Characterization of Kloeckera apiculata strains from the Friuli regionin Northern Italy

Forty-nine strains of Kloeckera apiculata, isolated from the Friuli region in Italy, were differentiated on the basis of fermentation behaviour and production of secondary compounds in two different grape musts at 18 °C. The isolates exhibited a controlled production of acetic acid, only in a few cases more that 1 g/l. In Moscato grape must the strains exhibited a more uniform behaviour for the production of higher alcohols, ethyl acetate and acetoin than in red grapes. In general, higher levels of ethanol, glycerol and acetic acid were produced in red grape must fermentation. Apiculate strains behaved differently in the two musts, with different metabolic phenotypes dominating the fermentation process. The existence of different metabolic phenotypes correlated with the must composition underlines the need to perform a selection of indigenous apiculate yeasts to obtain the desired consistent products.     

Evolution of Yeasts and Lactic Acid Bacteria During Fermentation and Storage of Bordeaux Wines

The levels of yeasts and lactic acid bacteria that naturally developed during the vinification of two red and two white Bordeaux wines were quantitatively examined. Yeasts of the genera Rhodotorula, Pichia, Candida, and Metschnikowia occurred at low levels in freshly extracted grape musts but died off as soon as fermentation commenced. Kloeckera apiculata (Hanseniaspora uvarum), Torulopsis stellata, and Saccharomyces cerevisiae, the dominant yeasts in musts, proliferated to conduct alcoholic fermentation. K. apiculata and eventually T. stellata died off as fermentation progressed, leaving S. cerevisiae as the dominant yeast until the termination of fermentation by the addition of sulfur dioxide. At least two different strains of S. cerevisiae were involved in the fermentation of one of the red wines. Low levels of lactic acid bacteria (Pediococcus cerevisiae, Leuconostoc mesenteroides, and Lactobacillus spp.) were present in grape musts but died off during alcoholic fermentation. The malolactic fermentation developed in both red wines soon after alcoholic fermentation and correlated with the vigorous growth of at least three different strains of Leuconostoc oenos.     

Impact of different malolactic fermentation inoculation scenarios on Riesling wine aroma

During malolactic fermentation (MLF), lactic acid bacteria influence wine aroma and flavour by the production of volatile metabolites and the modification of aroma compounds derived from grapes and yeasts. The present study investigated the impact of different MLF inoculation strategies with two different Oenococcus oeni strains on cool climate Riesling wines and the volatile wine aroma profile. Four different timings were chosen for inoculation with bacteria to conduct MLF in a Riesling must/wine with a high acidity (pH 2.9–3.1). Treatments with simultaneous inoculation showed a reduced total fermentation time (alcoholic and malolactic) compared to the sequential inoculations. No negative impact of simultaneous alcoholic and malolactic fermentation on fermentation success and on the final wine volatile aroma composition was observed. Compared to sequential inoculation, wines with co-inoculation tended to have higher concentrations of ethyl and acetate esters, including acetic acid phenylethylester, acetic acid 3-methylbutylester, butyric acid ethylester, lactic acid ethylester and succinic acid diethylester. Results of this study provide some alternatives to diversify the number of wine styles by safely conducting MLF in low-pH, cool-climate white musts with potential high alcohol content.     

产香酵母Pichia myanmarensis LX15的分离纯化及对精酿啤酒风味物质形成的影响

为了适应精酿啤酒对个性化风味的需求,能产生特定风味化合物的产香酵母成为研究者的研究重点。从精酿啤酒原液中分离到1株产香酵母LX15菌,该菌细胞呈圆形或卵圆形、多极芽殖生长;LX15菌在玉米粉培养基上培养7~10 d不形成假菌丝,在酵母膏蛋白胨培养基上培养3 d能够形成子囊孢子。经生理生化特征和系统发育分析,确认该生香酵母为Pichia myanmarensis菌中的一个菌株,所产主要风味化合物包括乙酸乙酯、乙酸异戊酯、己酸乙酯和辛酸乙酯。当LX15菌与啤酒酵母C1菌共发酵时,能够产生协同效应,提高酯类化合物和高级醇类的含量,并与LX15菌的接种比例正相关,但并不影响啤酒酿造的整体发酵速率和发酵能力。因此,LX15菌是一株适于提高精酿啤酒风味的产香酵母菌。     

Evolution of C6, C8 and C10 acids and their ethyl esters in cells and musts during fermentation with threeSaccharomyces cerevisiae races

Summary The evolution of the cell and must contents of three short-chain fatty acids (C₆, C₈ and C₁₀) and their ethyl esters during fermentations withSaccharomyces cerevisiae racescerevisiae, bayanus andcapensis were studied. The former is a fermentative yeast and the last two are flor film yeasts. The acid concentrations in the musts increased throughout the alcoholic fermentations, and maximum cell concentrations of the fatty acids were reached after 48 h of fermentation. Maximum ester concentrations in the cells were attained after 48–72 h of fermentation. In the musts, ethyl octanoate and ethyl decanoate reached a peak also at this point, and ethyl hexanoate after 10 days. After 134 days,S. cerevisiae racecapensis formed a thick flor film whileS. cerevisiae racebayanus developed a thin film andS. cerevisiae racecerevisiae formed no film. At this point, acid contents remained constant in the wines produced byS. cerevisiae racescerevisiae andbayanus, and decreased in those obtained with racecapensis. The ethyl ester contents tended to decrease with the exception of ethyl decanoate in the fermentations carried out byS. cerevisiae racescerevisiae andbayanus.     

酒醅来源酵母菌合成异戊醇能力与途径解析

【背景】异戊醇是酵母菌在白酒发酵过程中通过氨基酸合成代谢途径和氨基酸分解代谢途径合成的主要高级醇,其含量影响白酒饮用的舒适度。【目的】分析和比较分离自浓香型白酒酒醅中的酵母菌合成异戊醇的能力,揭示酵母菌合成异戊醇的途径。【方法】从酒醅中分离具有异戊醇合成能力的酵母菌株,比较不同生长时期酵母菌合成异戊醇的能力,通过前体物代谢分析它们合成异戊醇的途径。【结果】分离自酒醅的5株酵母的异戊醇合成能力从强到弱依次为Naumovozyma castellii JP3-1、Saccharomyces cerevisiae JP3、Pichia fermentans JP22、Pichia kudriavzevii JP1和Naumovozyma dairenensis CBS421。这些酵母合成异戊醇的时期主要在对数生长期,N. castellii JP3-1、P. fermentans JP22和N. dairenensis CBS421在稳定生长期也合成异戊醇。S. cerevisiae JP3、N. castellii JP3-1和N. dairenensis CBS421在整个生长时期主要通过Harris途径合成异戊醇;P. kudriavzevii JP1在整个时期主要通过Ehrlich途径合成异戊醇;P. fermentans JP22在对数生长期通过Harris途径和Ehrlich途径合成异戊醇的能力接近,在稳定生长期主要通过Harris途径合成异戊醇。【结论】本研究揭示了酒醅来源5个属种酵母合成异戊醇的途径、能力与其生长时期的关系,研究结果可为解析浓香型白酒发酵过程异戊醇合成、积累机制及实施白酒发酵过程异戊醇合成的精准调控提供理论依据。     

The use of killer biotyping in an ecological survey of yeast in an old patagonian winery

An ecological study of Saccharomyces cerevisiae strains in spontaneous alcoholic fermentation has been made in the same winery on two consecutive years (1993 and 1994) with Merlot type musts, and with Malbec type must on a third year (1998). Saccharomyces cerevisiae strains associated with winery surfaces were also analysed. Differential killer sensitivity patterns related to a killer reference panel of 10 killer yeasts belonging to nine species of four genera were used as a quick and simple procedure to discriminate between indigenous S. cerevisiae isolates at the strain level. Although a great diversity of wild strains was observed, two main indigenous S. cerevisiae strains, designated as S. cerevisiae 9 and S. cerevisiae 13, took over the Merlot type fermentation in both years. These strains also appeared in Malbec must fermentation during the year 1998 and they were again found on the winery surface the next year. These results show that some few and stable indigenous S. cerevisiae strains remained in the environmental winery over the considered period of time (1993–1999) and they represent an additional evidence of the taking over of musts by local strains of S. cerevisiae.     

Increase of acetate ester-hydrolysing esterase activity in mixed cultures of Saccharomyces cerevisiae and Pichia anomala

Aim: To examine the efficacy of mixed cultures with Saccharomyces cerevisiae and Pichia anomala on flavour profiles of alcoholic beverages, a Pichia mutant with low levels of ethyl acetate that negatively impact on the sensory quality was isolated. Methods and Results: A petite mutant isolated from P. anomala NBRC 10213 treated with ethidium bromide had the lower activity of ethyl acetate‐hydrolysing esterase (EAHase) than the wild‐type in crude extracts. In the fermentation tests of pure cultures, the P. anomala mutant produced less ethanol, acetate and ethyl acetate than the wild‐type. In mixed cultures with S. cerevisiae, the P. anomala mutant died quicker and produced lower amounts of ethyl acetate than the wild‐type. Mixed cultures of S. cerevisiae and P. anomala showed higher activities of EAHase than pure culture of S. cerevisiae throughout the fermentation periods. The transition to the formation of acetate esters was considerably analogous to the transition to the activity of acetate ester‐hydrolysing esterase with little time lag. Conclusions: The P. anomala mutant was superior to the wild‐type in flavour profiles. The higher ethyl acetate concentrations formed mainly by P. anomala in mixed cultures are the primary stimulus for the EAHase in S. cerevisiae and the activity of acetate ester‐hydrolysing esterase is crucial to the formation of acetate esters in mixed cultures of S. cerevisiae and P. anomala. Significance and Impact of the Study: An application of non‐Saccharomyces yeast, P. anomala to enhance the sensory quality in alcoholic beverage and a mechanism of the formation of acetate esters in mixed cultures with S. cerevisiae and P. anomala were offered.     

刺梨自然发酵过程中非酿酒酵母多样性分析

【目的】分析刺梨果实自然发酵过程中非酿酒酵母菌群特征,为筛选优质刺梨非酿酒酵母提供参考。【方法】基于Illumina MiSeq高通量测序技术和WL营养琼脂鉴定培养基纯种分离技术,分析刺梨果实自然发酵1 d (F1)、3 d (F3)、5 d (F5)和15 d (F15) 4个阶段及YPD培养基富集培养样本中非酿酒酵母种群组成和多样性。【结果】高通量测序分析结果共获得182个OTUs (operational taxonomic units,OTUs),归属于81个属107个种;物种多样性分析结果表明,刺梨果实自然发酵前期,优势非酿酒酵母为汉逊酵母(Hanseniasporasp.)和伯顿丝孢毕赤酵母(Hyphopichiaburtonii),二者在样本F1中分别占42.59%和26.85%;随着自然发酵的不断进行,二者的比例逐渐降低,在第15天(F15),Hanseniaspora sp.和H. burtonii比例降低至7.73%和0.52%。相反,Pichia sporocuriosa和未培养的酵母,随着自然发酵不断进行所占比例逐渐增大,分别由F1中的0.23%和0.33%增至F15中的37.26%和32.62%。此外,采用WL营养琼脂鉴定培养基纯种分离和鉴定技术,从刺梨上分离到Hanseniasporasp.、H.burtonii、克鲁维毕赤酵母(Pichia kluyveri)、P. sporocuriosa和异常威克汉姆酵母(Wickerhamomyces anomalus) 5种类型的可培养非酿酒酵母。【结论】刺梨果实上存在着丰富的非酿酒酵母菌资源,研究刺梨自然发酵过程中非酿酒酵母多样性,为酵母资源开发和利用奠定基础。     

Development of alcoholic and malolactic fermentations in highly acidic and phenolic apple musts

This work reports the influence of the high acidity and high phenolic content in apple musts on the development of alcoholic and malolactic fermentations and on the final chemical and microbiological composition of the ciders. Four different musts were obtained by pressing several varieties and proportions of cider apples from the Basque Country (Northern Spain). Specially acidic and phenolic varieties were selected. Three musts were obtained in experimental stations and the fourth one, in a cider factory following usual procedures. The evolution of these musts was monitored during five months by measuring 18 parameters throughout eight samplings. In the most acidic of the three experimental musts, yeasts were added to complete the alcoholic fermentation. In the rest of the musts, alcoholic and malolactic fermentations took place spontaneously due to natural microflora and no chemical was added to control these processes. Malolactic fermentation (MLF) finished before alcoholic fermentation in the three tanks obtained in experimental stations, even in the most acidic and phenolic one (pH 3.18, 1.78 g tannic acid/l). After four months, these ciders maintained low levels of lactic acid bacteria (10(4)CFU/ml) and low content of acetic acid (<0.60 g/l). Both fermentations began simultaneously in the must obtained in the cider factory, but MLF finished 10 days after alcoholic fermentation. Subsequently, this must maintained a high population of lactic acid bacteria (>10(6)CFU/ml), causing a higher production of acetic acid (>1.00 g/l) than in the other ciders. These results show the possible advantages of MLF finishing before alcoholic fermentation.     

Growth and fermentation behaviour of Brettanomyces/Dekkera yeasts under different conditions of aerobiosis

Brettanomyces/Dekkera yeasts grow in wine and their presence is often associated with spoiling activity. In this report, we investigated on the influence of different conditions of aerobiosis on growth and fermentation behaviour of these spoilage yeasts in wine. Results showed that in all conditions tested the Brettanomyces strain consumed all sugars, taking wine fermentation to completion. Strict-anaerobic conditions limited the growth of Brettanomyces. Both anaerobiosis (using a fermentation trap) and strict anaerobiosis did not negatively affect the principal by-products of fermentation whereas semi-anaerobiosis caused an increase of acetic acid, acetaldehyde and ethyl acetate that negatively affected the fermentation profile of resulting products.     

Controlled formation of volatile components in cider making using a combination of Saccharomyces cerevisiae and Hanseniaspora valbyensis yeast species

The effect of pure and mixed fermentation by Saccharomyces cerevisiae and Hanseniaspora valbyensis on the formation of major volatile components in cider was investigated. When the interaction between yeast strains of S. cerevisiae and H. valbyensis was studied, it was found that the two strains each affected the cell growth of the other upon inoculation of S. cerevisiae during growth of H. valbyensis. The effects of pure and mixed cultures of S. cerevisiae and H. valbyensis on alcohol fermentation and major volatile compound formation in cider were assessed. S. cerevisiae showed a conversion of sugar to alcohol of 11.5%, while H. valbyensis produced alcohol with a conversion not exceeding 6%. Higher concentrations of ethyl acetate and phenethyl acetate were obtained with H. valbyensis, and higher concentrations of isoamyl alcohol and isobutyl were formed by S. cerevisiae. Consequently, a combination of these two yeast species in sequential fermentation was used to increase the concentration of ethyl esters by 7.41–20.96%, and to decrease the alcohol concentration by 25.06–51.38%. Efficient control of the formation of volatile compounds was achieved by adjusting the inoculation time of the two yeasts.     

Performance evaluation of Pichia kluyveri, Kluyveromyces marxianus and Saccharomyces cerevisiae in industrial tequila fermentation

Traditionally, industrial tequila production has used spontaneous fermentation or Saccharomyces cerevisiae yeast strains. Despite the potential of non-Saccharomyces strains for alcoholic fermentation, few studies have been performed at industrial level with these yeasts. Therefore, in this work, Agave tequilana juice was fermented at an industrial level using two non-Saccharomyces yeasts (Pichia kluyveri and Kluyveromyces marxianus) with fermentation efficiency higher than 85 %. Pichia kluyveri (GRO3) was more efficient for alcohol and ethyl lactate production than S. cerevisiae (AR5), while Kluyveromyces marxianus (GRO6) produced more isobutanol and ethyl-acetate than S. cerevisiae (AR5). The level of volatile compounds at the end of fermentation was compared with the tequila standard regulation. All volatile compounds were within the allowed range except for methanol, which was higher for S. cerevisiae (AR5) and K. marxianus (GRO6). The variations in methanol may have been caused by the Agave tequilana used for the tests, since this compound is not synthesized by these yeasts.     

Yeast communities associated with artisanal mezcal fermentations from <Emphasis Type="Italic">Agave salmiana</Emphasis>

The aims of this work were to characterize the fermentation process of mezcal from San Luis Potosi, México and identify the yeasts present in the fermentation using molecular culture-dependent methods (RFLP of the 5.8S-ITS and sequencing of the D1/D2 domain) and also by using a culture-independent method (DGGE). The alcoholic fermentations of two separate musts obtained from Agave salmiana were analyzed. Sugar, ethanol and major volatile compounds concentrations were higher in the first fermentation, which shows the importance of having a quality standard for raw materials, particularly in the concentration of fructans, in order to produce fermented Agave salmiana must with similar characteristics. One hundred ninety-two (192) different yeast colonies were identified, from those present on WL agar plates, by RFLP analysis of the ITS1-5.8S- ITS2 from the rRNA gene, with restriction endonucleases, HhaI, HaeIII and HinfI. The identified yeasts were: Saccharomyces cerevisiae, Kluyveromyces marxianus, Pichia kluyveri, Zygosaccharomyces bailii, Clavispora lusitaniae, Torulaspora delbrueckii, Candida ethanolica and Saccharomyces exiguus. These identifications were confirmed by sequencing the D1-D2 region of the 26S rRNA gene. With the PCR-DGGE method, bands corresponding to S. cerevisiae, K. marxianus and T. delbrueckii were clearly detected, confirming the results obtained with classic techniques.     

Characteristics of a rice beer (zutho) and a yeast isolated from the fermented product in Nagaland,India

Rice beer, known locally as zutho was collected in the Kohima district in Nagaland, India, and subjected to analytical and microbiological characterization. Zutho was a whitish porridge-like slurry containing 5.0% (v/v) ethanol. Volatile esters and higher alcohols, such as ethyl acetate and 3-methylbutanol, were detected in this indigenous alcoholic beverage by gas chromatography. The pH and acidity of zutho were 3.6 and 5.1, respectively. Zutho had a fruity aroma and sour taste and its unique aroma had characteristics similar to those of Japanese sake and sprouted rice sake. A fermentation yeast isolated from zutho was identified as being a strain of Saccharomyces cerevisiae and was found to be suitable as the brewing yeast for ethanol fermentation.     

Influence of Williopsis saturnus yeasts in combination with Saccharomyces cerevisiae on wine fermentation

Aim: To examine the growth and survival of Williopsis saturnus strains along with wine yeast Saccharomyces cerevisiae in grape must. Methods and Results: For this study, fermentations were performed in sterilized grape must at 18°C. Inoculum level was 5 × 10⁶ cells per ml for each yeast. The results showed that W. saturnus yeasts exhibited slight growth and survival depending on the strain, but they died off by day 5. Saccharomyces cerevisiae, however, dominated the fermentation, reaching the population of about 8 log CFU ml^?1. It was observed that ethanol formation was not affected. The concentrations of acetic acid, ethyl acetate and isoamyl acetate were found higher in mixed culture experiments compared to control fermentation. The results also revealed that higher alcohols production was unaffected in general. Conclusion: Fermentations did not form undesirable concentrations of flavour compounds, but production of higher levels of acetic acid in mixed culture fermentations may unfavour the usage of W. saturnus in wine making. Significance and Impact of the Study: This study provides information on the behaviour of W. saturnus together with S. cerevisiae during the alcoholic fermentation.     

Improvement of Nitrogen Assimilation and Fermentation Kinetics under Enological Conditions by Derepression of Alternative Nitrogen-Assimilatory Pathways in an Industrial Saccharomyces cerevisiae Strain

Metabolism of nitrogen compounds by yeasts affects the efficiency of wine fermentation. Ammonium ions, normally present in grape musts, reduce catabolic enzyme levels and transport activities for nonpreferred nitrogen sources. This nitrogen catabolite repression severely impairs the utilization of proline and arginine, both common nitrogen sources in grape juice that require the proline utilization pathway for their assimilation. We attempted to improve fermentation performance by genetic alteration of the regulation of nitrogen-assimilatory pathways in Saccharomyces cerevisiae. One mutant carrying a recessive allele of ure2 was isolated from an industrial S. cerevisiae strain. This mutation strongly deregulated the proline utilization pathway. Fermentation kinetics of this mutant were studied under enological conditions on simulated standard grape juices with various nitrogen levels. Mutant strains produced more biomass and exhibited a higher maximum CO₂ production rate than the wild type. These differences were primarily due to the derepression of amino acid utilization pathways. When low amounts of dissolved oxygen were added, the mutants could assimilate proline. Biomass yield and fermentation rate were consequently increased, and the duration of the fermentation was substantially shortened. S. cerevisiae strains lacking URE2 function could improve alcoholic fermentation of natural media where proline and other poorly assimilated amino acids are the major potential nitrogen source, as is the case for most fruit juices and grape musts.     

Identification and characterization of yeasts in brem, a traditional Balinese rice wine

Fifty-one yeast strains isolated from fermented mash of Balinese rice wine, brem, fermented using five different types of starters, ragi tape, were identified on the basis of their internal transcribed spacer (ITS) regions and their 18S rDNA sequences. The results revealed that Saccharomyces cerevisiae(35 strains), Candida glabrata(six strains), Pichia anomala(three strains) and Issatchenkia orientalis(seven strains) were the main yeasts in the fermentation of the rice wine. These yeasts undergo succession during the fermentation in which S. cerevisiae was mostly found as the principal yeast at the end of fermentation. Phylogenetic analysis based on the 18S rDNA sequences of selected strains placed the isolated S. cerevisiae strains in the Saccharomyces sensu stricto group. Karyotype analysis of the S. cerevisiae strains resolved using pulsed field gel electrophoresis (PFGE) showed that the strains are typically associated with different types of starters.     

The Formation of Higher Alcohols in the Fermentation of Amino Acids by Yeast

We have found that some straight-chained α-amino acids are converted by yeast to the alcohols with correspondingly longer carbon chains in the alcoholic fermentation contrary to F. Ehrlich’s scheme, i.e., isobutyl alcohol from alanine and active amyl alcohol from α-amino-n-butyric acid or threonine.

In this report, we confirmed this fact in the alcoholic fermentation of many aliphatic amino acids by 2 yeast strains using gas chromatography. Moreover, n-propyl alcohol was proved to come from α-amino-n-butyric acid or threonine. Small quantities of n-propyl, isobutyl, active amyl and isoamyl alcohols were found in all the fermented solutions. There was some difference in the composition of higher alcohols of the alcoholic solutions fermented by different yeasts.     

Kinetics of growth and sugar consumption in yeasts

An overview is presented of the steady- and transient state kinetics of growth and formation of metabolic byproducts in yeasts.Saccharomyces cerevisiae is strongly inclined to perform alcoholic fermentation. Even under fully aerobic conditions, ethanol is produced by this yeast when sugars are present in excess. This so-called Crabtree effect probably results from a multiplicity of factors, including the mode of sugar transport and the regulation of enzyme activities involved in respiration and alcoholic fermentation. The Crabtree effect inS. cerevisiae is not caused by an intrinsic inability to adjust its respiratory activity to high glycolytic fluxes. Under certain cultivation conditions, for example during growth in the presence of weak organic acids, very high respiration rates can be achieved by this yeast.S. cerevisiae is an exceptional yeast since, in contrast to most other species that are able to perform alcoholic fermentation, it can grow under strictly anaerobic conditions.Non-Saccharomyces yeasts require a growth-limiting supply of oxygen (i.e. oxygen-limited growth conditions) to trigger alcoholic fermentation. However, complete absence of oxygen results in cessation of growth and therefore, ultimately, of alcoholic fermentation. Since it is very difficult to reproducibly achieve the right oxygen dosage in large-scale fermentations, non-Saccharomyces yeasts are therefore not suitable for large-scale alcoholic fermentation of sugar-containing waste streams. In these yeasts, alcoholic fermentation is also dependent on the type of sugar. For example, the facultatively fermentative yeastCandida utilis does not ferment maltose, not even under oxygen-limited growth conditions, although this disaccharide supports rapid oxidative growth.