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 : Previous Degree :

Thai Bachelor of Science in Biotechnology King Mongkuts Institute of Technology Ladkrabang Bangkok, Thailand RTG Fellowship

Scholarship Donor :

Asian Institute of Technology School of Environment, Resources and Development Thailand May 2011

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 authors 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.

ii

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 95C 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 (p0.05) 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.

iii

Table of Contents CHAPTER TITLE Title Page Acknowledgements Abstract Table of Contents List of Tables List of Figures List of Abbreviations PAGE i ii iii iv vi vii viii

Introduction 1.1 General background 1.2 Statement of problem 1.3 Scope of study 1.4 Objective Literature Review 2.1 Probiotics 2.2 Turmeric 2.3 Identification of probiotics 2.4 Antimicrobial components from lactic acid bacteria 2.5 Classification of bacteriocins 2.6 Edible packaging 2.7 Film composition 2.8 Antimicrobial packaging Materials and Methods 3.1 Materials 3.2 Chemicals 3.3 Equipments 3.4 Methodology Result and Discussion 4.1 Isolation and identification of Lactic acid bacteria (LAB) From turmeric rhizome 4.2 Growth of L. plantarum and L. casei TISTR 1463 4.3 Antimicrobial and bacteriocin activities 4.4 Purification of bacteriocin 4.5 Effect of temperature and pH on bacteriocin activity 4.6 Molecular mass determination of bacteriocins 4.7 The physical properties of edible film based on cassava starch
iv

1 1 2 2 2 3 3 3 3 5 7 12 13 15 21 21 21 22 24 36 36 37 38 39 40 42 43

4.8 Tensile strength and Elongation at break of edible film based on cassava starch 4.9 Water vapor transmission and water vapor transmission rate of edible film based on cassava starch 4.10 Effect of antimicrobial of edible film based on cassava starch on bacteria strains 4.11 Effect of antimicrobial packaging in fermented pork 4.12 Effect of bacteriocin in orange juice 4.13 Effect of bacteriocin in pasteurized milk Conclusions and Recommendations 5.1 Conclusions 5.2 Recommendation for further studies References Appendices

46 49 51 52 54 56 58 58 59 60 68

List of Tables TABLE 2.1 2.2 2.3 4.1 4.2 4.3 4.4 4.5 4.6 4.7 TITLE PAGE 11 12 19 38 41 44 47 49 52 53

4.8 4.9 4.10 4.11

Classification of bacteriocins for Lactic acid bacteria Summarization of bacteriocins, purification methods and molecular weight of bacteriocin Application of bactericidal food packaging systems Inhibition zones by Agar well diffusion test The bacteriocin activity of L. plantarum at 16, 18, 20, 22, 24 and 26 h against L. casei TISTR 1463 The thickness of edible film based on cassava starch The tensile strength and elongation at break of various types of edible film based on cassava starch Water vapor transmission (WVT) and water vapor transmission rate (WVTR) of various types of edible film based on cassava starch Antimicrobial activity of edible film based on cassava starch on the indicator strains The number of microorganism in the fermented pork which were wrapped with plastic bag and three type of edible films based on cassava starch The pH value of various types of orange juice during storage period The number of microorganism in orange juice The pH value of various types of pasteurize milk during storage period The number of microorganism in various types of pasteurize milk

55 56 57 57

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List of Figures FIGURE TITLE 2.1 3.1 3.2 3.3 3.4 3.5 3.6 3.7 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11 PAGE 12 23 26 27 28 30 34 35 36 37 40 41 41 43 44 45 46 46 48

4.12

4.13 4.14 4.15

Life cycle of biodegradable packaging Flow-chart of overall experiment Show the purification step; the bacteriocin was purified by using dialysis membrane (1,000 MWCO) Flow-chart of purification of the bacteriocin Flow-chart of sensitivity of bacteriocin to temperature and pH Flow-chart of cassava starch based films formation Flow-chart of orange juice experiment Flow-chart of pasteurized milk experiment Cell morphology of lactic acid bacteria isolated from fresh turmeric rhizomes Batch culture profile of Lactobacillus plantarum and Lactobacillus casei in MRS medium at 37C Bacteriocin activity of L. plantarum against L. casei TISTR 1463 at 18 h of culture The effect of temperature on bacteriocin activity The effect of pH on bacteriocin activity Molecular mass determination by SDS-PAGE Appearance of edible films based on cassava starch Scanning electron micrograph of the surface of cassava starch films Scanning electron micrograph of the surface of cassava starch films incorporated with partially purified bacteriocin Scanning electron micrograph of the surface of cassava starch film incorporated with the whole extracts Tensile strength of cassava starch films, cassava starch film incorporated with the whole extracts and cassava starch films incorporated with partially purified bacteriocin Elongation at break of cassava starch films cassava starch film incorporated with the whole extracts and cassava starch films incorporated with partially purified bacteriocin Water vapor transmission (WVT) of edible film based on cassava starch Water vapor transmission rate (WVTR) of edible film based on cassava starch The number of microorganism versus time of plastic bag and three types of edible films based on cassava starch

48

50 50 53

vii

List of Abbreviations C cfu Da EB FAO G g h kDa LAB log min ml mM mm MRS NB nm OD PCA SD TS V v/v w/v WVT WVTR C l m WHO Cytosine Colony Forming Unit Dalton Elongation at break Food and Agriculture Organization Guanine Gram Hour Kilodalton Lactic Acid Bacteria Logarithm Minute Milli liter Milli molar Milli meter Man Rogosa Sarpe media Nutrient Broth Nama Meter Optical density Plate Count Agar Standard deviation Tensile strength Voltage Volume by volume Weight by volume Water Vapor Transmission Water Vapor Transmission Rate Degree Celsius Micro liter Micro meter World Health Organization

viii

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 microorganisms. Bacteriocins are non-toxic and do not alter the nutritional properties. They are effective at low concentrations and remain active even under refrigerated

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.

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 nondairy 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,

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, nonmotile 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 37C to 41C (least temperature growth for bifidobacteria: 25-28C; highest temperature for bifidobacteria: 42-45C) 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,
4

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).
5

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):
lactoperoxidase

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

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 gramnegative 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 posttranslationally to some degree, with the secreted mature peptides size usually between 20 to 60 amino acids, and possessing bactericidal activity (OConnor 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 (OConnor et al, 2005).

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 -methyllanthionine), 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 (Glvez 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.

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.

Table 2.1 Classification of bacteriocins for Lactic acid bacteria

Class Class I: Lantibiotics Subgroup/subclass Subgroup I Nisin Subgroup II Lacticin 481 Subgroup III Mersacidin Subgroup IV LtnA2 Subgroup V Cytolysis group Subgroup VI Lactocin S Class II: Bacteriocins Subclass IIa Pediocin-like Subclass IIb Two peptides Nisin Plantaricin C Plantaricin plwa Plantaricin plwb Cytolysin CyILL&CyILS Lactosin S Bifidocin 1454 Lactocin 705 plantaricin A Subclass IIc Sec-dependent Class III: Bacteriocins Class III Acidocin B Lactacin B Bacteriocin Microorganism 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 Reference(s) 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

10

Table 2.2 Summarization of bacteriocins, purification methods and molecular weight of bacteriocins
Bacteriocin A5-11A A5-11B Strain Enterococcus durans Medium M17 3 steps - Cation-exchange chromatography - Reverse phase chromatography - RP-HPLC Compare between 2 methods - Amberlite resin - 55% Ammonium Sulphate 55% Ammonium Sulphate give a higher antimicrobial activity (AU/ml) MRS Ammonium sulphate precipitation (400g/l) followed by sequential cation exchange and hydrophobic interaction chromatography. 60% Ammonium Sulphate precipitation followed by RP-HPLC Purification Molecular weight 5206 Da 5218 Da References Batdorj et al., 2006

162W

L. curvatus L. Plantarum

MRS

8.1 kDa 15.5 kDa

11
AM09 Acidocin D20079 Enterococcin EFS2

L. acidophilus DSM 20079

6.6 kDa

Deraz et al., 2005

Enterococcus faecalis

APT

6756 Da

Maisnier-Patin et al., 1996

11

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

12

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
13

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.

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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
15

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 foodgrade 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
16

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-foodgrade 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, Gbilmez et al. (2007) studies the edible zein films incorporated with lysozyme, albumin proteins and disodium EDTA. They found that zein films effective on
17

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 nonpeptide 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). Rle 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.

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Table 2.3 Application of bactericidal food packaging systems Bacteriocins Nisin Packaging materials HPMC Foods Culture media Microorganisms L. monocytogenes, S. aureus Nisin HPMC, stearic acid Culture media L. monocytogenes, S. aureus Nisin Nisin 19 Nisin Nisin Nisin Nisin, lacticins Corn zein Corn zein, wheat gluten Agar coating Alginate Tapioca starch LDPE, Poly-amide Shredded cheese Culture media Fresh poultry Beef Culture media Culture media Total aerobes Lactobacillus plantarum S. typhimurium S. aureus L. innocua M. flacus, L. monocytogenes Nisin, lacticins Nisin, EDTA Nisin, lauric acid LDPE, Poly-amide PE, PE-PE oxide Zein Oyster, beef Beef Simulants Total aerobes, coli-form B. thermosphacta Migration test Kim et al., 2002 Cutter et al., 2001 Hoffman et al., 2001 Cooksey et al., 2000 Dawson et al., 2003 Natrajan and Sheldon, 1995 Millette et al., 2007 Sanjurjo et al., 2006 An et al., 2000 Sebti et al.,2002 References Coma et al., 2001

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Table 2.3 Application of bactericidal food packaging systems (Cont.) Bacteriocins Nisin, lauric acid Nisin, pediocin Nisin, grape seed extract, EDTA Packaging materials Soy protein Cellulose casing Soy protein Foods Turkey bologna Turkey breast, ham, beef Drop culture on film and enumerate after 1 h (25C) Microorganisms L. monocytogenes L. monocytogenes L. monocytogenes, E. coli O157:H7, Salmonella typhimurium 20 Enterocins Alginate, zein, polyvinyl alcohol (biodegradable film) Low-density polyethylene Ham L. monocytogenes Marcos et al., 2007 References Dawson et al., 2002 Ming et al.,1997 Sivarooban et al., 2008

Enterocins 416K1

Culture media, frankfurters and fresh cheeses

L. monocytogenes

Iseppi et al., 2008

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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 21

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 (BagMixer400, 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, 0.45m (Whatman, Japan)

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Bacteria culture Agar spot test Screen for bacteriocin production Well diffusion agar test Inhibition test of bacteriocins

Effect of temperature control on bacteriocin production

Purification of the bacteriocins by ammonium sulphate and then dialysis bag

Assay for bacteriocin activity

Partially purified bacteriocin

Effect of temperature and pH on bacteriocin activity Detection of molecular weight

Cassava starch partially purified bacteriocin based edible film

Biopreservative test

Physical properties, mechanical properties and antibacterial activities of edible film

Orange juice

Milk

Edible packaging for fermented pork

Figure 3.1 Flow-chart of overall experiment

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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 37C 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 (bioMrieux 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 37C for 18 h., Lactobacillus casei was at 37C for 18 h. and pathogen strains were at 37C 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 l of each culture was spot onto 20 ml MRS agar plates (1.5% w/v agar) and incubated for 24 h at 37C 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 37C for 24 h and the appearance of inhibitory zones was observed and measured.

24

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 4C 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 37C for 1 h. The aliquots of supernatant (50 l) were placed into each well of the seed plates. After pre-diffusion at 25C for 30 min, the plates were incubated at 37C. 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 37C 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 4C 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 37C Five hundred milliliter of MRS broth was inoculated with L. plantarum with 1% inocula and incubated at 37C. 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 4C, and ammonium sulphate (ANALAR B.D.H. grade, England) (65% of saturation, at 25C) was added to the supernatants to precipitate proteins. The mixtures had been stirred for 2 h at 4C, the protein precipitates were collected by centrifugation at 12,000 g for 45 min at 4C. 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 M (Whatman, Japan) and stored at -20C 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.
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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 4C and latter centrifuged at 12000g for 45 min at 4C. 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 m (Whatman, Japan) and stored at -20C until further use.

Figure 3.2 Show the partially purification step; the bacteriocin was purified by using dialysis membrane (1,000 MWCO)

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Purification of the bacteriocin

Bacteriocin precipitation by ammonium sulphate 65% (NH4)2SO4

Resuspend in potassium phosphate buffer

Dialyze overnight with 1 kDa MWCO

Filter with 0.45 M and store at -20C

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 l 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 25C for 30 min, the plates were incubated at 37C. 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.
27

3.4.9 Sensitivity of bacteriocin to heat Bacteriocin was treated at the various temperatures (40, 60, 80, 95C) 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 25C. Activities were assayed by the agar well diffusion method as mentioned previously in section 3.4.7.

Sensitivity of bacteriocin to heat and pH

Heat

pH

40C 0 min

60C 30 min

80C 60 min

95C 90 min

Adjust pH with 5M NaOH and 5M HCl pH 3 pH 5 pH 7 pH 9

Agar well diffusion test

Agar well diffusion test

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 Schgger 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 R250 (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,
28

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 10 5 CFU/ml of L. casei TISTR 1463. After incubation at 30C 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 70 oC 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.

29

Control film Mixture of cassava starch, glycerine and water at ratio of 5.0: 2.0: 93.0 to obtain film forming solution

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

Film forming solution was heated on a magnetic stirrer with hot plate until the system entered in the gelatinization step (70C) for 20 minutes

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 50C 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

Figure 3.5 Flow-chart of cassava starch based films formation

30

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 Rodrguez 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 18 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 x d = = =

maximum force at break, N wide of film, m 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 t l = = = =

head speed, 50 mm/min time for film extension until break, min initial length of film, m
31

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 = Where; w x A = = = (g H2O mm/cm2)

the weight of water absorbed in the cup (g) the average thickness of the film (mm) the permeation area (cm2) WVTR = (g H2O mm/h cm2)

Where

WVTR = x/t =

Water Vapor Transmission Rate 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 37 oC 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.

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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 (-18C). 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 4C 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 l) were taken and spread on plate count agar (PCA). The plates were incubated at 37C 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 Second treatment Third treatment Forth treatment Fifth treatment : without partially purified bacteriocin (Control) : 0.1% Sodium benzoate : 0.5% partially purified bacteriocin : 1% partially purified bacteriocin : 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 90C for 15 second and then cooled down immediately by put the samples into ice water bath. The samples were stored at room temperature (25C). 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).

33

For microbiological testing, the samples were taken at sampling time. 100 l of sample were taken and spread on Plate Count Agar (PCA). The plates were incubated at 37C for 48 hours.

Orange juice

Orange juice without bacteriocin (Control)

Orange juice with 0.1% Sodium benzoate

Orange juice with 0.5% bacteriocin

Orange juice with 1% bacteriocin

Orange juice with 2.5% bacteriocin

Pasteurization at 90C for 15 seconds

Cool down

Store at room temperature 25C Take the sample

pH measurement

Microbiological testing

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)
34

Second treatment Third treatment Forth treatment

: 0.5% partially purified bacteriocin : 1% partially purified bacteriocin : 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 4C 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 l of samples were taken and spread on Plate Count Agar (PCA). The plates were incubated at 37C for 24-48 hours.

Pasteurize milk

Pasteurize milk without bacteriocin (Control) Pasteurize milk 0.5% bacteriocin

Pasteurize milk 1% bacteriocin

Pasteurize milk 2.5% bacteriocin

Store at 4C

Take the sample

pH measurement

Microbiological testing

Figure 3.7 Flow-chart of pasteurize milk experiment

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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 Gramstaining reaction and catalase test. Further identification was carried out by using API kits (bioMrieux 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: Long-rods shape

(ii) Enterococcus faecium morphology: 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
36

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 10 11 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 37C. : 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.

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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 37C, L. casei TISTR 1463 produced showed the highest bacteriocin at 30C. 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 Diameter of inhibition zone of L. plantarum (mm) (18 h, 37C) No pH With pH adjustment and adjustment and no catalase catalase enzyme enzyme 18.0 0.00 10.5 0.50 18.0 0.00 18.0 0.00 10.0 0.00 10.0 1.00 Diameter of inhibition zone of L. casei (mm) (24 h, 30C) No pH With pH adjustment and adjustment and no catalase catalase enzyme enzyme 18.0 1.00 10.0 0.87 17.5 1.04 18.0 0.00 10.0 1.00

strain

E. coli TISTR 780 S. typhimurium TISTR 292 S. aureus TISTR 029

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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-offdialysed 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) 16 18 20 22 24 26 Bacteriocin activity (AU/ml) 2,560 5,120 2,560 2,560 2,560 2,560

39

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 40C, 60C and 80C, and after 10 min at 100C. The bacteriocin activity was found decreasing at 100C 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 (p0.05).

40

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

41

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, SDSpolyacrylamide 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.

42

Molecular weight (Daltons) 14,437 6,512 3,496 1,423

12-14 kDa Inhibition zone 1-2 kDa B Figure 4.6 Molecular mass determination by SDS-PAGE (A) Polypeptide SDS-PAGE Molecular Weight Standards of one part of SDSpolyacrylamide gel electrophoresis extracts peptide from the L. plantarum (B) The clear inhibition bands of the protein in another part of SDSpolyacrylamide 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.

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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 Cassava starch film Cassava starch film incorporated with the whole extracts Cassava starch film incorporated with partially purified Thickness (mm) 0.100 0.005 0.152 0.006 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
44

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

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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
46

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 filmforming 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 Tensile strength (MPa) 3.43a 0.15 0.56b 0.10 3.88a 0.20 Elongation at break (%) 41.61a 6.32 197.12b 3.93 50.60a 3.33

Type of edible film Cassava starch film Cassava starch film incorporate with the whole extracts Cassava starch film incorporate with bacteriocin

47

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

48

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 Cassava starch cassava starch film incorporated with the whole extracts Cassava starch film incorporated with partially purified bacteriocin

WVT (g H2O mm cm-2) 0.0007846a 0.0000641 0.0010239b 0.0000703

WVTR (g H2O mm h-1 cm-2) 0.0003923a 0.0000321 0.0005119b 0.0000351

0.0009789b 0,0000390

0.0004895b 0.0000195

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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

50

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.

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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) Salmonella Staphylococcus typhimurium aureus TISTR Listeria TISTR 292 029 monocytogenes 13.50 0.50 16.83 0.76 21.5 0.50

Cassava starch cassava starch film incorporated with the whole extracts Cassava starch film incorporated with partially purified bacteriocin

Escherichia coli TISTR 780 14.77 0.58

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 4C. 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.
52

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) cassava starch film incorporated with the whole extracts 2.001054.2104 9.631032.5103 3.47103 1.3103 5.89103 3.1103 5.50103 9.2102 Cassava starch incorporated with partially purified bacteriocin 2.001054.2104 1.421047.2103 5.831034.8103 1.071042.6103 1.321041.7103

(day) 0 1 3 7 10

plastic bag 2.001054.2104 8.531033.0103 1.101043.4103 1.231043.4103 2.321043.8103

cassava starch film 2.001054.2104 1.241042.7103 1.331042.5103 1.071042.1103 1.371043.3103

Figure 4.15 The number of microorganism versus time of plastic bag and three types of edible films based on cassava starch
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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 (25C). 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 37C. 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.

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Table 4.8 The pH value of various types of orange juice during storage period pH Pasteurized orange juice with 0.5% partially purified bacteriocin 3.40 0.0058 3.39 0.0058 3.38 0.0100 3.38 0.0100 3.41 0.0153 3.41 0.0058 3.41 0.0058 3.41 0.0068 3.45 0.0153 3.43 0.0100

Time Pasteurized (day) orange juice 3.36 0 0.0058 3.36 1 0.0115 3.37 3 0.0100 3.38 6 0.0058 3.36 10 0.0100 3.37 13 0.0000 3.37 21 0.0058 3.36 29 0.0058 3.40 36 0.0379 3.37 42 0.0000

Pasteurized orange juice with 0.1% Sodium benzoate 3.64 0.0058 3.66 0.0058 3.65 0.0000 3.67 0.0153 3.67 0.0058 3.66 0.0058 3.66 0.0058 3.68 0.0100 3.71 0.0153 3.70 0.0058

Pasteurized orange juice with 1.0% partially purified bacteriocin 3.40 0.0059 3.39 0.0058 3.40 0.0000 3.40 0.0100 3.41 0.0115 3.41 0.0100 3.41 0.0058 3.41 0.0000 3.45 0.0208 3.44 0.0200

Pasteurized orange juice with 2.5% partially purified bacteriocin 3.42 0.0058 3.41 0.0000 3.42 0.0000 3.42 0.0153 3.42 0.0153 3.41 0.0058 3.42 0.0058 3.42 0.0000 3.45 0.0100 3.46 0.0153

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Table 4.9 The number of microorganism in orange juice Number of microorganism (CFU/ml) Pasteurized Pasteurized Pasteurized orange juice orange juice orange juice with 0.5% with 1.0% with 0.1% partially partially Sodium purified purified benzoate bacteriocin bacteriocin 3 5.77 -

Time Pasteurized (day) orange juice 0 1 3 6 6.67 5.77 10 20.00 21 20.00 60.00 29 30.00 13.33 36 15.28

Pasteurized orange juice with 2.5% partially purified bacteriocin -

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 4C. 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.
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In case of microbiological testing, the samples were taken and spread on Plate Count Agar (PCA) plate and incubated at 37C. 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 pH Pasteurized milk with 0.5% partially purified bacteriocin 6.72 0.0000 6.72 0.0000 6.72 0.0058 6.71 0.0058 6.71 0.0058 6.69 0.0115 6.71 0.0100 6.71 0.0100 Pasteurized milk with 1.0% partially purified bacteriocin 6.72 0.0000 6.72 0.0000 6.73 0.0153 6.71 0.0000 6.73 0.0058 6.69 0.0100 6.71 0.0058 6.72 0.0058 Pasteurized milk with 2.5% partially purified bacteriocin 6.72 0.0000 6.72 0.0153 6.73 0.0100 6.73 0.0058 6.73 0.0058 6.70 0.0000 6.72 0.0058 6.72 0.0058

Time (day) 0 3 8 16 23 31 35 41

Pasteurized milk 6.72 0.0000 6.71 0.0231 6.72 0.0058 6.67 0.0100 6.69 0.0058 6.63 0.0379 6.55 0.0153 6.43 0.0208

Table 4.11 The number of microorganism in various types of pasteurize milk Number of microorganism (CFU/ml) Pasteurized milk Pasteurized milk with 0.5% with 1.0% partially purified partially purified bacteriocin bacteriocin -

Time (day) 0 3 8 16 35

Pasteurized milk 33.33 5.77 16.67 28.87

Pasteurized milk with 2.5% partially purified bacteriocin -

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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 95C 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.

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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 shelflife of product

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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.

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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 80l 10% SDS 80l 10% APS 8 l 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 l of 10% SDS 5. When ready to use, add 8 l of TEMED and mix 6. Immediately before pouring the gel, add 80 l 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 80l 10% SDS 80l 10% APS 8 l 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 l of 10% SDS 5. When ready to use, add 8 l of TEMED and mix 6. Immediately before pouring the gel, add 80 l 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 l 10% SDS 50 l 10% APS 5 l TEMED
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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 l of 10% SDS 5. When ready to use, add 5 l of TEMED and mix 6. Immediately before pouring the gel, add 50 l of 10% APS and mix

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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.

71

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 Triosephosphate isomerase Myoglobin -Lactalbumin Aprotinin Insulin b chain, oxidized Bacitracin Molecular Weight 26,625 16,950 14,437 6,512 3,496 1,423

72

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) Optical density Replication L. plantalum L. casei 1 0.162 0.105 2 0.161 0.103 3 0.156 0.106 Average 0.1597 0.1047 SD 0.003215 0.001528 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 1 1.462 0.671 2 1.516 0.686 3 1.464 0.665 Average 1.4807 0.674 SD 0.030616 0.010817 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 1 4.67 2.615 2 4.67 2.805 3 4.67 2.615 Average 4.67 2.6783 SD 0 0.109697 1 7.064 4.032 2 7.064 4.368 3 7 4.522 Average 7.0427 4.3073 SD 0.03695 0.25057 1 8.2195 5.53 2 8.194 5.6 3 7.888 5.89 Average 8.1005 5.6733 SD 0.184472 0.190875
73

Viable cell count (cfu/ml) L. plantarum L. casei 1.40E+08 1.32E+08 1.48E+08 1.15E+08 1.59E+08 1.22E+08 1.49E+08 1.23E+08 9539392 8544004 1.20E+09 1.03E+09 1.31E+09 1.18E+09 1.41E+08 1.57E+10 1.35E+10 1.31E+10 1.41E+10 1.4E+09 6.40E+10 3.50E+10 6.00E+10 5.30E+10 1.57E+10 1.36E+09 1.53E+09 1.19E+09 1.36E+09 1.7E+08 1.21E+10 9.80E+09 9.30E+09 1.04E+10 1.49E+09 6.70E+10 2.46E+10 1.94E+10 3.70E+10 2.61E+10

10

12

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) Optical density L. plantalum L. casei 8.41 6.516 8.54 6.66 8.36 6.804 8.4367 6.66 0.092916 0.144 8.82 7.469 8.75 7.975 8.61 6.908 8.7267 7.4507 0.106927 0.533736 8.77 7.568 8.81 7.711 8.79 7.744 8.79 7.6743 0.02 0.093554 8.964 7.728 8.88 7.584 8.532 7.776 8.792 7.696 0.22905 0.09992 Viable cell count (cfu/ml) L. plantarum L. casei 2.40E+11 2.03E+11 2.26E+11 2.23E+11 1.87E+10 1.91E+11 2.14E+11 1.86E+11 1.97E+11 1.49E+10 -

14

16

18

20

Replication 1 2 3 Average SD 1 2 3 Average SD 1 2 3 Average SD 1 2 3 Average SD

74

Appendix C2: Antimicrobial activity and bacteriocin activity Antimicrobial and bacteriocin activity of L. plantarum and L casei TISTR 1463 at 18 h, 30C

Diameter of inhibition zone of L. plantarum (mm) (18 h, 30C) No pH adjustment and no catalase enzyme 16.0 16.0 15.0 15.7 0.5774 12.0 10.0 11.5 11.2 1.0408 16.0 16.0 15.0 15.7 0.5774 With pH adjustment and catalase enzyme -

Diameter of inhibition zone of L. casei TISTR 1463 (mm) (18 h, 30C) No pH adjustment and no catalase enzyme 13.0 12.0 14.0 13.0 1.0000 12.0 11.0 12.0 11.7 0.5774 16.0 17.0 15.5 16.2 0.7638 With pH adjustment and catalase enzyme -

strain

E. coli TISTR 780

Salmonella typhimurium TISTR 292

Staphylococc us aureus TISTR 029

replicate 1 2 3 Average SD 1 2 3 Average SD 1 2 3 Average SD

75

Antimicrobial and bacteriocin activity of L. plantarum and L casei TISTR 1463 at 24 h, 30C Diameter of inhibition zone of L. plantarum (mm) (24 h, 30C) No pH adjustment and no catalase enzyme 19.0 18.0 19.0 18.7 0.5774 19.0 19.0 19.0 19.0 0.0000 18.0 18.0 19.0 18.3 0.5774 With pH adjustment and catalase enzyme 10.0 9.0 8.5 9.2 0.7638 8.5 7.0 8.0 7.8 0.7638 Diameter of inhibition zone of L. casei TISTR 1463 (mm) (24 h, 30C) No pH adjustment and no catalase enzyme 18.0 19.0 17.0 18.0 1.0000 17.5 16.0 18.0 17.5 1.0408 18.0 18.0 18.0 18.0 0.0000 With pH adjustment and catalase enzyme 9.5 9.5 11.0 10.0 0.8660 10.0 11.0 9.0 10.0 1.0000

strain

E. coli TISTR 780

replicate 1 2 3 Average SD 1 2 3 Average SD 1 2 3 Average SD

Salmonella typhimurium TISTR 292

Staphylococc us aureus TISTR 029

76

Antimicrobial and bacteriocin activity of L. plantarum and L casei TISTR 1463 at 18 h, 37C

Diameter of inhibition zone of L. plantarum (mm) (18 h, 37C) No pH adjustment and no catalase enzyme 18.0 18.0 18.0 18.0 0.0000 18.0 18.0 18.0 18.0 0.0000 18.0 18.0 18.0 18.0 0.0000

Diameter of inhibition zone of L. casei TISTR 1463 (mm) (18 h, 37C) No pH adjustment and no catalase enzyme 17.5 15.0 17.0 16.5 1.3229 18.0 17.0 16.0 17.0 1.0000 18.0 18.0 18.0 18.0 0.0000

strain

E. coli TISTR 780

replicate 1 2 3 Average SD 1 2 3 Average SD 1 2 3 Average SD

With pH adjustment and catalase enzyme 10.5 11.0 10.0 10.5 0.5000 10.0 10.0 10.0 10.0 0.0000 10.0 11.0 9.0 10.0 1.0000

With pH adjustment and catalase enzyme 7.5 7.0 8.0 7.5 0.5000 7.5 7.0 7.0 7.2 0.2887

Salmonella typhimurium TISTR 292

Staphylococc us aureus TISTR 029

77

Antimicrobial and bacteriocin activity of L. plantarum and L casei TISTR 1463 at 24 h, 37C

Diameter of inhibition zone of of L. plantarum (mm) (23 h, 37C) No pH adjustment and no catalase enzyme 18.0 18.0 18.0 18.0 0.0000 18.0 18.0 18.0 18.0 0.0000 18.0 18.0 18.0 18.0 0.0000

Diameter of inhibition zone of L. casei TISTR 1463 (mm) (24 h, 37C) No pH adjustment and no catalase enzyme 17.5 15.0 17.0 16.5 1.3229 18.0 17.0 16.0 17.0 1.0000 18.0 18.0 18.0 18.0 0.0000

strain

E. coli TISTR 780

replicate 1 2 3 Average SD 1 2 3 Average SD 1 2 3 Average SD

With pH adjustment and catalase enzyme 10.5 11.0 10.0 10.5 0.5000 10.0 10.0 10.0 10.0 0.0000 10.0 11.0 9.0 10.0 1.0000

With pH adjustment and catalase enzyme 7.5 7.0 8.0 7.5 0.5000 7.5 7.0 7.0 7.2 0.2887

Salmonella typhimurium TISTR 292

Staphylococc us aureus TISTR 029

78

Appendix C3: Effect of temperature on the bacteriocin activity Temperature (C) Replication 1 2 40C 3 Average SD 1 2 60C 3 Average SD 1 2 80C 3 Average SD 1 2 95C 3 Average SD Bacteriocin activity (AU/ml) 10 20 30 5,120 5,120 5,120 5,120 5,120 5,120 5,120 5,120 5,120 5,120 5,120 5,120 0 0 0 5,120 5,120 5,120 5,120 5,120 5,120 5,120 5,120 5,120 5,120 5,120 5,120 0 0 0 5,120 5,120 5,120 5,120 5,120 5,120 5,120 5,120 5,120 5,120 5,120 5,120 0 0 0 5,120 2,560 1,280 5,120 2,560 1,280 5,120 2,560 1,280 5,120 2,560 1,280 0 0 0

0 5,120 5,120 5,120 5,120 0 5,120 5,120 5,120 5,120 0 5,120 5,120 5,120 5,120 0 5,120 5,120 5,120 5,120 0

60 5,120 5,120 5,120 5,120 0 5,120 5,120 5,120 5,120 0 5,120 5,120 5,120 5,120 0 640 640 640 640 0

79

Appendix C4: Effect of pH treatment on the bacteriocin activity Bacteriocin activity (AU/ml) replicate 2 replicate 3 Average 2,560 2,560 2,560 5,120 5,120 5,120 5,120 5,120 5,120 2,560 2,560 2,560

pH 3 5 7 9

replicate 1 2,560 5,120 5,120 2,560

SD 0 0 0 0

80

Appendix C5: The thickness of edible film based on cassava starch Replication Thickness of edible film (mm) Cassava starch film Cassava starch film Cassava starch film incorporated with the incorporated with whole extracts bacteriocin 0.100 0.157 0.114 0.109 0.149 0.117 0.096 0.143 0.111 0.098 0.157 0.120 0.097 0.152 0.113 0.100 0.152 0.115 0.005 0.006 0.004

1.000 2.000 3.000 4.000 5.000 Avg SD

81

Appendix C6: The mechanical properties of edible film based on cassava starch At Break Points Force L (mm) (N) 13.775 3.2953 17.0583 3.5893 12.8583 3.4051 14.5639 3.4299 69.3417 67.475 70.1583 68.9917 17.0833 16.9917 19.0583 17.7111 0.7733 1.0202 0.7651 0.8529 4.297 4.7297 4.3534 4.46

Type of Films

Cassava starch

Cassava starch incorporate with the whole extracts Cassava starch incorporate with partially purified bacteriocin

Replication 1 2 3 Average SD 1 2 3 Average SD 1 2 3 Average SD

Time (s.) 16.53 20.47 15.43 17.48 83.21 80.97 84.19 82.79 20.5 20.39 22.87 21.25

Elongation at break (%) 39.36 48.74 36.74 41.61 6.3094 198.12 192.79 200.45 197.12 3.9267 48.81 48.55 54.45 50.6 3.3338

Tensile strength (Mpa) 3.30 3.59 3.41 3.43 0.1464 0.51 0.67 0.5 0.56 0.0954 3.74 4.11 3.79 3.88 0.2007

82

Appendix C7: The water vapor transmission and the water vapor transmission rate of edible film based on cassava starch

Type of edible film Cassava starch

Replicate 1 2 3

Average SD cassava starch film incorporated with the whole extracts

WVT (g H2O mm cm-2) 0.0008216 0.0008216 0.0007106 0.0007846 0.0000641

WVTR (g H2O mm h-1 cm-2) 0.0004108 0.0004108 0.0003553 0.0003923 0.0000321

1 2 3

Average SD Cassava starch film incorporated with partially purified bacteriocin

0.0010801 0.0010464 0.0009451 0.0010239 0.0000703

0.0005401 0.0005232 0.0004726 0.0005119 0.0000351

1 2 3

Average SD

0.0010215 0.0009704 0.0009449 0.0009789 0.0000390

0.0005107 0.0004852 0.0004724 0.0004895 0.0000195

83

Appendix C8: Antimicrobial activity of edible film based on cassava starch on the indicator strains

Antimicrobial activities Type of edible film replicate Escherichia coli TISTR 780 15.00 14.00 15.00 14.67 0.5774 Inhibition zone (mm) Salmonella Staphylococcus typhimurium aureus TISTR Listeria TISTR 292 029 monocytogenes 13.50 17.00 22.00 13.00 16.00 21.00 14.00 17.50 21.50 13.50 16.83 21.50 0.5000 0.7638 0.5000 -

1 2 Cassava starch 3 Average SD 1 Cassava starch 2 film incorporated 3 with antimicrobial Average substance SD 1 2 Cassava starch film incorporated 3 with bacteriocin Average SD

84

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 Number of microorganism (CFU/ml) Cassava starch cassava starch film incorporated with plastic cassava incorporated with the partially purified bag starch film whole extracts bacteriocin 143,000 176,000 220,000 251,000 240,000 250,000 173,000 142,000 181,000 240,000 240,000 183,000 199,909 41,555.88 9,100 9,300 11,700 5,900 11,200 13,100 10,400 19,200 5,300 14,700 6,800 17,500 8,533 12,367 9,633 14,200 2,991 2,701 2,538 7,238 8,200 11,500 3,500 11,400 14,800 8,300 4,800 3,200 9,900 13,300 2,110 2,900 10,967 13,300 3,470 5,833 3,427 2,532 1,345 4,823 15,300 11,600 2,480 9,700 8,600 8,300 6,800 13,600 13,100 12,300 8,400 8,800 12,333 10,733 5,893 10,700 3,415 2,136 3,062 2,551 19,500 11,000 4,500 11,300 23,200 17,400 6,300 14,600 27,100 12,800 5,700 13,700 23,233 13,733 5,500 13,200 3,800 3,301 917 1,706

(day) Replicate 1 2 0 3 Average SD 1 2 1 3 Average SD 1 2 3 3 Average SD 1 2 7 3 Average SD 1 2 10 3 Average SD

85

Appendix C10: Orange juice The pH value of various types of orange juice during storage period pH Pasteurize orange juice with 0.1% Sodium benzoate 3.64 3.64 3.65 3.64 0.0058 3.65 3.66 3.66 3.66 0.0058 3.65 3.65 3.65 3.65 0 3.65 3.67 3.68 3.67 0.0153 3.67 3.66 3.67 3.67 0.0058 3.66 3.67 3.66 3.66 0.0058
86

Time (day) Replication 1 2 3 Average 0 SD 1 2 3 Average 1 SD 1 2 3 Average 3 SD 1 2 3 Average 6 SD 1 2 3 Average 10 SD 1 2 3 Average 13 SD

Pasteurize orange juice 3.35 3.36 3.36 3.36 0.0058 3.35 3.37 3.37 3.36 0.0115 3.36 3.37 3.38 3.37 0.01 3.38 3.37 3.38 3.38 0.0058 3.37 3.36 3.35 3.36 0.01 3.37 3.37 3.37 3.37 0

Pasteurize orange juice with 0.5% bacteriocin 3.4 3.4 3.39 3.4 0.0058 3.39 3.39 3.4 3.39 0.0058 3.38 3.37 3.39 3.38 0.01 3.38 3.37 3.39 3.38 0.01 3.41 3.4 3.43 3.41 0.0153 3.42 3.41 3.41 3.41 0.0058

Pasteurize orange juice with 1.0% bacteriocin 3.39 3.4 3.4 3.4 0.0058 3.4 3.39 3.39 3.39 0.0058 3.4 3.4 3.4 3.4 0 3.39 3.4 3.41 3.4 0.01 3.42 3.42 3.4 3.41 0.0115 3.42 3.41 3.4 3.41 0.01

Pasteurize orange juice with 2.5% bacteriocin 3.43 3.42 3.42 3.42 0.0058 3.41 3.41 3.41 3.41 0 3.42 3.42 3.42 3.42 0 3.41 3.42 3.44 3.42 0.0153 3.43 3.42 3.4 3.42 0.0153 3.41 3.41 3.42 3.41 0.0058

The pH value of various types of orange juice during storage period (Cont.) pH Pasteurize orange juice with 0.1% Sodium benzoate 3.67 3.66 3.66 3.66 0.0058 3.68 3.69 3.67 3.68 0.01 3.7 3.73 3.71 3.71 0.0153 3.7 3.69 3.7 3.7 0.0058 Pasteurize orange juice with 0.5% bacteriocin 3.42 3.41 3.41 3.41 0.0058 3.41 3.42 3.41 3.41 0.0058 3.46 3.45 3.43 3.45 0.0153 3.43 3.44 3.42 3.43 0.01 Pasteurize orange juice with 1.0% bacteriocin 3.41 3.41 3.42 3.41 0.0058 3.41 3.41 3.41 3.41 0 3.47 3.46 3.43 3.45 0.0208 3.46 3.44 3.42 3.44 0.02 Pasteurize orange juice with 2.5% bacteriocin 3.42 3.42 3.41 3.42 0.0058 3.42 3.42 3.42 3.42 0 3.46 3.45 3.44 3.45 0.01 3.47 3.46 3.44 3.46 0.0153

Time (day) Replication 1 2 3 Average 21 SD 1 2 3 Average 29 SD 1 2 3 Average 36 SD 1 2 3 Average 42 SD

Pasteurize orange juice 3.37 3.38 3.37 3.37 0.0058 3.36 3.36 3.37 3.36 0.0058 3.44 3.37 3.38 3.4 0.0379 3.37 3.37 3.37 3.37 0

87

The number of microorganism in orange juice Time (Day) 10 13 10 10 0 6.666667 5.773503 -

sample 1 1 1 Average SD 2 2 2 Average SD 3 3 3 Average SD 4 4 4 Average SD 5 5 5 Average SD

0 -

1 -

3 -

6 -

21 20 40 0 20 20 -

29 30 90 60 60 30 -

36 30 10 0 13.33333 15.27525 10 0 0 3.333333 5.773503 -

88

Appendix C11: Pasteurized milk The pH value of various types of pasteurized milk during storage period pH Pasteurized milk Pasteurized milk with 1.0% with 2.5% bacteriocin bacteriocin 6.72 6.72 6.72 6.72 6.72 6.72 6.72 6.72 0 0 6.72 6.73 6.72 6.72 6.72 6.7 6.72 6.72 0 0.0153 6.74 6.74 6.71 6.73 6.73 6.72 6.73 6.73 0.0153 0.01 6.71 6.73 6.71 6.72 6.71 6.73 6.71 6.73 0 0.0058 6.73 6.73 6.74 6.72 6.73 6.73 6.73 6.73 0.0058 0.0058 6.68 6.7 6.69 6.7 6.7 6.7 6.69 6.7 0.01 0

Time (day)

Pasteurized milk Replication 1 2 3 Average SD 1 2 3 Average SD 1 2 3 Average SD 1 2 3 Average SD 1 2 3 Average SD 1 2 3 Average SD 6.72 6.72 6.72 6.72 0 6.68 6.72 6.72 6.71 0.0231 6.72 6.73 6.72 6.72 0.0058 6.68 6.66 6.67 6.67 0.01 6.7 6.69 6.69 6.69 0.0058 6.6 6.61 6.67 6.63 0.0379

16

23

31

Pasteurized milk with 0.5% bacteriocin 6.72 6.72 6.72 6.72 0 6.72 6.72 6.72 6.72 0 6.73 6.72 6.72 6.72 0.0058 6.7 6.71 6.71 6.71 0.0058 6.71 6.7 6.71 6.71 0.0058 6.68 6.68 6.7 6.69 0.0115

89

The pH value of various types of pasteurized milk during storage period (Cont.) pH Pasteurized milk Pasteurized milk with 1.0% with 2.5% bacteriocin bacteriocin 6.71 6.72 6.72 6.72 6.71 6.71 6.71 6.72 0.0058 0.0058 6.71 6.71 6.72 6.72 6.72 6.72 6.72 6.72 0.0058 0.0058

Time (day)

Pasteurized milk Replication 1 2 3 Average SD 1 2 3 Average SD 6.55 6.57 6.54 6.55 0.0153 6.45 6.41 6.42 6.43 0.0208

35

41

Pasteurized milk with 0.5% bacteriocin 6.72 6.71 6.7 6.71 0.01 6.72 6.71 6.7 6.71 0.01

90

The number of microorganism in pasteurized milk Time (Day) 16 23 40.0 20.0 30.0 0.0 30.0 0.0 33.3 6.7 5.774 11.547 -

Sample Pasteurize milk

Pasteurize milk with 0.5% bacteriocin Pasteurize milk with 1.0% bacteriocin Pasteurize milk with 2.5% bacteriocin

replicate 1 2 3 Average SD 1 2 3 Average SD 1 2 3 Average SD 1 2 3 Average SD

0 -

3 -

31 35 50.0 0.0 0.0 16.7 28.868 -

41 -

91