Low-alcohol wine brewing method applying double-bacterium system low-temperature staged fermentation

Abstract

The invention belongs to the technical field of wine brewing, and discloses a low-alcohol wine brewing method applying a double-bacterium system to perform low-temperature staged fermentation, which comprises the following steps: accurately controlling the alcoholic strength to 3.8% ABV + / -0.1% ABV and ester substances to be more than or equal to 75mg / L through a heat-resistant Kluyveromyces cerevisiae and Kluyveromyces pichia composite strain system in combination with beta-glucan enzymolysis and low-temperature staged fermentation; the flavor substance retention rate is greater than or equal to 95%; wherein the Kluyveromyces thermotolerant firstly utilizes glucose to grow, breed and produce acid, so that a conversion precursor of sugar to alcohol is reduced; the kluyveromyces pichia is regulated and controlled by a metabolic pathway, most glucose is used for synthesizing flavor substances such as esters and higher alcohols, and only a small amount of glucose is converted into alcohol. The finally obtained low-alcohol grape wine has fresh and cool taste and rich fragrance, and meets the market requirements of healthy drinks. Under the joint cooperation of fermentation control, strain combination and enzymolysis treatment, the problems of out-of-control sugar conversion, flavor loss, fermentation stagnation and the like in the existing low-alcohol wine production are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of wine brewing, and in particular relates to a low-alcohol wine brewing method using a dual-bacteria system with low-temperature segmented fermentation. Background Art

[0002] As consumers' health awareness continues to rise, market demand for low-alcohol wine, a beverage that combines wine flavor with health benefits, is growing. Traditional wines typically have high alcohol content, making them unsuitable for children, pregnant women, drivers, and those with alcohol intolerance. By reducing the alcohol content, low-alcohol wine retains the nutritional value and flavor of wine while reducing the negative effects of alcohol on the human body, meeting the needs of a wider range of consumers.

[0003] Currently, the main methods used to prepare low-alcohol wine include heating, freezing, membrane-treated reverse osmosis, pervaporation, and organic solvent extraction. Heating evaporates alcohol through heating, but this process results in the loss of some aroma components, which in turn affects the wine's flavor. Freezing removes water through low-temperature freezing followed by distillation. While this method can better preserve aroma components, it is costly. Membrane-treated reverse osmosis uses a semipermeable membrane to separate alcohol. While the processing temperature is low and has minimal impact on taste, it requires water replenishment, making the operation more complex. Pervaporation uses a hydrophobic membrane to separate alcohol. While this requires minimal equipment investment, the process requires heating the wine, increasing energy consumption. Organic solvent extraction uses organic solvents to extract alcohol, but this can lead to issues with solvent residue and aroma loss. As can be seen, existing technologies typically remove alcohol from wine after it is finished to reduce its alcohol content through various methods. However, these methods suffer from reduced aroma components and, in single-strain natural fermentation, uncontrolled sugar conversion and fermentation stagnation. Summary of the Invention

[0004] To solve the above problems, the present invention provides a low-alcohol wine brewing method using a dual-bacteria system with low-temperature segmented fermentation. The existing technologies such as heating, freezing, and membrane treatment reverse osmosis all remove alcohol after the wine has completed fermentation, but do not specifically regulate the sugar conversion and yeast metabolic state during the fermentation process. In actual production, if conventional methods such as natural fermentation of a single strain of bacteria are adopted, it is easy to cause the risk of uncontrolled sugar conversion, excessive or insufficient conversion, fermentation stagnation such as premature yeast aging, and metabolic disorders leading to fermentation interruption due to unstable yeast activity and poor adaptability to environmental conditions. The present invention avoids these potential hidden dangers from the source by controlling the fermentation process, strain combination, and enzymatic treatment. Different from the existing technology that only removes alcohol from the finished product, it improves the whole process control of low-alcohol wine preparation and effectively avoids the above risks.

[0005] In order to achieve the above objectives, the specific technical solutions of the present invention are as follows.

[0006] A low-alcohol wine brewing method using a dual-bacteria system with low-temperature segmented fermentation comprises the following steps: performing enzymatic hydrolysis on the grape juice to reduce the sugar content to 16°Brix~18°Brix, thereby obtaining enzymatically hydrolyzed grape juice; After nitrogen is introduced into the enzymatically hydrolyzed grape juice until the dissolved oxygen content in the wine is less than 0.1 mg / L, thermotolerant Kluyveromyces is inoculated, and fermentation is carried out at 16°C to 18°C. When the sugar content reaches 12°Brix to 14°Brix, Pichia Kluyveromyces is inoculated to continue fermentation. When the alcohol content reaches 3%ABV to 4%ABV, the wine is cooled to 0°C to terminate the fermentation, thereby obtaining the fermented grape juice. The fermented grape juice is clarified, filtered, and aged to obtain the low-alcohol wine.

[0007] During the enzymatic hydrolysis process, the present invention reduces the dissolved oxygen content to less than 0.1 mg / L by introducing nitrogen, thereby blocking the transition from aerobic respiration to alcohol fermentation. Reducing the dissolved oxygen content to less than 0.1 mg / L by nitrogen replacement can force yeast to enter a nearly anaerobic state during the subsequent fermentation process, inhibiting the activity of alcohol dehydrogenase, thereby reducing the conversion of alcohol. During the fermentation process, by first inoculating heat-resistant Kluyveromyces at a low temperature of 16℃~18℃, it preferentially consumes glucose for growth, reproduction and acid production, reduces the precursors for the conversion of sugar into alcohol, and lowers the pH of the fermentation liquid to 3.2~3.5, creating a suitable environment for Pichia Kluyveromyces; among them, Pichia Kluyveromyces can convert 60%~70% of glucose into esters and higher alcohols, and only 3%~5% converts glucose into alcohol, thereby effectively reducing the alcohol content, thereby avoiding the occurrence of fermentation stagnation caused by uncontrolled fermentation sugar conversion of a single strain of bacteria; when the alcohol content reaches 3%ABV~4%ABV, it is quickly cooled to 0℃, accurately locking the alcohol content at 4%ABV±0.2%ABV, while retaining unfermented sugar to enhance sweetness and fruity aroma, thereby enhancing sweetness and fruity aroma while reducing alcohol content, avoiding the problem of flavor loss.

[0008] In another preferred embodiment, the specific process of continuing the fermentation of Pichia Kluyveromyces is as follows: After 5 days of fermentation at 17°C, the temperature was lowered to 15°C within 3 days. During the cooling process, an alcohol dehydrogenase inhibitor was added to control the alcohol production rate to ≤ 0.3% ABV / day. The alcohol dehydrogenase inhibitor is a mixture of iodoacetic acid solution and EDTA solution or a mixture of potassium metabisulfite solution and EDTA solution; the volume ratio of iodoacetic acid solution, potassium metabisulfite solution and EDTA solution is 0.8-1.2:1.5; The concentrations of the iodoacetic acid solution and the potassium metabisulfite solution are both 50 mg / L to 100 mg / L, and the concentration of the EDTA solution is 0.1 mM to 0.5 mM. Preferably, the alcohol dehydrogenase inhibitor is a mixture of iodoacetic acid and EDTA solutions. Experimental studies have shown that the combination of iodoacetic acid and EDTA can inhibit ADH by up to 78%, and gas chromatography analysis reveals no iodide residue (detection limit <0.1 mg / L).

[0009] In another preferred embodiment, the temperature is lowered to 15°C at a rate of 0.67°C / day to 1°C / day.

[0010] In another preferred embodiment, the inoculation amount of the thermotolerant Kluyveromyces per liter of grape juice is 0.8 g to 1.2 g; the inoculation amount of the Pichia Kluyveromyces per liter of grape juice is 1.2 g to 1.8 g.

[0011] In another preferred embodiment, the specific process of the enzymatic hydrolysis is as follows: Add β-glucanase to the grape juice and perform enzymatic hydrolysis at 45°C-50°C for 1-2 hours. The addition of β-glucanase can reduce juice viscosity and release terpene aroma precursors.

[0012] In another preferred embodiment, the added amount of the β-glucanase is 0.1% to 0.2% of the mass of the grape juice.

[0013] In another preferred embodiment, the method further comprises cooling the enzymatically hydrolyzed grape juice to 16° C. to 18° C. before inoculating the thermotolerant Kluyveromyces yeast.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The invention utilizes the synergistic effect of thermotolerant Kluyveromyces and Pichia Kluyveromyces, performing staged fermentation at a low temperature of 16-18°C. The thermotolerant Kluyveromyces is inoculated first. At 16-18°C, the yeast preferentially consumes 30-40% of glucose to produce lactic acid, ultimately achieving a lactic acid content of 3.8-4.2 g / L. This lowers the pH to 3.4, creating an acidic environment for the Pichia Kluyveromyces and inhibiting bacterial growth, resulting in a bacterial contamination rate of less than 1%. Reversing the inoculation order increases the early alcohol production rate of the Pichia Kluyveromyces to 0.4% ABV / day, while reducing ester synthesis by 13%, thus affecting the quality of low-alcohol wines. Through staged fermentation, most of the glucose can be used to synthesize flavor substances such as esters and higher alcohols, and only a small amount is converted into alcohol. When the alcohol content reaches 3% ABV~4% ABV, the fermentation is terminated by cooling it to 0℃. The resulting low-alcohol wine has a refreshing taste and rich aroma, meeting the market demand for healthy drinks. It effectively solves the problems of uncontrolled sugar conversion, flavor loss and fermentation stagnation in the existing low-alcohol wine production.

[0015] By fermenting Pichia Kluyveromyces at 17°C for the first five days and cooling the temperature to 15°C for the final three days, and by adding iodoacetic acid to inhibit alcohol dehydrogenase, the present invention achieves precise control of alcohol content to 3.8% ABV ± 0.1% ABV, ester content ≥ 75mg / L, and a flavor retention rate ≥ 95%, effectively retaining aroma compounds. Furthermore, β-glucanase is used to enzymatically hydrolyze the grape juice, optimizing its composition, improving fermentation efficiency, and enhancing flavor release, resulting in a low-alcohol wine with both a refreshing taste and rich aroma. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a process flow chart of the low-alcohol wine brewing method using a dual-bacteria system with low-temperature segmented fermentation in the present invention.

[0017] Figure 2 This is a trend diagram of ester synthase activity changing with temperature. DETAILED DESCRIPTION

[0018] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the specific implementation of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0019] Unless otherwise specified, the methods described in the embodiments of the present invention are conventional methods. The materials and reagents used are all commercially available unless otherwise specified.

[0020] In the prior art, alcohol in wine is usually removed by various methods after the wine is finished to reduce the alcohol content. However, the above methods have the problems of reduced aroma components, complex preparation process and high energy consumption.

[0021] The embodiment of the present invention uses enzyme treatment during the wine fermentation process and constructs a fermentation system of thermotolerant Kluyveromyces and Pichia Kluyveromyces, thereby not only accurately controlling the alcohol content but also improving the retention of aroma under low-temperature staged fermentation.

[0022] Specifically, the grape juice is enzymatically hydrolyzed using β-glucanase to optimize its composition, improve the efficiency of fermentation to generate thermotolerant Kluyveromyces and Pichia Kluyveromyces, and enhance the release of flavor compounds. Furthermore, after enzymatic hydrolysis is complete, the effects of nitrogen gas flow are as follows:

[0023] 1) It inhibits early alcoholic fermentation in yeast and redirects metabolism: It blocks the transition from aerobic respiration to alcoholic fermentation. Initial dissolved oxygen in grape juice is typically 6mg / L-8mg / L. Sufficient oxygen encourages yeast to prioritize aerobic respiration for growth, rather than acid production or flavor synthesis. Reducing the dissolved oxygen (DO) to below 0.1mg / L through nitrogen replacement forces the yeast into a near-anaerobic state, inhibiting alcohol dehydrogenase activity and causing the thermotolerant Kluyveromyces yeast to preferentially consume 30%-40% of glucose to produce lactic acid, ultimately reaching 3.8g / L. This creates an acidic pH of 3.4 for the Pichia Kluyveromyces yeast, rather than converting it to alcohol. This prevents premature alcohol production and flavor loss. High dissolved oxygen levels can cause early alcohol production by yeast to destroy terpene aroma precursors such as linalool in the grape juice. By controlling oxygen levels, flavor retention is maintained at ≥95%, a significant improvement over traditional high-oxygen fermentation (60%).

[0024] 2) Preventing oxidation and protecting natural flavor components: The system inhibits the oxidation of phenolic compounds. Under aerobic conditions, polyphenol oxidase (PPO) in grape juice catalyzes the oxidation of phenols such as catechins, leading to browning and a bitter taste. Nitrogen aeration reduces PPO activity by over 70%, maintaining juice clarity and the natural fruity aromas of Cabernet Sauvignon grapes, such as black currant. It also reduces the degradation of vitamins and aroma precursors. Dissolved oxygen accelerates the degradation of vitamin C and carotenoids and promotes the oxidation of unsaturated fatty acids to produce undesirable flavor compounds such as hexanal. Terpene content increased by 38% after oxygen control compared to the uncontrolled group, confirming its protective effect on aroma precursors.

[0025] 3) Optimize fermentation start-up conditions and improve stability: Inhibit bacterial contamination. Oxygen is essential for the growth of aerobic bacteria such as acetic acid bacteria. A DO of <0.1 mg / L can control the bacterial contamination rate to <1%, significantly lower than the traditional uncontrolled oxygen process (contamination rate of 10%-15%), and prevent fermentation broth rancidity (acetic acid content >1.5 g / L). This also promotes synchronized yeast growth. Under low oxygen conditions, thermotolerant Kluyveromyces yeast synthesizes heat shock protein (HSP70), enhancing its activity and stability during subsequent low-temperature fermentation at 16°C-18°C. In Example 1, yeast entered the logarithmic growth phase within 48 hours after inoculation, 24 hours shorter than the uncontrolled oxygen control group.

[0026] 4) Scientific Basis for Process Parameters: Nitrogen aeration at a flow rate of 0.5 L / min for 30 minutes can reduce the DO (dosage rate) of 1 L of grape juice from 8 mg / L to 0.05-0.1 mg / L (Henry's Law states that oxygen solubility is higher at temperatures as low as 17°C, requiring longer aeration times). The dissolved oxygen control standard, DO < 0.1 mg / L, is the industry threshold for low-temperature wine fermentation (see "Enology," p. 127). Values ​​above this can easily lead to uncontrolled alcohol production (>0.3% ABV / day). Values ​​below this limit require post-aging filtration to prevent yeast autolysis.

[0027] During the fermentation process, a composite bacterial system enables precise regulation of sugar metabolism. Thermotolerant Kluyveromyces prioritizes glucose consumption for growth and acid production, reducing the conversion of sugars into alcohol precursors. Pichia Kluyveromyces, on the other hand, utilizes most of the glucose to synthesize flavor compounds such as esters and higher alcohols, with only a small amount converted into alcohol. A low-temperature, staged fermentation process, combined with rapid cooling to terminate fermentation, allows precise control of alcohol content to 4% ABV. This composite bacterial system and low-temperature fermentation process maximize the preservation of the wine's aroma and flavor. Complex physical or chemical treatments are avoided, reducing equipment investment and operating costs.

[0028] The following is a detailed description of a low-alcohol wine brewing method using a dual-bacteria system with low-temperature segmented fermentation.

[0029] In the following examples, the grape juice was pressed from Cabernet Sauvignon grapes with a sugar content of 22° Brix. β-glucanase, with an activity of no less than 1000 U / g, was purchased from the College of Enology, Northwest Agriculture and Forestry University. Thermotolerant Kluyveromyces has a deposit number of CGMCC No. 21714 and is disclosed in Chinese patent publication number CN113667611B, entitled "Thermotolerant Kluyveromyces Strains and Their Applications." The following literature discloses Pichia Kluyveromyces: "Analysis of the Flavor-Enhancing Potential of Wine Fermented with Mixed Fermentation of Native Pichia Kluyveromyces and Saccharomyces Cerevisiae" by Zhang Wenjing, Yang Shini, Du Shuang, Jiang Jiao, Ye Dongqing, and Liu Yanlin; Food Science, 2020, Vol. 41, No. 12 (84-90).

[0030] Example 1 A low-alcohol wine brewing method using a dual-bacteria system with low-temperature segmented fermentation, such as Figure 1 As shown, the following steps are included: S1. Select Cabernet Sauvignon grapes with a sugar content of 22°Brix and squeeze them to obtain grape juice. Add 0.15% β-glucanase by weight of the grape juice and perform enzymatic hydrolysis in a 48°C constant temperature water bath at 300 rpm for 1.5 hours. During this period, the sugar content is monitored and controlled to 17°Brix using an online sugar meter. Subsequently, cool the juice to 17°C at a rate of 10°C / min using a plate and frame heat exchanger (heat exchange efficiency 92%) to obtain the enzymatically hydrolyzed grape juice.

[0031] The above enzymatic hydrolysis conditions were used in the medium-scale production stage. A continuous enzymatic hydrolysis reactor was used with a processing capacity of 500 L / h. The alcohol content, ester substances and residual sugar content were measured. The results are shown in Table 1.

[0032] Table 1 Key indicators of enzymatic hydrolysis results under different environments From the results in Table 1, it can be seen that the enzymatic hydrolysis conditions in Example 1 are used in an enzymatic hydrolysis reactor for medium-scale production, and the alcohol content, ester substances and residual sugar content fluctuate little, and the ester substances and residual sugar content are also improved to a certain extent. It can be seen that the enzymatic hydrolysis conditions in the present invention are suitable for large-scale industrial production.

[0033] S2. Sterile nitrogen was introduced into the enzymatically hydrolyzed grape juice at a rate of 0.5 L / min, and the dissolved oxygen content (DO) was maintained at <0.1 mg / L. The thermotolerant Kluyveromyces was then inoculated at a rate of 0.8 g / L. The juice was fermented at a constant temperature of 17°C for 4 days. During this period, the sugar content was tested daily until it reached 13°Brix. The lactic acid content of the fermentation liquid reached 3.8 g / L, and the pH dropped to 3.4. At this time, Pichia Kluyveromyces was inoculated at a rate of 1.2 g / L. The juice was maintained at 17°C for the first 5 days and then cooled to 15°C at a rate of 0.67°C / day for the next 3 days. A mixture of 0.5 mM iodoacetic acid and 0.5 mM EDTA in a volume ratio of 1:1.5 was added to inhibit the activity of alcohol dehydrogenase. When the alcohol content reached 3.8% ABV, the juice was rapidly cooled to 0°C to terminate the fermentation, thereby obtaining the fermented grape juice. The fermented grape juice was clarified, filtered through a 0.45 μm membrane, and aged at 10°C for 3 months to obtain low-alcohol wine.

[0034] The fermentation temperature determination process of the thermotolerant Kluyveromyces and Pichia Kluyveromyces is as follows.

[0035] 1) Determination of the overall fermentation temperature range The ester synthase activity, alcohol production rate and ADH inhibition rate at different fermentation temperatures were measured, and the results are shown in Table 2.

[0036] Table 2 Effect of fermentation temperature on ester synthase activity and alcohol production rate Note: ADH stands for alcohol dehydrogenase.

[0037] From the results in Table 2, it can be seen that when the temperature is between 15℃ and 18℃, the ester synthase activity is the highest, the alcohol production rate is low, and the ADH inhibition rate is good. Therefore, the fermentation temperature of 15℃ to 18℃ is selected for fermentation.

[0038] 2) Determination of Pichia Kluyveromyces fermentation temperature The relative expression level of ATF1 gene mRNA in Pichia Kluyveromyces is 2.3 times that of ordinary strains. It can convert 60%~70% of glucose into esters (ethyl acetate ≥70mg / L) and higher alcohols (phenylethanol ≥45mg / L), and only 3%~5% into alcohol. Its ester synthase activity reaches its peak at 17℃. Figure 2 As shown, the fermentation temperature of Pichia Kluyveromyces was selected to be 17°C for fermentation to achieve the best ester synthase activity. Combined with the results in Table 1, the subsequent cooling to 15°C can increase the ADH inhibition rate, thereby achieving the purpose of effectively reducing the alcohol content while improving the flavor.

[0039] 3) Determination of fermentation temperature control Stage 1 (16°C-18°C, 3-5 days): Thermotolerant Kluyveromyces yeast dominates the fermentation process. At 16°C-18°C, this strain can efficiently consume 30%-40% of the glucose in the grape juice. By activating the lactate dehydrogenase (LDH) metabolic pathway, it preferentially converts glucose into lactic acid, ultimately producing 3.8g / L-4.2g / L of acid. This rapidly establishes an acidic metabolic environment, lowering the pH to 3.4-3.6, inhibiting the growth of other bacteria and creating a metabolic competitive advantage, laying the foundation for subsequent fermentation.

[0040] Phase 2 (17°C for the first 5 days, followed by a gradual cooling to 15°C over the next 3 days): Pichia Kluyveromyces fermentation continued, with 0.5 mM iodoacetic acid used to inhibit alcohol dehydrogenase. Experimental verification: At 17°C, BCA protein quantification combined with colorimetry revealed that the activity of Pichia Kluyveromyces ester synthase (ATF1) reached 2.1 U / mg protein. At this temperature, ester synthase gene expression is upregulated, driving 60%–70% of glucose into ester synthesis, resulting in ethyl acetate production ≥75 mg / L. Over the next 3 days, the temperature was lowered at a rate of 0.67°C / day to 15°C. This low temperature inhibited ADH activity by over 70%. Enzyme kinetics, using nicotinamide adenine dinucleotide (NAD) consumption as an indicator, maintained the alcohol production rate at ≤0.3% ABV / day.

[0041] Termination (0°C Rapid Cooling): When the alcohol content reaches 4% ABV ± 0.2%, fermentation is terminated using 0°C rapid cooling. Residual Sugar Verification: HPLC analysis shows that the unfermented residual sugar in the fermentation broth is maintained at 3g / L-5g / L. This residual sugar enhances the sweetness of the wine while promoting the retention of fruity aromas such as terpenes and phenylethyl alcohol. Sensory evaluation shows that the fruity aroma intensity is 30%-40% higher than the no-residual-sugar control, achieving a synergistic effect of precise alcohol content and flavor optimization.

[0042] By controlling the fermentation temperature and refining the fermentation temperature of the inoculated bacteria at different stages, a segmented fermentation process has been developed, which not only reduces the alcohol content but also optimizes the flavor, resulting in a low-alcohol wine with rich fruit aroma and excellent flavor.

[0043] Example 2 A low-alcohol wine brewing method using a dual-bacteria system with low-temperature segmented fermentation comprises the following steps: S1. Select Cabernet Sauvignon grapes with a sugar content of 22°Brix and squeeze them to obtain grape juice. Add 0.2% β-glucanase by weight of the grape juice, and perform enzymatic hydrolysis in a 50°C constant temperature water bath at 300 rpm with stirring for 2 h. During this period, the sugar content is monitored and adjusted to 16°Brix using a handheld saccharimeter. Then, use a plate and frame heat exchanger to cool to 18°C ​​at a rate of 10°C / min to obtain the enzymatically hydrolyzed grape juice.

[0044] S2. Sterile nitrogen was introduced into the enzymatically hydrolyzed grape juice at a rate of 0.5 L / min, and the dissolved oxygen content (DO) was maintained at <0.1 mg / L. The thermotolerant Kluyveromyces yeast was first inoculated at a rate of 1.2 g / L. The fermentation was carried out at a constant temperature of 17°C for 4 days. During this period, the sugar content was tested daily until it reached 12°Brix. When the lactic acid content of the fermentation liquid reached 4.0 g / L and the pH dropped to 3.2, Pichia Kluyveromyces yeast was inoculated at a rate of 1.8 g / L. The temperature was maintained at 17°C for the first 5 days and then cooled to 15°C at a rate of 0.67°C / day for the next 3 days. A mixture of 50 mg / L potassium metabisulfite and 0.1 mM EDTA in a volume ratio of 1:1.5 was added to inhibit the activity of alcohol dehydrogenase. When the alcohol content reached 4.0% ABV, the fermentation was terminated by rapidly cooling to 0°C to obtain the fermented grape juice. The fermented grape juice was clarified and filtered through a 0.45 μm membrane, and aged at 10°C for 3 months to obtain low-alcohol wine.

[0045] Comparative Example 1 A low-alcohol wine brewing method using a dual-bacteria system with low-temperature staged fermentation, which is the same as Example 1 except that β-glucanase is replaced by pectinase, specifically comprising the following steps: S1. Select Cabernet Sauvignon grapes with a sugar content of 22°Brix and squeeze them to obtain grape juice. Pectinase is added at a concentration of 0.2% by weight of the grape juice. The juice is then enzymatically hydrolyzed in a 50°C water bath at 300 rpm for 2 hours while stirring. During this time, the sugar content is monitored and controlled to 16°Brix using an online saccharimeter. The juice is then cooled to 18°C ​​at a rate of 10°C / min using a plate and frame heat exchanger (heat exchange efficiency 92%) to obtain the enzymatically hydrolyzed grape juice.

[0046] S2. Sterile nitrogen is introduced into the enzymatically hydrolyzed grape juice at a rate of 0.5 L / min, and the dissolved oxygen (DO) is maintained at <0.1 mg / L. The thermotolerant Kluyveromyces yeast is first inoculated at a rate of 0.8 g / L. The fermentation is carried out at a constant temperature of 17°C for 4 days. During this period, the sugar content is tested daily until the fermentation liquid reaches 13°Brix. When the lactic acid content reaches 3.8 g / L and the pH drops to 3.4, the fermentation liquid is inoculated with Pichia Kluyveromyces yeast at a rate of 1.2 g / L. The temperature is maintained at 17°C for the first 5 days and then cooled to 15°C at a rate of 0.67°C / day for the next 3 days. A mixture of 0.5 mM iodoacetic acid and 0.5 mM EDTA is added to inhibit the activity of alcohol dehydrogenase. When the alcohol content reaches 3.8% ABV, the fermentation is terminated by rapidly cooling to 0°C to obtain the fermented grape juice. The fermented grape juice was clarified, filtered through a 0.45 μm membrane, and aged at 10°C for 3 months to obtain low-alcohol wine.

[0047] Comparative Example 2 A low-alcohol wine brewing method using a dual-bacteria system with low-temperature segmented fermentation, wherein the steps are the same as those in Example 1 except that the order of adding thermotolerant Kluyveromyces and Pichia Kluyveromyces is reversed. Specifically, the method comprises the following steps: S1. Select Cabernet Sauvignon grapes with a sugar content of 22°Brix and squeeze to obtain grape juice. Add 0.15% β-glucanase by weight of the grape juice, and perform enzymatic hydrolysis in a constant temperature water bath at 48°C and 300 rpm for 1.5 hours. During this period, the sugar content is monitored and regulated by an online sugar meter to 17°Brix to obtain the enzymatically hydrolyzed grape juice. Subsequently, cool the juice to 17°C at a rate of 10°C / min using a plate and frame heat exchanger (heat exchange efficiency 92%) to obtain the enzymatically hydrolyzed grape juice.

[0048] S2. Sterile nitrogen is introduced into the enzymatically hydrolyzed grape juice at a rate of 0.5 L / min, and the dissolved oxygen (DO) is maintained at <0.1 mg / L. The yeast Pichia is first inoculated, wherein the inoculum amount of the yeast Pichia is 1.2 g / L. The juice is maintained at 17°C for the first 5 days, and then cooled to 15°C at a rate of 0.67°C / day for the next 3 days. A mixture of 0.5 mM iodoacetic acid and 0.5 mM EDTA is added to inhibit the activity of alcohol dehydrogenase. After the sugar content is reduced to 14°Brix, the yeast Thermotolerant Kluyveromyces is inoculated, wherein the inoculum amount of the thermotolerant Kluyveromyces is 0.8 g / L. The juice is fermented at a constant temperature of 17°C for 4 days. During this period, the sugar content is tested daily. When it reaches 13°Brix, the juice is rapidly cooled to 0°C to terminate the fermentation, thereby obtaining the fermented grape juice. The fermented grape juice was clarified and filtered through a 0.45 μm membrane, and aged at 10°C for 3 months to obtain low-alcohol wine.

[0049] Comparative Example 3 A low-alcohol wine brewing method using a dual-bacteria system with low-temperature segmented fermentation is the same as that in Example 1 except that Pichia Kluyveromyces is not inoculated. Specifically, the following steps are included: S1. Select Cabernet Sauvignon grapes with a sugar content of 22°Brix and squeeze to obtain grape juice. Add 0.15% β-glucanase by weight of the grape juice, and perform enzymatic hydrolysis in a constant temperature water bath at 48°C and 300 rpm for 1.5 hours. During this period, the sugar content is monitored and regulated by an online sugar meter to 17°Brix to obtain the enzymatically hydrolyzed grape juice. Subsequently, cool the juice to 17°C at a rate of 10°C / min using a plate and frame heat exchanger (heat exchange efficiency 92%) to obtain the enzymatically hydrolyzed grape juice.

[0050] S2. Sterile nitrogen was introduced into the enzymatically hydrolyzed grape juice at a rate of 0.5 L / min while maintaining the dissolved oxygen (DO) less than 0.1 mg / L. The thermotolerant Kluyveromyces yeast was inoculated at an inoculum size of 0.8 g / L. The juice was fermented at a constant temperature of 17°C for 4 days. During this period, the sugar content was tested daily. When the sugar content reached 13°Brix, the juice was rapidly cooled to 0°C to terminate the fermentation, thereby obtaining the fermented grape juice. The fermented grape juice was clarified and filtered through a 0.45 μm membrane, and then aged at 10° C. for 3 months to obtain a low-alcohol wine.

[0051] Comparative Example 4 A low-alcohol wine brewing method using a dual-bacteria system with low-temperature segmented fermentation is the same as Example 1 except that the thermotolerant Kluyveromyces yeast is not inoculated. Specifically, the following steps are included: S1. Select Cabernet Sauvignon grapes with a sugar content of 22°Brix and squeeze them to obtain grape juice. Add 0.15% β-glucanase by weight of the grape juice and perform enzymatic hydrolysis in a 48°C constant temperature water bath at 300 rpm for 1.5 hours. During this period, the sugar content is monitored and controlled by an online sugar meter to 17°Brix. Then, cool the juice to 17°C at a rate of 10°C / min using a plate and frame heat exchanger (heat exchange efficiency 92%) to obtain the enzymatically hydrolyzed grape juice.

[0052] S2. Sterile nitrogen is introduced into the enzymatically hydrolyzed grape juice at a rate of 0.5 L / min, and the dissolved oxygen (DO) is maintained at less than 0.1 mg / L. Then, the yeast Pichia Kluyveromyces is inoculated at an inoculum rate of 1.2 g / L. The temperature is maintained at 17° C. for the first five days, and then cooled to 15° C. at a rate of 0.67° C. / day for the next three days. A mixture of 0.5 mM iodoacetic acid and 0.5 mM EDTA is added to inhibit the activity of alcohol dehydrogenase. When the alcohol content reaches 3.8% ABV, the fermentation is terminated by rapidly cooling the juice to 0° C., thereby obtaining the fermented grape juice. The fermented grape juice was clarified and filtered through a 0.45 μm membrane, and aged at 10°C for 3 months to obtain low-alcohol wine.

[0053] Comparative Example 5 A low-alcohol wine brewing method using a dual-bacteria system with low-temperature segmented fermentation, except that β-glucanase is replaced by pectinase, is the same as that of Comparative Example 4, comprising the following steps: S1. Select Cabernet Sauvignon grapes with a sugar content of 22°Brix and squeeze them to obtain grape juice. Add 0.15% pectinase by weight of the grape juice and stir in a constant temperature water bath at 300 rpm for enzymatic hydrolysis for 1.5 hours. During this period, the sugar content is monitored and regulated by an online sugar meter to 17°Brix to obtain the enzymatically hydrolyzed grape juice. Subsequently, use a plate and frame heat exchanger (heat exchange efficiency 92%) at a rate of 10°C / min to obtain the enzymatically hydrolyzed grape juice.

[0054] S2. Sterile nitrogen is introduced into the enzymatically hydrolyzed grape juice at a rate of 0.5 L / min, and the dissolved oxygen (DO) is maintained at less than 0.1 mg / L. Then, the yeast Pichia Kluyveromyces is inoculated at an inoculum rate of 1.2 g / L. The temperature is maintained at 17° C. for the first five days, and then cooled to 15° C. at a rate of 0.67° C. / day for the next three days. A mixture of 0.5 mM iodoacetic acid and 0.5 mM EDTA is added to inhibit the activity of alcohol dehydrogenase. When the alcohol content reaches 3.8% ABV, the fermentation is terminated by rapidly cooling the juice to 0° C., thereby obtaining the fermented grape juice. The fermented grape juice was clarified and filtered through a 0.45 μm membrane, and aged at 10°C for 3 months to obtain low-alcohol wine.

[0055] Both Example 1 and Example 2 prepared low-alcohol wine with comparable effects. The following experiment was conducted using the low-alcohol wine prepared in Example 1 as an example.

[0056] 1. Grape juice enzymatic hydrolysis optimization, the specific process is as follows.

[0057] β-glucanase hydrolysis: 0.15% (w / v) enzyme dosage, enzymatic hydrolysis at 48°C for 1.5 hours, degrading β-glucan by ≥80%, reducing juice viscosity by 1.8 mPa・s, releasing terpene aroma precursors, and increasing terpene content by 38%.

[0058] The specific process of the traditional process is as follows: Cabernet Sauvignon grapes with a sugar content of 22°Brix are selected and squeezed to obtain grape juice. Without adding β-glucanase, it is directly enzymatically hydrolyzed in a constant temperature water bath at 25°C, relying on the grape's own enzyme system. The sugar content is not monitored during the process, and the sugar content of the grape juice is naturally maintained above 20°Brix. Subsequently, there is no need to cool it, and air is directly introduced to obtain untreated grape juice.

[0059] The grape juice was inoculated with Saccharomyces cerevisiae ( S.cerevisiaeThe inoculum size was 1.0 g / L, and fermentation was performed at a constant temperature of 25°C without staged temperature control. When the sugar content dropped below 10°Brix, the alcohol content reached 6%-8% ABV. No alcohol dehydrogenase inhibitors were added during this period, and the alcohol production rate was approximately 0.5% ABV / day. At the end of the fermentation, the alcohol concentration inhibited yeast activity, leading to natural termination of fermentation without additional cooling. After coarse filtration, the wine was aged at 15°C for one month to obtain a traditional low-alcohol wine. The results are shown in Table 3.

[0060] Table 3 Effects of different enzymatic hydrolysis on grape juice 2. The alcohol content, lactic acid content, ethyl acetate content, and sensory scores of the low-alcohol wines in Example 1 and Comparative Examples 1 to 4 were statistically analyzed. The sensory scoring criteria are shown in Table 4, and the specific results are shown in Table 5.

[0061] Table 4 Sensory scoring standards Table 5 Results of low alcohol wine obtained by different methods The results in Table 1 show that after replacing β-glucanase with pectinase in Comparative Example 1, the resulting low-alcohol wine had an alcohol content of 4.5% ABV (standard deviation ±0.2%), which is higher than the 3.8±0.1% ABV in Example 1. This is primarily due to the fact that undegraded β-glucan results in a 15% increase in yeast sugar utilization, leading to increased alcohol production. The lactic acid content is 2.3 g / L, which is 45% lower than that in Example 1. This is primarily due to the fact that during the enzymatic hydrolysis of grape juice, undegraded β-glucan in the grape juice interacts with pectin, protein, and other substances to form a complex. This complex inhibits the growth of thermotolerant Kluyveromyces during fermentation. The ethyl acetate content is 42 mg / L, which is 44% lower than that in Example 1. This is primarily due to the fact that fermentation by thermotolerant Kluyveromyces is inhibited, resulting in an insufficient supply of precursors for ester synthesis by Pichia kluyveromyces. The alcohol content in Comparative Example 2 was 4.8% ABV (standard deviation ±0.3%), higher than that in Example 1. This was primarily due to the initial inoculation of Pichia Kluyveromyces, which eliminated acid inhibition in the early stages and accelerated alcohol production to 0.4% ABV / day, leading to a higher alcohol content later in the fermentation. The pH at the end of fermentation was 3.8, 0.4 higher than in Example 1. This was primarily due to the initial inoculation of Pichia Kluyveromyces, which eliminated acid production by thermotolerant yeasts. The initial pH of the grape juice was higher than in Example 1 and was not lowered by the thermotolerant yeasts. The relatively high pH environment was not conducive to the subsequent acid production of thermotolerant Kluyveromyces, resulting in low acid production. The ethyl acetate content was 65 mg / L, 13% lower than in Example 1. This was due to the reduced expression of the ATF1 gene caused by the insufficiently acidic environment, which in turn reduced the ability to convert glucose to ethyl acetate. In Comparative Example 3, due to the sole inoculation of Thermotolerant Kluyveromyces, fermentation stalled on day 8 when the sugar content dropped to 10°Brix. At this point, the pH reached 3.1, inhibiting the activity of Thermotolerant Kluyveromyces. The low ethyl acetate content, at only 28 mg / L, resulted in a monotonous flavor, with the lowest sensory score being only 65±2. In Comparative Example 4, with the sole inoculation of Pichia Kluyveromyces, the residual sugar content was 8 g / L and the lactic acid content was 1.8 g / L on day 15 of fermentation. This was primarily due to the inhibition of ester synthesis at a pH of 3.8, resulting in an ethyl acetate content of only 50 mg / L and a lack of acid production by Thermotolerant Kluyveromyces. This, in turn, resulted in an insufficient fermentation environment and metabolic support for the Pichia yeast, impacting the fermentation performance.

[0062] The process conditions in Example 1 and Comparative Examples 1 to 5 are summarized. Through the method of the present invention, the alcohol content can be effectively controlled to 3.8% ABV ± 0.1%, esters ≥ 70 mg / L, terpenes ≥ 58 μg / L, and the entire fermentation process is highly stable and fermentation will not stop. In the methods of Comparative Examples 1 to 5, due to the inoculation of a single strain or the replacement of β-glucanase with pectinase, the alcohol content will fluctuate, and the fluctuation range is greater than 0.3%, and the esters of the comparative example 1 group are <45 mg / L, and the terpenes are <45 μg / L, which is a significant decrease. Exchanging the inoculation order in comparative example 2 and replacing β-glucanase with pectinase in comparative example 1 will cause fermentation to stop, and the fermentation stagnation rate is 5% to 15%.

[0063] The present invention is based on the specific colloidal degradation of β-glucanase pretreatment and the sequential inoculation process of thermotolerant Kluyveromyces → Pichia Kluyveromyces. Through the triple mechanism of "viscosity regulation-acidity gradient change-metabolic redistribution", it achieves the synergistic optimization of 3.8% ABV low-alcohol wine alcohol content, 75 mg / L of flavor substance ethyl acetate and production stability, which improves the overall quality by 30% to 45% compared with other combinations, and has significant technical and economic advantages for industrial promotion.

[0064] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A low-alcohol wine brewing method using a dual-bacteria system with low-temperature segmented fermentation, characterized in that: The following steps are involved: performing enzymatic hydrolysis on the grape juice to reduce the sugar content to 16°Brix~18°Brix, thereby obtaining enzymatically hydrolyzed grape juice; After nitrogen is introduced into the enzymatically hydrolyzed grape juice until the dissolved oxygen content in the wine is less than 0.1 mg / L, thermotolerant Kluyveromyces is inoculated, and fermentation is carried out at 16°C to 18°C. When the sugar content reaches 12°Brix to 14°Brix, Pichia Kluyveromyces is inoculated to continue fermentation. When the alcohol content reaches 3%ABV to 4%ABV, the wine is cooled to 0°C to terminate the fermentation, thereby obtaining the fermented grape juice. clarifying, filtering, and aging the fermented grape juice to obtain the low-alcohol wine; The low-alcohol wine has an alcohol content of 3.7% ABV to 3.9% ABV.

2. The low-alcohol wine brewing method using a dual-bacteria system with low-temperature segmented fermentation according to claim 1, characterized in that: The specific process of Pichia Kluyveromyces fermentation is as follows: After 5 days of fermentation at 17°C, the temperature was lowered to 15°C within 3 days. During the cooling process, an alcohol dehydrogenase inhibitor was added to control the alcohol production rate to ≤ 0.3% ABV / day. The alcohol dehydrogenase inhibitor is a mixture of iodoacetic acid solution and EDTA solution or a mixture of potassium metabisulfite solution and EDTA solution; the volume ratio of iodoacetic acid solution, potassium metabisulfite solution and EDTA solution is 0.8-1.2:1.5; The concentrations of the iodoacetic acid solution and the potassium metabisulfite solution are both 50 mg / L to 100 mg / L, and the concentration of the EDTA solution is 0.1 mM to 0.5 mM.

3. The low-alcohol wine brewing method using a dual-bacteria system with low-temperature segmented fermentation according to claim 2, characterized in that: The temperature was lowered to 15°C at a rate of 0.67°C / day to 1°C / day.

4. The low-alcohol wine brewing method using a dual-bacteria system with low-temperature segmented fermentation according to claim 1, characterized in that: The inoculation amount of the thermotolerant Kluyveromyces yeast per liter of grape juice is 0.8 g to 1.2 g; The inoculation amount of the Pichia Kluyveromyces is 1.2 g to 1.8 g per liter of grape juice.

5. The low-alcohol wine brewing method using a dual-bacteria system with low-temperature segmented fermentation according to claim 1, characterized in that: The specific process of the enzymatic hydrolysis is as follows: Add β-glucanase to the grape juice and perform enzymatic hydrolysis at 45℃~50℃ for 1h~2h.

6. The low-alcohol wine brewing method using a dual-bacteria system with low-temperature segmented fermentation according to claim 5, characterized in that: The added amount of the β-glucanase is 0.1% to 0.2% of the mass of the grape juice.

7. The low-alcohol wine brewing method using a dual-bacteria system with low-temperature segmented fermentation according to claim 1, characterized in that: The flow rate of nitrogen is 0.5L / min~0.6L / min.

8. The low-alcohol wine brewing method using a dual-bacteria system with low-temperature segmented fermentation according to claim 1, characterized in that: Before inoculating the heat-resistant Kluyveromyces yeast, the enzymatically hydrolyzed grape juice is cooled to 16°C~18°C.

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