Species Identification of Potential Probiotic Lactic Acid Bacteria Isolated from Malaysian Fermented Food Based on 16S Ribosomal RNA (16S rRNA) and Internal Transcribed Spacer (ITS) Sequences

Yaacob Ilyanie; Nur Huda Faujan; Md Yasin Ida Muryany

doi:10.55230/mabjournal.v52i4.c146

Authors

Yaacob Ilyanie School of Biology, Universiti Teknologi MARA Cawangan Negeri Sembilan, Kampus Kuala Pilah, 72000, Kuala Pilah, Negeri Sembilan, Malaysia; BioMECs, Universiti Teknologi MARA Cawangan Negeri Sembilan, Kampus Kuala Pilah, 70450, Kuala Pilah, Negeri Sembilan, Malaysia; Food Biotechnology Programme, Faculty of Science and Technology, Universiti Sains Islam Malaysia, 71800, Nilai, Negeri Sembilan, Malaysia
Nur Huda Faujan Food Biotechnology Programme, Faculty of Science and Technology, Universiti Sains Islam Malaysia, 71800, Nilai, Negeri Sembilan, Malaysia
Md Yasin Ida Muryany School of Biology, Universiti Teknologi MARA Cawangan Negeri Sembilan, Kampus Kuala Pilah, 72000, Kuala Pilah, Negeri Sembilan, Malaysia; BioMECs, Universiti Teknologi MARA Cawangan Negeri Sembilan, Kampus Kuala Pilah, 70450, Kuala Pilah, Negeri Sembilan, Malaysia

Keywords:

16S rRNA gene, ITS gene, lactic acid bacteria, Malaysian fermented food, PCR

Abstract

Taxonomic identification of potential probiotic lactic acid bacteria (LAB) in fermented foods is essential as the microorganisms’ benefits are varied and often strain-specific. Using biochemical and physiological methods alone is inadequate to precisely distinguish each strain. In this study, molecular techniques were employed in the identification of 17 LAB isolated from three Malaysian fermented foods: belacan (BE), bosou (BO), and budu (BUM). The respective sizes of polymerase chain reaction (PCR) products from the isolates were approximately 1500 bp and 750 bp when amplified with the 16S ribosomal RNA (16S rRNA) and internal transcribed spacer (ITS) gene primers. The phylogenetic analysis using both gene sequences revealed that all BE and BO isolates were identified as Lactiplantibacillus plantarum, while all BUM isolates were identified as Lacticaseibacillus paracasei. Both 16S rRNA and ITS genes could disclose the identity of the isolates up to the species level. In summary, the use of the ITS gene in conjunction with the 16S rRNA gene can help with the more effective identification of potential probiotic LAB strains isolated from fermented food.

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References

Abbasiliasi, S., Ramanan, R.N., Tengku Ibrahim, T.A., Mustafa, S., Mohamad, R., Hj Mohd Daud, H. & Ariff, A. 2011. Effect of medium composition and culture condition on the production of bacteriocin-like inhibitory substances (BLIS) by Lactobacillus paracasei LA07, a strain isolated from budu. Biotechnology & Biotechnological Equipment, 25(4): 2652-2657. DOI: https://doi.org/10.5504/BBEQ.2011.0101

Adesulu-Dahunsi, A.T., Sanni, A.I. & Jeyaram, K. 2017. Rapid differentiation among Lactobacillus, Pediococcus and Weissella species from some Nigerian indigenous fermented foods. LWT - Food Science and Technology, 77: 39-44. DOI: https://doi.org/10.1016/j.lwt.2016.11.007

Azrin, N.A.R., Yuzine, E., Ina-Salwany, M.Y., Suzana, R., Fathiah, M. & Murni, K. 2017. Characterization of potential probionts from blue swimming crab Portunus pelagicus and its antagonistic activity against Vibrio harveyi. International Journal of Biosciences, 11(4): 292-303. DOI: https://doi.org/10.12692/ijb/11.4.292-303

Bell, V., Ferrão, J. & Fernandes, T. 2017. Nutritional guidelines and fermented food frameworks. Foods, 6(8): 65. DOI: https://doi.org/10.3390/foods6080065

Chiu, T-H., Chen, T-R., Hwang, W-Z. & Tsen, H-Y. 2005. Sequencing of an internal transcribed spacer region of 16S-23S rRNA gene and designing of PCR primers for the detection of Salmonella spp. in food. International Journal of Food Microbiology, 97: 259- 265. DOI: https://doi.org/10.1016/j.ijfoodmicro.2004.04.005

Dang, H.T., Park, H.K., Myung, S.C. & Kim, W. 2012. Development of a novel PCR assay based on the 16S-23S rRNA internal transcribed spacer region for the detection of Lactococcus garvieae. Journal of Fish Diseases, 35: 481-487. DOI: https://doi.org/10.1111/j.1365-2761.2012.01382.x

Daroonpunt, R., Uchino, M., Tsujii, Y., Kazami, M., Oka, D. & Tanasupawat, S. 2016. Chemical and physical properties of Thai traditional shrimp paste (Ka-pi). Journal of Applied Pharmaceutical Science, 6 (5): 58-62. DOI: https://doi.org/10.7324/JAPS.2016.60509

Diyana-Nadhirah, K.P & Ina-Salwany, M.Y. 2016. Molecular characterization of 16S rRNA and internal transcribed spacer (ITS) regions of Aeromonas spp. isolated from cultured freshwater fishes in Malaysia. International Journal of Current Microbiology and Applied Sciences, 5(9): 431-440. DOI: https://doi.org/10.20546/ijcmas.2016.509.046

Felsberg, J., Jelínková, M. Kubizniaková, P. & Matoulková, D. 2015. Development of a PCR assay based on the 16S-23S rDNA internal transcribed spacer for identification of strictly anaerobic bacterium Zymophilus. Anaerobe, 33: 85-89. DOI: https://doi.org/10.1016/j.anaerobe.2015.02.004

Felsenstein, J. 1981. Evolutionary trees from DNA sequences: a maximum likelihood approach. Journal of Molecular Evolution, 17: 368-376. DOI: https://doi.org/10.1007/BF01734359

Fijan, S. 2014. Microorganisms with claimed probiotic properties: an overview of recent literature. International Journal of Environmental Research and Public Health, 11: 4745-4767. DOI: https://doi.org/10.3390/ijerph110504745

Haitham, A.R. 2017. Characterisation Of Probiotic Lactic Acid Bacteria Isolated from Malaysian Fermented Shrimp Pastes. (Ph.D). Universiti Sains Islam Malaysia, Malaysia.

Hajep, P. & Jinap, S. 2012. Fermented shrimp products as source of umami in Southeast Asia. Journal of Nutrition & Food Sciences, S10(006).

Halder, D., Mandal, M., Chatterjee., S.S., Pal, N.K. & Mandal, S. 2017. Indigenous probiotic Lactobacillus isolates presenting antibiotic like activity against human pathogenic bacteria. Biomedicines, 5(2): 31. DOI: https://doi.org/10.3390/biomedicines5020031

Han, Q., Kong, B., Chen, Q., Sun, F. & Zhang, H. 2017. In vitro comparison of probiotic properties of lactic acid bacteria isolated from Harbin dry sausages and selected probiotics. Journal of Functional Foods, 32: 391-400. DOI: https://doi.org/10.1016/j.jff.2017.03.020

Herbel, S.R., Vahjen, W., Wieler, L.H. & Guenther, S. 2013. Timely approaches to identify probiotic species of the genus Lactobacillus. Gut Pathogens, 5: 27. DOI: https://doi.org/10.1186/1757-4749-5-27

Huang, C-H., Li, S-W., Huang, L. & Watanabe, K. 2018. Identification and classification for the Lactobacillus casei group. Frontiers in Microbiology, 9: 1974. DOI: https://doi.org/10.3389/fmicb.2018.01974

Ida Muryany, M.Y. 2017. Pengenalpastian dan Pencirian Probiotik Novel dari Sumber Ikan Terfermentasi Serta Fungsinya dalam Inflamasi. (Ph.D). Universiti Kebangsaan Malaysia, Malaysia.

Ida Muryany, M.Y., Ina Salwany, M.Y., Ghazali, A.R., Hing, H.L. & Nor Fadilah, R. 2017. Identification and characterization of the lactic acid bacteria isolated from Malaysian fermented fish (pekasam). International Food Research Journal, 24 (2): 868-875.

Ina-Salwany, M.Y., Hishammuddin, H., Zulperi, Z., Salema, M., Karim, M. & Natrah, F.M.I. 2015. Elucidating the probiotic potential of Malaysian Paenibacillus pabuli against Vibrio alginolyticus in Artemia culture. Asian Journal of Agricultural Research, 9(5): 223-236. DOI: https://doi.org/10.3923/ajar.2015.223.236

Janda, J.M. & Abbott, S.L. 2007. 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls. Journal of Clinical Microbiology, 45(9): 2761-2764. DOI: https://doi.org/10.1128/JCM.01228-07

Khalil, E.S., Manap, M.Y., Mustafa, S., Amid, M., Alhelli, A.M. & Aljoubori, A. 2018. Probiotic characteristics of exopolysaccharides-producing Lactobacillus isolated from some traditional Malaysian fermented foods. CyTA-Journal of Food, 16(1): 287-298. DOI: https://doi.org/10.1080/19476337.2017.1401007

Kilinc, B., Cakli, S., Tolasa, S. & Dincer, T. 2006. Chemical, microbiological and sensory changes associated with fish sauce processing. European Food Research and Technology, 222: 604-613. DOI: https://doi.org/10.1007/s00217-005-0198-4

Kobayashi, T., Kajiwara, M., Wahyuni, M., Kitakado, T., Hamada-Sato, N., Imada, C. & Watanabe, E. 2003. Isolation and characterization of halophilic lactic acid bacteria isolated from "terasi" shrimp paste: A traditional fermented seafood product in Indonesia. Journal of General and Applied Microbiology, 49: 279-286. DOI: https://doi.org/10.2323/jgam.49.279

Kopermsub, P. & Yunchalard, S. 2010. Identification of lactic acid bacteria associated with the production of plaa-som, a traditional fermented fish product of Thailand. International Journal of Food Microbiology, 138: 200-204. DOI: https://doi.org/10.1016/j.ijfoodmicro.2010.01.024

Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. 2018. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35: 1547-1549. DOI: https://doi.org/10.1093/molbev/msy096

Lane, D.J. 1991. 16S/23S rRNA sequencing. In: Nucleic Acid Techniques in Bacterial Systematics. E. Stackebrandt & M. Goodfellow. John Wiley and Sons, New York.

Le, B. & Yang, S.H. 2018. Probiotic potential of novel Lactobacillus strains isolated from salted-fermented shrimp as antagonists for Vibrio parahaemolyticus. Journal of Microbiology, 56 (2): 138-144. DOI: https://doi.org/10.1007/s12275-018-7407-x

Liasi, S.A., Azmi, T.I., Hassan, M.D., Shuhaimi, M., Rosfarizan, M. & Ariff, A.B. 2009. Antimicrobial activity and antibiotic sensitivity of three isolates of lactic acid bacteria from fermented fish product, Budu. Malaysian Journal of Microbiology, 5(1): 33-37. DOI: https://doi.org/10.21161/mjm.15008

Liguori, A.P., Warrington, S.D., Ginther, J.L., Pearson, T., Bowers, J., Glass, M.B., Mayo, M, Wuthiekanun, V., Engelthaler, D., Peacock, S.J., Currie, B.J., Wagner, D.M., Keim, P. & Tuanyok, A. 2011. Diversity of 16S-23S rDNA internal transcribed spacer (ITS) reveals phylogenetic relationships in Burkholderia pseudomallei and its near-neighbors. PLoS ONE, 6(12): e29323. DOI: https://doi.org/10.1371/journal.pone.0029323

Lim, F.T., Lim, S.M. & Ramasamy, K. 2017. Pediococcus acidilactici LAB4 and Lactobacillus plantarum LAB12 assimilate cholesterol and modulate ABCA1, CD36, NPC1L1 and SCARB1 in vitro. Beneficial Microbes, 8(1): 97-109. DOI: https://doi.org/10.3920/BM2016.0048

Man, S.M., Kaakoush, N.O., Octavia, S. & Mitchell, H. 2010. The internal transcribed spacer region, a new tool for use in species differentiation and delineation of systematic relationships within the Campylobacter genus. Applied and Environmental Microbiology, 76(10): 3071-3081. DOI: https://doi.org/10.1128/AEM.02551-09

Martínez Álvarez, Ó., López Caballero, M.E., Gómez Guillén, M.C. & Montero García, P. 2017. Fermented seafood products and health. In: Fermented Foods in Health and Disease Prevention. Elsevier. DOI: https://doi.org/10.1016/B978-0-12-802309-9.00009-1

Nuraida, L. 2015. A review: Health promoting lactic acid bacteria in traditional Indonesian fermented foods. Food Science and Human Wellness, 4: 47-55. DOI: https://doi.org/10.1016/j.fshw.2015.06.001

Panghal, A., Janghu, S., Virkar, K., Gat, Y., Kumar, V. & Chhikara, N. 2018. Potential non-dairy probiotic products - a healthy approach. Food Bioscience, 21: 80-89. DOI: https://doi.org/10.1016/j.fbio.2017.12.003

Perera, O.D.A.N., Weerahewa, H.L.D. & Vidanagamage, S. 2017. Role and current trends of developing fruit, vegetable and cereal based probiotic foods: a review. International Journal of Fermented Foods, 6(2): 85-93.

Putra, T.F., Suprapto, H., Tjahjaningsih, W. & Pramono, H. 2018. The antagonistic activity of lactic acid bacteria isolated from peda, an Indonesian traditional fermented fish. IOP Conference Series: Earth and Environmental Science, 137. DOI: https://doi.org/10.1088/1755-1315/137/1/012060

Shehata, M.G., El Sohaimy, S.A., El-Sahn, M.A. & Youssef, M.M. 2016. Screening of isolated potential probiotic lactic acid bacteria for cholesterol lowering property and bile salt hydrolase activity. Annals of Agricultural Science, 61(1): 65-75. DOI: https://doi.org/10.1016/j.aoas.2016.03.001

Sim, K.Y., Chye, F.Y. & Anton, A. 2012. Probiotic potential and antimicrobial activities of micro-organisms isolated from an indigenous fish sauce. Borneo Science, 31: 57-63.

Singh, V., Mani, I. & Chaudhary, D.K. 2012. Molecular assessment of 16S-23S rDNA internal transcribed spacer length polymorphism of Aeromonas hydrophila. Advances in Microbiology, 2: 72-78. DOI: https://doi.org/10.4236/aim.2012.22009

Song, Y-L., Kato, N., Liu, C-X., Matsumiya, Y., Kato, H. & Watanabe, K. 2000. Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA. FEMS Microbiology Letters, 187: 167-173. DOI: https://doi.org/10.1016/S0378-1097(00)00196-8

Todorov, S.D., Stojanovski, S., Iliev, I., Moncheva, P., Nero, L.A. & Ivanova, I.V. 2017. Technology and safety assessment for lactic acid bacteria isolated from traditional Bulgarian fermented meat product "lukanka". Brazillian Journal of Microbiology, 48: 576-586. DOI: https://doi.org/10.1016/j.bjm.2017.02.005

Tokajian, S., Issa, N., Salloum, T., Ibrahim, J. & Farah, M. 2016. 16S-23S rRNA gene intergenic spacer region variability helps resolve closely related sphingomonads. Frontiers in Microbiology, 7: 149. DOI: https://doi.org/10.3389/fmicb.2016.00149

Wells, M.L., Potin, P., Craigie, J.S., Raven, J.A., Merchant, S.S., Helliwell, K.E., Smith, A.G., Camire, M.E. & Brawley, S.H. 2017. Algae as nutritional and functional food sources: revisiting our understanding. Journal of Applied Phycology, 29: 949-982. DOI: https://doi.org/10.1007/s10811-016-0974-5

Yasiri, A., Vannaxay, E., Kiatmontri, J. & Seubsasana, S. 2018. Isolation and determination of bile salt hydrolase-producing lactic acid bacteria from fermented spider plant. Journal of Pure and Applied Microbiology, 12(3): 1055-1060. DOI: https://doi.org/10.22207/JPAM.12.3.03

Zheng, J., Wittouck, S., Salvetti, E., Franz, C.M.A.P., Harris, H.M.B., Mattarelli, P., O'Toole, P.W., Pot, B., Vandamme, P., Walter, J., Watanabe, K., Wuyts, S., Felis, G.E., Gänzle, M.G. & Lebeer, S. 2020. A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. International Journal of Systematic and Evolutionary Microbiology, 70: 2782-2858. DOI: https://doi.org/10.1099/ijsem.0.004107