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.     

Fermentation rates of grape juice: v. Biotin content of juice and its effect on alcoholic fermentation rate

Microbiological analysis showed that juices from white grapes had less biotin than juices from red grapes. The biotin content of the juices of some varieties was significantly different from that of other varieties. We did not note any regional effects on the biotin content of the juices. Biotin content of the Cabernet Sauvignon grapes increased significantly with maturity, whereas the biotin content of a white variety did not. The biotin content, with the total nitrogen, can be used to estimate indirectly the yeast growth potential and hence to predict the fermentation rate of the juice. About 84% of the rate variation can be accounted for by the calculated regression equations.     

Chemical Characterization of Wines Fermented with Various Malo-lactic Bacteria

Six malo-lactic strains of lactic acid bacteria were isolated from California wines and identified as Lactobacillus delbrueckii, L. buchneri, L. brevis, Leuconostoc citrovorum, and two strains of Pediococcus cerevisiae. Malo-lactic fermentation was induced in separate lots of wine by inoculation of each lot with one of the strains of bacteria. Malo-lactic fermentation had occurred in each inoculated wine within 2 months. The resultant wines were subjected to chemical analysis, including gas chromatographic examination of concentrated extracts of the wines. Only a few differences in composition were found when the malo-lactic wines were compared one with another. The differences that were found were in volatile acidity and in concentrations of acetoin (plus diacetyl) and probably diethyl succinate.     

Alcohol Dehydrogenase Activities of Wine Yeasts in Relation to Higher Alcohol Formation

Alcohol dehydrogenase activities were examined in cell-free extracts of 10 representative wine yeast strains having various productivities of higher alcohols (fusel oil). The amount of fusel alcohols (n-propanol, isobutanol, active pentanol, and isopentanol) produced by the different yeasts and the specific alcohol dehydrogenase activities with the corresponding alcohols as substrates were found to be significantly related. No such relationship was found for ethanol. The amounts of higher alcohols formed during vinification could be predicted from the specific activities of the alcohol dehydrogenases with high accuracy. The results suggest a close relationship between the control of the activities of alcohol dehydrogenase and the formation of fusel oil alcohols. Also, new procedures for the prediction of higher alcohol formation during alcoholic beverage fermentation are suggested.     

Stimulatory Effect of Malo-Lactic Fermentation on the Growth Rate of Leuconostoc oenos

Although l-malic acid is not an energy source for the malo-lactic organism Leuconostoc oenos (L. citrovorum) ML 34, the growth rate of the organism was found to be greatly increased by malo-lactic fermentation (the decarboxylation of l-malic acid to l-lactic acid). The stimulation was especially striking at the low pH (below pH 4) of wine, the natural habitat of this bacterium. The stimulation of growth did not result from changes in pH that accompany malo-lactic fermentation. Thus, these results suggest a biological function of malo-lactic fermentation.     

Multiplicity and control of alcohol dehydrogenase isozymes in various strains of wine yeasts

An investigation has been made of selffertile (homothallic) progeny which are frequently encountered in matings of heterothallic Phytophthora species. The pattern of segregation of self-sterility from these homothallic isolates during vegetative growth, asexual and sexual reproduction has been studied in some detail.Heterokaryosis was shown not be the cause of this secondary homothallism in a number of cases investigated since homothallism could be transmitted by single uninucleate zoospores.The homothallic cultures (A¹ A²) showed different degrees of stability. One culture, obtained as a very fertile sector from an original A¹ A² gave only A¹ A² phenotypes on repeated single zoospore analysis, whilst two other sectors from the same A¹ A² culture gave in addition to A¹ A², some A² and a few A¹ phenotypes. The A² types derived from this homothallic were also unstable, giving some A¹ A² and occasional A¹ types as well as A² types in single zoospore cultures. This contrasts with the stability of the parental A² culture.Self-fertility was transmitted through the oospore to the sexual progeny. Most progeny were A¹ A² but heterothallic A¹ and A² types segregated.It is suggested that the self-fertile condition was due to the presence of an extra chromosome containing the mating type locus, the A¹ A² type being Aaa. A cytological investigation to test this trisomic hypothesis is being reported separately.     

Multiplication and Fermentation of Saccharomyces cerevisiae under Carbon Dioxide Pressure in Wine

Conditions for rapid fermentation of sugar in wine under pressure were sought for use in continuous production of naturally fermented sparkling wine. Wine yeast growth and fermentation were measured under CO(2) pressure. The medium was white wine with added glucose. Pressure was very inhibitory to growth, especially at low pH or high alcohol concentration. Use of various strains of wine yeast, cultures of various ages, or cells adapted to wine did not give more rapid growth. Addition of nutrients increased growth, but under no conditions was growth rapid enough to bring about sufficiently rapid fermentation rates. Conditions for rapid fermentation were sought by use of high levels of cells as inocula. Fermentation rates in wine also were inhibited by pressure, and were dependent on pH and alcohol levels. Addition of nutrients did not increase the fermentation rate, but rapid fermentation rates were obtained, under pressure, by inoculation with high levels of cells adapted several weeks to the base wine. Thus, continuous sparkling-wine production might be practical with proper amounts of adapted cells used as inocula, or perhaps with reuse of the fermentation culture.     

Carbonic acid from decarboxylation by "malic" enzyme in lactic acid bacteria

Carbonic anhydrase studies were used to determine the primary form of carbonic acid produced from decarboxylation of l-malic acid by "malic" enzyme in malolactic strains of five different species of lactic acid bacteria. Addition of carbonic anhydrase to the reaction mixture containing crude bacterial extract and l-malic acid, at pH 7, in all five cases resulted in an increase (13 to 23%) in the rate of carbon dioxide evolution over the control. The results indicated that the primary form of carbonic acid released from "malic" enzyme was not anhydrous carbon dioxide as previously supposed and as has been shown for other decarboxylating enzymes. The standard free-energy changes of the malo-lactic reaction with the various forms of carbonic acid as the primary decarboxylation product were calculated. The reaction is less exergonic when carbonic acid, bicarbonate ion, or carbonate ion is the primary decarboxylation product compared to anhydrous carbon dioxide. The free-energy of the reaction is not biologically available to the bacteria; with carbon dioxide not the primary decarboxylation product, the potential energy lost in a malo-lactic fermentation is not as great as previously considered. Endogenous carbonic anhydrase activity was not found.     

Cloning the Gene for the Malolactic Fermentation of Wine from Lactobacillus delbrueckii in Escherichia coli and Yeasts

The gene responsible for the malolactic fermentation of wine was cloned from the bacterium Lactobacillus delbrueckii into Escherichia coli and the yeast Saccharomyces cerevisiae. This gene codes for the malolactic enzyme which catalyzes the conversion of l-malate to l-lactate. A genetically engineered yeast strain with this enzymatic capability would be of considerable value to winemakers. L. delbrueckii DNA was cloned in E. coli on the plasmid pBR322, and two E. coll clones able to convert l-malate to l-lactate were selected. Both clones contained the same 5-kilobase segment of L. delbrueckii DNA. The DNA segment was transferred to E. coli-yeast shuttle vectors, and gene expression was analyzed in both hosts by using enzymatic assays for l-lactate and l-malate. When grown nonaerobically for 5 days, E. coli cells harboring the malolactic gene converted about 10% of the l-malate in the medium to l-lactate. The best expression in S. cerevisiae was attained by transfer of the gene to a shuttle vector containing both a yeast 2-mum plasmid and yeast chromosomal origin of DNA replication. When yeast cells harboring this plasmid were grown nonaerobically for 5 days, ca. 1.0% of the l-malate present in the medium was converted to l-lactate. The L. delbrueckii controls grown under these same conditions converted about 25%. A laboratory yeast strain containing the cloned malolactic gene was used to make wine in a trial fermentation, and about 1.5% of the l-malate in the grape must was converted to l-lactate. Increased expression of the malolactic gene in wine yeast will be required for its use in winemaking. This will require an increased understanding of the factors governing the expression of this gene in yeasts.