KEANEKARAGAMAN BAKTERI ASAM LAKTAT PENGHASIL ANTIMIKROBIA SELAMA PROSES FERMENTASI BAKASANG

Disertasi

Oleh:
HELEN JOAN LAWALATA 03/1314/PS

FAKULTAS BIOLOGI UNIVERSITAS GADJAH MADA YOGYAKARTA 2012

KEANEKARAGAMAN BAKTERI ASAM LAKTAT PENGHASIL ANTIMIKROBIA SELAMA PROSES FERMENTASI BAKASANG

Disertasi untuk memperoleh Derajat Doktor dalam Ilmu Biologi pada Fakultas Biologi Universitas Gadjah Mada

Dipertahankan di hadapan Dewan Penguji Program Pascasarjana Fakultas Biologi Universitas Gadjah Mada Pada tanggal : 06 Desember 2012

Oleh Helen Joan Lawalata 03/1314/PS

Lahir Di Manado, Sulawesi Utara Indonesia

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SUMMARY

Lactic acid bacteria (LAB) is a group of Gram-positive bacteria, nonsporing formed, coccus or rod and produce lactic acid as the major end product during the fermentation of carbohydrates (Axelsson, 1998; 2004) . The genera belonging to the group of lactic acid bacteria classified based on conventional phenotypic approach are Lactobacillus, Leuconostoc, Pediococcus, and

Streptococcus (Sneath et al., 1987). Nowadays, taxonomic revision of these genera and the description of a new genera based on molecular characteristics (sequence 16S rRNA gene) suggest that the lactic acid bacteria consist of 12 genera, namely Aerococcus, Carnobacterium, Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus,

Tetragenococcus, Vagococcus, and Weisella (Schleifer & Ludwig, 1998).. LAB is natural microbe that exists in the raw material of food and has an important role in the fermentation of traditional foods such as tempe, soy sauce, shrimp paste, fermented milk, and pickled products (Tanasupawat & Komagata, 1999). The ability of LAB in producing antimicrobial compounds showing the importance of the role of LAB in protecting food from spoilage caused by bacteria and fungi and can inhibit the growth of pathogenic bacteria, and bacteria bioamine producing that may contaminate food products of fermentation. In the fermentation process of fish, LAB will produce an organic acid as an end product of carbohydrate metabolism and therefore it lower will pH value and it can inhibit the growth of bacteria that live in the neutral condition (Ostergaard et al., 1998). In addition to organic acids, LAB also produces

antagonistic components, namely hydrogen peroxide, diasetil, and bacteriocins (Ouwehand, 1998; De Vuyst & Leroy, 2007).. In general, traditional fermentation takes place spontaneously by microbes that exist in the raw materials so that there are different types of microbes that can grow in accordance with the changing environment so that microbial growth is not expected to cause failure in the process of fermentation (Rahayu et al., 1995). Therefore, the quality of fermented foods will decrease and even often contaminated with pathogenic and putrefying microbes so that the shelf life of products is getting shorter and it is not safe for consumption. Based on the potency of antimicrobial of LAB isolated from fermented fish products, it is necessary to conduct research on the diversity of LAB that can be isolated during the fermentation process of bakasang that has the ability to produce antimicrobial compounds that can inhibit the growth of bacteria that contaminate the food fermentation. Therefore, LAB isolates obtained from bakasang could be used as starter culture to improve the quality of fermented foods into healthy foods and safe for consumption. The objective of this research were (i) to obtain LAB isolates during the fermentation process of bakasang, (ii) to determine the ability of the isolates to inhibit pathogenic bacteria, putrefying bacteria, and bioamine producing bacteria, (iii) to obtain LAB isolates isolated during the fermentation process of bakasang that have a high antagonistic ability, and (iv) to determine the identity, diversity, phylogenetic relationship, and novelty status LAB isolates with antagonistic ability based on a polyphasics systematic approach. the highest

Isolation and screening of LAB were carried out from three kinds samples of bakasang namely (i) Sample 1, bakasang made of the viscera, eggs, fish fillet of Cakalang, salt, and rice (ii) Sample 2, bakasang made from made from viscera, egg of Cakalang fish, salt, and rice (iii) Sample 3, commercial bakasang made from the viscera, eggs, and salt obtained from the traditional market Karombasan Manado, North Sulawesi. The isolation was done by using media of MRS agar containing CaCO3 and 1 ppm sodium azide. The method used was the pour plate and incubation was performed at 37 C for 24-72 hours. Isolation of LAB was performed on days 0, 1, 3, 5, and 7. Screening to determine LAB was based on (i) porsitive Gram, (ii) cell shape coccus or basil, (iii) negative catalase, (iv) nonsporing, and (v) non-motil. Screening of LAB isolates was based on its ability to inhibit growth of pathogenic bacteria, putrefying bacteria, and bioamine producing bacteria. Bacterial inhibition assay was performed by using cell culture, the sour supernatant and neutralized supernatant based on wells method (Well diffusion) (Davidson & Parish., 1989). Cell culture, the sour supernatant, and neutralized supernatant which could produce clear zone was regarded to have ability of antagonistic nature on the test bacteria. The inhibition zone diameter produced by the isolates was measured by the diameter of produced clear zone. Selection of LAB isolate was based on the highest inhibition potency against the test bacteria as well as representative of isolation source. Test bacteria used were including bacteria putrefying bacteria (St. aureus ATCC 25923), pathogenic bacteria (E. coli ATCC 35218) were obtained from the Laboratory of Microbiology Faculty of Medicine UGM, Yogyakarta and bioamine

producing bacteria (P. fluorescens FNCC 0070 was obtained from FNCC (Food Nutrition Culture Collection) Gadjah Mada University, Yogyakarta. Selected LAB isolates which produced antimicrobial compounds were subsequently characterized and identified by profile matching method in order to identify at the genus level (generic assignment). Further more species identification was carried out by using profile maching as well as probabilistic approach API system to determine reference strain. In effort to guaranty the result of identification polyphasic systematic approach were used including numericalfenetic systematic, chemical systematic, and molecular systematic covering DNA fingerprinting (ARDRA) and phylogenetic based on 16S rRNA gene sequence. Characterization and identification based on numerical-fenetic systematic were performed using phenotypic characters ranging from cell morphological, biochemical, as well as physiological characters. Further more chemosystematics application was performed by using total profile protein produced by SDS-PAGE method. Molecular characterization and identification were performed by application of DNA fingerprinting (ARDRA) and molecular phylogenetic analysis based on 16S rRNA gene sequence. Results of the study showed that from tree kinds of sample, 200 isolates of circular colony morphology, white or yellowish white, acid-producing bacteria indicated by clear zones around colonies were obtained. All of the acid producing bacteria isolates were screened based on (i) Gram-positive, (ii) catalase negative, (iii) non-sporing, and (iv) non-motile in order to ensure LAB. The result of screening showed that 125 among 200 isolates were found to be LAB and 75 isolated were non LAB.

Subsequently, 125 isolates were characterized and identify by profile matching using convensional method to identify genus level (generic assignment). The result of the generic assignment indicated that most of the isolates (114 isolates) belonged to genus Pediococcus. However 5 isolates could ether belong to genus Enterococcus or Streptococcus, and 5 isolates were identified to the member of the genus Lactobacillus and only one isolate was found to be member of genus Leuconostoc. The results of screening based on the ability to inhibit the growth of bacteria test showed that only one LAB isolate (BksC12) was not able to inhibit the growth of St. aureus ATCC 25923, two LAB isolates (BksJ50 and BksJ53) were not able to inhibit the growth of P. fluorescens FNCC 0070 and all of the LAB isolates were able to inhibit the growth of E. coli ATCC 35218. The ability to inhibit growth of bacteria test was obtained from the cell culture and the sour supernatant. The neutralized supernatant was unable to inhibit the growth of bacteria test. The ability of the cell culture, the sour supernatant, and the neutralized supernatant LAB isolates to inhibit the growth of bacteria test be measured by diameter of clear zone on the media Nutrient Agar (NA). Diameter size of clear zone formed around the colony was the diameter of inhibition (mm). Based on the inhibition of the cell culture and the sour supernatant suspected that inhibition of bacterial growth caused by organic acids other than bacteriocin. The lack of inhibition by the neutralized supernatant showed that the antimicrobial compound was not bacteriosin. Subsequently, selection of LAB isolates which producing the highest antimicrobial compounds based on inhibition of the greatest diameter was 15.0 mm (cell culture) and 13 mm (the sour supernatant).

Based on the selection, obtained four LAB isolates that have the highest inhibition and representative of isolation source namely BksC24 isolate obtained from sample 1 which able to inhibit the growth of St. aureus ATCC 25923 (14mm) and P. fluorescens FNCC 0070 (15mm), BksJ21 isolate obtained from sample 2 which able to inhibit the growth of P. fluorescens FNCC 0070 (15mm), BksJ43 isolate obtained from sample 2 which able to inhibit the growth of E. coli ATCC 35218 (15mm), and BksK25 isolate obtained from samples 3 which able to inhibit the growth of P. fluorescens FNCC 0070 (15mm). The results of conventional characterization and identification of four LAB isolates producing-antimicrobial showed that selected LAB isolates which produced antimicrobial (BksC24, BksJ21, BksJ43 and BksK25 isolates) belonged to member of genus Pediococcus. Characterization and identification of genus level by the profile matching method showed that the four LAB isolates have accordingly characters with the key characters in description of genus Pediococcus. Thereby, reference strains that were set to be used in the

identification of selected the four LAB isolates was species members belong to genus Pediococcus. Based on species identification which used profile matching method and probabilistic identification methods therefore that selected reference strains were Pediococcus acidilactici FNCC 0110 and P. pentosaceus FNCC 0019. Characterization and identification based on systematic numerical-fenetik showed that isolates BksC24, BksJ21, BksJ43, and BksK25 have high index similarity with P. acidilactici FNCC 0110. The result of identification by using data base software apiwebTM V5.1 (bioMe'rieux, France) showed different

results, namely the four LAB isolates producing antimicrobial have similarities with the species of P. Pentosaceus. The results based on the chemicals systematic identification showed that BksJ43 isolates and BksC24 isolates have the high similarities with the reference strain P. acidilactici FNCC 0110, while BksJ21 isolates and BksK25 isolates have low similarity with P. acidilactici FNCC

0110 reference strain and P. pentosaceus FNCC 0019 reference strain. The results of molecular analysis by DNA fingerprinting (ARDRA) with restriction endonuclease HaeIII showed that isolates BksC24, BksJ21, BksJ43 and BksK25 have high similarity index with P. acidilactici FNCC 0110. The results of molecular phylogenetic analyzes based on 16S rRNA sequences also showed that all of these four strains have the closely relationship with Pediococcus acidilactici DSM 20284T. However, based on the number of nucleotide differences between the four strains LAB and reference strain members of the genus Pediococcus showed that isolates BksJ21 and BksK25 isolates belonged to the members of the genus Pediococcus and suspected as member of new species (novel species) whereas BksC24 isolates and BksJ43 isolates suspected to be new strain (novel strain) members of the species P. acidilactici. Thus, the characterization and identification with a polyphasic systematic approach can reveal the identity, diversity, close relationship (phylogenetic) and the newly status of the selected LAB isolates producing antimicrobial compounds. Based on study, it can be concluded that BksJ21 isolates and BksK25 isolates suspected as a new species (novel species) members of the genus Pediococcus while BksC24 isolates and BksJ43 isolates suspected as a new strain (novel strain) members of the species P. acidilactici. However, for more ensure

that the four LAB isolates were found in bakasang is a novel species and novel strains, it is necessary to do further research using molecular methods based on pheS dan rpoA gene sequence analysis and analysis of DNA-DNA relatedness. The fourth LAB strains producing antimicrobial compound have potential as a natural preservative that can be applied to traditional fermented foods which might be expected to make a traditional fermented food into safety food.