Method for optimizing Chinese yeast through response surface based on temperature control program

Abstract

The invention discloses a Chinese yeast based on a temperature control program and a preparation method thereof, and belongs to the technical field of Chinese yeast brewing. According to the Chinese yeast fermentation process obtained through response surface method optimization (verification is carried out by taking dregs vinegar as an example), the yield of key products is remarkably increased, and a standardized temperature control program is introduced; reliable process basis and theoretical basis are provided for transformation and upgrading of various traditional fermented products including dregs vinegar, table vinegar, yellow wine and the like from traditional workshop type production to standardization and industrialization.

Description

Technical Field

[0001] This invention relates to a method for optimizing wine yeast based on a temperature control program response surface methodology, belonging to the field of wine yeast brewing technology. Background Technology

[0002] Yeast is the starting agent for many traditional fermented products, such as rice wine, vinegar, and huangjiu (yellow wine). Rice wine, originating from Hainan, is a traditional acidic fermented condiment that has seen significant market performance in recent years due to its unique flavor, particularly in hot pot. This product uses rice as its raw material and relies on traditional yeast for multi-stage fermentation, resulting in a refreshingly sour and aromatic product rich in various beneficial components. Current research largely focuses on the microbial community and flavor compounds in the later stages of rice wine fermentation. Compared to huangjiu and rice wine, there is less systematic research on yeast, the starting agent for rice wine. Traditional yeast uses natural fermentation, which suffers from unstable microbial communities, reliance on experience in the process, and significant batch-to-batch variations, severely hindering standardization and industrialization of production. Therefore, optimizing and establishing a standardized yeast fermentation process is crucial for improving product consistency and promoting the transformation of traditional processes towards industrialization. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing and controlling the temperature of the yeast, which makes the yeast cultivation conditions simple, easy to control, and easy to operate. Using this yeast to brew fermented products can standardize the process, effectively increase the total acid and ethanol content of the fermented products, retain the traditional flavor and taste, and help increase the market share of related products.

[0004] The first technical solution provided by this invention is a method for constructing wine-making agents using response surface methodology, comprising the following steps: S1. Single-factor experiments were used to determine the influencing factors and their levels. Among them, the factors were water addition, mixing water temperature, MQ (mother starter) addition, herbal addition, and FX (high enzyme active wine yeast) addition. The herbal ingredients consisted of nine-knot thorn and purple jade plate in a 1:1 ratio. Based on the single-factor experimental results of S1, an experimental design using the Plackett-Burman model was conducted. The independent variables were water addition (A), mixing water temperature (B), MQ addition amount (C), herbal addition amount (D), and FX addition amount (E), and the response values ​​were total acid (Y1) and ethanol (Y2). The three factors that significantly affected total acid and ethanol were obtained, namely mixing water temperature (X1), MQ addition amount (X2), and FX addition amount (X3). Based on the Plackett-Burman test results of S2, the Box-Behnken model was used for experimental design. The mixing water temperature (X1), MQ addition amount (X2), and FX addition amount (X3) were used as independent variables, and the comprehensive score of total acid and ethanol OD (Y) was used as the response value. Response surface analysis was conducted to obtain the optimal parameters for the preparation of the wine-making agent.

[0005] In some embodiments, in S1, the amount of water added is selected as 50%, 52.5%, 55%, 57.5%, or 60%, based on the amount of rice flour added.

[0006] In some embodiments, in S1, the mixing water temperature is selected as 40℃, 45℃, 50℃, 55℃, or 60℃, based on the amount of rice flour added.

[0007] In some embodiments, in S1, based on the amount of indica rice flour added, the amount of MQ added is selected as 0.5%, 1%, 1.5%, 2%, or 2.5%.

[0008] In some embodiments S1, the amount of herbal medicine added is selected as 0%, 0.25%, 0.5%, 0.75%, or 1%, based on the amount of rice flour added.

[0009] In some embodiments, in S1, based on the amount of rice flour added, the amount of FX added is selected as 0.5%, 1%, 1.5%, 2%, or 2.5%.

[0010] In some embodiments, in S2, the quadratic polynomial regression equation for total acid (Y1) is as follows: Y1 = 9.16 + 0.4426A + 0.01B + 0.8440C - 0.0731D + 0.6253E + 1.31BE; and the quadratic polynomial regression equation for ethanol (Y2) is as follows: Y2 = 1.5 + 0.0625A - 0.0167B + 0.0875C - 0.0500D + 0.1917E + 0.3125BE.

[0011] In some implementations, in S3, the quadratic polynomial regression equation of mixing water temperature (X1), MQ addition amount (X2), and FX addition amount (X3) on OD(Y) is as follows: Y=0.7898-0.0666X1-0.0619X2+0.0195X3+0.0257X1X2-0.0822X1X3-0.0522X2X3-0.1171X1 2 -0.3065X2 2 -0.1805X3 2 ; The established comprehensive scoring formula is as follows: ; ; in: For each indicator, the original value, , For each index, the maximum and minimum values ​​are given in its response surface experimental group. , These are the normalized values ​​for total acid and ethanol production, respectively.

[0012] In some implementations, the optimal parameters in S3 are: mixing water temperature 48°C, MQ addition amount 1.9%, and FX addition amount 1.6%.

[0013] This invention optimizes the response surface methodology of fermentation starter culture based on a temperature control program. This approach not only establishes a standardized production process, addressing the insufficient industrialization of fermentation starter culture, but also ensures the quality of the fermented product and the starter culture itself, thus providing technical support. It is hoped that this research will provide reference and support for improving the development and utilization technology of fermentation starter culture, and offer a reference basis for its industrial production. By controlling the mixing water temperature, MQ (methyl methacrylate) addition amount, and FX (fermented xylene) addition amount within specific values, this invention improves the quality of the fermentation starter culture, which is of great significance for promoting the transformation of fermentation starter culture technology and industrial development.

[0014] The second technical solution provided by this invention is a method for preparing a wine yeast. The method involves adding 0.25% herbal ingredients, 1.9% mother koji, 1.6% high-enzyme-active wine yeast, and 55% 48°C warm water, based on the amount of rice flour added. The mixture is stirred evenly, formed into pellets, rolled into rounds, and then fermented at a controlled temperature for 48 hours to obtain a wine yeast with high total acid and ethanol production. The herbal ingredients include *Nine-knot Thorn* and *Purple Jade Plate*. *Nine-knot Thorn*, also known as *Jiuji Thorn*, *Dongfeng Ju*, *Jiubing Le*, *Xiaoji Cao*, or *Cier Cai*, grows in Hainan. *Purple Jade Plate*, also known as *Jiupo Bing*, *Youzhui*, or *Jiubing Mu*, are characteristic herbs of Hainan wine yeast.

[0015] In some implementations, the temperature control process is divided into four stages: Phase I (Fermentation Start-up and Adaptation Period, 1-11h): In the early stage of this phase (1-4h), the yeast starts fermentation at room temperature, and the temperature gradually rises to above 25℃. During this period, its surface is in a high humidity state of 92-98%. Subsequently (4-11h), the lid is opened and dehumidification is performed. The yeast continues to ferment, and the surface temperature rises to 30℃, while the surface humidity decreases slightly.

[0016] Phase II (Main Fermentation and Regulation Period, 11-28h): In this phase, the fermentation process is driven by finely controlling the surface temperature of the starter culture. After dehumidification, the lid is closed and fermentation continues with increased temperature (11-21h), controlling the surface temperature to not exceed 35℃ to facilitate mold growth. During this phase, moisture is transferred from the inside of the starter culture to the surface, and the surface humidity rises to about 95%. Subsequently (21-23h), the temperature is maintained at about 35℃. When the mycelium (white mold) on the surface of the starter culture grows vigorously, the starter culture is turned over. After turning over (23-27h), the surface temperature continues to rise to 35-37℃ and is maintained for 4h. Finally (27-28h), the lid is closed to maintain the surface temperature of the starter culture at 38-40℃ for a short period, and the surface humidity drops below 75% due to the temperature increase.

[0017] Phase III (Cooling and Stabilization Period, 28-48h): The core of this phase is cooling to end fermentation. At 28h, the surface temperature of the yeast is lowered to below 35℃ by opening the lid and ventilation, and its surface humidity recovers slightly; then until 48h, the surface temperature is stabilized below 30℃ by continuous ventilation, during which time the surface humidity is close to the indoor humidity.

[0018] Phase IV (drying period, 48-72h): The final stage is a 2-day drying period, during which the finished wine has a moisture content of 10-12%.

[0019] The third technical solution provided by the present invention is a wine-based medicine prepared using the method described in the second technical solution.

[0020] The fourth technical solution provided by the present invention is a method for preparing rice lees vinegar. The method involves steaming rice at a rice-to-water ratio of 1:1.8, cooling it, mixing it evenly with a small amount of water, spices, and the yeast described in the third technical solution, fermenting it at 30°C for 2 days, then adding an appropriate amount of rinsing water, and fermenting it at 40°C for 2 days to obtain the finished rice lees vinegar.

[0021] In some embodiments, the amount of the wine yeast added is 1%.

[0022] Nine-knot thorn, small thistle, or sorrel are all medicinal and edible herbs in traditional Chinese medicine. Purple jade plate is a raw material in the traditional production method of fermented vinegar. The yeast used in the fermentation process is not consumed directly; the fermented vinegar itself is consumed directly, and the yeast used only accounts for approximately 0.1% of the total composition and is classified as a traditional Chinese medicine.

[0023] The fourth technical solution provided by this invention is the application of the method described in the first technical solution, the method described in the second technical solution, or the wine yeast described in the third technical solution in brewing and fermentation products.

[0024] In some embodiments, the fermented food is vinegar, edible vinegar, or wine.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention systematically constructs and optimizes the solid-state fermentation process for winemaking. Through a combination of single-factor experiments, Plackett-Burman design, and Box-Behnken response surface methodology, the optimal process parameters, with total acidity and ethanol yield as comprehensive evaluation indicators (OD value), were determined to be: mixing water temperature 48℃, MQ addition 1.9%, and FX addition 1.6%. Under these conditions, the water addition and herbal medicine addition were fixed at 55% and 0.25%, respectively.

[0026] Verification experiments showed that the optimized process was stable and reliable, and the total acid and ethanol content of the fermented wine product were significantly higher than those of the original process, with a comprehensive OD value of 1.021 > 0.919 (model predicted value). This invention successfully constructed a programmed temperature control process suitable for wine fermentation, which effectively ensured the stability and reproducibility of the fermentation process between different batches, and significantly improved the consistency of product quality.

[0027] In summary, the wine fermentation process optimized by response surface methodology (using lees vinegar as an example) not only significantly increases the yield of key products, but also provides a reliable technological foundation and theoretical basis for the transformation and upgrading of various traditional fermented products, including lees vinegar, vinegar, and rice wine, from traditional workshop-style production to standardized and industrialized production by introducing standardized temperature control procedures. Attached Figure Description

[0028] Figure 1 The effect of different amounts of water added (A) on the quality of the wine yeast (using total acid and ethanol as evaluation indicators); Figure 2 The effect of different mixing water temperatures (B) on the quality of the brewing agent (using total acid and ethanol as evaluation indicators); Figure 3 The effect of different MQ addition amounts (C) on the quality of the wine yeast (using total acid and ethanol as evaluation indicators); Figure 4 The effect of different amounts of added herbs (D) on the quality of the wine (using total acid and ethanol as evaluation indicators); Figure 5 The effect of different FX addition amounts (E) on the quality of the wine yeast (using total acid and ethanol as evaluation indicators); Figure 6 Temperature variation graphs of the fermentation process based on temperature control program for different amounts of water (A), mixing water temperature (B), MQ addition amount (C), herbal addition amount (D), and FX addition amount (E); Figure 7 Fill-in-the-area plot of temperature changes during fermentation based on a temperature-controlled program for the Plackett-Burman experimental group; Figure 8A filled-area plot showing the temperature changes during the fermentation process based on a temperature-controlled program in the Box-Behnken model experimental group; Figure 9 To verify the temperature change graph of the fermentation process based on the temperature control program; Figure 10 The temperature change graph is shown for the fermentation process based on the temperature control program of the original process (comparative verification experiment). Figure 11 A three-dimensional response surface plot of the interaction between mixing water temperature (X1) and MQ addition amount (X2); Figure 12 A three-dimensional response surface plot of the interaction between mixing water temperature (X1) and FX addition amount (X3); Figure 13 A three-dimensional response surface plot of the interaction between the added amount (X2) to MQ and the added amount (X3) to FX. Detailed Implementation

[0029] Reference Appendix Figures 1-13 The preferred embodiments of the present invention will be described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0030] Test method: Total acidity: Using an automatic titrator, referring to GB / T 13662-2018 Yellow rice wine, the supernatant of the original liquid was diluted 10 times, and 10 mL of the diluted solution was mixed with 60 mL of deionized water and then transferred into the sample cup of the automatic titrator for testing.

[0031] Ethanol: The biosensor method was used for determination. 1 mL of the supernatant of the original solution was placed in a colorimetric tube, 20 mL of deionized water was added first, the pH range was adjusted to 6-8, and the volume was adjusted to 50 mL. The solution was then transferred to the biosensor detection tube for detection.

[0032] Raw materials used in the examples: Distillery starter, Chinese medicinal herbs *Nine-knot Thorn*, and *Purple Jade Plate*, from Anding Awao Food Technology Co., Ltd. FX high-enzyme active wine starter, from Fangxian Yellow Wine Starter Factory. Indica rice flour, from Wuhu Fengxi Food Co., Ltd. Japonica glutinous rice, from Wuxi Xinmishi. Star anise and cinnamon, from Cangzhou Huahai Shunda Grain and Oil Seasoning Co., Ltd. Atractylodes macrocephala, from Lixing County Jiushuo Agricultural Development Co., Ltd.

[0033] Example 1: Preparation of Distilled Rice Wine (1) The specific process for preparing the lees vinegar wine yeast is as follows: ① Weigh out 94% rice flour, 4 / 5 of the starter culture powder, and herbs according to the proportions in Table 1, and mix them evenly in a mixer. ② While mixing, slowly add an appropriate amount of warm water and mix for 5 minutes. During this process, the material temperature rises, and the starch slightly gelatinizes, increasing viscosity. ③ Pour the mixed material into a starter culture forming machine and select the best-shaped balls. ④ Place the balls in a rounding machine to round them, and evenly sprinkle the remaining 6% rice flour and 1 / 5 of the starter culture powder on the surface of the balls. ⑤ Line the starter culture box with straw mats, spread the starter culture balls evenly in the box, ensuring appropriate spacing between each ball, and measure the fermentation temperature between the balls. ⑥ Ferment for 48 hours, controlling the temperature as closely as possible to Table 2. ⑦ At the end of fermentation, place the balls in an oven at 38 degrees Celsius and dry for 1-2 days.

[0034] Table 1 Ingredients for Wine Yeast and Distillers' Vinegar

[0035] Table 2. Temperature Program Control for Fermentation of Wine Yeast

[0036] (2) The specific process for brewing vinegar from fermented grains using yeast is as follows: ① Cooking: The ratio of japonica rice to steaming water is 1:1.8. Steam the rice in a rice steamer for 30 minutes. Ensure the rice is cooked through but not mushy, and has no hard center. ② Cooling: Spread the cooked rice out and let it cool to room temperature. Weigh an appropriate amount of rice into a 2000ml fermentation cup. ③ Mixing: Weigh out the spices, yeast, water, etc., according to the proportions in Table 1, and mix well. ④ Pre-fermentation: Seal with plastic wrap and place in a 30℃ fermentation chamber for 2 days. ⑤ Post-fermentation: Add rinsing water, seal with plastic wrap, and change the fermentation chamber temperature to 40℃. Ferment for 2 days. ⑥ Sterilization: Treat in a water bath at 70℃ for 30 minutes. ⑦ Measure the acidity and ethanol content of the finished product.

[0037] (3) Determination of total acid and ethanol Total acidity: Using an automatic titrator, referring to GB / T 13662-2018 Yellow rice wine, the supernatant of the original liquid was diluted 10 times, and 10 mL of the diluted solution was mixed with 60 mL of deionized water and then transferred into the sample cup of the automatic titrator for testing.

[0038] Ethanol: The biosensor method was used for determination. 1 mL of the supernatant of the original solution was placed in a colorimetric tube, 20 mL of deionized water was added first, the pH range was adjusted to 6-8, and the volume was adjusted to 50 mL. The solution was then transferred to the biosensor detection tube for detection.

[0039] Example 2: Optimization of the process for making vinegar, wine, and yeast from fermented grains. (1) Single-factor experiment Based on the basic process in Table 1, the controlled variable method was adopted. Under the condition that other conditions remained unchanged, the single factors and experimental conditions were set as follows (the following proportions are based on the amount of rice flour added): water addition (50%, 52.5%, 55%, 57.5%, 60%), mixing water temperature (40℃, 45℃, 50℃, 55℃, 60℃), mother koji (MQ) addition (0.5%, 1%, 1.5%, 2%, 2.5%), herbal medicine addition (0%, 0.25%, 0.5%, 0.75%, 1%, with Jiujieci and Ziyupan added at a 1:1 ratio), and high enzyme active wine yeast (FX) addition (0.5%, 1%, 1.5%, 2%, 2.5%), for a total of 5 optimization indicators. The total acid (Y1) and ethanol (Y2) were used as the comprehensive response values.

[0040] like Figure 1 As shown, the highest total acid and ethanol yields are obtained when the water content is 55% during the fermentation of fermented grain vinegar. During the pelleting process, it was observed that as the water content decreased during mixing, the surface of the grains became less viscous due to water shortage, resulting in a higher crack rate. It is speculated that the presence of cracks allows microorganisms in the open fermentation environment to more easily penetrate the grains, competing for key microorganisms and leading to low yields of total acid and ethanol. With increasing water content, the viscosity of the fermented grain vinegar starter changed significantly, and the reduced crack rate resulted in a more compact internal structure, affecting oxygen permeability. This inhibited the growth of some saccharification-related microorganisms, such as molds, consequently affecting the production of total acid and ethanol in the fermented grain vinegar. Therefore, the optimal water content is 55%.

[0041] like Figure 2 As shown, with increasing mixing water temperature, the viscosity of the yeast pellets increases, affecting the overall yield of acid and alcohol. However, when the mixing water temperature is below 50℃, the yeast pellet formation is poor. Although a higher total acid yield is achieved at 45℃, environmental microorganisms compete with ethanol-producing microorganisms such as yeast for growth, resulting in insufficient ethanol production in the fermentation product. When the mixing water temperature is 50℃, the yeast pellet formation is good, the acidity is moderate, and the ethanol production capacity is improved. When the mixing water temperature is above 50℃, the growth activity of microorganisms begins to be affected, especially the alcohol-producing microorganisms, leading to a significant decrease in the ethanol content of the fermentation product. Therefore, considering all production needs, the optimal mixing water temperature is 50℃.

[0042] Figure 3 The effects of the amount of mother koji added on the yield of acetic acid and alcohol from the fermentation residue were investigated. During fermentation, it was observed that with increasing mother koji addition, the time required for fermentation to begin was shorter, manifested by an earlier change in the surface temperature of the fermentation residue, indicating more vigorous microbial metabolism. The highest yields of total acid and ethanol were achieved when the mother koji addition was 2%. Above 2%, the temperature rose too quickly and was difficult to control, affecting the growth of other temperature-sensitive microorganisms such as molds, thus decreasing the yields of acid and alcohol. Therefore, the optimal mother koji addition was 2%.

[0043] like Figure 4 As shown, when the amount of herbs (Nine-knot Thorn, Purple Jade Plate) added is 0%, the lack of their regulatory effect leads to the growth of other microorganisms inhibiting the activity of alcohol-producing microorganisms, resulting in low alcohol production. When the amount of herbs added is 0.25%, the growth of alcohol-producing microorganisms is promoted, and the overall alcohol and acid production capacity reaches its maximum. With the increase of the amount of herbs added, the antibacterial properties of the herbs themselves affect the activity of most microorganisms in the wine yeast, thus affecting the fermentation capacity of the wine yeast. Therefore, the optimal amount of herbs added is 0.25%.

[0044] like Figure 5 As shown, with the increase of FX addition, the fermentation capacity of the fermented grain vinegar first increases and then decreases, especially improving the alcohol production capacity of the fermented grain vinegar. When the FX addition is 1.5%, the alcohol production is the highest; although the acidity is not the highest at this point, it is second only to the FX addition of 2%. Therefore, the optimal FX addition is 1.5%.

[0045] (2) Plackett-Burman test Using Plackett Using the Burman design principle, two levels, high (1) and low (-1), were selected as independent variables to screen and determine the key factors affecting the total acetic acid (Y1) and ethanol yield (Y2) of the waste meal.

[0046] Based on the results of the single-factor experiment in (1), an experimental design with N=12 was selected. The key factors in the fermentation process of the lees vinegar wine yeast were screened according to the Plackett-Burman experimental design. The significance of five factors, namely, water addition (A), mixing water temperature (B), MQ addition amount (C), herbal addition amount (D), and high enzyme activity wine yeast FX addition amount (E), was analyzed with total acid (Y1) and ethanol (Y2) as response values.

[0047] Analysis of the experimental results identified factors that significantly affected total acid and ethanol yield. The p-value for the total acid model was 0.0331 < 0.05, and the p-value for the ethanol model was 0.0367 < 0.05, indicating that both models were significant and could be used for further analysis.

[0048] The significant factors affecting total acidity were: the amount of MQ added (C, P = 0.0176), the amount of FX added (E, P = 0.0385), and their interaction term BE (P = 0.0050); the significant factors affecting ethanol were: the amount of FX added (E, P = 0.0182) and its interaction BE (P = 0.0059). Other factors, such as the amount of water added and the amount of herbal medicine added, did not show significant effects under the conditions of this experiment.

[0049] The model showed good fit, with R² = 0.8796 for total acid and R² = 0.8741 for ethanol, indicating that the model can explain the changes in response values ​​well. The coefficients of variation (CV) were 8.48% and 12.86%, respectively, and the Adeq precision was greater than 7, indicating good repeatability and predictive ability.

[0050] By performing multiple regression fitting on the data, the regression equation for total acid was obtained as follows: Y1 = 9.16 + 0.4426A + 0.01B + 0.8440C - 0.0731D + 0.6253E + 1.31BE; and the regression equation for ethanol was obtained as follows: Y2 = 1.5 + 0.0625A - 0.0167B + 0.0875C - 0.0500D + 0.1917E + 0.3125BE.

[0051] In summary, the Plackett-Burman experiment identified the amount of MQ, the amount of FX, and the BE interaction as key factors affecting the total acid and ethanol yield of fermentation products, providing a basis for subsequent response surface optimization.

[0052] (3) Response surface optimization Using the Box-Behnken design principle and a three-factor, three-level response surface methodology, the effects of mixing water temperature, MQ addition amount, and FX addition amount on the overall score (OD) were studied, and a predictive model was established using a quadratic polynomial regression equation.

[0053] To obtain the overall score (OD), the two response values ​​are normalized. The overall score formula is established to calculate the OD value: ; .

[0054] In the formula, For each indicator, the original value, , For each index, these are the maximum and minimum values ​​in its response surface experimental group. , These are the normalized values ​​for total acid and ethanol production, respectively.

[0055] The Box-Behnken experimental design results were fitted using Design-Expert 13 software through multiple regression analysis. The quadratic regression equations for the overall wine-drug score (OD) on the mixing water temperature (X1), MQ addition amount (X2), and FX addition amount (X3) are as follows: Y=0.7898-0.0666X1-0.0619X2+0.0195X3+0.0257X1X2-0.0822X1X3-0.0522X2X3-0.1171X12 -0.3065X2 2 -0.1805X3 2 .

[0056] Analysis of variance (ANOVA) of the model showed that the model P < 0.0001, indicating high significance; the lack-of-fit term P = 0.8985 > 0.05, which was not significant, indicating a good model fit. The model's coefficient of determination R² = 0.9768, and the adjusted R² = 0.9471, indicating that the model can explain the variation in response values ​​well. The coefficient of variation (CV) = 10.01%, indicating good experimental repeatability.

[0057] The order of influence of each factor on the OD value is: MQ addition amount (X2) > mixing water temperature (X1) > FX addition amount (X3). Among them, X1, X2, X1X3, X1², X2², and X3² have significant effects on the OD value (P<0.05).

[0058] pass Figure 12 Analysis shows that the mixing water temperature and the amount of FX added (X1X3) have a significant interaction, manifested by a steep slope of the response surface and elliptical contour lines, indicating a strong interaction between the two, which is consistent with the results obtained from the Plackett-Burman experiment in 3.1.2. As the mixing water temperature increases, the effect of the amount of FX added on the OD value gradually weakens, indicating that the promoting effect of FX is limited under high temperature conditions.

[0059] Under optimized process conditions, the temperature change trend during the fermentation of the wine yeast is basically consistent with the preset program (see...). Figure 8 The temperature rose steadily in the early stage of fermentation, remained at 35–37℃ in the middle stage, and gradually decreased and stabilized in the later stage. The stability of temperature control provided a favorable environment for microbial metabolism, which contributed to the accumulation of total acid and ethanol, further verifying the reliability of temperature program control.

[0060] Example 3 Verification Experiment Based on the experimental results of the Box-Behnken design in Example 2, and taking into account actual production needs, a set of optimal process parameters for the fermented grain vinegar starter were obtained. Following the starter process constructed in Table 1, with water addition at 55% and herb addition at 0.25%, mixing water temperature at 48℃, MQ addition at 1.9%, and FX addition at 1.6%, and with the temperature change trend controlled in the same manner, three parallel experiments were conducted. The OD values ​​of total acid and ethanol obtained from the verification experiments were calculated.

[0061] Based on the optimization results using response surface methodology and considering actual production conditions, the optimal process parameters for the wine-making process were determined to be: mixing water temperature 48℃, MQ addition 1.9%, and FX addition 1.6%, with a water addition of 55% and herbal medicine addition of 0.25%. Three parallel verification experiments were conducted under these conditions, and the results are shown in Table 3. The average OD value obtained from the verification experiments was 1.021 > 0.919 (model prediction value). Compared to the original process, the total acid yield increased by 22.7%, the ethanol yield increased by 67.6%, and the RSD was less than 2%, indicating that the optimized process is stable, reliable, and has good repeatability.

[0062] Table 3 Comparison of total acid, ethanol and OD values ​​in the verification test and the original process

[0063] To evaluate the superiority of the optimized process, it was compared with the original processes AB, CD, and FQ (data from single-factor experimental groups). The total acid and ethanol content of the optimized process group were significantly higher than those of the original process group, and the OD value was also significantly improved, indicating that the optimized brewing process has a significant advantage in improving the overall quality of the product.

[0064] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for constructing wine-making agents using response surface methodology, characterized in that, Includes the following steps: S1, using single-factor experiments, determined the influencing factors and their levels. These factors included water addition, mixing water temperature, mother koji addition, herbal medicine addition, and high-enzyme active wine yeast addition. The herbal medicine consisted of *Nine-knot Thorn* and *Purple Jade Plate* in a 1:1 ratio. S2, based on the results of S1, used a Plackett-Burman model experiment with water addition (A), mixing water temperature (B), mother koji addition (C), herbal medicine addition (D), and high-enzyme active wine yeast addition (E) as independent variables, and total acidity (Y1) and ethanol (Y2) as response factors. In response, a climbing experiment was conducted to identify three factors that significantly affected total acidity and ethanol: mixing water temperature (X1), MQ addition amount (X2), and FX addition amount (X3). In S3, based on the Plackett-Burman experiment results of S2, a Box-Behnken model experiment was designed with mixing water temperature (X1), mother koji addition amount (X2), and high enzyme activity yeast addition amount (X3) as independent variables, and the comprehensive score OD (Y) of total acidity and ethanol as the response value. Response surface analysis was then performed to obtain the optimal parameters for yeast preparation.

2. The method according to claim 1, characterized in that, In S1, based on the amount of indica rice flour added, the mixing water temperature was selected as 40℃, 45℃, 50℃, 55℃, and 60℃; the amount of mother koji added was selected as 0.5%, 1%, 1.5%, 2%, and 2.5%; the amount of herbal medicine added was selected as 0%, 0.25%, 0.5%, 0.75%, and 1%; and the amount of high-enzyme active wine yeast added was selected as 0.5%, 1%, 1.5%, 2%, and 2.5%.

3. The method according to claim 1, characterized in that, In S2, the quadratic polynomial regression equation for total acid (Y1) is as follows: Y1 = 9.16 + 0.4426A + 0.01B + 0.8440C - 0.0731D + 0.6253E + 1.31BE; the quadratic polynomial regression equation for ethanol (Y2) is as follows: Y2 = 1.5 + 0.0625A - 0.0167B + 0.0875C - 0.0500D + 0.1917E + 0.3125BE.

4. The method according to claim 1, characterized in that, In S3, the quadratic multinomial regression equations of mixing water temperature (X1), MQ addition amount (X2), and FX addition amount (X3) on OD(Y) are as follows: Y=0.7898-0.0666X1-0.0619X2+0.0195X3+0.0257X1X2-0.0822X1X3-0.0522X2X3-0.1171X1 2 -0.3065X2 2 -0.1805X3 2 ; The established comprehensive scoring formula is as follows: ; ; in: For each indicator, the original value, , For each index, the maximum and minimum values ​​are given in its response surface experimental group. , These are the normalized values ​​for total acid and ethanol production, respectively.

5. A method for preparing a wine-based medicine, characterized in that, The method involves adding 0.25% of herbal ingredients, 1.9% of mother koji, 1.6% of high-enzyme active wine yeast, and 55% of 48℃ warm water, based on the amount of rice flour added. The mixture is stirred evenly, formed into balls, rolled into rounds, and then fermented at a controlled temperature for 48 hours. After drying for 1-2 days, the finished wine yeast is obtained. The herbal ingredients consist of nine-knot thorn and purple jade plate in a 1:1 ratio.

6. The method according to claim 5, characterized in that, The temperature control process is divided into four stages: The first stage is the fermentation start-up and adaptation period: the initial stage is 1-4 hours, fermentation is started at room temperature, and the temperature gradually rises to above 25℃. During this period, the surface is in a high humidity state of 92-98%; then the lid is opened and dehumidification is performed for 4-11 hours, the wine yeast continues to ferment, the surface temperature rises to 30℃, and the surface humidity decreases slightly. Phase II is the main fermentation and regulation period: After dehumidification, the lid is closed and fermentation continues for 11-21 hours with the temperature rising. The surface temperature is controlled to not exceed 35℃ to facilitate mold growth. During this stage, moisture is transferred from the inside of the yeast to the surface, and the surface humidity rises to about 95%. The temperature is then maintained at 35℃ for 21-23 hours. When the mycelium on the surface of the yeast grows vigorously, the yeast is turned over. After turning, the surface temperature continues to rise to 35-37℃ for 23-27 hours and is maintained for 4 hours. Finally, the lid is closed for 27-28 hours to maintain the surface temperature of the yeast at 38-40℃ for a short period, and the surface humidity drops below 75% due to the temperature rise. Phase III is the cooling and stabilization period: In 28 hours, the surface temperature of the wine and yeast is reduced to below 35°C by opening the lid and ventilation, and the surface humidity recovers slightly; then until 48 hours, the surface temperature is stabilized below 30°C by continuous ventilation, during which the surface humidity is close to the indoor humidity. Stage IV is the drying period: the final stage is a 2-day drying period, which yields a finished wine with a moisture content of 10-12%.

7. A wine-based medicine prepared using the method described in any one of claims 5 to 6.

8. A method for preparing vinegar made from fermented grains, characterized in that, The method involves steaming rice at a rice-to-water ratio of 1:1.8, cooling it, mixing it evenly with water, spices, and the yeast starter described in claim 7, fermenting it at 30°C for 2 days, then adding an appropriate amount of rinsing water and fermenting it at 40°C for 2 days to obtain the finished product, lees vinegar; wherein the amount of yeast starter added is 1%.

9. The method according to any one of claims 1 to 4, the method according to any one of claims 5 to 6, or the wine yeast according to claim 7 in the brewing and fermentation of products.

10. The application according to claim 9, characterized in that, The fermented food is vinegar made from fermented grains, vinegar, or wine.

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