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Influence of Ph, Malic Acid and Glucose Concentrations on Malic Acid Consumption by Saccharomyces Cerevisiae F

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Influence of Ph, Malic Acid and Glucose Concentrations on Malic Acid Consumption by Saccharomyces Cerevisiae F Influence of pH, malic acid and glucose concentrations on malic acid consumption by Saccharomyces cerevisiae F. Delcourt, Patricia Taillandier, F. Vidal, Pierre Strehaiano To cite this version: F. Delcourt, Patricia Taillandier, F. Vidal, Pierre Strehaiano. Influence of pH, malic acid and glucose concentrations on malic acid consumption by Saccharomyces cerevisiae. Applied Microbiology and Biotechnology, Springer Verlag, 1995, 43 (2), pp.321-324. 10.1007/BF00172832. hal-02143212 HAL Id: hal-02143212 https://hal.archives-ouvertes.fr/hal-02143212 Submitted on 29 May 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Open Archive Toulouse Archive Ouverte (OATAO) OATAO is an open access repository that collects the work of some Toulouse researchers and makes it freely available over the web where possible. This is an author's version published in: https://oatao.univ-toulouse.fr/22860 Official URL : https://doi.org/10.1007/BF00172832 To cite this version : Delcourt, F. and Taillandier, Patricia and Vidal, F. and Strehaiano, Pierre Influence of pH, malic acid and glucose concentrations on malic acid consumption by Saccharomyces cerevisiae. (1995) Applied Microbiology and Biotechnology, 43 (2). 321-324. ISSN 0175-7598 Any correspondence concerning this service should be sent to the repository administrator: [email protected] F. Delcourt · P. Taillandier · F. Vidal · P. Strehaiano Influence of pH, malic acid and glucose concentrations on malic acid consomption by Saccharomyces cerevisiae Abstract Malic acid consumption by Saccharomyces The ability of Saccharomyces cerevisiae to use malic cerevisiae was studied in a synthetic medium. The ex­ acid during the fermentation of a synthetic medium si­ tent of malic acid degradation is affected by its initial mulating grape must has been known for many years concentration, the extent and the rate of deacidification (Vezinhet and Barre 1982), but few studies deal with increased with initial malate concentration up to 10 g/1. the kinetic aspects of malate degradation. It should be For malic acid consumption, an optimal pH range of noted that, with S. cerevisiae, the malic acid is never 3-3.5 was found, confirming that non-dissociated or­ completely consumed (Usseglio-Tomasset 1989). The ganic acids enter S. cerevisiae cells by simple diffusion. extent of malic acid degradation depends on the me­ A full factorial design has been employed to describe a dium composition. It is generally admitted that non-dis­ statistical model of the effect of sugar and malic acid on sociated organic acids enter yeast cells by simple diffu­ the quantity of malate degraded (g/1) by a given sion (Salmon 1987a). Baranowski and Radier (1984) amount of biomass (g/1). The results indicated that the have discovered a specific proteic uptake system of initial malic acid concentration is very important for the malic acid in Saccharomyces bailii, but not in S. cerevi­ ratio of malate consumption to quantity of biomass. siae. Salmon (1987b) showed that the malic enzyme of The yeast was found to be most efficient at higher lev­ S. cerevisiae catalyses the NAD(P) + dependent-decar­ els of malate. boxylation of L-malate into pyruvate. This enzyme is characterised by its ability to decarboxylate oxaloace­ tate and its strong dependence on Mn2 + ions (Puck Introduction and Radler 1974; Kuckzynski and Radier, 1982). Malic acid could play a role in supplying the C4 carbon skele­ Malic acid is one of the main organic acids in grapes, ton through malate dehydrogenase and the oxidative and generally it is necessary to remove it from wines in reactions of the tricarboxylic acid cycle when the usual order to improve their organic qualities and to ensure pathway for carbon skeleton synthesis is deficient (Sal­ their biological stability. The malic acid concentration mon et al. 1987). usually decreases slightly during alcoholic fermenta­ The objective of this work was to increase the level tion, but the consumption of this organic acid depends of deacidification of an enological strain of S. cerevisiae. on the genus and species of the yeasts used. The yeast On the one hand we tried to elucidate the effect of ini­ Schizosaccharomyces pombe has been reported to me­ tial malic acid and glucose concentrations on the level tabolize malic acid efficiently (100%) (Ribereau-Gayon of acid utilisation by the yeast. We were also interested and Peynaud 1962; Mayer and Temperli 1963; Taillan­ in the effect of pH on malic acid metabolism (deacidifi­ dier et al. 1988).The intensity of malic degradation was cation) during alcoholic fermentation. The pH range of not affected by the concentration of malic acid in the enological conditions is 3-4, we therefore studied the medium (Taillandier et al. 1988) unlike the situation in effect of pH on deacidification activity in this range. other yeast genera such as Saccharomyces. On the other hand, we studied the linkage between the ratio of malic acid consumption (g/1) to the amount F. Delcourt P. Taillandier (IBJ) · F. Vidal · P. Strehaiano of biomass (g/1) and initial concentrations of glucose ENSIGC, Laboratoire Génie Chimique, URA CNRS 192, and malic acid. Experimental design techniques have 18 chemin de la loge, F 31078 Toulouse Cedex, France, been used to measure the effect of each factor and the Fax: +33-62 25 23 18 interactions with a relatively small number of experi­ F. Delcourt F. Vidal ments. CRITT BAC, BP 52, 97152 Pointe à Pitre Cedex, France Table 1 Experimental design defining glucose (x1) and malic acid Materials and methods (x2) initial concentration conditions. Glucose: -1 =50 g/1 and + 1 = 100 g/1; malic acid: -1 =2.5 g/1 and + 1 = 7 g/1 Microorganism and culture medium Test Design matrix Work The strain of Saccharomyces cerevisiae 236 employed in this study number matrix was supplied by National Institute of Agronomie Research X1 X2 X1X2 y (INRA) of Guadeloupe. The standard culture medium contained glucose malic acid interaction KH2PO4 5 g/1, MgSO4 ·7H2O 0.4 g/1, (NH4)2SO4 2 g/1, yeast ex­ tract 2 g/1, glucose 100 g/1, malate 4.5 g/1, pH 3.4. For some experi­ 1 -1 -1 +1 0.29 ments the glucose, the malate and pH were varied as described in 2 +1 -1 -1 0.12 Results. 3 -1 +1 -1 0.30 4 +1 +1 +1 0.28 5 0 0 0 0.25 6 0 0 0 0.25 Culture conditions 7 0 0 0 0.25 Batch experiments were performed in 2-1 fermenters (Setric Ge­ nie Industriel, Toulouse). The volume of the medium in the fer­ menter was 1.2 1, the initial concentration of biomass was 3 x 106 cells/ml. The inoculum was prepared in 250-ml conical flasks with Results 150 ml standard culture medium. The temperature was regulated ° at 30 C, the pH was not regulated. Agitation rate was 150 rpm. Effect of initial concentration of malic acid Analytical determinations Experiments were conducted in fermenters with stand­ ard medium except that the malate concentration var­ Biomass was estimated gravimetrically. The cell dry weight was ied from 2.5 g/1 to 11 g/1. Figure 1 clearly demonstrates obtained by filtration of a known sample volume through a cellu­ lose acetate membrane filter (Sartorius 0.45 µm), washed with that the concentration of malic acid (2.5-11 g/1) does distilled water and dried to constant weight. Cell viability was de not affect the specific rate of growth (µ,). For the con­ termined by methylene blue staining. centrations studied, malic acid does not restrict the L Malic acid concentrations were measured using an enzymat yeast's production of biomass. ic procedure from Boehringer Mannheim no 648357. Glucose The quantity of malate consumed increased with the concentrations were estimated by immobilized glucose oxidase (Yellow Springs Instrument) and ethanol concentrations were de initial malate concentration up to 9.5 g/1 (Fig. 2). Mal­ termined by gas chromatography (Intersmat, Pavillon sous bois, ate was not entirely consumed by the yeasts, but with France) with a Porapak-Q column, using !-propanol (1% v/v) as high initial levels of malate (9.5 g/1), 2.2 g/1 malic acid the internai standard and a flame ionisation detector. The carrier was degraded and with low levels (2.5 g/1) only 0.8 g/1 gas was nitrogen (1.8 bars; 30 ml min 1). The temperature of the column was 170° C, that of the detector 250° C and that of the was degraded, that is to say about 60% less. injector 250 ° C. Effect of initial concentration of glucose Experimental design The degradation of 4.5 g/1 malic acid was studied for A full factorial design of experiments was used (Goupy 1988). A different initial glucose concentrations. The sugar con­ linear model was used to show the influence of two factors, glu cose and malate concentrations, on the ratio of malate consump centration was varied from 50 g/1 to 200 g/1 and, all tion (g/1) to the quantity of biomass (g/1) in the culture. This bio cases, glucose was exhausted. We noted that both the logical characteristic (y) is represented on this mode! by Eq. 1, where y is the estimated response and x1(glucose) and x2 (malic acid) are the factor levels: 0.6 (1) □ 2.5 g/l EB 4.5 g/l x1 and x2 are the coded values of each variable, and b0, bi, bii are coefficients of the polynomial estimated by the least-squares a 7 g/l the 0.4 method of fitting with a Statitcf multivariable linear regression <> o 9.5 g/l 1:1 analysis with constant.
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