Identification and biotechnological characterisation of yeast microbiota involved in spontaneous fermented wholegrain sourdoughs.

BACKGROUND: New strategies in the cereal-based industry has brought about the elaboration of new sourdoughs with better microbial stability and safety as well as nutritional value such as those based on wholegrain flours. This has led to an increasing interest in the selection of adapted yeasts for using them as new starters. Therefore, this study aimed to isolate, identify, and characterise diverse yeast strains from wholegrain spontaneous sourdoughs. RESULTS: Three wholegrain sourdoughs (wheat, rye, and oat) were fermented and monitored for 96 h. Minimum pH values ranged from 3.1 to 3.5 while maximum yeast counts were reached at 72 h. A total of 76 yeast isolates were identified by polymerase chain reaction random amplification of polymorphic DNA (PCR-RAPD) and catalogued in six different species by sequencing the internal transcribed spacer (ITS) region. The major species were Candida glabrata, Saccharomyces cerevisiae, Kazachstania unispora, and Wickerhamomyces anomalus. The studied kinetic parameters of the growth curves (λ, G, ODmax , and µmax ) and the fermentation capacity allowed to ascertain that 12 and 5 strains, respectively, were better than baker's yeast control. The fibre assimilation ability (cellulose, xylose, and ß-glucan) was observed in the 27% of the strains and only four strains showed phytase activity. CONCLUSIONS: The yeast population in the three wholegrain sourdoughs were variable along the fermentation time. Genetic identification showed that strains and species presented a different trend for each sourdough although common species were determined (e.g., W. anomalus). Candida glabrata (4T1) and Saccharomyces cerevisiae (3A6) showed, respectively, better kinetics and impedance results than the positive control, while W. anomalus (C4) was notorious in fibre assimilation and phytase degradation. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Use of probiotic yeasts with biocontrol activity for fermentation of ewe's milk.

BACKGROUND: There are sufficient scienctific studies that support the benefit that fermented dairy products produce in those who consume them. Traditionally, cow's milk has been the most commonly used milk but there is a growing interest in the development of new dairy products, substituting cow's milk with milk from other sources, as well as in the use of microorganisms in fermentation to replace artificial preservatives or treatments that may affect the chemical and organoleptic characteristics of the product. For these reasons, the aim of the present work was to understand the behavior of five potential probiotic yeasts during the fermentation of ewe's milk and to consider their potential use as biocontrol agents. RESULTS: Saccharomyces cerevisiae 3 and Hanseniaspora osmophila 1056 provided the most promising kinetic parameters in the different salt, temperature and pH conditions tested in their technological characterization. The profiles of organic acids and volatile compounds after the fermentation period was noteworthy for contributing to the final aroma of the dairy product. Sensory analysis revealed the sour taste of all samples, and S. cerevisiae 3, Lachancea thermotolerans 1039, and H. osmophila 1056 stood out for an accentuated cheese flavor. In addition, all strains showed biocontrol activity; they reduced the mycelium of the mycotoxigenic molds. CONCLUSION: Saccharomyces cerevisiae 3 and H. osmophila 1056 could be inoculated along with bacterial starters to provide a functional fermented beverage with improved flavor. These strains also have an added value as they act as biocontrol agents. © 2022 Society of Chemical Industry.

Study of potential probiotic and biotechnological properties of non-Saccharomyces yeasts from fruit Brazilian ecosystems.

Yeast isolates from flowers and fruits from a Brazilian forest were studied. The yeasts were identified at species and strain level by PCR-RFLP and PCR-RAPD, respectively. The 46 isolated yeasts were classified into 11 different species belonging to the genera Candida, Diutina, Hanseniaspora, Meyerozyma, Pichia, Rhodotorula, and Torulaspora. A total of 20 different strains were found. In order to ascertain the probiotic potential, the resistance to gastrointestinal conditions, autoaggregation, and hydrophobicity assays were studied, along with the capacity to form biofilm. The results indicate that, although most of the strains presented better results than Saccharomyces boulardii (the only strain recognized as a probiotic yeast), four strains were the most promising, namely, Rhodotorula mucilaginosa 32, Meyerozyma caribbica 35, and Diutina rugosa 12 and 45, according to the Duncan test. Several biotechnological properties were evaluated. D. rugosa inhibited Dekkera bruxellensis. The assimilation or fermentation of seven sugars was tested, and only five of the yeasts did not show a capacity to assimilate any of the sugars under aerobic conditions. However, all strains were able to ferment at least one of the sugars under anaerobic conditions. As far as enzyme production is concerned, positive results were only found for the enzymes' amylase, pectinase, and protease. D. rugosa 42 and Hanseniaspora opuntiae 18, followed of Pichia kluyveri 26, showed high values for the production of melatonin. In conclusion, the results of this study show that several non-Saccharomyces present probiotic characteristics, and these have good potential for industrial applications in the food or biotechnology industries.

Free and Immobilised ß-Glucosidases in Oenology: Biotechnological Characterisation and Its Effect on Enhancement of Wine Aroma.

In grapes, monoterpenes and norisoprenoids are in the form of non-volatile compounds, flavourless glycosides which could enhance the aroma of wines after its hydrolysis using ß- glucosidases enzymes. It is known that the use of immobilised enzymes offers advantages such as reusability and easy recuperation. In this study, a commercial ß-glucosidase was immobilised by absorption in sodium alginate. Biotechnological characteristics and terpen hydrolysis (hydrolysis aroma precursors) in muscat wines were studied after treatment with both free and immobilised commercial ß- glucosidase with two different concentrations. It was revealed that both forms shared an optimal pH (4.5) and a maximum temperature (64°C), even an increment on the activity between 40and 60°C. A similar Km value has been determined while Vmax from the immobilised enzyme was higher than the free (3.35 and 2.52 µmol min-1 mg-1, respectively). Additionally, the immobilised enzyme showed a better hydrolytic activity during 24 h, and its reusability has been proven. Regarding enzymatic hydrolysis in grape must, the best results were observed for the highest concentration of free ß-glucosidase although glucose release was also determined for the immobilised enzyme along the days. In contrast, maximum activity was reached by the immobilised ß-glucosidase in less time but in no case equalled the free ones. Finally, volatile compound liberation in wines treated with free or immobilised enzymes was analysed using HRGC-MS. Liberation for both enzymes and the greatest concentrations of some volatiles were detected when a double dose of the free ß-glucosidase was used. Nevertheless, the wines treated with the immobilised ß-glucosidase showed a high concentration of some volatile compounds such as nerol or geraniol.

Safety Evaluation of Yeasts With Probiotic Potential.

This work has evaluated the safety aspects of 20 yeast strains, isolated from food environments, selected in previous works due to their probiotic potential. Among the different strains, there are Saccharomyces and non-Saccharomyces yeasts. Before safety evaluation, differentiation of Saccharomyces cerevisiae strains was done by PCR amplification of inter-δ region with pairs of primers δ2-12 and δ12-21, which showed that they were all different from each other and also had different profiles to Saccharomyces boulardii (the only commercial probiotic yeast). The non-Saccharomyces ones were already known. The evaluation tests carried out were antibiotic and antifungal resistance, production of biogenic amines, deconjugation activity of bile salts, and different enzymatic activities: coagulase, deoxyribonuclease, hemolysin, proteolytic, and phospholipase. None of the studied strains demonstrated coagulase, hemolytic or DNase capacity (clear virulence factors), although all of them showed protease activity, some showed phospholipase activity, and half of the yeasts were capable of conjugating bile salts. Regarding antimicrobial compounds, all were resistant to antibiotics but showed sensitivity to the antimycotics used. Nevertheless, only one strain of Hanseniaspora osmophila was excluded for use in the food industry, due to its high production of tyramine.

Potential Probiotic Strains of Saccharomyces and Non-Saccharomyces: Functional and Biotechnological Characteristics.

Due to the evident demand for probiotic microorganisms, a growing number of scientific studies have involved the preliminary selection of new strains, but deeper studies for knowing specific functional and biotechnological properties are needed. In the present work, twenty yeasts (Saccharomyces and non-Saccharomyces) with potential probiotic characteristics, selected in previous works, were evaluated. The following assays were realized: adhesion to Caco-2/TC7 cells, prebiotic metabolisms, assimilation of cholesterol, enzymatic and antioxidant activity, and antifungal resistance. In addition, the effect of ultrasonic treatment was evaluated for attenuating the cultures before their possible incorporation into a food or supplement. In all of the cases, the unique commercial probiotic yeast (S. boulardii CNM I-745) was used as positive control. Results show different capabilities depending on the property studied. In general, no Saccharomyces yeasts were better in the adhesion to Caco cells, prebiotic metabolism, and presented higher variability of enzymatic activities. The ones related to cholesterol assimilation and antioxidant capability did not show a marked trend, and with respect to the attenuation process, the Saccharomyces yeasts were more resistant. For selecting the potential probiotic yeasts with better balance among all characteristics, a principal component analysis (PCA) was carried out. The most promising yeasts for use as health-promoting probiotics are Hanseniaspora osmophila 1056 and 1094, Lachancea thermotolerans 1039, and S. cerevisiae 3 and 146.

Potential probiotic and food protection role of wild yeasts isolated from pistachio fruits (Pistacia vera).

BACKGROUND: The biotechnological potential of yeasts from nuts such as pistachio, not only for health applications but also for industry use, has been scarcely studied. Interest in the probiotic capability of yeasts has increased in the past years as well as their utilization as food or feed preservatives. Their capabilities as biocontrol against problematic (spoilage or toxigenic) microorganisms or as antioxidants have been revalued. As a result, both abilities would be desirable to develop a new potential probiotic microorganism which could be added to food or feed to improve their properties. RESULTS: Molecular techniques allowed the identification of a total of seven different species and 15 strains. A screening of the probiotic potential of these strains was carried out. It was found that 65% of the strains resisted the gastrointestinal conditions as well as presented a generation time of < 22 h. Additionally, some strains showed better kinetic parameters than Saccharomyces boulardii (positive control). Complementary tests were done to determine their auto-aggregation capacity, cell surface hydrophobicity, behaviour in a sequential simulated digestion, biofilm formation capability and carbon source assimilation. Finally, 67% and 13% of the studied yeasts showed biocontrol and antioxidant activities, respectively. CONCLUSIONS: Diutina rugosa 14 followed by Diutina rugosa 8 were the best wild yeast from Pistacia vera as potential probiotic and in carbon source utilization. However, Hanseniaspora guilliermondii 6 and Aureobasidium proteae 5 could be used to improve food or feed product preservation because of their notable biocontrol and antioxidant capabilities. © 2020 Society of Chemical Industry.

Saccharomyces cerevisiae and Hanseniaspora osmophila strains as yeast active cultures for potential probiotic applications.

This work allowed the evaluation of the gastrointestinal resistance of five yeasts (Saccharomyces and non-Saccharomyces) in order to assess some biotechnological characteristics linked to the potential probiotics, using a dynamic gastrointestinal simulator (simgi®). The best results obtained were for strains Saccharomyces cerevisiae 3 and Hanseniaspora osmophila 1056. Having optimised the method, the yeasts were subsequently lyophilised, and the one that showed the least loss of viability, S. cerevisiae 3, was used in a freeze-dried form to obtain a new functional food. On the other hand, some characteristics of the product were compared with those of probiotic supplements and other commercial probiotic foods. The obtained functional product showed better parameters than the rest of the samples containing yeasts which, together with the great acceptance shown after the consumer tests, means that it can be presented as a possible commercial functional product.