Partial Analysis 16S rRNA Gene in Lactobacillus spp. from Natural Fermented Milk

Year 2014, Volume: 39 Issue: 1, 99 - 104, 22.07.2014

Nur Kusmiyati Nur Hidayat Fatchiyah Fatchiyah

https://doi.org/10.17826/cutf.82552

Abstract

Purpose: The aim of this research is to analysis 16S rRNA gene sequence of Lactobacillus spp. from natural fermented milk. Material and Methods: The methods that used in this study are total DNA isolation using alkaline lysis, PCR amplification carried out using of specific primers, and identification of amplified DNA using sequencing. The result of PCR process is a nuclotide with approximately 585 bp length which contained V1-V3 variable regions. 16S rRNA gene sequence then compared and aligned with existing 16S rRNA data sequence from GenBank. Results: The conserved region of 16S rRNA gene from nuclotide number 189-358 and 384-445 were 95,9% and 94,9% respectively, and the total conserved region of 16S rRNA gene is 78.8%. From total 7 isolates that isolated from natural fermented milk , 2 isolates were identified as L. plantarum with similarity value 98.28% and 97.78%, the other 5 isolates were identified as L. rhamnosus (2 isolates) with similarity value 98.38% and 97.51%, L. zeae (2 isolates) with similarity value 97.36% and 98.38%, and L. casei (1 isolate) with similarity value 98.20%. Conclusion: These results indicate the test isolates are members of the genus Lactobacillus, but classified in different species

Keywords

16S rRNA gene, Lactobacillus, natural fermentation milk, partial analysis

Doğal Fermente Olan Sütteki Lactobacillus Spp.16S rRNA Geninin Kısmi Analizi

Abstract

Amaç: Bu çalışmanın amacı doğal olarak fermente olan sütten Lactobacillus spp. türüne ait 16S rRNA ların gen sekansı yapmaktır. Materyal ve Metod: Kullanılan metodlar; alkali lizis yöntemiyle total DNA izolasyonu, spesifik primerlerle PCR amplifikasyonu ve amplifiye edilmiş DNA ların sekans analizlerinin yapılması şeklindedir. Elde edilen PCR ürünleri yaklaşık 585 bp uzunluğunda olup V1-V3 değişken bölgelerini içermektedir. Sekanslanan 16s rRNA daha sonra gen bankasıyla karşılaştırıldı. Bulgular: 16s rRNA geninin korunmuş bölgeleri; 189-358 ve 384-445 sırasıyla %95,9 ile %94,9 oranında korunurken, 16s rRNA geni totalde %78.8 oranında korunmuştur. Doğal sütün fermentasyonundan izole edilen bu 7 izolattan 2 tanesi L.Plantarum(benzerlik oranları %98.28 ve %97.78 diğer 5 izolattan 2"si L. Rhamnosus (benzerlik oranları (98.38 ve %97.51), diğer 2"si L. zeae (benzerlik oranları %97.36 ve % ve %98.38), biri ise L. case (benzerlik oranı%98.20) şeklinde tanımlanmıştır. Sonuç: Çalışmanın sonuçları test edilen izolatın Laktobailus genusuna ait olduğunu, fakat farklı türde sınıflandırıldığını göstermiştir.

Keywords

laktobacillus, doğal fermente süt, 16s rRNA, parsiyal analiz

References

• Tannock GW. A special fondness for Lactobacillus. Appl Environ Microbiol. 2004;70:3189–94.
• Dellaglio F, Felis GE & Torriani S. Is the genus Lactobacillus a single genus In LAB8 Symposium on Lactic Acid Bacteria . Egmond aan Zee, Netherlands: FEMS. 2005.
• Guarner F. & Schaafsma GJ. Probiotics. Int J Food Microbiol. 1998;39: 237–8.
• Klaenhammer TR, Barrangou R, Buck BL, AzcaratePeril MA & Altermann E. Genomic features of lactic acid bacteria effecting bioprocessing and health. FEMS Microbiol Rev. 2005;29:393–409.
• Dellaglio F & Felis GE. Taxonomy of Lactobacillus and bifidobacteria. Probiotics and Prebiotics: Scientific Aspects , pp. 25–49. Edited by G. W. Tannock. Norfolk, UK: Caister Academic Press. 200 Hammes WP & Hertel C. The Genera Lactobacillus and Carnobacterium .InThe Prokaryotes , release Edited by M. Dworkin. 2005.
• Villalobos DP, Bautista R, Canovas FM & Claros G. Isolation of Bacterial Artificial Chromosome DNA by Means of Improved Alkaline Lysis and Double Potassium Acetate Precipitation. Plant Molecular Biology Reporter. 2004;22:1–7.
• Randazzo CL, Torriani S, Akkermans ADL, de Vos WM and Vaughan EE. Diversity , Dynamics , and Activity of Bacterial Communities during Production of an Artisanal Sicilian Cheese as Evaluated by 16S rRNA Analysis. Appl. Environ. Microbiol. 2002;68:1882–92.
• Drancourt M, Bollet C, Cariioz A, Martelin JP, Gayral, & Raoult D. 16S Ribosomal DNA Sequence Analysis of A Large Collection of Environmental and Clinical Unidentifiable Bacterial Isolates. J. Gin. Microbial. 2000;38:3623-30.
• Bhattacharya D, Sarma S, Krishnan S, Mishra & Lal B. Evaluation of Genetic Diversity among Pseudomonas citronellolis Strains Isolated from Oily Sludge-Contaminated Sites. Appl. Environ. Microbiol. 2003;69:1435-41.
• Collins MD, Ash JAE, Farrow S, Wallbanks & Williams AM. 16S ribosomal ribonucleic acid sequence analyses of Pediacoccus and related taxa. Description of Vagococcus fluvialis gen. J. Appl. Bacteriol. 2006;67:453-60.
• Petrosino JF, Highlander S, Luna RA, Gibbs RA, & Versalovic J. Metagenomic pyrosequencing and microbial identification. Clinical Chemistry. 2009;55:856-66.
• Clarridge JE. Impact of 16S rRNA gene sequence analysis for identification of bacterial on clinical microbiology and infectious diseases. Clin Microbiol Rev. 2004;17:840-62.
• Heilig HGHJ, Erwin G, Zoetendal EG, Vaughan EE, Marteau P, Akkermans ADL & Vos WM. Molecular diversity of Lactobacillus spp. and other lactic acid bacteria in the human intestine as determined by specific amplification of 16S ribosomal DNA. Appl. & Envir. Microbiol. 2002;68:114–23.
• Davis JL. Phylogenetitic, molecular variations, and spesies concepts. Bioscience. 1996;46:502-10.
• Yazışma Adresi / Address for Correspondence: Dr. Fatchiyah Fatchiyah Dept of Biology, Faculty of Mathematics and Natural Sciences, Brawijaya University Malang, East Java, INDONESIA. e-mail: [e-mail], and [e-mail] g eliş tarihi/received :05.09.2012 kabul tarihi/accepted:24.09.2013