Method for preparing dregs vinegar based on artificial composite flora fermentation and product

Abstract

The invention relates to dregs vinegar prepared based on artificial composite flora fermentation and a preparation method thereof, and belongs to the technical field of food fermentation. The method takes sticky rice as a main raw material, and sequentially comprises the core steps of raw material treatment, alcoholic fermentation, lactic acid fermentation, acetic acid fermentation and the like. The method is characterized in that the fermentation time sequence is accurately controlled by sequentially inoculating saccharomyces cerevisiae, Wickerhamomyces anomalus, lactic acid bacteria and acetic acid bacteria and combining dynamic regulation and control of specific physicochemical indexes in the fermentation process. According to the method disclosed by the invention, the technical problems of long period, large flavor volatility, uncontrollable process and the like of the traditional natural fermentation process are effectively solved by virtue of the synergistic metabolism effect of multiple strains, and the standardized and stable output of the flavor of the dregs vinegar product and the controllable management of the whole fermentation process are realized; and a technical scheme and industrial support are provided for industrial and large-scale production of the dregs vinegar.

Description

Technical Field

[0001] This invention relates to the field of food fermentation technology, specifically to a method for preparing vinegar from fermented grains based on artificial complex microbial fermentation, and the vinegar product obtained by this method. Background Technology

[0002] Zhaobo vinegar, a unique traditional fermented food, is beloved for its complex flavor profile of sour, refreshing, umami, and fragrant. Rich in organic acids and various vitamins, it has long been popular with consumers due to its effectiveness in boosting immunity and maintaining gut health. However, traditional Zhaobo vinegar production relies heavily on natural fermentation, which depends entirely on the natural microbial flora in the environment. This lack of active intervention and precise control over the microbial community results in inherent instability during fermentation, leading to a series of key challenges hindering the industry's development. Specifically: first, the long fermentation cycle and low production efficiency limit large-scale production capacity; second, significant fluctuations in flavor and quality, with inconsistent taste and aroma across different batches and even within the same batch, making product stability difficult to guarantee; and third, weak controllability in the production process, making it susceptible to contamination by other microorganisms and increasing quality and safety risks.

[0003] While some researchers have attempted to improve production using pure microbial fermentation, existing methods are mostly limited to simple combinations of a few strains, failing to establish an effective artificial complex microbial community for synergistic metabolism and lacking precise control mechanisms for the fermentation process. This results in a single material transformation pathway during fermentation, a lack of flavor complexity in the final product, and a deficiency in the unique flavor depth and complexity of traditional vinegar made from fermented grains. Furthermore, there is still significant room for improvement in fermentation efficiency, making it difficult to simultaneously meet the dual industrial demands of standardized production and the preservation of traditional flavors.

[0004] Therefore, the key to overcoming the bottlenecks of existing technologies and improving the fermentation efficiency and product quality stability of vinegar made from fermented grains lies in developing a fermentation technology that can precisely control the fermentation process and the synergistic metabolism of microbial communities. This invention constructs a highly efficient artificial composite microbial community system by using specific strains with defined functions, achieving precise inoculation at different stages and synergistic control of fermentation parameters, thereby guiding the efficient transformation of fermentation raw materials and the precise formation of distinctive flavors. This technological innovation has significant practical implications and substantial industrial value for promoting the standardization of vinegar production, enhancing product flavor richness, reducing quality and safety risks, and facilitating the technological upgrading and large-scale development of the entire industry. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing vinegar from fermented grains based on artificial compound microbial communities. This method can achieve the technical effects of stable flavor and shortened fermentation cycle in vinegar from fermented grains. At the same time, the vinegar from fermented grains product obtained by the above preparation method is also one of the protection objectives of this invention.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A method for preparing vinegar from fermented grains based on artificial complex microbial communities includes the following steps: S1. Raw material processing: Wash the glutinous rice, soak it, steam it, and cool it down; S2. Alcoholic fermentation: Add saccharifying enzyme, brewer's yeast and abnormal Wickham yeast seed liquid, and water to the cooled glutinous rice, mix well, and carry out saccharification and alcoholic fermentation at 28~30℃ to obtain alcoholic fermentation mash. S3, Lactic acid fermentation: Seed liquids of Lactobacillus fermentatus and Lactobacillus plantarum are inoculated into the alcoholic fermentation mash, and lactic acid fermentation is carried out at 28~32℃; S4. Acetic acid fermentation: When the total acid of the fermentation system reaches 2.0~3.0 g / L (calculated as lactic acid), the seed liquid of Acetobacter pasteurellium is inoculated into it, and aerobic acetic acid fermentation is carried out at 28~32℃ to obtain acetic acid fermentation mash. S5. Sterilization and bottling: The acetic acid fermentation mash is homogenized, sterilized, and bottled to obtain the finished vinegar product.

[0007] Preferably, in step S1, high-quality glutinous rice raw materials with plump grains and no mold are selected, cleaned, and soaked in purified water for 6-8 hours, with the soaking liquid level 12-16 cm above the rice layer, until the glutinous rice can be easily crushed.

[0008] Preferably, in step S1, after soaking, the glutinous rice is drained, and purified water is added to a level 3 cm above the rice layer. It is then steamed for 35-40 minutes until the glutinous rice grains are crystal clear and have no white core. After steaming, the rice is cooled to a temperature of 30-35°C.

[0009] Preferably, in step S2, the activity of the saccharifying enzyme is 100,000 U / g, and the amount added is 0.005%~0.01% of the dry glutinous rice mass; the alcoholic fermentation time is 36~48 h, the temperature is 28~30℃, and the alcohol content of the fermented mash is 4%vol~6%vol.

[0010] Preferably, in step S2, the seed culture concentration of both *Saccharomyces cerevisiae* and *Wickhamia lanceolata* is 1×10⁻⁶. 7 ~5×10 7 The inoculum volume is 1% to 2% (v / v) of the fermentation mash volume, with a concentration of CFU / mL.

[0011] Preferably, in step S3, the seed culture concentration of both *Lactobacillus mucilaginosus* and *Lactobacillus plantarum* is 1×10⁻⁶. 7 ~5×10 7 The inoculum concentration was CFU / mL, and the inoculum amount was 0.5%~1.0% (v / v) based on the volume of the fermentation mash. The fermentation temperature was 28~32℃, and the fermentation time was 24~36 h. The fermenting *Lactobacillus mucilaginosus* and *Lactobacillus plantarum* were both deposited at the China Industrial Microbiological Culture Collection Center, with strain numbers CICC21800 and CICC 20871, respectively.

[0012] Preferably, in step S4, the seed culture concentration of *Acetobacter pasteurellium* is 1 × 10⁻⁶. 7 ~5×10 7 The inoculum concentration was CFU / mL, and the inoculum volume was 0.5%~1.0% (v / v) of the fermentation mash. The acetic acid fermentation conditions were 28~32℃ for 4~5 days. The *Acetobacter pasteurellii* strain was deposited at the China Industrial Microbial Culture Collection Center, with strain number CICC20874.

[0013] Preferably, in step S5, the acetic acid fermentation mash is homogenized, sterilized at 75-80℃ for 15-20 minutes, and bottled to obtain the finished product, lees vinegar.

[0014] The present invention further discloses a vinegar product made from lees using the above-described method.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Shortened Fermentation Cycle: By inoculating highly active specialized functional microbial strains, an artificial composite microbial fermentation system is constructed, eliminating the dependence of natural fermentation on environmental microorganisms and the resulting uncontrollability. Simultaneously, combined with precise control strategies for fermentation timing, the fermentation cycle is significantly shortened, production efficiency is improved, and technical support is provided for large-scale production capacity release.

[0016] 2. Excellent and stable flavor: Through the synergistic metabolism of brewing yeast and ester-producing yeast to produce alcohol and characteristic ester aroma, and the fermentation of lactic acid bacteria and acetic acid bacteria to produce acid and enhance aroma, the balance of key flavor substances such as acid, ester, and alcohol is achieved, making the product full-bodied and harmonious in taste, effectively solving the pain point of large flavor fluctuations in traditional processes.

[0017] 3. High degree of process controllability and standardization: The clear physicochemical indicators of each stage serve as the core basis for the transformation of fermentation processes, replacing the vague judgment mode that relies on human experience in traditional production. This makes the entire fermentation process quantifiable and replicable, laying a solid foundation for achieving industrialized and standardized production. Attached Figure Description

[0018] Figure 1Here is the liquid chromatogram of the organic acid components in the vinegar sample from Example 1; Figure 2 This is a liquid chromatogram of the organic acid components in commercially available vinegar products made from fermented grains. Detailed Implementation

[0019] The technical solution of the present invention is further illustrated below through specific implementation methods. Unless otherwise specified, the technical means used in the present invention are all methods known to those skilled in the art. Furthermore, the implementation schemes should be understood as illustrative, not limiting the scope of the present invention; the essence and scope of the present invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these implementation schemes without departing from the essence and scope of the present invention also fall within the protection scope of the present invention.

[0020] Unless otherwise specified, all reagents or instruments used in the examples are legitimate products that can be purchased through official channels.

[0021] Example 1 This embodiment describes a method for preparing vinegar based on artificial compound microbial fermentation, comprising the following steps: S1. Raw material processing: Select high-quality glutinous rice with plump grains and no mold. After washing it clean, soak it in purified water for 8 hours, with the water level 14 cm above the rice layer, until the glutinous rice can be easily crushed. Drain the water, add purified water 3 cm above the rice layer, and steam for 38 minutes until the glutinous rice grains are crystal clear and have no white core. After steaming, spread it out to cool until the temperature of the glutinous rice is 32℃.

[0022] S2. Alcoholic fermentation: Add saccharifying enzyme with an activity of 100,000 U / g to the cooled glutinous rice, at a dosage of 0.008% of the dry glutinous rice mass. Inoculate with seed cultures of *Saccharomyces cerevisiae* and *Wickham's abnormal* yeast, at concentrations of 3.0 × 10⁻⁶ U / g and 3.0 × 10⁻⁶ U / g respectively. 7 CFU / mL and 2.8×10 7 The inoculum concentration was CFU / mL, and the inoculum amount was 1.5% (v / v) based on the volume of the fermentation mash. An appropriate amount of purified water was added and thoroughly mixed with the materials. Fermentation was carried out at 29℃ for 42 h to obtain fermentation mash with an alcohol content of 5.2% vol.

[0023] S3, Lactic acid fermentation: Inoculate the alcoholic fermentation mash with Lactobacillus fermentum CICC 21800 seed culture (concentration 3.5 × 10⁻⁶). 7 CFU / mL) and Lactobacillus plantarum CICC 20871 seed culture (concentration 3.2×10⁻⁶ CFU / mL) 7 The inoculum volume was 0.8% (v / v) of the fermentation mash, and the inoculum concentration was CFU / mL. Lactic acid fermentation was carried out at 30°C. After 36 h of fermentation, the total acid (calculated as lactic acid) was measured to be 2.8 g / L.

[0024] S4, Acetic acid fermentation: When the total acid content (calculated as lactic acid) reaches 2.8 g / L, inoculate with *Acetobacter pasteurellium* CICC 20874 seed culture (concentration 3.0 × 10⁻⁶). 7 The inoculum volume was 0.7% (v / v) of the fermentation mash. Aerobic acetic acid fermentation was carried out at 30°C for 4.5 days.

[0025] S5. Sterilization and bottling: After the acetic acid fermentation is completed, the acetic acid fermentation mash is homogenized, sterilized at 78℃ for 18 minutes, and then bottled to obtain the finished product of lees vinegar.

[0026] Example 2 This embodiment describes a method for preparing vinegar based on artificial compound microbial fermentation, comprising the following steps: S1. Raw material processing: Select high-quality glutinous rice with plump grains and no mold. After washing it clean, soak it in purified water for 6 hours, with the water level 12 cm above the rice layer, until the glutinous rice can be easily crushed. Drain the water, add purified water to a level 3 cm above the rice layer, and steam for 40 minutes until the glutinous rice grains are crystal clear and have no white core. After steaming, let it cool until the temperature of the glutinous rice is 30℃.

[0027] S2. Alcoholic fermentation: Add saccharifying enzyme with an activity of 100,000 U / g to the cooled glutinous rice at a rate of 0.005% of the dry glutinous rice mass. Inoculate with seed cultures of *Saccharomyces cerevisiae* and *Wickham's abnormal* yeast at concentrations of 1.5 × 10⁻⁶ U / g. 7 CFU / mL and 1.2×10 7 The inoculum concentration was CFU / mL, and the inoculum amount was 2% (v / v) based on the volume of the fermentation mash. An appropriate amount of purified water was added and thoroughly mixed with the materials. Fermentation was carried out at 28℃ for 48 h to obtain fermentation mash with an alcohol content of 4.5% vol.

[0028] S3, Lactic acid fermentation: Inoculate the alcoholic fermentation mash with Lactobacillus fermentatus CICC 21800 seed culture (concentration 1.5 × 10⁻⁶). 7 CFU / mL) and Lactobacillus plantarum CICC 20871 seed culture (concentration 1.5×10⁻⁶ CFU / mL) 7 The inoculum volume was 1.0% (v / v) of the fermentation mash, and the inoculum concentration was CFU / mL. Lactic acid fermentation was carried out at 28°C. After 30 h of fermentation, the total acid (calculated as lactic acid) was measured to be 2.1 g / L.

[0029] S4, Acetic acid fermentation: When the total acid content (calculated as lactic acid) reaches 2.1 g / L, inoculate with *Acetobacter pasteurellium* CICC 20874 seed culture (concentration 1.2 × 10⁻⁶). 7 The inoculum volume was 1.0% (v / v) of the fermentation mash. Aerobic acetic acid fermentation was carried out at 28°C for 5 days.

[0030] S5. Sterilization and bottling: After the acetic acid fermentation is completed, the acetic acid fermentation mash is homogenized, sterilized at 75℃ for 20 minutes, and then bottled to obtain the finished product of lees vinegar.

[0031] Example 3 This embodiment describes a method for preparing vinegar based on artificial compound microbial fermentation, comprising the following steps: S1. Raw material processing: Select high-quality glutinous rice with plump grains and no mold. After washing it clean, soak it in purified water for 8 hours, with the water level 16 cm above the rice layer, until the glutinous rice can be easily crushed. Drain the water, add purified water 3 cm above the rice layer, and steam for 35 minutes until the rice grains are crystal clear and there is no white core inside. After steaming, let it cool until the temperature of the glutinous rice is 35℃.

[0032] S2, Alcoholic Fermentation: Add saccharifying enzyme with an activity of 100,000 U / g to the cooled glutinous rice at a rate of 0.01% of the dry glutinous rice mass. Inoculate with seed cultures of *Saccharomyces cerevisiae* and *Wickham's Aberrantia* at concentrations of 4.8 × 10⁻⁶ U / g. 7 CFU / mL and 5.0×10 7 The inoculum concentration was 1% (v / v) of the fermentation mash, with an appropriate amount of purified water added and thoroughly mixed with the materials. Fermentation was carried out at 30°C for 36 h to obtain fermentation mash with an alcohol content of 5.6% vol.

[0033] S3, Lactic acid fermentation: Inoculate the alcoholic fermentation mash with Lactobacillus fermentum CICC 21800 seed culture (concentration 4.5 × 10⁻⁶). 7 CFU / mL) and Lactobacillus plantarum CICC 20871 seed culture (concentration 4.5×10⁻⁶ CFU / mL) 7 The inoculum volume was 0.5% (v / v) of the fermentation mash, and the inoculum concentration was 0.5% of the total lactic acid (CFU / mL). Lactic acid fermentation was carried out at 32°C. After 24 h of fermentation, the total acid (calculated as lactic acid) was measured to be 3.0 g / L.

[0034] S4, Acetic acid fermentation: When the total acid content (calculated as lactic acid) reaches 3.0 g / L, inoculate with *Acetobacter pasteurellium* CICC 20874 seed culture (concentration 4.5 × 10⁻⁶). 7 The inoculum volume was 0.5% (v / v) of the fermentation mash. Aerobic acetic acid fermentation was carried out at 32°C for 4 days.

[0035] S5. Sterilization and bottling: After the acetic acid fermentation is completed, the acetic acid fermentation mash is homogenized, sterilized at 80℃ for 15 minutes, and then bottled to obtain the finished product of lees vinegar.

[0036] Comparative Example 1 This comparative example simulates the most common traditional production method of vinegar made from fermented grains, and its preparation steps are as follows: S1. Raw material processing: Select high-quality glutinous rice with plump grains and no mold, wash it clean, soak it in purified water until the glutinous rice can be easily crushed, steam it with water, and then cool it to about 30~35℃.

[0037] S2. Fermentation: Mix the cooled glutinous rice with the yeast starter, add an appropriate amount of water, stir gently, and transfer to a clean fermentation vat. Place in a cool, shady environment at 25-30℃ for natural fermentation. The fermentation period is 25-30 days, ending when the sour taste is strong and the flavor has developed.

[0038] S3. Sterilization and bottling: After fermentation, the fermented product is homogenized, sterilized at 80℃ for 15 min, and then bottled to obtain the finished product, vinegar made from fermented grains.

[0039] Comparative Example 2 This comparative example uses a one-time inoculation and fermentation method to prepare vinegar from fermented grains. The preparation steps are as follows: S1. Raw material processing: The method is the same as in Example 1.

[0040] S2. Inoculation and Fermentation: Add saccharifying enzyme with an activity of 100,000 U / g to the cooled glutinous rice, at a dosage of 0.008% of the dry glutinous rice mass. Simultaneously, inoculate with all of the following seed cultures at once: Saccharomyces cerevisiae seed culture, concentration 3.0 × 10⁻⁶. 7 CFU / mL, inoculum size 1.5% (v / v) of fermentation mash volume; abnormal Wickham yeast seed culture, concentration 2.8 × 10⁻⁶. 7 CFU / mL, inoculum amount 1.5% (v / v) based on fermentation mash volume; fermentation with Lactobacillus mucilaginosus CICC 21800 seed culture, concentration 3.5×10⁻⁶. 7 CFU / mL, inoculum amount 0.8% (v / v) of fermentation mash volume; Lactobacillus plantarum CICC 20871 seed culture, concentration 3.2×10⁻⁶. 7 CFU / mL, inoculum size 0.8% (v / v) of fermentation mash volume; Acetobacter pasteurellium CICC 20874 seed culture, concentration 3.0 × 10⁻⁶. 7 CFU / mL, inoculum amount is 0.7% (v / v) of fermentation mash volume. Add the same amount of purified water as in Example 1, mix thoroughly with all materials, and ferment at 32°C for a total time of about 7 days.

[0041] S3. Sterilization and bottling: After fermentation, homogenization, sterilization (78°C, 18 min) and bottling operations are carried out in the same manner as in Example 1 to obtain the finished product of lees vinegar.

[0042] Comparative Example 3 This comparative example is the same as Example 1, except that the fermentation order has been changed. Specifically, lactic acid fermentation is performed first, followed by alcoholic fermentation, and finally acetic acid fermentation. The preparation steps are as follows: S1. Raw material processing: The method is the same as in Example 1.

[0043] S2, Lactic Acid Fermentation: Add saccharifying enzyme with an activity of 100,000 U / g to the cooled glutinous rice, at a dosage of 0.008% of the dry glutinous rice mass. Inoculate with *Lactobacillus mucilaginosus* CICC 21800 seed culture (concentration 3.5 × 10⁻⁶). 7 CFU / mL) and Lactobacillus plantarum CICC 20871 seed culture (concentration 3.2×10⁻⁶ CFU / mL) 7 The inoculum volume was 0.8% (v / v) based on the volume of fermentation mash. Lactic acid fermentation was carried out at 30°C for 36 h.

[0044] S3, Alcoholic Fermentation: After 36 hours of fermentation, seed cultures of *Saccharomyces cerevisiae* and *Wickhamia lanceolata* were inoculated at concentrations of 3.0 × 10⁻⁶. 7 CFU / mL and 2.8×10 7 The inoculum concentration was CFU / mL, and the inoculum amount was 1.5% (v / v) based on the volume of fermentation mash. Fermentation was carried out at 29°C for 42 h.

[0045] S4. Acetic acid fermentation: After the alcohol content of the fermentation mash stabilizes, inoculate with Acetobacter pasteurella CICC 20874 seed culture (concentration 3.0 × 10⁻⁶). 7 The inoculum volume was 0.7% (v / v) of the fermentation mash. Aerobic acetic acid fermentation was carried out at 30°C for 4.5 days.

[0046] S5. Sterilization and Bottling: After acetic acid fermentation, the acetic acid fermentation mash is homogenized. It is then sterilized at 78℃ for 18 minutes and bottled to obtain the finished vinegar product.

[0047] I. Total acidity and pH of fermentation products The total acid in the fermentation products was determined in accordance with the national food safety standard GB 12456-2021, "Determination of Total Acid in Food". The pH of the fermentation products was determined using a pH meter.

[0048] The total acid (calculated as lactic acid) and pH values ​​of the final fermentation products are shown in Table 1 below.

[0049] surface Total acidity and pH of fermentation products Note: Different letters indicate that the embodiments are significantly different from the comparative examples. P <0.05).

[0050] As shown in Table 1, the total acid content of the products from the embodiments of the present invention ranged from 13.95 to 14.85 g / L. The total acid contents of Comparative Example 1 (conventional fermentation), Comparative Example 2 (single-stage inoculation fermentation), and Comparative Example 3 (altered fermentation sequence) were approximately 10.01 g / L, 9.41 g / L, and 8.99 g / L, respectively. The vinegar made from fermented grains using the present invention (Examples 1-3) had significantly higher total acid content (calculated as lactic acid) than all the comparative examples. P <0.05). The method described in this invention can increase the total acid content of the product by approximately 40% to 50% compared to traditional processes. This indicates that the present invention, through the sequential inoculation and precise control of artificial complex microbial communities, accelerates the acid-producing metabolic pathways of microorganisms, achieving a significant improvement in acid production efficiency. This invention overcomes the shortcomings of low efficiency in natural fermentation microbial communities, achieving a highly efficient acidification process. Although the exact same strains were used, Comparative Example 3 showed the worst acid production effect, followed by Comparative Example 2. This indicates that changing the fermentation sequence or inoculating all strains at once leads to metabolic interference and competitive inhibition between strains, preventing the formation of a highly efficient acid-producing system. This result confirms that sequential inoculation is a key technical feature for ensuring the phased and efficient collaboration of functional microbial communities, thereby achieving high acid production. Through the synergistic effect of artificial complex microbial communities, this invention greatly improves acid production efficiency, resulting in a final product with a richer sour taste and a lower pH value. This not only contributes to flavor formation but also provides better microbial stability.

[0051] II. Sensory Evaluation To further illustrate the effects of the present invention, the vinegar products from Example 1 and Comparative Examples 1-3 were used as examples for sensory evaluation. The sensory requirements scores are shown in Table 2 below.

[0052] Sensory evaluation method: Twenty sensory evaluators conducted quantitative scoring of each item in Table 2 in a sensory laboratory with uniform lighting and no odor. The average of the total scores of each indicator was used as the sensory score.

[0053] surface Sensory evaluation rating sheet The sensory evaluation results are shown in Table 3 below.

[0054] surface Sensory evaluation results table Sensory evaluation results showed that the vinegar prepared by the present invention (Example 1) had the highest total sensory evaluation score. Compared with the vinegar prepared by the present invention, the traditionally fermented vinegar (Comparative Example 1) was inferior in color and overall quality; the vinegar prepared by single inoculation and fermentation (Comparative Example 2) was inferior in taste, texture, and fermentation aroma; the vinegar prepared by changing the fermentation order (Comparative Example 3) was inferior in fermentation aroma, acidity level, taste, texture, and overall quality; and commercially available products performed poorly in color, acidity level, and overall quality. The reasons for these issues are as follows: Traditional natural fermentation relies on complex and uncontrollable natural microbial communities, which easily leads to uneven fermentation products and dull colors. Although Comparative Example 2 used the exact same strains as the embodiments of this invention, the single-stage mixed inoculation method caused different strains to engage in disordered competition and mutual inhibition in the early stages of fermentation, resulting in metabolic disorders, the production of astringent substances, and an inability to effectively synthesize harmonious ester and vinegar aromas, leading to weak or chaotic aromas. Comparative Example 3, using a pre-lactic acid fermentation sequence, severely inhibited yeast activity due to the initial acidic environment, resulting in insufficient alcohol production and inadequate formation of key flavor components such as esters. Furthermore, premature acidification may also hinder the formation and transformation of certain flavor compounds, resulting in a monotonous aroma, a single, sharp sour taste, and a lack of aftertaste. Existing commercially available fermentation technologies (possibly based on a single or few strains) have limitations in terms of flavor complexity and layering. This invention, through the precise temporal coordination of multiple strains, creates a richer aroma, a softer sour taste, and a more layered flavor profile, enhancing the sensory quality and market potential of the product.

[0055] III. Determination of Organic Acid Content The content of organic acid components in the fermented grain vinegar product obtained in the examples was determined by high performance liquid chromatography (HPLC), and compared with commercially available fermented grain vinegar. The results are shown in Table 4. Figure 1 and Figure 2 As shown. The specific determination conditions for organic acid components are as follows: Sample pretreatment: The fermented lees vinegar product was centrifuged at 4,000 rpm / min for 15 min, the supernatant was collected and filtered through a 0.22 μm microporous membrane, and each sample was measured three times.

[0056] Preparation of standard curve: Accurately weigh the standards of lactic acid, acetic acid, citric acid, D-malic acid, and tartaric acid, and precisely prepare mixed standard solutions with concentrations of 0.5, 1, 5, 10, 20, 50, 100, and 200 μg / mL. Quantify using the external standard method. Plot the standard curve and linear regression equation with the mass concentration of the standard solution as the abscissa and the peak area as the ordinate.

[0057] Chromatographic conditions: Agilent ZORBAX StableBond SB-Aq column (4.6 mm × 250 mm, 5.0 μm); mobile phase: methanol: 0.1% phosphoric acid water = 96:4; isocratic elution; column temperature: 30℃; injection volume: 10 μL; flow rate: 1 mL / min; photodiode array detector; detection wavelength: 210 nm.

[0058] surface Results of organic acid content determination (unit: μg / mL) Note: ND: Not detected; different letters indicate that the examples are significantly different from commercially available samples. P <0.05).

[0059] Quantitative analysis of organic acid components was performed using high-performance liquid chromatography. The contents of major organic acids such as lactic acid, acetic acid, and citric acid in the product of this invention were significantly higher than those in the commercially available control. P <0.05). Furthermore, the lactic acid / acetic acid ratio of the product of this invention is 1.49 times that of commercially available products. Lactic acid and acetic acid are important sources of the sour taste in fermented vinegar. Lactic acid contributes a mellow, full-bodied sour taste, while acetic acid contributes a sharp, pungent sour taste. This indicates that compared to commercially available fermented vinegar, the product of this invention has a more mellow and full-bodied sour taste. In addition, the significant increase in citric acid content in the product of this invention, together with lactic acid and acetic acid, constitutes a richer, more harmonious, and more layered complex sour taste system. A certain amount of D-malic acid and tartaric acid were also detected in the product of this invention; the synergistic effect of these organic acids further enriches the flavor profile of the product. This invention, through the aforementioned preparation method, can produce fermented vinegar products with a high lactic acid / acetic acid ratio and a more complete organic acid spectrum. This demonstrates that this invention has outstanding substantial characteristics and significant progress compared to traditional methods in improving the flavor quality of fermented vinegar (making it more mellow, full-bodied, and complex). In summary, the present invention provides a method for preparing vinegar from fermented grains based on artificial complex microbial communities. Through the time-series control of multiple microbial strains and the precise optimization of fermentation conditions, it achieves simultaneous improvement in product flavor richness, fermentation efficiency, microbial stability, and quality consistency, demonstrating significant industrial application value and market competitiveness.

[0060] The above description is a further detailed explanation of the present invention in conjunction with specific preferred embodiments. It should not be considered that the specific embodiments of the present invention are limited to this. For those skilled in the art, several simple deductions or substitutions can be made without departing from the present invention, and all of these should be considered to fall within the scope of patent protection determined by the submitted claims.

Claims

1. A method for preparing vinasse vinegar based on artificial complex microbial flora fermentation, characterized in that, The method comprises the following steps: S1, raw material treatment: clean glutinous rice, soak, steam, cool down; S2, alcohol fermentation: add saccharifying enzyme, wine yeast, and seed liquid of abnormal Wickerhamomyces anomalus and water to the cooled glutinous rice, mix evenly, and conduct saccharification and alcohol fermentation at 28-30℃ to obtain alcohol fermentation mash; S3, lactic acid fermentation: inoculate seed liquid of Lactobacillus mucosus and Lactobacillus plantarum into the alcohol fermentation mash, and conduct lactic acid fermentation at 28-32℃; S4, acetic acid fermentation: when the total acid of the fermentation system reaches 2.0-3.0 g / L in terms of lactic acid, inoculate seed liquid of Pediococcus acidilactici into the fermentation system, and conduct aerobic acetic acid fermentation at 28-32℃ to obtain acetic acid fermentation mash; S5, sterilization and packaging: homogenize, sterilize, and package the acetic acid fermentation mash to obtain finished product of vinasse vinegar.

2. The method for preparing vinasse vinegar based on artificial complex microbial population fermentation according to claim 1, characterized in that, In step S1, select high-quality glutinous rice with full grains and no mildew, clean it, and then soak it in pure water for 6-8 h, with the soaking liquid level being 12-16 cm higher than the rice layer.

3. The method for preparing vinasse vinegar based on artificial complex microbial population fermentation according to claim 1, characterized in that, In step S1, drain the water from the soaked glutinous rice, add pure water to be 3 cm higher than the rice layer, and steam for 35-40 min until the glutinous rice grains are crystal clear and have no white core; after steaming, spread and cool the glutinous rice until the temperature of the glutinous rice is 30-35℃.

4. The method for preparing vinasse vinegar based on artificial complex microbial population fermentation according to claim 1, characterized in that, In step S2, the activity of the saccharifying enzyme is 100,000 U / g, and the addition amount is 0.005%-0.01% of the mass of dry glutinous rice; the alcohol fermentation time is 36-48 h, and the temperature is 28-30℃, until the alcohol content of the fermentation mash is 4%vol-6%vol.

5. The method for preparing vinasse vinegar based on artificial complex microbial population fermentation according to claim 1, characterized in that, The seed liquid concentration of the Saccharomyces cerevisiae and the Wickerhamomyces anomalus in step S2 is 1 x 10 7 5 x 10 7 CFU / mL, and the inoculation amount is 1%~2% (v / v) of the volume of the fermentation mash.

6. The method for preparing vinasse vinegar based on artificial complex microbial population fermentation according to claim 1, characterized in that, The seed liquid concentration of the L. fermentum and L. plantarum in step S3 is 1×10 7 5×10 7 CFU / mL, the inoculation amount is 0.5%-1.0% (v / v) of the volume of the fermentation mash, the fermentation temperature is 28-32℃, and the fermentation time is 24-36 h.

7. In the method, the Lactobacillus mucosus and the Lactobacillus plantarum are Lactobacillus mucosus CICC 21800 and Lactobacillus plantarum CICC 20871, respectively.

8. The method for preparing vinasse vinegar based on artificial complex microbial population fermentation according to claim 1, characterized in that, The seed liquid concentration of the Acetobacter pasteurii in step S4 is 1×10 7 5×10 7 CFU / mL, the inoculation amount is 0.5%-1.0% (v / v) of the volume of the fermentation mash; the acetic acid fermentation conditions are 28-32°C for 4-5 days, and the Acetobacter pasteurii is Acetobacter pasteurii CICC 20874.

9. The method for preparing vinasse vinegar based on artificial complex microbial population fermentation according to claim 1, characterized in that, In step S5, homogenize the acetic acid fermentation mash, sterilize it at 75-80℃ for 15-20 min, and then package it to obtain finished product of vinasse vinegar.

10. Finished product of vinasse vinegar prepared by the method of any one of claims 1-8.

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