Resistance to imidazoles and triazoles in Saccharomyces cerevisiae as a new dominant marker.

The imidazole and triazole fungicides inhibit cytochrome P450 14 alpha-lanosterol demethylase (P45014DM) implicated in the ergosterol biosynthesis pathway, which is specific to fungi and yeasts. Two plasmids were obtained which allow triazole and imidazole resistance in Saccharomyces cerevisiae. The low copy number plasmid (pFD 383) encodes cytochrome P450 C14 alpha lanosterol demethylase under the control of phospho-glycerate-kinase promoter. S. cerevisiae transformed by the pFD 383 plasmid are resistant to imidazoles and triazoles. Moreover, this transformed strain shows increased levels of P45014DM mRNA and of cytochrome P450. A second low copy number plasmid (pFD 384) carries a mutant cytochrome P450 14 alpha lanosterol demethylase gene, which increases imidazole and triazole resistance. These constructions can be used on a dominant selection marker to transform wild-type yeasts and to confer imidazole and triazole resistance in industrial fermentation.

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.

Evolution of acetic Acid bacteria during fermentation and storage of wine.

Acetic acid bacteria were present at all stages of wine making, from the mature grape through vinification to conservation. A succession of Gluconobacter oxydans, Acetobacter pasteurianus, and Acetobacter aceti during the course of these stages was noted. Low levels of A. aceti remained in the wine; they exhibited rapid proliferation on short exposure of the wine to air and caused significant increases in the concentration of acetic acid. Higher temperature of wine storage and higher wine pH favored the development and metabolism of these species.

Inhibition of alcoholic fermentation of grape must by Fatty acids produced by yeasts and their elimination by yeast ghosts.

In a complete nutritive medium rich in sugar, such as grape must, the inhibition of alcoholic fermentation is caused by substances produced by the yeast which, acting synergistically with ethanol, are toxic to the yeasts themselves. Among these are decanoic and octanoic acids and their corresponding ethyl esters. Their adsorption by yeast ghosts permits the prevention and treatment of fermentation stoppages.

Occurrence of lactic Acid bacteria during the different stages of vinification and conservation of wines.

We showed that the growth of lactic acid bacteria during alcoholic fermentation depends on the composition of the must. We illustrated how the addition of sulfur dioxide to the must before fermentation and the temperature of storage both affect the growth of these bacteria in the wine. Whereas species of Lactobacillus and Leuconostoc mesenteroides were isolated from grapes and must, Leuconostoc oenos was the only species isolated after alcoholic fermentation. This organism was responsible for the malolactic fermentation. Isolates of this species varied in their ability to ferment pentoses and hexoses. The survival of Leuconostoc oenos in wines after malolactic fermentation depended on wine pH, alcohol concentration, SO(2) concentration, and temperature of storage.

Relationship between the sterol content of yeast cells and their fermentation activity in grape must.

In grape must of high sugar concentration, yeast growth, the viability rate of "resting" yeast cells, and fermentation activity were stimulated under certain conditions of aeration and temperature. This stimulation might be interpreted as being a result of the yeast cell sterol content. The addition of certain sterols to the fermenting medium was able to increase this sterol content. According to aeration conditions of the medium, which determined the sterol content of yeasts, the sterols added in the medium acted as (i) growth factors, (ii) fermentation inhibitors, and (iii) survival factors for the yeast.

[Occurrence of an extracellular beta-(1 to 3)-glucanase from Botrytis cinerea]. / Mise en évidence d'une bêta-(1 à 3)-glucanase exocellulaire chez Botrytis cinera.

Like other fungi, Botrytis cinera produces an exocellular beta-(1 leads to 3)-glucanase. This constitutive enzyme is produced only when Botrytis is cultivated in a glucose depleted medium. The enzyme is precipitated by ammonium sulfate from 20 to 80% saturation. Maximum activity is at pH between 5 and 6 and temperature between 40 and 50 degrees C; stability is good up to 30 degrees C. This enzyme cannot be applied to the treatment of wines containing Botrytis glucan.

Evidence for the existence of "survival factors" as an explanation for some peculiarities of yeast growth, especially in grape must of high sugar concentration.

The retardation and arrest of fermentation, observed before the complete sugar consumption of high-sugar grape must, come from an inhibition of the yeast metabolism during its decline phase and are variable with the strain. The addition of nutritional growth factors stimulates the initial growth of the yeast but is ineffective in the decline phase. Some substances, known previously as yeast anaerobic growth factors (sterols, oleanolic acid, oxytocin), in some conditions (initially aerated grape must and aerobically cultivated yeast) act by increasing the viability of the resting cells and prolonging their fermentation activity. These substances have been named "survival factors."

[The various functions of steroids on the yeast metabolism in grape must during fermentation: the notion of survival factor (author's transl)]. / Les différents rôles fonctionnels des stéroïdes sur les levures dans le moût de raisin en fermentation: notion de facteur de survie.

The physiological effects of steroids supplied to the yeasts in grape must is complicated. These substances may function as inhibitors, as growth factors or survival factors. The steroids have a "survival factor" action when they act on aerobically cultivated yeasts, fermenting under anaerobic conditions, in grape must of high sugar concentration. In this case, the substances have no effect on the cellular multiplication but they maintain viability and fermentation activity in the resting cells. Consequently, a greater quantity of sugar is consumed and the content of secondary products is modified by the end of the fermentation. Certain growth conditions during fermentation affect the survival factor action of steroids: high temperature, low pH, vitamin deficiency, low amount of inoculum, insufficient aeration, excessive sugar concentration.

[Trehalose, the principle disaccharide of wine]. / Le tréhalose, principal diholoside des vins

By gas chromatography of trimethylsilylated derivatives of sugars, trehalose has been shown to be the main disaccharide in wines; its amount can reach 600 mg/l. Other disaccharides identified (sucrose, isomaltose, lactose and turanose) are present in small amounts, seldom above 50 mg/l, sometimes below 5 mg/l. Traces of melibiose and gentiobiose are possible. Conditions of trehalose formation by yeast during fermentation are described. Also technological applications of these results are discussed.