Evaluation of probiotic and antifungal properties of the yeast isolated from buckwheat sourdough

Evaluation of antimicrobial and probiotic properties of the microbiota isolated from fermented pseudo-cereals is important in order to prepare adjunct and starter cultures. Probiotics are live and active microorganisms that, if used in sufficient numbers, help the microbial balance of the gastrointestinal tract and improve its function. Probiotics are also used as a substitute for antibiotics and synthetic preservatives in the prevention and treatment of complications of many infectious and pathogenic gastrointestinal pathogens. Probiotic microorganisms include lactic acid bacteria (LAB) and yeasts, and despite numerous reports on the probiotic properties of LAB, the probiotic properties of yeasts are less reported. Probiotic yeasts are organisms resistant to antibacterial compounds and effective against pathogens, which can rapidly increase their population in the gastrointestinal tract. These yeasts have several properties such as antimicrobial effects, resistance to acid and bile, binding to mucosal surfaces, inhibitory activity against pathogens and also the inability to transmit antibiotic resistance genes. In the present study, after isolation of the predominant yeast from buckwheat sourdough, the probiotic and antifungal properties of the isolate were investigated. Based on the literature review, no study has been presented to evaluate the probiotic and antifungal capabilities of yeasts isolated from buckwheat sourdough. 

In the present study, predominant yeast was isolated from buckwheat sourdough, and then it was identified using PCR. Subsequently, probiotic properties of the isolate including survival in present of low pH and bile salt, antibacterial effect, antibiotic susceptibility assay, aggregation, hydrophobicity, haemolytic activity as well as its antifungal activity against Aspergillus niger and Aspergillus flavus were studied. After spontaneous fermentation of buckwheat, predominant yeast was isolated using ten-fold dilution of the sourdough sample and its spread plating. The predominant isolate was identified through PCR amplification of a 650 bp target sequence from its ribosomal gene and sequencing of the PCR product. Then, survival of the yeast was determined at pH=2 and 0.3% bile salt as simulated gastrointestinal conditions. Subsequently, simultaneous culture of the yeast with some food-borne indicator bacteria in chromogenic media was used to investigate the inhibitory activity of the isolate against the studied bacteria. After that, resistance of the yeast isolate against the common antibiotics and some antimycotic agents was evaluated using disc method. Co-aggregation ability and hydrophobicity capability of the isolate were also determined based on the absorbance tests. In vitro safety of the yeast isolate was checked through its cultivation on blood agar containing sheep blood. Next stage, overlay bioassay was conducted to investigate antifungal effect of the yeast on the selected fungi. Finally, one way analysis of variance (ANOVA) with the least significant differences (LSD) post hock at p<0.05 was used for statistical analysis of the data. 

Sequencing results of the PCR products led to the identification of Pichia kudriavzevii as predominant yeast isolated from buckwheat sourdough. Survival rate of the isolate after treatment in simulated gastrointestinal conditions was 79.26% in comparison with the control. Antibacterial effect of the isolate on Escherichia coli was also significantly (P<0.05) higher than the other studied food-borne agents. Meanwhile, the growth of Listeria monocytogenes was decreased 19.50% in the present of the isolate. Whereas, the yeast isolate had no inhibitory effect on Salmonella enterica. Hydrophobicity and auto-aggregation capabilities of the isolate were also 64.07 and 67.40%, respectively. Furthermore, the isolate showed resistance towards all of the antibiotics tested, while it was resistant against ketoconazole and fluconazole, and the yeast was semi-sensitive towards itraconazole as antimycotic agents. The yeast isolate had no hemolytic activity, and its antifungal activity against A. niger and A. flavus was also verified. Accordingly, it is concluded that P. kudriavzevii isolate exhibits proper potential to be used as probiotic or protective culture in fermentation industries.In accordance with the results, probiotic characteristics of P. kudriavzevii (as the predominant yeast isolated from buckwheat sourdough) were approved. Accordingly, the isolate can be used as a potential probiotic culture in food industries.