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Probiotic Attributes of a Yeast-Like Fungus, Geotrichum Klebahnii

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Probiotic Attributes of a Yeast-Like Fungus, Geotrichum Klebahnii Vol. 8(20), pp. 2037-2043, 14 May, 2014 DOI: 10.5897/AJMR2013.6175 Article Number: D05D28C44722 ISSN 1996-0808 African Journal of Microbiology Research Copyright © 2014 Author(s) retain the copyright of this article http://www.academicjournals.org/AJMR Full Length Research Paper Probiotic attributes of a yeast-like fungus, Geotrichum klebahnii Poonam Syal and Ashima Vohra* Department of Microbiology, Institute of Home Economics, University of Delhi, HauzKhas Enclave, New Delhi, India. Received 13 July, 2013; Accepted 6 May, 2014 Geotrichum klebahnii, a filamentous yeast-like fungus, was isolated from a cheese sample. Several in vitro tests were carried out for its probiotic characterization. This isolate showed high survival rate of 100 ± 1.8% at low pH (pH 2) and 100 ± 0.9% at high oxbile concentration (1%) and also grew well at 37°C. It showed an auto-aggregation ability of 100.00 ± 1.5% after 20 h of incubation at 37°C, as well as 36.43 ± 0.70% and 52.13 ± 1.50% cell surface hydrophobicity with xylene and n-hexadecane, respectively. It had inhibitory activity against food-borne pathogens such as Salmonella sp., Vibrio sp. and Staphylococcus aureus. G. klebahnii produced the enzymes phytase and inulinase. It was a producer of vitamin B12 and exoploysaccharides. It assimilated up to 29.42 ± 2.1% cholesterol after 48 h of incubation at 37°C. The organism did not produce gelatinase and DNase assay, indicating its safety as a probiotic microorganism. This is the first report of the probiotic potential of yeast like fungus, G. klebahnii. Key words: Probiotics, yeast, antimicrobial, phytase, inulinase. INTRODUCTION Yeasts contribute to the fermentation of a broad range of environment, creates a selective environment for yeast other commodities, where various species may work growth (Fleet, 1990). Saccharomyces cerevisiae, which together with bacteria and/or filamentous fungi. Fer- according to The European Food Safety Authority (EFSA) mented milk products that are manufactured using starter has a Qualified Presumption of Safety (QPS) status, is cultures containing yeasts include acidophilus-yeast milk, the most common yeast used in food fermentation where kefir, Koumiss, Leban and cheese (Lang and Lang, it has shown various technological properties. 1975). Saccharomyces spp. For example Saccharomyces Probiotics have been defined as ‘live microorganisms burnetii, Saccharomyces kluveri, Saccharomyces which when administered in adequate amounts confer a byanus, Saccharomyces rosinii, Saccharomyces health benefit on the host’ (Fuller, 1989). S. boulardii is cerevisiae and Saccharomyces boulardii may be isolated the only yeast with clinical effects and the only yeast from a variety of dairy products including milk, yogurt, preparation with proven probiotic efficiency in double- cream, dahi, cheese and kefir. Saccharomyces spp. blind studies (Sazawal et al., 2006). cannot ferment lactose so they develop in milk as a Geotrichum sp., filamentous yeast-like fungus, is secondary flora, after bacterial growth. Lactic acid ubiquitous and is found in a wide range of habitats such produced by lactic acid bacteria creates a high acid as plant tissues, silage and soil (Jacques and *Corresponding author. E-mail: [email protected]. Tel: +919811642765. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 2038 Afr. J. Microbiol. Res. Caseregola, 2008). This genus belongs to the class presence of bile salt as compared to that without bile salt. Hemiascomycetaceae, order Saccharomycetales and family Dipodascaceae. It is also a component of the Comparison of growth at 30 and 37°C natural flora of human mouth, skin and gastrointestinal tract. Species belonging to this genus have been commonly Growth at both temperatures was compared by determining the isolated from dairy products such as cheese, fermented number of viable cells by the plate count method after incubation for milk and also from juices. One of its species, Geotrichum 48 h. candidum has been reported by the International Dairy Federation and European Food and Feed Culture Auto-aggregation assay Association as a microorganism with a documented history of use in dairy products (Mogensen et al., 2002). Sarkar G. klebahnii was grown for 48 h at 37°C in MYPG broth. The cells et al. (1994) reported the population of Geotrichum were harvested by centrifugation at 7000 rpm for 10 min, washed candidum in 40-50% of market samples of Kinema (a twice and resuspended in PBS. Cell suspensions (4 mL) were soyabean fermented food) to be 0.8-4 x 104cfu/g. Nago et mixed by vortexing for 10 s and auto-aggregation was determined after 3 and 20 h of incubation at 37°C. An aliquot (0.1 mL) of the al. (1998) isolated 54 strains of yeasts from Beninese ogi, upper suspension of PBS after incubation was transferred to out of which Geotrichum spp. accounted for 26% of the another tube with 3.9 mL of PBS and the absorbance (A) was yeast isolates. Geotrichum penicillatumand Geotrichum measured at 600 nm (Del et al., 2000). The auto-aggregation candidum have been reported to be isolated from Boza, a percentage is expressed as: 1-(At/A0) X 100, where At represents beverage consumed in Bulgaria, Albania (Vasudha and the absorbance at time t = 3, or 20 h and A0 the absorbance at t = 0. Mishra, 2013; Gotcheva et al., 2000). Geotrichum klebahnii ATCC 42397 (previously named Cell surface hydrophobicity Trichosporon penicillatumSNO-3) is a yeast-like fungus originally isolated from mandarin peel (Rojas et al., 2008). One milliliter of the hydrocarbon, that is, n-hexadecane and xylene This study is the first report of probiotic properties of G. were added to tubes containing cell suspensions (3 ml). The cells klebahnii, isolated from cheese sample. were vortexed for 120 s. The suspension was then kept undisturbed at 37°C for 5 min to allow phase separate, and the hydrocarbon layer was allowed to rise completely. After 5 min, the aqueous phase was removed carefully and the absorbance (OD) was MATERIALS AND METHODS measured using a spectrophotometer at 600 nm (Rosenberg et al., 1980). The decrease in the absorbance was taken as a measure of All the chemicals used were of AR grade and procured from the cell surface hydrophobicity (hydrophobicity (%)), calculated Qualigens and Himedia (Mumbai, India). using the equation given below: Hydrophobicity (%) = (ODinitial - ODfinal) / ODinitial x 100 Isolation procedure and Identification Where, ODinitial and ODfinal are the absorbances (at 600 nm) before A 25 g sample of the cheese sample was homogenized in 225 ml of and after extraction with the hydrocarbons. sterile phosphate buffered saline (PBS; g/L: NaCl 8.0, KCl 0.2, disodium phosphate 1.44 and potassium phosphate 0.24), pH 7. 10-fold serial dilutions of the sample were prepared in sterile PBS, Inhibitory action against enteric pathogens pH 7. The dilutions were spread plated on MYPG medium (g/L: malt extract 3.0, yeast extract 3.0, peptone 5.0, glucose 10.0 and agar Double layer method, was used to evaluate the antagonistic activity 20.0, pH 5.6) and the plates were incubated at 30°C for 24-48 h of G. klebahnii against enteric pathogens, that is, Escherichia coli, and observed for microorganisms with yeast-like morphology under Salmonella sp., Staphylococcus aureus, Vibrio cholerae and microscope. Pseudomonas sp. obtained from the Departmental Culture The culture was identified at IMTECH, Chandigarh, India, using Collection Centre. An overnight culture of G. klebahnii was BioLog identification kits. prepared in MYPG broth at 30°C and inoculated onto plates by swabbing a 1 inch by 1.5-inch area in the center of each plate. The plates were incubated at 30°C for 48 h. The growth in each plate Acid tolerance test was then overlaid with 10 mL of molten nutrient agar (0.7% agar) previously inoculated with 1 mL of the prepared test pathogen The culture was inoculated (1%) into PBS maintained at low pH (2, cultures. The agar was allowed to solidify and the plates were 2.5 and 3) using 1 N HCl. Samples were taken after 5 h of incubated aerobically at 37°C for 24 h and examined for growth incubation at 37°C and spread plated on MYPG. The survival rate inhibition (Maia et al., 2001). was calculated as the percentage of colonies grown on MYPG medium after exposure to low pH as compared to the initial cell concentration. Enzyme based screening Phytase Bile salt tolerance test Phytase assay was performed according to the method described PBS tubes containing 0.3, 0.5 and 1% oxbile were inoculated with by Vohra and Satyanarayana (2001) using phytate minimal medium the yeast culture and incubated at 37°C for 5 h. The survival rate of (g/L: glucose 15.0, sodium phytate 5.0, (NH4)2SO4 5.0, MgSO4.7H2O each strain was expressed as the percentage of viable cells in the 5.0, KCl 5.0, FeSO4.7H2O 0.01, MnSO4.4H2O 0.01 and agar 20.0). Syal and Vohra 2039 The plate was incubated at 30°C for 48 h. After incubation, the plate DNase enzyme and was streaked with G. klebahnii culture, followed was flooded with 2% cobalt chloride and then kept at room by plate incubation at 30°C for 48 h. After incubation, a clear temperature for 5 min. After removing cobalt chloride solution, a pinkish zone around the colonies against dark blue background was freshly prepared solution containing equal volume of 6.25% considered positive for DNase production (Gupta and Malik, 2007). ammonium molybdate and 0.42% ammonium vanadate was flooded onto the plates. They were incubated at room temperature for 5 min after which, the solution was removed. The plates were Statistical analysis observed for zone of hydrolysis.
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