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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. References

Aims: The reversion of the metabolic changes induced in wine yeasts by stressors.

Methods: Six strains of Saccharomyces were inoculated in grape must containing over 400 g l−1 of sugar and incubated at 35 °C, both with and without the addition of 100 mg l−1 of catechin, inositol or SO2.

Results: Significant correlations between addition of the stress-protectants and change in the metabolic behaviour of the wine yeasts were observed. Depending on strain and protectant, and expressing data as a percentage of increase or decrease compared to the control, fermentation vigour after 3 d increased by up to 10%, titratable acidity of the wines increased by up to 7%, ethanol content increased by up to 20%, unitary acetic acid production decreased by up to 35%, and unitary glycerol production decreased by up to 20%.

Impact of Study: By using protective agents it is possible to minimize the abnormal fermentation performance that wine yeasts exhibit under thermal and osmotic stress.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. References

Changes in environmental conditions such as temperature, aeration and nutrients can significantly affect yeast performance during alcoholic fermentation. Such changes serve to either exert or alleviate stress, thus affecting the response of the cells. This stress response causes significant changes in cellular composition that may either directly or indirectly impact on their fermentative performance (Majara et al. 1996).

In alcoholic fermentation at high temperature of must from dried grapes, yeasts are subjected, in addition, to thermal and osmotic stresses. In this case, wine yeasts modify their metabolic behaviour and, probably as a defence mechanism, give low ethanol yield and abnormally high acetic acid production (Caridi et al. 1999a).

It is well known that under stress Saccharomyces cerevisiae synthesizes and accumulates some derivatives of inositol (Dove et al. 1997). Moreover, it was noticed that similar stress-protective effects were obtained by supplying cells with catechin (Prior and Cao 1999) and antioxidants (Kagan and Tyurina 1998).

It is possible that protectants such as inositol, catechin and SO2 may minimize the abnormal fermentation performance of S. cerevisiae in the presence of stressors, thus improving wine quality.

The present research aims to minimize the metabolic consequences of stress caused to wine yeasts during alcoholic fermentation at high temperature of must at very high osmotic strength. Thus, it was decided to investigate the effect of three stress-protectants on the winemaking performance of six wine yeasts under concomitant thermal and osmotic stress.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. References

Three thermotolerant strains of S. cerevisiae– TT51, TT77 and TT141 – selected for winemaking of musts from dried grapes (Caridi et al. 1999b) and three strains of SaccharomycesS. cerevisiae 220 from the DIPROVAL collection, Coviolo (RE), Italy, S. cerevisiae VL1 from Lallemand and S. bayanus from Fermichamp (abbreviated as Ferm) – selected for winemaking were employed.

The sample of must from dried grapes utilized had the following characteristics: cultivar Greco Bianco, pH 3·14, titratable acidity 5·76 g l−1, sugar content adjusted to over 400 g l−1 by glucose monohydrate. The grape must was filtered through gauze, clarified at 4 °C for 24 h, and divided into four lots. To three lots a different protectant was added: (±) catechin produced by Sigma, inositol, and SO2 as potassium metabisulphite. The fourth lot was used as a control and no protectant was added. The three protective compounds were added at 100 mg l−1; this value was based on the dose of SO2 usually employed in winemaking. Each lot of grape must was divided into aliquots of 100 ml, dispensed into 180-ml flasks, and immediately inoculated in duplicate with 5% of 48 h-preculture of each strain in pasteurized grape must. To each flask, 10 ml of liquid paraffin was added to minimize the evaporation of water and of other volatiles and to avoid the surface coming into contact with oxygen. The flasks were then incubated at 35 °C.

The fermentation vigour after 3 days was determined by measuring the loss in weight of each flask, and expressed as g of CO2 100 ml−1 of must. After 30 days the samples were clarified at 4 °C for 24 h, decanted and analysed. Titratable acidity expressed as g l−1 tartaric acid, and ethanol content, expressed as percentage (v/v), were determined using standard methods (Ough and Amerine 1988). Acetic acid and glycerol were tested with specific Boehringer kits on diluted samples. Unitary production of these two parameters was calculated by correlating the values with ethanol content; data were expressed as g 100 ml−1 of ethanol. All parameters were elaborated by ANOVA analysis.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. References

Table 1 reports the values of fermentation vigour after 3 days of the six wine yeasts utilized. When protectants were added to the grape must, two strains did not show any significant effects; however, strain Ferm and all the three thermotolerant yeasts showed significant (< 0·01 or < 0·05) increases when catechin or inositol were added. The increase in fermentation vigour varied, compared to the control, from 0·07 (strain 220) to 0·43 g 100 ml−1 of must (strain TT77) when catechin was added (mean 0·25) and from 0·26 (strain VL1) to 0·50 g 100 ml−1 of must (strain TT51) when inositol was added (mean 0·40). The addition of SO2 induced an increase in this parameter in only two of the six strains, due to the partial sensibility of the other four strains. The increase in fermentation vigour induced by the addition of catechin or inositol indicates a better adaptation of yeasts to stressors. From the technological point of view, the higher fermentation rate reduces the possibility that, during the start of the winemaking, extraneous or harmful thermophilic or osmophilic microorganisms could develop.

Table 1.  Fermentation vigour after 3 d (g of CO2 100 ml−1 of must) of the six strains of wine yeasts Thumbnail image of

Table 2 reports the values of titratable acidity of the wines produced by the six wine yeasts. Apart from strain TT141, which did not show any significant effects, all the strains produced wines with significant (< 0·01 or < 0·05) increases in titratable acidity when protectants were added. The increase varied, compared to the control, from 0·15 (strain 220) to 0·41 g l−1 (strain Ferm) when catechin was added (mean 0·29), from 0·14 (strain TT141) to 0·46 g l−1 (strain Ferm) when inositol was added (mean 0·34), and from 0·21 (strain TT141) to 0·44 g l−1 (strain TT51) when SO2 was added (mean 0·32). The wine yeasts in the presence of the protectants produce wines with higher levels of titratable acidity; however, from the technological point of view, this increase is of scarce interest because differences are rather small.

Table 2.  Titratable acidity (g l−1 tartaric acid) of the wines produced by the six strains of wine yeasts Thumbnail image of

Table 3 reports the values of ethanol content of the wines produced by the six tested strains. Protectants induced a significant (< 0·01 or < 0·05) increase in ethanol yield. The increase varied, compared to the control, from 0·80 (strain TT77) to 1·30% v/v (strain TT51) when catechin was added (mean 1·08), from 0·60 (strain TT141) to 1·60% v/v (strain 220) when inositol was added (mean 0·88), and from 0·05 (strain Ferm) to 0·90% v/v (strain TT51) when SO2 was added (mean 0·44). The increase in ethanol content induced by addition of the protectants shows that wine yeasts acquire a higher ability to resist ethanol stress. From the technological point of view this is useful, because the increase in the ethanol content of the wines guarantees that wine yeasts will carry out a more complete winemaking process, in spite of an environment where thermal and osmotic stresses are exacerbated by ethanol stress.

Table 3.  Ethanol content (% v/v) of the wines produced by the six strains of wine yeasts Thumbnail image of

Table 4 reports the unitary acetic acid production of the wines produced by the six tested strains. All the protectants induced a very significant (< 0·01) decrease in the unitary acetic acid production. The decrease varied, compared to the control, from 0·18 (strain Ferm) to 0·72 g 100 ml−1 of ethanol (strain TT51) when catechin was added (mean 0·47), from 0·19 (strain Ferm) to 0·53 g 100 ml−1 of ethanol (strain TT77) when inositol was added (mean 0·38), and from 0·07 (strain Ferm) to 0·53 g 100 ml−1 of ethanol (strain TT77) when SO2 was added (mean 0·30). Recently catechin was reported to cause a significant (P=0·01) decrease in acetic acid production by yeasts in strongly clarified grape musts, probably due to a direct inhibition of the aldehyde dehydrogenase (Moruno et al. 1993). The marked reduction of the unitary acetic acid production observed in the present study validates the role in stress protection of catechin and, therefore, possibly other phenolic compounds. From the technological point of view, it seems possible to minimize the abnormal production of acetic acid by wine yeasts under concomitant thermal and osmotic stress using catechin, inositol or SO2.

Table 4.  Unitary acetic acid production (g 100 ml−1 of ethanol) of the wines produced by the six strains of wine yeasts Thumbnail image of

Table 5 reports the unitary glycerol production of the wines produced by the six tested strains. All the protectants induced a very significant (< 0·01) decrease in the unitary glycerol production. The decrease varied, compared to the control, from 2·79 (strain VL1) to 3·87 g 100 ml−1 of ethanol (strain TT51) when catechin was added (mean 3·34), from 1·83 (strain VL1) to 3·81 g 100 ml−1 of ethanol (strain 220) when inositol was added (mean 2·89), and from 0·41 (strain VL1) to 4·30 g 100 ml−1 of ethanol (strain TT77) when SO2 was added (mean 2·04). The glycerol content indicates the degree of microbial stress: the higher the level of glycerol, the higher the level of microbial stress. Indeed, glycerol has specific functions in stress protection (Shen et al. 1999); enhanced production and accumulation of glycerol was observed under stress in S. cerevisiae (Sunder et al. 1996). The protectants induce marked reductions of the unitary glycerol production, thus showing their ability to increase the stress-tolerance of wine yeasts.

Table 5.  Unitary glycerol production (g 100 ml−1 of ethanol) of the wines produced by the six strains of wine yeasts Thumbnail image of

Depending on strain and protectant, and expressing data as a percentage of increase or decrease compared to the control, fermentation vigour after 3 days increased by up to 10%, titratable acidity of the wines increased by up to 7%, ethanol content increased by up to 20%, unitary acetic acid production decreased by up to 35%, and unitary glycerol production decreased by up to 20%. Analysing the global behaviour of the six strains, it should be noted that:

1 protectants increase fermentation vigour, titratable acidity, and ethanol yield;

2 protectants decrease unitary acetic acid and glycerol yields;

3 inositol gives the best overall performances, followed by catechin.

These results show significant correlations between the addition of protectants and the change in metabolic behaviour of yeasts under concomitant thermal and osmotic stress. So, by adding the proper protectant, in particular inositol or catechin, it is possible to minimize the abnormal fermentation performance that wine yeasts exhibit in winemaking of must at very high sugar content fermented at high temperature.

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. References

The research was supported by a grant from the Ministry of Scientific Research and Technology – Research fund 60%: A. Caridi.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. References
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