Evaluation of probiotic properties of yeasts isolated from different foods and development of functional foods

This PhD thesis investigated the probiotic potential of 85 yeast strains isolated from 19 homemade natural cottage cheese, belly cream cheese, butter, kefir, yoghurt, string cheese, pickled mushrooms, pickled cucumbers, sauerkraut, bean pickles, taflan pickles, wilddag buyer vinegar, persimmon vinegar, Ekşidağ apple vinegar, red beet, cabbage, carrot pepper vinegar. , apple cider vinegar, grape vinegar, flower honey vinegar and mountain cranberry vinegar samples. The obtained yeast strains demonstrated the potential to survive gastrointestinal conditions after being exposed to simulated conditions during stomach and intestinal transit. It was determined that 10.58% of the isolates were moderate biofilm producers, 34.11% were weak biofilm producers, and 55.29% did not have biofilm formation capability. Catalase enzyme production was detected in all isolates. It was found that 83.52% of the isolates exhibited moderate protease activity, while 16.47% exhibited strong protease activity. Esterase activity was not observed in 2.35% of the isolates, 95.29% had moderate esterase activity, and 2.35% showed strong esterase activity. Moderate amylase activity was detected in 95.29% of the isolates, strong amylase activity in 2.35%, and no amylase activity in 2.35%. Strong β-glucosidase activity was observed in 50.58% of the isolates, while no activity was observed in 49.41%. It was concluded that the adhesion ability to hydrocarbons was species and strain-specific. Additionally, hydrophobicity varied depending on the type of hydrocarbon. Our study determined that the autoaggregation percentages increased with the extension of the incubation period for all strains. After 24 hours, the autoaggregation percentage was over 60% in all isolates. The coaggregation abilities of the yeast strains with E. coli ATCC 25922 ranged from 0.0% to 95.7%. The coaggregation values with S. Typhimurium ATCC 14028 ranged from 0.0% to 91.9%, with S. aureus ATCC 25923 from 0.0% to 95.1%, and with L. monocytogenes ATCC 7644 from 0.0% to 88.7%. It was concluded that the coaggregation ability depended on the incubation time and strain. Most of the obtained yeasts exhibited antimicrobial effects against E. coli ATCC 25922, E. coli O157H7, B. cereus, C. sakazakii ATCC 29544, L. monocytogenes ATCC 7644, S. aureus ATCC 25923, and S. Typhimurium ATCC 140828. The results confirmed resistance to all tested antibiotics. The 1,1-Diphenyl-2-picrylhydrazyl radical scavenging activity indicated that the antioxidant activity of the cells was higher than that of the intracellular extracts in all tested yeast strains. Yeasts identified by MALDITOF were subsequently determined by 18S-28S rRNA analysis. One of the yeast strains with the highest probiotic potential, Saccharomyces boulardii, was used to produce sugar-free prebiotic and probiotic chocolate containing 62.2% cocoa mass, 7% cocoa butter, 30% inulin, 0.50% stevioside, and 0.30% soy lecithin using the encapsulation method. In this study, the mixture of S. boulardii cocoa powder: Na-alginate: fructooligosaccharides in the ratio (10:1:1) was encapsulated by freeze dryer. The encapsulating mixture showed an encapsulation efficiency of 93.57 ± 1.02%. At least 107 log CFU/g of live probiotic count was achieved in the final products. When the crystal morphologies of the chocolate samples were examined, it was found that bulking agents (inulin) caused a rough texture, which was attributed to differences in viscosity. There was no significant change (P ≥ 0.05) in the moisture and hardness of the chocolate samples after 120 days of storage at 25 ˚C. Additionally, no blooming was observed in any of the samples during storage. It was determined that in the presence of probiotics in chocolate samples, a significant (P < 0.05) increase in pH values and a decrease in water activity values (P < 0.05) were observed during storage at 25 ˚C for 120 days. Scanning electron microscopy revealed that encapsulated yeasts were trapped within the chocolate. The round-shaped yeasts were uniformly distributed, and the appearance of the encapsulated probiotic cells was consistent across all samples. SEM images showed that probiotic bacteria's average size and shape did not change during freeze-drying with the encapsulating mixture or in encapsulated chocolates. On the first day of storage, all chocolate applications had similar initial probiotic counts (>11 log). Notably, the probiotic count remained above 8.08 log CFU/g throughout the 120-day storage period at 25 °C in the chocolate enriched with encapsulated probiotics. In contrast, chocolates enriched with non-encapsulated probiotics showed a significant (P < 0.05) and gradual decrease in probiotic counts by approximately 5.97 logs during storage. When the protective effects of encapsulations in chocolates were compared, a higher number of probiotics (2.51 log) were detected at the end of 120 days in encapsulated chocolates compared to non-encapsulated probiotics, demonstrating the protective effects of encapsulants. In vitro analysis, after one day of storage at 25°C, revealed a significant reduction (6.42 ± 0.0 log, P < 0.05) in chocolates supplemented with non-encapsulated probiotics when exposed to 2-hour gastric digestion (pH 3). However, only a 3.99 ± 0.2 log reduction was observed in samples containing encapsulated probiotics. The lower log reduction of probiotics in chocolates indicated that encapsulation and using chocolate as a carrier protected probiotics during in vitro digestion. It was concluded that pH 7 in the intestine was more suitable for probiotic growth compared to pH 3 in the stomach, which has harsh gastric conditions. These results demonstrated that probiotic chocolate enriched with encapsulated probiotics could maintain minimum therapeutic limits during long-term storage. Additionally, it was concluded that the complex mixture of chocolate components served as a good nutrient source for probiotic growth in the gastrointestinal tract. Fatty acid analysis of the chocolate samples was determined using GC-FID, with stearic acid being the most abundant in all samples. When the results are examined, depending on storage in encapsulated chocolates, Caproic (C6:0), Palmitic (C16:0), Stearic (C18:0), Oleic (C18:1n9c), Linoleic (C18:2n6c), Arachidonic (C20:0), cis -15-Tetracosenoic acid (C24:1n9) fatty acids showed no significant change (P>0.05), but unlike the 120th age of storage, Lauric (C12:0) fatty acid was detected at a rate of 1.649%. In non-encapsulated chocolates, Palmitic (C16:0), Stearic (C18:0), Oleic (C18:1n9c), Linoleic (C18:2n6c), Arachidonic (C20:0), Lignoceric (C24:0), cis-15-Tetracosenoic acid. No significant change was detected in (C24:1n9) fatty acids (P>0.05). On the 120th day of storage, Behenik (C22:0) fatty acids were detected at a different rate of 0.955%. Unlike unencapsulated chocolates, caproic acid (C6:0) was detected at a rate of 0.714% in encapsulated chocolates. In conclusion, this PhD thesis demonstrated that yeast strains possess promising probiotic potential and provided significant insights into the functional properties of probiotics found in traditionally produced products. Based on the findings of this study, it was concluded that probiotic chocolate enriched with S. boulardii could be considered a functional snack with potential prebiotic functions and a good nutrient source for human health.