Ms. Dilumi Bimaya Wickramaratne

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Diversity of heterofermentative autochthonous Lactobacillus strains in spontaneously fermented buffalo curd from Kanthale, Sri Lanka

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1 Diversity of heterofermentative autochthonous Lactobacillus strains in spontaneously fermented buffalo curd from Kanthale , Sri Lanka Dilumi Bimaya Wickramaratne University of Kelaniya , Sri Lanka

Diversity of heterofermentative autochthonous Lactobacillus strains in spontaneously fermented buffalo curd from Kanthale, Sri Lanka   :

Diversity of heterofermentative autochthonous Lactobacillus strains in spontaneously fermented buffalo curd from Kanthale, Sri Lanka   Dilumi Bimaya Wickramaratne Department of Microbiology, Faculty of Science. University of Kelaniya Dr (Mrs.) Deepthi K. Gunasena BSc (Kelaniya), PhD (Reading, UK), FI. Biol. (Sri Lanka) Senior Lecturer Department of Microbiology, Faculty of Science. University of Kelaniya

Introduction :

Introduction

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Highly heterogeneous genus Wide range of biochemical and physiological properties Naturally present or added intentionally to fermented dairy products Generally Recognized as Safe (GRAS) Genus Lactobacillus

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Lactobacillus strains isolated from traditional , spontaneously fermented food products High species diversity High diversity of metabolic activities Because of increasing interest in beneficial effects, the genus Lactobacillus is essential to modern food and feed technologies Recent trend - Isolation of autochthonous Lactobacillus strains from spontaneously fermented food to be used as starter cultures

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Buffalo Curd -“Meekiri” From Kanthale , Sri Lanka - Well known for Traditional production Spontaneous fermentation Backsloping to enhance the natural microbial flora.

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According to Taxonomic Outline of the Burgeys manual of Systematic Bacteriology (2009) T he genus Lactobacillus belongs to the P hylum - Firmicutes Class - Bacilli O rder - Lactobacillales Family - Lactobacillaceae

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At present, lactobacilli were grouped based solely on their fermentation types. Accepted ‘‘modern’’ definition - L actobacilli is divided as O bligate homofermentative F acultative heterofermentative O bligate heterofermentative, based on the type of fermented sugars and fermentation products (Hammes and Hertel, 2009).

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Metabolic group A or the homofermentative lactobacilli ferment hexoses almost exclusively (85 %) to lactic acid via the Embden-Meyerhof-Parnas pathway (EMP) or glycolysis; pentoses and gluconate are not fermented. Metabolic group B , the Facultative heterofermentative lactobacilli ferment hexoses to lactic acid via EMP and have the ability to degrade pentoses and gluconate via an inducible phosphoketolase, an enzyme of the pentosephosphate (PP) pathway, with a resulting production of acetic acid, ethanol and formic acid under glucose limitation. Metabolic group C, the obligate heterofermentative lactobacill i ferment hexoses to lactic acid, acetic acid (ethanol), and CO2 via the phosphogluconate pathway. Pentoses are fermented to lactic acid and acetic acid by the related pentose phosphate pathway.

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Objectives To isolate and identify heterofermentative Lactobacillus spp from Spontaneous fermented Buffalo Curd , as an initial step of introducing them as starter/probiotic strains

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Methods and results

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Randomly Representing the entire area Sample Collection

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Isolation of Lactobacillus strains from curd

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The initial identification of obtained pure cultures is based on colony and cell morphology determined by light microscopy, Gram staining, and catalase and oxidase reaction Species were classified based on the 80%-90% of similarity with the standard reference test results given Bergy’s Manual of Systematic Bacteriology (2009)

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Gram positive Non motile Rods -Vary from longer, slender, bent rods to short or cocobacilli. Chains and ring formation

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Biochemical characterization Oxidase Test Negative Catalase Test Negative Arginine Hydrolysis Arginine MRS Broth Production of NH 3 detected by Nessler’s reagent

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CO2 production from Glucose in Gibson’s semisolid media was used to determine the heterofermentative and homofermentative Lactobacilli. A B C Positive (Heterofermentative) Negative (Homofermentative) Negative Control ( Lactobacillus delbrueckii subsp. bulgaricus )

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Carbohydrate fermentation tests MRS fermentation broth ( Arabinose , Cellobiose , Esculin , Galactose , Maltose , Mannose, Melibiose , Raffinose , Ribose, Sucrose, Trehalose , Xylose , Salicin , Sorbitol , Mannitol , Rhamnose ,Lactose, Fructose , Amygdalin)

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72 pure cultures of Lactobacillus were isolated from 20 curd samples 52 different cultures (14 different species ) of heterofermentative Lactobacillus identified Among them there were 13 cultures of facultatively heterofermentative Lactobacillus and the most abundant facultatively heterofermentative species was Lactobacillus plantarum subsp. Plantarum (53.84%) Also there were 39 cultures of obligately heterofermentative Lactobacillus and the most abundant obligately heterofermentative species was Lactobacillus fermentum (61.53%)

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Conclusion Buffalo curd , a spontaneously fermented , traditional Sri Lankan dairy product contains a high diversity of heterofermentative Lactobacillus species. Relative abundance of obligately heterofermentative Lactobacillus (75%)is higher than the abundance of facultatively heterofermentative Lactobacillus (25%) . The most abundant facultatively heterofermentative species was Lactobacillus plantarum subsp. Plantarum (53.84%) and the most abundant obligately heterofermentative species was Lactobacillus fermentum (61.53%). These identified strains could be developed as starter cultures after investigating further for bioactivities that have human health benefits and technologically important characteristics.  

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References   Ayad, E., Verheul , A., Wouters , J. and Smit , G. (2002). Antimicrobial-producing wild lactococci isolated from artisanal and non-dairy origins. International Dairy Journal, 12(2-3), pp.145-150. Coeuret, V., Dubernet , S., Bernardeau , M., Gueguen , M. and Vernoux , J. (2003). Article. Le Lait , 83(4), pp.269-306. Çon , A. and Gökalp , H. (2000). Production of bacteriocin -like metabolites by lactic acid cultures isolated from sucuk samples. Meat Science, 55(1), pp.89-96. de Jong , H., Parry, C., van der Poll, T. and Wiersinga , W. (2012). Host–Pathogen Interaction in Invasive Salmonellosis . PLoS Pathog , 8(10), p.e1002933. De Vuyst , L. and Leroy, F. (2007). Bacteriocins from Lactic Acid Bacteria: Production, Purification, and Food Applications. Journal of Molecular Microbiology and Biotechnology, 13(4), pp.194-199. Dunne, C., Mahony , L. and Morrissey, D. (2001). In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings. The American Journal Of Clinical Nutrition, 73(2), pp.386S-392S .

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Felis, G. and Dellaglio , F. (2007). Taxonomy of lactobacilli and Bifidobacteria . Current Issues in Intestinal Microbiology, 8(2), pp.44-61. Food and Agriculture Organization of the United Nations and World Health Organization, (2002).  Guidelines for the Evaluation of Probiotics in Food . [online] London Ontario, Canada, pp.4-5.Availableat: http://www.who.int/foodsafety/fs_management/en/probiotic_guidelines.pdf?ua=1 [Accessed 03 Oct. 2016]. Giraffa , G. (2002). Enterococci from foods. FEMS Microbiology Reviews, 26(2), pp.163-171. Kadariya , J., Smith, T. and Thapaliya , D. (2014). Staphylococcus aureus and Staphylococcal Food-Borne Disease: An Ongoing Challenge in Public Health. BioMed Research International, 2014, pp.1-9. Oliveira, G., Favarin , L., Luchese , R. and McIntosh, D. (2015). Psychrotrophic bacteria in milk: How much do we really know?. Brazilian Journal of Microbiology, 46(2), pp.313-321. Schelin , J., Wallin-Carlquist , N., Thorup Cohn, M., Lindqvist , R. and Barker, G. (2011). The formation of Staphylococcus aureus enterotoxin in food environments and advances in risk assessment. Virulence, 2(6), pp.580-592.

Acknowledgement :

Acknowledgement Doctors at Veterinary regional office Kanthale Dr. Pradeepika Siriwardena Dr. G.G.M.D. Seneviratna

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Thank You !

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