The Lactic Acid Bacteria in Fermented Durian (D. zibethinus)

Lactic acid (C2H5COOH) is the organic acid that can serve as a food preservation. The group of bacteria which can produce lactic acid in their fermentation process known as Lactic Acid Bacteria (LAB). A fundamental biochemical change of fermentation is that an acidic environment is created. Many harmful organisms cannot exist in acidic solution so the fermentation productions are save to eat. Fermented Durian is the fermented food prepared from spontaneous fermentation of durian (Durio zibethinus) with or without salt by wild bacteria. The research was conducted to isolate and identify lactic acid bacteria in tempoyak. The data in this study were laboratory analysis. Samples were collected weekly in a month analyzed microbiologically from traditionl markets in Bengkulu. There were four species of lactic acid bacteria involved in fermented durian namely Leuconostoc mesentroides, Pediococcus acidilactici, Lactobacillus plantarum, and Lactobacillus curvatus.


INTRODUCTION
Lactic acid bacteria are group of bacteria have their ability to produce lactic acid from substrate of sugars in fermentation process. According to Stamer (1979) the term of lactic acid bacteria is including a group of microorganisms which have their ability to produce lactic acid from carbohydrates resources of their fermentation processes. Lactic acid bacteria involve in many food fermentation products such as vegetables, milk and meat. Furthermore, Dyson and McShane (2009) reported that a fundamental chemical underpinning of fermentation is that an acidic environment is created. Many harmful microorganisms cannot exist in an acidic solution. While lactic acid bacteria go to work on carbohydrates found in flour, grain, dairy products and vegetables to produce an acidic environment that is both suitable for the preservation of the food and for the changing the nature of the flavour of the food. Lactic acid bacteria also can turn the cyanide compounds into cyanic gases which than escape from fermenting food, thus rendering it harmless.
Fermented durian is a kind of traditional spontaneous fermented food prepared from durian (durio zibethinus) by wild microorganisms. Fermented durian is normally prepared from excess, poor quality or over-ripe of durian. The processing of fermented durian is very simple, prepared by mixing the durian pulp with or without salt and left to ferment anaerobic condition at room temperature for week or ten days.
Contamination with spontaneous wild microorganisms caused biochemical changes of substrate into lactic acid. There are many benefits from food fermentation that make it so widespread in almost every culture throughout the world, high profit from the fermentation procedure is low-cost, low-energy process, and production efficiency in food preservation.
Fermented durian is found not only in Bengkulu but also as long as Sumatera island with different names, and also in Malaysia. Until now, very litle study has been done , recent study reported that the dominant microorganisms in fermented durian was Lachtobacillus plantrum (Leisner et al, 2001). According to Keith (2010) indigenous food fermentations, fermentation in which organic acids are major products, in this category includes Korean kimchi, sauerkraut, fermented mild and cheeses. African ogi and uji, idli, dosai, tape and also tempe, in which acidification occurs during the initial soaking of the soybeans. Meanwhile, according to Leisner et al (2001). fermented durian is a traditional food made from tradision Malaysian durian pulp with or without the addition of salt. Furthermore, Amin et al (2004) reported that in Malaysia fermented durian normally prepared from the poor quality or over-ripe durian with the addition of 1.3% salt to support the growth of lactic acid bacteria and left to ferment at room temperature for 3-7 days.
The typical processing of durian use the traditional methods are well known as traditional foods. The term of traditional food is food, ingredients, naturally including herbs, food, and beverages traditionally prepared and consumed by local Indonesian community (Dewan Riset Nasional, 1995). Growth and development of commercial food processing industry are caused by several things including: (1). To increase the seasonal level of the availability of fruits. (2) Fermented fruits and vegetables provide a mechanism that causes the fresh ingredients transformed into the components of their respective acid without reducing the nutritional value and physical properties of the products.
(3) Can be applied with minimal equipment mechanically and economically so that the processing process can be applied cheaply (Fleming, 1988;Prescott and Dunn, 1958).

This research was conducted at
Laboratory of Agricultural Technology, Faculty of Agriculture University of Bengkulu. The samples were collected weekly in a month from traditional markets in Bengkulu. The sample was diluted with distilled water with various serial dilutions. Each of the samples was taken 1 ml and cultured on Glucose Yeast Peptone Agar (GYP), and incubated at 35 0 C for 48 hours. The single colony was collected and purified by the method of Streak Plate Technique and was grown on slant agar for further observation. Isolates from the various samples was identified gram reaction, shape, size, and composition of the cell, presence or absence of spores, capsule and flagella, test of temperature, catalyst, NaCl solution, homo / hetero-fermentation, oxygen requirement, and acid production from various carbon resources. Based on test results, the isolates were grouped into similar characteristics. This identification method refers to methods of Buhanan and Gibbon (1976) and Speck (1976).

RESULTS AND DISCUSSION
The observation of cell shape, gram reaction, catalyst, gas from glucose, the ability to live on 15% NaCl, the ability to live at a temperature of 10 ° C, 45 o C and 50 o C and acid production from various carbon resources, the isolates were classified into 4 groups (Table 1, 2, 3 and 4). Group I represent strains which sphere or ovoid cells in chains, and having cell size ranging 0.9 to 1 micron in diameter. They were gram positive, becoming gram negative with increasing age and acidity. Twenty-four hours cultures to 48 four hours cultures were gram positive and gram negative for 72 hours culture. These homo-fermentative isolates were catalase negative grew best at microaerophilic condition. This group has morphological characteristics such as filliform in stab and slant culture, and irregular surface growth in stab culture, acid on 24 hours lactose agar broth, nonmotile and non-spore formers. The optimum temperture between 10 o to 45 o C. No growth occurred at 50 o C. The strains can produce acid from glucose, sucrose, fructose, lactose, maltose, mannose and arabinose. They did not produce gas from glucose and did not grow in 15% NaCl solution. Based on these characteristics, the strains which belonged to Group I was Pediococcus acidilactici (Fig.1).

Figure 1. Photo micrographs of bacteria
Pediococcus acidilactici isolated from fermented durian The second group consisted of strains which were gram positive rods less than 5 micron wide with square ends and formed chain. Flagella were absent and non-motile, catalase negative and microaerophilic oxygen requirement. Filliform growth in stab and slant and irregular surface growth on lactose stab culture were observed. Gram positive, became gram negative after 48 hours of growth and with increasing acidity of media. No gas formation was produced from glucose. The homofermentative lactic acid bacteria formed acid from glucose, sucrose , fructose, lactose , maltose, mannose and arabinose. The strains grew in 15% NaCl solution and optimum growth between 10 o to 45 o C. Based on these characteristics, the strains which belonged to Group II was Lactobacillus plantarum (Fig.2).  The characteristics of group III were gram positive rods less than 1.5 micron wide with square ends and formed chain. Flagella were absent and non-motile, catalase negative and microaerophilic oxygen requirement. Filliform growth in stab and slant and irregular surface growth on lactose stab culture were observed. Gram positive, became gram negative after 48 hours of growth and with increasing acidity of media. No gas formation was produced from glucose. The homofermentative lactic acid bacteria formed acid from glucose , sucrose , fructose, lactose , maltose, mannose, but did not form from arabinose. The strains grew in 15% NaCl solution and optimum growth between 10 o to 40 o C. Based on these characteristics, the strains which belonged to Group III was Lactobacillus curvatus (Fig. 3).  Group IV represent strains which sphere or ovoid cells in chains, and having cell size ranging 0.5 to 0.75 micron in diameter. They were gram positive, becoming gram negative with increasing age and acidity. Twenty-four hours culture to 48 were gram positive and gram negative for 72 hours culture. These isolates were catalase negative grew best at microaerophilic condition. This group has morphological characteristics such as filliform in stab and slant culture, and irregular surface growth in stab culture, acid on 24 hours lactose agar broth, nonmotile and non-spore formers. The optimum temperture from 10 o to 45 o C. No growth occured at 50 o C. The strains can produce acid from glucose, sucrose, fructose, lactose , maltose, mannose and arabinose. They produced gas from glucose and grew in 15% NaCl solution. Based on these characteristics, the strains which belonged to Group IV was Leuconostoc mesentroides (Fig.4).  While According to Stammer (1979) genus Pediococcus and Leuconostoc are the types of lactic acid bacteria commonly found in fermenting fruits and vegetables.

CONCLUSION
There werere four species of lactic acid bacteria identified in fermented durian namely: Pediococcus acidilactici, Lactobacillus plantarum, Lactobacillus curvatus and Leuconostoc mesentroides. The four species have positive role in the fermented durian.