ANALYSIS OF PHYLOGENETIC LACTIC ACID IN GLUTINOUS RICE “TAPE KETAN” BY BIOINFORMATIC TOOLS IN RELATION WITH ITS BACTERIOCIN PRODUCT

1RINI NUR’AZIZAH, 2ISTIQOMAH SARI KUMARAWATI

1,2Faculty of Biology, Gadjah Mada University, Yogyakarta, Indonesia E-mail: [email protected]

Abstract— Glutinous rice or Tape Ketan is fermented food in Indonesia with prepared using ketan or Oryza sativa glutinosa using ragi as a starter. Tape ketan consumed for its taste, flavor, nutrients, and easy digestibility, it has a sweet-acid and mild alcoholic flavor. It producing asLactobacillus curvatus, Lactobacillus fermentum, Pediococcus pentosaceous, Enterococcus villorum, Confusa, Weissella paramesenteroides, Enterococcus faecium and Weissella kimchii. Analysis of philogenetic was done by Bioinformatic tool, it use 16S rRNA gene sequences retrieved from NCBI database with the distance based and character based methods. It was used ClustalX 2.1 for multiple sequences aligenment and Mega 6 software to build phylogenetic tree.

Keywords— Lactic Acid Bacteria, Tape ketan, Glutinous rice, Bioinformatic.

I. INTRODUCTION represented by treelike diagrams, it is estimated pedigree of the inherited relationships with overall Tape ketan or glotinous rice is popular fermented similirity [12]. Cladistic analysis of morphological food in Indonesia. Tape ketan is prepared using ketan characters and phenetic, cladistic or maximum- or Oryza sativa glutinosa using ragi as a starter. Tape likehood analysis of molecular character are the key ketan has a sweet-acid taste and mild alcoholic flavor. methods for phylogenies [13]. Bioinformatics is It is because fermentation. Fermentation can increase combination science between biology and the taste, nutritional and organoleptic like flavors, information technique or computer science. The dutty aromas and textures [1]. Tape ketan is produced on of bioinformatics is to solve problems molecular home industry in Indonesia with solid-state biology in computing. These problems is not only fermentation in traditional scale[2,3]. Tape ketan also fundamental things as solve of enzyme mechanism, produce Lactic Acid Bacteria (LAB). LAB is group protein metabolism or identification of microbes but of gam positive bacteria, nonsporulating, rods or also biomedical problems, design of medicine, cocci shape, nonaerobic, aerotolerant and producing primary, vaccine can be resolved [14]. Bioinformatics lactic acid as a result of fermentation [4]. LAB is used to phylogenetic analysis also. It use computer produce bacteriocins, bacteriocins are synthesized in software, as Clustal X for multiple sequences ribosome form peptides as antimicrobial those are aligenment and Mega 6 software to build active against othe bacteria, like pathogen microbia phylogenetic tree. Taxonomic studies on LAB from [5].Bacteriocins are divided into several classes. tape ketan are not yet complete.Thus, it is important Group I, also known as lantibiotic. Group II is to classify and identify LAB from tape ketan on the subdivided into three groups : subgroup basis not only of phenotypic characteristics but also IIa,bacteriocins active against Listeria phylogenetic analysis by 16S rRNA sequencing and monocytogenes, and pediocin PA-1,sakacins A and P, bioinformatic tools leucocin A, bavaricin MN and curvacin A are members of this group; then subgroup IIb require two II. MATERIALS AND METHOD different peptides for activity andlactococcins G and M, and lactacin F are members of it; and subgroup Samples were collected by literature searched both IIc, such as lactacin B, group IId such as lacticin. online or offline. Then, 16S rRna sequence were Group III are such as helveticins J andV, and retrieving from NCBI, there are Lactobacillus lactacins A and B. Leuconocin S, lactocin and curvatus(AB025183), Lactobacillus pediocin SJ-1 have lipid or carbohydrate moieties and fermentum(AF477498), Pediococcus are classified into group IV [6, 7]. The LABs pentosaceous(JX141316), Enterococcus produced by tape ketan areLactobacillus curvatus, villorum(AF335596), Weissella Confusa(LC063164), Lactobacillus fermentum, Pediococcus pentosaceous, Weissella paramesenteroides(KM392070), Enterococcus villorum, Weissella Confusa, Weissella Enterococcus faecium (AF039901) and Weissella paramesenteroides, Enterococcus faecium and kimchii(AF312874).In these sequences little changes Weissella kimchii [8, 9, 10, 11]. occurduring evolution. The sequences are colected in Phylogenetic is study of relationship specieses based one notepad. Then, sequences alignment were done on evolution, these relationship is estimated by by ClustalX, the result of allignment is used to the phylogenetic analysis.It generates branching, and next step, build a phylogenetic tree by software

Proceedings of 24th ISERD International Conference, Seoul, South Korea, 13th January 2016, ISBN: 978-93-82702-10-8 26 Analysis of Phylogenetic Lactic Acid Bacteria in Glutinous Rice “Tape Ketan” by Bioinformatic Tools in Relation With its Bacteriocin Product MEGA6, insertion and deletion are treated for belong to filum , classes , family missing datas, all character have same values and all Lactobacilalles, order (L. curvatus, data is analyzed with kimura 2-parameter model with L. fermentum, P. pentosaceous), Enterococcaceae (E. 1000 bootstrap. villorum, E. faecium) and Leuconostocaceae (W. confusa, W. paramesenteroidesand W. kimchii) [] III. RESULTS AND DISCUSSIONS

There are 8 species Lactic Acid Bacteria that found in Tape ketan after the literature survey, those are

Table 1. Lactic Acid Bacteria and its Bacteriocins Product

Bacteriocins produced Lactic Acid Bacteria, can as fermentumproduce class I bacteriocins, that is probiotics. Bacteria produce this bacteriocins during Fermentacin, it is lantibiotics containing, has nisin as their growth, substances ofprotein structure (either the first and known bacteriocin while L. proteins or polypeptides), possessing antimicrobial curvatusproduce IIa bacteriocins, A circular activities. Nowadays, bacteriocins have been widely bacteriocins [6,7]. L. curvatus, E. fillorum and E. utilised especially in food preservation[15].One of faecium grouping into one clade, genus Enterococcus food that containing LAB and produce bacteriocins is produce enterocins. That is include class IIb tape ketan, a traditional food from Indonesia that bacteriocins, two-peptide bacteriocins[6]. made from glutinous rice with solid stade fermentation method. CONCLUSION

Based on the research, can be conclused that tape ketan or glotinous rice is popular fermented food in Indonesia, produce bacteriocins from LAB. Taxonomy of LAB is not only based on phenetic analysis, but also with phylogenetic analysis. Analysis phylogenetic LAB in Tape ketan using bioinformatic tools generates two clades.

REFERENCES

[1] Nuraida, L. 2015. A review: Health promoting lactic acid bacteria in traditional Indonesian Figure 1. Phylogenetic tree of the LAB was prepared by fermented foods. Food Science and Human Neighbour Joining method (bootstrap analysis) Wellness, 6(1), 1-9. [2] Djien, K. S. 1972. Tape Fermentation. Applied Figure 1 showed that LAB from tape ketan have two Microbiology, 23(5), 976-978. clades, clade one is a group of W. kimchii,W. confusa, [3] Law, S. V., Abu Bakar, F., Mat Hashim, D. And W. paramesenteroides, andL. fermentumwith 100 Abdul Hamid, A. 2011. Mini Review: Popular fermented food and beverages in southeast Asia. values of bootstrap and second clade is group of L. International Food Research Journal, 18, 475-484. curvatus, E. villorum and E. faecium with 100 values [4] Gautam, P., Anjhurda, V. 2014. Phylogenetic of bootstrap. W. kimchiiis closest sequence with W. analysis of selected acid bacteria by using paramesenteroidesand W. confusawith similirity bioinformatic tools. International Journal of Engineering and Technical Research, 2(8), 53-58. 99%, they are one genus, equally produce Class IId [5] Yang, E., Lihua, F., Yueming, J., Craig, D., Sherry, bacteriosins, that is non-lanthionine-containing F. 2012. Antimicrobial activity of bacteriocin- bacteriocins, and unmodified, linear, non-pediocin- producing lactic acid bacteria isolated from cheeses like bacteriocins [7].The research result indicates and yoghurts. AMB Express, 2(48), 1-12. [6] Quinto, E. J., Pilar, J., Irma, C., Jesus, T., Javier, M. thatL. fermentum and L. curvatus which is one genus Tomas, G. 2014. Probiotic lactic acid bacteria: a are in different clade, ir is because of previous studies review. Food and Nutrition Sciences, 5, 1765-1775. have shown that the genus Lactobacillus isa [7] Perez, R. H., Takeshi, Z., Kenji, S. 2014. Novel heterogeneous group with unstable taxonomy [16].L. bacteriocins from lactic acid bacteria (LAB):

Proceedings of 24th ISERD International Conference, Seoul, South Korea, 13th January 2016, ISBN: 978-93-82702-10-8 27 Analysis of Phylogenetic Lactic Acid Bacteria in Glutinous Rice “Tape Ketan” by Bioinformatic Tools in Relation With its Bacteriocin Product Various structure and applications. Microbial Cell [12] Baxevanis, A. D., Oullette, B. F. F. 2001. Factories 2014, 13(1), 1-13. Bioinformatics: A practical guide to the analysis of [8] Hadisepoetro, E.S.S., Takada, N. and Oshima, Y. genes and proteins, second edition. John Wiley & 1979.Microflora in ragi and usar. Journal of Sons. London. Pp: 323-325. Fermentation Technology, 57, 251-259. [13] Benton, M. J. 2000. Stems, nodes, crown clades, [9] Hasseltine, C. W., Ray, M. L. 1988. Lactic acid in and rank-free lists: is Linnaeus dead?. Biol. Rev, 75, murcha and ragi. Journal of Applied Bacteriology, 633-648. 64, 395-401. [14] Clevarie, J. M., Notredame, C. 2006. [10] Ardana, M.M. and Fleet, G.H. 1989. The microbial Bioinformatics for Dummies. John Willey and Sons, ecologyof tape ketan fermentation. International New Jersey. Journal of Food Microbiology 9, 157-165. [15] Zacharof, M. P., Lovitt, R. W. 2012. Bacteriocins [11] Sujaya, I. N., Nocianitri, K. A., Asano, K. 2010. produced by lactic acid bacteria: a review article. Diversity of bacterial flora of Indonesian ragi tape APCBEE procedia, 2, 50-56. and their dynamics during the tape fermentation as [16] Schleifer, K.H. and Ludwig, W. (1995) Phylogeny determined by PCR-DGGE. International Food of the genus Lactobacillus and related genera. Research Journal, 17, 239-245. Systematic and Applied Microbiology, 18, 461-467.



Proceedings of 24th ISERD International Conference, Seoul, South Korea, 13th January 2016, ISBN: 978-93-82702-10-8 28