sign in sign up
<<
Home , Malic acid

Czech J. Food Sci. Vol. 27, 2009, Special Issue

Glucose, l- and pH Effect on Products in Biological Deacidification

A. Kunicka-Styczyńska*

Institute of Fermentation Technology and Microbiology, Technical University of Lodz, 90-924 Lodz, Poland, *E-mail: [email protected]

Abstract: Industrial yeasts Saccharomyces cerevisiae Syrena, an interspecies hybrid (S. cerevisiae × S. bayanus) HW2-3 and Schizosaccharomyces pombe met 3–15 h+ were examined to determine changes in fermentation profiles in different environmental conditions in YG medium with different concentrations of (2, 6, 40 or 100 g/l), l-malic acid (4, 7 or 11 g/l) and at pH 3.0, 3.5 and 5.0. The results were obtained by HPLC method (organic acids, acetaldehyde, glycerol, diacetyl) and enzymatically (l-malic acid, ethanol). In anaerobic conditions (100 g/l glucose), the optimal parameters for l-malic acid decomposition for S. cerevisiae Syrena and the hybrid HW2-3 were 11 g/l l-malic acid and pH 3.0 and 3.5, respectively. S. pombe expressed the highest demalication activity at 40 and 100 g/l glucose, 7 g/l l-malic acid and pH 3.0. The fermentation profiles of selected metabolites of yeast were unique for specific industrial strains. These profiles may help in the proper selection of yeast strains to fermentation and make it possible to predict the organoleptic changes in the course of fruit must fermentation.

Keywords: wine yeast; Saccharomyces cerevisiae; Schizosaccharomyces pombe; l-malic acid; biological deacidification

Introduction changes in the fermentation profiles (selected organic acids, acetaldehyde and glycerol) of two The excessive acidity of fruit musts is one of the wine yeast strains Saccharomyces sp. in contrast to main problems in the wine industry, so biological fission yeasts S. pombe. The impact of glucose and methods of acidity adjustment are of great inter- l-malic acid initial concentrations as well as pH on est to wine producers. l-Malic acid is one of the these fermentation products was investigated. dominant organic acids in musts and it can be both aerobically and anaerobically decomposed by wine yeasts. The ability of Saccharomyces yeasts to Material and Methods decompose extracellular l-malic acid differs from insignificant to 45% (Rodriguez & Thornton Microorganisms. Wine yeasts S. cerevisiae Syrena 1990). In turn, Schizosaccharomyces pombe can and interspecies hybrids S. cerevisiae × S. baya- decompose l-malate completely (Taillandier & nus HW2-3 obtained by natural hybridisation are Strehaiano 1991), but its application in wine- deposited in the Pure Culture Collection at the making is limited due to the production of a spe- Institute of Fermentation Technology and Micro- cific off-flavour (Taillandier et al. 1995). The biology, Technical University of Lodz LOCK 105. biological deacidification of musts will enable Fission yeast S. pombe met 3–15 h+ was obtained producing wine with the right sensory properties from the Pure Culture Collection at the Institute with a balance between sugar, acids and aroma of Microbiology, Wroclaw University. components (Volschenk et al. 2003). l-Malic acid Media and . Fermentations were can also promote the growth of bacteria carried out in YG medium composed of glucose (2, causing wine spoilage after bottling (Redzepovic 6, 40 or 100 g/l), l-malic acid (4, 7 or 11 g/l), yeast et al. 2003). Wine chemical composition is highly extract (5 g/l), KH2PO4 (5 g/l) and MgSO4∙7H2O dependent on yeast species and environmental (0.8 g/l) at 28°C during 12 hours. Different initial conditions. The aim of this study was to determine glucose and l-malic acid concentrations at pH

S319 Vol. 27, 2009, Special Issue Czech J. Food Sci.

3.0, 3.5, 5.0 in a batch culture in 2.5 l fermentor malic acid between S. cerevisiae and were studied. The medium was inoculated with S. bayanus, which confirms the differentiation of precultures of yeasts to a final concentration of 5% fermentation profiles. Similar results were obtained (w/v). The precultures were prepared in the YG in studies of S. bayanus and S. paradoxus yeast medium with 4 g/l glucose and without l-malic (Redzepovic et al. 2003). Cultures of S. pombe acid and incubated at 28°C during 24 h before revealed an increased efficiency in l-malic acid inoculation. decomposition with increased glucose concentra- Chemical analysis. l-Malic acid and ethanol tions (Table 1). The malic enzyme of S. pombe is were determined enzymatically with specialised dependent on NAD+ and located in cytosol, and kits (Boehringer Mannheim, GmbH Germany). its regulation is different from Saccharomyces Citric, succinic, lactic and acetic acids, acetal- (Volschenk et al. 2003). An increased expression dehyde and glycerol were determined by HPLC of this enzyme under high glucose concentrations method (Frayne 1986). was also observed in other studies (Groenewald Statistical analysis. Results were presented as & Viljoen-Bloom 2001; Redzepovic et al. 2003). an arithmetic mean of 6 assays and were analysed The production of glycerol gradually increased using a 3-way ANOVA test at a confidence level of with glucose concentration in the hybrid HW2-3 P < 0.05. Calculations were conducted by means cultures. According to the literature, yeast respond of STATISTICA 5.5. Software. to osmotic stress with higher production of glycerol (Groenewald & Viljoen-Bloom 2001; Vol- schenk et al. 2003), which is consistent with the Results and Discussion results of the studies presented. The permanent, low concentration of glycerol in the cultures of The fermentation profiles of wine yeast S. cere- S. cerevisiae Syrena revealed that this strain was visiae Syrena, interspecies hybrids S. cerevisiae × not very sensitive to osmotic stress. The ethanol S. bayanus HW2-3 and S. pombe were investigated content depended on the glucose concentration in at four different glucose concentrations (2, 6, 40, the medium and no straight correlation between and 100 g/l), with a presence of l-malic acid at l-malic acid consumption and ethanol production the initial concentration of 7 g/l. S. cerevisiae was observed. yeast showed the highest demalication activity In anaerobic conditions (100 g/l glucose), demali- in the medium with 2 g/l glucose (Table 1). No cation activity increased with the initial concentra- statistically significant differences (P < 0.05) were tion of l-malic acid only for S. cerevisiae Syrena observed in the concentration of l-malic acid in and the hybrid HW2-3 (Table 1). These results are the cultures of the hybrid HW2-3 in the presence consistent with previous studies into the activity of of glucose at 2, 6, and 100 g/l. S. cerevisiae yeast S. cerevisiae (Delcourt et al. 1995; Volschenk has a mixed respiro-fermentative metabolism et al. 2003) and S. pombe (Taillandier & Stre- when the glucose concentration in the growth haiano 1991). medium exceeds 1 mM (Verduyn et al. 1984). A gradual increase in the content of Thus, already at a glucose concentration of 2 g/l (statistically significant differences at P < 0.05) was glucose repression is observed, while an increase observed for S. cerevisiae Syrena with increased in the share of fermentative metabolism occurs concentrations of l-malic acid up to 7 g/l and for with increased sugar concentrations. In glucose- the hybrid HW2-3 in the studied range of 4–11 g/l. repressed cells the number of mitochondria de- In Saccharomyces sp. cells, there are two possible creases (Dejean et al. 2000), which negatively routes for malic acid decomposition. Malate can affects the malic enzyme located in them and be transformed into pyruvate via malic enzyme results in decreasing the demalication activity or into fumarate and then succinate via of Saccharomyces yeast. The results presented and fumarate reductase, respectively (Radler are consistent with studies by Redzepovic et al. 1986). Both the results presented and the literature (2003), where a decrease in the expression of the (Ramon-Portugal et al. 1999) confirm that an malic enzyme gene under glucose repression was increase in the malic acid concentration results in observed. Differences in the deacidification activity the higher production of succinic acid. At the same of S. cerevisiae Syrena and the interspecies hybrid time, other researchers report differences in the HW2-3 indicate differences in the regulation of concentration of succinic acid in fermented

S320 Czech J. Food Sci. Vol. 27, 2009, Special Issue

f c c c c e e e b b b b,c a,b a,b c,d glucose 11 nd 100 2 3.40 0.18 3.86 0.44 0.65 1.14 7.85 0.07 0.31 0.10 11.09 2.92 0.06 0.06 0.22 f i c c c c c c e e e e e a b b d d d d e,f c,d 7 40 nd nd 5.0 1.71 3.11 0.19 3.40 0.18 4.79 3.02 0.65 8.50 0.65 1.14 0.60 0.01 0.08 0.07 0.97 0.27 0.24 0.93 9.09 11.09 0.22 f g e e e e a a a a a a g b b b b b d d d d d S. pombe 6 4 nd 3.5 0.69 1.46 0.68 1.40 0.90 0.08 0.10 0.03 0.04 0.04 0.82 2.87 0.21 0.10 0.07 1.39 5.76 3.93 0.21 3.90 0.27 11.63 15.96 -malic acid 7 g/l, pH 3.0; l 1 3 f f c c c c c e e a a a a a a b b b d b,c b,c a,b c,d 2 0 nd 3.0 < 0.05); 1.74 3.39 0.15 3.40 0.18 0.65 1.14 0.17 0.01 0.04 0.02 0.02 0.03 2.40 0.83 0.07 0.54 and pH 10.42 11.09 P 2.98 0.13 0.06 0.22 2 i h f f f c c e e e e g g b d a,b 11 -malic acid 100 0.51 0.77 0.52 3.30 3.43 0.63 0.35 2.27 0.40 8.31 2.30 0.07 5.68 l 24.59 0.06 20.75 , 1 i i g h f f c c c c c e e e a a a g g d d d h a,b 7 40 5.0 6.88 0.51 3.15 0.49 3.30 3.12 0.42 0.31 0.63 0.37 0.01 0.35 0.01 2.27 2.25 0.24 0.94 5.68 1.07 24.59 22.75 15.80 0.06 12.68 g g f c c c c c c e e a a a a b b b b d d d d d 6 4 3.5 1.60 3.02 0.17 3.20 0.36 5.29 3.07 1.41 0.33 0.05 0.02 0.02 0.03 0.23 0.40 3.18 0.07 5.77 0.24 0.25 0.59 0.26 16.19 16.60 Inoculated yeast strain Interspecies hybrid HW2-3 i g f f c e e a a a g g b b b d d d a,b 2 0 nd nd nd nd 3.0 3.30 1.67 0.51 3.00 0.63 0.35 0.16 0.04 0.23 0.76 2.27 0.22 0.11 0.09 5.72 3.85 5.68 24.59 16.07 0.06 h f f f f e b b b d d d d g,h 11 nd nd 100 0.43 7.61 0.48 1.60 0.69 2.80 0.16 2.76 5.79 0.54 0.25 0.22 19.36 17.62 f g h f f f c e e e a g b b b b b d d d h a,b 7 40 nd nd 5.0 0.43 6.82 0.43 3.22 6.25 0.64 0.60 0.02 0.63 2.69 0.24 0.10 2.80 0.16 0.30 5.79 0.25 2.97 12.76 14.58 0.11 19.36 Syrena g h f f c c e a a a a a a b b b d d d d h b,c a,b a,b 6 4 3.5 6.53 1.75 3.21 0.35 6.25 0.16 0.02 0.03 0.03 0.04 0.02 2.71 3.06 0.08 4.77 0.52 0.29 0.27 2.71 2.98 16.39 0.06 0.06 19.34 S. cerevisiae -malic acid 7 g/l j l h f f c c a a a a a a a b d d d d b,c b,c a,b 2 0 nd nd nd 3.0 0.48 0.43 4.94 0.43 0.11 0.01 0.01 0.01 0.03 0.13 2.19 2.80 1.04 5.79 0.54 0.25 36.23 2.92 0.13 0.16 19.36 glucose 100 g/l, 3 (g/l) 2 (g/l) Concentrations of selected yeast metabolites (g/l) in medium with different initial levels of glucose 1 alic acid 3 -Malic acid -Malic acid -Malic acid -M Glucose l Succinic acid Lactic acid Acetic acid Acetaldehyde Glycerol Ethanol l l Citric acid Succinic acid Lactic acid Acetic acid Acetaldehyde Glycerol Ethanol pH l Citric acid Succinic acid Lactic acid Acetic acid Acetaldehyde Glycerol Ethanol nd – not detected; all values are an arithmetic mean of 6 assays; different letters inthe same line indicate100 g/l, significant pH 3.0; differences ( Table 1. Table

S321 Vol. 27, 2009, Special Issue Czech J. Food Sci. by different strains (Heerde & Radler 1978; Frayne R.F. (1986): Direct analysis of the major organic Redzepovic et al. 2003), which is also confirmed components in must and wine using HPLC. by the results presented in this paper. American Journal of Enology and Viticulture, 37: The changes in pH affected l-malic acid con- 281–287. sumption by the tested yeast. Optimal pH for the Groenewald M., Viljoen-Bloom M. (2001): Factors metabolism of this acid by S. cerevisiae Syrena and involved in the regulation of the Schizosaccharomy- S. pombe was 3.0, and 3.5 for hybrid HW2-3. It is ces pombe malic enzyme gene. Current Genetics, 39: known that l-malic acid enters S. cerevisiae cells 222–230. by simple diffusion and the optimal pH range for Heerde E., Radler F. (1978): Metabolism of the anaero- this process is 3.0–3.5 (Delcourt et al. 1995; bic formation of succinic acid by Saccharomyces cere- Volschenk et al. 2003), so the presented findings visiae. Archives of Microbiology, 117: 269–276. are consistent with these data. The observed sta- Radler F. (1986): Microbial . Experientia, tistically significant changes (P < 0.05) in glycerol 42: 884–892. formation by S. cerevisiae Syrena and the hybrid Ramon-Portugal F., Seiller I., Taillandier P., HW2-3 were the result of even a slight pH shift Favarel J.L., Nepveu F., Strehaiano P. (1999): Ki- from 3.0 to 3.5. Vigorous glycerol production is netics of production and consumption of organic ac- usually the effect of adverse environmental condi- ids during alcoholic fermentation by Saccharomyces tions during acidic must fermentation (Soufleros cerevisiae. Food Technology and Biotechnology, 37: et al. 2001); therefore, its concentration in young 235–240. wines may be elevated. The tested strains are sensi- Redzepovic S., Orlic S., Majdak A., Kozina B., Vol- tive to pH changes, which is also reflected in the schenk H., Viljoen-Bloom M. (2003): Differential patterns of their fermentation profiles. malic acid degradation by selected strains of Saccha- In anaerobic conditions (100 g/l glucose), optimal romyces during alcoholic fermentation. International parameters for l-malic acid decomposition for Journal of Food Microbiology, 83: 49–61. S. cerevisiae Syrena and the hybrid HW2-3 were Rodriquez S.B., Thornton R.J. (1990): Factors in- 11 g/l l-malic acid and pH 3.0 and 3.5, respectively. fluencing the utilization of l-malate by yeasts. FEMS S. pombe expressed the highest demalication ac- Microbiology Letters, 72: 17–22. tivity at 40 and 100 g/l glucose, 7 g/l l-malic acid Soufleros E.H., Pissa I., Petridis D., Lygerakis and pH 3.0. The results presented showed that M., Mermelas K., Boukouvalas G., Tsimitakis the fermentation profiles of selected metabolites E. (2001): Instrumental analysis of volatile and other of yeast are unique for specific industrial strains compounds of Greek kiwi wine; sensory evaluation used for acidic must fermentation. These profiles and optimisation of its composition. , may help in selecting the right yeast strain for 75: 487–500. fermentation and make it possible to predict the Taillandier P., Strehaiano P. (1991): The role of organoleptic changes in the course of fruit must l-malic acid in the metabolism of Schizosaccharomyces fermentation. pombe: substrate consumption and cell growth. Ap- plied Microbiology and Biotechnology, 35: 541–543. Taillandier P., Gilis M., Strehaiano P. (1995): Reference s Deacidification by Schizosaccharomyces: interactions with Saccharomyces. Journal of Biotechnology, 40: Dejean L., Beauvoit B., Guérin B., Rigoulet M. 199–205. (2000): Growth of the yeast Saccharomyces cerevisiae Verduyn C., Zomerdijk T.P.L.J., Van Dijken P., Schef- on a non-fermentable substrate: control of energetic fers W.A. (1984): Continuous measurement of etha- yield by the amount of mitochondria. Biochimica et nol production by aerobic yeast suspensions with an Biophysica Acta, 1457: 45–56. enzyme electrode. Applied Microbiology and Biotech- Delcourt F., Taillandier P., Vidal F., Strehaiano nology, 19: 181–185. P. (1995): Influence of pH, malic acid and glucose Volschenk H., Van Vuuren H.J.J., Viljoen-Bloom concentrations on malic acid consumption by Sac- M. (2003): Malo-ethanolic fermentation in Saccha- charomyces cerevisiae. Applied Microbiology and Bio- romyces and Schizosaccharomyces. Current Genetics, technology, 43: 321–324. 43: 379–391.

S322