L-MALIC ACID DEGRADING YEAST FOR WINE MAKING

Abstract: This invention refers to yeasts obtained by fusion of spheroplasts capable of maintaining in a single genotype the demalifying trait of a Schizosaccharomyces pombe parental strain and the growth rate of the parental strain Saccharomyces cerevisiae, 3 new yeasts obtained from protoplast fusion between the fusion product initially obtained (MB7TC alpha ) and the parental strain, and two other Saccharomyces cerevisiae yeasts, and the use of these yeasts for obtaining demalification of acid wines.

Description: L-MALTC ACID DEGRADING YEAST FOR WINE MAKIN G Descriptive Report of the Invention Patent "L- MALIC ACID DEGRADING YEAST FOR WINE MAKING " : This invention deals with a new yeast obtained by fusion between Saccharomyces cerevisiae and Schizosaccharo myces pombe (MB7TC<x- ) , which has the ability to degrade ma¬ lic acid, and of another new yeast obtained by retrofusion between the above fusion product and the parental strain Sac¬ charomyces cerevisiae , as well as new yeasts obtained by fu¬ sion between product MB7TC and a yeast with the Killer pheno- type and another pectinase-producing Saccharomyces cerevisiae yeast ,all of them capable of degrading 1-malic acid. These new yeasts were respectively designated MB7TC-- , MBII, MBK and MBO. The invention also concerns a process for the reduction of malic acidity in wine making. A wine is considered acid when it contains an elevated concentration of 1-malic acid, which in turn is pro¬ duced by fermentation of musts from not fully ripe grapes.L - malic acid is one of the acids present in musts and wines,to- gether with tartaric acid,citric acid, and much smaller amoun ts of oxalic acid, uronic acid,succinic acid,lactic acid, ace tic acid,glioxalic acid,glyceric acid,pyruvic acid.oxaloace - tic acid, alpha-ketoglutaric acid, etc. The two major acids in terms of concentration, tartaric and malic acids, are metabolized to sugar during the process of grape maturation on the vine. At the end of matura tion, malic acid undergoes the greatest rate of degradation , with concentrations being reduced from 200 ιιιEq/1 to less than 50 mEq/1 , while tartaric acid is degraded from 150-200 mEq/1 to 100-150 mEq/1. The me abolization of tartaric acid leads to its lowest concentrations almost one month before the end of grape maturation, and malic acid degradation continues to increase up to full grape maturation. As mentioned earlier , acid musts result from the harvesting of grapes that did not go through the full maturation process. Thus, when fermented, these musts also result in acid wines. Since tartaric acid present in must is stable to the action of bacteria and yeasts, the correction of acidity' in wines by biological processes is limited to malic acid de¬ gradation. Microbial degradation of malic acid can occur through the action of bacteria of the genera Pediococcus,Lac- tobacillus and Leuconostoc via malolactic fermentation,with the formation of lactic acid, or by degradation to ethanol via the action of yeasts that develop maloalcoholic fermentation. Malolactic fermentation primarily occurs in wines with acidity of less than 1 g/1 tartaric acid and pH higher ' than 3.1. Fermentation with homofermen ing bacteria of highes interest for wine should occur at pH between 3.2 and 3.3 (MEN- DOZA,1979) . Schizosaccharomyces pombe yeasts have been used * to decrease malic acid in wine by CASTELLI (1969) ,CΛSTEL I and HAZNEDARI (1971) , BENDA and SCHIMITT (1966) , BENDA (1974) HU- GLIN et al.(1976), GALANDER (1977.) and CARRAU (1981) .However, owing to problems related to the growth rate of Schizosaccha¬ romyces pombe wich require the use of large inocula for wine fication is not practical for common application in wineries. Indeed,one of the objectives of the present invention was to construct yeasts by fusing spheroplasts of Saccharomyces cere visiae ( having a greater growth rate ) and Schizosaccharomy- ces * . These yeasts can solve the problems outlined above becau se their growth rate is more rapid than that of the parental strain Schizosaccharomyces pombe and because they express the ability to degrade 1-malic acid in addition to presenting part of the genome of yeasts traditionally used in wine fermenta - tion. A decrease in malic acid content also favors the' natural development of malolactic fermentation which is known to be a determinant of the biological stabilization of wines. The present invention was based on the use of mi- crobial genetics, to whose methods protoplast fusion was added during the 1970's. This method permits the exchange of genetic material between cells that are not compatible for crossing. For yeasts, protoplast fusion utilizes lytic enzymes from the digestive juice of Helix aspersa which produce the formation of cells fully (protoplasts) or partially (spheroplasts) devoid of walls. Both protoplasts and spheroplasts can be easily fused in the presence of polyethyleneglycol , as initially observed by VAN SOLINGEN and VAN DER PLAAT (1977). The fusion products are then submitted to conditions that will lead to the reestablish ment of the cell wall. These conditions include a regenerating medium containing Ca and a large osmotic concentration, ith' inoculation onto pour-plates yielding the best percentage of regeneration. This technique has been used previously to obtain fusion products of technological interest (RUSSEL and STEWART, 1979 ; TUBB,1979). Modern wine making outfits use selected ' starter. Carrau (1982) reported the demalifying ability of 6 different strains of Schizosaccharomyces pombe , which degra¬ ded 55 to 75% of malic acid within 24 hours and 83.8 to 98.8% after 72 hours. On the basis of this ability of Schizosaccha- romyces pombe to degrade malic acid, (CARRAU,1979) proposed ' large-scale fermentations using this yeast to degrade malic a cid in wine. This process would utilize inocula of 500 x 10 cells of Schizosaccharomyces pombe in order to obtain parcial malic acid degradation during wine fermentation. The methodologies that constitute the inventions * are reported below: 1. Selection of genotypes to obtain the first fu - sion product (MB7TCd) . A strain of Schizosaccharomyces pombe called Benda I was selected as the yeast with the most effective ability to degrade malic acid. As a yeast of enologic use which produces good organoleptic characteristics in wines, we selected the strain Saccharomyces cerevisiae denominated Montrachet. 2. Selection of markers for obtaining MB7TC . The Schizosaccharomyces pombe strain does not grow on galactose as the single source of carbon and its growth on WLN (Difco) gives origin to greenish colonies. The Montrachet strain of Saccharomyces cerevisiae forms white colonies when growing in WLN medium. 3. Production of spheroplasts. This invention was based on the technique of ' GOODEY (1980), except that 0.8 M KCl was substituted for 1 M sorbitol as osmotic buffer. 4. Spheroplast fusion and regeneration. After the appearance of spheroplast levels higher by centrifugation and the pellets pooled in a single tube and treated with 2 ml of a solution of 37% polyethyleneglycol • 4,000 ( GOODEY,1980) . 5. Recovery of fusion products. After the fusion treatment, the cells were regene rated in regenerating medium (6.67 g YNB, 20 g glucose, 180 g sorbitol, 1.11 g CaCl- , and distilled water to 1,000 ml). The colonies formed were observed microscopically to determine the occurrence of colonies with irregular bor ders, called " hairy " colonies. Colony coloration was also ' observed in WLN medium and colonies with irregular borders ' that also showed colors differing from those of the parental' strains in WLN were studied for capacity to degrade malic a - cid. 6. Production of MBO, MBII and MBK. In order to increase the genotypic contribution of Saccharomyces to the fusion products,strain MB7TC was fu¬ sed with strains O Mendoza and KI, both of them Saccharomyces cerevisiae, and also with the parental strain Montrachet. The methods for obtaining new fusion products ' were the same as those described for MB7TC 7. Evaluation of demalifying ability. The clones obtained by spheroplast fusion were studied in test tubes and in 1,000-ml Erlenmayer flasks contai ning grape must. A racemic mixture of malic.acid (10 g/1) was added to the musts for the study of malic acid degradation a- bility. The results for clone MB7TC with respect to fer en - ting ability, as indicated by C0 2 release with the use of Mil ler valves by the method of CASTELLI (1969) , and malic acid degradation are shown in Figure I and Table I, respectively . than that of the parental Schizosaccharomyces pombe strain , a d that its demalifying ability is good. 8. How these fusion products can be used by the wine making industry. In wine making, the concentrations of native yeast cells after the extraction of grape juice often reach values ' higher than 10 cells/ml , a fact implying that the yeasts des cribed in the present invention need to be inoculated at simi¬ lar concentrations for their demalifying ability to be expres- sed. The present invention proposes a wine making process uti- 5 lizing inocula of 100 x 10 cells/ml fusion products at the be ginning of fermentation in order to obtain wines with partial' degradation of 1-malic acid. TABLE I - Malic acid degrada - dation by the fusion product M B7TC*/ and the parental strains Montrachet and Benda I,expres¬ sed as mEq/1 and % total. 72 hours 144 hours Strains mEq/1 mEq/1 Montrachet Benda I MB7TCoC + Montrachet FIGURE I- Fermentation ra¬ te of yeast MB7TC- * * - compa¬ red to that of the paren¬ tal strains Montrachet and Benda I.

Claims: CLAIMS 1. New yeasts characterized by the fact that they are obtained by fusion between Saccharomyces cerevisiae (Mon¬ trachet) and Schizosaccharomyces pombe (Benda I) and that they have the ability to degrade 1-malic acid. 2. New yeasts according to claim 1 characterized by the fact that they are denominated MB7TC<-_ . 3. New yeast according to claims 1 and 2 characte rized by the fact that it is utilized in wine-making proces - ses. 4. New yeast characterized by the fact that it is obtained by fusion between MB7TC**-* and the parental strain Sac charomyces cerevisiae (Montrachet) and has the ability to de - grade 1-malic acid. 5. New yeast according to claim 4 characterized by the fact of being denominated MBII. 6. New yeast according to claim 4 characterized by the fact that it is utilized in wine making processes. 7. New yeast characterized by the fact that it is obtained by fusion between MB7TC - and a Saccharomyces cerevi- siae yeast with killer phenotype and has the ability to degra - de 1-malic acid. 8. New yeast according to claim 7 characterized by the fact that it is denominated MBK. 9. New yeast according to claim 7 characterized by the fact that it is utilized in wine making processes. 10. New yeast characterized by the fact that it is obtained by fusion between MB7TC and a Saccharomyces cerevisi ae yeast denominated 0 Mendoza. 11. New yeast according to claim 10 characterized by the fact that it is denominated MBO. 12. New yeast according to claim 10 characterized by the fact that it is utilized in wine making processes. 13. Process for the reduction of malic acidity in wi¬ ne making characterized by the fact that it utilizes yeasts MB 1TC °<- ,MBII,MBK and MBO obtained according to claims 1,4,7 and 11. 14. Process for the reduction of malic acidity accor¬ ding to claim 13 characterized by the fact that it utilizes con 5 centrations of 100 x 10 cells of any of the yeasts MB7TC txc , MBII,MBK and MBO , as an inoculum to be added to the must at the beginning of fermentation.

Inventors: CARRAU JUAN L, DILLON ALDO JOSE PINHEIRO, PASQUAL MIRIAN SALVADOR, SERAFINI LUCIANA ATTI, CARRAU, JUAN, L., DILLON, ALDO, JOSE, PINHEIRO, PASQUAL, MIRIAN, SALVADOR, SERAFINI, LUCIANA, ATTI
Applicants: UNIV CAXIAS DO SUL, UNIVERSIDADE DE CAXIAS DO SUL
Application year: 1989
Main classification: C12N15
IPC codes: C12G1, C12N1, C12N15, C12P1, C12R1