Study On The Characteristic Analysis And Antibacterial Activity Of Lactobacillus Plantarum TWK10 Fermented Soy Milk - Unika Repository

  

STUDY ON THE CHARACTERISTIC ANALYSIS AND

ANTIBACTERIAL ACTIVITY OF Lactobacillus plantarum

TWK10 FERMENTED SOY MILK

  

Practical Training at Fu Jen Catholic University, Taipei, Taiwan

By:

ALFIANA SAVITRI

Student ID : 14.I1.0116

  

Faculty: Agricultural Technology

This Practical training report has been approved and supported by examiner in

th

  

Practical Training Exam on June 8 2017

th

  Semarang, June 8 2017 Department of Food Technology Faculty of Agricultural Technology Soegijapranata Catholic University

  Practical Training Advisor I Practical Training Advisor II Dr. Tsung-Yu Tsai Dr. Ir. Lindayani, MP.

  

Dean

Dr. Victoria Kristina Ananingsih, ST., Msc.

  

PREFACE

  Praise the Lord because of His grace and blessing, the author had the opportunity to undergo the practical training and finished the report. This report is the complete accountability from the practical training which was done in Fu Jen Catholic University,

  th th New Taipei City, Taiwan that took place from January 4 until February 28 2017.

  During the training, the author did the research entitled: “Study on the Characteristic Analysis and Antibacterial Activity of Lactobacillus plantarum TWK10 Fermented Soy Milk”. This report was written as a requirement to acquire Bachelor Degree of Food Technology in Soegijapranata Catholic University, Semarang, Indonesia. The author would not be able to finish this task alone, and only by support and guidance given by people around the author that, this report could be finished. Special thanks for: 1.

  Allah SWT who always blessed, saved and guided the author in every step of practical training in Taiwan.

  2. Dr.V. Kristina Ananingsih, ST, MSc. for giving the author the opportunity to join the internship program.

  3. Dr. Tsung-Yu Tsai as author’s advisor for giving advices and supporting the author all the time during the practical training.

  4. Dr. Ir. Lindayani, MP. as author’s advisor for taking care of the author during the practical training and making the report.

  5. Joanne, Ruby, and Jessica who always helped the author to do the research.

  6. My family, Mom, Dad, and Brother, who always support and cheer for the author.

  7. Jessica, Denny, Irfan and all my friends that cannot be mentioned one by one who always support the author during the practical training until finishing the report.

  The author realizes that this report is still far from perfect and there are still many shortcomings due to the limitation of the author. However, the author hopes that this report can be an inspiration and provide useful information for all the reader.

  th

  Semarang, June 8 2017 Alfiana Savitri

  TABLE OF CONTENTS

  APPROVAL PAGE ......................................................................................... i PREFACE ........................................................................................................ ii TABLE OF CONTENTS ................................................................................. iii LIST OF TABLES ........................................................................................... iv LIST OF FIGURES ......................................................................................... v LIST OF APPENDIXES ................................................................................. vi 1.

  1 INTRODUCTION........................................................................................

  1.1.

  1 Background of Practical Training.........................................................

  1.2.

  2 Purpose of Practical Training................................................................

  1.3.

  2 Time and Place of Practical Training....................................................

  2.

  3 INTITUTION PROFILE...............................................................................

  2.1.

  3 Fu Jen Catholic University.....................................................................

  2.2.

  3 Department of Food Science..................................................................

  3.

  4 RESEARCH PROJECT................................................................................

  3.1.

  4 Background of Research........................................................................

  3.2.

  5 Literature Review...................................................................................

  3.2.1.

  5 Fermented Soy Milk........................................................................

  3.2.2.

  6 Lactobacillus plantarum TWK 10..................................................

  3.2.3.

  7 Extraction........................................................................................

  3.2.4.

  8 Antibacterial Agents........................................................................

  4.

  10 RESEARCH METHODOLOGY..................................................................

  4.1.

  10 Materials.................................................................................................

  4.2.

  10 Methods..................................................................................................

  4.2.1.

  10 Sterilization of Soy Milk.................................................................

  4.2.2.

  10 Fermentation of Soy Milk...............................................................

  4.2.3.

  11 Microbial Count..............................................................................

  4.2.4.

  11 pH Analysis.....................................................................................

  4.2.5.

  11 Acidity Value..................................................................................

  4.2.6.

  11 Extraction for Water and Ethanol...................................................

  4.2.7.

  12 Prepare for Antibacterial Test.........................................................

  5.

  13 RESULT AND DISCUSSION......................................................................

  6.

  19 CONCLUSION AD SUGGESTION.............................................................

  6.1.

  19 Conclusion..............................................................................................

  6.2.

  19 Suggestion...............................................................................................

  7.

  20 REFERENCES...............................................................................................

  8.

  23 APPENDIX....................................................................................................

  LIST OF TABLES

  Table 1. Composition of soymilk unfortified in 100 gram..................................... 6 Table 2. pH and acidity value of Lactobacillus plantarum TWK 10 fermented soymilk..................................................................................... 15 Table 3. Antibacterial activity of water and ethanol extracts from L. plantarum

  TWK10 fermented soy milk against Sthaphylococcus aureus BCRC 10451 and Escherichia coli BCRC11634...................................... 16

  Table 4. Antibacterial activity of Lactobacillus plantarum TWK 10 in different medium................................................................................... 17

  LIST OF FIGURES Figure 1. Growth curve of bacteria...................................................................

  14 Figure 2. The cell number of Lactobacillus plantarum TWK 10 fermented soymilk............................................................................

  14 Figure 3. The pH value of Lactobacillus plantarum TWK 10 fermented soymilk............................................................................

  15 Figure 4. The titratable acidity of Lactobacillus plantarum TWK 10 fermented soymilk............................................................................

  15 Figure 5. Map of Fu Jen Catholic University ....................................................

  23

  LIST OF APPENDIXES Appendix 1. Place of Practical Training .......................................................

  23 Appendix 2. List of Attendance …….........................................................

  24

1. INTRODUCTION

1.1. Background of Practical Training

  Food is important in the sustainability of human life. People do not only think to eat delicious food but also the nutrition that comes from it. They begin to expect healthy food to maintain their health. Nowadays, many food industries with their great technology put concern about food nutrition, quality, and safety. We know that food technology has an important role to develop food industry. In this era we begin to place concerns about food in many aspects like product development, safety, packaging, nutrition, waste management, and also food microbiology. Department of Food Technology, Soegijapranata Catholic University have a training program for student to improve their ability, knowledge, and skills. There are two programs available for this training, i.e student can improve their skill on food industry or do research on laboratory. Students who choose to train in the industry will get experience about how food technologist has the role about product development. Students who choose to do research will get experience on doing research in the university. Department of Food Science, Fu Jen Catholic University, Taiwan is choosing as one of University selected for research training in biotechnology and microbiology.

  The title of the research was, “Study on the Characteristic Analysis and Antibacterial Activity of Lactobacillus plantarum

  TWK10 fermented soy milk”. The advisor of this research was Dr Tsung-Yu Tsai, as the Associate Professor of Food Science Department, Fu Jen Catholic University, Taiwan. The mentor of this research was Joanne as a student of master degree program of Food Science Department, Fu Jen Catholic University, Taiwan.

  1.2. Purpose of Practical Training

  This practical training have purpose : a.

  To give experience to explore food in the research, so the student can implement the knowledge that they learned in the scientist world or real food industry.

  b.

  To give an opportunity to adapt with new circumtances and neighborhood.

  c.

  To boarden the knowledge and experience of international exposure.

  1.3. Time and Place of Practical Training

  This practical training took place in department of food science, Fu Jen Catholic University, Xinzhuang District, New Taipei City, Taiwan. This research was conducted

  th th during 4 January to 28 February 2017.

  .

2. INSTITUTION PROFILE

  2.1. Fu Jen Catholic University

  Fu Jen Catholic University (FJCU) is the first catholic university in Taiwan. Establish in 1925 by Benedictines of St. Vincent Archabbey in Latrobe, Pennsylvania and re- establish in 1961. Located in Zhongzheng Road, Xinzhuang District, New Taipei City, Taiwan which is strategic location and have an easy access to social activities on the capital of Taiwan. There are 1.942 staff academic, with 22.395 undergraduate students and 4.041 graduated students (master program). They provide 11 colleges with 48 department, 47 master program, 23 in service master program, 11 program doctoral program, and 16 departments in school of continuing education. FJCU on the basis of truth, goodness, beauty, and holiness committed to a dialogue leading to the integration of Chinese culture and Christian faith, to academic research and the promotion of genuine knowledge, to the development of society and the advancement of humankind. Fu Jen have seven goals for their, there are human dignity, meaning of life, academic research, community awareness, dialogue with cultures, religious cooperation, and spirit of service.

  2.2. Department of Food Science

  Department of family studies and nutrition science was established in 1963. In 1971 nutrition science was combined with the food sciences as the department of nutrition and food science. And in 2006 food sciences became an individual department that offers bachelor’s degree program and master’s degree program. With committed to promote the healthier, tastier, and safer foods for improving eating quality, human health and wellness.

3. RESEARCH PROJECT

3.1. Background of Research

  Antibacterial is a substance that can prevent, manage, and treat infection caused by bacteria and is known as antibiotics. Antibiotics in natural or synthetic form can interfere, inhibit the growth of bacteria, or kill the bacteria. Bacteria become resistant to antibacterial by natural or acquired mechanism. Natural resistant occur because of gene mutation and acquired resistant happen because adaptation of bacteria with conditional environment. This bacterial resistant represent a health problem. Hence we need to find new sources of antibacterial by testing active compound from plant or functional food (Stefanovic et al, 2012).

  Soybean contains bioactive constituents like protein, isoflavone and trypsin inhibitor. Cooking soybean can cause degradation of protein and nutritive value by heating. The highest protein value is given by 4-10 % residual trypsin inhibitory activity in the heated soy milk (Yuan et al, 2008). Soymilk is an extract from soybean which is an emulsion that contains soluble protein and other nutrition. It has the same appearance with cow’s milk but it has a beanie flavor. It can be easily consume for lactose intolerant person. The nutrition inside is not different with cow’s milk. To increase the nutrition value, soymilk can be fermented by lactic acid bacteria like Lactobacillus plantarum TWK 10.

  By fermenting soymilk, it can increase the anti oxidative value and also increase activity of lactic acid bacteria on it. We know that lactic acid has the ability to surpress bacteria. It has prime functions for a strong antimicrobial activity against many spoilage bacteria and food

• –born pathogens. It also produces inhibitory compound like hydrogen peroxide, acetaldehyde, diacetyl, and bacteriocin. In biology, bacteriocin is an active protein which exhibits bactericidal effect against bacteria (Blazenka, 1995). Extraction is an important step for isolation of bioactive compound. The extraction of bioactive compound depends on time of extraction, solid
• – liquid ratio, and the types of solvents (Cacace & Mazza, 2003). The solubility of bioactive compound is regulateds by the polarity of solvent used. Some solvent used for the extraction of bioactive

compound are methanol, ethanol, acetone, water, ethyl acetate and propanol. The right solvent makes maximum yield of compound optimal (Nazck & Shahidi, 2004).

  This experiment are to analyze the characteristic of Lactobacillus plantarum TWK 10 fermented soymilk in certain condition and to investigate the antibacterial activity of

  

Lactobacillus plantarum TWK 10 fermented soymilk and antibacterial activity of L.

plantarum TWK10 in different medium. Characteristic analysis involves recovery of

  water and ethanol extract, cell number, pH value, and titratable acidity. The inhibition zone diameters determine the antibacterial activity on sample. For different medium, there are MRS broth, Isolation soy protein, and Casein. We used E. coli for gram negative bacteria and S. aureus for gram positive bacteria.

3.2. Literature Review 3.2.1. Fermented Soy Milk

  Soymilk is a beverage from soybean. It contains of high vegetable protein, unsaturated fatty acid, soluble and insoluble dietary fibers and isoflavone (Božanić et al, 2011).

  Soymilk was extracted from soybean and through pasteurization process. Extract of soybean have a s imilar appearance with cow’s milk but it has a beany or soy flavor.

  Therefore, soymilk can be used as alternative for dairy product. It has similar protein content (3.5- 4.0%) with cow’s milk and pattern of amino acid, has 60-90% nutritional value of cow’s milk. Soymilk is deficient in sulfur containing amino acid. Soymilk contains protein amino acid Arginine and Glycine, that can reduce cholesterol. Soymilk can be a good alternative for people who are allergic to cow’s milk. It contains free lactose and easy for digestion. Soymilk is suitable for diabetic people (Ahaniah et al,

  2014) . Besides that soymilk becomes a great antioxidant when fermented. Fermenting soymilk can remove undesirable flavor like beany taste and makes protein more digestible. In some products, fermenting can increase their shelf life. In soymilk, fermentation will produce lactic acid because one of the bacteria use in fermenting is from genera

  

Lactobacillus. We usually know it as Lactic Acid Bacteria (LAB). Bifidobacterium is

  another main microorganism that is currently used (Božanić et al, 2011). LAB is a gram positive bacteria, do not have ability to form spore, and have coccus or rod shape. It ferment all carbohydrate into lactic acid. Soymilk contains galactooligosaccharides that can be used as prebiotic for probiotic. But fermentation can reduce galactooligosaccharides content in soymilk. During fermentation, hydrolysis of milk protein, decrease of pH, and increase of viscosity occurs. After fermentation, the characteristic of soymilk changes to curd, this is because sugars are fermented by bacteria into lactic acid (Horackova et al, 2015) . Composition of soymilk unfortified show in Table 1.

  Table 1. Composition of soymilk unfortified in 100 gram Composition of milk Unit Composition of milk Unit Water 88.05 g Potassium, K 118 mg Energy 54 kcal Sodium, Na 51 mg Protein 3.27 g Zinc, Zn 0.12 mg Fat (total lipid) 1.75 g Vitamin C (ascorbic acid) 0.0 mg Fatty acids, saturated 0.205 g Thiamin (vitamin B1) 0.06 mg Sugar total 3.99 g Riboflavin (vitamin B2) 0.069 mg Carbohydrates 6.28 g Niacin (vitamin B3) 0.513 mg Fiber 0.6 g Vitamin B6 0.077 mg Calcium, Ca 25 mg Folic acid 18 µg Iron, Fe 0.64 mg Vitamin B12 0.0 µg Magnesium, Mg 25 mg Vitamin A 0 µg Phosphorus, Mg 52 mg Vitamin E 0.11 mg [Source: USDA Nutrient Database for Standard Reference 2016] 3.2.2.

   Lactobacillus plantarum TWK 10

Lactobacillus plantarum is a gram positive, it has rod or coccus shape and can grow at

  temperatures 15

• –45°C with low pH around 3.2 (Kleerebezem et al., 2003). It has the ability to grow in aerobe or anaerobe condition. L. plantarum ferment sugar into lactic

acid, ethanol, acetic acid, and carbon dioxide in selective substrate and under certain condition (Siezen & Johan, 2011). It is acid and salt tolerant. Lactobacillus plantarum TWK10 have potential effect in probiotics: its metabolites inhibit pathogen growth, blocks melanin production, suppresses serum cholesterol, exhibits anti-obesity effects, modulates the immune system, reduce the risk of atherosclerosis and related cardiovascular disease when used to ferment in soymilk (Liu et al, 2016). This bacteria is considered as a safe probiotic and has become a current interest for researcher especially in food industry. As a probiotic component, it can help to limit the amount of pathogenic bacteria and decrease negative impacts on humans. L. plantarum TWK 10 strain can be found in Taiwanese fermented cabbage (Liu, 2016). Lactic acid bacteria are typically bacteria that can survive in wide ranges of temperature and pH in some easy spoilage food. L. plantarum that produce lactic acid has antibacterial activity and can be used as preservative in food product. L. plantarum have more effectiveness in inhibiting some microorganism than preservative like sodium benzoate and potassium sorbate (Crowley et al., 2012).

3.2.3. Extraction

  Extraction is a method to separate active compound based on differences in distribution of solvent between two solvent. The basic step of extraction process may contain pre washing, drying of material or freeze drying, blending or grinding to increase ability of kinetics, making a homogenous sample, and improving contact between surfaces of sample with the solvent. Appropriate actions must be taken to minimize lost or collapse of the potential active constituents during preparation of the extract from sample. Using different solvent systems is available to extract bioactive compound from natural plant. Extractions have various methods, depending on the aim, solvent, and desired compound. Some extraction methods use sonification, heating under reflux, soxhlet extraction, solid phase, and supercritical-fluid extraction (Sasidharan et al, 2011). Types of solvent can affect quality of an extract. Appropriate solvents use

  ‘like dissolves like’ principle, it means that non polar solvent will easily extract non polar substance and polar constituent will be extracted out by polar solvent. Extraction by differences of solvent is popular method of extraction. Ethanol is one of non polar solvent that can extract tannin, polyphenol, flavonol, terpenoid, sterol, alkaloid, polyacetylene, and propolis. Thus water which has characteristic as polar solvent can dissolve Sugar, amino acid, saponin, tannin, lectine, terpenoid, anthocyanin, starches, and polypeptide (Gupta

  

et al , 2012). To move molecule from high concentration to low concentration it needs

  driving force. Shaking is one of driving force that has principle the increasing of driving force followed by increasing of mass transfer rate and make consequently prompted diffusion rate of solute (Kain et al, 2009). Sampath (2013) study present that shaking can give effect on yield increasing of phenolic compound extraction until maximum.

3.2.4. Antibacterial Agents

  Antibacterial agent is used to help preventing spoilage caused by bacteria. Antibacterial agent can be differentiated by their mechanism of action such as antibacterial agent can inhibit growth of cell wall, inhibit synthesis of protein, and inhibit synthesis of nucleic acid cell. Activity of antibacterial agent can divided into 2 various there are bacteriostatic and bactericidal. Bacteriostatic is antibacterial that can inhibit growth of bacteria but cannot kill the pathogen, but bactericidal can kill pathogens (Brooks et al., 2013) Antibacterial activity test performed with diffusion and dilution method. Diffusion test is performed by measuring diameter of clear zone. This clear zone showed inhibition of bacterial growth by antibacterial agent. Mueller Hinton Agar (MHA) is used for antimicrobial testing by diffusion test. This agar commonly performed on aerobic and facultative anaerobic bacteria. MHA contains beef extract (2 gram), acid hydrolysate of casein (1.75 gram), and starch (1.5 gram) (Wayne, 2006).

  Diffusion method is done in three methods there are agar disc diffusion, agar plug diffusion, and agar well diffusion. The agar disc method work by inoculating agar plate with inoculum of the test microorganism then paper disc (diameter 6mm) that have been soaked in the test compound are placed on the surface of agar plate. The agar plate is incubated in certain condition. During incubation, antibacterial agent in the disc will diffuse into agar and work to inhibit growth of the test microorganism. The result is determined by measuring inhibition zone or clear zone (Salama et al, 2010).

  Agar plug diffusion is almost similar with agar disc. It is often used to know the antagonism characteristic between microorganisms (Jiménez et al, 2005). Agar plate media is inoculated with test microorganism. During incubation, diffusion happens when microbial cell secrete molecule and their secretion diffuse into agar medium. Then agar plot or cylinder plug is taken aseptic and deposited onto the surface of the other agar media that previously inoculated with the test microorganism. Antibacterial activity can be detected by the presence of inhibition zone around the agar media. It happens because there is a diffusion of substance from agar plot into agar media (Balouiri et al., 2016). Agar well diffusion method is used to evaluate antibacterial activity of microbial or plant extract (Magaldi et al., 2004). This method have a similarity with disk diffusion, inoculated microorganism is spreading on surface of agar plate. Well with diameter of

  6

• –8 mm is made with sterile tip or pipette plastic. The amount of well is made as needed, each well filled with 20
• –100 µl liquid extract or antibacterial agent. Agar plate is incubated under certain condition depending on condition of microorganism. Diffusion happened when antibacterial diffuse into agar plate and affect the growth of microorganism. It can inhibit the growth of microorganism by showing a clear zone (Balouiri et al., 2016).

4. RESEARCH METHODOLOGY

  4.1. Materials

  The materials that used were soymilk, Lactobacillus plantarum TWK10,

  

Staphylococcus aureus BCRC 10451, Escherichia coli BCRC 11634, gentamicin, MRS,

  MHA (Mueller Hinton Agar), NaCl 0.85%, NaOH 0.1N, ethanol, dimethylsulphoxide (DMSO), water, phenolphthalein indicator, micropipette, tips, tubes, plate dish, inoculating loop, 0.22µm syringe filter, sterile cotton bud, sterile bottle, centrifuge bottle, centrifuge, vortex, burette, water bath, shaker water bath, refrigerator, freezer, incubator, laminar flow, rotary evaporator, pH meter (glass electrode pH), and freeze drying.

  4.2. Methods 4.2.1. Sterilization of Soy Milk o

  Pour 1L soymilk into sterile bottle. Put it in the water bath with temperature 93 C for 1 hour. Then let it cool down until it reached room temperature and store at 4°C in the refrigerator (Yuan et al, 2008).

4.2.2. Fermentation of Soy Milk

  Using inoculating loop, Lactobacillus plantarum TWK10 was taken and put into 10 ml

  o

  MRS media. Then put it in the incubator at 37 C for 24 hours. Pour 10 ml MRS media contain Lactobacillus plantarum TWK10 into 1L soymilk. Then put the bottle of

  o

  soymilk in the incubator 37

  C. Take it out every 8 hours, 24 hours, 32 hours, 48 hours, 56 hours, and 72 hours.

  4.2.3. Microbial Count

  Plate that has been incubated was taken out. Do observation by counting the white spot in each dilution. Then make it into curve. It will show microbial rate on living.

  4.2.4. pH Analysis

  pH analysis was measured using glass electrode pH meter. It was calibrated with liquid that have pH 4, 7, and 10. The electrode was dipped in the sample till number of pH showed constantly and recorded it. Each sample was tested three times. pH meter is washes by pure water with very change of samples (Khodke et al, 2015).

  4.2.5. Acidity Value

  Each sample will be tested three times. 10ml of fermented soy milk was put in the Erlenmeyer with 3

• –4 drops of Phenolphthalein indicator. Burette was filled with 0.1 NaOH as titrant. The solution was titrated until the color of solution change into pink. Then burette reading was recorded as acidity value (Khodke et al, 2015).

  4.2.6. Extraction for Water and Ethanol

  Fermented soymilk was dried using freeze dryer for 3 days. Each 40 grams of dry soy milk was extracted with 170ml of ethanol for ethanol extract and 125ml of water for

  o

  water extract. Then put it in the water bath 37 C for 30 minutes. After that, using shaker

  o o

  bath, shake in 37 C 150rpm for 30 minutes (Salama et al,2010). Centrifuge at 4 C 12000 rpm for 30 minutes to separate water and ethanol contain. Collect the supernatant in each samples, freeze the supernatant of water extract. For supernatant of ethanol extract, was evaporated using rotary evaporator then put into freeze dryer for 3 days.

4.2.7. Prepare for Antibacterial Test

  Culture stock of Escherichia coli as gram negative and Sthaphylococcus aureus as gram positive was prepared by inoculating 1 loop of each bacterial into nutrient agar and incubated for 24 hours at 37°C. From each culture, single colony (from plate dish) was transferred into nutrient broth and shaken using vortex until reach equal density with

  0.05 Mcfarland standard. Agar well diffusion method was used to see antibacterial activity. Stock of water extract and ethanol extract were prepared as samples. 0.1 gram of water extract and ethanol extract was dissolved in 1mL sterilized water for water extract and DMSO for ethanol extract. Then filter using 0.22 µm syringe filter. Use sterilized cotton swab to spread each bacteria on MHA (Mueller Hinton Agar). Make 7 mm diameter well in MH agar using sterile pipette plastic. Load 50 µ L samples in each well, and 20 µ L for gentamicin as positive control. Incubate for 24 hours and measure the inhibition zone. The measurement was done by measuring the diameter of inhibition zone, then minus with diameter of the well (Pushparaj, 2014).

5. RESULT AND DISCUSSION

  In this study, soymilk was fermented by Lactobacillus plantarum TWK 10. It took 72

  o

  hours for fermentation at 37 C. Lactobacillus is a microbe that can produce lactic acid. It is a rod shape, gram positive bacteria. It is commonly found in gastrointestinal tracts,

  o

  and various food products. Lactobacillus can grow at temperature of 15 C and in

• –45 low pH. This bacteria works by changing sugar into lactic acid, ethanol, and carbon dioxide under certain condition. It can grow with or without presence of oxygen (Kleerebezem et al. 2003).This bacteria is interesting because it has safe probiotic characteristic that can help limit pathogen bacteria in the gastrointestinal tract. Soymilk contain high protein from extracted soy bean and it is free of lactose. Soymilk is fermented using Lactobacillus plantarum TWK10 for 72 hours. According to Bremer (1996), when microorganism is cultivated in liquid media, they will grow in a batch culture, it has growth curve in four phases: lag phase, log phase, stationary phase, and death phase (Figure 1). In the lag phase usually there is no increase of microbe, as they try to adapt with new environment. During log phase microbes start growing and dividing itself into media. At this point, the grow rate depends on the nutrient that the media has, and their environmental condition. They will grow quickly until reaching the top. Then microbes will be in stationary phase in which no observed. In this phase there will be balanced growth and microbe will be in constant amount. But this condition depends on nutrient levels that they have. If condition or environment change, there will be unbalanced result and it will not give stationary curve. During stationary phase,

  6

  usually only culture that have maximum concentration 10 /ml can be found. In this phase, microbial does not grow because of nutrient limitation factor. In death phase, microbe do not lyse, but loses their ability to produce a microbe. In the experiment it was found that the initial growth time of bacteria in the log phase is 8.778 CFU/ml in 24 hours. Stationary phase occurred during fermentation from 24 to 72 hours. No significant enhancement during that time. The final growth reach 9.322 number of CFU/ml. There is no death phase in this condition showed that bacteria grow according to the growth curve that shown in Figure 2.

  Figure 1. Growth curve of bacteria Figure 2. The cell number of Lactobacillus plantarum

  TWK 10 fermented soymilk Soymilk which has been fermented for 72 hours was then tested by pH meter (Table 2, Figure 3, and Figure 4), of the initial pH is 6.58. During fermentation until at the end of fermentation there is a decrease in pH value to 4.16. During fermentation time, bacteria

  

Lactobacillus plantarum TWK10 produced a lactic acid that can increase acidity and

affect pH value. It also happen to acidity value, there is an increase during fermentation.

  Table 2. pH and acidity value of Lactobacillus plantarum TWK 10 fermented soymilk Hours pH Acidity (unit)

  6.58 2.15 ± 0.52

  8 5.95 2.60 ± 0.18

  24 5.55 3.02 ± 0.03

  32 5.40 4.67 ± 0.58 6.35 ± 0.98

  48

  4.40

  56 4.37 6.85 ± 0.35

  72 4.16 8.17 ± 0.29

  The data are presented as means of the inhibition zone diameters ±SD (n = 3)

  Figure 3. The pH value of Lactobacillus Figure 4. The titratable acidity of

  plantarum TWK 10 fermented Lactobacillus plantarum TWK 10

  soymilk fermented soymilk When acidity value increases then pH value decreases during fermenting time. From Table 2, initial time of acidity showed lowest value was 2.15 then after 72 hours acidity increases to 8.17. This result is suitable with Bozanic

  ’s (2011) opinion, during fermentation time, there are changes of hydrolysis in milk protein that causes a decrease in pH value. When pH value decreased, there is an increase in acidity value. Extraction is one of method which is use to separate substance from a solid or liquid mixture with a liquid solvent. The solvent is or not miscible with the solid or liquid substance. Solid or liquid mixture will have intensive contact into solvent. With mixing process, two phases will be separated by gravity and centrifugal force. It will give extract and supernatant. To get pure active agent or recovery of the solvent, there is a need for further separation process. Evaporation is one of further separation processes. This process is used in condition that substance has very high volatile and solvent should have a low boiling temperature and low heat of evaporation. It is used to accelerate process to get pure substance. Evaporation can use rotary evaporator. Crystallization is another method to remove all water content by cooling substance in very low temperature and make substance falls out. Usually freeze drying is used in this process. Freeze drying also known as lyophilization, which is a drying process in which the solvent or the suspension medium is crystallized at a low temperature and thereafter sublimated from the solid state directly into vapor phase (Ciurzynska, 2011). Freeze drying has key benefits such as lower temperature, high recovery of volatiles, and high yield (Dincer, 2003). It shows how many percent (%) of extract from initial weight of sample. From this study, the recovery rate of water extract is 12.25 % and ethanol extract 2.93%. Water extract can be more recovered than ethanol extract.

  Table 3. Antibacterial activity of water and ethanol extracts from L. plantarum TWK 10 fermented soy milk against Sthaphylococcus aureus BCRC 10451 and

  Escherichia coli BCRC 11634

  Zone of inhibition (mm) Microorganism

  Ethanol extract Water extract Gentamicin

  4.7±0.6 17.5±4.9

• – S. aureus BCRC 10451

  E. coli BCRC 11634

  19.5±0.7

• – –

  The data are presented as means of the inhibition zone diameters ±SD (n = 3)

  Key :

  No presence of inhibition zone

• – = Table 3 showed that there is an inhibition zone in ethanol extract of Lactobacillus

  

plantarum TWK 10 fermented soymilk against S. aureus. And there is no antibacterial

  activity on Lactobacillus plantarum TWK 10 fermented soymilk against S. aureus on water extract. Different solvents have the capacity to extract different phytochemical dependings on their polarity in solvent, this indicates that most of bioactive compound that plays a role as antibacterial agent on sample have a moderately non polar characteristic (Salama et al, 2010). For activity against E. coli, in the water extract and ethanol extract there is no presence of inhibition zone. This indicates that Lactobacillus

  

plantarum TWK 10 fermented soymilk cannot inhibit E. coli. Gentamicin is used for

• 8 - -

  48 8.0 ± 0.0 8.0 ± 1.0 Isolation soy protein UN - -

  48 8.0 ± 0.0 6.3 ± 0.6 The data are presented as means of the inhibition zone diameter ±SD (n = 3) Keys: UN = Unfermented

  8.0 ± 0.0 6.3 ± 0.6

  40

  7.7 ± 0.6 5.7 ± 0.6

  32

  6.7 ± 0.6 5.7 ± 0.6

  24

  40 8.0 ± 0.0 7.0 ± 0.0 48 7.3 ± 1.1 6.7 ± 0.6 Casein UN - -

  7.0 ± 0.0 7.7 ± 0.6

  32

  7.3 ± 0.6 5.7 ± 0.6

  24

  7.7 ± 0.6 4.7 ± 1.5

  40

  6.3 ± 0.6 6.7 ± 0.6

  32

  6.3 ± 0.6 6.0 ± 1.0

  24

  0.8 23.4 ± 1.1 MRS broth UN - -

  E. coli BCRC 11634 Gentamicin 21.6 ±

  Zone of Inhibition (mm) S. aureus BCRC 10451

  Table 4. Antibacterial activity of Lactobacillus plantarum TWK 10 in different medium

  growth by specific inhibition. Gentamicin is effective to prevent infection of gram negative and positive bacteria such as E. coli and Staphylococcus sp.

  

E. coli is 19.5±0.7 mm. Gentamicin is a bacterial antibiotic which can affects bacterial

  controlling growth of bacteria. The inhibition zone for S. aureus is 17.5±4.9 mm and for

• 8 - -
• 8 - -
• – = No presence of inhibition zone

  Based on Table 4, MRS broth, isolation soy protein, and Casein are used instead of the protein in MRS broth. Using different type of MRS broth to cultivate lactic acid bacteria was to determine which cultivating substrate produce more antibacterial agent. In this experiment, there were 7 time points (0, 8, 24, 32, 40, 48h) of lactic acid bacteria grown in different medium. In this study we compared antibacterial activity in different incubation time of sample. For inhibition of both bacteria (Table 4), Gentamicin is used as an antibacterial agent that has inhibition zone 21.6±0.8 mm against S. aureus and 23.4±1.1 mm against E. coli. There is no inhibition zone for 0 hour until 8 hours in all medium for both bacteria. It means that L. plantarum TWK 10 incubated for 0 hour until 8 hours cannot produce antibacterial compounds against S. aureus and E. coli. To inhibit S. aureus incubation with MRS media for 48 hours or incubation with isolated soy protein and casein for 40 hours is suitable. In 40 hours incubation time found that isolation soy protein was the best medium for L. plantarum TWK 10 against S. aureus. For activity against E. coli, MRS media showed the largest inhibition zone in 48 hours of incubation with 8.0±1 mm. This makes it a suitable medium to against E. coli for that period of time.

6. CONCLUSION AND SUGGESTION

  6.1.Conclusion

   Water extract of Lactobacillus plantarum TWK 10 fermented soymilk more recovered than ethanol extract. There are increasing of cell number during fermentation followed with decreasing of pH value and increasing of acidity value.

   Activity of antibacterial on Lactobacillus plantarum TWK 10 fermented soymilk can against S. aureus BCRC 10451 with 4.7±0.6 mm of inhibition zone on ethanol extract. Isolate soy protein and casein are the best medium that can against S. auerus BCRC 10451 in 40 hours incubation time with 8.0±0.0 mm of inhibition zone. For against E. coli BCRC 11634 the best medium is MRS broth in 48 hours of incubation with 8.0±1.0 mm of inhibition zone.

  6.2.Suggestion

  For future studies, the effect of fermentation to the ability of producing antibacterial agents need to be more evaluated.