Exploring volatile compounds and microbial dynamics: Kluyveromyces marxianus and Hanseniaspora opuntiae reduce Forastero cocoa fermentation time.

Traditional cocoa bean fermentation is a spontaneous process and can result in heterogeneous sensory quality. For this reason, yeast-integrated starter cultures may be an option for creating consistent organoleptic profiles. This study proposes the mixture of Hanseniaspora opuntiae and Kluyveromyces marxianus (from non-cocoa fermentation) as starter culture candidates. The microorganisms and volatile compounds were analyzed during the cocoa fermentation process, and the most abundant were correlated with predominant microorganisms. Results showed that Kluyveromyces marxianus, isolated from mezcal fermentation, was identified as the dominant yeast by high-throughput DNA sequencing. A total of 63 volatile compounds identified by HS-SPME-GC-MS were correlated with the more abundant bacteria and yeast using Principal Component Analysis and Agglomerative Hierarchical Clustering. This study demonstrates that yeasts from other fermentative processes can be used as starter cultures in cocoa fermentation and lead to the formation of more aromatic esters, decrease the acetic acid content.

Isolation and identification of native yeasts from the spontaneous fermentation of grape musts.

Recently, there has been growing interest in the characterization of native yeasts for their use in production of wines with regional characteristics. This study aimed to investigate Saccharomyces and non-Saccharomyces yeasts present in the spontaneous fermentation of Tannat and Marselan grape musts collected from Concordia (Entre Ríos, Argentina) over 2019, 2020, and 2021 vintages. The evolution of these fermentative processes was carried out by measuring total soluble solids, total acidity, volatile acidity, pH, ethanol concentration, and total carbon content. Isolated Saccharomyces and non-Saccharomyces yeasts were identified based on colony morphology in WL medium, 5.8S-ITS-RFLP analysis, and 26S rDNA D1/D2 gene sequencing. Two hundred and ten yeast colonies were isolated and identified as Pichia kudriavzevii, Saccharomyces cerevisiae, Hanseniaspora uvarum, Metschnikowia pulcherrima, Candida albicans, Candida parapsilosis, Pichia occidentalis, Pichia bruneiensis, Hanseniaspora opuntiae, Issatchenkia terricola, and Hanseniaspora vineae. P. kudriavzevii isolated from all vintages was associated with the spontaneous fermentation of grape musts from the Concordia region.

Brewing a Craft Belgian-Style Pale Ale Using Pichia kudriavzevii 4A as a Starter Culture.

There is an expanding market for beer of different flavors. This study aimed to prepare a craft Belgian-style pale ale with a non-Saccharomyces yeast. Pichia kudriavzevii 4A was used as a sole starter culture, and malted barley as the only substrate. The ingredients and brewing process were carefully monitored to ensure the quality and innocuousness of the beverage. During fermentation, the yeast consumed 89.7% of total sugars and produced 13.8% v/v of ethanol. The product was fermented and then aged for 8 days, adjusted to 5% v/v alcohol, and analyzed. There were no traces of mycotoxins, lead, arsenic, methanol, or microbiological contamination that would compromise consumer health. According to the physicochemical analysis, the final ethanol concentration (5.2% v/v) and other characteristics complied with national and international guidelines. The ethyl acetate and isoamyl alcohol present are known to confer sweet and fruity flavors. The sensory test defined the beverage as refreshing and as having an apple and pear flavor, a banana aroma, and a good level of bitterness. The judges preferred it over a commercial reference sample of Belgian-style pale ale made from S. cerevisiae. Hence, P. kudriavzevii 4A has the potential for use in the beer industry.

Isolation of microorganisms from the feces of ring-tailed coati related to the production of "misha coffee" in the central forest of Peru and evaluation of some features of technological importance.

A diversity of yeasts and lactic bacteria were isolated from the feces of ring-tailed coaties bred in captivity and related to the production of "misha coffee". Isolation of yeasts was carried out using oxytetracycline-glucose-yeast extract agar containing 100 mg/L oxytetracycline and, lactic bacteria using de Man-Rogosa and Sharpe agar containing 20 mg/L of vancomicin. Then, isolates were biochemically analysed using API strips (ID 32C for yeasts and 50CHL for lactic bacteria) followed by 16S and 26S rRNA gene sequencing. Among the yeasts, Debaryomyces hansenii, Pichia kluyveri, Pichia kudriavzevii, and Candida sorboxilosa were the most frequent, whereas Weissella cibaria, Weissella paramesenteroides, Enterococcus thailandicus and Enterococcus faecalis were the most important lactic bacteria. Cultivation of the isolated yeasts under agitated conditions, showed that Pichia kluyveri LBFT.Lev3 (0.15 ± 0.01 h-1) and Pichia kudriavzevii LBTF.Lev7 (0.14 ± 0.01 h-1) had higher specific growth rates than Debaryomyces hansenii LBFT.Lev9 (0.09 ± 0.01 h-1), whereas cultivation of lactic bacteria under static fashion showed that Weisella paramesenteroides LBTF.Bal2 (0.16 ± 0.01 h-1) and Weisella cibaria LBTF.Bal3 (0.18 ± 0.01 h-1) had better growth than Enterococcus thailandicus LBTF.Bal1 (0.1 ± 0.015 h-1) and Enterococcus faecalis LBTF.Bal7 (0.14 ± 0.01 h-1). Additionally, evaluation of pectinolytic activity revealed that Pichia kudriavzevii LBTF.Lev7 and Debaryomyces hansenii LBFT.Lev9 were able to use pectin as carbon source for their growth. On the other hand, W. cibaria LBTF.Bal3, E. thailandicus LBTF.Bal1 and W. paramesenteroides LBTF.Bal2 showed inhibitory activity against S. mutans ATCC 35668, B. subtilis subsp. spizizenii ATCC 6633 and Staph. epidermidis ATCC 14990. Results of this study are useful for the search of potential application of the isolated yeasts and lactic bacteria in coffee and other food fermentations.

Evaluation of yeasts from Ecuadorian chicha by their performance as starters for alcoholic fermentations in the food industry.

Yeasts involved in the spontaneous fermentation of traditional beverages like chicha (indigenous Andean beer) may have the potential to be used as starter cultures to improve the quality and microbiological safety of these products, but also as non-conventional alternatives to other food alcoholic fermentations. In this research, we isolated, identified and characterised yeast strains from four Ecuadorian chichas made by using four different raw materials: rice (RC), oat (OC), grape (GC) and a mixture of seven corn varieties (yamor, YC). Finally, 254 yeast isolates were obtained and identified by molecular methods. Eleven yeast genera and 16 yeast species were identified with relatively few isolates belonging to Saccharomyces cerevisiae (9.1% belonging to 6 strains) and Torulaspora delbrueckii (18.6% belonging to 2 strains). In order to select good candidates for fermentative starter production, different analyses were performed. The results of the stress response tests showed a wide variability between species and strains, and identified some yeasts displaying high stress tolerance, similarly to commercial wine strains. Amylase production was screened as being indicative of the capacity to degrade and ferment starch-rich substrates. A Cryptococcus sp. isolate showed the highest amylase activity. The growth rate and fermentative capacity in molasses medium was measured for three S. cerevisiae, T. delbrueckii and Candida sp. strains as tests for yield and performance in biomass production. Based on their excellent behaviour, three S. cerevisiae strains and one T. delbrueckii strain were selected for further analyses, including dehydration tolerance and invertase activity as additional desired traits for chicha starters. All the S. cerevisiae strains exhibited high invertase activity and one also displayed high resistance to dehydration. The yeasts selected in this study can thus be suitably used as dry starters for the microbiologically controlled production of traditional beverages, and also for other alcoholic fermentations.

Adaptive Laboratory Evolution of Native Torulaspora delbrueckii YCPUC10 With Enhanced Ethanol Resistance and Evaluation in Co-inoculated Fermentation.

Torulaspora delbrueckii is a yeast species typically present in the early stages of the fermentation process. T. delbrueckii positively modifies the aromatic properties of wines. However, its contribution to the final quality of the wine is restricted by its low tolerance to ethanol. T. delbrueckii is capable of fermenting and tolerating an ethanol concentration ranging from 7.4% (v/v) to slightly higher than 9% (v/v). For this reason, it cannot complete fermentation, when alcohol reach levels higher than 12% (v/v), limiting their use in the industry. The objective of this work was to obtain new variants of T. delbrueckii with improved resistance to ethanol through adaptive laboratory evolution. Variants capable of tolerating ethanol levels of 11.5% (v/v) were obtained. These presented improved kinetic parameters, and additionally showed an increase in resistance to SO2 in ethanol compared to the original strain. Co-inoculated fermentations were performed with the original strain (FTd/Sc) and with the evolved strain (FTdF/Sc), in addition to a control fermentation using only Saccharomyces cerevisiae EC1118 (FSc). The results obtained show that FTdF/Sc present higher levels of 2-Ethylhexanol, compared to FTd/Sc and FSc. Furthermore, FTdF/Sc presents higher levels of total alcohols, total aldehydes, total phenolic derivatives, and total sulfur compounds with significant differences with FSc. These results provide a T. delbrueckii YCPUC10-F yeast with higher resistance to ethanol, which can be present throughout the fermentation process and be used in co-inoculated fermentations. This would positively impact the performance of T. delbrueckii by allowing it to be present not only in the early stages of fermentation but to remain until the end of fermentation.

Selection of non-Saccharomyces yeasts to be used in grape musts with high alcoholic potential: a strategy to obtain wines with reduced ethanol content.

Ethanol content of wine has increased over the last decades as consequence of searching phenolic maturity, requiring increased grape maturity. This may result in the production of wines with excessive alcohol levels (sometimes more than 15% (v/v)), sluggish and stuck fermentations and excessive volatile acidity. Many strategies to reduce ethanol in wines are being studied, and microbial methods have some additional advantages. However, because of the broad intra- and interspecies variability, new selection criteria should be included. Therefore, the goal of the present work was to design and evaluate a simple and integral procedure for non-Saccharomyces yeast selection. This strategy allowed selection of yeasts that presented successful implantation in grape must with high alcohol potential and their use in co-cultures could reduce the ethanol in wines. A total of 114 native non-Saccharomyces yeasts were assayed to determine their respiratory, fermentative and physiological characteristics of enological interest. Hanseniaspora uvarum BHu9 and BHu11, H. osmophila BHo51, Starmerella bacillaris BSb55 and Candida membranaefaciens BCm71 were selected as candidates to design co-culture starters.

Enzymatic activities produced by mixed Saccharomyces and non-Saccharomyces cultures: relationship with wine volatile composition.

During certain wine fermentation processes, yeasts, and mainly non-Saccharomyces strains, produce and secrete enzymes such as ß-glucosidases, proteases, pectinases, xylanases and amylases. The effects of enzyme activity on the aromatic quality of wines during grape juice fermentation, using different co-inoculation strategies of non-Saccharomyces and Saccharomyces cerevisiae yeasts, were assessed in the current study. Three strains with appropriate enological performance and high enzymatic activities, BSc562 (S. cerevisiae), BDv566 (Debaryomyces vanrijiae) and BCs403 (Candida sake), were assayed in pure and mixed Saccharomyces/non-Saccharomyces cultures. ß-Glucosidase, pectinase, protease, xylanase and amylase activities were quantified during fermentations. The aromatic profile of pure and mixed cultures was determined at the end of each fermentation. In mixed cultures, non-Saccharomyces species were detected until day 4-5 of the fermentation process, and highest populations were observed in MSD2 (10% S. cerevisiae/90% D. vanrijiae) and MSC1 (1% S. cerevisiae/99% C. sake). According to correlation and multivariate analysis, MSD2 presented the highest concentrations of terpenes and higher alcohols which were associated with pectinase, amylase and xylanase activities. On the other hand, MSC1 high levels of ß-glucosidase, proteolytic and xylanolytic activities were correlated to esters and fatty acids. Our study contributes to a better understanding of the effect of enzymatic activities by yeasts on compound transformations that occur during wine fermentation.