Pakoba fruit samples used in this study were obtained from North Minahasa Regency.  Occurrence of lactic acid bacteria (LAB) performed on Pakoba Fruit (Syzygium sp.)  has succeeded in obtaining 35 colonies of acid-producing bacteria indicated by the formation of clear zones around the colonies grown on MRS Agar selective media containing calcium carbonate (CaCO₃) 1% and sodium azide 1 ppm (Figure 1).  MRS is a growth medium for acid-producing bacteria because in MRS media there is glucose which is a source of carbon and energy for the growth of these bacteria. Calcium carbonate (CaCO₃) suspension that appears cloudy in the medium so that it will be dissolved by acids produced by acid-producing bacteria including LAB to form a clear zone around the colony.

The presence of calcium carbonate (CaCO3) is an indication that the bacteria growing on MRS media are acid-producing bacteria which include lactic acid bacteria (LAB). Before the isolation process, enrichment is carried out to obtain isolates of acid-producing bacteria which were incubated for 2 x 24 hours at room temperature.^5,6  Furthermore, purification was carried out on 35 isolates obtained as much as 2x with the quadran streak technique on MRSA+ CaCO₃ media.

Early detection of LAB colonies is based on the presence of clear zones formed around the colony on MRSA+CaCO₃ 1% isolation media.  Furthermore, screening of LAB was carried out by conducting a confirmatory test on isolates suspected of being LAB by conducting a Gram reaction test, catalase reaction test, endospore test, and motility test.

LAB screening of 35 colonies of acid-producing bacteria that have a circular shape with white and yellowish-white colony colors, gram positive, catalase negative, non-motile, without spores showed that seventeen colonies of acid-producing bacteria were confirmed as members of the lactic acid bacteria group and eighteen colonies of acid-producing bacteria were not included in the lactic acid bacteria group because they showed characteristics gram negative, catalase positif, motile and has spores (Table 1). This indicates that LAB isolates have been successfully obtained from samples of Pakoba fruit (Syzygium sp.). In addition to LAB, bacteria members of the genus Micrococcus, Bacillus, Pseudomonas, Staphylococcus, Salmonella, Shigella, Vibrio, Klepsiella are usually found in fruits and fruit products such as fresh fruit juice (Mango fruit and Avocado fruit), fresh guava fruit juice, banana fruit, papaya fruit, Jujube fruit, fruit, Plum fruit, Apple fruit, Cantaloupe fruit, Watermelon fruit, Dragon fruit.^20-23

Table (1):
Screening of LAB and Non LAB Isolates Growing on MRSA-CaCO3 Medium Obtained from Buah Pakoba (Syzygium sp.)

Based on Table 2, it is known that the morphology of the seventeen colony of LAB isolates has almost the same character. The morphological observations of the colonies of the seventeen isolates show that the seventeen isolates have a circular colony shape, yellowish-white color, entire edge (flat) and opaque inner structure (not translucent). The morphological character of LAB colonies from Pakoba fruit obtained in this study is the same as the results of research by²⁴  who reported that lactic acid bacteria isolates from Sweet Arum mangoes have colony characteristics, namely yellowish-white color, round shape, entire edge, smooth surface and convex elevation, and opaque inner structure.

Table (2):
Morphological characteristics of BAL colonies of Pakoba Fruit (Syzygium sp.)

Based on the results of Gram stain, it is known that the seventeen LAB isolates from Pakoba fruit are bacil (stem) shaped and purple cells. This shows that all seventeen bacteria are Gram positive. Gram positive bacteria have the ability to retain the main crystal violet paint after washing with alcohol, so the cells are purple
(Figure 2). According to,¹⁷ bacteria that can withstand purple paint (crystal violet) even though they have been denatured with alcohol or acetone and still give purple color to bacterial cells are called Gram positive bacteria. Conversely, bacteria that cannot withstand crystal violet dye after decolorization with alcohol and will absorb safranin paint with red characteristics are Gram negative bacteria. The ability of Gram positive bacteria to maintain crystal violet paint is due to the fact that the cell wall of Gram positive bacteria has a lower lipid content than Gram negative bacteria, so that the bacterial cell wall will be more easily degraded due to treatment with alcohol. The degraded cell wall causes the size of the cell pores to be small and the permeability is reduced so that the violet crystal dye cannot leave the cell and the cell will remain purple. While Gram negative bacteria look red because bacteria lose crystal violet dye when rinsing with alcohol but can absorb the last dye, safranin. Gram-negative bacteria contain lipids, or substances such as fat in a higher percentage than those contained in Gram-positive bacteria.²⁵

The characteristics of the seventeen LAB isolates obtained from Pakoba fruit as shown in Table 3 and Table 4, correspond to the key characters of lactic acid bacteria.⁴  In general, lactic acid bacteria have featured characters, namely including the group of Gram positive bacteria, non-spore, bacil or cocci shaped, catalase negative, single or pair structure cell (two-two)non-motile and produce lactic acid as the main end product during carbohydrate fermentation.³ The four genera most commonly found as the BAL group are Lactobacillus, Leuconostoc, Pediococcus and Streptococcus.^3,26 The genus Lactobacillus is a group of Gram positive bacteria in the form of bacil (rods), while the genera Leuconostoc, Pediococcus and Streptococcus are Gram positive bacteria in the form of coccus (round). Based on the data in Table 4 which shows that the seventeen LAB isolates from Pakoba fruit are Gram positive bacteria in the form of bacil, so it is indicated that the seventeen LAB isolates belong to the genus Lactobacillus. The main characteristics for differentiating LAB isolates to the genus level are cell shape, cell structure, and gas from glucose. Previous research also found LAB genus Lactobacillus from fruits, including guava rice, tome-tome, mangosteen, honey pineapple, salak, soursop and banana goods.^6,27-29

Table (3):
Characterization (Confirmation Test) Cell of LAB Isolates obtained from Pakoba Fruit (Syzygium sp.)

Table (4):
Morphological, Biochemical, and Physiological Characterization Cell of LAB from Pakoba (Syzygium sp.)

*Key character description genera Lactobacillus, Leuconostoc and Pediococcus based on I^a Lactobacillus

Fruit is one of the natural habitats for bacteria, including LAB. This is because the fruit contains various chemical compounds such as water, carbohydrates, fats, vitamins, organic acids, and various minerals needed as nutrients for LAB. Some types of fruit that are thought to be the habitat of LAB include Langsat fruit (Lactobacillus sp.),⁵ Salak fruit, banana, soursop fruit, mangosteen fruit, guava rice-rice  fruit (Lactobacillus, Pediococcus, Lactococcus and Leuconostoc),²⁸ Kersen fruit (Lactobacillus),²⁸ and sweet arum mango fruit (Lactobacillus).²⁴

Crude production of exopolysaccharides (EPS) LAB  in Pakoba fruit (Syzygium sp.)
In this study, crude production of exopolysaccharides (EPS) produced by each LAB isolate obtained from Pakoba fruit was carried out. The Seventeen isolates of LAB from Pakoba fruit (Syzygium sp.) It has the ability to produce various amounts of exopolysaccharides, ranging from 102 mg / L  to 1570 mg / L.

Based on Figure 3, the lowest EPS value produced by PM7.11 isolate was 102 mg/L while the highest EPS value was produced by PM6.10 isolate was 1570 mg / L. EPS value of LAB PM6.10 isolate from pakoba fruit was higher when compared to commercial LAB isolate Lactobacillus casei with the same treatment of 1340 mg / L.¹⁶ The results of previous studies that isolated EPS-BAL from various types of products, such as from fermented carp (Dekke Na Niura) obtained EPS levels of 2490-3490 mg / L,¹⁹ Kersen Fruit obtained EPS levels of 870-1910 mg / L,¹² and fermented Kefir Kolostrum obtained EPS levels of 1600-4840 ml / L,¹⁶ compared to the results of EPS dry weight obtained from this study was quite large.

EPS production by seventeen LAB isolates from Pakoba fruit showed varying amounts although the seventeen isolates were identified in the same genus  Lactobacillus. This is due to the influence of its genetic diversity. Species differences refer to differences in genes carried so that they are closely related to the metabolic processes carried out and the number of metabolites produced. Each gene is responsible for overseeing the formation, function, and ability of an enzyme to work, so that when there are different genes even though they are still classified as the same species but with different strains, the final results of metabolism can be different.¹⁹ Lactic acid bacteria produce secondary metabolite products in the form of exopolysaccharides that are secreted outside the cell when under unfavorable conditions. Differences in the ability to produce EPS due to differences in species or strains.¹⁶

The difference in the amount of EPS obtained in this study with other studies is thought to be caused by several factors, one of which is the incubation time used. in this study EPS production by LAB was harvested at the 48th hour after being grown on 10 mL of liquid media.¹⁶ The maximum production of EPS produced by a culture will be influenced by the growth factors of the culture, where the optimum EPS production occurs during maximum cell production, namely in the stationary phase or the final phase of LAB.³⁰ The stationary phase of LAB isolates begins in the range of 20-24 hours and in the next stage of growth there will be degradation of EPS. This happens because EPS produced by microbes in the stationary phase can be reused as a source of carbon in the near-death phase because microbes have enzymes that can degrade the EPS. As a result, the extension of incubation time will actually decrease EPS production.¹⁹

Other factors that can affect the amount of EPS produced by LAB in addition to incubation time are excess carbohydrates in the media, the type of media used, fermentation conditions such as temperature, pH, and oxygen levels, and the physiology of each bacterium,³¹ the optimum temperature of LAB is known to be 37°C, so in this study, to obtain maximum polysaccharides, the incubation temperature was lowered to 35°C.

EPS also tends to be influenced by the bacterial environment such as pH, growth phase, availability of nutrients such as carbon and nitrogen sources, temperature, and fermentation conditions.¹⁴ This test used MRS Broth medium which added 5% Sucrose as a growing medium from lactic acid bacteria because this media contains many nutrients favored by LAB. The added sucrose is an excellent substrate for EPS synthesis with abundant yields.

EPS production increases with increasing sucrose concentrations and involves extracellular sucrase-type enzymes used by LAB isolates to polymerize EPS, namely fructansucrase and glucansucrase.¹³  In this test LAB isolates aged 24 hours. The use of 24-hour age isolates because the logarithmic phase ends at that time and will enter the stationary phase where in this phase bacteria produce EPS optimally. Bacteria begin to experience extreme conditions such as reduced growth nutrients resulting in secondary metabolite products in the form of exopolysaccharides that are released outside the cell when conditions are unfavorable. The principle of this test is to separate exopolysaccharides from bacterial cells using centrifugation at 4°C to prevent protein denaturation. The pellets obtained from the results of the process are dried and weighed until they reach a constant weight. The results obtained are then analyzed quantitatively.

The filtrate obtained was added with 96% cold ethanol by twice the volume of the sample used to precipitate EPS. The use of ethanol with double the sample aims to facilitate the rate of diffusion so that the distribution of particles will be greater with the greater surface area. 96% ethanol is used as a precipitator for polysaccharides and has a relatively small ability to dissolve polysaccharides, although the ability to dissolve other substances is relatively large.³²

Ethanol has a much lower dielectric constant than water so it has lower polarity than water, whereas polysaccharides contain many hydroxyl groups that give their polar characteristics, as a result of which ethanol concentrations increase in solution and cause a decrease in polysaccharide solubility or cause precipitation.³²

Exopolysaccharides produced by microbes have great potential for the future, the use of microbes to produce polysaccharides is very profitable in economic terms compared to plant polysaccharides. Producing microbial polysaccharides can be done continuously on a large scale and does not require large areas of land such as plants.³²