrograph of the surface of cassava starch films

incorporated with partially purified bacteriocin (A) cassava starch film

incorporated with partially purified bacteriocin at 1500 magnification (B)

cassava starch film incorporated with partially purified bacteriocin at 4000

magnification

Figure 4.10 Scanning electron micrograph of the surface of cassava starch film

incorporated with the whole extracts from L. plantarum at 60 magnification

4.8 Tensile strength and Elongation at break of edible film based on cassava starch

The tensile strength and percentage elongation of cassava starch film, cassava

starch film incorporated with the whole extracts and cassava starch film incorporated with

A B

47

partially purified bacteriocin were measured by Texture analyzer (Model TA.XT2 Texture

Technologies Corp., NY. USA). The tensile strength and elongation at break of the films are

highly related with film composition, plasticizer, its concentration, and the pH of film-

forming solution (Jia et al., 2009). The data of tensile strength and percentage elongation of

various types of cassava starch film are given on figure 4.11 and 4.12, respectively and also shown

in table 4.4 and in Appendix C6.

Figure 4.11 illustrates that the tensile strength (TS) of the cassava starch film

decreased when the cassava starch film incorporated with the whole extracts. On the other

hand, the tensile strength (TS) of the cassava starch film incorporated with partially

purified bacteriocin increases from the cassava starch. Therefore the substances that added

into the film affect the tensile strength of the film. The result of this experiment shows that

the addition of the whole extracts into cassava starch significantly (p<0.05) reduced the

tensile strength (TS) and the addition of bacteriocin into the cassava starch film was not

significant difference at 95% level of confidence when compared with cassava starch film.

Decrease in tensile strength (TS) may result from the weaker intermolecular interactions of

the composition of the film.

Figure 4.12 illustrates that the percentage elongation at break (%EB) of the cassava

starch film incorporated with the whole extracts increased from the cassava starch film.

The result shows that the addition of the whole extracts significantly (p<0.05) increased

elongation at break (%EB). On the other hand, the addition of bacteriocin into the cassava

starch film was not significant difference at 95% level of confidence when compared with

cassava starch film.

Table 4.4 The tensile strength and elongation at break of various types of edible film based

on cassava starch

Type of edible film

Tensile strength

(MPa)

Elongation at break

(%)

Cassava starch film 3.43a ± 0.15 41.61

a ± 6.32

Cassava starch film incorporate with the

whole extracts 0.56b ± 0.10 197.12

b ± 3.93

Cassava starch film incorporate with

bacteriocin 3.88a ± 0.20 50.60

a ± 3.33

48

Figure 4.11 Tensile strength of cassava starch films, cassava starch film incorporated with

the whole extracts and cassava starch films incorporated with partially

purified bacteriocin

Figure 4.12 Elongation at break of cassava starch films, cassava starch film incorporated

with the whole extracts and cassava starch films incorporated with partially

purified bacteriocin

49

4.9 Water vapor transmission and water vapor transmission rate of edible film based

on cassava starch

It is well known that a proper barrier to water vapor would have a significant effect

on the shelf-life of food product. Water vapor transport properties of edible film packaging

materials are often influenced by edible film composition. The water transport properties

of packaging materials are responsible for the product quality deterioration and shelf-life

reduction.

The water vapor transferred through the film was determined according to ASTM.

The film water vapor transmission (WVT) and water vapor transmission rate (WVTR)

were calculated according to equation in the section 3.4.12.2. The results of WVT and

WVTR are shown in table 4.5 and in Appendix C7.

Figure 4.13 and 4.14 Illustrate that the whole extracts and partially purified

bacteriocin which added to the film affected the water vapor transmission (WVT) and

water vapor transmission rate (WVTR). The water vapor transmission (WVT) and water

vapor transmission rate (WVTR) of cassava starch film was significant difference at 95%

level of confidence. The addition of the whole extracts and partially purified bacteriocin

into the cassava starch film increase significantly (p<0.05) the water vapor transmission

(WVT) and water vapor transmission rate (WVTR). However the water vapor transmission

(WVT) and water vapor transmission rate (WVTR) of cassava starch film incorporated

with the whole extracts and cassava starch film incorporated with partially purified

bacteriocin were not significant difference at 95% level of confidence. The increasing in

water vapor transmission and water vapor transmission rate mean the structure of the film

has more porous.

Table 4.5 Water vapor transmission (WVT) and water vapor transmission rate (WVTR) of

various types of edible film based on cassava starch

Type of edible film WVT

(g H2O mm cm-2

)

WVTR

(g H2O mm h-1

cm-2

)

Cassava starch 0.0007846a ± 0.0000641 0.0003923

a ± 0.0000321

cassava starch film

incorporated with the whole

extracts

0.0010239b ± 0.0000703 0.0005119

b ± 0.0000351

Cassava starch film

incorporated with partially

purified bacteriocin

0.0009789b ± 0,0000390 0.0004895

b ± 0.0000195

50

Figure 4.13 Water vapor transmission (WVT) of edible film based on cassava starch

Figure 4.14 Water vapor transmission rate (WVTR) of edible film based on cassava starch

51

4.10 Effect of antimicrobial of edible film based on cassava starch on bacteria strains

One of the best properties of the edible film is the antimicrobial packaging. The

antimicrobial packaging is a system that can kill or inhibit the growth of microorganisms.

The growth of microorganism is the major problem of food spoilage leading to shorten

shelf life, quality degradation, change in nature of microflora and pathogen problem. The

experiment in this part was performed to test the efficiency of three types of cassava starch

film. The antimicrobial activity of three types of cassava starch film was determined by

agar diffusion method. The inhibitory microorganism strains in this study were Escherichia

coli TISTR 780, Salmonella typhimurium TISTR 292, Staphylococcus aureus TISTR 029,

and Listeria monocytogenes. All of these strains are caused the spoilage of foods and food

poisoning. Table 4.6 shows the data of antimicrobial activity of three types of cassava

starch film on the indicator strains.

The control film is the cassava starch film. The antimicrobial activity of cassava

starch film did not have the property to inhibit the growth of Escherichia coli TISTR 780,

Salmonella typhimurium TISTR 292, Staphylococcus aureus TISTR 029 and Listeria

monocytogenes.

The cassava starch film incorporated with the whole extracts active against all of

four types of indicator strains. When the films were tested with Escherichia coli TISTR

780, the inhibition zones were 14.77 ± 0.58 mm. The inhibition zones of Salmonella

typhimurium TISTR 292 were 13.50 ± 0.50 mm. For Staphylococcus aureus TISTR 029,

the inhibition zones were the range of 16.83 ± 0.76 mm. In case of the inhibition zone of

Listeria monocytogenes were 21.5 ± 0.50 mm.

The cassava starch film incorporated with partially purified bacteriocin did not

active against all of four types of indicator strains. However it does not mean that the

cassava starch film incorporated with partially purified bacteriocin is not good

antimicrobial packaging. Because in this experiment used only 4 indicator strains, it is not

cover all microorganism which cause the foodborne spoilage.

52

Table 4.6 Antimicrobial activity of edible film based on cassava starch on the indicator

strains

Type of edible film Antimicrobial activities

Inhibition zone (mm)

Escherichia coli

TISTR 780

Salmonella

typhimurium

TISTR 292

Staphylococcus

aureus TISTR

029

Listeria

monocytogenes

Cassava starch - - - -

cassava starch film

incorporated with

the whole extracts 14.77 ± 0.58 13.50 ± 0.50 16.83 ± 0.76 21.5 ± 0.50

Cassava starch film

incorporated with

partially purified

bacteriocin - - - -

4.11 Effect of antimicrobial packaging in fermented pork

Cassava starch films were used to produce edible packaging and apply on freshly

prepared fermented pork for prolonging the shelf life. The fermented pork is the popular

product in Thailand. The fermented pork was wrapped with the various types of edible

packaging which are cassava starch film, cassava starch film incorporated with the whole

extracts, and cassava starch film incorporated with partially purified bacteriocin. For the

control, the fermented pork was wrapped with plastic bag packaging. The fermented pork

was stored in the refrigerator at 4°C.

The samples were taken at 0, 1, 3, 7 and 10 days for evaluation of the growth of

microorganisms. Plate Count Agar (PCA) was used in this experiment to observe the

number of microorganisms. Table 4.7 shows the number of microorganism in the

fermented pork. At the beginning time, it has large quantity of microorganisms. After one

day, the amount of microorganism decreased. This is because the spicy in the ingredient

affects the growth of microorganism. However some bacteria still survived in the

fermented pork. As seen from the slowly increasing of the quantity of microorganisms

after one day.

Figure 4.15 shows the number of microorganism versus time of plastic bag

packaging, cassava starch film, cassava starch film incorporated with the whole extracts

and cassava starch film incorporated with partially purified bacteriocin. The data of 0 day

did not show on this graph. From the graph, it is indicated that the amount of

microorganisms in the fermented pork which wrapped with plastic bag has the highest

number of microorganisms. On the other hand, the cassava starch film incorporated with

the whole extracts can inhibit or slow down the growth of microorganism in the fermented

pork product.

53

In case of the texture of the fermented pork, the texture of the fermented pork

which was wrapped with various types of edible film based on cassava starch has a

problem on the quiet dry texture when compare with the texture of the fermented pork

which was wrapped with plastic bag after 10 days. Therefore the experiment was done

only 10 days because the barrier property problem of edible film. It can summarize that the

cassava starch film incorporated with the whole extracts can inhibit or slow down the

growth of microorganisms, but it cannot prevent the migration of water from the food

product to the atmosphere.

Table 4.7 the number of microorganism in the fermented pork which were wrapped with

plastic bag and three type of edible films based on cassava starch

Time Number of microorganism (CFU/ml)

(day) plastic bag cassava starch film

cassava starch film

incorporated with

the whole extracts

Cassava starch

incorporated with

partially purified

bacteriocin

0 2.00×105±4.2×10

4 2.00×10

5±4.2×10

4 2.00×10

5±4.2×10

4 2.00×10

5±4.2×10

4

1 8.53×103±3.0×10

3 1.24×10

4±2.7×10

3 9.63×10

3±2.5×10

3 1.42×10

4±7.2×10

3

3 1.10×104±3.4×10

3 1.33×10

4±2.5×10

3 3.47×10

3 ± 1.3×10

3 5.83×10

3±4.8×10

3

7 1.23×104±3.4×10

3 1.07×10

4±2.1×10

3 5.89×10

3 ± 3.1×10

3 1.07×10

4±2.6×10

3

10 2.32×104±3.8×10

3 1.37×10

4±3.3×10

3 5.50×10

3 ± 9.2×10

2 1.32×10

4±1.7×10

3

Figure 4.15 The number of microorganism versus time of plastic bag and three types of

edible films based on cassava starch

54

4.12 Effect of bacteriocin in orange juice

The partially purified bacteriocin from L. plantarum was used in this study to

determine the ability of bacteriocin to prolong the shelf-life of the orange juice. In this

experiment, the partially purified bacteriocin was compared with sodium benzoate in case

of shelf life of product. Sodium benzoate usually use as chemical preservative in the food

industrial to avoid the product contamination from microorganism. The orange juice was

separated into five study treatments which were orange juice (control), orange juice with

0.1% sodium benzoate, orange juice with 0.5% partially purified bacteriocin, orange juice

with 1.0% partially purified bacteriocin and orange juice with 2.5% partially purified

bacteriocin. The orange juice was kept in the room temperature (25°C). The samples were

taken at the interval time during the period of storage to determine the pH and the amount

of microorganism. The data of pH of orange juice and the amount of microorganisms

shows in table 4.8 and 4.9, respectively.

As shown in Table 4.8, it shows that the pH of all types of the samples at the

beginning time was quiet difference. The pH values at the beginning time of pasteurized

orange juice, pasteurized orange juice with 0.1% sodium benzoate, pasteurized orange

juice with 0.5% bacteriocin, pasteurized orange juice with 1.0% bacteriocin and

pasteurized orange juice with 2.5% bacteriocin were 3.36 ± 0.0058, 3.64 ± 0.0058, 3.40 ±

0.0058, 3.40 ± 0.0059 and 3.42 ± 0.0058, respectively. During periods of storage, the pH

values of pasteurized orange juice did not change much from the beginning time, but in

case of the others had little change from the beginning time

In case of microbiological testing, the samples were taken and spread on Plate

Count Agar (PCA) plate and incubated at 37°C. The microorganisms were first found in

pasteurized orange juice after 10 days of storage periods. For pasteurized orange juice with

0.1% sodium benzoate found microorganism after 36 days of period storage and after that

the microorganism was not detected. In case of pasteurized orange juice with 0.5%, 1.0%

and 2.5% partially purified bacteriocin were not detected any microorganism on PCA

plate. However, it does not mean that all of the microorganisms can be detected from the

sample because the PCA is not suitable for all types of microorganisms. From this part, it

could summarize that pasteurized orange juice tends to spoilage first.

55

Table 4.8 The pH value of various types of orange juice during storage period

Time

(day)

pH

Pasteurized

orange juice

Pasteurized

orange juice

with 0.1%

Sodium

benzoate

Pasteurized

orange juice

with 0.5%

partially

purified

bacteriocin

Pasteurized

orange juice

with 1.0%

partially

purified

bacteriocin

Pasteurized

orange juice

with 2.5%

partially

purified

bacteriocin

0

3.36 ±

0.0058 3.64 ± 0.0058 3.40 ± 0.0058 3.40 ± 0.0059 3.42 ± 0.0058

1

3.36 ±

0.0115 3.66 ± 0.0058 3.39 ± 0.0058 3.39 ± 0.0058 3.41 ± 0.0000

3

3.37 ±

0.0100 3.65 ± 0.0000 3.38 ± 0.0100 3.40 ± 0.0000 3.42 ± 0.0000

6

3.38 ±

0.0058 3.67 ± 0.0153 3.38 ± 0.0100 3.40 ± 0.0100 3.42 ± 0.0153

10

3.36 ±

0.0100 3.67 ± 0.0058 3.41 ± 0.0153 3.41 ± 0.0115 3.42 ± 0.0153

13

3.37 ±

0.0000 3.66 ± 0.0058 3.41 ± 0.0058 3.41 ± 0.0100 3.41 ± 0.0058

21

3.37 ±

0.0058 3.66 ± 0.0058 3.41 ± 0.0058 3.41 ± 0.0058 3.42 ± 0.0058

29

3.36 ±

0.0058 3.68 ± 0.0100 3.41 ± 0.0068 3.41 ± 0.0000 3.42 ± 0.0000

36

3.40 ±

0.0379 3.71 ± 0.0153 3.45 ± 0.0153 3.45 ± 0.0208 3.45 ± 0.0100

42

3.37 ±

0.0000 3.70 ± 0.0058 3.43 ± 0.0100 3.44 ± 0.0200 3.46 ± 0.0153

56

Table 4.9 The number of microorganism in orange juice

Time

(day)

Number of microorganism (CFU/ml)

Pasteurized

orange juice

Pasteurized

orange juice

with 0.1%

Sodium

benzoate

Pasteurized

orange juice

with 0.5%

partially

purified

bacteriocin

Pasteurized

orange juice

with 1.0%

partially

purified

bacteriocin

Pasteurized

orange juice

with 2.5%

partially

purified

bacteriocin

0 - - - - -

1 - - - - -

3 - - - - -

6 - - - - -

10 6.67 ± 5.77 - - - -

21

20.00 ±

20.00 - - - -

29

60.00 ±

30.00 - - - -

36

13.33 ±

15.28 3 ± 5.77 - - -

4.13 Effect of bacteriocin in pasteurized milk

The partially purified bacteriocin from L. plantarum was used in this study to

determine the ability of bacteriocin to prolong the shelf-life of the dairy product.

Pasteurized milk is an example of dairy products which was used in this study. The

pasteurized milk (CP-Meiji) was bought from the convenience store (108 shop in AIT,

Thailand). The various concentrations of partially purified bacteriocin which were 0.5%,

1% and 2.5% were added into the pasteurized milk. The pasteurized milks were kept in the

refrigerator temperature at 4°C. The samples were taken at the interval time during the

period of storage. The samples were determined the pH and the amount of

microorganisms. The data of the pH and the amount of microorganisms shows in table 4.10

and 4.11, respectively.

Generally, the pH of most samples of milk is 6.6-6.8. In this study, the pH of

pasteurized milk was 6.72 at the beginning time. In case of pasteurized milk in table 4.10,

the pH value decreased lower than 6.6 in 35 days during storage. On day 41, the samples

were taken as usual but one tube of the pasteurized milk form a curd in the test tube.

However in cases of pasteurized milk with 0.5%, 1.0% and 2.5% partially purified

bacteriocin were not form a curd in the test tube and the pH value still stable in the range

of 6.71-6.72. From the pH measurement experiment of pasteurized milk, it can summarize

that the partially purified bacteriocin from L. plantarum has ability to prolong the shelf-life

of pasteurized milk.

57

In case of microbiological testing, the samples were taken and spread on Plate

Count Agar (PCA) plate and incubated at 37°C. This part of experiment did not successful

as hoped because the number of the microorganism found only some periods of time. This

may be because the PCA do not suitable for the microorganism in the pasteurized milk.

However from the table 4.11 could imply that the bacteriocin has effective against

microorganism in pasteurized milk because it was not found the microorganism on the

plate of the pasteurized milk sample which incorporated with partially purified bacteriocin.

Table 4.10 The pH value of various types of pasteurize milk during storage period

Time

(day)

pH

Pasteurized milk

Pasteurized milk

with 0.5%

partially purified

bacteriocin

Pasteurized milk

with 1.0%

partially purified

bacteriocin

Pasteurized milk

with 2.5%

partially purified

bacteriocin

0 6.72 ± 0.0000 6.72 ± 0.0000 6.72 ± 0.0000 6.72 ± 0.0000

3 6.71 ± 0.0231 6.72 ± 0.0000 6.72 ± 0.0000 6.72 ± 0.0153

8 6.72 ± 0.0058 6.72 ± 0.0058 6.73 ± 0.0153 6.73 ± 0.0100

16 6.67 ± 0.0100 6.71 ± 0.0058 6.71 ± 0.0000 6.73 ± 0.0058

23 6.69 ± 0.0058 6.71 ± 0.0058 6.73 ± 0.0058 6.73 ± 0.0058

31 6.63 ± 0.0379 6.69 ± 0.0115 6.69 ± 0.0100 6.70 ± 0.0000

35 6.55 ± 0.0153 6.71 ± 0.0100 6.71 ± 0.0058 6.72 ± 0.0058

41 6.43 ± 0.0208 6.71 ± 0.0100 6.72 ± 0.0058 6.72 ± 0.0058

Table 4.11 The number of microorganism in various types of pasteurize milk

Time

(day)

Number of microorganism (CFU/ml)

Pasteurized milk

Pasteurized milk

with 0.5%

partially purified

bacteriocin

Pasteurized milk

with 1.0%

partially purified

bacteriocin

Pasteurized milk

with 2.5%

partially purified

bacteriocin

0 - - - -

3 - - - -

8 - - - -

16 33.33 ± 5.77 - - -

35 16.67 ± 28.87 - - -

58

CHAPTER 5

CONCLUSIONS AND RECOMMENDATIONS

5.1 Conclusions

1. The antimicrobial activity of both L. plantarum and L. casei TISTR 1463 showed

significant antibacterial activities against indicator strains which were Escherichia

coli TISTR 780, Salmonella typhimurium TISTR 292 and Staphylococcus aureus

TISTR 029. In case of bacteriocin activity, the bacteriocin of both strains could

inhibit the growth of indicator strains but lesser than antimicrobial substance.

However, L. plantarum can produce more efficient than L. casei TISTR 1463.

2. The L. plantarum was chosen to purify the bacteriocin which was tested with L.

casei TISTR 1463 as indicator strain. The result shown that the activity of partially

purified bacteriocin from L. plantarum is 5,120 AU/ml.

3. The bacteriocin activity of L. plantarum was not affected when treated at 95°C for

10 min, and stable at pH 5 and pH 7.

4. The molecular weight of bacteriocin extracted from L. plantarum isolated from

turmeric rhizome is approximately 1-2 kDa and 12-14 kDa.

5. The surface of all type of edible film based on cassava starch was wrinkled with

small folding.

6. The addition of the whole extracts into the film significantly reduced the tensile

strength (TS) and increased elongation at break (%EB). On the other hand, there

was not significant difference on tensile strength (TS) and elongation at break

(%EB) incorporated with partially purified bacteriocin into the cassava starch film.

7. The addition of the whole extracts and the partially purified bacteriocin into the

cassava starch film increases significantly the water vapor transmission (WVT) and

water vapor transmission rate (WVTR).

8. The various types of cassava starch based film were applied on the fresh fermented

pork product. The result shows that the cassava starch film incorporated with the

whole extracts could inhibit the growth of microorganism. However the cassava

starch based film can be applied on the products for the short period because of the

barrier properties problem.

59

9. The partially purified bacteriocin from L. plantarum has ability to prolong the

shelf-life of product. In case of pasteurized orange juice and pasteurized milk, there

were not observed the growth of microorganism during period of storage on both

products when incorporated with bacteriocin. On the other hand, for the control of

both pasteurized orange juice and pasteurized milk, there were observed the growth

of microorganism during period of storage.

5.2 Recommendation for further studies

1. Isolation of new species of probiotic bacteria from non-dairy products which may

have more efficient antibacterial compounds of bacteriocin.

2. Alternate and improved method of the bacteriocin purification and quantification of

the purity.

3. Even though the cassava starch film incorporated with the whole extracts has more

effectiveness, it needs to improve the mechanical properties of the film.

4. Bacteriocin from L. plantarum could be studies more in case of increase the shelf-

life of product

60

REFERENCES

An, D. S., Kim, Y. M., Lee, S. B., Paik, H. D. and Lee, D. S. (2000). Antimicrobial low

density polyethylene film coated with bacteriocins in binder medium. Journal of

Food Science and Biotechnology, 9, 14-20.

Anal, A.K., Stevens, W.F. and Remuñán-López, C. (2006). Ionotropic cross-linked

chitosan microspheres for controlled release of ampicillin. International Journal

of Pharmaceutics, 312: 166-173.

Arthur, C. O. and Satu, V. (2004). Antimicrobial components from lactic acid bacteria. In

S. Salminen, A. V. Wright, & A. Ouwehand (Eds.), Lactic acid bacteria:

Microbiological and functional aspects (3rd

ed.) (pp.375-395). NY: Marcel Dekker,

Inc.

Baldwin, E. A., Nispero-Carriedo, M. O. and Baker, R. A. (1995). Edible coatings for

lightly processed fruits and vegetables. Horticultural Science, 30, 35-38.

Barefoot, S. F. and Klaenhammer, T. R. (1984). Purification and characterization of the

Lactobacillus acidophilus bacteriocin lactacin B. Antimicrobial Agents and

Chemotherapy, 26, 328-334.

Batdorj, B., Dalgalarrondo, M., Choiset, Y., Pedroche, J., Métro, F., Prévost, H., Chobert,

J.-M. and Haertlé. (2006). Purification and characterization of two bacteriocins

produced by lactic acid bacteria isolated from Mongolian airag. Journal of Applied

Mocrobiology, 101, 837-848.

Bhunia, A. K., Johnson, M. C. andRay, B., (1987). Direct detection of an antimicrobial

peptide of Pediococcus acidilactici in sodium dodecyl sulfate-polyacrylamide gel

electrophoresis. Journal Ind. Microbiology, 2, 319-322.

Bujalance, C., Jiménez-Valera, M., Moreno, E. and Ruiz-Bravo, A. (2006). A selective

differential medium for Lactobacillus plantarum. Journal of Microbiological

Methods, 66, 572-575.

Charteris, W. P., Kelly, P. M., Morelli, L. and Collins, J. K. (1997). Selective detection,

enumeration and identification of potentially probiotic Lactobacillus and

Bifidobacterium species in mixed bacterial populations. International Journal of

Food Microbiology, 35, 1-27.

Cleveland, J., Montcille, T. J., Nes, I. F. and Chikindas, M. L. (2001). Bacteriocins: safe,

natural antimicrobials for food preservation. International Journal of Food

Microbiology, 71, 1-20.

61

Coma, V., Sebti, I., Pardon, P., Deschamps, A. and Pichavant, F. H. (2001). Antimicrobial

edible packaging based on cellulosic ethers, fatty acids, and nisin incorporation to

inhibit Listeria innocua and Staphylococcus aureus. Journal of Food Protection,

64, 470-475.

Cooksey, D. K., Gremmer, A. and Grower, J. (2000). Characteristics of nisin-containing

corn zein pouches for reduction of microbial growth in refrigerated shredded

cheddar cheese. In Book of Abstracts, 2000 Annual Meeting, (pp. 188). Chicago,

IL: Institute of Food Technologists.

Cuozzo, S. A., Sesma, F., Palacios, J. M., de Ruiz Holgado, A. P. and Raya, R. R. (2000).

Identification and nucleotide sequence of genes involved in the synthesis of

lactocin 705, a two-peptide bacteriocin from Lactobacillus casei CRL 705. FEMS

Microbiology Letters, 185, 157-161.

Cutter, C. N., Willett, J. L. and Siragusa, G. R. (2001). Improved antimicrobial activity of

nisin-incorporated polymer films by formulation change and addition of food grade

chelator. Letters in Applied Microbiology, 33,325-328.

Daeschel, M.A. (1989). Antimicrobial substances from lactic acid bacteria for use as food

preservatives. Food Technol, 43, 164–166.

Dawson, P. L., Carl, G. D., Acton, J. C. and Han, I. Y. (2002). Effect of lauric acid and

nisin-impregnated soy-based films on the growth of Listeria monobytogenes on

turkey bolona, Poultry Science, 81, 721-726.

Dawson, P. L., Hirt, D. E., Rieck, J. R., Acton, J. C. and Sotthibandhu, A. (2003). Nisin

release from films is affected by both protein type and film-forming method. Food

Research International, 36, 959-968.

Debaufort, F., Quezada-Gallo, J. A. and Voilley, A. (1998). Edible films and coatings:

tomorrow’s packaging: a review. Critical Reviews in Food Science and Nutrition,

38, 299-313.

Deraz, S. F., Karlsson, E. N., Hedström, M., Andersson, M. M. and Mattiasson, B. (2005).

Purification and characterization of acidocin D20079, a bacteriocin produced by

Lactobacillus acidophilus DSM 20079. Journal of Biotechnology, 117, 343-354.

Gálvez, A., Abriouel, H., López, R. L. and Omar, N. B. (2007) Bacteriocin-based

strategies for food biopreservation. International Journal of Food microbiology,

120, 51-70.

Gilmore, M. S., Segarra, R. A., Booth, M. C., Bogie, C. P., Hall, L. R. and Clewell, D. B.

(1994). Genetic structure of the Enterococcus faecalis plasmid pAD1-encoded

62

cytolytic toxin system and its relationship to lantibiotic determinants. Journal of

Bacteriology, 176, 7335-7344.

Goktepe, I. (2006). Probiotic as biopreservatives for enhancing food safety. Probiotics in

food safety and human health. Boca Raton,. Florida: CRC Press.

Gonzalez, B., arca, P., Mayo, B. and Suarea, J. E. (1994). Detection, purification, and

partial characterization of plantaricin C, a bacteriocin produced by a Lactobacillus

plantarum strain of dairy origin. Applied and Environmental Microbiology, 60,

2158-2163.

Güçbilmez Ç. M. (2007). Antimicrobial and antioxidant activity of edible zein films

incorporated with lysozyme, albumin proteins and disodium EDTA. Food Research

International, 40, 80-91.

Guilbert, S. and Gontard, N. (1995). Edible and biodegradable food packaging. In P.

Ackermann, M. Jägerstad, & T. Ohlsson (Eds.), Foods and Packaging Materials –

Chemical Interactions (pp. 159-168). Cambridge: The Royal Society of Chemistry.

Guilbert, S., Gontard, N. and Gorris, L. G. M. (1996). Prolongation of the shelf-life of

perishable food products using biodegradable films and coatings. Lebensmittel-

Wissenschaft und-Technologie, 29, 10-17.

Han, J. H. (2002). Protein-based edible films and coatings carrying antimicrobial agents. In

A. Gennadios (Ed.), Protein-based Films and Coatings (pp. 485-499). Boca Raton,

Florida: CRC Press.

Han, J. H. (2003). Antimicrobial food packaging. In R. Ahvenainen (Ed.), Novel Food

Packaging Techniques (pp. 50-70). Cambridge: Woodhead Publishing Lts.

Han, J. H. (2005). Antimicrobial packaging systems. In J. H. Han (Ed.), Innovations in

Food Packaging (pp. 80-107). California: Elsevier Academic Press.

Han, J. H. and Gennadios, A. (2005). Edible films and coatings: a review. In J. H. Han

(Ed.), Innovations in Food Packaging (pp. 240-262). California: Elsevier Academic

Press.

Hata, T., Tanaka, R. and Ohmomo, S. (2010). Isolation and characterization of plantaricin

ASM1: A new bacteriocin produced by Lactobacillus plantarum A-1. International

Journal of Food Microbiology, 137, 94-99.

Hoffman, K. L., Han, I. Y. and Dawson, P. L. (2001). Antimicrobial effects of corn zein

films impregnated with nisn, lauric acid, and EDTA. Journal of Food Protection,

64, 885-889.

63

Holo, H., Jeknic, Z., Daeschel, M., Stevanovic, S. and Nes, I. F. (2001). Plantaricin W

from Lactobacillus plantarum belongs to a new family of two-peptide lantibiotics.

Microbiology, 147, 643-651.

Howard, L. R. and Gonzales, A. R. (2001). Food safety and produce operations: what is the

future? Horticultural Science, 36, 33-39.

Iamareerat, B. (2010). Reinforced cassava starch based edible film with essential oil and

clay nanoparticles. Thesis Report of Asian Institute of Technology, Bangkok.

Iseppi, R., Pilati, F., Marini, M., Toselli, M., de Niederhäusern, S., Guerrieri, E., Messi,

P., Sabia, C., Manicardi, G., Anacarso, I. and Bondi, M. (2008). Anti-listerial

activity of a polymeric film coated with hybrid coatings doped with Enterocin

416K1 for use as bioactive food packaging. International Journal of Food

Microbiology, 123, 281-287.

Jia, D., Fang, Y. and Yao, K. (2009). Water vapour barrier and mechanical properties of

konjac glucomanna-chitosam-soyprotein isolate edible films. Food and

Bioproducts Processing, 87, 7-10.

Joerger, M.C. and Klaenhammer, T.R. (1986). Characterization and purification of

helveticin J and evidence for a chromosomally determined bacteriocin produced by

Lactobacillus helveticus 481. Journal of Bacteriology, 167, 439-446.

Kamau, D. N., Doores, S. and Pruitt, K. M. (1990). Enhanced thermal destruction of

Listeria monocytogenes and Staphylococcus aureus by the lactoperoxidase system.

Applied and Environmental Microbiology, 56, 2711-2716.

Kester, J. J. and Fennema, O.R. (1986). Edible films and coatings: A review. Food

Technology, 48, 47-59.

Kim, Y. M., Paik, H. D. and Lee, D. S. (2002). Shelf-life characteristics of fresh oysters

and ground beef as affected by bacteriocins-coated plastic packaging film. Journal

of the Science of Food and Agriculture, 82, 998-1002.

King, A. D. J. and Nagel, C. W. (1975). Influence of carbon dioxide upon the metabolism

of Pseudomonas aeruginosa. Journal of Food Science, 40, 362-366.

Krochta, J. M. (2002). Proteins as raw materials for films and coatings: definitions, current

status, and opportunities. In A. Gennadios, (Ed). Protein-based Films and Coatings

(pp. 1-41). Boca Raton, Florida: CRC Press.

Kumari, A. and Garg, A.P. (2007). A bacteriocin from Lactococcus lactis CCSUB94

isolated from milk and milk products. Research Journal of Microbiology, 2, 375-

380.

64

Leal, M. V., Baras, M., Ruiz-Barba, J.L., Floriano, B. and Jiménez-Díaz, R. (1998).

Bacteriocin production and competitiveness of Lactobacillus plantarum LPCO10 in

olive juice broth, a culture medium obtained from olives. International Journal of

Food Microbiology, 42, 129-134.

Leer, R. J., van der Vossen, J. M., van Giezen, M., van Noort, J. M. and Pouwels, P. H.

(1995). Genetic analysis of acidocin B, a novel bacteriocin produced by

Lactobacillus acidophilus. Microbiology, 141, 1629-1635.

Lindgren, S. E. and Dobrogosz, W. J. (1990) Antagonistic activities of lactic acid bacteria

in food and feed fermentations. FEMS Microbiol. Rev, 87, 149-163.

Marcos, B., Jofré, A., Aymerich, T., Monfort, J. M. and Garriga, M. (2008). Combined

effect of natural antimicrobials and high pressure processing to prevent Listeria

monobytogenes growth after a cold chain break during storage of cooked ham.

Food Control, 19(1), 76-81.

Maisnier-Patin, S., Forni, E. and Richard, J. (1996). Purification, Partial characterization

and mode of action of enterococcin EFS2, an antilisterial bacteriocin produced by a

strain of Enterococcus faecalis isolated from a cheese. Journal of Food

Microbiology, 30, 255-270.

Millette, M., Tien, C. L., Smoragiewicz, W. and Lacroix, & M. (2007). Inhibition of

Staphylococcus aureus on beef by nisin-coataining modified films and beads. Food

Control, 18(7), 878-884.

Ming, X., Weber, G. H., Ayres, J. W. and Sandine, W. E. (1997). Bacteriocins applied to

food packaging materials to inhibit Listeria monobytogenes on meats. Journal of

Food Science, 62, 413-415.

Moorthy, S.N. (2004). Tropical sources of starch. In Eliasson, A.C., Starch in Food:

structure, function and applications, Woodhead Publishing Limited, England, pp.

326-334.

Muriana, P. (1996). Bacteriocins for control of Listeria spp. in food. Journal of Food

Protection, 56, 54-63.

Natrajan, N. and Sheldon, B. W. (1995). Evaluation of bacteriocin-based packaging and

edible film delivery systems to reduce Salmonella in fresh poultry. Journal of

Poultry Science, 74, 31.

Nissen-Meyer, J., Larson, A. G., Sletten, K., Daeschel, M. and Nes, I. F. (1993).

Purification and characterization of plantaricin A, a Lactobacillus plantarum

bacteriocin whose activity depends on the action of two peptides. Journal of

General Microbiology, 139, 1973-1978.

65

O’Connor, E. B., Barrett, E., Fitzgerald, G., Hill, C., Stanton, C. and Ross, R. P. (2005).

Production of vitamins, Exopolysaccharides and Bacteriocins by probiotic bacteria.

Probiotic Dairy Products. Oxford, UK: Blackerll Publishing Ltd.

Peyron, A. (1991). L’enrobage et les produits filmogenes: un nouveau mode d’emballage

(Coatings and product films: new method of packaging). Viandes Prod, 12, 41-46.

Pranoto, Y. (2004). Application of Alginate and Chitosan Edible Films Incorporating

Natural Antimicrobial Agents. Thesis Report of Asian Institute of Technology,

Bangkok.

Rößle, C., Auty, M. A. E., Brunton, N., Gormley, R. T. and Butler, F. (2010). Evaluation

of fresh-cut apple slices enriched with probiotic bacteria. Innovative Food Science

and Emerging Technologies, 11, 201-209.

Robertson, G. L. (2006). Edible and biobased food packaging materials. In G. L. Robertson

(Ed.), Food Packaging: Principles and Practice (pp. 43-54). Boca Raton, Florida:

CRC Press.

Rodríguez, M., Osés, J., Ziani, K. and Maté, J.I. (2006). Combined effect of plasticizers

and surfactants on the physical properties of starch based edible films. Journal of

Food Research International, 39(8): 840-846.

Roger, L. A. and Whitter, E. D., (1928). Limiting factors in lactic acid fermentation.

Journal of Bacteriology, 16, 211-229.

Sambrook and Russell, (2001). Molecular Cloning: A Laboratory Manual (3 rd ed.). Cold

Spring Harbor Laboratory Press. New York.

Sanjurjo, K., Flores, S., Gerschenson, L. and Jagus, R. (2006). Study of the performance of

nisin supported in edible films. Food Reasearch International, 39, 749-754.

Schägger, H. and von Jagow, G. (1987). Tricine-sodium dodecyl sulphate-polyacrilamide

gel electrophoresis for the separation of proteins in the range of 1 to 100 kDa.

Analytical Biochemistry,166, 368-379.

Sebti, I., Pichavant, F. H. and Coma, V. (2002). Edible bioactive fatty acid-cellulosic

derivative composites used in food-packaging applications. Journal of agricultural

and food chemistry, 50, 4290-4294.

Sindhu, S., Chempakam, B., Leela, N. K., Suseela Bhai, R. (2011). Chemoprevention by

essential oil of turmeric leaves (Curcuma longa L.) on the growth of Aspergillus

flavus and aflatoxin production. Food and chemical toxicology, 45(5), 1188-1192.

66

Sivarooban, T., Hettiarachchy, N. S. and Johnson, M. G. (2008). Physical and

antimicrobial properties of grape seed extract, nisin, ans EDTA incorporated soy

protein edible films. Food Research International, 41, 781-785.

Skaugen, M., Nissen Meyer, J., Jung G., Stevanovic, S., Sletten, K., Inger, C., Abildgaard,

M. and Nes, I.F. (1994). In vivo conversion of L-serine to D-alanine in a

ribosomally synthesized polypeptide. Journal of Biological Chemistry, 269, 27183-

27185.

Sothornvit, R. and Krochta, J. M. (2000). Oxygen permeability and mechanical properties

of films from hydrolyzed whey protein. Journal of Agricultural and Food

Chemistry, 48, 6298-6302.

Sothornvit, R. and Krochta, J. M. (2001). Plasticizer effect on mechanical properties of β-

lactoglobulin films. Journal of Food Engineering, 50, 149-155.

Thomas, L. V., Clatksom, M. R. and Delves-Broughton, J. (2000). Nisin. In A. S. Naidu

(Ed.), Natural food antimicrobial systems (pp.463-524). Boca-Raton, FL: CRC

press.

Vuyst, L. D., Avonts, L. and Makras, L. (2004). Probiotics, prebiotics and gut health. In C.

Remacle, & B. Reusens (Eds.), Functional foods, ageing and degenerative disease.

Florida: CRC Press LLC.

Villani, F., Salzano, G., Sorrentino, E., Pepe, O., Marino, P. and Coppola, S. (1993).

Enterocin 226NWC, a bacteriocin produced by Enterococcus faecalis 226, active

against Listeria monocytogenes. Journal Applied Bacteriology, 74, 380-387.

Were, L., Hettiarachcky, N. S. and Coleman, M. (1999). Properties of cysteine-added soy

protein-wheat gluten films. Journal of Food Science, 64, 514-518.

Wu, Y., Weller, C. L., Hamouz, F., Cuppett, S. L. and Schnepf, M. (2002). Development

and application of multicomponent edible coatings and films: a review. Advances in

Food and Nutrition Research, 44, 347-394.

Yin, L-J., WU. C-W. and Jiang. S-T. (2007). Biopreservative effect of pediocin ACCEL on

refrigerated seafood, Fisheries science. 73. 907-912.

Yildirim, M. and Hettiarachchy, N. S. (1997). Biopolymers produced by cross-linking soy

bean 11S globulin with whey proteins using transglutaminase. Journal of Food

Science, 62, 270-275.

Yildirim, Z. and Johnson, M. G. (1998). Characterization and antimicrobial spectrum of

bifidocin B, a bacteriocin produced by Bifidobacterium bifidum NCFB 1454.

Journal of Food Protection, 61, 47-51.

67

Zhong, Q.P. and Xia, W.S. (2008). Physicochemical properties of edible and preservative

films from chitosan/cassava starch/gelatin blend plasticized with glycerol. Food

Technol. Biotechnol.,46(3): 262-269.

68

APPENDICES

APPENDIX A

50 mM potassium phosphate buffer pH 7.0

Prepare a 500 mM K2HPO4 stock (100 ml)

K2HPO4 8.7090 g.

Adjust volume to 100 ml with distil water.

Prepare a 500 mM KH2PO4 stock (100 ml)

KH2PO4 6.8045 g

Adjust volume to 100 ml with distil water.

50 ml of 500 mM K2HPO4 stock waas adjusted pH to 7 by using 500 mM KH2PO4

stock. After that, the 500 mM potassium phosphate buffer pH 7.0 was diluted with

distil water until the final concentration was 50 mM potassium phosphate buffer pH

7.0.

69

APPENDIX B

Appendix B1: Preparation of SDS-polyacrylamide gel electrophoresis

16% Separating Gel

2.6 ml ddH2O

3.2 ml 40%Acrylamide

2 ml 1.5 M Tris pH 8.8

80μl 10% SDS

80μl 10% APS

8 μl TEMED

Making the separating gel

1. Begin with 2.6 ml ddH2O

2. Add 3.2 ml 40%Acrylamide/bis-acrylamide solution

3. Add 2 ml of 1.5 M Tris pH 8.8 and mix

4. Mix in 80 μl of 10% SDS

5. When ready to use, add 8 μl of TEMED and mix

6. Immediately before pouring the gel, add 80 μl of 10% APS and mix

10% Spacer Gel

3.8 ml ddH2O

2 ml 40%Acrylamide

2 ml 1.5 M Tris pH 8.8

80μl 10% SDS

80μl 10% APS

8 μl TEMED

Making the spacer gel

1. Begin with 3.8 ml ddH2O

2. Add 2 ml 40%Acrylamide/bis-acrylamide solution

3. Add 2 ml of 1.5 M Tris pH 8.8 and mix

4. Mix in 80 μl of 10% SDS

5. When ready to use, add 8 μl of TEMED and mix

6. Immediately before pouring the gel, add 80 μl of 10% APS and mix

4% Stacking Gel

3.1 ml ddH2O

0.5 ml 40%Acrylamide

1.25 ml 1.5 M Tris pH 8.8

50 μl 10% SDS

50 μl 10% APS

5 μl TEMED

70

Making the stacking gel

1. Begin with 3.1 ml ddH2O

2. Add 0.5 ml 40%Acrylamide/bis-acrylamide solution

3. Add 1.25 ml of 1.5 M Tris pH 8.8 and mix

4. Mix in 50 μl of 10% SDS

5. When ready to use, add 5 μl of TEMED and mix

6. Immediately before pouring the gel, add 50 μl of 10% APS and mix

71

Appendix B2: Coomassie staining and Destain

Coomassie staining

1 g Coomassie brilliant blue R-250

50 ml glacial acetic acid

500 ml MeOH

450 ml H2O

Stain the gel 20 min in a tray on a rotating platform. Longer staining helps see faint bands

but then you have to destain longer. Pour the used stain down the sink. Rinse gel in a little

water to get excess stain off. Destain by soaking gel in destain solution in a tray on a

rotator.

Destain

10% glacial acetic acid

5-10% MeOH

With 5% MeOH you can destain overnight. With 20% MeOH can destain a 0.8 mm thick

gel in 1.5 h at room temp. For faster destaining, use more methanol, or do it in a warm

room. Brief pulsing of the gel in destain in the microwave also speeds it up.

72

Appendix B3: Polypeptide SDS-PAGE Molecular Weight Standards (BIO-RAD)

Fig. B1. SDS polyacrylamide gels run in the Mini-PROTEIN® II cell according to the

method of Schagger and von Jagow. Polypeptide SDS-PAGE standards run on a 16.5%

Tris-Tricine gel, stained with Coomassie G-250.

Protein Molecular weights (Daltons)

Protein Molecular Weight

Triosephosphate isomerase

Myoglobin

Α-Lactalbumin

Aprotinin

Insulin b chain, oxidized

Bacitracin

26,625

16,950

14,437

6,512

3,496

1,423

73

APPENDIX C

Appendix C1: The optical density and viable cell count of the growth of L. plantarum

and L. casei TISTR 1463 in MRS broth medium

Time

(hour) Replication

Optical density Viable cell count (cfu/ml)

L. plantalum L. casei L. plantarum L. casei

0

1 0.162 0.105 1.40E+08 1.32E+08

2 0.161 0.103 1.48E+08 1.15E+08

3 0.156 0.106 1.59E+08 1.22E+08

Average 0.1597 0.1047 1.49E+08 1.23E+08

SD 0.003215 0.001528 9539392 8544004

2

1 0.396 0.22 - -

2 0.408 0.221 - -

3 0.396 0.217 - -

Average 0.4 0.2193 - -

SD 0.006928 0.002082

4

1 1.462 0.671 1.20E+09 1.36E+09

2 1.516 0.686 1.03E+09 1.53E+09

3 1.464 0.665 1.31E+09 1.19E+09

Average 1.4807 0.674 1.18E+09 1.36E+09

SD 0.030616 0.010817 1.41E+08 1.7E+08

6

1 2.472 1.434 - -

2 2.802 1.472 - -

3 2.796 1.464 - -

Average 2.69 1.4567 - -

SD 0.188817 0.020033

8

1 4.67 2.615 1.57E+10 1.21E+10

2 4.67 2.805 1.35E+10 9.80E+09

3 4.67 2.615 1.31E+10 9.30E+09

Average 4.67 2.6783 1.41E+10 1.04E+10

SD 0 0.109697 1.4E+09 1.49E+09

10

1 7.064 4.032 - -

2 7.064 4.368 - -

3 7 4.522 - -

Average 7.0427 4.3073 - -

SD 0.03695 0.25057

12

1 8.2195 5.53 6.40E+10 6.70E+10

2 8.194 5.6 3.50E+10 2.46E+10

3 7.888 5.89 6.00E+10 1.94E+10

Average 8.1005 5.6733 5.30E+10 3.70E+10

SD 0.184472 0.190875 1.57E+10 2.61E+10

74

The optical density and viable cell count of the growth of L. plantarum and L. casei TISTR

1463 in MRS broth medium (Cont.)

Time

(hour) Replication

Optical density Viable cell count (cfu/ml)

L. plantalum L. casei L. plantarum L. casei

14

1 8.41 6.516 - -

2 8.54 6.66 - -

3 8.36 6.804 - -

Average 8.4367 6.66 - -

SD 0.092916 0.144

16

1 8.82 7.469 2.40E+11 1.91E+11

2 8.75 7.975 2.03E+11 2.14E+11

3 8.61 6.908 2.26E+11 1.86E+11

Average 8.7267 7.4507 2.23E+11 1.97E+11

SD 0.106927 0.533736 1.87E+10 1.49E+10

18

1 8.77 7.568 - -

2 8.81 7.711 - -

3 8.79 7.744 - -

Average 8.79 7.6743 - -

SD 0.02 0.093554

20

1 8.964 7.728 - -

2 8.88 7.584 - -

3 8.532 7.776 - -

Average 8.792 7.696 - -

SD 0.22905 0.09992

75

Appendix C2: Antimicrobial activity and bacteriocin activity

Antimicrobial and bacteriocin activity of L. plantarum and L casei TISTR 1463 at 18 h,

30°C

strain replicate

Diameter of inhibition zone

of L. plantarum (mm) (18 h, 30°C)

Diameter of inhibition zone

of L. casei TISTR 1463 (mm) (18 h, 30°C)

No pH

adjustment

and no

catalase

enzyme

With pH

adjustment

and catalase

enzyme

No pH

adjustment

and no

catalase

enzyme

With pH

adjustment

and catalase

enzyme

E. coli TISTR

780

1 16.0 - 13.0 -

2 16.0 - 12.0 -

3 15.0 - 14.0 -

Average 15.7 - 13.0 -

SD 0.5774 - 1.0000 -

Salmonella

typhimurium

TISTR 292

1 12.0 - 12.0 -

2 10.0 - 11.0 -

3 11.5 - 12.0 -

Average 11.2 - 11.7 -

SD 1.0408 - 0.5774 -

Staphylococc

us aureus

TISTR 029

1 16.0 - 16.0 -

2 16.0 - 17.0 -

3 15.0 - 15.5 -

Average 15.7 - 16.2 -

SD 0.5774 - 0.7638 -

76

Antimicrobial and bacteriocin activity of L. plantarum and L casei TISTR 1463 at 24 h,

30°C

strain replicate

Diameter of inhibition zone

of L. plantarum

(mm) (24 h, 30°C)

Diameter of inhibition zone

of L. casei TISTR 1463

(mm) (24 h, 30°C)

No pH

adjustment

and no

catalase

enzyme

With pH

adjustment

and catalase

enzyme

No pH

adjustment

and no

catalase

enzyme

With pH

adjustment

and catalase

enzyme

E. coli TISTR

780

1 19.0 10.0 18.0 9.5

2 18.0 9.0 19.0 9.5

3 19.0 8.5 17.0 11.0

Average 18.7 9.2 18.0 10.0

SD 0.5774 0.7638 1.0000 0.8660

Salmonella

typhimurium

TISTR 292

1 19.0 - 17.5 -

2 19.0 - 16.0 -

3 19.0 - 18.0 -

Average 19.0 - 17.5 -

SD 0.0000 - 1.0408 -

Staphylococc

us aureus

TISTR 029

1 18.0 8.5 18.0 10.0

2 18.0 7.0 18.0 11.0

3 19.0 8.0 18.0 9.0

Average 18.3 7.8 18.0 10.0

SD 0.5774 0.7638 0.0000 1.0000

77

Antimicrobial and bacteriocin activity of L. plantarum and L casei TISTR 1463 at 18 h,

37°C

strain replicate

Diameter of inhibition zone

of L. plantarum

(mm) (18 h, 37°C)

Diameter of inhibition zone

of L. casei TISTR 1463

(mm) (18 h, 37°C)

No pH

adjustment

and no

catalase

enzyme

With pH

adjustment

and catalase

enzyme

No pH

adjustment

and no

catalase

enzyme

With pH

adjustment

and catalase

enzyme

E. coli TISTR

780

1 18.0 10.5 17.5 7.5

2 18.0 11.0 15.0 7.0

3 18.0 10.0 17.0 8.0

Average 18.0 10.5 16.5 7.5

SD 0.0000 0.5000 1.3229 0.5000

Salmonella

typhimurium

TISTR 292

1 18.0 10.0 18.0 -

2 18.0 10.0 17.0 -

3 18.0 10.0 16.0 -

Average 18.0 10.0 17.0 -

SD 0.0000 0.0000 1.0000 -

Staphylococc

us aureus

TISTR 029

1 18.0 10.0 18.0 7.5

2 18.0 11.0 18.0 7.0

3 18.0 9.0 18.0 7.0

Average 18.0 10.0 18.0 7.2

SD 0.0000 1.0000 0.0000 0.2887

78

Antimicrobial and bacteriocin activity of L. plantarum and L casei TISTR 1463 at 24 h,

37°C

strain replicate

Diameter of inhibition zone

of of L. plantarum

(mm) (23 h, 37°C)

Diameter of inhibition zone

of L. casei TISTR 1463

(mm) (24 h, 37°C)

No pH

adjustment

and no

catalase

enzyme

With pH

adjustment

and catalase

enzyme

No pH

adjustment

and no

catalase

enzyme

With pH

adjustment

and catalase

enzyme

E. coli TISTR

780

1 18.0 10.5 17.5 7.5

2 18.0 11.0 15.0 7.0

3 18.0 10.0 17.0 8.0

Average 18.0 10.5 16.5 7.5

SD 0.0000 0.5000 1.3229 0.5000

Salmonella

typhimurium

TISTR 292

1 18.0 10.0 18.0 -

2 18.0 10.0 17.0 -

3 18.0 10.0 16.0 -

Average 18.0 10.0 17.0 -

SD 0.0000 0.0000 1.0000 -

Staphylococc

us aureus

TISTR 029

1 18.0 10.0 18.0 7.5

2 18.0 11.0 18.0 7.0

3 18.0 9.0 18.0 7.0

Average 18.0 10.0 18.0 7.2

SD 0.0000 1.0000 0.0000 0.2887

79

Appendix C3: Effect of temperature on the bacteriocin activity

Temperature

(°C) Replication

Bacteriocin activity (AU/ml)

0 10 20 30 60

40°C

1 5,120 5,120 5,120 5,120 5,120

2 5,120 5,120 5,120 5,120 5,120

3 5,120 5,120 5,120 5,120 5,120

Average 5,120 5,120 5,120 5,120 5,120

SD 0 0 0 0 0

60°C

1 5,120 5,120 5,120 5,120 5,120

2 5,120 5,120 5,120 5,120 5,120

3 5,120 5,120 5,120 5,120 5,120

Average 5,120 5,120 5,120 5,120 5,120

SD 0 0 0 0 0

80°C

1 5,120 5,120 5,120 5,120 5,120

2 5,120 5,120 5,120 5,120 5,120

3 5,120 5,120 5,120 5,120 5,120

Average 5,120 5,120 5,120 5,120 5,120

SD 0 0 0 0 0

95°C

1 5,120 5,120 2,560 1,280 640

2 5,120 5,120 2,560 1,280 640

3 5,120 5,120 2,560 1,280 640

Average 5,120 5,120 2,560 1,280 640

SD 0 0 0 0 0

80

Appendix C4: Effect of pH treatment on the bacteriocin activity

pH

Bacteriocin activity (AU/ml)

replicate 1 replicate 2 replicate 3 Average SD

3 2,560 2,560 2,560 2,560 0

5 5,120 5,120 5,120 5,120 0

7 5,120 5,120 5,120 5,120 0

9 2,560 2,560 2,560 2,560 0

81

Appendix C5: The thickness of edible film based on cassava starch

Replication Thickness of edible film (mm)

Cassava starch film Cassava starch film

incorporated with the

whole extracts

Cassava starch film

incorporated with

bacteriocin

1.000 0.100 0.157 0.114

2.000 0.109 0.149 0.117

3.000 0.096 0.143 0.111

4.000 0.098 0.157 0.120

5.000 0.097 0.152 0.113

Avg 0.100 0.152 0.115

SD 0.005 0.006 0.004

82

Appendix C6: The mechanical properties of edible film based on cassava starch

Type of

Films Replication

At Break Points Elongation

at break

(%)

Tensile

strength

(Mpa) Δ L (mm)

Force Time

( N ) (s.)

Cassava

starch

1 13.775 3.2953 16.53 39.36 3.30

2 17.0583 3.5893 20.47 48.74 3.59

3 12.8583 3.4051 15.43 36.74 3.41

Average 14.5639 3.4299 17.48 41.61 3.43

SD 6.3094 0.1464

Cassava

starch

incorporate

with the

whole

extracts

1 69.3417 0.7733 83.21 198.12 0.51

2 67.475 1.0202 80.97 192.79 0.67

3 70.1583 0.7651 84.19 200.45 0.5

Average 68.9917 0.8529 82.79 197.12 0.56

SD 3.9267 0.0954

Cassava

starch

incorporate

with

partially

purified

bacteriocin

1 17.0833 4.297 20.5 48.81 3.74

2 16.9917 4.7297 20.39 48.55 4.11

3 19.0583 4.3534 22.87 54.45 3.79

Average 17.7111 4.46 21.25 50.6 3.88

SD 3.3338 0.2007

83

Appendix C7: The water vapor transmission and the water vapor transmission rate

of edible film based on cassava starch

Type of edible film Replicate

WVT

(g H2O mm cm-2

)

WVTR

(g H2O mm h-1

cm-2

)

Cassava starch 1 0.0008216 0.0004108

2 0.0008216 0.0004108

3 0.0007106 0.0003553

Average 0.0007846 0.0003923

SD 0.0000641 0.0000321

cassava starch film

incorporated with the whole

extracts 1 0.0010801 0.0005401

2 0.0010464 0.0005232

3 0.0009451 0.0004726

Average 0.0010239 0.0005119

SD 0.0000703 0.0000351

Cassava starch film

incorporated with partially

purified bacteriocin 1 0.0010215 0.0005107

2 0.0009704 0.0004852

3 0.0009449 0.0004724

Average 0.0009789 0.0004895

SD 0.0000390 0.0000195

84

Appendix C8: Antimicrobial activity of edible film based on cassava starch on the

indicator strains

Type of edible

film replicate

Antimicrobial activities

Inhibition zone (mm)

Escherichia

coli TISTR

780

Salmonella

typhimurium

TISTR 292

Staphylococcus

aureus TISTR

029

Listeria

monocytogenes

Cassava starch

1 - - - -

2 - - - -

3 - - - -

Average - - - -

SD - - - -

Cassava starch

film incorporated

with antimicrobial

substance

1 15.00 13.50 17.00 22.00

2 14.00 13.00 16.00 21.00

3 15.00 14.00 17.50 21.50

Average 14.67 13.50 16.83 21.50

SD 0.5774 0.5000 0.7638 0.5000

Cassava starch

film incorporated

with bacteriocin

1 - - - -

2 - - - -

3 - - - -

Average - - - -

SD - - - -

85

Appendix C9: the number of microorganism in the fermented pork which were wrapped

with plastic bag and three type of edible films based on cassava starch

Time

Replicate

Number of microorganism (CFU/ml)

(day)

plastic

bag

cassava

starch film

cassava starch film

incorporated with the

whole extracts

Cassava starch

incorporated with

partially purified

bacteriocin

0

1 143,000 176,000 220,000 251,000

2 240,000 250,000 173,000 142,000

3 181,000 240,000 240,000 183,000

Average 199,909

SD 41,555.88

1

1 9,100 9,300 11,700 5,900

2 11,200 13,100 10,400 19,200

3 5,300 14,700 6,800 17,500

Average 8,533 12,367 9,633 14,200

SD 2,991 2,701 2,538 7,238

3

1 8,200 11,500 3,500 11,400

2 14,800 8,300 4,800 3,200

3 9,900 13,300 2,110 2,900

Average 10,967 13,300 3,470 5,833

SD 3,427 2,532 1,345 4,823

7

1 15,300 11,600 2,480 9,700

2 8,600 8,300 6,800 13,600

3 13,100 12,300 8,400 8,800

Average 12,333 10,733 5,893 10,700

SD 3,415 2,136 3,062 2,551

10

1 19,500 11,000 4,500 11,300

2 23,200 17,400 6,300 14,600

3 27,100 12,800 5,700 13,700

Average 23,233 13,733 5,500 13,200

SD 3,800 3,301 917 1,706

86

Appendix C10: Orange juice

The pH value of various types of orange juice during storage period

Time

(day) Replication

pH

Pasteurize

orange

juice

Pasteurize

orange juice

with 0.1%

Sodium

benzoate

Pasteurize

orange juice

with 0.5%

bacteriocin

Pasteurize

orange juice

with 1.0%

bacteriocin

Pasteurize

orange juice

with 2.5%

bacteriocin

0

1 3.35 3.64 3.4 3.39 3.43

2 3.36 3.64 3.4 3.4 3.42

3 3.36 3.65 3.39 3.4 3.42

Average 3.36 3.64 3.4 3.4 3.42

SD 0.0058 0.0058 0.0058 0.0058 0.0058

1

1 3.35 3.65 3.39 3.4 3.41

2 3.37 3.66 3.39 3.39 3.41

3 3.37 3.66 3.4 3.39 3.41

Average 3.36 3.66 3.39 3.39 3.41

SD 0.0115 0.0058 0.0058 0.0058 0

3

1 3.36 3.65 3.38 3.4 3.42

2 3.37 3.65 3.37 3.4 3.42

3 3.38 3.65 3.39 3.4 3.42

Average 3.37 3.65 3.38 3.4 3.42

SD 0.01 0 0.01 0 0

6

1 3.38 3.65 3.38 3.39 3.41

2 3.37 3.67 3.37 3.4 3.42

3 3.38 3.68 3.39 3.41 3.44

Average 3.38 3.67 3.38 3.4 3.42

SD 0.0058 0.0153 0.01 0.01 0.0153

10

1 3.37 3.67 3.41 3.42 3.43

2 3.36 3.66 3.4 3.42 3.42

3 3.35 3.67 3.43 3.4 3.4

Average 3.36 3.67 3.41 3.41 3.42

SD 0.01 0.0058 0.0153 0.0115 0.0153

13

1 3.37 3.66 3.42 3.42 3.41

2 3.37 3.67 3.41 3.41 3.41

3 3.37 3.66 3.41 3.4 3.42

Average 3.37 3.66 3.41 3.41 3.41

SD 0 0.0058 0.0058 0.01 0.0058

87

The pH value of various types of orange juice during storage period (Cont.)

Time

(day) Replication

pH

Pasteurize

orange

juice

Pasteurize

orange juice

with 0.1%

Sodium

benzoate

Pasteurize

orange juice

with 0.5%

bacteriocin

Pasteurize

orange juice

with 1.0%

bacteriocin

Pasteurize

orange juice

with 2.5%

bacteriocin

21

1 3.37 3.67 3.42 3.41 3.42

2 3.38 3.66 3.41 3.41 3.42

3 3.37 3.66 3.41 3.42 3.41

Average 3.37 3.66 3.41 3.41 3.42

SD 0.0058 0.0058 0.0058 0.0058 0.0058

29

1 3.36 3.68 3.41 3.41 3.42

2 3.36 3.69 3.42 3.41 3.42

3 3.37 3.67 3.41 3.41 3.42

Average 3.36 3.68 3.41 3.41 3.42

SD 0.0058 0.01 0.0058 0 0

36

1 3.44 3.7 3.46 3.47 3.46

2 3.37 3.73 3.45 3.46 3.45

3 3.38 3.71 3.43 3.43 3.44

Average 3.4 3.71 3.45 3.45 3.45

SD 0.0379 0.0153 0.0153 0.0208 0.01

42

1 3.37 3.7 3.43 3.46 3.47

2 3.37 3.69 3.44 3.44 3.46

3 3.37 3.7 3.42 3.42 3.44

Average 3.37 3.7 3.43 3.44 3.46

SD 0 0.0058 0.01 0.02 0.0153

88

The number of microorganism in orange juice

sample

Time (Day)

0 1 3 6 10 13 21 29 36

1 - - - - 10 - 20 30 30

1 - - - - 10 - 40 90 10

1 - - - - 0 - 0 60 0

Average - - - - 6.666667 - 20 60 13.33333

SD - - - - 5.773503 - 20 30 15.27525

2 - - - - - - - - 10

2 - - - - - - - - 0

2 - - - - - - - - 0

Average - - - - - - - - 3.333333

SD - - - - - - - - 5.773503

3 - - - - - - - - -

3 - - - - - - - - -

3 - - - - - - - - -

Average - - - - - - - - -

SD - - - - - - - - -

4 - - - - - - - - -

4 - - - - - - - - -

4 - - - - - - - - -

Average - - - - - - - - -

SD - - - - - - - - -

5 - - - - - - - - -

5 - - - - - - - - -

5 - - - - - - - - -

Average - - - - - - - - -

SD - - - - - - - - -

89

Appendix C11: Pasteurized milk

The pH value of various types of pasteurized milk during storage period

Time

(day) Replication

pH

Pasteurized Pasteurized milk

with 0.5%

bacteriocin

Pasteurized milk Pasteurized milk

with 2.5%

bacteriocin

milk with 1.0%

bacteriocin

0

1 6.72 6.72 6.72 6.72

2 6.72 6.72 6.72 6.72

3 6.72 6.72 6.72 6.72

Average 6.72 6.72 6.72 6.72

SD 0 0 0 0

3

1 6.68 6.72 6.72 6.73

2 6.72 6.72 6.72 6.72

3 6.72 6.72 6.72 6.7

Average 6.71 6.72 6.72 6.72

SD 0.0231 0 0 0.0153

8

1 6.72 6.73 6.74 6.74

2 6.73 6.72 6.71 6.73

3 6.72 6.72 6.73 6.72

Average 6.72 6.72 6.73 6.73

SD 0.0058 0.0058 0.0153 0.01

16

1 6.68 6.7 6.71 6.73

2 6.66 6.71 6.71 6.72

3 6.67 6.71 6.71 6.73

Average 6.67 6.71 6.71 6.73

SD 0.01 0.0058 0 0.0058

23

1 6.7 6.71 6.73 6.73

2 6.69 6.7 6.74 6.72

3 6.69 6.71 6.73 6.73

Average 6.69 6.71 6.73 6.73

SD 0.0058 0.0058 0.0058 0.0058

31

1 6.6 6.68 6.68 6.7

2 6.61 6.68 6.69 6.7

3 6.67 6.7 6.7 6.7

Average 6.63 6.69 6.69 6.7

SD 0.0379 0.0115 0.01 0

90

The pH value of various types of pasteurized milk during storage period (Cont.)

Time

(day) Replication

pH

Pasteurized Pasteurized milk

with 0.5%

bacteriocin

Pasteurized milk Pasteurized milk

with 2.5%

bacteriocin

milk with 1.0%

bacteriocin

35

1 6.55 6.72 6.71 6.72

2 6.57 6.71 6.72 6.72

3 6.54 6.7 6.71 6.71

Average 6.55 6.71 6.71 6.72

SD 0.0153 0.01 0.0058 0.0058

41

1 6.45 6.72 6.71 6.71

2 6.41 6.71 6.72 6.72

3 6.42 6.7 6.72 6.72

Average 6.43 6.71 6.72 6.72

SD 0.0208 0.01 0.0058 0.0058

91

The number of microorganism in pasteurized milk

Sample replicate

Time (Day)

0 3 8 16 23 31 35 41

Pasteurize

milk

1 - - - 40.0 20.0 - 50.0 -

2 - - - 30.0 0.0 - 0.0 -

3 - - - 30.0 0.0 - 0.0 -

Average - - - 33.3 6.7 - 16.7 -

SD - - - 5.774 11.547 - 28.868 -

Pasteurize

milk with

0.5%

bacteriocin

1 - - - - - - - -

2 - - - - - - - -

3 - - - - - - - -

Average - - - - - - - -

SD - - - - - - - -

Pasteurize

milk with

1.0%

bacteriocin

1 - - - - - - - -

2 - - - - - - - -

3 - - - - - - - -

Average - - - - - - - -

SD - - - - - - - -

Pasteurize

milk with

2.5%

bacteriocin

1 - - - - - - - -

2 - - - - - - - -

3 - - - - - - - -

Average - - - - - - - -

SD - - - - - - - -