Optimization of honey-must preparation and alcoholic fermentation by Saccharomyces cerevisiae for mead production

Mead fermentation is a time-consuming process, often taking several months to complete. Despite of the use of starter cultures several problems still persist such as lack of uniformity of the final products, slow or premature fermentation arrest and the production of off-flavors by yeast. Thus the aim of this study was to optimize mead production through the use of an appropriate honey-must formulation to improve yeast performance alcoholic fermentation and thereby obtain a high quality product. Honey-must was centrifuged to reduce insoluble solids, pasteurized at 65 °C for 10 min, and then subjected to different conditions: nitrogen supplementation and addition of organic acids. Although the addition of diammonium phosphate (DAP) reduced fermentation length, it did not guarantee the completeness of the fermentation process, suggesting that other factors could account for the reduced yeast activity in honey-must fermentations. Sixteen yeast-derived aroma compounds which contribute to the sensorial quality of mead were identified and quantified. Global analysis of aromatic profiles revealed that the total concentration of aroma compounds in meads was higher in those fermentations where DAP was added. A positive correlation between nitrogen availability and the levels of ethyl and acetate esters, associated to the fruity character of fermented beverages, was observed whereas the presence of potassium tartrate and malic acid decreased, in general, their concentration.This study provides very useful information that can be used for improving mead quality.     

Effects of Saccharomyces cerevisiae culture and Saccharomyces cerevisiae live cells on in vitro mixed ruminal microorganism fermentation

The objective of this study was to examine the effects of a Saccharomyces cerevisiae live cell product and a S. cerevisiae culture product on the in vitro mixed ruminal microorganism fermentation of ground corn, soluble starch, alfalfa hay, and Coastal bermudagrass hay. In the presence of ground corn, neither concentration (0.35 or 0.73 g/L) of S. cerevisiae culture nor live cells had any effect on final pH, H2, CH4, propionate, or butyrate. The S. cerevisiae culture had no effect on acetate, but both concentrations of S. cerevisiae live cells decreased acetate and the acetate:propionate ratio. When soluble starch was the substrate, both concentrations of S. cerevisiae live cells and 0.73 g/L of S. cerevisiae culture decreased the acetate:propionate ratio. Although the treatment effects were not statistically significant, both concentrations of live cells and 0.73 g/L of the culture decreased lactate concentrations compared with the control incubations. When alfalfa hay served as the substrate, neither the S. cerevisiae culture nor the live cells had an effect on propionate, butyrate, or the acetate:propionate ratio. Both concentrations of S. cerevisiae culture decreased the final pH and in vitro dry matter disappearance, and the 0.73 g/L treatment decreased the amount of acetate. However, both treatments of S. cerevisiae live cells increased final pH and decreased acetate and in vitro dry matter disappearance. Neither yeast treatment had much effect on the Coastal bermudagrass hay fermentations. In general, both S. cerevisiae supplements seemed to have similar effects on the mixed ruminal microorganism fermentation.     

酒酒球菌和酿酒酵母共接种发酵动力学模型建立

为研究酿酒酵母(Saccharomyces cerevisiae)和酒酒球菌(Oenococcus oeni)共发酵过程中菌体生长、底物消耗和产物生成的动力学变化,选取本土O.oeni ZX-1、MG-1分别与S.cerevisiae VW、AW同时接种,每隔24 h测定发酵体系内S.cerevisiae活菌数、还原糖含量、乙醇体积分数、O.oeni活菌数和L-苹果酸含量,采用经典Logistic、Boltzmann、SGompertz和DoseResp模型对测定值进行非线性拟合。结果表明,Boltzmann模型可以较好地反映S.cerevisiae生长和乙醇体积分数变化;Logistic模型对还原糖含量变化拟合效果最佳;4个处理组的O.oeni生长和L-苹果酸含量变化动力学模型中,Boltzmann模型和DoseResp模型拟合系数R²一致,均能较好预测共发酵过程中O.oeni生长和L-苹果酸含量的变化趋势。综合分析,供试本土O.oeni分别与S.cerevisiae VW、AW共接种均可顺利完成发酵,建立的动力学模型可为本土菌株的工业化酿酒工艺优化控制提供数据...     

Development of a multilocus variable number of tandem repeat typing method for Oenococcus oeni

Oenococcus oeni is responsible for the malolactic fermentation of wine. Genomic diversity has already been established in this species. In addition, winemakers usually report varying starter culture efficiency. The monitoring of indigenous and selected strains is essential for understanding strain survival and implantation during the winemaking process. In this study, we report the development of the first typing scheme for O. oeni using multiple-locus variable number of tandem repeat analysis (VNTR). The discriminatory power of 14 out of 44 tandem repeat loci in the genome of the PSU-1 strain was initially evaluated with a test collection of 18 genotypically distinct starter strains. Then five VNTR loci, which can be easily scored with the technology used here, were identified and used to genotype a collection of 236 strains, previously classified by restriction endonuclease analysis-pulsed-field gel electrophoresis (REA-PFGE) and multilocus sequence typing (MLST) into 136 REA-PFGE types or 110 MLST types. The discriminatory power of VNTR (as determined by Simpson's index of discrimination) was higher than that of the other two methods, with 201 VNTR types. The targeted VNTR markers were found to be stable and did not change for the clones of the same strain deposited in a collection at intervals of several years. Strains isolated from the different wine producing areas or the products were assigned to phylogenetic groups and were statistically linked with the VNTR profiles. Another interesting observation was that the loci were found in sequences homologous to regions encoding for membrane-anchored proteins.     

RNA fingerprinting analysis of Oenococcus oeni strains under wine conditions

Oenococcus oeni is a lactic acid bacterium of economic interest used in winemaking. This bacterium is the preferred species for malolactic fermentation (MLF) due its adaptability to the chemically harsh wine environment. MLF enhances the organoleptic properties and ensures deacidification of wines.     

Antimicrobial activity of pediocin PA-1 against Oenococcus oeni and other wine bacteria

Pediocin PA-1 is an antimicrobial peptide produced by lactic acid bacteria (LAB) that has been sufficiently well characterised to be used in food industry as a biopreservative. Sulphur dioxide is the traditional antimicrobial agent used during the winemaking process to control bacterial growth and wine spoilage. In this study, we describe the effect of pediocin PA-1 alone and in combination with sulphur dioxide and ethanol on the growth of a collection of 53 oenological LAB, 18 acetic acid bacteria and 16 yeast strains; in addition, production of pediocin PA-1 by Pediococcus acidilactici J347-29 in presence of ethanol and grape must is also reported. Inhibitory concentrations (IC) and minimal bactericide concentrations of pediocin PA-1 were determined against LAB, and revealed a bacteriostatic effect. Oenococcus oeni resulted more sensitive to pediocin PA-1 (IC₅₀ = 19 ng/ml) than the other LAB species (IC₅₀ = 312 ng/ml). Cooperative inhibitory effects of pediocin PA-1 and either sulphur dioxide or ethanol were observed on LAB growth. Moreover, the pediocin PA-1 producing P. acidilactici strain J347-29 was able to grow and produce the bacteriocin in presence of ethanol (up to 4% ethanol in the fermentation broth) and grape must (up to 80%), which indicated that pediocin PA-1 can be considered as a potential biopreservative in winemaking.     

Influence of freeze-drying conditions on survival of Oenococcus oeni for malolactic fermentation

Malolactic fermentation (MLF) is an important process in wine production. Oenococcus oeni is most often responsible for MLF. Starter culture technology, involving the inoculation of O. oeni into wines, has been developed for inducing MLF. In this study, the effects of cell washing, pH of suspension medium, preincubation in sodium glutamate, initial cell concentration and freezing temperature on viability of freeze-dried O. oeni H-2 were investigated. The cell viability of samples without washing with potassium phosphate buffer was significantly lower than those samples undergone washing. When pH of suspension medium was 7.0 the cell survival was the highest. The cell viability was enhanced when the cells were preincubated at 25 °C before freezing. When 2.5% sodium glutamate was used as protective agent in suspension medium, the optimal initial cell concentration was 10⁹ CFU/ml. The cell viability increased by 21.6% as freezing temperature decreased from − 20 °C to − 65 °C. However, when the cells were frozen in liquid nitrogen (− 196 °C), the cell survival significantly decreased.     

Intraspecific genetic diversity of lactic acid bacteria from malolactic fermentation of Cencibel wines as derived from combined analysis of RAPD-PCR and PFGE patterns

Three molecular techniques, randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), pulsed field gel electrophoresis (PFGE) and differential display-polymerase chain reaction (DD-PCR) have been used to assess the intraspecific diversity of the lactic microbiota responsible for spontaneous malolactic fermentation (MLF) in Cencibel wines made at a cellar in Castilla-La Mancha (Spain). The results indicate that RAPD-PCR and PFGE are of value in typing this microbiota. Better discrimination was achieved by RAPD-PCR. Reproducibility using DD-PCR was not good, which makes this method unsuitable. Combined numerical analysis of the patterns obtained from RAPD-PCR and PFGE allowed a better discrimination; this would therefore be a suitable tool to discriminate the diversity of bacterial communities like those found in MLF of wines. Genetic diversity data from combined numerical analysis suggest that there is considerable microbial diversity within MLF of Cencibel wines, with some genotypes coinciding in the two vinifications analysed.     

Hydrolysis of glycosidically bound flavour compounds from oak wood by Oenococcus oeni

Malolactic fermentation (MLF), which is conducted by lactic acid bacteria (LAB), has a significant influence on the stability and organoleptic quality of wine. Recent studies have shown that when MLF is carried out in oak wood barrels, LAB were also able to interact with wood and increase volatile compound contents such as vanillin during MLF. The release of these compounds indicates that LAB may convert vanillin precursors present in oak wood. In this work, the effect of commercial glycosidases on the released vanillin was firstly studied. This aldehyde is present in wood extracts in monoglycosidic forms where the major glycones are arabinose and xylose. Other aglycons released during MLF in barrels, syringaldehyde and whisky-lactones, can be considered as other sources of aroma. Secondly, strains selected with high activities toward glycoside substrates could hydrolyse vanillin glycoside precursors from oak wood with the same efficiency as commercial enzymes.     

Expanding the diversity of oenococcal bacteriophages: Insights into a novel group based on the integrase sequence

Temperate bacteriophages are a contributor of the genetic diversity in the lactic acid bacterium Oenococcus oeni. We used a classification scheme for oenococcal prophages based on integrase gene polymorphism, to analyze a collection of Oenococcus strains mostly isolated in the area of Bordeaux, which represented the major lineages identified through MLST schemes in the species. Genome sequences of oenococcal prophages were clustered into four integrase groups (A to D) which were related to the chromosomal integration site. The prevalence of each group was determined and we could show that members of the int_B- and int_C-prophage groups were rare in our panel of strains. Our study focused on the so far uncharacterized members of the int_D-group. Various int_D viruses could be easily isolated from wine samples, while int_D lysogens could be induced to produce phages active against two permissive O. oeni isolates. These data support the role of this prophage group in the biology of O. oeni. Global alignment of three relevant int_D-prophages revealed significant conservation and highlighted a number of unique ORFs that may contribute to phage and lysogen fitness.     

Evidence of mixed wild populations of Oenococcus oeni strains during wine spontaneous malolactic fermentations

Two hundred and four bacterial isolates from Rioja red wines undergoing spontaneous malolactic fermentation (MLF) were studied. Bacterial species was determined both by microbiological identification methods and by specific PCR analysis. Oenococcus oeni was shown to be the predominant species (98.5% of total isolates). Pulsed field gel electrophoresis (PFGE) of chromosomal DNA digested with SfiI was used to differentiate individual strains of O. oeni. A wide variety of restriction digest patterns were detected, which indicated a rich biodiversity of indigenous strains. Most fermentations (37 out of 41) showed from 2 to 6 clones growing in the same tank. Five O. oeni strains were the most frequently found, appearing in more than three of the 13 studied wineries, and most times in combination with other less frequently found strains. PFGE was shown to be a suitable method for strain differentiation, for monitoring individual strains and determining which strains actually survive and carry out MLF. A high genotypic heterogeneity of wild O. oeni strains was demonstrated and 90% of the studied wines showed mixed populations of O. oeni strains during MLF.     

Influence of pH and ethanol on malolactic fermentation and volatile aroma compound composition in white wines

The present study investigated the influences of pH and ethanol on malolactic fermentation (MLF) and the volatile aroma profile of the subsequent white wines from Riesling and Chardonnay inoculated with two different Oenococcus oeni strains. In all cases MLF was induced after completion of alcoholic fermentation (AF). Partial MLF occurred under low pH 3.2 and high alcohol (118.3 g/L) conditions. In the cases with complete MLF, the time required for each strain varied from 13 to 61 days and was dependent on bacterial culture, cultivar and wine parameter. Chemical properties of each wine were determined after AF, complete and partial MLF. The wines showed significant differences in total higher alcohols, esters and acids that are important for the sensory profile and quality of wine. This work demonstrated that the wine matrix as well as the pH and alcohol concentration affects MLF and the final volatile aroma profile. Results indicate that changes in volatile aroma composition are not necessarily related to complete MLF and that partial MLF already has distinct influences on the wine aroma profile of white wines.     

Inducing malolactic fermentation in Chardonnay musts and wines using different strains of Oenococcus oeni

Malolactic fermentations (MLF) were induced in a commercially prepared Washington State Chardonnay must to evaluate the influence of timing of inoculation and pre-culture conditions of Oenococcus oeni strains MCW, EQ-54, and WS-8. The must (pH 3.62, 21.5°Brix) was divided into lots and inoculated with Saccharomyces cerevisiae strain CY3079. Strains of O. oeni were pre-cultured by growing in diluted juice or by re-hydration of freeze-dried strains. Bacteria were inoculated into the musts before (Day 0) or after completion of the alcoholic fermentation (Day 22). Yeast populations exceeded 10⁷cfu/mL in all fermentations that proceeded to dryness. However, the viability of most strains of O. oeni quickly declined after inoculation regardless of the timing of inoculation or the strain used. MLF was induced in the wines inoculated with strains EQ-54 and WS-8 but not with MCW, and the rate depended on the time of inoculation. The method used to prepare bacterial starter cultures had no apparent influence on the completion of MLF. Values for volatile acidity were slightly higher (P< 0.05) in wines inoculated with O. oeni before alcoholic fermentation compared with those inoculated after alcoholic fermentation.     

Acetic acid removal by Saccharomyces cerevisiae during fermentation in oenological conditions. Metabolic consequences

The commercial Saccharomyces cerevisiae strains used in champagne winemaking were tested for their ability to metabolise acetic acid during alcoholic fermentation. Fermentation tests were performed in conditions close to oenological ones using a Chardonnay grape juice supplemented with acetic acid. The amount of acetic acid metabolised by wine yeast increased with increasing initial acetic acid concentration and this elimination occurred during the second part of the exponential growth phase. When the initial acetic acid concentration exceeds 1 g/l, and whatever the yeast strain used, the concentration of acetic acid in the resulting wine cannot be reduced to an acceptable level according to the current legislation. Acetic acid removal modified yeast metabolism, since more acetaldehyde, less glycerol and less succinic acid were produced. Considering the reduction of the NADPH/NADP⁺ ratio following acetic acid consumption, we propose, as a new hypothesis, that acetic acid could modify yeast metabolism by reducing the activity of the NADP⁺ dependent aldehyde dehydrogenase Ald6p.     

Ethanol yield and volatile compound content in fermentation of agave must by Kluyveromyces marxianus UMPe-1 comparing with Saccharomyces cerevisiae baker's yeast used in tequila production

In tequila production, fermentation is an important step. Fermentation determines the ethanol productivity and organoleptic properties of the beverage. In this study, a yeast isolated from native residual agave must was identified as Kluyveromyces marxianus UMPe-1 by 26S rRNA sequencing. This yeast was compared with the baker's yeast Saccharomyces cerevisiae Pan1. Our findings demonstrate that the UMPe-1 yeast was able to support the sugar content of agave must and glucose up to 22% (w/v) and tolerated 10% (v/v) ethanol concentration in the medium with 50% cells survival. Pilot and industrial fermentation of agave must tests showed that the K. marxianus UMPe-1 yeast produced ethanol with yields of 94% and 96% with respect to fermentable sugar content (glucose and fructose, constituting 98%). The S. cerevisiae Pan1 baker's yeast, however, which is commonly used in some tequila factories, showed 76% and 70% yield. At the industrial level, UMPe-1 yeast shows a maximum velocity of fermentable sugar consumption of 2.27g·L(-1)·h(-1) and ethanol production of 1.38g·L(-1)·h(-1), providing 58.78g ethanol·L(-1) at 72h fermentation, which corresponds to 96% yield. In addition, the major and minor volatile compounds in the tequila beverage obtained from UMPe-1 yeast were increased. Importantly, 29 volatile compounds were identified, while the beverage obtained from Pan1-yeast contained fewer compounds and in lower concentrations. The results suggest that the K. marxianus UMPe-1 is a suitable yeast for agave must fermentation, showing high ethanol productivity and increased volatile compound content comparing with a S. cerevisiae baker's yeast used in tequila production.     

Influence of assimilable nitrogen on volatile acidity production by Saccharomyces cerevisiae during high sugar fermentation

We analyzed the variability of volatile acidity and glycerol production by Saccharomyces cerevisiae on a large sample of high sugar musts. The production of volatile acidity was inversely correlated with the maximum cell population and the assimilable nitrogen concentration. The higher the nitrogen concentration, the less volatile acidity was produced. An approach to minimize volatile acidity production during high sugar fermentations by adjustment of assimilable nitrogen in musts was investigated in terms of both quantity and addition time. It was found that the optimal nitrogen concentration in the must is 190 mgN.l(-1). The best moment for nitrogen addition was at the beginning of fermentation. Addition at the end of the growth phase had less effect on volatile acidity reduction. We suggest that by stimulating cell growth, nitrogen addition provides NADH in the redox-equilibrating process, which in turn reduces volatile acidity formation.     

Bioethanol production from rice straw by a sequential use of Saccharomyces cerevisiae and Pichia stipitis with heat inactivation of Saccharomyces cerevisiae cells prior to xylose fermentation

In order to establish an efficient bioethanol production system from rice straw, a new strategy to ferment the mixture of glucose and xylose by a sequential application of Saccharomyces cerevisiae and Pichia stipitis was developed, in which heat inactivation of S. cerevisiae cells before addition of P. stipitis was employed. The results showed that heating at 50°C for 6h was sufficient to give high xylose fermentation efficiency. By application of the inactivation process, 85% of the theoretical yield was achieved in the fermentation of the synthetic medium. At the same time, the xylitol production was reduced by 42.4% of the control process. In the simultaneous saccharification and fermentation of the lime-pretreated and CO(2)-neutralized rice straw, the inactivation of S. cerevisiae cells enabled the full conversion of glucose and xylose within 80 h. Finally, 21.1g/l of ethanol was produced from 10% (w/w) of pretreated rice straw and the ethanol yield of rice straw reached 72.5% of the theoretical yield. This process is expected to be useful for the ethanol production from lignocellulosic materials in the regions where large-scale application of recombinant microorganisms was restricted.     

Genetic diversity of Oenoccoccus oeni isolated from wines treated with phenolic extracts as antimicrobial agents

Molecular techniques have been applied to study the evolution of wine-associated lactic acid bacteria from red wines produced in the absence and presence of antimicrobial phenolic extracts, eucalyptus leaves and almond skins, and to genetically characterize representative Oenococcus oeni strains. Monitoring microbial populations by PCR-DGGE targeting the rpoB gene revealed that O. oeni was, as expected, the species responsible for malolactic fermentation (MLF). Representative strains from both extract-treated and not-treated wines were isolated and all were identified as O. oeni species, by 16S rRNA sequencing. Typing of isolated O. oeni strains based on the mutation of the rpoB gene suggested a more favorable adaptation of L strains (n = 63) than H strains (n = 3) to MLF. Moreover, PFGE analysis of the isolated O. oeni strains revealed 27 different genetic profiles, which indicates a rich biodiversity of indigenous O. oeni species in the winery. Finally, a higher number of genetic markers were shown in the genome of strains from control wines than strains from wines elaborated with phenolic extracts. These results provide a basis for further investigation of the molecular and evolutionary mechanisms leading to the prevalence of O. oeni in wines treated with polyphenols as inhibitor compounds.