Determining Glucose Isomerase Activity in Different Wine Environments to Prevent Sluggish or Stuck Fermentations by Using Glucose Isomerase

Abstract

The objective of this study was to determine glucose isomerase activity in different prepared original or synthetic wine media to prevent sluggish or stuck fermentation, which may be caused by sugar uptake deficiency in yeast. The unfermented grape juice contains almost equal amounts of glucose and fructose. After fermentation, the residual sugar is mostly fructose, this is called glucose/fructose discrepancy (GFD) and is caused by the affinity decrease of hexose transporters towards fructose as ethanol accumulates. This results in stuck fermentation and is unwanted as the wine is sweet and risks microbial spoilage. Converting remaining fructose to glucose by glucose isomerase may be a solution so we tested the activity of this enzyme in synthetic and original wine media. Glucose formation, 0.5 % w/v, from 1% w/v fructose took place in synthetic wine medium containing 13 % v/v ethanol, 1% w/v glycerol and at pH 3.3. In original wine medium glucose formation did not take place except when wine was diluted at least five folds and at pH values equal or higher than 6 whether if tartaric acid was present or not. Since neither dilution, nor pH adjustment can be applicable, other ways to employ this enzyme should be tried.

1. Introduction

Wine fermentation is a complicated biochemical process in which yeasts play an active role in the production of ethanol, CO₂, and other metabolites from glucose and fructose of grapes [1]. Wine fermentation spontaneously takes place by yeast strains that are present on the grape surface or winery equipment. By today’s technology, to achieve complete fermentation, good oenological properties and high production yield commercially produced yeast strains, and mostly Saccharomyces cerevisiae are used for wine fermentation as starter microorganisms [2].

S. cerevisiae strains derived from industrial wine have hexose transporters (HXT 1–7) that are responsible for wine fermentation. It is mentioned that there is no growth or fermentation when HXT 1–7 are deleted from the genes of this yeast [3]. Ethanol formation in the wine medium causes a change in the affinities of hexose transporters and the change in the affinities of hexose transporters causes stuck fermentation. In this study, stuck fermentation due to ethanol formation was discussed and the experiments were conducted to prevent stuck fermentation.

The hexose sugars, glucose and fructose, are the main reducing monosaccharides present in grapes or grape musts. The amounts of total sugars in grapes or grape musts change between 160 and 300 g/L that consist of almost equal amounts of glucose and fructose before fermentation [4]. During wine fermentation, yeasts, especially S. cerevisiae, coferment these monosaccharides and produce wine components [3, 4, 5]. Since yeasts have glucophilic character, which is the preference of fermenting glucose to fructose [5], the utilization rate of glucose is higher than that of fructose during fermentation [4]. The glucophilic character of yeasts may be due to transportation across the plasma membrane of yeast by hexose transporters or phosphorylation inside the cell of yeast by hexose kinases has different affinities through glucose and fructose [4]. These different utilization rates result in glucose/fructose discrepancy (GFD) and residual fructose amount higher than 2 g/L [6] when the fermentation process is completed [4]. Since the sweetness of fructose is approximately twice than that of glucose [7], it affects the final sweetness of wine and the wine fermentation results in higher sweetness, which is undesirable in the wine industry. Also, high residual fructose increases the risk of microbial spoilage [8, 9, 10] and decreases the ethanol yield in wine [4, 5, 6]. This has been informed that sluggish (incomplete) or stuck (depleted) fermentation in the literature [6].

Although the exact reason for stuck or sluggish fermentations has not been determined yet, there are more than 15 reported reasons such as nitrogen deficiency [3, 4, 6, 8, 9, 11], limitation or excess amount of oxygen [8, 9], too much clarification [8, 9], formation of by-products due to fermentation [9], high ethanol accumulation [4, 6, 8, 9, 12], vitamin and mineral deficiency [8, 9], toxic residues for yeasts from fermentation [5, 8, 9], deprivation of nutrients for yeasts [10], too high or low temperatures [10], environment with high acidity [10], the formation of inhibitors like phenols [10], change in the equation of ionic components [10], higher sulfite content [8], and so on.

There are some known reasons for stuck fermentation and also, there are some possible ways and improvement methods against stuck fermentation such as nitrogen supplementation, controlling the oxygen amount, controlling the temperature of the environment, selecting the yeast according to process, controlling nutrients for yeast growth, and so on. Although many techniques and improvements are developed, stuck fermentation is still a major problem for the wine industry since it causes product losses [9].

Usage of an enzyme in wine media for preventing or restarting the stuck fermentation was not studied before. Therefore, using glucose isomerase to prevent or restart the stuck fermentation was studied as a novel approach.

2. Materials and methods

In this part, the “synthetic media” term was defined for different experiments that were in other parts, too. This term means that media are prepared with different wine components such as ethanol, glycerol, tartaric acid, calcium, and so on. To mimic the wine environment and activate the enzyme, 0.06 g MgSO₄.7H₂O was added into 100 mL synthetic media solutions [13]. The experiments were conducted with 1 g of immobilized glucose isomerase added to 100 mL of solutions.

2.1 The effect of substrate type and temperature on the activity of glucose isomerase

In these experiments, either glucose or fructose was used as substrates of reactions at an amount of 1% w/v. The flasks were incubated in shakers at 60 or 30°C at 150 rpm. The pH values of media were 5.8 and 5.4 for glucose and fructose as substrates, respectively.

2.2 The effect of ethanol on the activity of glucose isomerase

To see the effect of ethanol on the enzyme, synthetic wine media were prepared by adding 13% v/v ethanol. Fructose was added to the media at an amount of 1% w/v. The pH of the solution was 5.5. The prepared flasks were incubated in shakers at 150 rpm. For comparing wine and wine without ethanol, samples were prepared with 1% fructose w/v. The flasks were incubated at 60°C for approximately 42 hours. The pHs of samples were 3.6 and 2.8 for wine with alcohol and without alcohol, respectively.

2.3 The effect of low pH values on the activity of glucose isomerase in synthetic wine medium

Different pH values, which were 3.3, 3.6, and 4, were adjusted with 1:1 acetic acid in synthetic wine media containing 1% w/v fructose. The flasks were incubated at 60°C and 150 rpm for almost 150 hours.

2.4 The effect of glycerol in synthetic wine media on the activity of glucose isomerase

The synthetic wine media were prepared with 1% w/v fructose, 13% v/v ethanol, and different glycerol contents. The flasks were incubated at 60°C for 47 hours. In order to simulate the wine environment, the pH was adjusted to 3.3 with 1:1 acetic acid solution.

2.5 The effect of sulfite content on the activity of glucose isomerase

The synthetic wine medium contained 1% w/v fructose as a substrate with different sulfite amounts. The flasks were incubated at 60°C and 150 rpm for 14.5 hours. The pH values of solutions were adjusted to 3.5 with 1:1 acetic acid solution.

2.6 The effect of tannins in red wine on the activity of glucose isomerase

In this experiment, 1 g of fructose was added to a 100 mL wine medium. Samples were incubated in flasks at 60°C and 150 rpm for almost 70 hours. The pH values of solutions were 3.37, 3.05, and 3.5 for Doluca Mistik Red, Turkey, 2016 (alcohol: 14.0% v/v) (w1), Doluca Mistik White, Turkey, 2016 (alcohol: 13.5% v/v) (w2), and Frontera, Chile, 2015 (alcohol: 12.5% v/v) (w3), respectively.

2.7 The effect of fermentation components on the activity of glucose isomerase

To obtain synthetic wine media with grape juice, 5% w/v fructose, 13% v/v ethanol, and 0.8% v/v glycerol were mixed with grape juice. Two types of grape juices were used during the experiments. The industrial white and red grape juices that were obtained from the market were Kavaklıdere brand. Also, the homemade grape juice was tested and used during the experiments. Samples were incubated in flasks at 60°C and 150 rpm. Since grape juice already contained Mg⁺² [14], the enzyme did not require an additional activator. The pH of homemade grape juice was 3.65 and 3.88 for synthetic wine media with homemade grape juice.

2.8 The effect of calcium content on the activity of glucose isomerase

In this experiment, 1% w/v fructose was used as a substrate, and 13% v/v ethanol and 0.8% v/v glycerol were added to provide synthetic wine environment. The pH values of the media were adjusted to 3.6 with 1:1 acetic acid solution. Since 100 mL red wine contains 8 mg calcium, the same amount of calcium must be present in the synthetic wine medium to provide the same conditions. Therefore, 0.015 g Ca (OH)₂ was added to 100 mL synthetic wine medium. The flasks were incubated at 60°C and 150 rpm for almost 65 hours. Another experiment was conducted by considering the ion retention capacity of EDTA to hold the calcium in homemade red wine media with alcohol content of 14% v/v. Different concentrations of EDTA were added to red wine media containing 1% w/v fructose as a substrate. The flasks were incubated at 60°C and 150 rpm. The pH of solutions with 0.1, 0.2, 0.6, 1, and 2% w/v EDTA were 3.46, 3.43, 3.53, 3.53, and 3.6, respectively. For the cation exchanger experiment, to test the enzyme activity in red wine media containing little amounts of calcium, 1% w/v fructose was added to sample numbers 0, 4, 6, and 10. The numbers of samples were named from 0 to 10. The number of 0 was the original wine sample, and other numbers were wine samples that were eluted through the resin at a rate of 40 mL per sample. While sample 0 contained almost 13 ppm calcium in it, other samples contained much less amounts of calcium with respect to sample 0. The pH values of samples were 3.6, and they were incubated at 60°C and 150 rpm for almost 80 hours.

2.9 The activity of glucose isomerase in diluted wine media

In the first experiment, homemade red wines were used containing 1% w/v fructose (added after dilution) as a substrate. Wines were diluted with distilled water at six different concentrations: 90, 70, 50, 30, 20, and 5% v/v and also 100% v/v red wine as a control. The prepared solutions were incubated at 60°C and 150 rpm for 42 hours. The pH values of solutions were 3.15, 3.15, 3.19, 3.3, 3.53, 3.59, and 4.45 for wines at concentrations of 100, 90, 70, 50, 30, 20and 5% v/v, respectively. Also, different brands and types of wines; w1, w2, and w3; at different concentrations, 100, 10, and 5% v/v, were used in another experiment. The samples containing 1% w/v fructose as a substrate were incubated at 60°C and 150 rpm for 27 hours. The pH values of solutions were 3.5, 3.45, and 3.03 for 100% v/v w3, w1, and w2, respectively.

2.10 The effect of tartaric acid on the activity of glucose isomerase

Synthetic wine environments with 1% w/v fructose and 0.3% w/v tartaric acid were prepared at pH values of 3.55 and 6.33 adjusted with 5 M NaOH and 24% w/v KOH solutions, respectively. Samples were incubated at 60°C and 150 rpm. For experiments, samples numbered as 0, 2, 5, 8, 9, 10, and 12 were chosen with the addition of 1% w/v fructose. The pH values of solutions, 0, 2, 5, 8, 9, 10, and 12, after fructose addition were 3.36, 9.85, 6.7, 4.8, 4.08, 3.83, and 3.66, respectively. Samples from 0 to 13 were passed through anion exchanger resin. The first 9 samples passed through an anion exchanger had higher pH values than the original sample. In the original sample, number 0, the tartaric acid content was equal to 0.248% w/v. Other samples from 1 to 13 did not contain any tartaric acid. The samples were incubated at 60°C and 150 rpm.

2.11 The effect of pH on the activity of glucose isomerase in red wine medium

Homemade wines at different pH values; 4, 5, 6, 7, and 8, containing 1% w/v fructose as a substrate were tested at temperatures of 60 and 30°C for this experiment. The pH adjustments of samples were done with a 5 M NaOH solution. The samples were incubated at 150 rpm, 60 and 30°C, for 70 hours.

2.12 Immobilized glucose isomerase

The enzyme that was used in this study was immobilized glucose isomerase and supplied from Cargill, Bursa. It was produced by Novozymes and the group of it was Sweetzyme IT Extra. The color of the enzyme was brown, and it was in the granulated form. Its approximate density value was 0.50 g/mL. Its typical activity range was above 400 IGIU/G (immobilized glucose isomerase unit per gram). The enzyme was stored at 4°C to avoid contamination.

2.13 Atomic absorption spectroscopy

Atomic absorption spectroscopy is a common method to detect metals and metalloids in liquid samples. Free atoms of gas are generated in the atomizer and they can absorb the radiation at a given frequency. By atomic absorption, the absorption of ground-state atoms in the gaseous state can be measured. The atoms make transitions to higher energy levels by absorbing the UV or visible light. The concentrations of metals or metalloids are determined from the absorption amount. To measure calcium content of the wine samples with an analytical method, atomic absorption unit (Jarrell Ash) was used at Middle East Technical University, Chemical Engineering Department.

2.14 HPLC (high-performance liquid chromatography)

In this study, the samples were analyzed to determine glucose, fructose, ethanol, glycerol, tartaric acid, malic acid, and acetic acid concentrations by using HPLC in Middle East Technical University, Food Engineering Department, Biotechnology Laboratory (Agilent Technologies, USA). The column and detector type of this HPLC was Rezex™ RFQ-Fast Acid H+ (8%) LC Column, 100 × 7.8 mm (length and internal diameter, respectively), and refractive index detector, respectively. The temperature of the refractive index detector and fast acid column was set to 30 and 25°C, respectively. 0.05 M H₂SO₄ was used as an eluent. For every sample, 10 μL of analyte was injected automatically with a flow rate of 0.6 mL/min.

3. Results and discussion

3.1 The effect of substrate type and temperature on the isomerization reaction of glucose isomerase

The equilibrium reaction from fructose to glucose or vice versa took place regardless of substrate type and temperature. According to the results of the experiments, the isomerization reactions were equilibrated at a faster rate when starting with glucose compared with fructose, especially at 30°C. Another important point resulting from experiments, the reactions took place at faster rates when conducted at 60°C compared with 30°C. Also, an equilibrium point was reached after 5 hours at 60°C while after 9 hours at 30°C (data not shown). As a result of these experiments, it can be understood that the enzyme is suitable for both substrate types at different temperatures.

3.2 The effect of ethanol on the isomerization reaction of glucose isomerase

According to the results, glucose formation and fructose depletion were shown at 60 and 30°C. Also, it was shown that glucose concentration was slightly higher than fructose concentration after 25 hours at 30°C. However, fructose concentration was slightly higher than glucose concentration after 25 hours at 60°C. As it was mentioned before, the reaction took place more slowly at 30°C compared with 60°C by looking at the data (data not shown). As a consequence of this experiment, it was interpreted that 13% v/v ethanol did not inhibit the activity of the enzyme regardless of different temperature values.

Since ethanol did not inhibit the isomerization reaction in synthetic environment, an experiment was conducted by vaporizing ethanol in red wine to show the effect of ethanol on red wine. The ethanol concentration was reduced approximately from 13 to 0.3% v/v by keeping wine at air temperature on the magnetic stirrer. At the end of 42 hours, glucose concentrations of four samples did not change (data not shown). This means that the enzyme did not isomerize fructose even if there was no ethanol in the wine.

3.3 The effect of low pH values on isomerization reaction in synthetic medium

As seen in Figure 1, it was noticed that fructose was converted to glucose regardless of the pH values. Also, it was clearly noticed that the reaction at pH 4 was the fastest one when compared to others, whereas the slowest reaction was at pH 3.3 by looking at glucose formation rate. Therefore, it was thought that the reason for the inhibition of enzyme in the wine medium was not because of the low pH values. The rate of reaction decreased proportionally with decreasing pH, the inhibition effect in the wine medium was not due to acidic medium, and the effect of ethanol with low pH values had to be tested.

Figure 1.

Change of glucose concentrations at different pH values without and with ethanol.

Under the same conditions in Figure 1, the glucose was formed under the same pH values with 13% v/v ethanol in medium as seen in Figure 1, too. The ethanol effect experiments were conducted with the same amount of ethanol as experiments above, 13% v/v. The rate of reaction was obviously slower at pH 3.3. When without and with ethanol conditions were compared, almost the same amounts of glucose were formed for pH values of 3.6 and 4, whereas the amount of glucose at pH 3.3 was clearly lower in ethanol medium. Also, the glucose amounts formed at pH 3.6 and 4 were equalized at about the 45th hour with ethanol in the environment. The formed glucose amounts from 1% w/v fructose were nearly 0.2 and 0.1% w/v in medium without and with ethanol, respectively, at pH 3.3. As a consequence, glucose formation was detected in acidic media even in the presence of ethanol. The rate of reaction at pH 3.3 was clearly slower than others regardless of the presence of ethanol.

3.4 The effect of glycerol in synthetic media on the isomerization reaction

According to Figure 2, the isomerization reaction took place in all synthetic media with different glycerol concentrations. The rates of reactions were close to each other for all glycerol concentrations until the glycerol concentration was approached to 1% v/v. That is, the slowest reaction rate was obtained at the concentration of 1% of glycerol. It was easily concluded that the existence of glycerol in synthetic media containing ethanol did not inhibit the isomerization reaction.

Figure 2.

Change of glucose concentrations in synthetic media with different glycerol contents during 47 hours.

3.5 The effect of sulfite content on isomerization reaction

Although it is known that glucose isomerase is used with sulfite [13], the experiments were also conducted in synthetic media with sulfite. After 14.5 hours of incubation, almost equal amounts of glucose were formed in all concentrations: 0.1, 0.04, and 0.01%, of sulfite (data not shown). Therefore, even higher sulfite contents compared to those present in wine did not inhibit the activity of glucose isomerase.

3.6 The effect of tannins in red wine on the isomerization reaction

It was thought that tannins might have an inhibitory effect on the isomerization reaction. Therefore, the comparison of glucose isomerase activity in red and white wine media was made. Glucose concentrations of all types of wines, w1, w2, and w3 remained stable during 70 hours of incubation. Normally, enzyme activity is seen in the first 2 or 3 hours of the experiments; in this case, no activity was observed even after 70 hours (data not shown). As a consequence, it was concluded that tannins had no inhibitory effect on the isomerization reaction since white wine also showed a similar behavior.

3.7 The effect of fermentation components on isomerization reaction

Red grape juice contains almost equal amounts of glucose and fructose before fermentation. Therefore, to determine enzyme activity in grape juices, 5% w/v fructose was added into grape juice. As a result, glucose and fructose concentrations remained stable for 2.5 days. The fructose concentrations of samples were higher than those of glucose. Although samples were kept for 2.5 days, the enzyme did not isomerize the substrate in solutions (data not shown). As a result, since enzyme activity was not observed in grape must before fermentation, it was thought that a component that is present before fermentation would be inhibiting the enzyme.

3.8 The effect of calcium content on isomerization reaction

Generally, red wine contains both calcium and magnesium in amounts of 80 ppm and 120 ppm, respectively [15]. The ratio of magnesium to calcium must be equal to 12 to provide activating conditions for glucose isomerase. It was thought that the inhibition in the wine medium was because of the calcium content. In light of this information, experiments were conducted in synthetic media containing ethanol, glycerol and calcium, and red wine with the increased magnesium content. The conversion reaction took place in a synthetic medium without calcium; however, there was no formation of glucose in the synthetic medium with calcium (data not shown). As a result of this experiment, it was thought that calcium in wine may be inhibiting the glucose isomerase.

After the addition of 840 ppm magnesium into red wine, there was no formation of glucose in the red wine medium even with increased magnesium content at pH 3.28. The glucose formation was observed in red wine media with increased pH with or without additional magnesium. However, the reaction rate was faster at pH 7.5 than at pH 8.0 since the medium at pH 7.5 contained additional magnesium. The reactions reached equilibrium after about 50 hours (data not shown). As a result, it was concluded that the additional magnesium had no effect on low pH wine medium; however, it speeded the reaction rate at high pH wine media. Therefore, the effect of pH on isomerization reaction in wine media must be considered.

Another experiment was conducted by considering the ion retention capacity of EDTA to hold the calcium in homemade red wine media. There was no formation of glucose after 70 hours (data not shown). Therefore, it was concluded that EDTA had no positive effect on isomerization reaction in red wine media.

To test the enzyme activity in red wine media containing little amounts of calcium, samples 0, 4, 6, and 10 were chosen. The glucose concentrations of samples 0, 4, 6, and 10 remained stable and isomerization reaction did not take place after 80 hours (data not shown). Whereas calcium in synthetic medium inhibited the activity of glucose isomerase, there was also an inhibition effect in red wine even if there was no calcium in the environment.

3.9 The activity of glucose isomerase in dilute wine media

In the first experiment, homemade red wines were diluted with distilled water at six different concentrations. As seen in Table 1, except for concentrations at 20 and 5% v/v, the glucose concentrations of samples remained stable. At concentrations of 5 and 20% v/v red wines, the glucose formation took place during incubation.

Time	100% hand made	90% hand made	70% hand made	50% hand made	30% hand made	20% hand made	5% hand made	100% w1	10% w1	5% w1	100% w2	10% w2	5% w2	100% w3	10% w3	5% w3
0	0.274	0.247	0.192	0.137	0.078	0.05	0	0.098	0	0	0.068	0	0	0.274	0.025	0.012
6							0.156
16	0.272	0.245	0.19
16.5				0.151	0.108	0.242
17								0.104	0.474	0.47	0.073	0.505	0.462	0.281	0.482	0.471
22	0.273	0.246	0.191
24							0.463
26								0.109	0.474	0.477	0.073	0.489	0.45	0.279	0.487	0.459
41.5				0.149	0.116	0.38

Table 1.

Glucose concentrations of different wines with different dilution rates.

Also, different brands and types of wines at different dilution concentrations were used in another experiment. As seen in Table 1, the glucose formation took place at concentrations of 10 and 5% v/v regardless of the brand type. Also, almost equal amounts of glucose formed at the same hours for different dilution factors and brand types.

As a result of these experiments, it was thought that a component or some components in wine coming from grapes may inhibit the isomerization reaction but this component or components may lose its or their effects in diluted wines but only at the levels of fivefold dilutions.

3.10 The effect of tartaric acid on the enzyme activity

The experiments were conducted with synthetic media containing fructose and tartaric acid and red wine containing no tartaric acid.

Fructose concentrations remained stable at 1% w/v at pH 3.55 and also, no glucose formation took place in flasks after 150 hours. However, glucose formation and fructose consumption were observed at pH 6.33. The reaction reached equilibrium within 30 hours with higher fructose contents (data not shown). As a result, L- (+)-tartaric acid inhibited the activity of glucose isomerase at pH 3.55; however, if the pH of the solution was 6.33, there was no inhibition with tartaric acid. Therefore, it was thought that if tartaric acid in wine is eliminated, the reaction would take place.

For experiments, samples numbered as 0, 2, 5, 8, 9, 10, and 12 were chosen. As seen in Figure 3, the 100% glucose formation took place at pH values of 9.85 and 6.7 and 50% glucose formation took place at 4.8, that is, sample numbers of 2, 5, and 8, respectively. Even if all samples did not contain tartaric acid, the conversion reaction took place at only higher pH values. Therefore, it was thought that the tartaric acid had an inhibitory effect at low pH values, under a pH of approximately 5. As a result, it had to be examined that glucose isomerase could or not convert fructose to glucose in wine media containing tartaric acid at high pH.

Figure 3.

Glucose concentrations of wine samples without tartaric acid.

3.11 The effect of pH on isomerization reactions in red wine medium

As seen in Figure 4, the glucose formation took place at pH values of 6, 7, and 8 regardless of temperature. If temperature values were compared, the glucose concentration increased from 0.2% to almost 0.9% w/v for 60°C; however, it was from 0.2% to almost 0.4% w/v for 30°C during 70 hours of incubation. If pH values were compared, the reaction rates were higher for pH 8, 7, and 6 in decreasing order at 60°C. However, the reaction rate was almost equal for pH 7 and 8 but lower for pH 6 at 30°C.

Figure 4.

Glucose concentrations of homemade red wines at different pH values at 60 and 30°C.

From the results of these experiments, the isomerization reactions took place in red wine media at pH values higher than 5 regardless of temperature. Therefore, it was thought that the pH of the medium had to be suitable in order for the glucose isomerase to be active in wine media.

4. Conclusion

In this study, the effects of different environmental and chemical factors on the activity of the enzyme glucose isomerase were tested with the final aim of using this enzyme for the conversion of fructose to glucose present in stuck wine fermentations.

To conclude, 0.5% w/v glucose formation from 1% w/v fructose took place in synthetic medium containing 13% v/v ethanol and 1% v/v glycerol at pH 3.3 and at temperatures of 60 or 30°C in approximately 48 hours. However, the glucose formation did not take place in synthetic medium if there was 0.3% w/v tartaric acid at pH 3.55, whereas glucose was formed at pH 6.33. In the original wine medium with dilution effect and at pH values equal or higher than 6, glucose was formed from fructose whether there was tartaric acid or not. Since dilution and increasing the pH of wine cannot be applicable, other ways to employ this enzyme to prevent stuck fermentation should be tried.

In final words, we can mention some methods that can be employed for stuck fermentations. A membrane system can be used for separating acetic acid from the wine medium [16]. This may increase glucose formation from fructose by using glucose isomerase. If a low pH resistant glucose isomerase can be searched and found, thanks to this enzyme, fructose be converted to glucose and stuck fermentation may be prevented. Finally, though not related to glucose isomerase, different yeast strains may be employed. The yeast strains usually employed in enology have glucophilic characters that prefer glucose against fructose. Fructophilic yeast strains, even if not employed at the start of the fermentations, may be added to stuck fermentations and may help consume the residual fructose in the medium.