Sour rot-damaged grapes are sources of wine spoilage yeasts

Yeast species of sound and sour rot-damaged grapes were analysed during fermentation and grape ripening in the vineyard, using general and selective culture media. During 2003 and 2004 vintages, microvinifications were carried out with sound grapes to which different amounts of grapes with sour rot were added. The wine spoilage species Zygosaccharomyces bailii was only recovered during fermentations with sour rot, reaching 5.00 log CFU mL(-1) (2003) and 2.48 log CFU mL(-1) (2004) at the end of fermentation. The study of yeast populations during the sour rot ripening process (2005 vintage) showed that the veraison-damaged grapes always exhibited higher total yeast counts and a much greater diversity of species. From a total of 22 ascomycetous species, 17 were present only in damaged grapes. The most frequent species were Issatchenkia occidentalis and Zygoascus hellenicus. The spoilage species Z. bailii and Zygosaccharomyces bisporus were consistently isolated exclusively from damaged grapes. This work demonstrates that one of the most dangerous wine spoilage species, Z. bailii, is strongly associated with sour rot grapes and survives during fermentation with Saccharomyces cerevisiae. The use of selective media provides a more accurate characterization of grape contamination species.     

田间施药对自然发酵葡萄酒酵母菌群落结构的影响

【背景】酵母菌是葡萄酒发酵过程中一类非常重要的微生物,其多样性及群体组成对葡萄酒的质量有重要贡献。影响葡萄酒中酵母菌组成的因素有很多,但目前尚未见葡萄园田管理对葡萄酒酵母菌群落结构影响方面的报道。【目的】探索田间施药对自然发酵葡萄酒酵母菌群落结构的影响。【方法】采用分离培养、常规分子生物学鉴定和Illumina MiSeq宏基因组测序结合的方法分析不同样品中的酵母菌群落结构情况。【结果】从不使用内吸收型化学农药的葡萄样品自然发酵液中分离鉴定出Pichia、Hanseniaspora、Schizosaccharomyces、Candida、Saccharomyces、Zygoascus、Issatchenkia等7个属8个种的酵母菌,宏基因组测序结果表明有Pichia(29.42%)、Saccharomyces(21.91%)、Issatchenkia(17.99%)、 Hanseniaspora(12.10%)、 Candida(7.47%)、 Zygosaccharomyces(5.32%)、Schizosaccharomyces (3.07%)、Aureobasidium (0.29%)等属的酵母菌参与发酵;使用常规化学农药的葡萄样品自然发酵液中分离鉴定出Pichia、Hanseniaspora、Schizosaccharomyces、Candida、Cryptococcus等5个属6个种的酵母菌,宏基因组测序结果表明有Pichia (41.66%)、Hanseniaspora (21.54%)、Candida(19.11%)、 Zygosaccharomyces(7.78%)、 Schizosaccharomyces(4.04%)、 Cryptococcus(3.21%)、Saccharomyces (1.12%)、Aureobasidium (0.49%)等属的酵母菌参与发酵。【结论】两样品中酵母菌比例有显著差异,表明在酿酒葡萄的园田管理中化学农药的使用对自然发酵葡萄酒的酵母菌群落结构有较大影响。     

Yeasts associated to Malbec grape berries from Mendoza, Argentina

AIMS: The aims of this work were to evaluate different pre-isolation treatments applied to complete yeast extraction from grapes and to identify the yeast microflora associated to Malbec grapes from two vineyards located in Mendoza, Argentina. METHODS AND RESULTS: The pre-isolation treatments evaluated were shaking, jet streaming with pressurized water and grape blending. The overall results clearly indicated that when a more vigorous and disruptive pre-isolation treatment was applied; larger numbers of yeast species were recovered. The yeast population on healthy and ripe Malbec grapes was in the order of 10(5)-10(6) CFU g(-1). Eight different yeast species were isolated from berries, including Kloeckera apiculata, Metschnikowia pulcherrima, Pichia membranifaciens, Saccharomycodes ludwigii, Candida species (Candida stellata and Candida raghi), Issatchenkia orientalis and Rhodotorula spp. CONCLUSIONS: Grape blending gave the highest yeast counts. Rainfall near grape harvest time quantitatively and qualitatively modifies the yeast microflora. The yeast species identified on ripe grapes from the Mendoza region, partially match those previously documented in different parts of the world related. S. ludwigii has not been previously reported in grapes. SIGNIFICANCE AND IMPACT OF THE STUDY: The report is on yeast microbiota in grapes from Mendoza, Argentina. Saccharomycodes ludwigii was found in high percentage (17%), this species has not been described before on grapes surface. The importance of pre-isolation steps to the recovery of high number of yeasts was shown. Influence of climatic conditions near harvest time on microflora was confirmed.     

Production of ethanol by alginate-entrapped Saccharomyces cerevisiae strain "14-12"

Cells of S. cerevisiae strain "14-12" of different ages were immobilized in sodium alginate and used for conversion of glucose to ethanol. Immobilized cells of 48 hr old were the most potential. Employment of high counts of alginate-entrapped cells shortened the period required for production of the maximal alcohol yield. However, the percentage surviving cells decreased with increasing initial cell counts. Maximal accumulation of ethanol (4.18 g/100 ml) was obtained after 4 days of static fermentation with 1.8 X 10(8) immobilized yeast cells. The residual viable cell count was found to represent 3-fold the surviving percentage in a control experiment using an inoculum of the free yeast cells. Immobilized yeast cells could convert about 85% of the available sugars to ethanol over 28 days of the repeated-batch fermentation. The immobilized cells retained 50% of their viability for 16 days. After 48 days of repeated fermentation only 6% of the yeast cells were viable, and on the 52nd day no viable cells could be detected.     

Ecological survey of Saccharomyces cerevisiae strains from vineyards in the Vinho Verde Region of Portugal

One thousand six hundred and twenty yeast isolates were obtained from 54 spontaneous fermentations performed from grapes collected in 18 sampling sites of three vineyards (Vinho Verde Wine Region in northwest Portugal) during the 2001-2003 harvest seasons. All isolates were analyzed by mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) and a pattern profile was verified for each isolate, resulting in a total of 297 different profiles, that all belonged to the species Saccharomyces cerevisiae. The strains corresponding to seventeen profiles showed a wider temporal and geographical distribution, being characterized by a generalized pattern of sporadic presence, absence and reappearance. One strain (ACP10) showed a more regional distribution with a perennial behavior. In different fermentations ACP10 was either dominant or not, showing that the final outcome of fermentation was dependent on the specific composition of the yeast community in the must. Few of the grape samples collected before harvest initiated a spontaneous fermentation, compared to the samples collected after harvest, in a time frame of about 2 weeks. The associated strains were also much more diversified: 267 patterns among 1260 isolates compared to 30 patterns among 360 isolates in the post- and pre-harvest samples, respectively. Fermenting yeast populations have never been characterized before in this region and the present work reports the presence of commercial yeast strains used by the wineries. The present study aims at the development of strategies for the preservation of biodiversity and genetic resources as a basis for further strain development.     

Seasonal movement and distribution of the grape mealybug (Homoptera: Pseudococcidae): developing a sample program for San Joaquin Valley vineyards

The grape mealybug, Pseudococcus maritimus (Ehrhorn), is an important pest of table grapes in California's San Joaquin Valley. The mealybug causes direct damage by infesting grape bunches, resulting in very low economic injury levels. To develop a sampling program to help growers predict damage and make control decisions, we destructively sampled six entire grapevines each month to determine mealybug abundance and within-vine distribution. These absolute counts were then used to evaluate several relative sampling methods: sticky tape barriers on canes, excised spur samples, standard-sized pieces of bark, timed counts, and nondestructive counts on spurs. At midseason we sampled additional vines to correlate mealybug numbers with economic damage at harvest. Finally, mealybug life stages and natural enemies were recorded throughout the study. Timed 5-min counts show the strongest correlation with total mealybug numbers, and a simple count of mealybugs on three spurs per vine at midseason is the best predictor of economic damage. Mealybugs completed approximately equals 2.5 generations in 1998. Comparison to data on mealybug development suggests that exceptionally long growing seasons could exacerbate infestations by allowing the completion of a third generation. No mealybugs were found on bunches before early August, when second-generation crawlers moved out of the bark. Grape bunches that touched old wood had significantly higher damage and mealybug densities. The majority of mealybugs were always found in protected locations (under the bark of the trunk, spurs or canes), indicating the need for chemical or biological controls that can penetrate these refugia.     

The inhibitory activity of wine yeast starters on malolactic bacteria

Malolactic fermentation is a process that is influenced by various factors that can inhibit the growth of the malolactic bacteria. Inhibitory metabolites produced by yeast may have an important role in the correct development of malolactic fermentation. For these reasons, we have investigated the effects of such metabolites on the growth of malolactic bacteria under different environmental conditions, to aid in our understanding of the significance of these interactions in the wine-making environment. Our screening methods to detect interactions between yeast and malolactic bacteria showed a variable and wide diffusion of yeast inhibitory activity on the growth of the malolactic bacteria. However, this first approach to determine this inhibitory activity of yeast gave an overestimation when compared to the results obtained under actual wine-making conditions. The evaluation of malic acid consumption indicated that under inhibitory conditions a partial L-malic acid degradation was seen, indicating that the malolactic activity continued without bacterial growth. However, these yeast-inhibiting effects in addition to other environmental factors could cause a complete failure of malolactic fermentation.     

Assessment of the yeast species composition of cocoa bean fermentations in different cocoa-producing regions using denaturing gradient gel electrophoresis

The yeast species composition of 12 cocoa bean fermentations carried out in Brazil, Ecuador, Ivory Coast and Malaysia was investigated culture-independently. Denaturing gradient gel electrophoresis of 26S rRNA gene fragments, obtained through polymerase chain reaction with universal eukaryotic primers, was carried out with two different commercial apparatus (the DCode and CBS systems). In general, this molecular method allowed a rapid monitoring of the yeast species prevailing during fermentation. Under similar and optimal denaturing gradient gel electrophoresis conditions, the CBS system allowed a better separated band pattern than the DCode system and an unambiguous detection of the prevailing species present in the fermentation samples. The most frequent yeast species were Hanseniaspora sp., followed by Pichia kudriavzevii and Saccharomyces cerevisiae, independent of the origin of the cocoa. This indicates a restricted yeast species composition of the cocoa bean fermentation process. Exceptionally, the Ivorian cocoa bean box fermentation samples showed a wider yeast species composition, with Hyphopichia burtonii and Meyerozyma caribbica among the main representatives. Yeasts were not detected in the samples when the temperature inside the fermenting cocoa pulp-bean mass reached values higher than 45 °C or under early acetic acid production conditions.     

Effect of hot trub and particle addition on fermentation performance of Saccharomyces cerevisiae

In this investigation, the effect of hot trub (a precipitation product of the wort boiling process in beer manufacturing) addition on fermentation performance was observed under variation of yeast vitality, and origin and the amount of hot trub. Its addition improved suspended cell concentrations for all yeast vitalities tested, and the more trub was added, the greater the effect. Further, pilot-scale fermentations showed significantly lower pH values and an accelerated extract degradation, thus, advancing fermentation by roughly 1 day for hot trub addition versus the fermentation of extremely bright wort. Since the positive effect of trub has often been associated with its particulate characteristics, fermentations with fractionated model particles, such as poly(vinylpyrrolidones) and kieselguhr, of different particle sizes were carried out under variation of yeast vitality and particle amounts. The addition of both particle types also improved fermentation performance, however, the effect was not as great as that of hot trub. Particulate material may improve the development of CO₂ from the fermenting medium, thus reducing its concentration and inhibitory effect on yeast metabolism. The most effective fraction of kieselguhr had a 40 μm peak which also occurred in particle size distributions of all hot trubs investigated. This could be of particular interest when discussing particle effects.     

Nachweis und Vorkommen von Mykophenolsäure und Citrinin in Rotwein

Due to infections with moulds already in the vineyards, the formation of mycotoxins is possible under certain circumstances during the process of red wine making. At this, metabolites ofPenicillium spp. are of major importance, as this species are to be found frequently on grapes. Beside the nephrotoxic citrinin, which is often co-occurring with ochratoxin A, the occurrence of mycophenolic acid (MPA), a substance of immunosuppressive action, was investigated since it is formed by a great number of Penicillium-species. The detection of these compounds was carried out by means of ELISA and LC-MS. As testing material 44 red wine samples of different provenience and vintages were used. Mycophenolic acid could be detected in 91 % of the samples. The maximum content amounted to 130 ng/ml, yet most of the samples resulted in much lower concentrations of between 3 and 20 ng/ml. The extent of contamination seems to depend rather on the origin of the wine than on the vintage. In particular samples from Southern Europe were most contaminated. This could be due to different practises in wine-making. Citrinin was not detectable in any sample (< 0,2 ng/ml). Regarding the detected concentrations of MPA and citrinin, there is probably no concern for consumers’ health. However, the degree of contamination of wine with MPA may well serve as an indicator for hygiene in production.     

Effect of Yeast Hulls on Stuck and Sluggish Wine Fermentations: Importance of the Lipid Component

The effect of yeast hulls (yeast ghosts) on sluggish or stuck white wine fermentations was studied. The enhancing effect on yeast growth and fermentation rate displayed by the hulls was shown to be similar to the effect provided by lipid extract from the same hulls. Unsaturated fatty acids and sterols were incorporated into the yeast from lipid extracts during fermentation carried out under oxygen-limited conditions. Adsorption of toxic medium-chain fatty acid (decanoic acid) onto the yeast hulls took place through a dialysis membrane. However, when the hulls were placed inside a dialysis bag, the increase in yeast growth and fermentation rate seen when freely suspended hulls were used did not occur. Accordingly, the effect of yeast hulls in preventing stuck fermentations cannot be attributed only to the adsorption and consequent removal of medium-chain fatty acids from the juice.     

The growth kinetics and fermentation behaviour of some non-Saccharomyces yeasts associated with wine-making

Summary The growth kinetics and fermentation behaviour of five non-Saccharomyces yeast species associated with wine-making were evaluated.The results showed that the Candida stellata and Torulspora delbrueckii species are interesting for biotechnological applications in wine-making, whereas small-size apiculate yeasts could be profitably used in the production of wine for vinegar manufacture.     

Oenological properties of non-Saccharomyces yeasts associated with wine-making

Several yeast cultures belonging to five non-Saccharomyces species associated with wine-making were evaluated for their oenological properties. Results showed that Candida stellata and Torulaspora delbrueckii could positively affect the taste and flavour of alcoholic beverages. Apiculate yeasts exhibited large amounts of negative byproducts, particularly ethyl acetate. Nevertheless, Kloeckera apiculata showed a significantly negative correlation between either acetic acid and ethyl acetate formation and ethanol production. Selected non-Saccharomyces yeast cultures could be applied profitably in wine-making for optimization of wine bouquet using new fermentation technologies.     

Oenological properties of non-Saccharomyces yeasts associated with wine-making

Several yeast cultures belonging to five non-Saccharomyces species associated with wine-making were evaluated for their oenological properties. Results showed that Candida stellata and Torulaspora delbrueckii could positively affect the taste and flavour of alcoholic beverages. Apiculate yeasts exhibited large amounts of negative byproducts, particularly ethyl acetate. Nevertheless, Kloeckera apiculata showed a significantly negative correlation between either acetic acid and ethyl acetate formation and ethanol production. Selected non-Saccharomyces yeast cultures could be applied profitably in wine-making for optimization of wine bouquet using new fermentation technologies.     

The survival of gamebird (Galliformes) chicks in relation to pesticide use on cereals

Field experiments were carried out to test the effects of cereal pesticides (herbicides, fungicides and insecticides) on chick survival of Grey Partridge Perdix perdix , Red-legged Partridge Alectoris rufa and Pheasant Phasianus colchicus. On fields in experimental plots the outer 6 m of cereal (the headland) were not sprayed with pesticides from 1 January 1984, whereas control plots were fully sprayed. Gamebird brood counts were carried out after the cereal harvest. In addition, nine Grey Partridge broods were radio-tracked for 21 days after hatching (four in sprayed plots and five in unsprayed plots) to determine their movements, home range size and survival in relation to pesticide spraying.
The mean brood size of Grey Partridge and Pheasant was significantly higher on plots where field edges were unsprayed than on fully sprayed control plots. Data for Red-legged Partridge were inconclusive. The survival of individually marked Grey Partridge broods was negatively related to the distance moved between successive nocturnal roost sites. Survival was significantly higher, the distance moved between roost sites significantly shorter and the proportion of home range including headland significantly greater for broods feeding in spring barley fields with unsprayed field edges compared with broods feeding in fully sprayed fields.     

Influence of aeration during propagation of pitching yeast on fermentation and beer flavor

The effect of yeast propagated at different aeration conditions on yeast physiology, fermentation ability, and beer quality was investigated using three strains of Saccharomyces cerevisiae. It was shown that yeast cells grown under continuous aeration conditions during propagation were almost two times higher as compared with discontinuous aeration conditions. The maximum of cell growth of all samples reached between 36 h and 48 h. The concentration of trehalose was increased under continuous aerated yeasts, whereas glycogen was decreased. It was also observed that the concentration of glycogen and trehalose in yeast cells had no direct effect on subsequent fermentation ability. The effect of yeast propagated under different aeration conditions on subsequent fermentation ability was different from yeast strains, in which the influence will be most pronounced at the first fermentation. Later, the yeasts might regain its original characteristics in the following fermentations. Generally, continuously propagated yeast had a positive effect on beer quality in subsequent fermentation. Hence, the concentration of aroma compounds obtained with yeast propagated under 6 1/h for 48 h aeration was lower than those grown under other aeration conditions in the bottom yeasts; in particular, the amounts of phenylethyl alcohol, ester, and fatty acids were decreased.     

Improvement of ethanol production in very high gravity fermentation by horse gram (Dolichos biflorus) flour supplementation

AIMS: To determine the effect of osmotic stress on yeast and to investigate the protective role of horse gram flour during very high gravity (VHG) ethanol fermentation. METHODS AND RESULTS: Saccharomyces cerevisiae was inoculated into high sugar (30-40%, w/v) containing medium with and without supplementation of horse gram flour. The fermentation experiments were carried out in batch mode. The effect of 4 or 6% of horse gram flour to the medium on the metabolic behaviour and viability of yeast was studied. Significant increase in ethanol yield up to 50% and dramatic decrease in glycerol production up to 100% was observed in the presence of horse gram flour. The fermentation rate was increased from 3 to 5 days with increased viable cell count. The physical and chemical factors of horse gram flour may aid in reducing the osmotic stress of high gravity fermentation of ethanol as well as enhancing ethanol yield. CONCLUSIONS: It was found that horse gram flour not only reduced fermentation time but also enhanced ethanol production by better utilization of sugar. SIGNIFICANCE AND IMPACT OF THE STUDY: Production of high ethanol concentration by using VHG sugar fermentation eliminates the expensive steps in the conventional process and saves time.     

Selective hybridisation of pure culture wine yeasts

Summary Pure culture wine yeasts vary in their efficiency of conversion of grape sugar to ethanol. Selective hybridisation over three generations gave significant in fermentation efficiency, i.e. from 84% to 93%. Sulphur dioxide tolerance was found to be under the control of dominant polymeric genes. Other wine-making characteristics were monitored during the hybridisation programme and selected hybrid strains had wine-making qualities comparable with those of the parent strains, but with increased sugar conversion efficiency.     

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.     

High-throughput sequencing of amplicons for monitoring yeast biodiversity in must and during alcoholic fermentation

We compared pyrosequencing technology with the PCR-ITS-RFLP analysis of yeast isolates and denaturing gradient gel electrophoresis (DGGE). These methods gave divergent findings for the yeast population. DGGE was unsuitable for the quantification of biodiversity and its use for species detection was limited by the initial abundance of each species. The isolates identified by PCR-ITS-RFLP were not fully representative of the true population. For population dynamics, high-throughput sequencing technology yielded results differing in some respects from those obtained with other approaches. This study demonstrates that 454 pyrosequencing of amplicons is more relevant than other methods for studying the yeast community on grapes and during alcoholic fermentation. Indeed, this high-throughput sequencing method detected larger numbers of species on grapes and identified species present during alcoholic fermentation that were undetectable with the other techniques.