Altered Patterns of Maltose and Glucose Fermentation by Brewing and Wine Yeasts Influenced by the Complexity of Nitrogen Source

Maltose and glucose fermentations by industrial brewing and wine yeasts strains were strongly affected by the structural complexity of the nitrogen source. In this study, four Saccharomyces cerevisiae strains, two brewing and two wine yeasts, were grown in a medium containing maltose or glucose supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low sugar concentration for brewing and wine strains, independent of nitrogen supplementation, and the type of sugar. At high sugar concentrations altered patterns of sugar fermentation were observed, and biomass accumulation and ethanol production depended on the nature of the nitrogen source and were different for brewing and wine strains. In maltose, high biomass production was observed under peptone and casamino acids for the brewing and wine strains, however efficient maltose utilization and high ethanol production was only observed in the presence of casamino acids for one brewing and one wine strain studied. Conversely, peptone and casamino acids induced higher biomass and ethanol production for the two other brewing and wine strains studied. With glucose, in general, peptone induced higher fermentation performance for all strains, and one brewing and wine strain produced the same amount of ethanol with peptone and casamino acids supplementation. Ammonium salts always induced poor yeast performance. The results described in this paper suggest that the complex nitrogen composition of the cultivation medium may create conditions resembling those responsible for inducing sluggish/stuck fermentation, and indicate that the kind and concentration of sugar, the complexity of nitrogen source and the yeast genetic background influence optimal industrial yeast fermentation performance.     

Stuck and slow fermentations in enology: statistical study of causes and effectiveness of combined additions of oxygen and diammonium phosphate

One hundred and seventy-eight musts from different regions in France were selected by enologists and fermented under standardized conditions. Two kinds of fermentation problems were distinguished: (a) slow and (b) sluggish (late-onset sluggish) and stuck (with residual sugar) fermentations. Slow fermentations, characterized by a low fermentation rate throughout the process, were always due to low assimilable nitrogen concentrations in the must. The advantages of using formol titration for measuring assimilable nitrogen are discussed. In contrast, sluggish and stuck fermentations, characterized by very low yeast viability at the end of fermentation, could not be predicted from the analytical data even though their probability was increased when the initial sugar concentration was high. All problems of stuck and sluggish fermentations (concerning 40% of the musts) were solved by supplying 7 mg/l oxygen and 300 mg/l diammonium phosphate at the halfway of the fermentation process, which confirmed (i) the importance of these two nutrients in enological practices and (ii) the importance of adding them at the right time.     

Alleviation of stuck wine fermentations using salt‐preconditioned yeast

The influence of salt (sodium chloride) on the cell physiology of wine yeast was investigated. Cellular viability and population growth of three wine‐making yeast strains of Saccharomyces cerevisiae, and two non‐Saccharomyces yeast strains associated with wine must microflora (Kluyveromyces thermotolerans and K. marxianus) were evaluated following salt pre‐treatments. Yeast cells growing in glucose defined media exposed to different sodium chloride concentrations (4, 6 and 10% w/v) exhibited enhanced viabilities compared with nontreated cultures in subsequent trial fermentations. Salt ‘preconditioning’ of wine yeast seed cultures was also shown to alleviate stuck and sluggish fermentations at the winery scale, indicating potential benefits for industrial fermentation processes. It is hypothesized that salt induces specific osmostress response genes to enable yeast cells to better tolerate the rigours of fermentation, particularly in high sugar and alcohol concentrations. Copyright © 2014 The Institute of Brewing & Distilling     

氮源对酿酒酵母GJ2008不同糖浓度甘蔗汁酒精分批发酵的影响

研究了不同糖浓度下氮源对酿酒酵母GJ2008甘蔗汁酒精发酵的影响,旨在为高浓度甘蔗汁酒精发酵提供研究基础。将2 g/L尿素加入150 g/L、200 g/L、250 g/L、300 g/L和350 g/L总糖质量浓度甘蔗汁培养基中,以对应糖浓度未添加氮源组为对照组,通过分析细胞生长、糖代谢和乙醇生成来探究氮源如何影响甘蔗汁酒精发酵。结果表明：与未添加氮源相比,氮源加快了发酵速度,发酵周期最高缩短为18 h,糖消耗速率和乙醇生成速率最大分别提高了55%和96%;使酒精发酵更彻底,总糖含量最高减少了40.42 g/L;增加了目的产物,乙醇含量最高提升了28.4%。说明氮源有效地促进了甘蔗汁酒精发酵,加强了酵母的无氧呼吸途径。     

Study of two wine strains rehydrated with different activators in sluggish fermentation conditions

Rehydration of active dry yeasts is essential in wine fermentation. To help yeast cells withstand the extreme conditions during alcoholic fermentation, adjuvants are added to the must. However, few commercial activators are added directly to the yeast rehydration water, even though this might render hydration more efficient due to a favorable osmotic gradient. The effect of five “metabolic activators” added directly to the water to rehydrate two commercial wine yeast strains in sluggish fermentation conditions was evaluated; these conditions were achieved by acid and osmotic stress. Cell vitality, sugar consumption, glycerol, ethanol and nitrogen contents at 8 and 15 days of process were determined. Furthermore, discrepancy in glucose and fructose consumption was also measured. Ergosterol at certain doses generally showed a favorable effect, so that it could be considered a good activator compared with other molecules involved in essential metabolic cycles.     

Impact of mixed Torulaspora delbrueckii-Saccharomyces cerevisiae culture on high-sugar fermentation

Conventional wine yeasts produce high concentrations of volatile acidity, mainly acetic acid, during high-sugar fermentation. This alcoholic fermentation by-product is highly detrimental to wine quality and, in some cases, levels may even exceed legal limits. In this study, a non-conventional species, Torulaspora delbrueckii, was used, in pure cultures and mixed with Saccharomyces cerevisiae yeast, to ferment botrytized musts. Fermentation rate, biomass growth, and the formation of volatile acidity, acetaldehyde, and glycerol were considered. This study demonstrated that T. delbrueckii, often described as a low acetic producer under standard conditions, retained this quality even in a high-sugar medium. Unlike S. cerevisiae, this species did not respond to the hyper-osmotic medium by increasing acetic production as soon as it is inoculated into the must. Nevertheless, this yeast produced low ethanol and biomass yields, and the fermentation was sluggish. As a result, T. delbrueckii fermentations do not reach the required ethanol content (14%vol.), although this species can survive at this concentration. A mixed culture of T. delbrueckii and S. cerevisiae was the best combination for improving the analytical profile of sweet wine, particularly volatile acidity and acetaldehyde production. A mixed T. delbrueckii/S. cerevisiae culture at a 20:1 ratio produced 53% less in volatile acidity and 60% less acetaldehyde than a pure culture of S. cerevisiae. Inoculating S. cerevisiae after 5 days' fermentation by T. delbrueckii had less effect on volatile acidity and acetaldehyde production and resulted in stuck fermentation. These results contribute to a better understanding of the behaviour of non-Saccharomyces and their potential application in wine industry.     

Indirect monitoring of TORC1 signalling pathway reveals molecular diversity among different yeast strains

Saccharomyces cerevisiae is the main species responsible for the alcoholic fermentation in wine production. One of the main problems in this process is the deficiency of nitrogen sources in the grape must, which can lead to stuck or sluggish fermentations. Currently, yeast nitrogen consumption and metabolism are under active inquiry, with emphasis on the study of the TORC1 signalling pathway, given its central role responding to nitrogen availability and influencing growth and cell metabolism. However, the mechanism by which different nitrogen sources activates TORC1 is not completely understood. Existing methods to evaluate TORC1 activation by nitrogen sources are time-consuming, making difficult the analyses of large numbers of strains. In this work, a new indirect method for monitoring TORC1 pathway was developed on the basis of the luciferase reporter gene controlled by the promoter region of RPL26A gene, a gene known to be expressed upon TORC1 activation. The method was tested in strains representative of the clean lineages described so far in S. cerevisiae. The activation of the TORC1 pathway by a proline-to-glutamine upshift was indirectly evaluated using our system and the traditional direct methods based on immunoblot (Sch9 and Rps6 phosphorylation). Regardless of the different molecular readouts obtained with both methodologies, the general results showed a wide phenotypic variation between the representative strains analysed. Altogether, this easy-to-use assay opens the possibility to study the molecular basis for the differential TORC1 pathway activation, allowing to interrogate a larger number of strains in the context of nitrogen metabolism phenotypic differences.     

咖啡渣酿酒酵母的筛选及氮源优化营养条件的研究

该文首先对比不同酵母菌酿造咖啡渣酒的产气、产酒精和产香能力,而后探究氮源的种类和浓度对酵母菌酿造咖啡渣酒的影响。结果表明,在备筛的4株酵母菌中,Saccharomyces cerevisiae D254产气能力、产酒精能力和发酵液的感官评分都较佳,适用于酿造咖啡渣酒;氮源的加入能有效解决咖啡渣酒发酵迟缓的问题,提高酵母菌生物量、酒体酒精度,降低残糖含量;其中添加0.20%磷酸氢二铵作氮源时效果最好,生物量由1.21 g/L增加至1.73 g/L,酒精度由7.3%vol增加至9.6%vol,残糖由19.52 g/L降低至5.73 g/L,且所得咖啡渣酒具有咖啡特有的芳香,酒体柔和协调。     

Implications of nitrogen nutrition for grapes, fermentation and wine

This review discusses the impacts of nitrogen addition in the vineyard and winery, and establishes the effects that nitrogen has on grape berry and wine composition and the sensory attributes of wine. Nitrogen is the most abundant soil‐derived macronutrient in a grapevine, and plays a major role in many of the biological functions and processes of both grapevine and fermentative microorganisms. Manipulation of grapevine nitrogen nutrition has the potential to influence quality components in the grape and, ultimately, the wine. In addition, fermentation kinetics and formation of flavour‐active metabolites are also affected by the nitrogen status of the must, which can be further manipulated by addition of nitrogen in the winery. The only consistent effect of nitrogen application in the vineyard on grape berry quality components is an increase in the concentration of the major nitrogenous compounds, such as total nitrogen, total amino acids, arginine, proline and ammonium, and consequently yeast‐assimilable nitrogen (YAN). Both the form and amount of YAN have significant implications for wine quality. Low must YAN leads to low yeast populations and poor fermentation vigour, increased risk of sluggish/stuck/slow fermentations, increased production of undesirable thiols (e.g. hydrogen sulfide) and higher alcohols, and low production of esters and long chain volatile fatty acids. High must YAN leads to increased biomass and higher maximum heat output due to greater fermentation vigour, and increased formation of ethyl acetate, acetic acid and volatile acidity. Increased concentrations of haze‐causing proteins, urea and ethyl carbamate and biogenic amines are also associated with high YAN musts. The risk of microbial instability, potential taint from Botrytis‐infected fruit and possibly atypical ageing character is also increased. Intermediate must YAN favours the best balance between desirable and undesirable chemical and sensory wine attributes. ‘Macro tuning’, of berry nitrogen status can be achieved in the vineyard, given genetic constraints, but the final ‘micro tuning’ can be more readily achieved in the winery by the use of nitrogen supplements, such as diammonium phosphate (DAP) and the choice of fermentation conditions. This point highlights the need to monitor nitrogen not only in the vineyard but also in the must immediately before fermentation, so that appropriate additions can be made when required. Overall, optimisation of vineyard and fermentation nitrogen can contribute to quality factors in wine and hence affect its value. However, a better understanding of the effect of nitrogen on grape secondary metabolites and different types of nitrogen sources on yeast flavour metabolism and wine sensory properties is still required.     

酿酒酵母低温耐受机制的研究进展

酿酒酵母（Saccharomyces cerevisiae）和发酵温度是影响葡萄酒产量和品质的关键性因素。低温发酵可以促进酯类、醇类、酮类、萜烯类等芳香物质的合成,提高葡萄酒质量。然而,低温发酵会延长酿酒酵母的潜伏期,降低细胞活性和发酵速率,甚至使发酵过程中止。因此,研究酿酒酵母的低温耐受机制为解决葡萄酒低温发酵这一工业难题提供了理论依据。介绍了低温对不同酿酒酵母生理特性、发酵性能、基因表达和细胞成分变化的影响,以及识别和响应低温信号的分子机制等方面的研究进展。     

Mead production: effect of nitrogen supplementation on growth,fermentation profile and aroma formation by yeasts in mead fermentation

Mead is an alcoholic beverage, produced since ancient times, resulting from an alcoholic fermentation of diluted honey by yeasts. When it is produced in a traditional manner, mead producers can encounter several problems related to a lack of essential nutrients, such as available nitrogen. Thus, the aim of this study was to evaluate the effect of nitrogen addition to honey‐must on the fermentation performance of two Saccharomyces cerevisiae wine yeasts, QA23 and ICV D47, as well as on the mead composition and production of volatile aroma compounds. A portion of honey‐must was supplemented with diammonium phosphate (DAP) to achieve the nitrogen concentration required by yeast to complete alcoholic fermentation. The supplementation with DAP reduced the fermentation length to around 7 days, but not all sugars were fully consumed, suggesting that other factors could be interfering with yeast growth. For both yeasts the specific growth rate and final biomass were higher in musts supplemented with DAP. Mead final composition was similar under the two experimental conditions. Analysis of the volatile profile revealed that the concentrations of the volatile fatty acids and volatile phenols were higher in meads supplemented with DAP. The concentrations of ethyl hexanoate, ethyl octanoate and isoamyl acetate were above their perception threshold and were higher in meads supplemented with DAP, which could contribute to the enhancement of the fruity character. This study could be useful for the optimization of mead production and quality improvement. Copyright © 2015 The Institute of Brewing & Distilling     

Nitrogen requirements of commercial wine yeast strains during fermentation of a synthetic grape must

Nitrogen deficiencies in grape musts are one of the main causes of stuck or sluggish wine fermentations. Currently, the most common method for dealing with nitrogen-deficient fermentations is adding supplementary nitrogen (usually ammonium phosphate). However, it is important to know the specific nitrogen requirement of each strain, to avoid excessive addition that can lead to microbial instability and ethyl carbamate accumulation. In this study, we aimed to determine the effect of increasing nitrogen concentrations of three different nitrogen sources on growth and fermentation performance in four industrial wine yeast strains. This task was carried out using statistical modeling techniques. The strains PDM and RVA showed higher growth-rate and maximum population size and consumed nitrogen much more quickly than strains ARM and TTA. Likewise, the strains PDM and RVA were also the greatest nitrogen demanders. Thus, we can conclude that these differences in nitrogen demand positively correlated with higher growth rate and higher nitrogen uptake rate. The most direct effect of employing an adequate nitrogen concentration is the increase in biomass, which involves a higher fermentation rate. However, the impact of nitrogen on fermentation rate is not exclusively due to the increase in biomass because the strain TTA, which showed the worst growth behavior, had the best fermentation activity. Some strains may adapt a strategy whereby fewer cells with higher metabolic activity are produced. Regarding the nitrogen source used, all the strains showed the better and worse fermentation performance with arginine and ammonium, respectively.     

新疆精河枸杞果酒发酵工艺的研究

该文以新疆精河新鲜枸杞为原料,通过初始糖量、发酵温度、发酵时间、发酵接种量、pH值等单因素对枸杞果酒发酵工艺的影响,结合响应面分析法研究初始糖量、发酵温度、接种量对枸杞果酒品质的影响,得出枸杞果酒品质与影响因素间的回归模型,根据模型进行工艺参数优化,结果表明,发酵最优条件为:初始糖量为18％,发酵温度28℃,发酵时间7d,pH值为4.5,果酒酵母接种量6％.     

氮源对葡萄酒酵母发酵性能影响的研究进展

酵母对于葡萄酒发酵至关重要,其在酒精发酵中有特定的营养需求。氮源作为葡萄的内源性营养物质,对酵母的生长代谢、发酵速率及香气等次级代谢物的产生有重要影响。在葡萄酒酿造过程中,酿酒酵母占据主导地位,其对氮源的利用和代谢已有广泛研究。近年来,由于非酿酒酵母的优良发酵特性被逐渐挖掘,相关研究激增;氮源对其生长与发酵性能的影响也成为研究热点。该文综述了近年来氮源对于酿酒酵母和非酿酒酵母生长、发酵及产香能力影响的研究进展,并讨论了未来潜在研究领域与方向,以期为我国葡萄酒酵母发酵特性及混合发酵中营养管理策略的深入研究分析提供借鉴与参考。     

模拟葡萄汁中可同化氮和还原糖对酵母发酵特性的影响

为研究葡萄汁中可同化氮和还原糖对酵母发酵特性的影响,设计150、240、330、420、500?mg/L可同化氮质量浓度和170、200、230?g/L还原糖质量浓度,共计15?个处理,测定了模拟葡萄汁酒精发酵过程中酵母生长、还原糖消耗和可同化氮消耗的变化。结果表明,模拟汁中可同化氮质量浓度过低（150?mg/L）则不能充分满足酵母生长的需要,同时限制了酵母的还原糖消耗速率,通过提高初始还原糖质量浓度至200?g/L可促进酒精发酵进行;酵母在初始可同化氮质量浓度高于240?mg/L的模拟汁中可以正常生长,此时初始还原糖、可同化氮质量浓度对酵母生长量均无显著影响,还原糖含量最直接影响酿酒酵母菌株的发酵特性,决定发酵时间长短,表现为在初始还原糖质量浓度较低（170?g/L）的模拟汁中,酵母生长速率随着模拟汁初始可同化氮质量浓度的升高而加快,在初始还原糖质量浓度较高（200～230?g/L）的模拟汁中,酵母生长速率不受初始可同化氮质量浓度的影响;当模拟汁初始可同化氮质量浓度高于330?mg/L时,酵母对可同化氮的消耗开始出现剩余,剩余量随着模拟汁初始可同化氮质量浓度的升高而增加,此时可同化氮质量浓度能够充分满足酵母可同化氮代谢的需要,且酵母对可同化氮消耗量随着初始还原糖质量浓度的增加而略有减少。     

Fermentation kinetics and chemical characterisation of vino tostado,a traditional sweet wine from Galicia (NW Spain)

BACKGROUND: Grapes after harvesting are air dried and pressed in order to concentrate sugars, acids and flavour compounds to produce vino tostado (toasted wine), a wine with intense aroma and flavour notes and high residual sugar concentration. In order to get a better knowledge of the difficulties involved, several fermentations were conducted at 12 and 28 °C using 0, 15 and 30 g hL^?1 ammonium sulfate and 0, 25 and 50 g hL^?1 exogenous commercial yeast (Saccharomyces cerevisiae var. bayanus) to study the kinetics of sugar consumption and ethanol, acetic acid and glycerol production. RESULTS: Fermentation kinetic parameters were calculated and metal concentrations and antioxidant activity were analysed. CONCLUSION: The spontaneous fermentation at 12 °C and all fermentations conducted with the commercial yeast gave vino tostado of adequate quality, while the spontaneous fermentation at 28 °C was sluggish. High‐temperature fermentations led to sweeter wines with higher volumetric productivities, although low‐temperature fermentations produced better wines in terms of higher glycerol and lower acetic acid levels. Fructose was the only sugar to be consumed during spontaneous fermentations, while both glucose and fructose were consumed during fermentations of the inoculated musts, with preference for each monosaccharide depending on temperature. Copyright © 2009 Society of Chemical Industry     

液态发酵法酿造野木瓜果醋

以野木瓜为原料,采用液态发酵法酿造野木瓜果醋。对酵母菌进行选择,当安琪葡萄酒干酵母与马克思克鲁维酵母菌GIM2.119以1∶1(体积比)比例混合时,最适合野木瓜酒精发酵。研究发酵温度、初始糖度、酵母量对酒精发酵影响,采用均匀设计试验确定酒精发酵最佳工艺条件:温度为28℃,酵母量为8.5%,初始糖度为17.0%,在此最佳条件下进行酒精发酵,酒精度可达8.13%vol。探讨了温度、酒精度、醋酸菌接种量、转速对醋酸发酵的影响,响应面分析法结果表明醋酸发酵最佳工艺条件,酒精度为7.0%vol,接种量为8.0%,温度为32℃,转速为115 r/min,在此条件下,总酸含量为7.60 g/100 mL。野木瓜果醋成品感官、理化、微生物指标符合国标。     

响应面法优化蜂蜜酒发酵工艺

以狼牙蜂蜜为原料,考察发酵液糖度、温度、pH值等参数对蜂蜜酒品质的影响。试验采用Box-Behnken试验设计和响应面法优化蜂蜜酒发酵工艺,确定最优的发酵条件。结果表明,当初始糖度为25 °Bx、发酵温度为25 ℃、pH值为5.0时所酿制蜂蜜酒的品质最佳,且产品具有蜂蜜独特的风味。     

Influence of addition of yeast autolysate on the formation of amines in wine

Amines, substances which can be toxic or have a negative effect on wine aroma, are formed during alcoholic and malolactic fermentations depending on various factors. In this work the influence of the addition of yeast autolysate to must on the concentration of amines in wine is studied. For this purpose Chardonnay must supplemented with the yeast autolysate was used; a second sample was also used without it (control sample). This autolysate provided amino acids and long‐chain fatty acids (C16 and C18). The enrichment of must by these nutrients did not produce an increase of amines during alcoholic fermentation despite the fact that consumption of amino nitrogen in general, and of some precursor amino acids in particular, was somewhat higher in the supplemented sample. After malolactic fermentation it was observed that the concentration of biogenic amines was higher in the wine from the supplemented must. Copyright © 2006 Society of Chemical Industry     

甘露糖蛋白对‘赤霞珠’葡萄酒中糖酸和香气的影响

为进一步探究甘露糖蛋白对葡萄酒感官品质的影响,以'赤霞珠'葡萄为原料酿造葡萄酒,分别在其酒精发酵前和酒精发酵后添加不同量的甘露糖蛋白,在酒精发酵结束后放置12个月检测葡萄酒常规理化指标和单体糖、有机酸、香气物质含量并进行感官品评。结果表明,酒精发酵前添加甘露糖蛋白对于葡萄酒感官品质无促进作用,而酒精发酵后添加0.3 g/L甘露糖蛋白可使葡萄酒中果糖含量降低20.7%,苹果酸和酒石酸分别降低4.4%和8.4%,同时成倍提高了香气组分中酯类、醇类的含量,从而提高葡萄酒的感官品质。因此,甘露糖蛋白的添加可作为提高葡萄酒感官品质的处理措施,推荐在酒精发酵后以0.3 g/L的剂量添加。