Influence of temperature and ethephon concentration on growth and composition of Cabernet Sauvignon grapevines

Potted Cabernet Sauvignon grapevines were acclimated to two different temperature regimes (25°C/15°C and 35°C/25°C day/night temperatures, respectively) until 100% bloom, when the vines were treated with either 0, 250, 500, or 750 ppm ethephon (Ethrel^®). Three days after ethephon application all vines were combined and held at 25°C/15°C in a phytotron room for 15 weeks. Growth was suppressed by a greater range of ethephon concentrations at the cool temperature, but effects were shorter-lived than at the high temperature. Generally, the 500 ppm treatment reduced vigor most effectively. The degree to which ethephon influences vine growth is mediated by temperature. Ethephon and temperature treatments caused significant differences in the concentrations of potassium, calcium, and magnesium in leaves.     

本土酵母NX11424对赤霞珠葡萄酒发酵中酵母菌多样性及香气成分的影响

【背景】NX11424酵母是一株具有良好发酵特性且能赋予赤霞珠葡萄酒浓郁果香的宁夏本土酿酒酵母。【目的】解析NX11424酵母发酵的赤霞珠葡萄酒中的水果香气特征。【方法】以赤霞珠为材料,设置3个发酵处理：自然发酵、灭菌接种NX11424的发酵和直接接种NX11424的发酵,利用26S rDNA D1/D2区测序分析法鉴定发酵过程中酵母菌的种类,并通过顶空固相微萃取和气相色谱-质谱联用技术定量测定不同发酵处理下赤霞珠葡萄酒的香气成分及含量。【结果】三个发酵处理下赤霞珠葡萄酒各理化指标无显著性差异。所分离到的酵母菌鉴定为2属3种：萄葡汁有孢汉逊酵母(Hanseniaspora uvarum)、酿酒酵母(Saccharomyces cerevisiae)和布拉迪酵母(Saccharomyces boulardii,S. boulardii);这2属3种均存在于自然发酵中,接种发酵中仅存在H. uvarum和S. cerevisiae两种酵母。三个发酵处理下的赤霞珠葡萄酒中香气物质种类无差异,均为69种;其中,酯类28种,醇类25种,有机酸5种,萜烯类2种和其他类化合物9种;但各发酵处理下香气物质的含量存在显著差异。聚类分析表明,69种香气成分被聚为3类。第1类香气物质包括香茅醇、丙醇等9种成分,其中7种香气物质的含量在灭菌接种发酵中较高;第2类香气物质包括己酸乙酯、棕榈酸乙酯等31种成分,其含量均在直接接种发酵中较高;第3类香气物质包括丁酸乙酯、乳酸乙酯等29种成分,其中27种香气物质的含量在自然发酵中较高。虽然直接接种发酵处理中酵母菌的多样性低于自然发酵处理,但是该处理的赤霞珠葡萄酒中酯类香气物质含量较多,水果香气浓郁,对赤霞珠葡萄酒香气的改善更明显。【结论】NX11424与发酵中的其他本土酵母间的相互作用可以改善葡萄酒的质量,为宁夏本土酵母NX11424在赤霞珠葡萄酒酿造过程中改善葡萄酒质量奠定了坚实的基础。     

Yeast species associated with spontaneous wine fermentation of Cabernet Sauvignon from Ningxia,China

The eastern base of the Helan Mountains in Ningxia is a fast developing wine production area in China. Of urgent necessity to the Ningxia wine industry is to be able to produce wines with typical regional characteristics. It is well known that autochthonous yeast species and strains play an important role in introducing local character or terroir into the winemaking practice. The aim of this study was to investigate indigenous yeast species diversity and preselect desirable S. cerevisiae strains in the Ningxia region. Four hundred wine-related yeast colonies were isolated from Cabernet Sauvignon musts in three vineyards at the beginning, middle and final stages of spontaneous fermentations. Yeast species were first classified according to colony morphologies on Wallerstein Laboratory Nutrient Agar (WL) and confirmed by sequencing of the D1/D2 domain of the 26S rRNA gene. Nine unique colony morphology types were profiled on WL agar and three new types were found. After sequence analysis of representative colonies from each WL group, nine yeast species were identified, namely H. uvarum, H. occidentalis, M. pulcherrima, C. zemplinina, H. vineae, I. orientalis, Z. bailii, P. kluyveri and S. cerevisiae. The non-Saccharomyces yeasts appeared mainly in the early stage of the fermentation while H. uvarum, I. orientalis and P. kluyveri were able to remain in the final stage. Fourty-six S. cerevisiae isolates were preselected according to physiological characteristics and twelve strains with valuable fermentation properties were obtained.     

Influence of wine fermentation temperature on the synthesis of yeast-derived volatile aroma compounds

The yeast Saccharomyces cerevisiae synthesises a variety of volatile aroma compounds during wine fermentation. In this study, the influence of fermentation temperature on (1) the production of yeast-derived aroma compounds and (2) the expression of genes involved in aroma compounds’ metabolism (ADH1, PDC1, BAT1, BAT2, LEU2, ILV2, ATF1, ATF2, EHT1 and IAH1) was assessed, during the fermentation of a defined must at 15 and 28°C. Higher concentrations of compounds related to fresh and fruity aromas were found at 15°C, while higher concentrations of flowery related aroma compounds were found at 28°C. The formation rates of volatile aroma compounds varied according to growth stage. In addition, linear correlations between the increases in concentration of higher alcohol and their corresponding acetates were obtained. Genes presented different expression profiles at both temperatures, except ILV2, and those involved in common pathways were co-expressed (ADH1, PDC1 and BAT2; and ATF1, EHT1 and IAH1). These results demonstrate that the fermentation temperature plays an important role in the wine final aroma profile, and is therefore an important control parameter to fine-tune wine quality during winemaking.     

遮阳网对酿酒葡萄果实及葡萄酒品质的影响

遮阳网可以通过调节微气候来影响果树的生长和果实的发育。该试验以酿酒葡萄‘赤霞珠’和‘西拉’为材料,于2015年在新疆和硕进行遮阳网试验,处理时间为转色期(8月5日)至采收前12d(9月5日),探究遮阳网对酿酒葡萄果实和相应葡萄酒相关品质指标的影响。结果表明:(1)遮阳网显著降低了日均温度、光照强度和光合有效辐射,增加了日均湿度,有效改善了葡萄生长的微气候。(2)与露地相比,遮网处理使葡萄果实的横径、纵径和单果重都有所增加;能够显著抑制葡萄果实糖分的过快积累和有机酸的快速降解,进而降低葡萄酒酒精度;能够显著提高葡萄果实和葡萄酒中总酚和总单宁含量,并显著降低总花色苷和总类黄酮含量。(3)采用HPLC法在葡萄和葡萄酒中共检测出9种单体花色苷,在‘赤霞珠’果实中除花青素-3-葡萄糖苷外,其余8种单体花色苷的含量都是露地对照高于遮阳网处理,在‘西拉’果实中除二甲花翠素-3-香豆酰化葡萄糖苷外,其余8种单体花色苷也是露地对照高于遮阳网处理。研究结果为解决炎热产区生产中葡萄成熟过快、葡萄酒酒精度过高等问题以及提高酿酒葡萄和葡萄酒品质具有一定的实际指导意义。     

三种酒曲酿造的崇明老白酒的挥发性风味物质分析及比较

分别采用八二酒曲、纯种米根霉曲及酿酒酵母曲、纯种米根霉曲和粘红酵母曲按崇明老白酒工艺酿造老白酒,应用顶空固相微萃取-气相色谱质谱联用法对样品进行挥发性物质的检测,并对其进行醇酯比、香气活力值(OAVs)的计算及主成分分析,筛选出较佳的酒曲,得出崇明老白酒的主体香气.辛酸乙酯、1-辛烯-3-醇、3-甲基丁酸乙酯、己酸乙酯和乙酸异戊酯的OAVs大于10,是崇明老白酒的主要呈香物质.由八二酒曲酿造的酒液挥发性物质总量最高,醇酯比为5.89∶1,呈香物质以具蘑菇香的1-辛烯-3-醇为主.米根霉和酿酒酵母曲酿造的酒液酯类、芳香族化合物种类最多,醇酯比3.24∶1,香气以丁酸乙酯的甜香、苯乙醛、苯乙醇和乙酸苯乙酯的玫瑰花香为主要特征.米根霉和粘红酵母曲酿造的酒液挥发性化合物总量最低,醇酯比为2.86,主要香气特征为辛酸乙酯的甜香和己酸乙酯的水果香.纯种曲酿造的酒液的香气类型、风味种类比例、呈香物质的丰富程度均优于八二酒曲酿造的酒液.     

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.     

酿酒葡萄‘赤霞珠’VvbHLH 93基因的克隆及分析北大核心CSCD

bHLH93转录因子参与调节植物的生长发育、应对各种胁迫等多种生理过程,该研究以酿酒葡萄‘赤霞珠’为试验材料,采用RT-PCR方法克隆葡萄VvbHLH 93基因全长,并进行生物信息学分析;用qRT-PCR法分析bHLH93在不同组织和果实不同发育时期的表达量,为进一步探索VvbHLH 93基因的功能及其机制奠定基础。结果表明:(1)成功克隆获得葡萄VvbHLH 93,该基因cDNA全长为1319 bp,开放阅读框长度为939 bp,编码312个氨基酸,相对分子量为35.18 kD,理论等电点为4.68,无信号肽和跨膜域,属于bHLH转录因子家族。(2)葡萄bHLH93蛋白与荷花的亲缘关系较近;VvbHLH93蛋白的C端为酸性结构区,富含丝氨酸、苏氨酸磷酸化位点;VvbHLH 93基因的启动子含有光响应元件和低温、干旱、赤霉素等应答元件。(3)qRT-PCR分析表明,VvbHLH 93在‘赤霞珠’中的表达具有组织特异性,在叶片中基本不表达,在茎中的表达量最高;随着赤霞珠葡萄果实的发育成熟,VvbHLH93相对表达量不断降低,到幼果期(直径>2 mm)后第5周开始相对表达量基本为0,总体呈现降低的变化趋势;与对照相比,在低温胁迫、盐胁迫、高温胁迫及充分灌溉胁迫下VvbHLH 93表达量显著降低。研究推测,VvbHLH 93基因可能是葡萄抗逆胁迫的负转录因子。     

葡萄品种赤霞珠采收期果穗和果粒性状的差异分析

作为发酵前的一个重要工艺过程,穗选和粒选在优质葡萄酒生产中被广泛采用,但人们对果穗之间和同一果穗内果粒之间品质差异的了解还非常有限。本研究对采收期大小和紧密度不同的4穗成熟赤霞珠葡萄进行了分析:对每穗果实的果穗紧密度、果穗重量、每果穗中果粒数量,每粒果的种子数量和重量、可溶性固形物浓度和果皮颜色等进行了检测,并在此基础上进行了穗选和粒选的模拟试验。结果表明:果粒中可溶性固形物的积累与果粒的曝光程度相关性较差,但果粒的曝光程度显著影响果皮着色,果穗紧密程度显著影响浆果果皮着色和种子成熟;每粒果实的可溶性固形物浓度与果粒重相关性不强,但随着果粒重的增加,可溶性固形物浓度有减小的趋势。穗选和粒选能提高中等大小果粒(0.76~1.50 g)所占的比例,使1粒种子果粒所占的比例降低,增加单位重量果实中的种子重量,降低果粒重的变异系数,但对整个浆果群体可溶性固形物浓度分布和平均值无实质影响。因此,影响酿酒葡萄果穗潜在质量的关键因素在于果穗紧密度、果粒大小和种子成熟度;建议在优质葡萄酒生产过程中先进行穗选,淘汰紧密度过紧的果穗,然后在此基础上再进行粒选。     

Two new grape cultivars, bud sports of Cabernet Sauvignon bearing pale-coloured berries, are the result of deletion of two regulatory genes of the berry colour locus

Bud sports are infrequent changes in phenotype affecting shoots of woody perennials but the molecular basis of these mutations has rarely been identified. In this report, we show that the bronze-coloured berries of the Malian cultivar, a documented bud sport of the wine grape Cabernet Sauvignon (Vitis vinifera L.), lack anthocyanins in the subepidermal cells compared to the red/black berried Cabernet Sauvignon in which both the epidermis and several subepidermal cell layers contain anthocyanin. The Malian phenotype is correlated with an alteration in the genome indicated by a reduction of hybridisation signal using a MYBA probe. In Shalistin, a white-berried bud sport of Malian, the red allele at the berry colour locus appears to have been deleted completely. These data suggest that Malian could be a L1/L2 periclinal chimera, which gave rise to Shalistin by an invasion of epidermal cells (L1) by the mutated subepidermal cells (L2). The red grape Pinot Noir has given rise to a number of pale coloured sports, although the provenance of the extant sports is not known. We show that a clone of Pinot Blanc (white-berried) does not have a deletion of the red allele of the same dimensions as that in Shalistin, though a small deletion is a likely explanation for the altered phenotype. However, the mechanism of deletion of the red allele of the berry colour locus is a possible means by which other red to white clonal mutations of grapevines have occurred.     

Investigations on the genetics of foaming in wine yeasts

Summary The phenomenon of foaming is an undesirable characteristic of some wine yeasts. Investigation of this phenotype indicated that it is under the control of at least two dominant genes 21 centimorgans apart on the same chromosome. Elimination of the foaming character by hybridisation demonstrates that pure-culture wine yeast material can be improved by this technique.     

Genetic instability of heterozygous, hybrid, natural wine yeasts

We describe a genetic instability found in natural wine yeasts but not in the common laboratory strains of Saccharomyces cerevisiae. Spontaneous cyh2(R)/cyh2(R) mutants resistant to high levels of cycloheximide can be directly isolated from cyh2(S)/cyh2(S) wine yeasts. Heterozygous cyh2(R)/cyh2(S) hybrid clones vary in genetic instability as measured by loss of heterozygosity at cyh2. There were two main classes of hybrids. The lawn hybrids have high genetic instability and generally become cyh2(R)/cyh2(R) homozygotes and lose the killer phenotype under nonselective conditions. The papilla hybrids have a much lower rate of loss of heterozygosity and maintain the killer phenotype. The genetic instability in lawn hybrids is 3 to 5 orders of magnitude greater than the highest loss-of-heterozygosity rates previously reported. Molecular mechanisms such as DNA repair by break-induced replication might account for the asymmetrical loss of heterozygosity. This loss-of-heterozygosity phenomenon could be economically important if it causes sudden phenotype changes in industrial or pathogenic yeasts and of more basic importance to the degree that it influences the evolution of naturally occurring yeast populations.     

Influence of glucose concentration on the physiology and lipid composition of some yeasts

The fatty acid contents of the “Crabtree-positive” yeastsSaccharomyces cerevisiae, S. carlsbergensis andS. delbrueckii decreased with increasing concentrations of glucose in the medium. These lower values were due to a lower content of sterol esters and phosphatides inS. cerevisiae, and of sterol esters inS. carlsbergensis. In contrast the fatty acid contents of the Crabtree-negativeS. fragilis, Schwanniomyces occidentalis andCandida utilis increased with increasing concentrations of glucose and inCandida utilis this was due almost entirely to a higher content of triglycerides. This work was supported in part by grant B/SR/5780 from the Science Research Council. We are grateful to the Brewer's Society for a Research Scholarship to Mr. B. Johnson. We thank Mr. A. Bradley for competent technical assistance.     

抗性砧木对赤霞珠葡萄叶片白藜芦醇合成过程及含量的影响

为了明确不同抗性砧木对赤霞珠葡萄叶片白藜芦醇含量及其合成过程中前体物质和相关代谢酶活性的影响,分析不同抗性砧木与白藜芦醇合成的关系,以获得提高接穗品种赤霞珠葡萄叶片白藜芦醇含量的抗性砧木。该研究选择弗卡(Fercal)、5C、140R、3309M、3309C、SO4、抗砧3号(Kangzhen3)、5BB为砧木与赤霞珠(CS)葡萄进行嫁接,以赤霞珠自根苗为对照(CK),采用高效液相色谱技术,测定成熟葡萄叶片白藜芦醇以及合成白藜芦醇前体物质苯丙氨酸、肉桂酸、香豆酸含量,并对合成白藜芦醇相关代谢酶苯丙氨酸解氨酶(PAL)、肉桂酸-4-羟化酶(C4H)、4-香豆-辅酶A连接酶(4CL)、过氧化物酶(POD)以及多酚氧化酶(PPO)的活性进行测定。结果表明:(1)不同砧木均能提高接穗赤霞珠葡萄叶片白藜芦醇含量11%~46%。(2)在各砧穗组合中,CS/140R嫁接苗叶片的白藜芦醇含量最高达到18.24μg/g,其合成白藜芦醇前体物质苯丙氨酸和香豆酸含量也最高,分别达到38.61和1.06μg/g。(3)在各砧穗组合中,白藜芦醇相关代谢酶PAL活性以CS/3309C嫁接苗叶片最高;嫁接苗叶片代谢酶C4H和4CL活性均高于赤霞珠自根苗;POD和PPO活性均以CS/Fercal的接穗赤霞珠叶片最强。研究发现,不同抗性砧木能显著提高赤霞珠葡萄叶片白藜芦醇含量,相关代谢酶C4H活性对葡萄白藜芦醇的合成至关重要,PPO活性与葡萄叶片白藜芦醇合成也密切相关,CS/140R是8个砧穗组合中提高赤霞珠葡萄叶片白藜芦醇含量最具优势的砧穗组合。     

Fermentation-based biotransformation of bioactive phenolics and volatile compounds from cashew apple juice by select lactic acid bacteria

Select lactic acid bacteria (LAB); Lactobacillus plantarum, L. casei and L. acidophilus were targeted for enhancing bioactives and flavor volatiles of cashew apple juice (CAJ) that is an underutilized byproduct from cashew nut processing in Tropical countries. Results indicated the vitamin C and phenolic metabolites such as condensed tannin can be increased at certain stages such as at 12 h over the 48 h fermentation period. Whereas antioxidant activity based on DPPH and ABTS radical scavenging activity generally decreased from initial unfermented stage range of (75%–95%) to consistently in the 50% range by 48 h of fermentation and this follows the decrease in viable counts. The fermentation process increased the condensed tannin contents in CAJ whereas hydrolysable tannins decreased. In this study the changes in flavor volatile types were also analyzed over the course of CAJ fermentation. The results indicated that LAB changed the flavor profiles of fermented CAJ and overall the fruity odor decreased, but the whiskey and acid odor increased. These results provide the foundation to further target the functional benefits of LAB-induced fermented CAJ for further human, animal, and plant health applications.     

Interspecies hybridization and recombination in Saccharomyces wine yeasts

The ascomycetous yeasts traditionally referred to as the Saccharomyces sensu stricto complex are a group of closely related species that are isolated by a postzygotic barrier. They can easily hybridize; and their allodiploid hybrids propagate by mitotic divisions as efficiently as the parental strains, but can barely divide by meiosis, and thus rarely produce viable spores (sterile interspecies hybrids). The postzygotic isolation is not effective in allotetraploids that are able to carry out meiosis and produce viable spores (fertile interspecies hybrids). By application of molecular identification methods, double (Saccharomyces cerevisiae x Saccharomyces uvarum and S. cerevisiae x Saccharomyces kudriavzevii) and triple (S. cerevisiae x S. uvarum x S. kudriavzevii) hybrids were recently identified in yeast populations of fermenting grape must and cider in geographically distinct regions. The genetic analysis of these isolates and laboratory-bred hybrids revealed great variability of hybrid genome structures and demonstrated that the alloploid genome of the zygote can undergo drastic changes during mitotic and meiotic divisions of the hybrid cells. This genome-stabilization process involves loss of chromosomes and genes and recombination between the partner genomes. This article briefly reviews the results of the analysis of interspecies hybrids, proposes a model for the mechanism of genome stabilization and highlights the potential of interspecies hybridization in winemaking.     

赤霞珠葡萄果实苹果酸生物合成关键转录因子的筛选与鉴定

该研究以宁夏贺兰山东麓酿酒葡萄种植区栽培面积最大的‘赤霞珠’为材料,在前期完成从果实形成至成熟不同发育时期的转录组测序以及关键有机酸含量测定基础上,进一步通过转录因子结合位点预测、差异表达基因分析、加权基因共表达网络关联分析(WGCNA),逐步筛选出与‘赤霞珠’果实苹果酸生物合成相关功能基因特异结合的、影响苹果酸生物合成的相应转录因子,并对其进行qRT PCR验证,以揭示这些关键功能基因及其关键转录因子在葡萄不同种植区、果实不同发育时期存在的相互调控作用机制,为以后培育优质酿酒葡萄提供新的理论依据与思路。结果表明：(1)GC/MS分析发现, ‘赤霞珠’果实在4个发育时期的延胡索酸和苹果酸含量变化趋势基本一致,两种酸含量均从果实硬果期到绿果期逐步升至最高(3.63和626.53 μg/g),之后缓慢下降,经转色期到成熟期后逐渐降至最低(2.14和244.26 μg/g),而草酰乙酸的变化趋势却相反,在硬果期含量最高(315.54 μg/g),经绿果期、转色期到成熟期逐渐降至最低值(126.11 μg/g)。(2)‘赤霞珠’果实发育时期样本转录组测序共获得可能与苹果酸生物合成途径12种功能基因结合的转录因子6 411个,其中延胡索酸水化酶(FH)的3个功能基因有86个转录因子,苹果酸脱氢酶(MDH)的10个功能基因有717个转录因子。(3)转录组测序数据及其与有机酸含量WGCNA关联结果的Veen分析确定了‘赤霞珠’果实成熟过程中与苹果酸生物合成相关度最高的3个FH基因(VIT_14s0060g01700、VIT_13s0019g03330、VIT_07s0005g00880)、2个MDH基因(VIT_10s0003g01000、VIT_13s0019g05250)及相应的18个关键转录因子。(4)qRT PCR验证及相关性分析表明, FH基因VIT_13s0019g03330与其转录因子VIT_01s0011g06200、VIT_08s0056g01230以及MDH基因VIT_13s0019g05250与其转录因子VIT_06s0004g04960、VIT_10s0003g02070的表达水平与苹果酸的积累存在显著正相关关系,推测这4个关键转录因子可能通过调控功能基因的转录,综合影响‘赤霞珠’果实苹果酸的生物合成。     

Influence of anxiety on the production of oral volatile sulfur compounds

Since many patients complain about halitosis without there being any clinical evidence of its cause, psychological symptoms have been pointed out as halitosis-inducing factors. The aim of this study was to evaluate the influence of anxiety on the production of volatile sulfur compounds (VSC). Seventeen undergraduate men in good oral and general health participated in this study, after approval by the ethics committee. The volunteers were requested to refrain from toothbrushing, using mouth rinse and eating on the experimental day. Before presenting the anxiogenic condition, the volunteer was asked to fill out the Beck Anxiety Inventory questionnaire, to check whether he had been exposed to stressors during the previous week. The Video-Recorded Stroop Color-Word Test (VRSCWT) was used to elicit anxiety. The VSC (halimeter), blood pressure, heart rate and salivary flow measurements were taken before and after the VRSCWT. The volunteers presented a minimal or slight level of anxiety before the test. There was an increase in the oral concentration of VSC, Systolic Blood Pressure and of heart rate (p < 0.05) after the VRSCWT, and no changes in the salivary flow. The results of the present study showed that the anxiogenic condition (VRSCWT) induced increases in VSC concentration, which might contribute to halitosis.