Yeast Starter Cultures Research Articles

Simultaneous Alcoholic and Malolactic Fermentations by Saccharomyces cerevisiae and Oenococcus oeni Cells Co-immobilized in Alginate Beads.

Malolactic fermentation (MLF) usually takes place after the end of alcoholic fermentation (AF). However, the inoculation of lactic acid bacteria together with yeast starter cultures is a promising system to enhance the quality and safety of wine. In recent years, the use of immobilized cell systems has been investigated, with interesting results, for the production of different fermented foods and beverages. In this study we have carried out the simultaneous immobilization of Saccharomyces cerevisiae and Oenococcus oeni in alginate beads and used them in microvinifications tests to produce Negroamaro wine. The process was monitored by chemical and sensorial analyses and dominance of starters and cell leaking from beads were also checked. Co-immobilization of S. cerevisiae and O. oeni allowed to perform an efficient fermentation process, producing low volatile acidity levels and ethanol and glycerol concentrations comparable with those obtained by cell sequential inoculum and co-inoculum of yeast and bacteria cells in free form. More importantly, co-immobilization strategy produced a significant decrease of the time requested to complete AF and MLF. The immobilized cells could be efficiently reused for the wine fermentation at least three times without any apparent loss of cell metabolic activities. This integrated biocatalytic system is able to perform simultaneously AF and MLF, producing wines similar in organoleptic traits in comparison with wines fermented following traditional sequential AF and MLF with free cell starters. The immobilized-cell system, that we here describe for the first time in our knowledge, offers many advantages over conventional free cell fermentations, including: (i) elimination of non-productive cell growth phases; (ii) feasibility of continuous processing; (iii) re-use of the biocatalyst.

Whole Genome Comparison Reveals High Levels of Inbreeding and Strain Redundancy Across the Spectrum of Commercial Wine Strains ofSaccharomyces cerevisiae

Humans have been consuming wines for more than 7000 yr . For most of this time, fermentations were presumably performed by strains of Saccharomyces cerevisiae that naturally found their way into the fermenting must . In contrast, most commercial wines are now produced by inoculation with pure yeast monocultures, ensuring consistent, reliable and reproducible fermentations, and there are now hundreds of these yeast starter cultures commercially available. In order to thoroughly investigate the genetic diversity that has been captured by over 50 yr of commercial wine yeast development and domestication, whole genome sequencing has been performed on 212 strains of S. cerevisiae, including 119 commercial wine and brewing starter strains, and wine isolates from across seven decades. Comparative genomic analysis indicates that, despite their large numbers, commercial strains, and wine strains in general, are extremely similar genetically, possessing all of the hallmarks of a population bottle-neck, and high levels of inbreeding. In addition, many commercial strains from multiple suppliers are nearly genetically identical, suggesting that the limits of effective genetic variation within this genetically narrow group may be approaching saturation.

It's Gettin’ Hot in Here: Breeding Robust Yeast Starter Cultures for Cocoa Fermentation

Cocoa beans have to undergo post-harvest fermentation and drying to develop the typical 'cocoa flavor' associated with chocolate. Yeasts play a pivotal role during the fermentation but are generally outcompeted early in the process. Meersman and colleagues describe an elegant breeding-based approach to generate robust yeast starter cultures for cocoa fermentation.

Effect of Maize/Millet Malt Hydrolysis and Baker’s Yeast/Burukutu Starter Fermentation on the Yield of Ethanol from Dioscorea Spp (Yam)

The ethanol production ability of baker’s yeast (Saccharomyces cerevisiae) and burukutu starter culture were examined on yam starch hydrolyzed with malts of maize and millet. Four substrate treatments which include yam starch (as control), yam starch hydrolyzed with millet malt (B), yam starch hydrolyzed with maize malt (C) and yam starch hydrolyzed with a mixture both millet and maize malt (D) were prepared in duplicate and made up to 600 ml. Baker’s yeast (4.40 g) was inoculated on one set of four while burukutu starter was inoculated on the other set of four and fermented for a period of four days at a temperature of 26°C. The pH was observed to drop as fermentation period increased, the highest ethanol yield of up to 74.5 g/Kg was obtained from starch hydrolyzed with maize malt and inoculated with baker’s yeast. Yam is a potential source of industrial scale ethanol production, there was a significant difference (p<0.05) between maize and millet malts in ethanol yield but between baker’s yeast and burukutu starter culture fermentation there was no significant difference (p<0.05), the interaction between malts and the inoculums was significant (p<0.05). Ethanol yield strongly depends on the hydrolysis of yam starch into fermentable sugar.

The impact of yeast starter cultures on the microbial communities and volatile compounds in cocoa fermentation and the resulting sensory attributes of chocolate.

Theobroma cacao seeds are the main raw material for chocolate production. During their fermentation, a succession of microorganisms are responsible for the physicochemical changes occurring in the pulp and inside the beans. The aim of this study was to investigate the effects of yeast inoculation (Saccharomyces cerevisiae UFLA CA11, Pichia kluivery CCMA0237, and Hanseniaspora uvarum CCMA0236) on the profile of the volatile compounds and microbial communities in cocoa fermentation. The resulting chocolate was also evaluated by temporal dominance of sensations (TDS) analyses. The dominant microorganisms during spontaneous fermentation were S. cerevisiae, H. uvarum, H. guilliermondii, Lactobacillus fermentum, Pediococcus sp., and Acetobacter pasteurianus. Similarly, S. cerevisiae, P. kluyveri, Candida sp., Pediococcus sp., and A. pasteurianus were the predominant microorganisms assessed by Denaturing Gradient Gel Electrophoresis (DGGE) in inoculated fermentation. Sixty-seven volatile compounds were detected and quantified by gas chromatography/mass spectrometry (GC/MS) at the end of fermentation and chocolates. The main group of volatile compound found after the inoculated and spontaneous fermentations was esters (41 and 39%, respectively). In the chocolates, the main group was acids (73 and 44% from the inoculated and spontaneous fermentations, respectively). The TDS analyses showed a dominance of bitter and cocoa attributes in both chocolates. However, in the inoculated chocolate, lingering fruity notes were more intense, while the chocolate produced by spontaneous fermentation was more astringent. Thus, the inoculation of yeast influenced the microbial profile, which likely affected the volatile compounds that affect sensory characteristics, resulting in chocolate with dominant bitter, cocoa, and fruity attributes.

Production, reactivation and nutrient requirements of active dried yeast in winemaking: theory and practice

Wine yeast, from production to application, traverse an incredible range of environments. Central to their ability to not only survive but to thrive throughout this journey is their capacity to transform themselves in response to the varied conditions encountered. Wine yeast resilience alone, however, is insufficient. Producers and winemakers must be assiduous in their use of wine yeast with an understanding of yeast requirements throughout the process to achieve optimal results. Active dried yeast (ADY) starter cultures for initiating grape must fermentations are convenient, reliable and available for a wide range of strains suited to different wine types. These benefits cannot be fully realised without optimisation of: their production; reactivation of the dried product prior to inoculation; and appropriate nutrient management during fermentation. This review discusses current knowledge on production of Saccharomyces ADY for greatest biological stability and activity, and factors that affect their activity following rehydration and inoculation into grape must. The efficacy of various types of commercial additives used in reactivation of ADY and, winemaking interventions and nutrients that are used to optimise fermentation are also discussed.

Ongoing domestication of wine yeast: past, present and future

Yeast starter cultures for winemaking have traditionally been developed from successful fermentation isolates. Over the past 10–20 years there has been a move to harness genetic techniques to improve production strains. The approaches to achieve this have been similar to those applied in the agricultural sector for crop and livestock development. Thus, domestication of wine yeasts now involves hybridisation, mutagenesis, selection and screening. There is also a growing interest in tapping into isolates of non-Saccharomyces yeasts to develop novel starter cultures that bring complexity and potentially enhance regionality of wines. This review will cover these developments and will look to the future opportunities arising from the application of genetic engineering and synthetic biology approaches for the introduction of novel phenotypes that are outside of the reach of traditional methods.

Genomic expression program of Saccharomyces cerevisiae along a mixed-culture wine fermentation with Hanseniaspora guilliermondii.

BackgroundThe introduction of yeast starter cultures consisting in a blend of Saccharomyces cerevisiae and non-Saccharomyces yeast strains is emerging for production of wines with improved complexity of flavor. The rational use of this approach is, however, dependent on knowing the impact that co-inoculation has in the physiology of S. cerevisiae. In this work the transcriptome of S.cerevisiae was monitored throughout a wine fermentation, carried out in single culture or in a consortium with Hanseniasporaguilliermondii, this being the first time that this relevant yeast–yeast interaction is examined at a genomic scale.ResultsCo-inoculation with H. guilliermondii reduced the overall genome-wide transcriptional response of S. cerevisiae throughout the fermentation, which was attributable to a lower fermentative activity of S. cerevisiae while in the mixed-fermentation. Approximately 350 genes S. cerevisiae genes were found to be differently expressed (FDR < 0.05) in response to the presence of H. guilliermondii in the fermentation medium. Genes involved in biosynthesis of vitamins were enriched among those up-regulated in the mixed-culture fermentation, while genes related with the uptake and biosynthesis of amino acids were enriched among those more expressed in the single-culture. The differences in the aromatic profiles of wines obtained in the single and in the mixed-fermentations correlated with the differential expression of S. cerevisiae genes encoding enzymes required for formation of aroma compounds.ConclusionsBy integrating results obtained in the transcriptomic analysis performed with physiological data our study provided, for the first time, an integrated view into the adaptive responses of S. cerevisiae to the challenging environment of mixed culture fermentation. The availability of nutrients, in particular, of nitrogen and vitamins, stands out as a factor that may determine population dynamics, fermentative activity and by-product formation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0318-1) contains supplementary material, which is available to authorized users.

Production of freeze-dried yeast culture for the brewing of traditional sorghum beer, tchapalo.

Freeze-drying is a well-known dehydration method widely used to preserve microorganisms. In order to produce freeze-dried yeast starter culture for the brewing purpose of African sorghum beer, we tested protective agents (sucrose, glucose, glycerol) in combination with support materials (millet, maize, sorghum, and cassava flours) at 1:1 ratio (v/v). The yeast strains Saccharomyces cerevisiae F 12-7 and Candida tropicalis C 0-7 previously isolated from sorghum beer were used in a mixed culture at a ratio of 2:1 (C.tropicalis/S.cerevisiae). After the freeze-drying, the residual water contents were between 0.78 -2.27%, 0.55 -4.09%, and 0.40-2.61%, respectively, with sucrose, glucose and glycerol. The dried yeasts viabilities were between 4.0% and 10.6%. Among the protective agents used, sucrose was found to be the best protectant giving cell viabilities of 8.4-10.6%. Considering the support materials, millet flour was the best support after drying. When the freeze-dried yeast powders were stored at 4°C and room temperature (25-28°C) for up to 3months, the survival rates were the highest with cassava flour as the support material.

Breeding Strategy To Generate Robust Yeast Starter Cultures for Cocoa Pulp Fermentations.

Cocoa pulp fermentation is a spontaneous process during which the natural microbiota present at cocoa farms is allowed to ferment the pulp surrounding cocoa beans. Because such spontaneous fermentations are inconsistent and contribute to product variability, there is growing interest in a microbial starter culture that could be used to inoculate cocoa pulp fermentations. Previous studies have revealed that many different fungi are recovered from different batches of spontaneous cocoa pulp fermentations, whereas the variation in the prokaryotic microbiome is much more limited. In this study, therefore, we aimed to develop a suitable yeast starter culture that is able to outcompete wild contaminants and consistently produce high-quality chocolate. Starting from specifically selected Saccharomyces cerevisiae strains, we developed robust hybrids with characteristics that allow them to efficiently ferment cocoa pulp, including improved temperature tolerance and fermentation capacity. We conducted several laboratory and field trials to show that these new hybrids often outperform their parental strains and are able to dominate spontaneous pilot scale fermentations, which results in much more consistent microbial profiles. Moreover, analysis of the resulting chocolate showed that some of the cocoa batches that were fermented with specific starter cultures yielded superior chocolate. Taken together, these results describe the development of robust yeast starter cultures for cocoa pulp fermentations that can contribute to improving the consistency and quality of commercial chocolate production.

Part I: the influence of serial repitching ofSaccharomyces pastorianuson the uptake dynamics of metal ions and fermentable carbohydrates during the fermentation of barley and gluten-free buckwheat and quinoa wort

Recently, research has been focusing on the use of alternative raw materials for brewing purposes and gluten-free beer-like beverages from malted buckwheat and quinoa are of commercial interest. A common commercial process involves the serial repitching of the yeast biomass, but this has not been described using buckwheat and quinoa wort fermentations. Our research studies (Parts I–III) explored the serial repitching of the yeast strain Saccharomyces pastorianus TUM 34/70 on the composition of a barley, buckwheat and quinoa fermentation medium. The present paper focuses on the fermentation performance and the uptake dynamics of metal ions and fermentable carbohydrates. Both pseudocereals showed high variations in all of the attributes examined during successive fermentations. In buckwheat the differences between successive fermentations were similar to those observed with barley, whereas differences in quinoa varied quite significantly from those observed with barley and showed a directional trend, suggesting a general weakening of the yeast from the sixth successive fermentation onward. In particular, the assimilation of the fermentable carbohydrates lessened and metal ion uptake appeared poorly controlled. It was concluded that buckwheat showed good potential for serial repitching of S. pastorianus TUM 34/70, whereas serial repitching of a quinoa wort appeared to be limited to five or six fermentations. Copyright © 2015 The Institute of Brewing & Distilling

Application of ISSR-PCR for rapid strain typing of Debaryomyces hansenii isolated from dry-cured Iberian ham

Yeast populations of dry-cured Iberian ham isolated from seven industries in the province of Badajoz were characterized by ISSR-PCR using the (CAG)4 primer and PCR-RFLP of the ITS1-5.8S rRNA-ITS2 fragment, and identified by DNA sequencing. A total of 242 isolates were analyzed, indicating the primary species present was Debaryomyces hansenii at 80.9% of the isolates followed by Candida zeylanoides at 10.3% of the isolates. The remainders of isolates were identified as Yamadazyma triangularis, Sporobolomyces roseus, Meyerozyma guilliermondii, Rhodotorula slooffiae, and Cryptococcus victoriae. The ISSR-PCR method was a fast and reliable method which was able to discriminate species at a level comparable to restriction analyses of the ITS1-5.8S rRNA-ITS2 region. This method allowed for strain typing of D. hansenii, yielding 29 different PCR patterns within 196 isolates. Moreover, ISSR-PCR using the (CAG)4 primer indicated that this technique could be a promising tool for rapid discrimination of yeast starter cultures and spoilage species in dry-cured Iberian ham.

Dynamics of Cocoa Bean Pulp Degradation during Cocoa Bean Fermentation: Effects of Yeast Starter Culture Addition

Fermentation is a crucial step in the post-harvest processing of cocoa beans. This process comprises mixed culture microbi al activities on the cocoa bean pulp, producing metabolites that act as import ant precursors for cocoa flavour development. Variations in the microbial po pulation dynamics during the fermentation process may induce changes in the overall process. Thus, the introduction of a specific microbial starter cultur e may improve the quality of the fermentation. This article discusses the effects of the addition of Saccharomyces cerevisae var. Chevalieri starter culture on cocoa bean ferm entation. The dynamics in the yeast concentration, sugary pulp co mpounds and metabolic products were measured during fermentation. The alterations in the dynamic metabolite profile were significant, although only a slight difference was observed in the yeast population. A higher fermenta tion index was measured for the cocoa bean fermentation with yeast starter cult ure, 1.13 compared to 0.84. In conclusion, this method can potentially be applied to shorten the cocoa bean fermentation time.

Optimization of carbon and nitrogen medium components for biomass production using non-Saccharomyceswine yeasts

The impact of different nitrogen and carbon sources on biomass production of the non-Saccharomyces wine yeast species Lachancea thermotolerans, Metschnikowia pulcherrima and Issatchenkia orientalis was assessed. Using a molasses-based medium, yeast extract and corn steep liquor as well as ammonium sulphate and di-ammonium phosphate (DAP) as nitrogen sources were compared in shake-flask cultures. A medium with 20 g l⁻¹ sugar (diluted molasses) and 500 mg l⁻¹ total yeast assimilable nitrogen, from yeast extract, gave the highest biomass concentrations and yields. Invertase pretreatment was required for cultures of M. pulcherrima and I. orientalis, and respective biomass yields of 0.7 and 0.8 g g⁻¹ were achieved in aerobic bioreactor cultures. The absence of ethanol production suggested Crabtree-negative behaviour by these yeasts, whereas Crabtree-positive behaviour by L. thermotolerans resulted in ethanol and biomass concentrations of 5.5 and 11.1 g l⁻¹, respectively. Recent studies demonstrate that non-Saccharomyces yeasts confer positive attributes to the final composition of wine. However, optimal process conditions for their biomass production have not been described, thereby limiting commercial application. In this study, industrial media and methods of yeast cultivation were investigated to develop protocols for biomass production of non-Saccharomyces yeast starter cultures for the wine industry.

Characterization of yeast starter cultures used in household alcoholic beverage preparation by a few ethnic communities of Northeast India

In this study, we report the characterization of traditional household starter cultures from a few ethnic groups of northeast India. Pure cultures obtained from the study have been deposited in the Microbial Type Culture Collection (MTCC), India. These isolates have been analyzed for their growth characteristics, sensitivity to temperature and pH, alcohol tolerance, alcohol production, and alcohol dehydrogenase (ADH) content. The pure cultures obtained from different starter cultures revealed the presence of Debaryomyces, Wickerhamomyces, and Candida along with the fermenting yeast Saccharomyces. The growth behavior at different temperatures, pH and alcohol tolerance revealed numerous facts and behavior of the yeast strains associated with traditional alcoholic fermentation. All the isolates were found to be thermotolerant up to 37 °C, fairly pH-resistant, good in ADH secretion, and with appreciable alcohol production. For all the strains studied, the Saccharomyces cerevisiae MTCC 3976 strain from the Tea Tribes of Assam and the Wickerhamomyces anomalus MTCC 3979 from the Apatani Tribe of Arunachal Pradesh were found to be exceptional in terms of thermotolerance, alcohol tolerance, alcohol production, and ADH activity, and hence may be identified as potential strains for industrial fermentation.

Effect of Lactic Acid Bacteria and Yeast Starter Cultures on the Soaking Time and Quality of “Ofada” Rice

Freshly harvested paddy rice was randomly obtained from three different farms in “Ofada” town, Ogun State, Nigeria and processed according to the traditional parboiling method. The rice was inoculated singly with cultures of Lactobacillus amylophilus, Leuconostoc mesenteroides, Saccharomyces uvarum and Saccharomyces cerevisiae which were isolated from the soak-water. In order to assess the effect of starter cultures on the soaking time of the rice, the pH and titratable acidity were determined at 12 h interval. The chemical composition and the sensory quality of the rice were also determined at the end of the soaking period while uninoculated rice served as control. The chemical composition of the rice as well as the pH and TTA of the soak-water were significantly (p < 0.05) different from the control while the processing time was shortened to 48 h. The rice inoculated with Saccharomyces uvarum and Saccharomyces cerevisae had the highest protein content and best sensory attributes.

Influence of specific fermentation conditions on natural microflora of pomace in “Grappa” production

As reported in the European Community regulation, grappa is a spirit beverage made in Italy from marc that has been steam distilled or distilled after the addition of water. Grape marc from red grapes has already undergone alcoholic fermentation with the must and can be distilled immediately. Grape marc from white grapes does not contain ethanol but contains sugars that are fermented by spontaneous anaerobic fermentation during a storage period. The characteristic aroma of grappa consists of a large number of volatile compounds, which arise from various sources, the most important of which is yeast. Very few studies have been undertaken to characterize the natural populations of yeast during the fermentation of grape marc. The goal of this study was to understand how different pHs, temperatures and yeast starter cultures affect the growth and dynamics of yeast species involved in pomace fermentation, which could be the basis for improving the final quality of grappa production. We found that a temperature of 15°C has the greatest effect on improving the quality of the product. Unfortunately, due to the solid state of the grape marc and the impossibility of its mixing, it appears that acidification and the addition of yeast starter cultures during the silage period are not effective.

TLR ligands of Lactobacillus sakei LK-117 isolated from seed mash for brewing sake are potent inducers of IL-12

Many studies have investigated the immunostimulatory effects of bacteria, such as the anti-allergic effects of lactic acid bacteria (LAB) and LAB-fermented milk. Importantly, these anti-allergic effects have been observed for both viable and nonviable bacteria. However, there are no reported immunological effects of LAB isolated from kimoto, the traditional yeast starter culture used for brewing sake, which also involves spontaneous lactate fermentation. In this study, we determined whether the Leuconostoc mesenteroides and Lactobacillus sakei bacterial strains obtained from kimoto affected the production of interleukin-12 (IL-12), an inducer of the T-helper type-1 immune response. By incubating autoclaved bacteria with J774.1 macrophage-like cells, we found that L. sakei LK-117 induced a sustained increase in IL-12p40 production. The IL-12-inducing ability of LK-117 was unaffected by anti-TLR2 neutralization and was entirely inhibited when the LK-117 cells were treated with RNase. When LK-117 cells were treated with M-1, an N-acetylmuramidase, at varying concentrations and for different periods of time, the ability of the bacteria to induce IL-12 decreased quickly. Although an active fraction could be prepared by chromatography from the soluble products of enzymolysis, the fraction's induction ability was <2% of that of intact organisms, and induction ability disappeared completely upon anti-TLR2 neutralization after treating the active fraction with RNase. These results suggest that single-stranded RNA released from cells that were disrupted by autoclaving might act as a TLR ligand and provide a novel mechanism in which heat-killed LAB could be used to regulate immune activity.

Optimizing the selection process of yeast starter cultures by preselecting strains dominating spontaneous fermentations

We propose an efficient and time-saving strategy for starter culture selection. Our approach is based on the accomplishment of 3 phases: (i) the selection of yeast strains dominating spontaneous fermentations, (ii) the selection among the dominant strains of those showing the best technological characteristics, and (iii) the final selection among good technological strains of those showing the desired qualitative traits. We applied this approach to wine fermentations, even though the same strategy has the potential to be employed for the selection of any type of starter culture. We isolated and identified yeast strains at the mid- and final stages of 6 spontaneous fermentations carried out in 3 different Spanish wineries. We identified all strains as Saccharomyces cerevisiae by restriction fragment length polymorphism of the ribosomal DNA internal transcribed spacer region, and subsequently distinguished each strain by analyzing the polymorphism of the inter-delta regions. Strains that were detected both at the mid- and final stages of the fermentation were considered dominant. Four dominant strains were finally selected and tested in pilot-scale fermentation, and their performance was compared with that of a commercial wine strain. All dominant strains showed good fitness and resulted suitable to be employed as starter cultures. One of the dominant strains isolated in this study is currently commercialized.

Identification of yeast population dynamics of spontaneous fermentation in Beijing wine region, China

The aim of this study was (i) to investigate changes occurring in the yeast population profile during spontaneous fermentation of grape juice; (ii) to assess the proliferation of commercial yeast starter culture strains in vineyards; and (iii) to identify indigenous wine strains for future development of starter strains that better reflect the yeast biodiversity of China’s grape-growing regions. To achieve this, yeasts were isolated at four different stages during fermentation of both hand-pressed and winery-sourced must samples ofVitis vinifera L. cv. Roussanne and Merlot. A total of 1600 yeast colonies were isolated and then grouped according to macroscopic and microscopic characteristics. A selection of 291 colonies from the different groups was subjected to species identification using the internal transcribed spacer regions of the 5.8S rRNA gene (ITS1-5.8S-ITS2 region) and the inter-delta (δ) sequence of the 26S rRNA D1/D2 region. In addition, 104Saccharomyces cerevisiae colonies were subjected to strain identification. Twelve species belonging to nine different genera were found amongst the isolates. During the early stages of fermentation, it was found thatHanseniaspora uvarum andCandida stellata numerically dominated the four to six yeast species present, including a region-specific yeast,Sporobolomyces beijingensis. TwoS. cerevisiae strains were isolated from the final stage of fermentation. These two indigenous strains, which were found to be different from the nine commercial yeast strains previously used as starter cultures in this particular Beijing-based winery, might possess potentially important region-specific oenological characteristics. This study provides the first essential step towards the preservation and exploitation of the hidden oenological potential of the untapped wealth of yeast biodiversity in China’s wine-producing regions.