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European Journal of Biotechnology and Bioscience 2014; 2 (5): 27-30 ISSN: 2321-9122 EJBB 2014; 2 (5): 27-30 Received: 21-10-2014 Accepted: 08-11-2014 Prasad. M. P Department of Microbiology/Biotechnology, Sangenomics Research Lab, Domlur Layout, Bangalore 560071, India. Correspondence: Prasad. M. P Department of Microbiology/Biotechnology, Sangenomics Research Lab, Domlur Layout, Bangalore 560071, India.

Evaluation of antimicrobial activity of yeast protein against clinical isolates

Prasad. M. P Abstract Preservative and antimicrobial agents are required to ensure that manufactured foods remain safe and unspoiled with that they get fermented to producer higher quality of food products. In the present study yeasts have been isolated from different sources and their antimicrobial activity has been studied so that the yeast isolates can a play a role in inhibiting the pathogenic microorganisms along with fermentation of the food production due to which the food can be preserved for a longer period of time. The antimicrobial activity for each of the isolate was checked against B.subtilis, Pseudomonas, E.coli, Klebseilla, Salmonella, Serratia, Staphyloccous, Streptococcus. Isolate 1 and 5 showed antimicrobial activity against most of the pathogens and had a greater zone of inhibition. Klebseilla was inhibited by all the isolates proving that S.cerevasiea can be used as of the antimicrobial agent against klebseilla. The proteins were purified from the isolates which showed better inhibition activity and they were checked whether the proteins were produced extracellular or intracellular. It was found that most of the intracellular proteins had a greater impact on the inhibition of the microorganisms and the phosphate buffer B method for the purification of the protein had a improved inhibition when compared to the other methods used to purify the proteins. Keywords: Antimicrobial, S. cerevasiae, Proteins, Klebseilla, ammonium sulphate precipitation. 1. Introduction Antimicrobial proteins play an important role during innate immunity in many living organisms like Plants, Insects, Fish, Amphibians and Mammals [1] [2] [3]. Yeasts are eukaryotic microorganisms classified in the kingdom Fungi, with 1,500 species currently described [4]. The useful physiological properties of yeast have led to their use in the field of biotechnology, fermentation of sugars by yeast being the oldest and largest application of this technology. Many types of yeasts are used for making many foods: baker's yeast in bread production; brewer's yeast in beer fermentation; yeast in wine fermentation and for xylitol production [5]. Some yeast like S. boulardii is used to maintain and restore the natural flora in the gastrointestinal tract. S. boulardii has been shown to reduce the symptoms of acute diarrhoea [6], reduce the chance of infection by Clostridium difficile [7], reduce bowel movements in diarrhoea-predominant IBS patients [8], and reduce the incidence of antibiotic traveller’s and HIV/AIDS-associated diarrhoeas [9]. Materials and Methodology Isolation and culture media Isolation and sub culturing of the yeast isolates were performed on media including yeast extract peptone dextrose (YPD) broth medium (glucose, 10; yeast extract, 3; peptone, 3 g/L), yeast extract peptone dextrose (YPD) agar medium (YPD broth + 15 g agar). pH of the media was adjusted at 4.5. The investigated yeast isolates were isolated from rotten fruits, foodstuffs, fruit juices and soils. Samples were introduced into YPD and YM broth media and incubated at 280C for 24 hr. The samples were diluted with distilled water and then 0.1ml of dilutes were plated onto YPD agar media. After incubation at 280C, the appearing yeast colonies were purified. Saccharomyces cerevisiae were identification according to [10], [11, 12]. Antimicrobial Activity The potential of the isolates to effect the growth of pathogenic microorganisms was

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Determined by Agar diffusion method. The pathogenic microorganisms tested for antimicrobial activity were B.subtilis, Pseudomonas, E.coli, Klebseilla, Salmonella, Serratia, Staphyloccous, Streptococcus. Protein Purification The isolates showing better zone of inhibition were selected and cultured on YPD broth and incubated for 24 hours at room temperature. The broth was centrifuged at 10,000rpm for 20min to separate the cells and the extracellular components. The cells and even the extracellular components were used for the further investigation to determine the proteins having the potential of antimicrobial activity were extracellular or intracellular. The proteins were purified from the cells and the extracellular broth with different methods wherein they the cells were lysed with Hepes buffer to breakdown the membrane. The intracellular and extracellular proteins were then purified by phosphate buffer A, Phosphate buffer B and 50 % ammonium

sulphate precipitation. The samples were dialysed and then they were checked for antimicrobial activity to determine if the proteins were extracellular or intracellular. Results Isolation of the Microorganisms A total of 6 isolates were isolated from 12 different sources. These isolates were cultured and maintained on YPD agar medium for further investigation. The isolates were mainly obtained from juice samples and soil. Antimicrobial Activity The antimicrobial activity for each of the isolate was checked against B.subtilis, Pseudomonas, E.coli, Klebseilla, Salmonella, Serratia, Staphyloccous, Streptococcus. Isolate 1 and 5 showed antimicrobial activity against most of the pathogens and had a greater zone of inhibition. Klebseilla was inhibited by all the isolates proving that S.cerevasiea can be used as of the antimicrobial agent against klebseilla.

Fig 1: Antimicrobial activity of the isolates

Fig 2: Antimicrobial assay by agar diffusion method Protein purification and antimicrobial activity: Isolate 1 and 6 were selected for further investigation as they showed greater zone of inhibition against most of the pathogenic microorganisms. The intracellular and extracellular proteins

were purified by 3 different ways and were checked for the antimicrobial activity to determine the proteins which play a role in reduction of the growth of the pathogens (Figure 1 and 2).

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Fig 3: Antimicrobial activity of intracellular protein from isolate 1.

The antimicrobial activity of the intracellular proteins from isolate 1 showed better activity when the proteins were purified by phosphate buffer B. the maximum zone of

inhibition was seen against serratia when the proteins were purified using phosphate buffer B (Figure 3).

Fig 4: Antimicrobial activity of intracellular protein from isolate 6. The intracellular protein from isolate 6 showed maximum zone of inhibition against Klebseilla. Ammonium sulphate method

had very little effect on the purification of antimicrobial protein (Figure 4).

Fig 5: Antimicrobial activity of Etracellular protein from isolate 1.

The antimicrobial activity of the Extracellular proteins from isolate 1 showed better activity when the proteins were purified by phosphate buffer B. the maximum zone of

inhibition was seen against Klebseilla with a inhibition zone of 16mm when the proteins were purified using phosphate buffer B (Figure 5).

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Fig 6: Antimicrobial activity of Extracellular protein from isolate 6.

The antimicrobial effect from the extracellular protein was seen maximum against serratia from the proteins purified from isolate 6 (Figure 6). Discussion [13], isolated twelve yeast strains were isolated and determined taxonomically from traditional dairy products, in which they occurred as additional microflora. Morphological, cultural and physiological tests were carried out resulting in determination of the strains to species. The recent study aims at establishing the species variety of the contaminants in different bulgarian dairy products, which caused disadvantages in them. [14] Isolated Sixteen yeast isolates from various sources including molasses, foodstuffs, concentrated juices and soils were identified using simplified API 20 C AUX yeast identification system and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). [15] Made a notable advance in the purification of pyrophosphatase obtained from baker's yeast. The method of purification used by these authors consisted chiefly in fractionation of a yeast autolysate with ammonium sulfate and fractional adsorption of the enzyme on calcium phosphate gel, followed by elution with strong salt solution. [16] Isolated and purified a Specific “Protector” Protein Which Inhibits Enzyme Inactivation by a Thiol/Fe (III)/02 Mixed-function Oxidation System. References 1. Andreu D, Rivas L. Biopolymers (Pept. Sci.). 1998; 47:

415-433. 2. Lehrer RI, Ganz T. Curr Opin Immunol 1999; 11:23-27. 3. Schrder JM. Biochem Pharmacol 1999; 57:121-134. 4. Cruickshank R, Duguid JP, Marmion BP, Swain RHA.

Medical microbiology. 12 edit. Vol. 2 Churchill Livingstone, Edinburgh, London and New York, 1975.

5. El-Eraqy NZ. Fungal infection in the female genital tract of buffaloes. M.V.Sc thesis (microbiology). Faculty of Vet Med Cairo Univ 1990.

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9. Kurtzman CP, Fell JW. Yeast Systematic and Phylogeny—Implications of Molecular Identification Methods for Studies in Ecology". Biodiversity and Ecophysiology of Yeasts. The Yeast Handbook, Springer 2006.

10. Johnson S, Maziade PJ, McFarland LV, Trick W, Donskey C, Currie B et al. Is primary prevention of Clostridium difficile infection possible with specific probiotics? International Journal of Infectious Diseases 2012; 16(11): e786–92. doi:10.1016/j.ijid.2012.06.005. PMID 22863358.

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12. Mc Farland LV. Systematic review and meta-analysis of Saccharomyces boulardii in adult patients. World Journal of Gastroenterology 2010; 16 (18):2202–22. doi:10.3748/wjg.v16.i18.2202. PMC 2868213. PMID 20458757.

13. Pedersen O, Andersen T, Christensen C. CO2 in planted aquaria". The Aquatic Gardener 2007; 20(3):24–33.

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Rhee, Earl R Stadtmann. The Isolation and Purification of a Specific “Protector” Protein Which Inhibits Enzyme Inactivation by a Thiol/Fe (III)/02 Mixed-function Oxidation System. The journal of biological chemistry Vol. 263, No. 10, Issue of April 5 , pp. 4704-4711, 1988.

16. Irena Savova, Maria Nikolova. Isolation and taxonomic study of yeast strains from Bulgarian dairy products. Journal of culture collections 2000-2002; 3:59-65.