Sugar and Alcohol Stabilization of Yeast in Sweet Wine

Secondary fermentation of sweet wine was prevented by the Delle stabilization procedure. For this procedure, advantage is taken of the inhibitory effects of high concentrations of sugar as well as of alcohol. Thus, relatively small amounts of wine spirits were added to fermenting musts to obtain stability, as compared to the conventional procedure in which larger amounts of alcohol are added and the inhibitory effect of alcohol only is considered. The Delle value is a function of the concentrations in the wine, after spirits addition, of alcohol and sugar. Delle values which gave stable wine were dependent on time of alcohol addition, on strain of wine yeast, and on composition of wine spirits. Fractional addition of spirits, concentration of SO(2), and clarity of must had little effect on the Delle value. Sensory comparison of wines especially prepared for tasting by the Delle procedure and by the conventional procedure showed the wines made by the Delle procedure to be superior in quality. Under proper storage conditions, the Delle wines were shown to be microbiologically stable and resistant to wine spoilage organisms.     

WL营养琼脂对葡萄酒相关酵母的鉴定效果验证

利用WL营养琼脂对采自葡萄园和葡萄汁发酵过程中的35株酵母菌进行了分类鉴定,同时进行了5.8S-ITS和26S rDNA D1/D2区的扩增与测序。结果表明利用WL营养琼脂的鉴定结果与测序结果基本符合。WL营养琼脂是一种较为有效的葡萄酒相关酵母菌的分类鉴定培养基。     

选择压力对染料脱色酵母菌活性影响的研究

一株对染料活性黑5(Reactive Black 5,RB5)具有高效降解活性的酵母菌—Debaryomyces polymor-phus,在传代培养过程中脱色能力及产锰依赖过氧化物酶(MnP)活性逐步降低。研究结果表明,采用向培养体系中反复投加新鲜染料的方法,可以对脱色酵母菌的生长形成持续性选择压力,从而使菌株的脱色和产MnP活性得以显著提高。     

Effect of Zinc Ions on Conformational Stability of Yeast Alcohol Dehydrogenase

Yeast alcohol dehydrogenase preparations were prepared with the conformational zinc ion removed (Apo-I YADH) and with both the conformational and catalytic zinc ions removed (Apo-II YADH). The unfolding of Apo-I YADH and Apo-II YADH during denaturation in urea solutions was then followed by fluorescence emission, circular dichroism, and second-derivative optical spectroscopies. Compared with the native enzyme, Apo-I YADH incurred some slight unfolding, and its stability against urea was markedly decreased, while Apo-II YADH incurred marked unfolding but contained residual ordered structure even at high urea concentrations. The results show that native YADH is more conformationally stable against urea denaturation than Apo-I YADH, indicating that the conformational Zn²⁺ plays an important role in stabilizing the conformation of the YADH molecule. However, unfolding of the region around the conformational zinc ion is shown not to be the rate limited step in the unfolding of the molecule by the fact that the unfolding and inactivation rate constants of native and Apo-I YADH are the same. It is suggested that the catalytic zinc ion is more important in maintaining the structure of YADH. YADH lost its cooperative unfolding ability after the zinc ions were removed. The shape of the transition curves of Apo-I YADH suggests the existence of an unfolding intermediate. For both native and Apo-I YADH, inactivation occurs at much lower urea concentrations than that needed to produce significant conformational changes of the enzyme molecule. At urea concentration above 4 M, the inactivation rate constants are much higher than those of the fast phase of the reaction of unfolding. These results support the suggestion of flexibility at the active site of the enzyme (C. L. Tsou (1986) Trends Biochem. Sci., 11, 427-429; (1993) Science, 262, 308-381).     

Purification and Some Properties of Alcohol Acetyltransferase from Banana Fruit

Isoamyl acetate, one of the major characteristic aroma compoundsof banana fruit (Musa sapientum L.), was synthesized by thecondensation of acetyl-CoA and isoamyl alcohol under the actionof alcohol acetyltransferase, which was found to be localizedin the soluble fraction of pulp cells. The activity of thisenzyme increased with the ripening of banana fruit. The enzyme was purified about 62-fold. The purified enzyme wasvery labile at pHs lower than pH 7.0 but relatively stable atpH 7.5{small tilde}9. The enzyme was most active at 30C andpH 8.5. The molecular weight was estimated to be about 40,000by gel filtration. Its K_m values for acetyl-CoA and isoamylalcohol were 50 µM and 0.4 mM, respectively. (Received January 28, 1985; Accepted May 30, 1985)     

Effect of Polyelectrolytes on Catalytic Activity of Alcohol Dehydrogenase

Fluorescent and optical spectroscopy were used to study the interaction of alcohol dehydrogenase (ADH) with negatively charged polystyrene sulfonate (PSS) and dextran sulfate (DS), as well as positively charged poly(diallyldimethylammonium) (PDADMA). As found, DS and PDADMA did not affect the structural and catalytic enzyme properties. In contrast, PSS slightly decreased the protein self-fluorescence over 1 h of incubation, which is associated with partial destruction of its quaternary (globular) structure. Investigation of the ADH activity with and without PSS showed its dependency on the incubation time and the PSS presence. Sodium chloride (2.0 and 0.2 M) or ammonium sulfate (0.1 M) added to the reaction mixture did not completely protect the enzyme quaternary structure from the PSS action. However ammonium sulfate or 0.2 M sodium chloride stabilized the enzyme and partially inhibited the negative PSS effect.     

Construction of Killer Wine Yeast Strain

A double-stranded RNA plasmid which confers the superkiller phenotype was transferred into a wine yeast (Montrachet strain 522) and its leucine-requiring derivative (strain 694) by cytoduction, using the protoplast fusion technique. The killer wine yeast constructed completely suppressed the growth of killer-sensitive strains of Saccharomyces cerevisiae in yeast extract-peptone-glucose medium at pH 4.5, whereas the killer effect was somewhat decreased at pH 3.5. The wine yeast harboring the killer factor also inhibited the growth of killer-sensitive cells satisfactorily when it was grown in grape juice.     

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.     

葡萄酒工业酵母生孢特性研究

孢子是单倍体配子,由于具有遗传型与表现型相一致的优点,是遗传学上常用的材料。用工业化葡萄酒活性干酵母进行生孢试验,结果表明,供试8支菌种均可生孢,但生孢率与孢子形态相差较大。其中,17#生孢最强,30#、29#、27#次之,28#、18#、26#渐弱,32#生孢最弱。子囊多为圆形大细胞,孢子多为球形或卵圆形,子囊内一般含2~4个孢子,但29#中可见5~6个孢子。     

Effect of Increased beta-Glucosidase Activity on Virulence of Erwinia amylovora

Plant tissues often contain beta-glucosides that can be enzymatically hydrolyzed to produce toxic aglycones. It has been suggested that the low beta-glucosidase activity found in Erwinia amylovora contributes to bacterial virulence by allowing the bacteria to infect plants that contain beta-glucosides without inducing the formation of toxic aglycones. To test this suggestion, we created strains of E. amylovora which had high beta-glucosidase activities and studied the ability of these strains to cause fire blight disease in pears (Pyrus communis). We isolated spontaneous mutants that were able to utilize beta-glucosides as the sole carbon source and showed that one class had about 10 times as much beta-glucosidase activity as the wild-type strain. In addition, we constructed several plasmids that carry the Escherichia coli bgl operon under the control of a transposon Tn5 promoter that is expressed in E. amylovora. These plasmids were introduced in E. amylovora by transformation. Pathogenesis studies in immature Bartlett pear fruits, etiolated sprouts, and young shoots showed that a 100-fold increase in beta-glucosidase activity does not interfere with normal development of fire blight disease in these model systems.     

Yeast Diversity and Persistence in Botrytis-Affected Wine Fermentations

Culture-dependent and -independent methods were used to examine the yeast diversity present in botrytis-affected (“botrytized”) wine fermentations carried out at high (~30°C) and ambient (~20°C) temperatures. Fermentations at both temperatures possessed similar populations of Saccharomyces, Hanseniaspora, Pichia, Metschnikowia, Kluyveromyces, and Candida species. However, higher populations of non-Saccharomyces yeasts persisted in ambient-temperature fermentations, with Candida and, to a lesser extent, Kluyveromyces species remaining long after the fermentation was dominated by Saccharomyces. In general, denaturing gradient gel electrophoresis profiles of yeast ribosomal DNA or rRNA amplified from the fermentation samples correlated well with the plating data. The direct molecular methods also revealed a Hanseniaspora osmophila population not identified in the plating analysis. rRNA analysis also indicated a large population (>10⁶ cells per ml) of a nonculturable Candida strain in the high-temperature fermentation. Monoculture analysis of the Candida isolate indicated an extreme fructophilic phenotype and correlated with an increased glucose/fructose ratio in fermentations containing higher populations of Candida. Analysis of wine fermentation microbial ecology by using both culture-dependent and -independent methods reveals the complexity of yeast interactions enriched during spontaneous fermentations.     

Flavor Activity of Sulfur-containing Compounds Related to Flavor Nucleotides

Substance B, the major component, isolated from rice plant treated with probenzaole and inoculated, having anti-conidial germination activity against blast fungus, was found to be a mixture of fatty acids, including palmitic acid, linoleic acid and linolenic acid. The main compound of substance B was linolenic acid, having strong anti-conidial germination activity. It was determined as α-linolenic acid by gas chromatographic analysis. The minor components showed little or no anti-conidial germination activity.     

Effect of Simultaneous Inoculation with Yeast and Bacteria on Fermentation Kinetics and Key Wine Parameters of Cool-Climate Chardonnay

Inoculating grape musts with wine yeast and lactic acid bacteria (LAB) concurrently in order to induce simultaneous alcoholic fermentation (AF) and malolactic fermentation (MLF) can be an efficient alternative to overcome potential inhibition of LAB in wines because of high ethanol concentrations and reduced nutrient content. In this study, the simultaneous inoculation of yeast and LAB into must was compared with a traditional vinification protocol, where MLF was induced after completion of AF. For this, two suitable commercial yeast-bacterium combinations were tested in cool-climate Chardonnay must. The time courses of glucose and fructose, acetaldehyde, several organic acids, and nitrogenous compounds were measured along with the final values of other key wine parameters. Sensory evaluation was done after 12 months of storage. The current study could not confirm a negative impact of simultaneous AF/MLF on fermentation success and kinetics or on final wine parameters. While acetic acid concentrations were slightly increased in wines after simultaneous AF/MLF, the differences were of neither practical nor legal significance. No statistically significant differences were found with regard to the final values of pH or total acidity and the concentrations of ethanol, acetaldehyde, glycerol, citric and lactic acids, and the nitrogen compounds arginine, ammonia, urea, citrulline, and ornithine. Sensory evaluation by a semiexpert panel confirmed the similarity of the wines. However, simultaneous inoculation led to considerable reductions in overall fermentation durations. Furthermore, differences of physiological and microbiological relevance were found. Specifically, we report the vinification of “super-dry” wines devoid of glucose and fructose after simultaneous inoculation of yeast and bacteria.