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Effect of yeast strain and fermentation conditions on the release of cell wall polysaccharides
International Journal of Food Microbiology 137 (2010) 303–307
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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o
Short Communication
Effect of yeast strain and fermentation conditions on the release of cell wall polysaccharides Giovanna Giovani, Valentina Canuti, Iolanda Rosi ⁎ Dipartimento di Biotecnologie Agrarie, Università di Firenze, Via Donizetti, 6, 50144, Firenze, Italy
a r t i c l e
i n f o
Article history: Received 31 August 2009 Received in revised form 24 November 2009 Accepted 6 December 2009 Keywords: Cell wall polysaccharides Mannoproteins S. cerevisiae Alcoholic fermentation Wine yeast
a b s t r a c t To improve our understanding of the factors involved in polysaccharide release during alcoholic fermentation, we investigated three Saccharomyces cerevisiae strains in fermentation trials conducted at two temperatures (25 °C and 32 °C) and three sugar concentrations (20%, 23.5%, and 27%), with or without supplementation of grape juice with diammonium phosphate (DAP) or microcrystalline cellulose. In two yeast strains, the release of cell wall polysaccharides increased significantly with an increase in fermentation temperature and sugar concentration of the grape juice; the polysaccharide release was greater in stressed conditions, in which the cells were less viable and less metabolically active. In the third strain, the average amount of polysaccharides released into the medium decreased significantly at 32 °C with 27% sugar, and increased in grape juice supplemented with DAP. Thus, this strain released more polysaccharides when conditions were nearer to optimal and the yeast cells were more viable and metabolically active. Our results suggest that the yeast strains released cell wall polysaccharides via different mechanisms, and that the cell wall integrity pathway may account for some of the differences in polysaccharide release among the strains. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The cell wall of the yeast Saccharomyces cerevisiae is composed of a 10 nm thick layer of polysaccharides (predominantly glucans and mannoproteins) and serves as the interface between the cell and the neighbouring environment. It provides osmotic and physical protection and determines the shape of the cell (Klis et al., 2002). Several papers have reported that S. cerevisiae releases cell wall polysaccharides, particularly mannoproteins, into the extracellular medium during yeast growth (Boivin et al., 1998; Dupin et al., 2000; Llauberes et al., 1987; Rosi et al., 2000). This release may be a consequence of cell wall-controlled hydrolysis of the mother cell to allow the emergence of the bud (Charpentier et al., 1986; Fleet, 1991). It has been suggested that secretion of most yeast mannoproteins and the enzyme involved in cell wall synthesis occurs at the bud site (Lipke and Ovalle, 1998). Wines aged on yeast lees may contain increased amounts of mannoproteins because the dead cells undergo autolysis and enzymatic cell wall degradation. However, not all wines are obtained by aging on lees; therefore, polysaccharide release by S. cerevisiae should also be investigated during alcoholic fermentation of grape must. The yeast strain is one of the factors controlling the amount of mannoproteins released during winemaking (Escot et al., 2001; Llauberes et al., 1987; Rosi et al., 1998; Rosi et al., 2000). Recent studies showed that using autolytic thermosensitive mutants (Giovani
⁎ Corresponding author. Tel.: + 39 055 3220322; fax: + 39 055 355995. E-mail address: rosiol@unifi.it (I. Rosi). 0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2009.12.009
and Rosi, 2007; Gonzalez et al., 2003; Nunez et al., 2005) or genetically engineered wine yeast strains of S. cerevisiae (Brown et al., 2007; Gonzalez-Ramos et al., 2008; Gonzalez-Ramos and Gonzalez, 2006) resulted in increased amounts of mannoproteins in the fermenting medium. In addition, environmental factors such as temperature, carbon source, or level of initial colloid content of the medium have been shown to influence the amount of cell wall polysaccharides secreted into the fermenting medium (Guilloux-Benatier et al., 1995; Rosi and Giovani, 2003). Mannoproteins derived from yeast cell walls have attracted much attention in the winemaking world because of their reported contribution to wine quality. Desirable oenological properties of mannoproteins include protection against protein and tartaric instability (Brown et al., 2007; Dupin et al., 2000; Lubbers et al., 1993; Moine-Ledoux and Dubourdieu, 1998; Waters et al., 1994), retention of aromatic compounds (Chalier et al., 2007; Lubbers et al., 1994), reduction of astringency (Escot et al., 2001), increased sweetness (Rosi et al., 1998), and increased body and mouth feel (Vidal et al., 2004), which are especially appreciated in red wines. Furthermore, mannoproteins stimulate the growth of lactic acid bacteria and consequently malolactic fermentation (Guilloux-Benatier et al., 1995; Rosi et al., 2000), and they improve the foam quality of sparkling wines (Moreno-Arribas et al., 2000; Nunez et al., 2006). Yeast cell wall polysaccharides may also adsorb mycotoxins, thus decreasing their toxic effects and mediating their removal from the medium (Caridi, 2007; Kogan and Kocher, 2007; Moruno et al., 2005). The objective of this study was to investigate the effect of several growth conditions, including temperature, sugar concentration, and supplementation of ammonium salts and microcrystalline cellulose to
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grape juice, on fermentation behaviour and polysaccharide release during alcoholic fermentation by three wine strains of S. cerevisiae. The results of our study may be relevant not only for improving wine quality, but also for more general biotechnological purposes, because there is increasing interest in the production of glucan and mannan for agrofood, pharmaceutical and cosmetic purposes.
grape juice and experimental wines were quantified by HPLC according to the method described by Giovani and Rosi (2007). Results reported are averages of duplicate determinations. The content of released polysaccharides was obtained from the difference between the total polysaccharide content of wines and the original grape juice.
2. Materials and methods
2.5. Statistical analysis
2.1. Chemicals
Treatment means were compared with Fisher's unrestricted least significant difference (FLSD) (STATGRAPHICS 4.1 Plus, 1999; Manugistics, Rockville, MD, USA).
The following chemicals were used: glucose, fructose, microcrystalline cellulose, and diammonium phosphate were obtained from Sigma-Aldrich (Milan, Italy). Yeast extract, peptone, and agar were obtained from Oxoid (Milan, Italy).
3. Results 3.1. Fermentation behaviour of the three wine yeast strains
2.2. Strains We used the two commercial S. cerevisiae strains BM45 (A) and D254 (B) (Lallemand, Montreal, Canada), and S. cerevisiae BLC83 (C), a yeast strain belonging to our wine yeast collection. The yeast strains were maintained on YPD medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, and 15 g/L agar), stored at 4 °C, and subcultured once a year. 2.3. Fermentation trials The same batch of commercial pasteurised white grape juice (marketed by Hero, Verona, Italy) was used for all of the experiments. Juice characteristics were: 200 g/L sugar, titratable acidity (5.1 g/L tartaric acid, pH 3.4), 70 mg/L free α-amino nitrogen, 30 mg/L ammonium nitrogen (100 mg/L total yeast-assimilable nitrogen [YAN]), and 138 mg/L total polysaccharides. Sulfur dioxide was added to the juice at 50 mg/L. The juice was separately adjusted to 235 g/L and 270 sugar by addition of glucose:fructose (1:1). Each lot with the same sugar concentration was divided into three aliquots: one was the control, one was supplemented with 200 mg/L microcrystalline cellulose, an inert support that improves yeast growth and metabolism, and one was supplemented with 300 mg/L diammonium phosphate (DAP). When DAP was added to the juice, the YAN content increased to 160 mg/L. Inocula were prepared by growing a loopful of cells from YPD slants in 100 mL of liquid YPD. Erlenmeyer flasks were incubated at 25 °C and shaken at 150 rpm on a gyratory shaker. After 24 h of growth, cells were inoculated into 1-L glass bottles containing 800 mL of juice, at OD 0.2 (λ = 600 nm) (approximately 1–2 × 106 colonyforming units [CFU]/mL). The flasks were closed with a Müller valve that had previously been filled with sulfuric acid, and incubated at two temperatures: 25 °C and 32 °C. The fermentations were carried out in duplicate. In total, 108 fermentations were performed (18 fermentations for each strain in duplicate). The fermentation rate was monitored daily by weight loss as a result of CO2 escaping from the system, until the weight was constant. 2.4. Analysis After 14 days of fermentation, a sample was withdrawn and different aliquots were used for: (1) viable cell count; (2) dry mass evaluation, after collecting cells on a filter; and (3) ethanol and total polysaccharide contents, after centrifugation to remove yeast cells. Yeast cell viability was checked by plating appropriate dilutions of cells on YPD plates, in triplicate. Plates were incubated at 30 °C for 2 days. Viability was expressed as CFU/mL. Cell dry weight was determined by filtration of a volume of sample on a preweighed filter, washing with distilled water, and drying at 105 °C for 24 h. Ethanol was measured by the Office International de la Vigne et du Vin official method of analysis (OIV, 1990). The polysaccharide contents of the
The alcohol and biomass contents of the wines at the end of alcoholic fermentation are reported in Table 1. At 25 °C, in juices with 20% and 23.5% sugar, all of the strains displayed a higher power to produce alcohol, while in juices with 27% sugar, cellulose and DAP supplementation enhanced alcohol production by strains A and B. At 32 °C, the ethanol production diminished for all strains. When grape juice was supplemented with cellulose, an increase in alcohol production was observed for all strains, whereas DAP supplementation increased alcohol production by strain C in juices with 23.5% and 27% sugar. At 25 °C, strains A and B produced more cells than strain C (Table 1). Cellulose and DAP supplementation stimulated growth of all three strains at 25 °C and 32 °C. However, when we compared the biomass values with the alcohol content of the wines, strain C cells demonstrated greater fermentative activity than strain A or B cells. 3.2. Polysaccharide release Data regarding the influence of the different fermentation conditions on polysaccharide release by the three yeast strains are reported in Table 1. Strains B and C produced, under all tested conditions, polysaccharide amounts that varied between 100 mg/L and 200 mg/L. In contrast, strain A showed a greater dependence on the environmental conditions. The amount of polysaccharides varied from 30 mg/ L in wine obtained from 20% sugar must and fermented at 25 °C, to approximately 200 mg/L in wine obtained from 23.5% sugar must, supplemented with DAP, and fermented at 32 °C. To investigate the effect of different environmental stimuli on the cell response in terms of polysaccharide release, we standardized the amount of polysaccharides based on the final biomass. The average amount of released polysaccharides as a function of temperature, sugar concentration, and must supplementation was determined and analyzed using the FLSD test (Table 2). In strains A and B, the release of macromolecules increased significantly with an increase in fermentation temperature. In particular, at 32 °C, strain A released an average amount of polysaccharides that was about 3-fold higher than was released at 25 °C. In addition, an increase in must sugar concentration stimulated the release of polysaccharides by strains A and B. In contrast, the average amount of polysaccharides released into the medium by strain C decreased significantly with increases in temperature and sugar concentration, and increased significantly in grape juice supplemented with DAP. For strains A and B, must supplementation did not have a significant effect on the release of polysaccharides. Fig. 1 displays the data that we collected to assess the existence of a relationship between the cell viability level measured at the end of fermentation and the cell wall polysaccharide content of the wines. Samples in which the number of viable cells at 25 °C were high had the lowest polysaccharide contents (strain A and strain B). This trend was not observed for strain C cultures, in which the amount of polysaccharides was as high in samples with high numbers of viable
DAP Cellulose
11.79def 130def 2.5e 12.67d 176il 2.5bc 12.72f 141cd 2.8fg
Control
11.53cd 114cde 1.7a 12.48d 121cde 2.5bc 11.73e 111ab 2.2ab
DAP
10.77b 170h 2.9g 11.35c 193lm 2.6cd 11.49de 168ef 3.1gh
Cellulose
11.35c 91c 2.5ef 11.53c 173hil 2.6cd 11.45d 203hi 2.7def
Control
10.63b 133efg 2.0bc 11.39c 144fg 2.1a 10.99c 128bc 2.6cde
DAP
10.29a 158gh 2.6ef 10.51b 207m 3.0f 10.37d 175fg 2.6cde
Cellulose
11.83ef 30a 2.0bc 13.33f 44a 2.3ab 14.48i 93a 2.4bc
DAP
10.27a 114cde 1.9ab 10.02a 156ghi 2.8def 10.01a 166ef 2.8ef 11.63cde 177h 2.2cd 13.87il 192lm 2.7cde 15.69o 156def 2.8fg 11.93f 154fgh 2.0bc 13.95l 154gh 2.3ab 15.63no 132c 2.5cd
Cellulose Control
11.53cd 107cd 1.9ab 13.69ghi 128def 2.1a 15.59no 111ab 2.0a 11.73def 122de 3.7i 13.58g 131def 3.8g 15.61no 217i 4.1l
DAP Cellulose
11.64de 93c 3.5h 13.66gh 114cd 3.8g 15.26lm 154de 3.2h 27
23.5
20
Alcohol (%v/v) Polysaccharides (mg/L) Cell dry weight (g/L) Alcohol (%v/v) Polysaccharides (mg/L) Cell dry weight (g/L) Alcohol (%v/v) Polysaccharides (mg/L) Cell dry weight (g/L)
DAP
11.52cd 106cd 2.4e 13.22ef 101bc 2.6cd 14.05h 109ab 2.8ef
Strain C Sugar (%)
11.79def 56b 2.6ef 13.84hil 103bc 2.9ef 15.03l 154de 2.9fg
Control Control Cellulose
11.82ef 52ab 2.5ef 13.69ghi 82b 2.7cde 15.39mn 109ab 2.8ef
Control
10.87b 128de 2.4de 10.49b 142efg 2.8def 11.06c 174efg 2.4bc
Strain C Strain B Temperature 32 °C
Strain A Strain B
Temperature 25 °C
Strain A
305
Table 2 Mean of the amount of polysaccharides released (mg/g dw) by three yeast strains as a function of temperature, sugar concentration and must supplementation. Data with the same letters are not significantly different. Variables
Strain A
Strain B
Strain C
Temperature (°C) 25 32 LSD
23.66a 62.06b 9.38***
38.44a 61.33b 13.11***
64.00b 58.44a 4.94*
Sugar (%) 20 23.5 27 LSD
34.15a 45.84b 48.58b 11.49***
44.16a 52.00ab 53.50b 8.97*
66.83b 64.00b 52.83a 10.82***
Supplementation Control Cellulose DAP LSD
43.16ns 40.28ns 45.13ns –
51.00ns 47.33ns 51.33ns –
56.33a 61.66ab 65.66b 8.15**
*P ≤ 0.05. **P ≤ 0.01. ***P ≤ 0.001.
ns
Not significant.
cells (25 °C) as it was in samples with lower numbers of viable cells (some of the trials at 32 °C). In general, at 32 °C, strain C maintained a higher cell viability than strains A or B. 4. Discussion
Response
Table 1 Content of alcohol, released polysaccharide and yeast cell dry weight in fermented wine samples. Data with the same letters are not significantly different (P ≤ 0.05).
11.58cde 175h 2.7fg 13.02e 179l 2.7cde 13.13g 193hi 3.4i
LSD
0.28 25.6 0.24 0.20 21.2 0.25 0.27 20.7 0.24
G. Giovani et al. / International Journal of Food Microbiology 137 (2010) 303–307
According to our results, the ability of the investigated yeast strains to grow and ferment in different environmental conditions translated into different quantities of polysaccharides released into the medium. However, the strains may have utilized different release mechanisms. While the mean amount of macromolecules released by strains A and B was greater under stressful conditions (i.e., higher sugar concentration and temperature), the opposite was true for strain C (Table 2). The latter strain released more polysaccharides when the conditions were closer to optimal (i.e., 25 °C, low sugar concentration, greater YAN content in the must). The relationship between cell viability at the end of fermentation and the quantity of released polysaccharides reinforced this observation. Indeed, at 25 °C, when viability remained high under all conditions, strain C released more polysaccharides than did strains A or B (Fig. 1). When cell viability was lower (trials at 32 °C), the polysaccharide content of the wines remained more or less the same for strain C, while it increased for strains A and B. These data suggest that in addition to release by actively growing cells (Guilloux-Benatier et al., 1995), passive release by dying or dead cells (Dupin et al., 2000) also contributed to the final amount of polysaccharides. Compared to strain C, more of the polysaccharides released by strains A and B appeared to be due to contributions by cells in a declining phase. A further explanation for the differences in polysaccharide release among the strains is that yeast strains may activate the cell wall integrity (CWI) pathway to varying degrees. This pathway is regulated through the cell cycle, but it is also activated in response to a variety of external stimuli that cause cell wall stress (Levin, 2005). Several studies (Aguilar-Uscanga and François, 2003; Klis et al., 2002; Klis et al., 2006; Liu et al., 2008) have provided ample evidence that the yeast cell wall is not a static entity; rather, it is dynamically remodelled in response to changes in environmental conditions. Indeed, cells can respond to environmental stress by changes in molecular architecture, changes in the relationship between cell wall polysaccharides, and increases in the amounts of several cell wall proteins. As a consequence of this cellular adaptation response, the cell wall is remodelled in an attempt by the cell to survive (Arroyo et al., 2009; Popolo et al., 2001). The release of polysaccharides by viable yeast cells could therefore be a consequence of cell wall remodelling. β(1,3)-glucanosyltranferase appears to play an important cross-linking function in cell walls. Interestingly, gas1Δ mutants, which are defective in genes coding for
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Fig. 1. Number of viable cells and polysaccharide content of wines at the end of alcoholic fermentation conducted by the three yeast strains (A, B, and C).
β(1,3)-glucanosyltranferase, have reduced amounts of β(1,3)-glucan and β(1,6) glucosylated GPI mannoproteins in their cell walls; these molecules are instead detected in the culture medium (Ram et al., 1998). Our results suggest that a greater capacity for cell wall remodelling, by way of CWI pathway activation, could have permitted strain C to release more cell wall polysaccharides under all environmental conditions, and to maintain, under more stressful conditions, a larger viable and metabolically active population. Our results regarding the influence of cellulose and nitrogen supplementation on the fermentative performance under mild and more stressful conditions show that both supplementations improved the osmotic and temper-
ature stress tolerance of all strains under all conditions (Table 1). As previously reported (Bisson, 1991; Groat and Ough, 1978; Thomas et al., 1994), the addition of cellulose and ammonium ions to grape juice, particularly clarified juice, stimulated yeast growth and the fermentation rate, and hastened the completion of fermentation. However, the degree of resistance to more stressful fermentation conditions was strain dependent; strain C had the highest fermentation rate and ethanol production. In conclusion, we found that the cells of three wine yeasts had variable responses to different types of environmental stress. Strains A and B released a greater quantity of polysaccharides when the
G. Giovani et al. / International Journal of Food Microbiology 137 (2010) 303–307
conditions were more osmotically and thermally stressful. Under these conditions, the cells were less viable and metabolically active. In contrast, strain C released more polysaccharides when the temperature and sugar concentration were lower and the must was nutritionally richer. Under these conditions, the cells were more viable and metabolically active. This different response leads us to conclude that the strains release cell wall polysaccharides by way of different mechanisms, and that the CWI pathway may be involved. From a practical point of view, however, the amount (mg/L) of cell wall polysaccharides released by the three yeast strains under most of the fermentation conditions that we tested are most likely sufficient to have a significant effect on some aspects of wine quality, such as aroma compound retention, colour stability, and mouth-feel properties (reduction of astringency), as reported previously (Chalier et al., 2007; Escot et al., 2001). A temporal analysis of cell wall changes in the three yeasts under different fermentation conditions and a chemical characterization of the released polysaccharides are necessary to determine if the release of polysaccharides into the medium is an integral part of the adaptation response to the environment. References Aguilar-Uscanga, B., François, J.M., 2003. A study of the yeast cell wall composition and structure in response to growth conditions and mode of cultivation. Letters in Applied Microbiology 37, 268–274. Arroyo, J., Bermejo, C., García, R., Rodríguez-Peña, J.M., 2009. Genomics in the detection of damage in microbial systems: cell wall stress in yeast. Clinical Microbiology and Infection 15, 44–46. Bisson, L.F., 1991. Influence of nitrogen on yeast and fermentation of grapes. In: Ranz, J.M. (Ed.), Proceedings of the International Symposium on Nitrogen in Grapes and Wine. American Society of Enology and Viticology, Davis, CA. Boivin, S., Feuillat, M., Alexandre, H., Charpentier, C., 1998. Effect of must turbidity on cell wall porosity and macromolecule excretion of Saccharomyces cerevisiae cultivated on grape juice. American Journal of Enology and Viticulture 49, 325–332. Brown, S.L., Stockdale, V.J., Pettolino, F., Pocock, K.F., de Barros Lopes, M., Williams, P.J., Bacic, A., Fincher, G.B., Høj, P.B., Waters, E.J., 2007. Reducing haziness in white wine by overexpression of Saccharomyces cerevisiae genes YOL155c and YDR055w. Applied Microbiology and Biotechnology 73, 1363–1376. Caridi, A., 2007. New perspectives in safety and quality enhancement of wine through selection of yeasts based on the parietal adsorption activity. International Journal of Food Microbiology 120, 167–172. Chalier, P., Angot, B., Delteil, D., Doco, T., Gunata, Z., 2007. Interactions between aroma compounds and whole mannoprotein isolated from Saccharomyces cerevisiae strains. Food Chemistry 100, 22–30. Charpentier, C., Nguyen Van Long, T., Bonaly, R., Feuillat, M., 1986. Alteration of cell wall structure in Saccharomyces cerevisiae and Saccharomyces bayanus during autolysis. Applied Microbiology and Biotechnology 24, 405–413. Dupin, I.V.S., McKinnon, B.M., Ryan, C., Boulay, M., Markides, A.J., Jones, G.P., Williams, P.J., Waters, E.J., 2000. Saccharomyces cerevisiae mannoproteins that protect wine from protein haze: their release during fermentation and lees contact and a proposal for their mechanism of action. Journal of Agricultural and Food Chemistry 48, 3098–3105. Escot, S., Feuillat, M., Dulau, L., Charpentier, C., 2001. Release of polysaccharides by yeasts and the influence of released polysaccharides on color stability and wine astringency. Australian Journal of Grape and Wine Research 7, 153–159. Fleet, G.H., 1991. Cell Wall. The yeasts. : Yeast organelles, vol. 4. Academic Press, London, pp. 199–277. Giovani, G., Rosi, I., 2007. Release of cell wall polysaccharides from Saccharomyces cerevisiae thermosensitive autolytic mutants during alcoholic fermentation. International Journal of Food Microbiology 116, 19–24. Gonzalez, R., Martinez-Rodriguez, A.J., Carrascosa, A.V., 2003. Yeast autolytic mutants potentially useful for sparkling wine production. International Journal of Food Microbiology 84, 21–26. Gonzalez-Ramos, D., Gonzalez, R., 2006. Genetic determinants of the release of mannoproteins of enological interest by Saccharomyces cerevisiae. Journal of Agricultural and Food Chemistry 54, 9411–9416.
307
Gonzalez-Ramos, D., Cebollero, E., Gonzalez, R., 2008. A recombinant Saccharomyces cerevisiae strain overproducing mannoproteins stabilizes wine against protein haze. Applied and Environmental Microbiology 74, 5533–5540. Groat, M., Ough, C.S., 1978. Effects of insoluble solids added to clarified musts on fermentation rate, wine composition, and wine quality. American Journal of Enology and Viticulture 29, 112–119. Guilloux-Benatier, M., Guerreau, J., Feuillat, M., 1995. Influence of initial colloid content on yeast macromolecule production and on the metabolism of wine microorganisms. American Journal of Enology and Viticulture 46, 486–492. Klis, F.M., Mol, P., Hellingwerf, K., Brul, S., 2002. Dynamics of cell wall structure in Saccharomyces cerevisiae. FEMS Microbiology Reviews 26, 239–256. Klis, F.M., Boorsma, A., De Groot, P.W.J., 2006. Cell wall construction in Saccharomyces cerevisiae. Yeast 23, 185–202. Kogan, G., Kocher, A., 2007. Role of yeast cell wall polysaccharides in pig nutrition and health protection. Livestock Science 109, 161–165. Levin, D.E., 2005. Cell Wall Integrity Signaling in Saccharomyces cerevisiae. Microbiology and Molecular Biology Reviews 69, 262–291. Lipke, P.N., Ovalle, R., 1998. Cell wall architecture in yeast: new structure and new challenges. The Journal of Bacteriology 180, 3735–3740. Liu, H.Z., Wang, Q., Liu, X.Y., Tan, S.S., 2008. Effects of spaceflight on polysaccharides of Saccharomyces cerevisiae cell wall. Applied Microbiology and Biotechnology 81, 543–550. Llauberes, R.M., Dubourdieu, D., Villetaz, J.C., 1987. Exocellular polysaccharides from Saccharomyces in wine. Journal of The Science of Food and Agriculture 41, 277–280. Lubbers, S., Léger, B., Charpentier, C., Feuillat, M., 1993. Effet colloïdes protecteurs d'extraits de parois de levures sur la stabilité tartrique d'un vin modèle. Journal of International Science Vigne Vin 27, 13–22. Lubbers, S., Charpentier, C., Feuillat, M., Voilley, A., 1994. Influence of yeast walls on the behavior of aroma compounds in a model wine. American Journal of Enology and Viticulture 45, 29–33. Moine-Ledoux, V., Dubourdieu, D., 1998. Interprétation moléculaire de l'amélioration de la stabilité protéique des vins blancs au cours de leur élevage sur lies. Revue des Oenologues 86, 11–14. Moreno-Arribas, V., Pueyo, E., Nieto, F.J., Martín-Álvarez, P.J., Polo, M.C., 2000. Influence of the polysaccharides and the nitrogen compounds on foaming properties of sparkling wines. Food Chemistry 70, 309–317. Moruno, E.G., Sanlorenzo, C., Boccaccino, B., Di Stefano, R., 2005. Treatment with yeast to reduce the concentration of ochratoxin A in red wine. American Journal of Enology and Viticulture 56, 73–76. Nunez, Y.P., Carrascosa, A.V., Gonzalez, R., Polo, M.C., Martinez-Rodriguez, A.J., 2005. Effect of accelerated autolysis of yeast on the composition and foaming properties of sparkling wines elaborated by a Champenoise method. Journal of Agricultural and Food Chemistry 53, 7232–7237. Nunez, Y.P., Carrascosa, A.V., Gonzalez, R., Polo, M.C., Martinez-Rodriguez, A., 2006. Isolation and characterization of a thermally extracted yeast cell wall fraction potentially useful for improving the foaming properties of sparkling wines. Journal of Agricultural and Food Chemistry 54, 7898–7903. Office International de la Vigne et du Vin, 1990. Recueil des méthodes internationales d'analyse des vins et des moûts. Paris. Popolo, L., Gualtieri, T., Ragni, E., 2001. The yeast cell-wall salvage pathway. Medical Mycology 39, 111–121. Ram, A.F., Kapteyn, J.C., Montijn, R.C., Caro, L.H., Douwes, J.E., Baginsky, W., Mazur, P., van den Ende, H., Klis, F.M., 1998. Loss of the plasma membrane-bound protein Gas1p in Saccharomyces cerevisiae results in the release of beta 1, 3-glucan into the medium and induces a compensation mechanism to ensure cell wall integrity. The Journal of Bacteriology 180, 1418–1424. Rosi, I., Giovani, G., 2003. Effect of some winemaking variables on the production of exopolysaccharides by Saccharomyces cerevisiae. In: Lonvaud-Funel, A., et al. (Ed.), 7th International Symposium of Œnology, pp. 324–328. Rosi, I., Gheri, A., Ferrari, S., 1998. Effets des levures produisant des polysaccharides pariétaux sur certain caractéristiques des vins rouges pendant la fermentation. Revue Francaise d' Œnologie 172, 24–26. Rosi, I., Gheri, A., Domizio, P., Fia, G., 2000. Production de macromolecules pariétales de Saccharomyces cerevisiae au cours de la fermentation et leur influence sur la fermentation malolactique. Revue des Oenologues 94, 18–20. Thomas, K.C., Hynes, S.H., Ingledew, W.M., 1994. Effects of particulate materials and osmoprotectants on very-high-gravity ethanolic fermentation by Saccharomyces cerevisiae. Applied and Environmental Microbiology 60, 1519–1524. Vidal, S., Francis, L., Williams, P., Kwiatkowski, M., Gawel, R., Cheynier, V., Waters, E., 2004. The mouth-feel properties of polysaccharides and anthocyanins in a wine like medium. Food Chemistry 85, 519–525. Waters, E.J., Pellerin, P., Brillouet, J.M., 1994. A Saccharomyces mannoprotein that protects wine from protein haze. Carbohydrate Polymers 23, 185–191.
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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o
Short Communication
Effect of yeast strain and fermentation conditions on the release of cell wall polysaccharides Giovanna Giovani, Valentina Canuti, Iolanda Rosi ⁎ Dipartimento di Biotecnologie Agrarie, Università di Firenze, Via Donizetti, 6, 50144, Firenze, Italy
a r t i c l e
i n f o
Article history: Received 31 August 2009 Received in revised form 24 November 2009 Accepted 6 December 2009 Keywords: Cell wall polysaccharides Mannoproteins S. cerevisiae Alcoholic fermentation Wine yeast
a b s t r a c t To improve our understanding of the factors involved in polysaccharide release during alcoholic fermentation, we investigated three Saccharomyces cerevisiae strains in fermentation trials conducted at two temperatures (25 °C and 32 °C) and three sugar concentrations (20%, 23.5%, and 27%), with or without supplementation of grape juice with diammonium phosphate (DAP) or microcrystalline cellulose. In two yeast strains, the release of cell wall polysaccharides increased significantly with an increase in fermentation temperature and sugar concentration of the grape juice; the polysaccharide release was greater in stressed conditions, in which the cells were less viable and less metabolically active. In the third strain, the average amount of polysaccharides released into the medium decreased significantly at 32 °C with 27% sugar, and increased in grape juice supplemented with DAP. Thus, this strain released more polysaccharides when conditions were nearer to optimal and the yeast cells were more viable and metabolically active. Our results suggest that the yeast strains released cell wall polysaccharides via different mechanisms, and that the cell wall integrity pathway may account for some of the differences in polysaccharide release among the strains. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The cell wall of the yeast Saccharomyces cerevisiae is composed of a 10 nm thick layer of polysaccharides (predominantly glucans and mannoproteins) and serves as the interface between the cell and the neighbouring environment. It provides osmotic and physical protection and determines the shape of the cell (Klis et al., 2002). Several papers have reported that S. cerevisiae releases cell wall polysaccharides, particularly mannoproteins, into the extracellular medium during yeast growth (Boivin et al., 1998; Dupin et al., 2000; Llauberes et al., 1987; Rosi et al., 2000). This release may be a consequence of cell wall-controlled hydrolysis of the mother cell to allow the emergence of the bud (Charpentier et al., 1986; Fleet, 1991). It has been suggested that secretion of most yeast mannoproteins and the enzyme involved in cell wall synthesis occurs at the bud site (Lipke and Ovalle, 1998). Wines aged on yeast lees may contain increased amounts of mannoproteins because the dead cells undergo autolysis and enzymatic cell wall degradation. However, not all wines are obtained by aging on lees; therefore, polysaccharide release by S. cerevisiae should also be investigated during alcoholic fermentation of grape must. The yeast strain is one of the factors controlling the amount of mannoproteins released during winemaking (Escot et al., 2001; Llauberes et al., 1987; Rosi et al., 1998; Rosi et al., 2000). Recent studies showed that using autolytic thermosensitive mutants (Giovani
⁎ Corresponding author. Tel.: + 39 055 3220322; fax: + 39 055 355995. E-mail address: rosiol@unifi.it (I. Rosi). 0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2009.12.009
and Rosi, 2007; Gonzalez et al., 2003; Nunez et al., 2005) or genetically engineered wine yeast strains of S. cerevisiae (Brown et al., 2007; Gonzalez-Ramos et al., 2008; Gonzalez-Ramos and Gonzalez, 2006) resulted in increased amounts of mannoproteins in the fermenting medium. In addition, environmental factors such as temperature, carbon source, or level of initial colloid content of the medium have been shown to influence the amount of cell wall polysaccharides secreted into the fermenting medium (Guilloux-Benatier et al., 1995; Rosi and Giovani, 2003). Mannoproteins derived from yeast cell walls have attracted much attention in the winemaking world because of their reported contribution to wine quality. Desirable oenological properties of mannoproteins include protection against protein and tartaric instability (Brown et al., 2007; Dupin et al., 2000; Lubbers et al., 1993; Moine-Ledoux and Dubourdieu, 1998; Waters et al., 1994), retention of aromatic compounds (Chalier et al., 2007; Lubbers et al., 1994), reduction of astringency (Escot et al., 2001), increased sweetness (Rosi et al., 1998), and increased body and mouth feel (Vidal et al., 2004), which are especially appreciated in red wines. Furthermore, mannoproteins stimulate the growth of lactic acid bacteria and consequently malolactic fermentation (Guilloux-Benatier et al., 1995; Rosi et al., 2000), and they improve the foam quality of sparkling wines (Moreno-Arribas et al., 2000; Nunez et al., 2006). Yeast cell wall polysaccharides may also adsorb mycotoxins, thus decreasing their toxic effects and mediating their removal from the medium (Caridi, 2007; Kogan and Kocher, 2007; Moruno et al., 2005). The objective of this study was to investigate the effect of several growth conditions, including temperature, sugar concentration, and supplementation of ammonium salts and microcrystalline cellulose to
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grape juice, on fermentation behaviour and polysaccharide release during alcoholic fermentation by three wine strains of S. cerevisiae. The results of our study may be relevant not only for improving wine quality, but also for more general biotechnological purposes, because there is increasing interest in the production of glucan and mannan for agrofood, pharmaceutical and cosmetic purposes.
grape juice and experimental wines were quantified by HPLC according to the method described by Giovani and Rosi (2007). Results reported are averages of duplicate determinations. The content of released polysaccharides was obtained from the difference between the total polysaccharide content of wines and the original grape juice.
2. Materials and methods
2.5. Statistical analysis
2.1. Chemicals
Treatment means were compared with Fisher's unrestricted least significant difference (FLSD) (STATGRAPHICS 4.1 Plus, 1999; Manugistics, Rockville, MD, USA).
The following chemicals were used: glucose, fructose, microcrystalline cellulose, and diammonium phosphate were obtained from Sigma-Aldrich (Milan, Italy). Yeast extract, peptone, and agar were obtained from Oxoid (Milan, Italy).
3. Results 3.1. Fermentation behaviour of the three wine yeast strains
2.2. Strains We used the two commercial S. cerevisiae strains BM45 (A) and D254 (B) (Lallemand, Montreal, Canada), and S. cerevisiae BLC83 (C), a yeast strain belonging to our wine yeast collection. The yeast strains were maintained on YPD medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, and 15 g/L agar), stored at 4 °C, and subcultured once a year. 2.3. Fermentation trials The same batch of commercial pasteurised white grape juice (marketed by Hero, Verona, Italy) was used for all of the experiments. Juice characteristics were: 200 g/L sugar, titratable acidity (5.1 g/L tartaric acid, pH 3.4), 70 mg/L free α-amino nitrogen, 30 mg/L ammonium nitrogen (100 mg/L total yeast-assimilable nitrogen [YAN]), and 138 mg/L total polysaccharides. Sulfur dioxide was added to the juice at 50 mg/L. The juice was separately adjusted to 235 g/L and 270 sugar by addition of glucose:fructose (1:1). Each lot with the same sugar concentration was divided into three aliquots: one was the control, one was supplemented with 200 mg/L microcrystalline cellulose, an inert support that improves yeast growth and metabolism, and one was supplemented with 300 mg/L diammonium phosphate (DAP). When DAP was added to the juice, the YAN content increased to 160 mg/L. Inocula were prepared by growing a loopful of cells from YPD slants in 100 mL of liquid YPD. Erlenmeyer flasks were incubated at 25 °C and shaken at 150 rpm on a gyratory shaker. After 24 h of growth, cells were inoculated into 1-L glass bottles containing 800 mL of juice, at OD 0.2 (λ = 600 nm) (approximately 1–2 × 106 colonyforming units [CFU]/mL). The flasks were closed with a Müller valve that had previously been filled with sulfuric acid, and incubated at two temperatures: 25 °C and 32 °C. The fermentations were carried out in duplicate. In total, 108 fermentations were performed (18 fermentations for each strain in duplicate). The fermentation rate was monitored daily by weight loss as a result of CO2 escaping from the system, until the weight was constant. 2.4. Analysis After 14 days of fermentation, a sample was withdrawn and different aliquots were used for: (1) viable cell count; (2) dry mass evaluation, after collecting cells on a filter; and (3) ethanol and total polysaccharide contents, after centrifugation to remove yeast cells. Yeast cell viability was checked by plating appropriate dilutions of cells on YPD plates, in triplicate. Plates were incubated at 30 °C for 2 days. Viability was expressed as CFU/mL. Cell dry weight was determined by filtration of a volume of sample on a preweighed filter, washing with distilled water, and drying at 105 °C for 24 h. Ethanol was measured by the Office International de la Vigne et du Vin official method of analysis (OIV, 1990). The polysaccharide contents of the
The alcohol and biomass contents of the wines at the end of alcoholic fermentation are reported in Table 1. At 25 °C, in juices with 20% and 23.5% sugar, all of the strains displayed a higher power to produce alcohol, while in juices with 27% sugar, cellulose and DAP supplementation enhanced alcohol production by strains A and B. At 32 °C, the ethanol production diminished for all strains. When grape juice was supplemented with cellulose, an increase in alcohol production was observed for all strains, whereas DAP supplementation increased alcohol production by strain C in juices with 23.5% and 27% sugar. At 25 °C, strains A and B produced more cells than strain C (Table 1). Cellulose and DAP supplementation stimulated growth of all three strains at 25 °C and 32 °C. However, when we compared the biomass values with the alcohol content of the wines, strain C cells demonstrated greater fermentative activity than strain A or B cells. 3.2. Polysaccharide release Data regarding the influence of the different fermentation conditions on polysaccharide release by the three yeast strains are reported in Table 1. Strains B and C produced, under all tested conditions, polysaccharide amounts that varied between 100 mg/L and 200 mg/L. In contrast, strain A showed a greater dependence on the environmental conditions. The amount of polysaccharides varied from 30 mg/ L in wine obtained from 20% sugar must and fermented at 25 °C, to approximately 200 mg/L in wine obtained from 23.5% sugar must, supplemented with DAP, and fermented at 32 °C. To investigate the effect of different environmental stimuli on the cell response in terms of polysaccharide release, we standardized the amount of polysaccharides based on the final biomass. The average amount of released polysaccharides as a function of temperature, sugar concentration, and must supplementation was determined and analyzed using the FLSD test (Table 2). In strains A and B, the release of macromolecules increased significantly with an increase in fermentation temperature. In particular, at 32 °C, strain A released an average amount of polysaccharides that was about 3-fold higher than was released at 25 °C. In addition, an increase in must sugar concentration stimulated the release of polysaccharides by strains A and B. In contrast, the average amount of polysaccharides released into the medium by strain C decreased significantly with increases in temperature and sugar concentration, and increased significantly in grape juice supplemented with DAP. For strains A and B, must supplementation did not have a significant effect on the release of polysaccharides. Fig. 1 displays the data that we collected to assess the existence of a relationship between the cell viability level measured at the end of fermentation and the cell wall polysaccharide content of the wines. Samples in which the number of viable cells at 25 °C were high had the lowest polysaccharide contents (strain A and strain B). This trend was not observed for strain C cultures, in which the amount of polysaccharides was as high in samples with high numbers of viable
DAP Cellulose
11.79def 130def 2.5e 12.67d 176il 2.5bc 12.72f 141cd 2.8fg
Control
11.53cd 114cde 1.7a 12.48d 121cde 2.5bc 11.73e 111ab 2.2ab
DAP
10.77b 170h 2.9g 11.35c 193lm 2.6cd 11.49de 168ef 3.1gh
Cellulose
11.35c 91c 2.5ef 11.53c 173hil 2.6cd 11.45d 203hi 2.7def
Control
10.63b 133efg 2.0bc 11.39c 144fg 2.1a 10.99c 128bc 2.6cde
DAP
10.29a 158gh 2.6ef 10.51b 207m 3.0f 10.37d 175fg 2.6cde
Cellulose
11.83ef 30a 2.0bc 13.33f 44a 2.3ab 14.48i 93a 2.4bc
DAP
10.27a 114cde 1.9ab 10.02a 156ghi 2.8def 10.01a 166ef 2.8ef 11.63cde 177h 2.2cd 13.87il 192lm 2.7cde 15.69o 156def 2.8fg 11.93f 154fgh 2.0bc 13.95l 154gh 2.3ab 15.63no 132c 2.5cd
Cellulose Control
11.53cd 107cd 1.9ab 13.69ghi 128def 2.1a 15.59no 111ab 2.0a 11.73def 122de 3.7i 13.58g 131def 3.8g 15.61no 217i 4.1l
DAP Cellulose
11.64de 93c 3.5h 13.66gh 114cd 3.8g 15.26lm 154de 3.2h 27
23.5
20
Alcohol (%v/v) Polysaccharides (mg/L) Cell dry weight (g/L) Alcohol (%v/v) Polysaccharides (mg/L) Cell dry weight (g/L) Alcohol (%v/v) Polysaccharides (mg/L) Cell dry weight (g/L)
DAP
11.52cd 106cd 2.4e 13.22ef 101bc 2.6cd 14.05h 109ab 2.8ef
Strain C Sugar (%)
11.79def 56b 2.6ef 13.84hil 103bc 2.9ef 15.03l 154de 2.9fg
Control Control Cellulose
11.82ef 52ab 2.5ef 13.69ghi 82b 2.7cde 15.39mn 109ab 2.8ef
Control
10.87b 128de 2.4de 10.49b 142efg 2.8def 11.06c 174efg 2.4bc
Strain C Strain B Temperature 32 °C
Strain A Strain B
Temperature 25 °C
Strain A
305
Table 2 Mean of the amount of polysaccharides released (mg/g dw) by three yeast strains as a function of temperature, sugar concentration and must supplementation. Data with the same letters are not significantly different. Variables
Strain A
Strain B
Strain C
Temperature (°C) 25 32 LSD
23.66a 62.06b 9.38***
38.44a 61.33b 13.11***
64.00b 58.44a 4.94*
Sugar (%) 20 23.5 27 LSD
34.15a 45.84b 48.58b 11.49***
44.16a 52.00ab 53.50b 8.97*
66.83b 64.00b 52.83a 10.82***
Supplementation Control Cellulose DAP LSD
43.16ns 40.28ns 45.13ns –
51.00ns 47.33ns 51.33ns –
56.33a 61.66ab 65.66b 8.15**
*P ≤ 0.05. **P ≤ 0.01. ***P ≤ 0.001.
ns
Not significant.
cells (25 °C) as it was in samples with lower numbers of viable cells (some of the trials at 32 °C). In general, at 32 °C, strain C maintained a higher cell viability than strains A or B. 4. Discussion
Response
Table 1 Content of alcohol, released polysaccharide and yeast cell dry weight in fermented wine samples. Data with the same letters are not significantly different (P ≤ 0.05).
11.58cde 175h 2.7fg 13.02e 179l 2.7cde 13.13g 193hi 3.4i
LSD
0.28 25.6 0.24 0.20 21.2 0.25 0.27 20.7 0.24
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According to our results, the ability of the investigated yeast strains to grow and ferment in different environmental conditions translated into different quantities of polysaccharides released into the medium. However, the strains may have utilized different release mechanisms. While the mean amount of macromolecules released by strains A and B was greater under stressful conditions (i.e., higher sugar concentration and temperature), the opposite was true for strain C (Table 2). The latter strain released more polysaccharides when the conditions were closer to optimal (i.e., 25 °C, low sugar concentration, greater YAN content in the must). The relationship between cell viability at the end of fermentation and the quantity of released polysaccharides reinforced this observation. Indeed, at 25 °C, when viability remained high under all conditions, strain C released more polysaccharides than did strains A or B (Fig. 1). When cell viability was lower (trials at 32 °C), the polysaccharide content of the wines remained more or less the same for strain C, while it increased for strains A and B. These data suggest that in addition to release by actively growing cells (Guilloux-Benatier et al., 1995), passive release by dying or dead cells (Dupin et al., 2000) also contributed to the final amount of polysaccharides. Compared to strain C, more of the polysaccharides released by strains A and B appeared to be due to contributions by cells in a declining phase. A further explanation for the differences in polysaccharide release among the strains is that yeast strains may activate the cell wall integrity (CWI) pathway to varying degrees. This pathway is regulated through the cell cycle, but it is also activated in response to a variety of external stimuli that cause cell wall stress (Levin, 2005). Several studies (Aguilar-Uscanga and François, 2003; Klis et al., 2002; Klis et al., 2006; Liu et al., 2008) have provided ample evidence that the yeast cell wall is not a static entity; rather, it is dynamically remodelled in response to changes in environmental conditions. Indeed, cells can respond to environmental stress by changes in molecular architecture, changes in the relationship between cell wall polysaccharides, and increases in the amounts of several cell wall proteins. As a consequence of this cellular adaptation response, the cell wall is remodelled in an attempt by the cell to survive (Arroyo et al., 2009; Popolo et al., 2001). The release of polysaccharides by viable yeast cells could therefore be a consequence of cell wall remodelling. β(1,3)-glucanosyltranferase appears to play an important cross-linking function in cell walls. Interestingly, gas1Δ mutants, which are defective in genes coding for
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Fig. 1. Number of viable cells and polysaccharide content of wines at the end of alcoholic fermentation conducted by the three yeast strains (A, B, and C).
β(1,3)-glucanosyltranferase, have reduced amounts of β(1,3)-glucan and β(1,6) glucosylated GPI mannoproteins in their cell walls; these molecules are instead detected in the culture medium (Ram et al., 1998). Our results suggest that a greater capacity for cell wall remodelling, by way of CWI pathway activation, could have permitted strain C to release more cell wall polysaccharides under all environmental conditions, and to maintain, under more stressful conditions, a larger viable and metabolically active population. Our results regarding the influence of cellulose and nitrogen supplementation on the fermentative performance under mild and more stressful conditions show that both supplementations improved the osmotic and temper-
ature stress tolerance of all strains under all conditions (Table 1). As previously reported (Bisson, 1991; Groat and Ough, 1978; Thomas et al., 1994), the addition of cellulose and ammonium ions to grape juice, particularly clarified juice, stimulated yeast growth and the fermentation rate, and hastened the completion of fermentation. However, the degree of resistance to more stressful fermentation conditions was strain dependent; strain C had the highest fermentation rate and ethanol production. In conclusion, we found that the cells of three wine yeasts had variable responses to different types of environmental stress. Strains A and B released a greater quantity of polysaccharides when the
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conditions were more osmotically and thermally stressful. Under these conditions, the cells were less viable and metabolically active. In contrast, strain C released more polysaccharides when the temperature and sugar concentration were lower and the must was nutritionally richer. Under these conditions, the cells were more viable and metabolically active. This different response leads us to conclude that the strains release cell wall polysaccharides by way of different mechanisms, and that the CWI pathway may be involved. From a practical point of view, however, the amount (mg/L) of cell wall polysaccharides released by the three yeast strains under most of the fermentation conditions that we tested are most likely sufficient to have a significant effect on some aspects of wine quality, such as aroma compound retention, colour stability, and mouth-feel properties (reduction of astringency), as reported previously (Chalier et al., 2007; Escot et al., 2001). A temporal analysis of cell wall changes in the three yeasts under different fermentation conditions and a chemical characterization of the released polysaccharides are necessary to determine if the release of polysaccharides into the medium is an integral part of the adaptation response to the environment. References Aguilar-Uscanga, B., François, J.M., 2003. A study of the yeast cell wall composition and structure in response to growth conditions and mode of cultivation. Letters in Applied Microbiology 37, 268–274. Arroyo, J., Bermejo, C., García, R., Rodríguez-Peña, J.M., 2009. Genomics in the detection of damage in microbial systems: cell wall stress in yeast. Clinical Microbiology and Infection 15, 44–46. Bisson, L.F., 1991. Influence of nitrogen on yeast and fermentation of grapes. In: Ranz, J.M. (Ed.), Proceedings of the International Symposium on Nitrogen in Grapes and Wine. American Society of Enology and Viticology, Davis, CA. Boivin, S., Feuillat, M., Alexandre, H., Charpentier, C., 1998. Effect of must turbidity on cell wall porosity and macromolecule excretion of Saccharomyces cerevisiae cultivated on grape juice. American Journal of Enology and Viticulture 49, 325–332. Brown, S.L., Stockdale, V.J., Pettolino, F., Pocock, K.F., de Barros Lopes, M., Williams, P.J., Bacic, A., Fincher, G.B., Høj, P.B., Waters, E.J., 2007. Reducing haziness in white wine by overexpression of Saccharomyces cerevisiae genes YOL155c and YDR055w. Applied Microbiology and Biotechnology 73, 1363–1376. Caridi, A., 2007. New perspectives in safety and quality enhancement of wine through selection of yeasts based on the parietal adsorption activity. International Journal of Food Microbiology 120, 167–172. Chalier, P., Angot, B., Delteil, D., Doco, T., Gunata, Z., 2007. Interactions between aroma compounds and whole mannoprotein isolated from Saccharomyces cerevisiae strains. Food Chemistry 100, 22–30. Charpentier, C., Nguyen Van Long, T., Bonaly, R., Feuillat, M., 1986. Alteration of cell wall structure in Saccharomyces cerevisiae and Saccharomyces bayanus during autolysis. Applied Microbiology and Biotechnology 24, 405–413. Dupin, I.V.S., McKinnon, B.M., Ryan, C., Boulay, M., Markides, A.J., Jones, G.P., Williams, P.J., Waters, E.J., 2000. Saccharomyces cerevisiae mannoproteins that protect wine from protein haze: their release during fermentation and lees contact and a proposal for their mechanism of action. Journal of Agricultural and Food Chemistry 48, 3098–3105. Escot, S., Feuillat, M., Dulau, L., Charpentier, C., 2001. Release of polysaccharides by yeasts and the influence of released polysaccharides on color stability and wine astringency. Australian Journal of Grape and Wine Research 7, 153–159. Fleet, G.H., 1991. Cell Wall. The yeasts. : Yeast organelles, vol. 4. Academic Press, London, pp. 199–277. Giovani, G., Rosi, I., 2007. Release of cell wall polysaccharides from Saccharomyces cerevisiae thermosensitive autolytic mutants during alcoholic fermentation. International Journal of Food Microbiology 116, 19–24. Gonzalez, R., Martinez-Rodriguez, A.J., Carrascosa, A.V., 2003. Yeast autolytic mutants potentially useful for sparkling wine production. International Journal of Food Microbiology 84, 21–26. Gonzalez-Ramos, D., Gonzalez, R., 2006. Genetic determinants of the release of mannoproteins of enological interest by Saccharomyces cerevisiae. Journal of Agricultural and Food Chemistry 54, 9411–9416.
307
Gonzalez-Ramos, D., Cebollero, E., Gonzalez, R., 2008. A recombinant Saccharomyces cerevisiae strain overproducing mannoproteins stabilizes wine against protein haze. Applied and Environmental Microbiology 74, 5533–5540. Groat, M., Ough, C.S., 1978. Effects of insoluble solids added to clarified musts on fermentation rate, wine composition, and wine quality. American Journal of Enology and Viticulture 29, 112–119. Guilloux-Benatier, M., Guerreau, J., Feuillat, M., 1995. Influence of initial colloid content on yeast macromolecule production and on the metabolism of wine microorganisms. American Journal of Enology and Viticulture 46, 486–492. Klis, F.M., Mol, P., Hellingwerf, K., Brul, S., 2002. Dynamics of cell wall structure in Saccharomyces cerevisiae. FEMS Microbiology Reviews 26, 239–256. Klis, F.M., Boorsma, A., De Groot, P.W.J., 2006. Cell wall construction in Saccharomyces cerevisiae. Yeast 23, 185–202. Kogan, G., Kocher, A., 2007. Role of yeast cell wall polysaccharides in pig nutrition and health protection. Livestock Science 109, 161–165. Levin, D.E., 2005. Cell Wall Integrity Signaling in Saccharomyces cerevisiae. Microbiology and Molecular Biology Reviews 69, 262–291. Lipke, P.N., Ovalle, R., 1998. Cell wall architecture in yeast: new structure and new challenges. The Journal of Bacteriology 180, 3735–3740. Liu, H.Z., Wang, Q., Liu, X.Y., Tan, S.S., 2008. Effects of spaceflight on polysaccharides of Saccharomyces cerevisiae cell wall. Applied Microbiology and Biotechnology 81, 543–550. Llauberes, R.M., Dubourdieu, D., Villetaz, J.C., 1987. Exocellular polysaccharides from Saccharomyces in wine. Journal of The Science of Food and Agriculture 41, 277–280. Lubbers, S., Léger, B., Charpentier, C., Feuillat, M., 1993. Effet colloïdes protecteurs d'extraits de parois de levures sur la stabilité tartrique d'un vin modèle. Journal of International Science Vigne Vin 27, 13–22. Lubbers, S., Charpentier, C., Feuillat, M., Voilley, A., 1994. Influence of yeast walls on the behavior of aroma compounds in a model wine. American Journal of Enology and Viticulture 45, 29–33. Moine-Ledoux, V., Dubourdieu, D., 1998. Interprétation moléculaire de l'amélioration de la stabilité protéique des vins blancs au cours de leur élevage sur lies. Revue des Oenologues 86, 11–14. Moreno-Arribas, V., Pueyo, E., Nieto, F.J., Martín-Álvarez, P.J., Polo, M.C., 2000. Influence of the polysaccharides and the nitrogen compounds on foaming properties of sparkling wines. Food Chemistry 70, 309–317. Moruno, E.G., Sanlorenzo, C., Boccaccino, B., Di Stefano, R., 2005. Treatment with yeast to reduce the concentration of ochratoxin A in red wine. American Journal of Enology and Viticulture 56, 73–76. Nunez, Y.P., Carrascosa, A.V., Gonzalez, R., Polo, M.C., Martinez-Rodriguez, A.J., 2005. Effect of accelerated autolysis of yeast on the composition and foaming properties of sparkling wines elaborated by a Champenoise method. Journal of Agricultural and Food Chemistry 53, 7232–7237. Nunez, Y.P., Carrascosa, A.V., Gonzalez, R., Polo, M.C., Martinez-Rodriguez, A., 2006. Isolation and characterization of a thermally extracted yeast cell wall fraction potentially useful for improving the foaming properties of sparkling wines. Journal of Agricultural and Food Chemistry 54, 7898–7903. Office International de la Vigne et du Vin, 1990. Recueil des méthodes internationales d'analyse des vins et des moûts. Paris. Popolo, L., Gualtieri, T., Ragni, E., 2001. The yeast cell-wall salvage pathway. Medical Mycology 39, 111–121. Ram, A.F., Kapteyn, J.C., Montijn, R.C., Caro, L.H., Douwes, J.E., Baginsky, W., Mazur, P., van den Ende, H., Klis, F.M., 1998. Loss of the plasma membrane-bound protein Gas1p in Saccharomyces cerevisiae results in the release of beta 1, 3-glucan into the medium and induces a compensation mechanism to ensure cell wall integrity. The Journal of Bacteriology 180, 1418–1424. Rosi, I., Giovani, G., 2003. Effect of some winemaking variables on the production of exopolysaccharides by Saccharomyces cerevisiae. In: Lonvaud-Funel, A., et al. (Ed.), 7th International Symposium of Œnology, pp. 324–328. Rosi, I., Gheri, A., Ferrari, S., 1998. Effets des levures produisant des polysaccharides pariétaux sur certain caractéristiques des vins rouges pendant la fermentation. Revue Francaise d' Œnologie 172, 24–26. Rosi, I., Gheri, A., Domizio, P., Fia, G., 2000. Production de macromolecules pariétales de Saccharomyces cerevisiae au cours de la fermentation et leur influence sur la fermentation malolactique. Revue des Oenologues 94, 18–20. Thomas, K.C., Hynes, S.H., Ingledew, W.M., 1994. Effects of particulate materials and osmoprotectants on very-high-gravity ethanolic fermentation by Saccharomyces cerevisiae. Applied and Environmental Microbiology 60, 1519–1524. Vidal, S., Francis, L., Williams, P., Kwiatkowski, M., Gawel, R., Cheynier, V., Waters, E., 2004. The mouth-feel properties of polysaccharides and anthocyanins in a wine like medium. Food Chemistry 85, 519–525. Waters, E.J., Pellerin, P., Brillouet, J.M., 1994. A Saccharomyces mannoprotein that protects wine from protein haze. Carbohydrate Polymers 23, 185–191.