understanding wine lees by Bruce Zoecklein

cewh Published the article • 0 comments • 225 views • 2016-08-31 20:22 • 来自相关话题

The Nature of Wine Lees
During aging sur lie, yeast components are released into the wine. These macromolecules can positively influence structural integration, phenols (including tannins), body, aroma, oxygen buffering, and wine stability. Some macromolecules can provide a sense of sweetness as a result of bridging the sensory sensations between the phenolic elements, acidity, and alcohol, aiding in harmony and integration.
Mannoproteins in the yeast cell wall are bound to glucans (glucose polymers), which exist in wines as polysaccharide and protein moieties (Feuillat, 2003). They are released from the yeast cell wall by the action of an enzyme, β-1,3-glucanase. β-1,3-glucanase is active during yeast growth (fermentation) and during aging in the presence of non-multiplying yeast cells. Stirring increases the concentration (Feuillat, 1998).
Lees and mannoproteins can impact the following:

integration of mouthfeel elements by interaction between structural/textural features
reduction in the perception of astringency and bitterness (Escot et al., 2001; Saucier, 1997)
increasing wine body
encouraging the growth of malolactic bacteria and, possibly, yeasts
preventing bitartrate instability (Lubbers et al., 1993; Moine-Ledoux, 1996; Moine-Ledoux and Dubourdieu, 2002; Waters et al., 1994)
interacting with wine aroma (Lubbers et al., 1994)

The amount of mannoprotein released during fermentation is dependent on several factors, including the following:

Yeast strain: Large differences are noted among yeasts in the amount of mannoproteins produced during fermentation and released during autolysis.
Must turbidity: Generally, the more turbid the must, the lower the mannoprotein concentration (Guilloux-Benatier et al., 1995). Mannoproteins released during fermentation are more reactive than those released during the yeast autolysis process in modifying astringency. This helps provide additional justification for measuring the non-soluble solids of juice pre-fermentation.

Wines aged on lees with no fining have mannoproteins present, while those fined prior to aging have a large percentage of mannoproteins removed. Periodic stirring sur lie increases the mannoprotein concentration, and increases the rate of β-1,3-glucanase activity. Generally, yeast autolysis is relatively slow (in the absence of glucanase enzyme addition) and may require months or years to occur, limiting the mannoprotein concentration (Charpentier and Feuillat, 1993).
The impact of lees components such as polysaccharides on astringency can cause an increase in the wine’s volume or body. Lees contact is particularly effective at modifying wood tannin astringency by binding with free ellagic tannins (harsh tannins). Sur lie storage can reduce the free ellagic acid by as much as 60% (via precipitation), while increasing the percentage of ellagic tannins bound to polysaccharides by 24% (Ribéreau-Gayon et al., 2000).
In the Burgundy and other regions, red wines are aged on their lees in conjunction with the addition of exogenous β ‑1,3-glucanase enzyme. This procedure is an attempt to release mannoproteins, which winemakers believe may enhance the suppleness of the wine, while reducing the perceived astringency.
Several alternative methods of increasing mannoprotein levels have been suggested (Feuillat, 2003), including the following:

selection and use of yeast which produce high levels of mannoproteins during the alcoholic fermentation
yeast which autolyze rapidly upon completion of alcoholic fermentation
addition of β-1,3-glucanase to wines stored on lees
addition of exogenous mannoproteins (proprietary products), prepared from yeast cell walls, to wines on lees

Lees Management Considerations.
Table 1 shows some important practical winemaking considerations regarding lees management.
During fermentation, the level of macromolecules continually rises, peaking at approximately 270 mg/L, by which time they contain 82% sugar and only 18% protein (Feuillat, 2003).
Guilloux‑Benatier et al. (1995) found a relationship between the degrees of must clarification and the amount of yeast macromolecules recovered in the wine. When the must was not clarified, there is no production of yeast macromolecules.
Table 1. Lees Management Considerations

Non-soluble solids level

Method of stirring

Frequency and duration of stirring

Type and size of vessel

Duration of lees contact

MLF

Timing and type of racking

SO2 timing and level of addition

Frequency of barrel topping

However, mild must clarification, such as cooling for 12 hours, increased the amount of yeast-produced macromolecule production by 76 mg/L, and heavy must clarification, such as bentonite fining, increased the production by 164 mg/L. Boivin et al. (1998) found that the amount of macromolecules produced will vary between 230 and 630 mg/L, and that they will contain 20 – 30% glucose and 70 – 80% mannose.
During lees contact, the composition of the wine changes as the yeast commence enzymatic hydrolysis of their cellular contents. One important feature is the process of proteolysis, whereby proteins are hydrolyzed to amino acids and peptides. These compounds result in an increase in the available nitrogen content of the wine. Amino acids can act as flavor precursors, possibly enhancing wine complexity and quality.
Yeast-derived macromolecules provide a sense of sweetness as a result of binding with wood phenols and organic acids, aiding in the harmony of a wine’s structural elements by softening tannins.
It is important to differentiate between light lees and heavy lees. Heavy lees can be defined as the lees which precipitate within 24 hours immediately post-fermentation. They are composed of large particles (greater than 100 micrometers) and consist of grape particulates, agglomerates of tartrate crystals, yeasts, bacteria, and protein-polysaccharide-tannin complexes.
Light lees, on the other hand, can be defined as those that precipitate from the wine more than 24 hours post-fermentation. These are composed mainly of small particles (1- 25 micrometers) of yeasts, bacteria, tartaric acid, protein-tannin complexes, and some polysaccharides.
There is no value in storing wine on heavy lees. Indeed, such storage can result in off aroma and flavors, and a depletion of sulfur dioxide. Light lees storage, however, can have a significant advantage in structural balance, complexity, and stability.
Lees stirring and the frequency of stirring is important, both as a practical and stylistic consideration. Feuillat and Charpentier (1998) have demonstrated that periodic stirring of the wine while on lees increases the mannoprotein level and the amount of yeast-derived amino acids, and that wines aged on their lees in barrel exhibit an increase in colloidal macromolecules.
Stirring generates an oxidative process which increases the acetaldehyde content, and which may increase the acetic acid concentration. Stirring also changes the sensory balance between fruit, yeast, and wood by enhancing the yeast component, and reducing the fruit and, to a lesser degree, the wood component.
Additionally, stirring may have the effect of enhancing secondary chemical reactions, possibly as the result of oxygen pick-up. Stuckey et al. (1991) demonstrated increases in both the total amino acid content and wine sensory score in wines stored for five months without stirring. The non-stirred wine was perceived to have greater fruit intensity.
MLF reduces the harshness of new oak and aids in the development of complex and mature flavors. Traditionally, stirring is continued until MLF is complete. After that, the lees are said to become more dense, which aids in clarification.
During barrel aging, what we are looking for is slow, well-managed, and controlled oxygenation. Some lees contact may allow for this oxygenation, and lees aid in the prevention of oxidation.
In Burgundy, wines are traditionally racked off the lees in March, usually the time when MLF is completed. Frequently this is an aerobic racking off the heavy lees, then back into wood on light lees, followed by an SO2 addition. Leaving the wine on the light lees helps to nourish the wine. The addition of SO2 helps to protect the wine from oxidation. A subsequent racking often occurs in early July, and is in the absence of air.
Timing of SO2 additions, and the quantity of SO2 added, are important stylistic considerations. Early use of SO2 increases the number of components that bind to subsequent additions of SO2. The addition of too much SO2 counters the wood flavors and limits oxidation reactions, while too little SO2 may allow the wine to become tired and over-aged.
Production considerations, such as the timing of MLF, the method of barrel storage, and time of bottling, are factors influencing SO2 levels. Barrel topping is an aerobic process that can result in excessive oxidation. Additionally, wines that spend a second winter in the cellar tend to lose their aroma unless the wine is particularly rich.
Delteil (2002) compared two red wines. One wine was barrel-stored on light lees for 9 months; the other, racked several times prior to barreling, was stored for the same period without lees. These two Syrah wines differed significantly in their palate and aroma profiles.
The wine stored sur lie had a much lower perception of astringency and a greater integration of the phenolic elements. The sur lie wine also had a lower perception of oak character, resulting in a higher perception of varietal fruit.
Lees contact is particularly effective at modifying wood tannin astringency by binding with free ellagic tannins, thus lowering the proportion of active tannins. Sur lie storage can reduce the free ellagic acid by as much as 60%, while increasing the percentage of ellagic tannins bound to polysaccharides by 24% (Ribéreau-Gayon et al., 2000).
The following is a review of the impact of lees on wines.

Lees, Color and Mouthfeel.High lees concentration can reduce color, as a function of adsorption onto the yeast cell surface.  Additionally, lees adsorb oxygen which can limit the anthocyanin-tannin polymerization, resulting in an increase in dry tannin perception. This may or may not be off-set by the release of lees components which can soften mouthfeel.

Lees and Wine Aroma. Aroma stabilization is dependent upon the hydrophobicity (ability to repel water molecules) of the aroma compounds. The protein component of the mannoprotein fraction is important for overall aroma stabilization (Lubbers et al., 1994). Such interactions can modify the volatility and aromatic intensity of wines.

When wine is aged on its lees with no fining, mannoproteins are present and are free to interact and to fortify the existing aroma components. When wines are fined prior to aging, mannoproteins are removed and will not be present to augment the existing aroma components. Additionally, when wines are cross‑flow filtered, eliminating a certain percentage of macromolecules, the loss of color intensity, aroma, and flavor can be noted.

Lees and Oak Bouquet. Lees modify oaky aromas, due to their ability to bind with wood-derived compounds such as vanillin, furfural, and methyl-octalactones.

Lees and Oxidative Buffering Capacity. Both lees and tannins act as reducing agents. During aging, lees release certain highly-reductive substances which limit wood-induced oxygenation. Wines have a higher oxidation-reduction potential in barrels than in tanks. Inside the barrel, this potential diminishes from the wine surface to the lees. Stirring helps to raise this potential.

This is a primary reason why wines stored in high-volume tanks should not be stored on their lees. Such storage can cause the release of “reductive” or sulfur-containing compounds. If there is a desire to store dry wines in tanks sur lie, it is recommended that the lees be stored in barrels for several months, then added back to the tank (Ribéreau-Gayon et al., 2000).

Lees and White Wine Protein Stability. The greater the lees contact, the lower the need for bentonite or other fining agents for protein stability. It is not believed that lees hydrolyze grape proteins, or that proteins are adsorbed by yeast. Rather, lees aging produces an additional mannoprotein, which somehow adds stability. The production of this mannoprotein is increased with temperature, time, and frequency of stirring.

Lees and Biological Stability. Guilloux‑Benatier et al. (2001) have studied the liberation of amino acids and glucose during barrel aging of Burgundy wine on its lees. Their studies were done with and without the addition of exogenous β‑1,3-glucanase preparations. They found little or no increase in amino acids in wine stored on lees, versus wine stored on lees with the addition of β‑1,3-glucanase.

Their most significant finding was an increase in glucose concentration, from 43 mg/L in the control wine, to 570 mg/L in wine stored on its lees, to 910 mg/L in wine stored on its lees with added β ‑1,3-glucanase. The finding of this relatively large amount of glucose led these authors to speculate that the growth of the spoilage yeast Brettanomycesin barreled wine may be stimulated by the availability of this carbon source.

Lees and Bitartrate Stability. Mannoproteins produced by yeast can act as crystalline inhibitors. The longer the lees contact time, the greater is the likelihood of potassium bitartrate stability.
 
reference:http://www.newworldwinemaker.com/2016/05/5715/ 查看全部
The Nature of Wine Lees
During aging sur lie, yeast components are released into the wine. These macromolecules can positively influence structural integration, phenols (including tannins), body, aroma, oxygen buffering, and wine stability. Some macromolecules can provide a sense of sweetness as a result of bridging the sensory sensations between the phenolic elements, acidity, and alcohol, aiding in harmony and integration.
Mannoproteins in the yeast cell wall are bound to glucans (glucose polymers), which exist in wines as polysaccharide and protein moieties (Feuillat, 2003). They are released from the yeast cell wall by the action of an enzyme, β-1,3-glucanase. β-1,3-glucanase is active during yeast growth (fermentation) and during aging in the presence of non-multiplying yeast cells. Stirring increases the concentration (Feuillat, 1998).
Lees and mannoproteins can impact the following:

integration of mouthfeel elements by interaction between structural/textural features
reduction in the perception of astringency and bitterness (Escot et al., 2001; Saucier, 1997)
increasing wine body
encouraging the growth of malolactic bacteria and, possibly, yeasts
preventing bitartrate instability (Lubbers et al., 1993; Moine-Ledoux, 1996; Moine-Ledoux and Dubourdieu, 2002; Waters et al., 1994)
interacting with wine aroma (Lubbers et al., 1994)

The amount of mannoprotein released during fermentation is dependent on several factors, including the following:

Yeast strain: Large differences are noted among yeasts in the amount of mannoproteins produced during fermentation and released during autolysis.
Must turbidity: Generally, the more turbid the must, the lower the mannoprotein concentration (Guilloux-Benatier et al., 1995). Mannoproteins released during fermentation are more reactive than those released during the yeast autolysis process in modifying astringency. This helps provide additional justification for measuring the non-soluble solids of juice pre-fermentation.

Wines aged on lees with no fining have mannoproteins present, while those fined prior to aging have a large percentage of mannoproteins removed. Periodic stirring sur lie increases the mannoprotein concentration, and increases the rate of β-1,3-glucanase activity. Generally, yeast autolysis is relatively slow (in the absence of glucanase enzyme addition) and may require months or years to occur, limiting the mannoprotein concentration (Charpentier and Feuillat, 1993).
The impact of lees components such as polysaccharides on astringency can cause an increase in the wine’s volume or body. Lees contact is particularly effective at modifying wood tannin astringency by binding with free ellagic tannins (harsh tannins). Sur lie storage can reduce the free ellagic acid by as much as 60% (via precipitation), while increasing the percentage of ellagic tannins bound to polysaccharides by 24% (Ribéreau-Gayon et al., 2000).
In the Burgundy and other regions, red wines are aged on their lees in conjunction with the addition of exogenous β ‑1,3-glucanase enzyme. This procedure is an attempt to release mannoproteins, which winemakers believe may enhance the suppleness of the wine, while reducing the perceived astringency.
Several alternative methods of increasing mannoprotein levels have been suggested (Feuillat, 2003), including the following:

selection and use of yeast which produce high levels of mannoproteins during the alcoholic fermentation
yeast which autolyze rapidly upon completion of alcoholic fermentation
addition of β-1,3-glucanase to wines stored on lees
addition of exogenous mannoproteins (proprietary products), prepared from yeast cell walls, to wines on lees

Lees Management Considerations.
Table 1 shows some important practical winemaking considerations regarding lees management.
During fermentation, the level of macromolecules continually rises, peaking at approximately 270 mg/L, by which time they contain 82% sugar and only 18% protein (Feuillat, 2003).
Guilloux‑Benatier et al. (1995) found a relationship between the degrees of must clarification and the amount of yeast macromolecules recovered in the wine. When the must was not clarified, there is no production of yeast macromolecules.
Table 1. Lees Management Considerations

Non-soluble solids level

Method of stirring

Frequency and duration of stirring

Type and size of vessel

Duration of lees contact

MLF

Timing and type of racking

SO2 timing and level of addition

Frequency of barrel topping

However, mild must clarification, such as cooling for 12 hours, increased the amount of yeast-produced macromolecule production by 76 mg/L, and heavy must clarification, such as bentonite fining, increased the production by 164 mg/L. Boivin et al. (1998) found that the amount of macromolecules produced will vary between 230 and 630 mg/L, and that they will contain 20 – 30% glucose and 70 – 80% mannose.
During lees contact, the composition of the wine changes as the yeast commence enzymatic hydrolysis of their cellular contents. One important feature is the process of proteolysis, whereby proteins are hydrolyzed to amino acids and peptides. These compounds result in an increase in the available nitrogen content of the wine. Amino acids can act as flavor precursors, possibly enhancing wine complexity and quality.
Yeast-derived macromolecules provide a sense of sweetness as a result of binding with wood phenols and organic acids, aiding in the harmony of a wine’s structural elements by softening tannins.
It is important to differentiate between light lees and heavy lees. Heavy lees can be defined as the lees which precipitate within 24 hours immediately post-fermentation. They are composed of large particles (greater than 100 micrometers) and consist of grape particulates, agglomerates of tartrate crystals, yeasts, bacteria, and protein-polysaccharide-tannin complexes.
Light lees, on the other hand, can be defined as those that precipitate from the wine more than 24 hours post-fermentation. These are composed mainly of small particles (1- 25 micrometers) of yeasts, bacteria, tartaric acid, protein-tannin complexes, and some polysaccharides.
There is no value in storing wine on heavy lees. Indeed, such storage can result in off aroma and flavors, and a depletion of sulfur dioxide. Light lees storage, however, can have a significant advantage in structural balance, complexity, and stability.
Lees stirring and the frequency of stirring is important, both as a practical and stylistic consideration. Feuillat and Charpentier (1998) have demonstrated that periodic stirring of the wine while on lees increases the mannoprotein level and the amount of yeast-derived amino acids, and that wines aged on their lees in barrel exhibit an increase in colloidal macromolecules.
Stirring generates an oxidative process which increases the acetaldehyde content, and which may increase the acetic acid concentration. Stirring also changes the sensory balance between fruit, yeast, and wood by enhancing the yeast component, and reducing the fruit and, to a lesser degree, the wood component.
Additionally, stirring may have the effect of enhancing secondary chemical reactions, possibly as the result of oxygen pick-up. Stuckey et al. (1991) demonstrated increases in both the total amino acid content and wine sensory score in wines stored for five months without stirring. The non-stirred wine was perceived to have greater fruit intensity.
MLF reduces the harshness of new oak and aids in the development of complex and mature flavors. Traditionally, stirring is continued until MLF is complete. After that, the lees are said to become more dense, which aids in clarification.
During barrel aging, what we are looking for is slow, well-managed, and controlled oxygenation. Some lees contact may allow for this oxygenation, and lees aid in the prevention of oxidation.
In Burgundy, wines are traditionally racked off the lees in March, usually the time when MLF is completed. Frequently this is an aerobic racking off the heavy lees, then back into wood on light lees, followed by an SO2 addition. Leaving the wine on the light lees helps to nourish the wine. The addition of SO2 helps to protect the wine from oxidation. A subsequent racking often occurs in early July, and is in the absence of air.
Timing of SO2 additions, and the quantity of SO2 added, are important stylistic considerations. Early use of SO2 increases the number of components that bind to subsequent additions of SO2. The addition of too much SO2 counters the wood flavors and limits oxidation reactions, while too little SO2 may allow the wine to become tired and over-aged.
Production considerations, such as the timing of MLF, the method of barrel storage, and time of bottling, are factors influencing SO2 levels. Barrel topping is an aerobic process that can result in excessive oxidation. Additionally, wines that spend a second winter in the cellar tend to lose their aroma unless the wine is particularly rich.
Delteil (2002) compared two red wines. One wine was barrel-stored on light lees for 9 months; the other, racked several times prior to barreling, was stored for the same period without lees. These two Syrah wines differed significantly in their palate and aroma profiles.
The wine stored sur lie had a much lower perception of astringency and a greater integration of the phenolic elements. The sur lie wine also had a lower perception of oak character, resulting in a higher perception of varietal fruit.
Lees contact is particularly effective at modifying wood tannin astringency by binding with free ellagic tannins, thus lowering the proportion of active tannins. Sur lie storage can reduce the free ellagic acid by as much as 60%, while increasing the percentage of ellagic tannins bound to polysaccharides by 24% (Ribéreau-Gayon et al., 2000).
The following is a review of the impact of lees on wines.

Lees, Color and Mouthfeel.High lees concentration can reduce color, as a function of adsorption onto the yeast cell surface.  Additionally, lees adsorb oxygen which can limit the anthocyanin-tannin polymerization, resulting in an increase in dry tannin perception. This may or may not be off-set by the release of lees components which can soften mouthfeel.

Lees and Wine Aroma. Aroma stabilization is dependent upon the hydrophobicity (ability to repel water molecules) of the aroma compounds. The protein component of the mannoprotein fraction is important for overall aroma stabilization (Lubbers et al., 1994). Such interactions can modify the volatility and aromatic intensity of wines.

When wine is aged on its lees with no fining, mannoproteins are present and are free to interact and to fortify the existing aroma components. When wines are fined prior to aging, mannoproteins are removed and will not be present to augment the existing aroma components. Additionally, when wines are cross‑flow filtered, eliminating a certain percentage of macromolecules, the loss of color intensity, aroma, and flavor can be noted.

Lees and Oak Bouquet. Lees modify oaky aromas, due to their ability to bind with wood-derived compounds such as vanillin, furfural, and methyl-octalactones.

Lees and Oxidative Buffering Capacity. Both lees and tannins act as reducing agents. During aging, lees release certain highly-reductive substances which limit wood-induced oxygenation. Wines have a higher oxidation-reduction potential in barrels than in tanks. Inside the barrel, this potential diminishes from the wine surface to the lees. Stirring helps to raise this potential.

This is a primary reason why wines stored in high-volume tanks should not be stored on their lees. Such storage can cause the release of “reductive” or sulfur-containing compounds. If there is a desire to store dry wines in tanks sur lie, it is recommended that the lees be stored in barrels for several months, then added back to the tank (Ribéreau-Gayon et al., 2000).

Lees and White Wine Protein Stability. The greater the lees contact, the lower the need for bentonite or other fining agents for protein stability. It is not believed that lees hydrolyze grape proteins, or that proteins are adsorbed by yeast. Rather, lees aging produces an additional mannoprotein, which somehow adds stability. The production of this mannoprotein is increased with temperature, time, and frequency of stirring.

Lees and Biological Stability. Guilloux‑Benatier et al. (2001) have studied the liberation of amino acids and glucose during barrel aging of Burgundy wine on its lees. Their studies were done with and without the addition of exogenous β‑1,3-glucanase preparations. They found little or no increase in amino acids in wine stored on lees, versus wine stored on lees with the addition of β‑1,3-glucanase.

Their most significant finding was an increase in glucose concentration, from 43 mg/L in the control wine, to 570 mg/L in wine stored on its lees, to 910 mg/L in wine stored on its lees with added β ‑1,3-glucanase. The finding of this relatively large amount of glucose led these authors to speculate that the growth of the spoilage yeast Brettanomycesin barreled wine may be stimulated by the availability of this carbon source.

Lees and Bitartrate Stability. Mannoproteins produced by yeast can act as crystalline inhibitors. The longer the lees contact time, the greater is the likelihood of potassium bitartrate stability.
 
reference:http://www.newworldwinemaker.com/2016/05/5715/

The Aromatic Thiols

cewh Published the article • 0 comments • 240 views • 2016-07-02 14:27 • 来自相关话题

At the AWITC technical conference in July, we attended an aroma and flavour compound workshop where we were given the opportunity to familiarize ourselves with a range of wine-related, good-bad-and-ugly aroma compounds. In a previous release we discussed the different compounds that contribute to green character in wine. This time we focus on one of the important fermentation aromas, the aromatic thiols.

These are sulphur-containing compounds, and are related by their chemistry to the negative ‘reductive aromas’ previously discussed. The most important fermentation thiols include:
3MHA: 3-mercaptohexylacetate. Passion fruit, gooseberry, guava and other tropical fruit aromas at lower levels, sweaty at higher levels. Sensory perception threshold 4ng/L3MH: 3-mercaptohexanol. Passion fruit, grapefruit and general citrus aromas. Sensory perception threshold 60ng/L4MMP: 4-mercapto-4-methylpentan-2-one. Box tree, broom, blackcurrant and cat urine aromas. Sensory perception threshold 0.8-3.0ng/L

These aromas contribute significantly to the aroma of Sauvignon blanc, but also form part of the fruit aromas of Cabernet Sauvignon, Merlot, Shiraz and Grenache, as well as other white varieties such as Chenin blanc, Riesling, Pinot gris and Gewurztraminer.

Fermentation thiols have their precursors in the grape and are released into wine to a greater or lesser degree depending on the way the grapes and juice are handled, both in the vineyard and the winery.

The 4MMP precursors develop earlier in the grape than those of 3MH and 3MHA. Thus timing of harvest can influence the relative concentration of the individual thiols in wine, and thus which of the aromas will be more dominant. Earlier harvesting favours higher concentrations of 4MMP in the resulting wine and the potential dominance of boxtree aromas, while later harvesting favours 3MH and 3MHA and the potential dominance of tropical and citrus aromas.

4MMP is found equally in skin and pulp while 3MH and 3MHA are found mainly near the skins. Thus skin damage e.g. by mechanical harvester, or during crushing may proportionately increase the levels of the 3MH and 3MHA precursors in the juice. The same is true for skin contact and the use of extraction enzymes. This proportionate increase in 3MH and 3MHA precursors may proportionately increase the levels of their thiols in the wine.

Yeast strain selection can significantly affect the concentration and relative proportion of the individual thiols. Many commercial strains have been specifically isolated to enhance thiol release into wine. Relatively higher fermentation temperatures may also favour the release of thiols.

Reductive processing and maturation conditions will favour the preservation of thiols in wine.

Note that, because they are chemically related to the volatile sulphur compounds, any CuSO4 fining will remove these desirable aromatics from wine.
reference: http://www.vinlab.com/blog/Details/4#.V3clf-wQjtg 查看全部
At the AWITC technical conference in July, we attended an aroma and flavour compound workshop where we were given the opportunity to familiarize ourselves with a range of wine-related, good-bad-and-ugly aroma compounds. In a previous release we discussed the different compounds that contribute to green character in wine. This time we focus on one of the important fermentation aromas, the aromatic thiols.

These are sulphur-containing compounds, and are related by their chemistry to the negative ‘reductive aromas’ previously discussed. The most important fermentation thiols include:
  • 3MHA: 3-mercaptohexylacetate. Passion fruit, gooseberry, guava and other tropical fruit aromas at lower levels, sweaty at higher levels. Sensory perception threshold 4ng/L
  • 3MH: 3-mercaptohexanol. Passion fruit, grapefruit and general citrus aromas. Sensory perception threshold 60ng/L
  • 4MMP: 4-mercapto-4-methylpentan-2-one. Box tree, broom, blackcurrant and cat urine aromas. Sensory perception threshold 0.8-3.0ng/L


These aromas contribute significantly to the aroma of Sauvignon blanc, but also form part of the fruit aromas of Cabernet Sauvignon, Merlot, Shiraz and Grenache, as well as other white varieties such as Chenin blanc, Riesling, Pinot gris and Gewurztraminer.

Fermentation thiols have their precursors in the grape and are released into wine to a greater or lesser degree depending on the way the grapes and juice are handled, both in the vineyard and the winery.

The 4MMP precursors develop earlier in the grape than those of 3MH and 3MHA. Thus timing of harvest can influence the relative concentration of the individual thiols in wine, and thus which of the aromas will be more dominant. Earlier harvesting favours higher concentrations of 4MMP in the resulting wine and the potential dominance of boxtree aromas, while later harvesting favours 3MH and 3MHA and the potential dominance of tropical and citrus aromas.

4MMP is found equally in skin and pulp while 3MH and 3MHA are found mainly near the skins. Thus skin damage e.g. by mechanical harvester, or during crushing may proportionately increase the levels of the 3MH and 3MHA precursors in the juice. The same is true for skin contact and the use of extraction enzymes. This proportionate increase in 3MH and 3MHA precursors may proportionately increase the levels of their thiols in the wine.

Yeast strain selection can significantly affect the concentration and relative proportion of the individual thiols. Many commercial strains have been specifically isolated to enhance thiol release into wine. Relatively higher fermentation temperatures may also favour the release of thiols.

Reductive processing and maturation conditions will favour the preservation of thiols in wine.

Note that, because they are chemically related to the volatile sulphur compounds, any CuSO4 fining will remove these desirable aromatics from wine.
reference: http://www.vinlab.com/blog/Details/4#.V3clf-wQjtg

what are Cava varieties?

Reply

cewh Replyed • 1 person concerned • 1 replies • 500 views • 2016-06-08 21:13 • 来自相关话题

Wine Industry Pumps

cewh Published the article • 0 comments • 259 views • 2016-06-07 17:50 • 来自相关话题

Pumps are used in many beverage and food process applications. For example, egg whites, honey, food oils, apple sauce, apple juice, donut glaze and pancake batter are all moved using pumps. Pumps can also be used to gently circulate fluid when fermenting high alcohol beer where oxygen is injected into the process to significantly reduce the fermentation time.

Pumps can provide a winemaker with the ability to transfer just-harvested grapes from a de-stemmer/crusher to the tank for fermentation. They can also be used for pump overs in fermentation tanks to allow for color enhancement on red wines and providing a way to move the juice from the tank to barrels for aging.

Pumps are also used to move the wine to the filtering process to remove sediment or solids and then to move the wine to the bottling line for packaging. Regardless of the style, pumps provide time savings to the winemaker and should be considered part of the wine production lifeline.

The winemaker should choose a pump that has the greatest versatility for the particular operation. A versatile pump—one that can run at variable speeds and provide a winery with multiple task fulfillment capabilities—is a cost advantage to a winemaker. Some other advantages of a versatile pump are self-priming, reversible flow, portability and ease of cleaning.

This article discusses some typical pumps found in the wine industry. However, they can also be used in other food and beverage industry segments. Pump styles can be offered in flow ranges from a trickle to hundreds of gallons per minute and with AC or DC voltages.

Pumps can be obtained as a pump alone, with the motor attached and or mounted on a cart for ease of movement within the winery. Some pumps offer low pressure and some can produce high discharge pressures. Picking the flow and pressure to meet the needs of the application is important for successful and continuous production.

Flexible Impeller Pumps
Flexible impeller pumps (FIPs) are self-priming with either wet or dry at start up. They offer gentle, smooth and variable flow rates. This design includes a flexible impeller that rotates in a fixed cavity. The use of an offset cam causes the vanes on the impeller to deflect, decreasing the cell volume initially.

When the vanes leave the cam contact, the volume increases between the vanes, and fluid is drawn into the larger cell cavity with the help of atmospheric pressure. As the impeller rotates, it reduces the cell volume at the discharge port on contact with the cam.

Each cavity then produces a nearly-even and perfect smooth flow and is repeated on each revolution of the impeller. These pumps can transfer solids suspended in liquid. They are reversible and can be mounted above or below the liquid source. The fluid has contact with the rubber flexible impeller and the interior of the body housing. Pump bodies and materials, preferably, should be manufactured from sanitary stainless steel with sanitary rubber compounds. These are positive displacement pumps.

A portable, flexible impeller pump used in wine production
Rotary Lobe Pumps & External Circumferential Piston Pumps
Rotary lobe pumps and external circumferential piston (ECP) pumps, positive displacement pumps, offer high efficiency, gentle pumping action and corrosion resistance. These pumps are reliable and can be cleaned in place (CIP) or steamed in place (SIP). Rotary lobe pumps are capable of handling thick or thin solids, liquids and paste products. Some models of rotary lobe pumps perform well on self-priming if wetted. They can produce significant pressure.

These pumps, like FIPs, can have the direction of fluid flow reversed. Run dry capability is possible if the seals are wetted during the run dry timeframe. Rotary lobe pumps have two alternating direction rotating rotors that mesh in operation. The fluid or product flows into the pump and is captured by the rotating lobes. The product is transferred in the cavities around the outside of the lobe body. The product does not effectively travel between the meshing actions of the two lobes. 

Centrifugal Pumps
Centrifugal pumps use gravity to push water into the pump cavity, and the high speed of the pump impeller then discharges the fluid from the discharge port. These pumps tend to be the most efficient with a smooth, pulse-free delivery. Minimal wear is associated with the pump components, the impeller and pump head are generally easily disassembled.

Most centrifugal pumps are small, but can produce a high volume of flow. Most can be obtained in AC and DC versions and are relatively inexpensive. The main draw back to centrifugal pumps is that they are not self-priming and may cavitate easily. The most common form of centrifugal pumps is a radial flow design.

Air-Operated Diaphragm Pumps
Air-operated diaphragm (AOD) pumps use air to power them. The pump design is self-priming, capable of handling high solids content, can run dry, is portable, explosion proof, has a high pumping efficiency and can deliver a variable flow rate and discharge pressure. One disadvantage is the requirement to have an air compressor on hand for use. This is a positive displacement pump.

Written by:
Keith Evans, Jabsco Flexible Impeller Pumps, Xylem, Inc.
Courtersy of: http://www.pump-zone.com/topics/pumps/pumps/wine-industry-pumps
 
reference: http://www.weg.net/nz/Media-Ce ... Pumps 查看全部
Pumps are used in many beverage and food process applications. For example, egg whites, honey, food oils, apple sauce, apple juice, donut glaze and pancake batter are all moved using pumps. Pumps can also be used to gently circulate fluid when fermenting high alcohol beer where oxygen is injected into the process to significantly reduce the fermentation time.

Pumps can provide a winemaker with the ability to transfer just-harvested grapes from a de-stemmer/crusher to the tank for fermentation. They can also be used for pump overs in fermentation tanks to allow for color enhancement on red wines and providing a way to move the juice from the tank to barrels for aging.

Pumps are also used to move the wine to the filtering process to remove sediment or solids and then to move the wine to the bottling line for packaging. Regardless of the style, pumps provide time savings to the winemaker and should be considered part of the wine production lifeline.

The winemaker should choose a pump that has the greatest versatility for the particular operation. A versatile pump—one that can run at variable speeds and provide a winery with multiple task fulfillment capabilities—is a cost advantage to a winemaker. Some other advantages of a versatile pump are self-priming, reversible flow, portability and ease of cleaning.

This article discusses some typical pumps found in the wine industry. However, they can also be used in other food and beverage industry segments. Pump styles can be offered in flow ranges from a trickle to hundreds of gallons per minute and with AC or DC voltages.

Pumps can be obtained as a pump alone, with the motor attached and or mounted on a cart for ease of movement within the winery. Some pumps offer low pressure and some can produce high discharge pressures. Picking the flow and pressure to meet the needs of the application is important for successful and continuous production.

Flexible Impeller Pumps
Flexible impeller pumps (FIPs) are self-priming with either wet or dry at start up. They offer gentle, smooth and variable flow rates. This design includes a flexible impeller that rotates in a fixed cavity. The use of an offset cam causes the vanes on the impeller to deflect, decreasing the cell volume initially.

When the vanes leave the cam contact, the volume increases between the vanes, and fluid is drawn into the larger cell cavity with the help of atmospheric pressure. As the impeller rotates, it reduces the cell volume at the discharge port on contact with the cam.

Each cavity then produces a nearly-even and perfect smooth flow and is repeated on each revolution of the impeller. These pumps can transfer solids suspended in liquid. They are reversible and can be mounted above or below the liquid source. The fluid has contact with the rubber flexible impeller and the interior of the body housing. Pump bodies and materials, preferably, should be manufactured from sanitary stainless steel with sanitary rubber compounds. These are positive displacement pumps.

A portable, flexible impeller pump used in wine production
Rotary Lobe Pumps & External Circumferential Piston Pumps
Rotary lobe pumps and external circumferential piston (ECP) pumps, positive displacement pumps, offer high efficiency, gentle pumping action and corrosion resistance. These pumps are reliable and can be cleaned in place (CIP) or steamed in place (SIP). Rotary lobe pumps are capable of handling thick or thin solids, liquids and paste products. Some models of rotary lobe pumps perform well on self-priming if wetted. They can produce significant pressure.

These pumps, like FIPs, can have the direction of fluid flow reversed. Run dry capability is possible if the seals are wetted during the run dry timeframe. Rotary lobe pumps have two alternating direction rotating rotors that mesh in operation. The fluid or product flows into the pump and is captured by the rotating lobes. The product is transferred in the cavities around the outside of the lobe body. The product does not effectively travel between the meshing actions of the two lobes. 

Centrifugal Pumps
Centrifugal pumps use gravity to push water into the pump cavity, and the high speed of the pump impeller then discharges the fluid from the discharge port. These pumps tend to be the most efficient with a smooth, pulse-free delivery. Minimal wear is associated with the pump components, the impeller and pump head are generally easily disassembled.

Most centrifugal pumps are small, but can produce a high volume of flow. Most can be obtained in AC and DC versions and are relatively inexpensive. The main draw back to centrifugal pumps is that they are not self-priming and may cavitate easily. The most common form of centrifugal pumps is a radial flow design.

Air-Operated Diaphragm Pumps
Air-operated diaphragm (AOD) pumps use air to power them. The pump design is self-priming, capable of handling high solids content, can run dry, is portable, explosion proof, has a high pumping efficiency and can deliver a variable flow rate and discharge pressure. One disadvantage is the requirement to have an air compressor on hand for use. This is a positive displacement pump.

Written by:
Keith Evans, Jabsco Flexible Impeller Pumps, Xylem, Inc.
Courtersy of: http://www.pump-zone.com/topics/pumps/pumps/wine-industry-pumps
 
reference: http://www.weg.net/nz/Media-Ce ... Pumps

Crabtree effect

cewh Published the article • 0 comments • 209 views • 2016-05-22 22:36 • 来自相关话题

From Wikipedia, the free encyclopediaSee also: Evolution of aerobic fermentation

Named after the English biochemist Herbert Grace Crabtree, the Crabtree effect describes the phenomenon whereby the yeast, Saccharomyces cerevisiae, produces ethanol (alcohol) in aerobic conditions and high external glucose concentrations rather than producing biomass via the tricarboxylic acid (TCA) cycle, the usual process occurring aerobically in most yeasts e.g. Kluyveromyces spp[1]. This phenomenon is observed in most species of the Saccharomyces, Schizosaccharomyces, Debaryomyces, Brettanomyces, Torulopsis, Nematospora, and Nadsonia genera.[2] Increasing concentrations of glucose accelerates glycolysis (the breakdown of glucose) which results in the production of appreciable amounts of ATP through substrate-level phosphorylation. This reduces the need of oxidative phosphorylation done by the TCA cycle via the electron transport chain and therefore decreases oxygen consumption. The phenomenon is believed to have evolved as a competition mechanism (due to the antiseptic nature of ethanol) around the time when the first fruits on Earth fell from the trees.[1] The crabtree effect works by repressing respiration by the fermentation pathway, dependent on the substrate.[2] 查看全部

From Wikipedia, the free encyclopediaSee also: Evolution of aerobic fermentation

Named after the English biochemist Herbert Grace Crabtree, the Crabtree effect describes the phenomenon whereby the yeast, Saccharomyces cerevisiae, produces ethanol (alcohol) in aerobic conditions and high external glucose concentrations rather than producing biomass via the tricarboxylic acid (TCA) cycle, the usual process occurring aerobically in most yeasts e.g. Kluyveromyces spp[1]. This phenomenon is observed in most species of the Saccharomyces, Schizosaccharomyces, Debaryomyces, Brettanomyces, Torulopsis, Nematospora, and Nadsonia genera.[2] Increasing concentrations of glucose accelerates glycolysis (the breakdown of glucose) which results in the production of appreciable amounts of ATP through substrate-level phosphorylation. This reduces the need of oxidative phosphorylation done by the TCA cycle via the electron transport chain and therefore decreases oxygen consumption. The phenomenon is believed to have evolved as a competition mechanism (due to the antiseptic nature of ethanol) around the time when the first fruits on Earth fell from the trees.[1] The crabtree effect works by repressing respiration by the fermentation pathway, dependent on the substrate.[2]

Brettanomyces Character in Wine ©Richard Gawel

cewh Published the article • 0 comments • 203 views • 2016-05-22 22:27 • 来自相关话题

Introduction

The desirability or otherwise of the wine character known as "Brett" is one of the most controversial issues of recent times. Arguments have been made for Brett character being a complexing and a legitimate expression of natural, uncomplicated winemaking, while others view it simply as an unattractive wine fault that results from poor winery hygiene and sloppy winemaking. 

[Brettanomyces forming pseudomycelium on oak]
Figure 1: Brettanomyces bruxellensis forming pseudomycelium
© 2004 High Power Ultrasonics Pty Ltd

The Aroma and Flavour of Brett Character

But what is Brett character and how and why does it appear in some wines? The wine character described as "Bretty" comes in various forms. It is the combined result of the creation of a number of compounds by the yeast Brettanomyces bruxellensis, and its close relative, Dekkera bruxulensis. The three most important known aroma active compounds are 1) 4-ethyl phenol (4-ep), which has been variously described as having the aromas of Band-aids®, antiseptic and horse stable 2) 4-ethyl guaiacol (4-eg) which has a rather pleasant aroma of smoked bacon, spice or cloves and 3) isovaleric acid which has an unpleasant smell of sweaty animals, cheese and rancidity. Other characters associated with Brett include wet dog, creosote, burnt beans, rotting vegetation, plastic and (but not exclusively caused by Brett) mouse cage aroma and vinegar.

The Formation of Brett Character in Wine

Brettanomyces has been isolated from the outside of grapes and from winery equipment. However its, favoured winery haunt is the oak barrel as it often provides for conditions that strongly favours its growth.

Certain conditions are known to favour the growth of Brettanomyces during winemaking. If low free sulfur dioxide levels are coupled with high wine pH and warm temperatures during barrel maturation, then issues may arise. If older oak is used and the wine has a reasonable amount of dissolved oxygen, …. look out! Furthermore it is thought that Brett can also multiply after bottling if the wine contains residual fermentable sugars, a situation made more likely if the wine was minimally filtered. Lets look at the why's of these factors.

Brettanomyces proliferates under warm cellaring conditions. Twenty degrees C is an ideal temperature, with even small reductions in temperature seriously hamper its growth. Sulfur dioxide is an anti-microbial agent that is added by winemakers throughout the winemaking process. If it is added in sufficient amounts, and the pH of the wine is reasonably high (SO2 is more effective at higher acidity levels), then the growth of Brett will be retarded. On the other hand, high alcohol levels and the existence of even small amounts of fermentable sugars such as glucose suit the growth of Brett, as they are its preferred source of energy for growth. Some recent research under laboratory conditions suggest that Brett does not grow at alcohol levels above 13%. However, this result is not consistent with the observation that many wines with alcohols far in excess of this have gone bretty under winery conditions.

Filtering the wine before bottling can reduce the numbers of Brett cells, and hence the incidence of Brett character that develops in the bottle. However, there is anecdotal evidence that filtered wines that are sound at the time of bottling can randomly become infected with Brettanomyces after a period of time, probably as a result of the bottled wine containing residual sugar and being stored in warm conditions.

It is widely acknowledged that the majority of wines with Brett character, became that way during the period of barrel maturation, particularly if second use (or older) oak barrels were used. Brett can colonise a barrel between fills, and can begin to reproduce when the barrel is refilled with new wine. Figure 1 shows Brett extending pseudomycilium into the surface of an oak stave. Topping up barrels with a wine which contains Brett cells, may also contribute to those barrels 'going Bretty'. Shaving and re-toasting the inside of re-used barrels significantly reduces the incidence of Brett growth. However, it is also worth noting that the use of new barrels does not guarantee that Brett will not appear. Recent work in California has shown that new barrels filled with sterilised wine can still sustain populations of Brett high enough to produce above threshold levels of 4-ep. 

But why does oak maturation particularly favour Brett growth? Firstly, Brett is a slow growing yeast that does not compete well against other micro-organisms. During alcoholic fermentation the wine yeast Saccharomyces out easily out-competes it. Two possible reasons are that it naturally grows slower than Saccharomyces, and that it prefers aerobic conditions for growth. During primary ferment, the wine is saturated with carbon dioxide which makes for a hostile environment for Brettanomyces. On the other hand, barrel maturation is a step in conventional winemaking that provides both the time and the lack of competition needed for Brett to successfully grow to levels which results in sensory modification to the wine. Wines stored in barrel are usually lower in SO2 and are kept warmer than at any other time (other than during ferment of course). This is necessary so as to encourage malolactic fermentation (MLF). Lastly, the necessary processes of racking off lees and regularly topping up barrels ensures that there are always reasonable levels of dissolved oxygen in the wine. For all these reasons, it is thought that the time between the completion of primary fermentation and the start of MLF this is the most likely time that Brett multiplies and produces brettiness in wine.



Brettanomyces Character is Seen Primarily in Red Wine. Why?

One final matter concerning Brett is rarely mentioned. It occurs almost exclusively in red wines. Why is this so? Red wines have a much higher level of tannin like substances called coumaric and ferulic acid than do white wines as they are extracted from the skins of grapes during red wine fermentation. The wine yeastSaccharomyces and some lactic acid bacteria such as Lactobacillis have enzymes which degrade these acids to weakly smelling intermediates called 4-vinyl phenol and 4-vinyl guaiacol (Step 1 of Figure 2). These compounds are then enzymatically degraded over a period of months by Brettanomyces to the strong smelling 4-ethyl phenol and 4-ethyl guaiacol respectively (Step 2 of Figure 2). Incidentally Brettanomyces is the only major micro-organism in wine that has the ability to transform 4-vinyl-phenol into the potent band-aid® smelling, 4-ethyl phenol. Hence 4-ethyl phenol is rightly considered to be the "trademark" aroma ofBrettanomyces growth in wine. Where you find 4-ethyl phenol you will invariably find Brett, and vice versa.

Surveys of Australian wines have shown that detectable levels of 4-ethyl phenol is more likely to be seen in darker coloured wines, with Shiraz and Cabernet wines than wines made from either Pinot noir and Grenache. The reason for this is unclear, but may involve the coumarates which are a form of coloured anthocyanins found in red wines.

[Formation of 4-ethyl phenol in wine]
Figure 2: Pathway to the formation of 4-ethyl phenol and 4-ethyl guaiacol in wine


The Prevalence of Brett Character

Has Brett character become apparently more prevalent in recent years? Some commentators believe that we have simply become more aware of it and that it has always been around. I am sure that there is some truth in this. Upon personal reflection, I feel that classic Hunter Shiraz with its 'sweaty saddle' aroma and flavour is a very likely case in point. However, in my opinion, the overpowering, fruit destroying, antiseptic like aromas and flavours that are now occasionally encountered in wines sourced from every winemaking region of Australia is a relatively new phenomenon. The trend in this country today is to produce red wines picked from riper grapes. In addition to maximizing flavour development in some varieties, this also results in wines that are on average higher in pH and alcohol. Furthermore, residual sweetness is being retained in some commercial red wines in an attempt to fill out the palate and to give it greater apparent fruitiness. These trends together with the use of minimal SO2 and filtration, has enhanced the conditions under which Brett is retained and thrives.

The Desirability or Otherwise of Brett Character in Wine

But is the action of Brett desirable? In my humble opinion, the answer depends on degree. As well as producing a band-aid aroma, Brett can create an array of 'interesting' smells that can excite those that are inclined to be excited by them. Furthermore, the ratio of the rather unattractive 4-ethyl phenol to the rather pleasant smelling 4-ethyl guaiacol varies substantially from wine to wine, with reports varying from 3:1 to over 40:1. In the latter case, it is highly likely that the wine would smell like the inside of a band-aid box, while in the former, the aroma would in all likelihood be far more spicy and savoury like. The reason for these differences between wines are not completely understood but are likely to be either due to differing ratios between wines coumaric and ferulic acids (the respective precursors of 4-ep and 4-eg), or to different strains of Brettanomyces being more effective in producing one compound relative to the other. Very recent research with five different strains of Brettanomyces has not lent much support to the latter possibility. Under laboratory conditions the different strains produced roughly equal proportions of 4-ep to 4-eg in the same red wine. But the search for strains of Brett which may be low 4-ep producers will no doubt continue.

In some wine growing regions such as Bordeaux, the Rhone and, dare I say it, the Hunter Valley, it is now acknowledged that some wine producers have developed 'house styles' over time that have actually been defined by some form of Brett character. Many of these producers, or the media, or both, have naively attributed these unusual and sometimes complexing characters to being 'an expression of the soil'. However, overwhelming scientific evidence in the form of elevated 4-ethyl phenol levels in their wines have forced them to admit to the less romantic notions of the microbiological origin of these characters. This is not to say that they necessarily will, or indeed should, do anything different in the future, as many Bretty house styles have become widely accepted and in some cases revered by the wine tasting public. But in the cases where a wine smells more of a hospital ward than it does wine, surely the wine-maker should begin to reflect on what wine drinkers seriously value. That is, real fruit and real complexity. Unfortunately some winemakers (possibly in an attempt to save their career), have attributed the accidental making of overtly Bretty wines as a serious attempt at making something different and complex. Wine diversity is a wonderful thing and should be encouraged in the face of continued 'internationalisation' of wines. But as Pascal Chattonet once argued. Brettyiness has nothing to do with a wines 'typicity' as claimed by some French wine producers. His counterclaim is that wines that are overly Bretty do indeed smell and taste much the same, so overt Brettyness mitigates against 'typicity' and diversity. I'm in Pascal's camp. Real 'typicity' and 'expression' indeed come from the fruit. A message that I hope is not lost on the winemaking fraternity.

This work was presented at the Australian Society of Wine Education National Convention. Hunter Valley, Australia. 4th-6th of June 2004. 
www.aswe.org.au 查看全部
Introduction

The desirability or otherwise of the wine character known as "Brett" is one of the most controversial issues of recent times. Arguments have been made for Brett character being a complexing and a legitimate expression of natural, uncomplicated winemaking, while others view it simply as an unattractive wine fault that results from poor winery hygiene and sloppy winemaking. 

[Brettanomyces forming pseudomycelium on oak]
Figure 1: Brettanomyces bruxellensis forming pseudomycelium
© 2004 High Power Ultrasonics Pty Ltd

The Aroma and Flavour of Brett Character

But what is Brett character and how and why does it appear in some wines? The wine character described as "Bretty" comes in various forms. It is the combined result of the creation of a number of compounds by the yeast Brettanomyces bruxellensis, and its close relative, Dekkera bruxulensis. The three most important known aroma active compounds are 1) 4-ethyl phenol (4-ep), which has been variously described as having the aromas of Band-aids®, antiseptic and horse stable 2) 4-ethyl guaiacol (4-eg) which has a rather pleasant aroma of smoked bacon, spice or cloves and 3) isovaleric acid which has an unpleasant smell of sweaty animals, cheese and rancidity. Other characters associated with Brett include wet dog, creosote, burnt beans, rotting vegetation, plastic and (but not exclusively caused by Brett) mouse cage aroma and vinegar.

The Formation of Brett Character in Wine

Brettanomyces has been isolated from the outside of grapes and from winery equipment. However its, favoured winery haunt is the oak barrel as it often provides for conditions that strongly favours its growth.

Certain conditions are known to favour the growth of Brettanomyces during winemaking. If low free sulfur dioxide levels are coupled with high wine pH and warm temperatures during barrel maturation, then issues may arise. If older oak is used and the wine has a reasonable amount of dissolved oxygen, …. look out! Furthermore it is thought that Brett can also multiply after bottling if the wine contains residual fermentable sugars, a situation made more likely if the wine was minimally filtered. Lets look at the why's of these factors.

Brettanomyces proliferates under warm cellaring conditions. Twenty degrees C is an ideal temperature, with even small reductions in temperature seriously hamper its growth. Sulfur dioxide is an anti-microbial agent that is added by winemakers throughout the winemaking process. If it is added in sufficient amounts, and the pH of the wine is reasonably high (SO2 is more effective at higher acidity levels), then the growth of Brett will be retarded. On the other hand, high alcohol levels and the existence of even small amounts of fermentable sugars such as glucose suit the growth of Brett, as they are its preferred source of energy for growth. Some recent research under laboratory conditions suggest that Brett does not grow at alcohol levels above 13%. However, this result is not consistent with the observation that many wines with alcohols far in excess of this have gone bretty under winery conditions.

Filtering the wine before bottling can reduce the numbers of Brett cells, and hence the incidence of Brett character that develops in the bottle. However, there is anecdotal evidence that filtered wines that are sound at the time of bottling can randomly become infected with Brettanomyces after a period of time, probably as a result of the bottled wine containing residual sugar and being stored in warm conditions.

It is widely acknowledged that the majority of wines with Brett character, became that way during the period of barrel maturation, particularly if second use (or older) oak barrels were used. Brett can colonise a barrel between fills, and can begin to reproduce when the barrel is refilled with new wine. Figure 1 shows Brett extending pseudomycilium into the surface of an oak stave. Topping up barrels with a wine which contains Brett cells, may also contribute to those barrels 'going Bretty'. Shaving and re-toasting the inside of re-used barrels significantly reduces the incidence of Brett growth. However, it is also worth noting that the use of new barrels does not guarantee that Brett will not appear. Recent work in California has shown that new barrels filled with sterilised wine can still sustain populations of Brett high enough to produce above threshold levels of 4-ep. 

But why does oak maturation particularly favour Brett growth? Firstly, Brett is a slow growing yeast that does not compete well against other micro-organisms. During alcoholic fermentation the wine yeast Saccharomyces out easily out-competes it. Two possible reasons are that it naturally grows slower than Saccharomyces, and that it prefers aerobic conditions for growth. During primary ferment, the wine is saturated with carbon dioxide which makes for a hostile environment for Brettanomyces. On the other hand, barrel maturation is a step in conventional winemaking that provides both the time and the lack of competition needed for Brett to successfully grow to levels which results in sensory modification to the wine. Wines stored in barrel are usually lower in SO2 and are kept warmer than at any other time (other than during ferment of course). This is necessary so as to encourage malolactic fermentation (MLF). Lastly, the necessary processes of racking off lees and regularly topping up barrels ensures that there are always reasonable levels of dissolved oxygen in the wine. For all these reasons, it is thought that the time between the completion of primary fermentation and the start of MLF this is the most likely time that Brett multiplies and produces brettiness in wine.



Brettanomyces Character is Seen Primarily in Red Wine. Why?

One final matter concerning Brett is rarely mentioned. It occurs almost exclusively in red wines. Why is this so? Red wines have a much higher level of tannin like substances called coumaric and ferulic acid than do white wines as they are extracted from the skins of grapes during red wine fermentation. The wine yeastSaccharomyces and some lactic acid bacteria such as Lactobacillis have enzymes which degrade these acids to weakly smelling intermediates called 4-vinyl phenol and 4-vinyl guaiacol (Step 1 of Figure 2). These compounds are then enzymatically degraded over a period of months by Brettanomyces to the strong smelling 4-ethyl phenol and 4-ethyl guaiacol respectively (Step 2 of Figure 2). Incidentally Brettanomyces is the only major micro-organism in wine that has the ability to transform 4-vinyl-phenol into the potent band-aid® smelling, 4-ethyl phenol. Hence 4-ethyl phenol is rightly considered to be the "trademark" aroma ofBrettanomyces growth in wine. Where you find 4-ethyl phenol you will invariably find Brett, and vice versa.

Surveys of Australian wines have shown that detectable levels of 4-ethyl phenol is more likely to be seen in darker coloured wines, with Shiraz and Cabernet wines than wines made from either Pinot noir and Grenache. The reason for this is unclear, but may involve the coumarates which are a form of coloured anthocyanins found in red wines.

[Formation of 4-ethyl phenol in wine]
Figure 2: Pathway to the formation of 4-ethyl phenol and 4-ethyl guaiacol in wine


The Prevalence of Brett Character

Has Brett character become apparently more prevalent in recent years? Some commentators believe that we have simply become more aware of it and that it has always been around. I am sure that there is some truth in this. Upon personal reflection, I feel that classic Hunter Shiraz with its 'sweaty saddle' aroma and flavour is a very likely case in point. However, in my opinion, the overpowering, fruit destroying, antiseptic like aromas and flavours that are now occasionally encountered in wines sourced from every winemaking region of Australia is a relatively new phenomenon. The trend in this country today is to produce red wines picked from riper grapes. In addition to maximizing flavour development in some varieties, this also results in wines that are on average higher in pH and alcohol. Furthermore, residual sweetness is being retained in some commercial red wines in an attempt to fill out the palate and to give it greater apparent fruitiness. These trends together with the use of minimal SO2 and filtration, has enhanced the conditions under which Brett is retained and thrives.

The Desirability or Otherwise of Brett Character in Wine

But is the action of Brett desirable? In my humble opinion, the answer depends on degree. As well as producing a band-aid aroma, Brett can create an array of 'interesting' smells that can excite those that are inclined to be excited by them. Furthermore, the ratio of the rather unattractive 4-ethyl phenol to the rather pleasant smelling 4-ethyl guaiacol varies substantially from wine to wine, with reports varying from 3:1 to over 40:1. In the latter case, it is highly likely that the wine would smell like the inside of a band-aid box, while in the former, the aroma would in all likelihood be far more spicy and savoury like. The reason for these differences between wines are not completely understood but are likely to be either due to differing ratios between wines coumaric and ferulic acids (the respective precursors of 4-ep and 4-eg), or to different strains of Brettanomyces being more effective in producing one compound relative to the other. Very recent research with five different strains of Brettanomyces has not lent much support to the latter possibility. Under laboratory conditions the different strains produced roughly equal proportions of 4-ep to 4-eg in the same red wine. But the search for strains of Brett which may be low 4-ep producers will no doubt continue.

In some wine growing regions such as Bordeaux, the Rhone and, dare I say it, the Hunter Valley, it is now acknowledged that some wine producers have developed 'house styles' over time that have actually been defined by some form of Brett character. Many of these producers, or the media, or both, have naively attributed these unusual and sometimes complexing characters to being 'an expression of the soil'. However, overwhelming scientific evidence in the form of elevated 4-ethyl phenol levels in their wines have forced them to admit to the less romantic notions of the microbiological origin of these characters. This is not to say that they necessarily will, or indeed should, do anything different in the future, as many Bretty house styles have become widely accepted and in some cases revered by the wine tasting public. But in the cases where a wine smells more of a hospital ward than it does wine, surely the wine-maker should begin to reflect on what wine drinkers seriously value. That is, real fruit and real complexity. Unfortunately some winemakers (possibly in an attempt to save their career), have attributed the accidental making of overtly Bretty wines as a serious attempt at making something different and complex. Wine diversity is a wonderful thing and should be encouraged in the face of continued 'internationalisation' of wines. But as Pascal Chattonet once argued. Brettyiness has nothing to do with a wines 'typicity' as claimed by some French wine producers. His counterclaim is that wines that are overly Bretty do indeed smell and taste much the same, so overt Brettyness mitigates against 'typicity' and diversity. I'm in Pascal's camp. Real 'typicity' and 'expression' indeed come from the fruit. A message that I hope is not lost on the winemaking fraternity.

This work was presented at the Australian Society of Wine Education National Convention. Hunter Valley, Australia. 4th-6th of June 2004. 
www.aswe.org.au

Removal from and addition of sulfur dioxide to must, juice and wine

cewh Published the article • 0 comments • 3936 views • 2016-05-17 21:36 • 来自相关话题

retrieved from ​http://www.awri.com.au/wp-content/uploads/TN06.pdf

Problem Fermentations

cewh Published the article • 0 comments • 180 views • 2016-05-17 21:34 • 来自相关话题

Problem fermentations can be divided into two broad categories: issues with fermentation rate progression and off-character formation. Both types of problems are sporadic and chronic, and display a dependence upon juice composition and strain variability. Both are easier to prevent than to treat. However, complete avoidance of these problems requires a sophisticated chemical analysis of juice composition that is generally beyond the scope of the typical winery. In many cases, fermentation progression appears completely normal immediately prior to the appearance of a problem. Fermentation behavior is inherently difficult to predict due to the number of potential variables. At present, a problem fermentation is only recognized once it has arisen. There are steps that can be taken to restore yeast vitality, but the success of such efforts is dependent upon correct diagnosis of the root cause of the problem

Problem Diagnosis: Fermentation Rate and Progression

Overview

There are several fermentation rate and progression issues that can arise during grape juice fermentation: long lags before onset of fermentation, a too-slow or too-rapid rate of fermentation, a sluggish maximal rate of fermentation, a slowing of fermentation, and actual cessation of sugar consumption. Careful analysis of fermentation conditions and of the fermentation profile can provide clues to the reason for poor fermentation performance.  Astute monitoring of the fermentation can assist the winemaker in early identification of problem fermentations.  Proper analysis of juice composition and careful attention to yeast nutritional and physiological needs can reduce the incidence of  fermentation arrest. Minimizing shocks to the cells during fermentation (super heating or super cooling; high competitive bioloads) will also reduce the incidence of fermentation arrest.

The conditions of fermentation, for example, temperature, pH, aeration, level of solids, inoculation practices, can all impact the “normal” fermentation rate without leading to an incomplete fermentation. What is typical for a particular strain or fermentation condition needs to be clearly established in order to be able to confidently identify abnormal behavior.  In many cases, a lack of information regarding normal expected fermentation performance seriously compounds the ability of the winemaker to quickly identify and correct problem fermentations.

Successful restarting of a stuck fermentation depends upon two critical factors: proper pre-conditioning of the yeast to be used as the inoculum and knowledge of the cause of the fermentation arrest.  The latter will directly impact the former, as the tolerances of the strain used in the re-inoculation must compensate for the specific stresses of the arrested fermentation.   Cells can rapidly lose viability in an arrested state, depending upon the nature of the inhibitory condition and the ethanol concentration at the time of arrest. When dealing with arrested fermentations it is important to keep in mind that the clock is ticking on culture health and vitality, and the longer the delay the higher the percentage of non-viable cells. Cell death leads to the release of components that are detected by viable cells, leading them to shut down metabolic activities rather than risk loss of viability. Fermentations can be challenging to restart, even with a fresh inoculum, if cell death has occurred.

Fermentation Progression: Typical Fermentations

The first step in identification of the cause of a decrease in fermentation rate is a thorough understanding of the characteristics of a normal fermentation profile.  The figure  displays a typical Brix fermentation curve for the commercial strain Cote des Blancs in Grenache noir must harvested at 26 Brix.

  [typical]

Sugar consumption initiates almost immediately upon inoculation. The highest rate of sugar utilization occurs after the cell population has reached maximal density and ethanol concentrations are too low to be inhibitory. The fermentation was complete at day 14. This is a typical profile for this commercial yeast strain.

  [typical2]

The blue circle shows the lag in initiation of fermentation. During the first 48 hours cells are adapting to the juice conditions, detoxifying juice SO2 and engaging in cell division.

  [typical3]

The blue circle now shows the steepest part of the sugar consumption curve. This coincides with the fastest rate of fermentation. In this particular fermentation, that high rate is sustained until the fermentation is well below 5 Brix. Given that the fermentation started at roughly 26 Brix, this indicates the strain has sustained ethanol tolerance until the external ethanol has reached approximately 11-12 % ethanol (v/v). As ethanol accumulates further, the fermentation progressively slows.

  [typical4]

There is a distinct transition to a slower rate of fermentation. The blue circle shows the change from the initial faster rate to the new slower rate.  The rate slows because ethanol in the medium forces an adaptation of the plasma membrane. The yeast can easily form a membrane that depends upon ethanol replacing water for its structure and functionality if sufficient survival factors are present to allow the new membrane to be made. Survival factors include nitrogen, sterols and fatty acids. If sufficient oxygen has been given to the fermentation early on, the cells will be able to make the necessary sterols and fatty acids. If not, then the sterols and fatty acids will need to be provided. They can be found in more complex nutrients made from yeast extracts or from yeast ghost addition (lysed yeast cells) as the fatty acids and sterols are associated with the membranes still attached to the cell walls in the yeast ghost preparations.

This slower rate is therefore the ethanol-adapted rate. At this point in the fermentation net cell growth has ceased and sugar is being consumed to provide energy to allow cells to remain resistant to ethanol. Typical fermentation curves are often characterized by two different linear phases of sugar consumption as indicated by the circles.

  [typical5]

This fermentation was conducted at 20°C with good temperature control. The fermentation is warmer during the most active phase of sugar consumption because heat is released during glycolysis. Periodic fluctuation in temperature coincides with pumpovers.

This same juice was allowed to undergo an uninocualted fermentation depicted in the following figure. The fermentation went to dryness, but the curve displays some key differences as compared to the fermentation inoculated with a commercial strain. The fermentation required an additional week for dryness to be attained. The initial rate of fermentation was more rapid than in the inoculated one suggesting that the wild strains initially present are more robust fermentors than Cote des Blancs.  This fermentation displays three distinct linear phases.

  [uninoculated]

The strains rapidly initiating fermentation appear to become displaced by more ethanol-tolerant strains around 10 Brix (roughly 7-9 % ethanol). Many wild strains are not tolerant to ethanol above this concentration. The second phase is conducted by strains of greater ethanol tolerance, with the fermentation rate again showing a decline around 11-12 % ethanol. The overall rates were slower than the inoculated fermentation but the fermentation was complete. There was less temperature fluctuation, also consistent with a slower overall rate of fermentation. In some cases, rather than a rapid start, the uninoculated fermentations will show a very slow start as the population of resident Saccharomyces strains build. This is particularly true if SO2 has been used as it can inhibit the native populations. Depending upon the initial bioload level of  Saccharomyces, uninoculated fermentations may lag for 7 to 10 days or longer. During this time the cells are actively dividing but many more generations are required to attain a high enough biomass level for noticeable sugar utilization to occur. Cell generation time is very much dependent upon juice nutrient content, temperature and pH. Uninoculated fermentations typically have on the order of 10 to 100 cells/mL. It will take 20  to 24 generations to reach the final cell density of 1 x 108 cells/mL compared to only 8 generations if an inoculation of 106 cells/mL is used. If the generation time is 3 hours, it will take 24 hours for the inoculated fermentation to reach final cell density but 60 to 72 hours for an uninoculated fermentation to do so.

Grape sugar is an equimolar mixture of glucose and fructose with trace amounts of sucrose, mannose and galactose. To illustrate the differences in utilization of glucose and fructose, a synthetic juice fermentation with monitoring of glucose, fructose and viable cell count is presented in the next figure.

  [fermsynth]

Glucose (pink line) is consumed at a faster rate than fructose (red line). In the beginning both sugars appear to be consumed at similar rates but after roughly 24 hours, the rates begin to deviate. Glucose is completely consumed by 70 hours, in this figure, while fructose is not completely consumed until roughly 120 hours. Thus, at the end of fermentation, the juice contains essentially pure fructose. The Brix curve represents the summation of the sugar values and drops below 0 because the specific gravity of an ethanol mixuter is below that of water.

The same synthetic juice fermentaiton was conducted under conditions limiting availability of fatty acids, diagrammed in the growth curves below. Unsaturated fatty acids are needed for ethanol tolerance and must be provided to the fermentation or sufficient oxygen must be available to allow their biosynthesis. In this case, cultures were anearobic, no oxygen provided, and the medium was not supplemented with fatty acids.

  [fermprof]

The growth curves indicate that the absence of fatty acids did not completely block growth, but the culture did not attain the same population density  as the culture given fatty acids. Further, both conditions demonstrate a faster consumption of glucose than of fructose. However, in the case of the fatty-acid-limited condition, the differences in glucose versus fructose consumption rates are almost immediately apparent and a rather high concentration of fructose is left at the end of the fermentation. Oxygen/fatty acid limitation results in high residual fructose concentrations in the medium.

Fermentation profiles may differ by the yeast strain and compositional conditions. In the following figure, the fermentation displays a sustained rate and does not appear to have the distinct transistion point indicating an inhibitory concentration of ethanol has arisen in the must.

  [mourvmust]  

The starting Brix of this must was just slightly above 20. In this situation, dryness is attained at a low ethanol level, thus the fermentation curve does not show an ethanol-induced transition to a slower rate of fermentation.

The following fermentation shows a long lag. The fermentation is slow to initiate as the inoculum becomes dominant, but once it is dominant, the rate of fermentation is sustained over the time course of the fermentation.

  [grenmust]

The long lag suggests that conditions were inhibitory early on, either due to high bioloads from the vineyard or use of too high a concentration of sulfite. But once the strain adapts to the conditions of the must, the fermentation proceeds to completion.

UCD522 was inoulated into the same Grenache noir must. This strain did not display a long lag but did show a more typical transition at a high ethanol concentration.

  [grenmust2]  

In general, an uninoculated fermentation will likely be slower than an inoculated one, but the profiles of fermentation may be quite similar depending upon the yeast inoculum used and its characteristics.

  [fermcomp]

There are several components of the fermentation profile that can be evaluated in order to define what is typical for a particular strain.  Length of lag, maximum fermentation rate and duration, transition point (point at which ethanol becomes inhibitory) post-transition rate of fermentation, comparison of pre- and post-transition fermentation rates and the overall time of fermentation (from lag to dryness) can all be measured easily from the graph and the information used to build a historical profile of normal for a particular strain.  This type of strategy can also be used for uninoculated fermentations as well.  The availability of such a database in the winery will allow more rapid determination of abnormal fermentation performance.

Types of Fermentation Progression Problems that Can Occur

The sugar consumption pattern of problem fermentations can be a useful diagnostic tool for the winemaker.  Slow fermentations can be broadly divided into four types:  sluggish initiation with rate eventually becoming normal; normal initiation becoming sluggish; sluggish throughout the entire time course; and an abrupt stop.  Typical types of fermentations are shown in the following figure.

  [types]

Fermentations of the first type, with a sluggish initiation, generally can go to dryness depending upon the cause of the problem and whether or not the lag in initiation generates a secondary problem such as high populations of competing non-Saccharomyces organisms.  The other three types of slow fermentations may eventually go to dryness or arrest and become stuck.  The different types of slow fermentation profiles are a consequence of distinct kinds of stresses imposed on the yeast and the timing of imposition of the stress.

Slow initiation of fermentation, rate becoming normal (in red below): Slow fermentation initiation generally reflects either the presence of a toxin, high viscosity, specific fermentation conditions (such as low juice temperature) or a deficient population of healthy starting yeast.

  [types2]

This type of fermentation profile may occur with either uninoculated or inoculated fermentations. In uninoculated fermentations, the sluggish start may simply be due to low numbers of Saccharomyces in the must, and not indicative of any particular problem other than an initial low biomass.  We have found that fermentations will finish reasonably well with as little as 100-1000 viable Saccharomyces cells/mL present at the beginning, depending upon juice conditions and the relative numbers of other types of microorganisms.  If it is desired that the fermentation initiate within 24-48 hours, then an inoculum of approximately 106viable cells/mL should be added. Fruit coming in from the vineyard may be deficient in Saccharomycespopulations (less than 100 viable cells/ mL), but we have found that after the first few weeks of crush, the passage of juices and musts through winery equipment has elevated the winery populations ofSaccharomyces.  Cell counts of juice and must post-processing can be raised to a desirable range (104 -105cells/mL) just by transit through winery equipment, depending upon sanitation practices and the particular strains of Saccharomyces that have colonized the winery.  Holding of juices and musts at low temperatures enriches for Kloeckera apicuIata (Hanseniaspora uvarum) and decreases the numbers of Saccharomycesstrains present. Saccharomyces is not as low-temperature-tolerant as other yeasts.   It should be noted that addition of SO2 may not impact the viability and persistence of non-Saccharomyces yeast strains unless the amount added is over 50 mg/L.   If Saccharomyces is able to eventually dominate the fermentation, these fermentations should be able to go to dryness, provided  that nutrient consumption by the non-Saccharomyces yeast and bacteria has not created a deficient situation nor have toxic substances been produced.  Nutrient supplementation of musts held at low temperature should be carefully evaluated. Such practices may feed the bacteria and non-Saccharomyces yeasts differentially, leading to high production of potentially toxic (to Saccharomyces) end products.  The lower the initial population of Saccharomyces, the more the juice will have to provide components necessary for cell growth.  For example, a juice with an initial concentration of Saccharomyces of 100 cells/mL will have to undergo 13  generations to reach a typical inoculum level of 106 cells/mL.  On the other hand, fermentations starting at 106 to 107cells/mL will largely be conducted by non-proliferative cells and the nutritional requirements of such cells appear to be different than that for growth.  With a high initial inoculum, deficiencies in stationary phase nutrition may be more apparent.

In the case of inoculated fermentations, poor starter culture preparation can often be a factor.  Fermentations may be inoculated in two ways: either directly using rehydrated active dry yeast or from an active fermentation or starter culture.  Both types of practices may be fraught with difficulty.

Use of Active Dry Yeasts:

If rehydrated active dry yeast are to be used, it is very important that manufacturer’s instructions be followed particularly with respect to both the medium and temperature used for rehydration. The first step of any winery standard operating procedure should be to check the instructions on the packet. Some yeast perform better if hydrated under the specific recommended conditions. The maximum temperatures listed for rehydration should not be exceeded as this will result in a lethal temperature shock to the yeast.  Similarly, if the rehydration temperature is too low, the viability of the starter culture will also decrease.  The temperature of rehydration should be between  35-40°C (95-105°F). It is likewise important to use appropriate rehydration media.  Some strains tolerate rehydration in water or juice, while others only rehydrate properly in water.  Use of wine for rehydration is not recommended as this frequently leads to problems with viability due to the ethanol shock to the yeast during rehydration.  Other conditions of the rehydration are equally important. The yeast should be added slowly to water that is being vigorously agitated, taking care that clumps do not form.  Propeller mixers are useful for this purpose. The yeast should not be allowed to sit longer than roughly 15 minutes in water before being thoroughly mixed with the must or juice. We routinely add the yeast inoculum to red must while doing a pumpover and, with white juice, we add it to bottom of the tank prior to filling from the press.  If the yeast is not mixed in well, it will not be dispersed in the must, and fermentation can initiate sluggishly. Some strains perform better if rehydrated in the presence of some nutrients, particularly sugars, as this provides a source of energy for the cells. Several studies have shown that if nitrogen is limited, but sugar is plentiful, the cells will start accumulating phosphate, sulfate and other organic and micronutrients in the medium. The rehydration step can be used to feed the inoculant population preferentially.  Manufacturer’s directions should always be followed.

Common Problems with Inoculations:

It is important for wineries to develop standard operating procedures for processes like rehydration and inoculation and to make sure all individuals with the responsibility for strain preparation know what procedure should be followed. Common mistakes are: adding the yeast to water that is too hot, mixing the yeast and SO2 together to save time, not bothering to rehydrate the yeast strain, using yeast packets past their pull dates and inadequate mixing of the inoculant or the tank following inoculation. If low temperature juices are inoculated, the yeast strain will have to adapt to the lower temperature. However, other wild yeasts present in the must or juice will be able to grow, putting the inoculum at a greater disadvantage. Generally, this just leads to a longer lag but does not prevent the Saccharomyces strain from dominating and completing the fermentation.

Some yeast strains are very sensitive to sulfur dioxide.  We find that it is important to mix the SO2 into the tank prior to inoculation with yeast and to make sure that the SO2 is well dispersed. If it is not, then the yeast may hit a layer of a toxic level of SO2.  Under no circumstances should the yeast and SO2 be mixed together in the inoculum!

Use of Fermenting Must as Inoculum:

Use of fermenting must as an inoculum can also be problematic.  If the ethanol content of the fermenting must is too high at the time that it is used as an inoculum, then the yeast may be subjected to osmotic shock upon addition to fresh must.  Cells that have already adapted membranes and protein content to high ethanol conditions will have to “de-adapt” upon abrupt dilution of the inhibiting ethanol in fresh medium.  We have found that the best results are obtained if the ethanol content of the starter is around 3-5%, but no higher than 7% (v/v).

Further, use of fermenting must as inocula may result in use of nutrient-depleted cultures.  In general, for a vitamin deficiency to become manifest in a culture of healthily growing yeast, roughly 40 generations of growth in the absence of the vitamin is required.  If active dry yeast is used as inoculum, micronutrient-deficient juices will not lead to a starvation situation for the yeast since these cultures have been enriched in micronutrients. However, if the inoculum is serially passaged through juices, micronutrient deficiencies can arise that will inhibit not only the initiation of fermentation but its progression as well. We have found that a starter can be passaged once to fresh juice without loss of fermentation initiation ability. With a second passage, some fermentation problems may arise, but a third passage of the inoculum can lead to a sluggish if not stuck fermentation.

Inoculation Management Techniques:

Sluggish initiation of fermentation may also be caused by poor strain management techniques.  If there is a dramatic difference between the temperature of the inoculum and that of the must, the yeast will receive a temperature shock which may impact continued viability.  This is frequently a problem with juices that have been cold-settled, then inoculated prior to reaching a temperature that is warm enough.  In such cases, it may be better to start the yeast in a fraction of pre-warmed juice, and once the yeast have started, use this mixture as the inoculum of a low temperature fermentation.

Over-clarified juices may also initiate slowly due to a low solids content.  A current fashion in the California wine industry is to reduce the solids content of the white juice so that the finished wines will not need much, if any, fining and filtration.  Wineries using this approach need to determine if such practices are associated with a higher incidence of stuck fermentations.

Similarly, heat treatments of musts and juices can lead to a loss of nutrients and over-settling and clarification. If such treatments are used on marginal fruit, particularly in cases of moldy clusters, the mold and other accompanying organisms may not be completely inactivated by the heat treatment. Their numbers may be reduced but not eliminated entirely. The bacteria, in particular, can rebound after such heat treatments, so addition of sulfite may be necessary. If nutrients have been lost in the heating process, nutrient additions can be made to compensate.

Slow initiation of fermentation, fermentation sluggish throughout (in red below): Problem fermentations displaying a sluggish initiation may not recover and may remain slow over the entire time course of sugar consumption.  Such fermentations generally reflect a problem with attainment of maximal viable biomass.  The causes of this type of problem are numerous and can indicate a problem in cell growth, maintenance of viability or both.

  [types3]  

Saccharomyces cell counts can easily be used to determine precisely if a culture is unhealthy.  Under normal conditions, at approximately 48-72 hours into the fermentation at 18 - 25°C, suspended cell counts should be around 107 to 108 cells/ mL, with a viability of 80 to 100%, depending upon the wine strain used and the conditions of inoculation and fermentation.  If the cell counts or percent viability are significantly below these values, and there is no extenuating circumstance such as heavy use of SO2 or a very low temperature of fermentation (12-14°C or below), then the fermentation will likely be at high risk for being sluggish throughout and may even arrest. The cell count may fluctuate during fermentation by as much as a factor of 100 (106 to 108) as subpopulations of yeast settle and new growth occurs, but the percent viability should remain high.  When determining cell counts, it is important to only evaluate the suspended population; the settled populations should not be mixed and resuspended.  It is also important to distinguish betweenSaccharomyces cells and those of other organisms, especially the non-Saccharomyces yeasts, in the fermentation.

Problems in attainment of maximal biomass typically indicate a nutrient deficiency or suboptimal growth conditions, such as a low pH.  Difficulty in maintenance of maximal viable biomass can indicate a severe deficiency of survival factors, increasing ethanol or acetaldehyde sensitivity, presence of zymocidal (toxic to yeast) substances, or poor strain tolerances to stress.

Normal initiation of fermentation, rate becoming sluggish (in red below): Frequently fermentations initiate normally, attaining both maximal biomass and fermentation rate, but fail to maintain hexose consumption, gradually slowing and becoming sluggish.

[types4]

This type of fermentation profile suggests that nutrients required for growth were plentiful and conditions fully permissive for proliferation.  These types of problematic fermentations appear to be due to difficulty in maintaining metabolic activity or viability during the non-proliferative or stationary phase.  This is the most common type of arrest seen – a normal initiation with no indication of an ensuing problem. A moderate deficiency in survival factors can lead to problems in ability to tolerate ethanol or acetaldehyde that will not be apparent until ethanol accumulates in the medium. More severe deficiencies may impact growth as well.   The role of fatty acids and sterols in ethanol tolerance is well known and preparations containing these components can be used as nutrient additions.  However, it is important to add these compounds to the must prior to the arrest of fermentation or it may be too late to rectify the problem. The current tendency to harvest fruit at high initial sugar content may result in inhibitory ethanol concentrations, and be a contributing factor to the appearance of this type of slow fermentation.  This may be alleviated by blending such musts and juices with those of a lower initial Brix.  The presence of zymostatic or zymocidal toxins that are more toxic at higher ethanol levels can also be a cause of a late arrest of fermentation.  An imbalance of potassium and hydrogen ions yields such a fermentation profile, suggesting that problems in regulation of hydrogen ion fluxes into the cell can seriously impact non-proliferative phase metabolism.  We have also seen very high inoculum levels of yeast (108 cells/mL) result in a sluggish fermentation.  These fermentations initiate very rapidly, but seem to progress slower than fermentations that build a population of yeast rather than starting at maximal cell density.  These fermentations display greatly elevated levels of yeast esters.

One area that merits further investigation is examination of the factors important in stabilizing soluble cytoplasmic functions in the presence of high ethanol.  Ethanol can disrupt enzyme activity and organelle function as well as perturb the cell’s permeability barrier.  Recent work suggests that trehalose, proline and glycine are all important factors in stabilizing internal components.  The first two, or their precursors (glucose for trehalose; arginine for proline), are generally plentiful in grape must. Glycine is not.  These compounds do not appear to be able to substitute for each other in very high gravity fermentations.  Nitrogen deficiency may impact the yeasts' ability to synthesize protective factors and lead to enhanced ethanol sensitivity of cytoplasmic functions.  Further, since ethanol induces leakage of several compounds from the cell, higher levels of these components than are required for growth may be necessary.  The impact of acetaldehyde accumulation on fermentation arrest also needs to be further studied.  Factors leading to a reduction in alcohol dehydrogenase activity may increase cytoplasmic acetaldehyde to toxic levels.

A final cause of this type of fermentation arrest may be a mild temperature shock.  Sensitivity to temperature increases at high ethanol concentrations, and, if a fermentation is being cooled, the temperature may drop below permissive levels, depending upon the type of refrigeration being used and its control.  It is also important to remember that most cooling jackets do not provide for a uniform temperature across the tank. The area immediately adjacent to the cooling system (jacket or insert) will likely be much cooler than the rest of the tank.

  [grennoir]

This graph shows an example of a fermentation that became too hot during the initial stages of sugar consumption. There is a too-rapid initiation of fermentation and a dramatic slow down of sugar utilization around 8 Brix. The ethanol content at this time is around 9%. Depending upon the strain, early heat shocks do not become manifest as a fermentation problem until ethanol accumulates to 8-11%, depending upon the strain. In this case, it appears that an ethanol tolerant subpopulation was able to eventually grow and complete the fermentation, but this is not always the case. The dramatic swings in temperature in this fermentation are due to the pumpover regimen and mixing of the tank, with the concurrent dissipation of heat during this process. Such hot starts of fermentations generally lead to reduced complexity of the wine.

Premature settling of the yeast population is also a good indicator of a problem in maintenance of metabolic activity during stationary phase.  We have observed an early settling in fermenting musts held too long at a low temperature (below 20°C) following the cessation of active fermentation.  In many cases, readjustment of the temperature does not result in an improved fermentation rate.

Fermentation normal, abrupt stop (in red below): An abrupt cessation of sugar consumption is usually indicative of a major traumatic shock to the fermenting yeast.  This might be due to exposure to extreme temperatures. Uncooled fermentations may attain an inhibitory temperature due to the release of heat during hexose catabolism, as shown above.  Similarly, fermentations can be overcooled depending upon the design of the system being used.

[types5]

Other types of shocks, such as a mistake in addition of sulfur dioxide, can also lead to an abrupt stop of the fermentation.  In one case, filtration of an incomplete fermentation through a pad filter contaminated withStreptomyces, led to an abrupt arrest of fermentation. Similarly, addition of a malolactic starter culture to a fermentation that is not yet dry can lead to a rather abrupt arrest, depending upon the biological activity, size of the inoculum, nutritional complement of the must, presence and nature of organic and fatty acids in the ML inoculum and the particular yeast and bacterial strains involved. Depending upon the pH, organic acids released by bacterial metabolism may be protonated and, therefore, simply diffuse across  the yeast plasma membrane. Once inside the cytoplasm, the acids release protons due to the higher internal pH. Yeast can use the plasma membrane ATPase to pump out the hydrogen ions coming from the acids. However, if the capacity of the pump is saturated by pumping out the hydrogen ions coming in from the enhanced passive proton flux due to ethanol, then the yeast will not be able to tolerate the acid addition and fermentation will rapidly arrest. Bacterial fatty acids can become inserted into the ethanol-adapted plasma membrane, disrupting sugar uptake and leading to cell death. Some yeast strains tolerate ML bacteria additions much more readily than others and some ML strains are less prone to cause a problem.

Premature addition of fining agents can also lead to loss of culture biomass and dramatically slow the fermentation.  These factors are all generally well within the control of the winemaker and should not routinely pose a problem.

Restarting Arrested Fermentations:

Restarting an arrested fermentation can be challenging, depending upon the cause of the arrest. If fermentation is inhibited due to a nutrient deficiency of either a macronutrient like nitrogen, a micronutrient like biotin, or a mineral ion, addition of the missing component is often enough to restart fermentation. Yeast frequently are easily able to recover from a cold shock if the tank is warmed and mixed to resuspend the population. It is more difficult to restart fermentations arrested due to ethanol intolerance, heat shock or presence of inhibitory organic and fatty acids. In the case of heat shock, survival of the elevated temperature forces changes in plasma membrane composition incompatible with tolerance of the membrane to ethanol. Thus, the alcohol tolerance level is reduced and cells will arrest at a lower than normal ethanol percent.

High ethanol can cause a phase change in the membrane that the cells cannot easily repair. Reducing the ethanol content via dilution likely will not restore the functionality of the membrane. The membrane can only be restored if cells are returned to fully permissive growth conditions so that a new membrane can be made for the daughter cells, which is difficult to do for a fermentation already above 10% ethanol.

If inhibitory acids are present the acids level will need to be reduced before growth can commence. If the acidity has lead to acidification of the cell cytoplasm the cells will not recover. A new inoculant will be just as susceptible to inhibition by the existing acids. Sometimes use of yeast ghosts or inactivated yeast cells to sop up the fatty acids or other inhibitors present in the medium followed by removal of the yeast biomass and reinoculation can help restart fermentations arrested due to the presence of inhibitors.

Regardless of the cause of the fermentation problem, it is frequently necessary to first rack the wine off of the settled yeast lees before attempting to restart the fermentation.  Some types of arrested fermentations may restart without addition of yeast following this treatment.  We have found from analysis of juice-like media wherein the cause of fermentation arrest can be strictly controlled, even moderate aeration will lead to a spontaneous restart of fermentations arrested due to sterol or fatty acid limitation, provided that is the only limitation and the restart is conducted in a timely fashion.  Nitrogen or micronutrient limitation on top of either an oxygen deficiency or temperature shock, will inhibit a spontaneous restart.

Another problem with reinitiating stuck fermentations concerns the timing at which intervention will have a positive outcome.  It has been well established that nitrogen limitation must be corrected before a cessation in fermentation occurs.  We have seen the same effect of potassium addition.  By the time a fermentation has arrested due to an imbalance of potassium and hydrogen ion concentrations, it is too late to correct the problem by adjustment of the ratio of the two ions.  If the stuck or sluggish fermentation is an adaptive response to adverse conditions, the readjustment of the medium must occur prior to commitment to that adaptation.  A decrease in fermentation rate is frequently a consequence of adaptation and not a cause.  Thus, it is desirable to develop diagnostic tools for the early identification of a problem fermentation, preferably prior to significant loss of transporter activity.  It would also be useful to develop better means by which to determine the precise cause of the stress so that it may be rectified.  Frequently the circumstances preceding the arrest of fermentation and the type of change of the fermentation profile can provide key information as to the likely cause of the problem.

Restarting Procedures:

There are three different types of strategies for restarting arrested fermentations. It is helpful if some idea of the cause of the arrest is known, but if not, there are options that can be used to complete the fermentation:

Rejuvenating the existing biomass
Reinoculation with a new adapted  inoculum
Use of activated encapsulated or yeast-in-bag processes

Rejuvenating Existing Biomass

If fermentation has ceased due to a reversible inhibition of the culture, then rejuvenating the biomass can lead to restoration and completion of fermentation. Reversible inhibition would be a low temperature shock, a mild deficiency of survival factors or a modest nutrient limitation. Raising the temperature, aerating the biomass and provision of nitrogen and cofactors can restore fermentation rates. Often, it is not known if a fermentation will restart from the existing biomass. The ability to restart from the existing biomass can often be determined by some quick bench trials. Samples of the tank can be taken and subjected to different treatments isolation and in combination, nutrient addition, aeration, temperature increase, to see if fermentation reinitiates. The successful treatment can then be applied to the entire tank. It is important that the bench trials be conducted under conditions that mimic what will happen in the larger production tank. There may be better mixing in the bench trial that will not be replicated in the larger tank.

If a microscope is available, it is also advantageous to examine the arrested population under the microscope and compare it to a healthy population from another tank. If the yeast from the arrested population appear lysed (popped open), or have granules inside that are moving by Brownian motion, then the population is in decline and will be much harder if not impossible to rejuvenate.

Before attempting rejuvenation, it is important to first check the tolerances of the strains and compare them to the fermentation conditions. A frequent cause of arrest of fermentation is use of a yeast strain that does not have the ethanol-tolerance level needed to complete a fermentation. The ethanol-tolerance level of commercial yeast strains is generally known. A good rule of thumb is to assume a worst-case scenario with respect to Brix yield of ethanol, 0.62 x Brix value, and be sure the strain has the ethanol tolerance capacity to attain this level. Fermentation of a 24 Brix juice would require an ethanol tolerance of 15%. 28 Brix would require a tolerance of 17%. Tolerance to ethanol is impacted by growth conditions, so the tolerances listed by manufacturers are not absolutes, but do provide a good estimate of the level of ethanol at which the strain can be expected to arrest. Strains also differ in tolerance of temperature shocks, nutrient limitation and bacterial competition. This information is also generally known for commercial strains. Before considering a rejuvenation strategy an assessment of the inoculant strain should be undertaken. If it does not have the tolerances needed to complete the fermentation then a reinoculation strategy with a more tolerant strain should be employed.

Reinoculation with a New Adapted Inoculum

If the fermentation arrest is due to ethanol intolerance, to temperature or acid shock, or to poor innate tolerances of the strain dominating the fermentation, it will be necessary to reinoculate. The new inoculants will have to be adapted to the conditions of the arrest. If the ethanol concentration is above 7% the new strain may have difficulty adapting to the fermentation conditions and will have to be introduced to the ethanol concentration in a gradual fashion. For example, mixing half of the arrested wine with fresh juice to drop the ethanol concentration to or below 5% will assure a healthy start for the new inoculant. If enough fresh juice is not available, water and commercial nutrients can be used to dilute both the juice and wine mixture to a permissive alcohol level. Once active fermentation is evident, meaning that obvious fermentative release of carbon dioxide is occurring, then more and more of the arrested ferment can be added in stepwise fashion taking care to not let the ferment go dry at any time in the process.

Several commercial strains are available that have been isolated specifically because of their low nutrient requirements, high ethanol, temperature and bacterial metabolite tolerances, and ease of rehydration. One of these strains should be considered instead of using the initial strain.  The availability of a microscope greatly enhances the ability to monitor the health and vitality of the new inoculant. It is also important to not add excessive sulfite when attempting a restart or to add the ML bacteria at the same time as the attempted restart.

Use of Activated Encapsulated or Yeast-in-Bag Processes

Growing a new inoculant, taking care to make sure it is adapted to the conditions of the arrested fermentation, can often be a time consuming process with no guarantee of success. It is often easy to let the new culture go too far, that is, it consumes all the available sugar in one mixture and arrests itself before a transfer can occur.  If all that is needed is to consume the rest of the sugar and complete a fermentation, then use of an encapsulated yeast may be a better approach. Encapsulated yeast are “trapped” in an alginate matrix. The alginate beads are fully permeable to substrates and endproducts but do not allow growth of the yeast cells. The reinforcement of the alginate matrix puts the yeast in a biofilm like mode of metabolism. Under these conditions substrates can be consumed efficiently and cells are not sensitive to factors inhibiting growth or disrupting membranes. The encapsulated yeast have been adapted to high ethanol and low nutrients during the encapsulation process and will work well in completing sugar consumption of an arrested fermentation, provided that other inhibitory conditions such as high SO2, high acetic acid, low pH, very high ethanol level, and/or high bacterial bioloads, do not exist or have not been rectified. The encapsulated yeast can be placed inside of a mesh bag for optimal distribution within the tank and for ease of removal. Again, if using one of these products, the manufacturer’s recommendations should be carefully followed. Extremes of temperature should be avoided as well as any other conditions that may adversely impact the viability of the yeast within the capsules.
 
retrieved from http://wineserver.ucdavis.edu/ ... .html 查看全部
Problem fermentations can be divided into two broad categories: issues with fermentation rate progression and off-character formation. Both types of problems are sporadic and chronic, and display a dependence upon juice composition and strain variability. Both are easier to prevent than to treat. However, complete avoidance of these problems requires a sophisticated chemical analysis of juice composition that is generally beyond the scope of the typical winery. In many cases, fermentation progression appears completely normal immediately prior to the appearance of a problem. Fermentation behavior is inherently difficult to predict due to the number of potential variables. At present, a problem fermentation is only recognized once it has arisen. There are steps that can be taken to restore yeast vitality, but the success of such efforts is dependent upon correct diagnosis of the root cause of the problem

Problem Diagnosis: Fermentation Rate and Progression

Overview

There are several fermentation rate and progression issues that can arise during grape juice fermentation: long lags before onset of fermentation, a too-slow or too-rapid rate of fermentation, a sluggish maximal rate of fermentation, a slowing of fermentation, and actual cessation of sugar consumption. Careful analysis of fermentation conditions and of the fermentation profile can provide clues to the reason for poor fermentation performance.  Astute monitoring of the fermentation can assist the winemaker in early identification of problem fermentations.  Proper analysis of juice composition and careful attention to yeast nutritional and physiological needs can reduce the incidence of  fermentation arrest. Minimizing shocks to the cells during fermentation (super heating or super cooling; high competitive bioloads) will also reduce the incidence of fermentation arrest.

The conditions of fermentation, for example, temperature, pH, aeration, level of solids, inoculation practices, can all impact the “normal” fermentation rate without leading to an incomplete fermentation. What is typical for a particular strain or fermentation condition needs to be clearly established in order to be able to confidently identify abnormal behavior.  In many cases, a lack of information regarding normal expected fermentation performance seriously compounds the ability of the winemaker to quickly identify and correct problem fermentations.

Successful restarting of a stuck fermentation depends upon two critical factors: proper pre-conditioning of the yeast to be used as the inoculum and knowledge of the cause of the fermentation arrest.  The latter will directly impact the former, as the tolerances of the strain used in the re-inoculation must compensate for the specific stresses of the arrested fermentation.   Cells can rapidly lose viability in an arrested state, depending upon the nature of the inhibitory condition and the ethanol concentration at the time of arrest. When dealing with arrested fermentations it is important to keep in mind that the clock is ticking on culture health and vitality, and the longer the delay the higher the percentage of non-viable cells. Cell death leads to the release of components that are detected by viable cells, leading them to shut down metabolic activities rather than risk loss of viability. Fermentations can be challenging to restart, even with a fresh inoculum, if cell death has occurred.

Fermentation Progression: Typical Fermentations

The first step in identification of the cause of a decrease in fermentation rate is a thorough understanding of the characteristics of a normal fermentation profile.  The figure  displays a typical Brix fermentation curve for the commercial strain Cote des Blancs in Grenache noir must harvested at 26 Brix.

  [typical]

Sugar consumption initiates almost immediately upon inoculation. The highest rate of sugar utilization occurs after the cell population has reached maximal density and ethanol concentrations are too low to be inhibitory. The fermentation was complete at day 14. This is a typical profile for this commercial yeast strain.

  [typical2]

The blue circle shows the lag in initiation of fermentation. During the first 48 hours cells are adapting to the juice conditions, detoxifying juice SO2 and engaging in cell division.

  [typical3]

The blue circle now shows the steepest part of the sugar consumption curve. This coincides with the fastest rate of fermentation. In this particular fermentation, that high rate is sustained until the fermentation is well below 5 Brix. Given that the fermentation started at roughly 26 Brix, this indicates the strain has sustained ethanol tolerance until the external ethanol has reached approximately 11-12 % ethanol (v/v). As ethanol accumulates further, the fermentation progressively slows.

  [typical4]

There is a distinct transition to a slower rate of fermentation. The blue circle shows the change from the initial faster rate to the new slower rate.  The rate slows because ethanol in the medium forces an adaptation of the plasma membrane. The yeast can easily form a membrane that depends upon ethanol replacing water for its structure and functionality if sufficient survival factors are present to allow the new membrane to be made. Survival factors include nitrogen, sterols and fatty acids. If sufficient oxygen has been given to the fermentation early on, the cells will be able to make the necessary sterols and fatty acids. If not, then the sterols and fatty acids will need to be provided. They can be found in more complex nutrients made from yeast extracts or from yeast ghost addition (lysed yeast cells) as the fatty acids and sterols are associated with the membranes still attached to the cell walls in the yeast ghost preparations.

This slower rate is therefore the ethanol-adapted rate. At this point in the fermentation net cell growth has ceased and sugar is being consumed to provide energy to allow cells to remain resistant to ethanol. Typical fermentation curves are often characterized by two different linear phases of sugar consumption as indicated by the circles.

  [typical5]

This fermentation was conducted at 20°C with good temperature control. The fermentation is warmer during the most active phase of sugar consumption because heat is released during glycolysis. Periodic fluctuation in temperature coincides with pumpovers.

This same juice was allowed to undergo an uninocualted fermentation depicted in the following figure. The fermentation went to dryness, but the curve displays some key differences as compared to the fermentation inoculated with a commercial strain. The fermentation required an additional week for dryness to be attained. The initial rate of fermentation was more rapid than in the inoculated one suggesting that the wild strains initially present are more robust fermentors than Cote des Blancs.  This fermentation displays three distinct linear phases.

  [uninoculated]

The strains rapidly initiating fermentation appear to become displaced by more ethanol-tolerant strains around 10 Brix (roughly 7-9 % ethanol). Many wild strains are not tolerant to ethanol above this concentration. The second phase is conducted by strains of greater ethanol tolerance, with the fermentation rate again showing a decline around 11-12 % ethanol. The overall rates were slower than the inoculated fermentation but the fermentation was complete. There was less temperature fluctuation, also consistent with a slower overall rate of fermentation. In some cases, rather than a rapid start, the uninoculated fermentations will show a very slow start as the population of resident Saccharomyces strains build. This is particularly true if SO2 has been used as it can inhibit the native populations. Depending upon the initial bioload level of  Saccharomyces, uninoculated fermentations may lag for 7 to 10 days or longer. During this time the cells are actively dividing but many more generations are required to attain a high enough biomass level for noticeable sugar utilization to occur. Cell generation time is very much dependent upon juice nutrient content, temperature and pH. Uninoculated fermentations typically have on the order of 10 to 100 cells/mL. It will take 20  to 24 generations to reach the final cell density of 1 x 108 cells/mL compared to only 8 generations if an inoculation of 106 cells/mL is used. If the generation time is 3 hours, it will take 24 hours for the inoculated fermentation to reach final cell density but 60 to 72 hours for an uninoculated fermentation to do so.

Grape sugar is an equimolar mixture of glucose and fructose with trace amounts of sucrose, mannose and galactose. To illustrate the differences in utilization of glucose and fructose, a synthetic juice fermentation with monitoring of glucose, fructose and viable cell count is presented in the next figure.

  [fermsynth]

Glucose (pink line) is consumed at a faster rate than fructose (red line). In the beginning both sugars appear to be consumed at similar rates but after roughly 24 hours, the rates begin to deviate. Glucose is completely consumed by 70 hours, in this figure, while fructose is not completely consumed until roughly 120 hours. Thus, at the end of fermentation, the juice contains essentially pure fructose. The Brix curve represents the summation of the sugar values and drops below 0 because the specific gravity of an ethanol mixuter is below that of water.

The same synthetic juice fermentaiton was conducted under conditions limiting availability of fatty acids, diagrammed in the growth curves below. Unsaturated fatty acids are needed for ethanol tolerance and must be provided to the fermentation or sufficient oxygen must be available to allow their biosynthesis. In this case, cultures were anearobic, no oxygen provided, and the medium was not supplemented with fatty acids.

  [fermprof]

The growth curves indicate that the absence of fatty acids did not completely block growth, but the culture did not attain the same population density  as the culture given fatty acids. Further, both conditions demonstrate a faster consumption of glucose than of fructose. However, in the case of the fatty-acid-limited condition, the differences in glucose versus fructose consumption rates are almost immediately apparent and a rather high concentration of fructose is left at the end of the fermentation. Oxygen/fatty acid limitation results in high residual fructose concentrations in the medium.

Fermentation profiles may differ by the yeast strain and compositional conditions. In the following figure, the fermentation displays a sustained rate and does not appear to have the distinct transistion point indicating an inhibitory concentration of ethanol has arisen in the must.

  [mourvmust]  

The starting Brix of this must was just slightly above 20. In this situation, dryness is attained at a low ethanol level, thus the fermentation curve does not show an ethanol-induced transition to a slower rate of fermentation.

The following fermentation shows a long lag. The fermentation is slow to initiate as the inoculum becomes dominant, but once it is dominant, the rate of fermentation is sustained over the time course of the fermentation.

  [grenmust]

The long lag suggests that conditions were inhibitory early on, either due to high bioloads from the vineyard or use of too high a concentration of sulfite. But once the strain adapts to the conditions of the must, the fermentation proceeds to completion.

UCD522 was inoulated into the same Grenache noir must. This strain did not display a long lag but did show a more typical transition at a high ethanol concentration.

  [grenmust2]  

In general, an uninoculated fermentation will likely be slower than an inoculated one, but the profiles of fermentation may be quite similar depending upon the yeast inoculum used and its characteristics.

  [fermcomp]

There are several components of the fermentation profile that can be evaluated in order to define what is typical for a particular strain.  Length of lag, maximum fermentation rate and duration, transition point (point at which ethanol becomes inhibitory) post-transition rate of fermentation, comparison of pre- and post-transition fermentation rates and the overall time of fermentation (from lag to dryness) can all be measured easily from the graph and the information used to build a historical profile of normal for a particular strain.  This type of strategy can also be used for uninoculated fermentations as well.  The availability of such a database in the winery will allow more rapid determination of abnormal fermentation performance.

Types of Fermentation Progression Problems that Can Occur

The sugar consumption pattern of problem fermentations can be a useful diagnostic tool for the winemaker.  Slow fermentations can be broadly divided into four types:  sluggish initiation with rate eventually becoming normal; normal initiation becoming sluggish; sluggish throughout the entire time course; and an abrupt stop.  Typical types of fermentations are shown in the following figure.

  [types]

Fermentations of the first type, with a sluggish initiation, generally can go to dryness depending upon the cause of the problem and whether or not the lag in initiation generates a secondary problem such as high populations of competing non-Saccharomyces organisms.  The other three types of slow fermentations may eventually go to dryness or arrest and become stuck.  The different types of slow fermentation profiles are a consequence of distinct kinds of stresses imposed on the yeast and the timing of imposition of the stress.

Slow initiation of fermentation, rate becoming normal (in red below): Slow fermentation initiation generally reflects either the presence of a toxin, high viscosity, specific fermentation conditions (such as low juice temperature) or a deficient population of healthy starting yeast.

  [types2]

This type of fermentation profile may occur with either uninoculated or inoculated fermentations. In uninoculated fermentations, the sluggish start may simply be due to low numbers of Saccharomyces in the must, and not indicative of any particular problem other than an initial low biomass.  We have found that fermentations will finish reasonably well with as little as 100-1000 viable Saccharomyces cells/mL present at the beginning, depending upon juice conditions and the relative numbers of other types of microorganisms.  If it is desired that the fermentation initiate within 24-48 hours, then an inoculum of approximately 106viable cells/mL should be added. Fruit coming in from the vineyard may be deficient in Saccharomycespopulations (less than 100 viable cells/ mL), but we have found that after the first few weeks of crush, the passage of juices and musts through winery equipment has elevated the winery populations ofSaccharomyces.  Cell counts of juice and must post-processing can be raised to a desirable range (104 -105cells/mL) just by transit through winery equipment, depending upon sanitation practices and the particular strains of Saccharomyces that have colonized the winery.  Holding of juices and musts at low temperatures enriches for Kloeckera apicuIata (Hanseniaspora uvarum) and decreases the numbers of Saccharomycesstrains present. Saccharomyces is not as low-temperature-tolerant as other yeasts.   It should be noted that addition of SO2 may not impact the viability and persistence of non-Saccharomyces yeast strains unless the amount added is over 50 mg/L.   If Saccharomyces is able to eventually dominate the fermentation, these fermentations should be able to go to dryness, provided  that nutrient consumption by the non-Saccharomyces yeast and bacteria has not created a deficient situation nor have toxic substances been produced.  Nutrient supplementation of musts held at low temperature should be carefully evaluated. Such practices may feed the bacteria and non-Saccharomyces yeasts differentially, leading to high production of potentially toxic (to Saccharomyces) end products.  The lower the initial population of Saccharomyces, the more the juice will have to provide components necessary for cell growth.  For example, a juice with an initial concentration of Saccharomyces of 100 cells/mL will have to undergo 13  generations to reach a typical inoculum level of 106 cells/mL.  On the other hand, fermentations starting at 106 to 107cells/mL will largely be conducted by non-proliferative cells and the nutritional requirements of such cells appear to be different than that for growth.  With a high initial inoculum, deficiencies in stationary phase nutrition may be more apparent.

In the case of inoculated fermentations, poor starter culture preparation can often be a factor.  Fermentations may be inoculated in two ways: either directly using rehydrated active dry yeast or from an active fermentation or starter culture.  Both types of practices may be fraught with difficulty.

Use of Active Dry Yeasts:

If rehydrated active dry yeast are to be used, it is very important that manufacturer’s instructions be followed particularly with respect to both the medium and temperature used for rehydration. The first step of any winery standard operating procedure should be to check the instructions on the packet. Some yeast perform better if hydrated under the specific recommended conditions. The maximum temperatures listed for rehydration should not be exceeded as this will result in a lethal temperature shock to the yeast.  Similarly, if the rehydration temperature is too low, the viability of the starter culture will also decrease.  The temperature of rehydration should be between  35-40°C (95-105°F). It is likewise important to use appropriate rehydration media.  Some strains tolerate rehydration in water or juice, while others only rehydrate properly in water.  Use of wine for rehydration is not recommended as this frequently leads to problems with viability due to the ethanol shock to the yeast during rehydration.  Other conditions of the rehydration are equally important. The yeast should be added slowly to water that is being vigorously agitated, taking care that clumps do not form.  Propeller mixers are useful for this purpose. The yeast should not be allowed to sit longer than roughly 15 minutes in water before being thoroughly mixed with the must or juice. We routinely add the yeast inoculum to red must while doing a pumpover and, with white juice, we add it to bottom of the tank prior to filling from the press.  If the yeast is not mixed in well, it will not be dispersed in the must, and fermentation can initiate sluggishly. Some strains perform better if rehydrated in the presence of some nutrients, particularly sugars, as this provides a source of energy for the cells. Several studies have shown that if nitrogen is limited, but sugar is plentiful, the cells will start accumulating phosphate, sulfate and other organic and micronutrients in the medium. The rehydration step can be used to feed the inoculant population preferentially.  Manufacturer’s directions should always be followed.

Common Problems with Inoculations:

It is important for wineries to develop standard operating procedures for processes like rehydration and inoculation and to make sure all individuals with the responsibility for strain preparation know what procedure should be followed. Common mistakes are: adding the yeast to water that is too hot, mixing the yeast and SO2 together to save time, not bothering to rehydrate the yeast strain, using yeast packets past their pull dates and inadequate mixing of the inoculant or the tank following inoculation. If low temperature juices are inoculated, the yeast strain will have to adapt to the lower temperature. However, other wild yeasts present in the must or juice will be able to grow, putting the inoculum at a greater disadvantage. Generally, this just leads to a longer lag but does not prevent the Saccharomyces strain from dominating and completing the fermentation.

Some yeast strains are very sensitive to sulfur dioxide.  We find that it is important to mix the SO2 into the tank prior to inoculation with yeast and to make sure that the SO2 is well dispersed. If it is not, then the yeast may hit a layer of a toxic level of SO2.  Under no circumstances should the yeast and SO2 be mixed together in the inoculum!

Use of Fermenting Must as Inoculum:

Use of fermenting must as an inoculum can also be problematic.  If the ethanol content of the fermenting must is too high at the time that it is used as an inoculum, then the yeast may be subjected to osmotic shock upon addition to fresh must.  Cells that have already adapted membranes and protein content to high ethanol conditions will have to “de-adapt” upon abrupt dilution of the inhibiting ethanol in fresh medium.  We have found that the best results are obtained if the ethanol content of the starter is around 3-5%, but no higher than 7% (v/v).

Further, use of fermenting must as inocula may result in use of nutrient-depleted cultures.  In general, for a vitamin deficiency to become manifest in a culture of healthily growing yeast, roughly 40 generations of growth in the absence of the vitamin is required.  If active dry yeast is used as inoculum, micronutrient-deficient juices will not lead to a starvation situation for the yeast since these cultures have been enriched in micronutrients. However, if the inoculum is serially passaged through juices, micronutrient deficiencies can arise that will inhibit not only the initiation of fermentation but its progression as well. We have found that a starter can be passaged once to fresh juice without loss of fermentation initiation ability. With a second passage, some fermentation problems may arise, but a third passage of the inoculum can lead to a sluggish if not stuck fermentation.

Inoculation Management Techniques:

Sluggish initiation of fermentation may also be caused by poor strain management techniques.  If there is a dramatic difference between the temperature of the inoculum and that of the must, the yeast will receive a temperature shock which may impact continued viability.  This is frequently a problem with juices that have been cold-settled, then inoculated prior to reaching a temperature that is warm enough.  In such cases, it may be better to start the yeast in a fraction of pre-warmed juice, and once the yeast have started, use this mixture as the inoculum of a low temperature fermentation.

Over-clarified juices may also initiate slowly due to a low solids content.  A current fashion in the California wine industry is to reduce the solids content of the white juice so that the finished wines will not need much, if any, fining and filtration.  Wineries using this approach need to determine if such practices are associated with a higher incidence of stuck fermentations.

Similarly, heat treatments of musts and juices can lead to a loss of nutrients and over-settling and clarification. If such treatments are used on marginal fruit, particularly in cases of moldy clusters, the mold and other accompanying organisms may not be completely inactivated by the heat treatment. Their numbers may be reduced but not eliminated entirely. The bacteria, in particular, can rebound after such heat treatments, so addition of sulfite may be necessary. If nutrients have been lost in the heating process, nutrient additions can be made to compensate.

Slow initiation of fermentation, fermentation sluggish throughout (in red below): Problem fermentations displaying a sluggish initiation may not recover and may remain slow over the entire time course of sugar consumption.  Such fermentations generally reflect a problem with attainment of maximal viable biomass.  The causes of this type of problem are numerous and can indicate a problem in cell growth, maintenance of viability or both.

  [types3]  

Saccharomyces cell counts can easily be used to determine precisely if a culture is unhealthy.  Under normal conditions, at approximately 48-72 hours into the fermentation at 18 - 25°C, suspended cell counts should be around 107 to 108 cells/ mL, with a viability of 80 to 100%, depending upon the wine strain used and the conditions of inoculation and fermentation.  If the cell counts or percent viability are significantly below these values, and there is no extenuating circumstance such as heavy use of SO2 or a very low temperature of fermentation (12-14°C or below), then the fermentation will likely be at high risk for being sluggish throughout and may even arrest. The cell count may fluctuate during fermentation by as much as a factor of 100 (106 to 108) as subpopulations of yeast settle and new growth occurs, but the percent viability should remain high.  When determining cell counts, it is important to only evaluate the suspended population; the settled populations should not be mixed and resuspended.  It is also important to distinguish betweenSaccharomyces cells and those of other organisms, especially the non-Saccharomyces yeasts, in the fermentation.

Problems in attainment of maximal biomass typically indicate a nutrient deficiency or suboptimal growth conditions, such as a low pH.  Difficulty in maintenance of maximal viable biomass can indicate a severe deficiency of survival factors, increasing ethanol or acetaldehyde sensitivity, presence of zymocidal (toxic to yeast) substances, or poor strain tolerances to stress.

Normal initiation of fermentation, rate becoming sluggish (in red below): Frequently fermentations initiate normally, attaining both maximal biomass and fermentation rate, but fail to maintain hexose consumption, gradually slowing and becoming sluggish.

[types4]

This type of fermentation profile suggests that nutrients required for growth were plentiful and conditions fully permissive for proliferation.  These types of problematic fermentations appear to be due to difficulty in maintaining metabolic activity or viability during the non-proliferative or stationary phase.  This is the most common type of arrest seen – a normal initiation with no indication of an ensuing problem. A moderate deficiency in survival factors can lead to problems in ability to tolerate ethanol or acetaldehyde that will not be apparent until ethanol accumulates in the medium. More severe deficiencies may impact growth as well.   The role of fatty acids and sterols in ethanol tolerance is well known and preparations containing these components can be used as nutrient additions.  However, it is important to add these compounds to the must prior to the arrest of fermentation or it may be too late to rectify the problem. The current tendency to harvest fruit at high initial sugar content may result in inhibitory ethanol concentrations, and be a contributing factor to the appearance of this type of slow fermentation.  This may be alleviated by blending such musts and juices with those of a lower initial Brix.  The presence of zymostatic or zymocidal toxins that are more toxic at higher ethanol levels can also be a cause of a late arrest of fermentation.  An imbalance of potassium and hydrogen ions yields such a fermentation profile, suggesting that problems in regulation of hydrogen ion fluxes into the cell can seriously impact non-proliferative phase metabolism.  We have also seen very high inoculum levels of yeast (108 cells/mL) result in a sluggish fermentation.  These fermentations initiate very rapidly, but seem to progress slower than fermentations that build a population of yeast rather than starting at maximal cell density.  These fermentations display greatly elevated levels of yeast esters.

One area that merits further investigation is examination of the factors important in stabilizing soluble cytoplasmic functions in the presence of high ethanol.  Ethanol can disrupt enzyme activity and organelle function as well as perturb the cell’s permeability barrier.  Recent work suggests that trehalose, proline and glycine are all important factors in stabilizing internal components.  The first two, or their precursors (glucose for trehalose; arginine for proline), are generally plentiful in grape must. Glycine is not.  These compounds do not appear to be able to substitute for each other in very high gravity fermentations.  Nitrogen deficiency may impact the yeasts' ability to synthesize protective factors and lead to enhanced ethanol sensitivity of cytoplasmic functions.  Further, since ethanol induces leakage of several compounds from the cell, higher levels of these components than are required for growth may be necessary.  The impact of acetaldehyde accumulation on fermentation arrest also needs to be further studied.  Factors leading to a reduction in alcohol dehydrogenase activity may increase cytoplasmic acetaldehyde to toxic levels.

A final cause of this type of fermentation arrest may be a mild temperature shock.  Sensitivity to temperature increases at high ethanol concentrations, and, if a fermentation is being cooled, the temperature may drop below permissive levels, depending upon the type of refrigeration being used and its control.  It is also important to remember that most cooling jackets do not provide for a uniform temperature across the tank. The area immediately adjacent to the cooling system (jacket or insert) will likely be much cooler than the rest of the tank.

  [grennoir]

This graph shows an example of a fermentation that became too hot during the initial stages of sugar consumption. There is a too-rapid initiation of fermentation and a dramatic slow down of sugar utilization around 8 Brix. The ethanol content at this time is around 9%. Depending upon the strain, early heat shocks do not become manifest as a fermentation problem until ethanol accumulates to 8-11%, depending upon the strain. In this case, it appears that an ethanol tolerant subpopulation was able to eventually grow and complete the fermentation, but this is not always the case. The dramatic swings in temperature in this fermentation are due to the pumpover regimen and mixing of the tank, with the concurrent dissipation of heat during this process. Such hot starts of fermentations generally lead to reduced complexity of the wine.

Premature settling of the yeast population is also a good indicator of a problem in maintenance of metabolic activity during stationary phase.  We have observed an early settling in fermenting musts held too long at a low temperature (below 20°C) following the cessation of active fermentation.  In many cases, readjustment of the temperature does not result in an improved fermentation rate.

Fermentation normal, abrupt stop (in red below): An abrupt cessation of sugar consumption is usually indicative of a major traumatic shock to the fermenting yeast.  This might be due to exposure to extreme temperatures. Uncooled fermentations may attain an inhibitory temperature due to the release of heat during hexose catabolism, as shown above.  Similarly, fermentations can be overcooled depending upon the design of the system being used.

[types5]

Other types of shocks, such as a mistake in addition of sulfur dioxide, can also lead to an abrupt stop of the fermentation.  In one case, filtration of an incomplete fermentation through a pad filter contaminated withStreptomyces, led to an abrupt arrest of fermentation. Similarly, addition of a malolactic starter culture to a fermentation that is not yet dry can lead to a rather abrupt arrest, depending upon the biological activity, size of the inoculum, nutritional complement of the must, presence and nature of organic and fatty acids in the ML inoculum and the particular yeast and bacterial strains involved. Depending upon the pH, organic acids released by bacterial metabolism may be protonated and, therefore, simply diffuse across  the yeast plasma membrane. Once inside the cytoplasm, the acids release protons due to the higher internal pH. Yeast can use the plasma membrane ATPase to pump out the hydrogen ions coming from the acids. However, if the capacity of the pump is saturated by pumping out the hydrogen ions coming in from the enhanced passive proton flux due to ethanol, then the yeast will not be able to tolerate the acid addition and fermentation will rapidly arrest. Bacterial fatty acids can become inserted into the ethanol-adapted plasma membrane, disrupting sugar uptake and leading to cell death. Some yeast strains tolerate ML bacteria additions much more readily than others and some ML strains are less prone to cause a problem.

Premature addition of fining agents can also lead to loss of culture biomass and dramatically slow the fermentation.  These factors are all generally well within the control of the winemaker and should not routinely pose a problem.

Restarting Arrested Fermentations:

Restarting an arrested fermentation can be challenging, depending upon the cause of the arrest. If fermentation is inhibited due to a nutrient deficiency of either a macronutrient like nitrogen, a micronutrient like biotin, or a mineral ion, addition of the missing component is often enough to restart fermentation. Yeast frequently are easily able to recover from a cold shock if the tank is warmed and mixed to resuspend the population. It is more difficult to restart fermentations arrested due to ethanol intolerance, heat shock or presence of inhibitory organic and fatty acids. In the case of heat shock, survival of the elevated temperature forces changes in plasma membrane composition incompatible with tolerance of the membrane to ethanol. Thus, the alcohol tolerance level is reduced and cells will arrest at a lower than normal ethanol percent.

High ethanol can cause a phase change in the membrane that the cells cannot easily repair. Reducing the ethanol content via dilution likely will not restore the functionality of the membrane. The membrane can only be restored if cells are returned to fully permissive growth conditions so that a new membrane can be made for the daughter cells, which is difficult to do for a fermentation already above 10% ethanol.

If inhibitory acids are present the acids level will need to be reduced before growth can commence. If the acidity has lead to acidification of the cell cytoplasm the cells will not recover. A new inoculant will be just as susceptible to inhibition by the existing acids. Sometimes use of yeast ghosts or inactivated yeast cells to sop up the fatty acids or other inhibitors present in the medium followed by removal of the yeast biomass and reinoculation can help restart fermentations arrested due to the presence of inhibitors.

Regardless of the cause of the fermentation problem, it is frequently necessary to first rack the wine off of the settled yeast lees before attempting to restart the fermentation.  Some types of arrested fermentations may restart without addition of yeast following this treatment.  We have found from analysis of juice-like media wherein the cause of fermentation arrest can be strictly controlled, even moderate aeration will lead to a spontaneous restart of fermentations arrested due to sterol or fatty acid limitation, provided that is the only limitation and the restart is conducted in a timely fashion.  Nitrogen or micronutrient limitation on top of either an oxygen deficiency or temperature shock, will inhibit a spontaneous restart.

Another problem with reinitiating stuck fermentations concerns the timing at which intervention will have a positive outcome.  It has been well established that nitrogen limitation must be corrected before a cessation in fermentation occurs.  We have seen the same effect of potassium addition.  By the time a fermentation has arrested due to an imbalance of potassium and hydrogen ion concentrations, it is too late to correct the problem by adjustment of the ratio of the two ions.  If the stuck or sluggish fermentation is an adaptive response to adverse conditions, the readjustment of the medium must occur prior to commitment to that adaptation.  A decrease in fermentation rate is frequently a consequence of adaptation and not a cause.  Thus, it is desirable to develop diagnostic tools for the early identification of a problem fermentation, preferably prior to significant loss of transporter activity.  It would also be useful to develop better means by which to determine the precise cause of the stress so that it may be rectified.  Frequently the circumstances preceding the arrest of fermentation and the type of change of the fermentation profile can provide key information as to the likely cause of the problem.

Restarting Procedures:

There are three different types of strategies for restarting arrested fermentations. It is helpful if some idea of the cause of the arrest is known, but if not, there are options that can be used to complete the fermentation:

Rejuvenating the existing biomass
Reinoculation with a new adapted  inoculum
Use of activated encapsulated or yeast-in-bag processes

Rejuvenating Existing Biomass

If fermentation has ceased due to a reversible inhibition of the culture, then rejuvenating the biomass can lead to restoration and completion of fermentation. Reversible inhibition would be a low temperature shock, a mild deficiency of survival factors or a modest nutrient limitation. Raising the temperature, aerating the biomass and provision of nitrogen and cofactors can restore fermentation rates. Often, it is not known if a fermentation will restart from the existing biomass. The ability to restart from the existing biomass can often be determined by some quick bench trials. Samples of the tank can be taken and subjected to different treatments isolation and in combination, nutrient addition, aeration, temperature increase, to see if fermentation reinitiates. The successful treatment can then be applied to the entire tank. It is important that the bench trials be conducted under conditions that mimic what will happen in the larger production tank. There may be better mixing in the bench trial that will not be replicated in the larger tank.

If a microscope is available, it is also advantageous to examine the arrested population under the microscope and compare it to a healthy population from another tank. If the yeast from the arrested population appear lysed (popped open), or have granules inside that are moving by Brownian motion, then the population is in decline and will be much harder if not impossible to rejuvenate.

Before attempting rejuvenation, it is important to first check the tolerances of the strains and compare them to the fermentation conditions. A frequent cause of arrest of fermentation is use of a yeast strain that does not have the ethanol-tolerance level needed to complete a fermentation. The ethanol-tolerance level of commercial yeast strains is generally known. A good rule of thumb is to assume a worst-case scenario with respect to Brix yield of ethanol, 0.62 x Brix value, and be sure the strain has the ethanol tolerance capacity to attain this level. Fermentation of a 24 Brix juice would require an ethanol tolerance of 15%. 28 Brix would require a tolerance of 17%. Tolerance to ethanol is impacted by growth conditions, so the tolerances listed by manufacturers are not absolutes, but do provide a good estimate of the level of ethanol at which the strain can be expected to arrest. Strains also differ in tolerance of temperature shocks, nutrient limitation and bacterial competition. This information is also generally known for commercial strains. Before considering a rejuvenation strategy an assessment of the inoculant strain should be undertaken. If it does not have the tolerances needed to complete the fermentation then a reinoculation strategy with a more tolerant strain should be employed.

Reinoculation with a New Adapted Inoculum

If the fermentation arrest is due to ethanol intolerance, to temperature or acid shock, or to poor innate tolerances of the strain dominating the fermentation, it will be necessary to reinoculate. The new inoculants will have to be adapted to the conditions of the arrest. If the ethanol concentration is above 7% the new strain may have difficulty adapting to the fermentation conditions and will have to be introduced to the ethanol concentration in a gradual fashion. For example, mixing half of the arrested wine with fresh juice to drop the ethanol concentration to or below 5% will assure a healthy start for the new inoculant. If enough fresh juice is not available, water and commercial nutrients can be used to dilute both the juice and wine mixture to a permissive alcohol level. Once active fermentation is evident, meaning that obvious fermentative release of carbon dioxide is occurring, then more and more of the arrested ferment can be added in stepwise fashion taking care to not let the ferment go dry at any time in the process.

Several commercial strains are available that have been isolated specifically because of their low nutrient requirements, high ethanol, temperature and bacterial metabolite tolerances, and ease of rehydration. One of these strains should be considered instead of using the initial strain.  The availability of a microscope greatly enhances the ability to monitor the health and vitality of the new inoculant. It is also important to not add excessive sulfite when attempting a restart or to add the ML bacteria at the same time as the attempted restart.

Use of Activated Encapsulated or Yeast-in-Bag Processes

Growing a new inoculant, taking care to make sure it is adapted to the conditions of the arrested fermentation, can often be a time consuming process with no guarantee of success. It is often easy to let the new culture go too far, that is, it consumes all the available sugar in one mixture and arrests itself before a transfer can occur.  If all that is needed is to consume the rest of the sugar and complete a fermentation, then use of an encapsulated yeast may be a better approach. Encapsulated yeast are “trapped” in an alginate matrix. The alginate beads are fully permeable to substrates and endproducts but do not allow growth of the yeast cells. The reinforcement of the alginate matrix puts the yeast in a biofilm like mode of metabolism. Under these conditions substrates can be consumed efficiently and cells are not sensitive to factors inhibiting growth or disrupting membranes. The encapsulated yeast have been adapted to high ethanol and low nutrients during the encapsulation process and will work well in completing sugar consumption of an arrested fermentation, provided that other inhibitory conditions such as high SO2, high acetic acid, low pH, very high ethanol level, and/or high bacterial bioloads, do not exist or have not been rectified. The encapsulated yeast can be placed inside of a mesh bag for optimal distribution within the tank and for ease of removal. Again, if using one of these products, the manufacturer’s recommendations should be carefully followed. Extremes of temperature should be avoided as well as any other conditions that may adversely impact the viability of the yeast within the capsules.
 
retrieved from http://wineserver.ucdavis.edu/ ... .html

what is acetaldehyde style in terms of winemaking practice?

Reply

cewh Post a question • 1 person concerned • 0 replies • 305 views • 2016-05-07 19:46 • 来自相关话题

when fermentation in stationary phase, TA value is 9.6, a problem for later fermentation?

Reply

cewh Post a question • 1 person concerned • 0 replies • 1860 views • 2016-05-07 19:42 • 来自相关话题