Method for rapidly enriching caproic acid bacteria from white wine pit mud

Abstract

The invention belongs to the technical field of biology, and particularly discloses a method for rapidly enriching caproic acid bacteria from white wine pit mud. The method comprises the following steps: adding normal saline into the white wine pit mud, and uniformly mixing to obtain pit mud suspension; performing constant-temperature water bath treatment on the pit mud suspension, inoculating and transferring the pit mud suspension into a saline bottle filled with a special ES culture medium, and performing constant-temperature culture; putting the cultured fermentation liquor into a test tube, carrying out constant-temperature water bath treatment, then inoculating into a saline bottle filled with a special ES culture medium, and carrying out constant-temperature culture; and repeating the steps, and subculturing to the third generation to the fourth generation to obtain the stable caproic acid-producing microbial flora. According to the method, the microbial community structure is optimized through heat treatment, specific guiding factors are combined, the ethanol utilization type caproic acid producing bacteria with Clostridium kluyveri as the main functional bacteria can be efficiently and directionally enriched, operation is easy and convenient, the period is short, the obtained flora is high in stability, the caproic acid yield is high, and the method is suitable for follow-up enlarged culture and large-scale intensified application in baijiu brewing.

Description

A method for rapidly enriching caproic acid bacteria from baijiu cellar mud Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for rapidly enriching caproic acid bacteria from baijiu cellar mud. Background Technology

[0002] Ethyl hexanoate is the main aroma compound in strong-aroma baijiu, imparting its typical floral and tropical fruit aromas and serving as a core indicator for its quality grading. Hexanoic acid, a key precursor to ethyl hexanoate, primarily originates from the metabolic activities of the microbial community in the fermentation pit mud, especially hexanoic acid-producing functional bacteria such as *Clostridium kluyveri*, *Caproiciproducens galactitolivorans*, and *Ruminococcaceae bacterium* CPB6. These microorganisms convert single or multiple substrates such as ethanol, lactic acid, and glucose into hexanoic acid in the fermentation pits, which is then esterified to produce ethyl hexanoate, thus shaping the typical style of baijiu. However, high-quality fermentation pit mud capable of efficiently supporting the growth and metabolism of these functional bacteria requires long-term aging to form a stable and enriched micro-ecosystem. This lengthy natural aging process severely restricts the production efficiency and capacity of high-quality strong-aroma baijiu, resulting in a high concentration of high-quality fermentation pit mud resources in a few enterprises with long-term continuous brewing capabilities. Therefore, overcoming the time barrier of natural fermentation pit mud formation has become a key challenge for improving the controllability of strong-aroma baijiu quality and the sustainability of the industry.

[0003] Hexanoic acid-producing microorganisms exhibit a clear metabolic preference in substrate utilization, generally falling into two main categories: ethanol-utilizing and lactic acid-utilizing. *Clostridium kluyveri* is a typical ethanol-utilizing bacterium, capable of efficiently synthesizing hexanoic acid via a reverse β-oxidation pathway using ethanol as an electron donor and acetic acid as a carbon source. *Caproiciproducens spp.*, on the other hand, is predominantly lactic acid-utilizing, primarily using lactic acid and other organic acids as substrates to produce hexanoic acid. Studies have shown that these two types of functional bacteria widely coexist in the cellar mud of high-quality strong-aroma baijiu, collectively forming the core microbial community basis for hexanoic acid production.

[0004] However, current enrichment methods for caproic acid-producing bacteria in fermentation pits mainly focus on lactic acid-utilizing bacteria, generally relying on yellow water (a byproduct of baijiu brewing, rich in lactic acid, acetic acid, and complex nutrients) or lactic acid as a guiding factor, and requiring strict anaerobic conditions—usually requiring cultivation in a highly airtight fermentation device with protective gases such as nitrogen. Mainstream methods include: i) Enrichment using activated culture medium: For example, Chinese invention patent CN112011419A discloses a method for preparing a multi-species compound bacterial solution rich in caproic acid bacteria and producing high caproic acid through a two-stage fermentation process of "first activation, then enrichment." This method requires fully mixed stirring cultivation in a sealed fermenter, with continuous replenishment of culture medium and continuous N2 purging to maintain a strictly anaerobic environment. This method can effectively enrich caproic acid-producing bacteria, with a maximum relative abundance of up to 40.9% (an increase of approximately 40% from the initial level), but its enrichment effect on Clostridium species is relatively limited, with a maximum relative abundance of only 19.5% (an increase of approximately 17%). ii) Continuous transfer enrichment method: Based on enhanced culture, guiding factors such as yellow water are introduced, and multiple rounds of dilution and transfer are combined to improve selectivity. For example, patent CN116286452A inoculates the pit mud suspension into a culture medium containing 15% (v / v) yellow water and enhanced Daqu, and cultures it at 37°C under N2 protection. After multiple rounds of expansion culture, it successfully promotes the synthesis of butyric acid and hexanoic acid. However, this method is cumbersome and takes more than 40 days. iii) Long-term simulated culture method: For example, the literature "Complete genome sequence of Ruminococcaceaebacterium CPB6: A newly isolated culture for efficient n-caproic acid production from lactate" (Journal of Biotechnology, 2017, 259, 91-94) simulates the natural aging process of pit mud by adding yellow water to the anaerobic fermenter. This method can obtain a stable and abundant hexanoic acid-producing bacterial community. However, this method requires high equipment and the experimental cycle usually exceeds 100 days, which is difficult to meet the needs of efficient research and development and industrial application. Moreover, the above-mentioned enrichment methods mostly rely on lactic acid or yellow water as guiding factors, which can easily lead to ethanol-utilizing caproic acid-producing bacteria (such as Clostridium kluyveri) being masked by competitors such as lactic acid-utilizing bacteria during the enrichment process. This not only limits their enrichment efficiency but also affects the systematic analysis of the microecology of the pit mud and the precise regulation of functional microbial communities.

[0005] Therefore, developing a targeted enrichment method for ethanol-utilizing caproic acid-producing bacteria will help fully tap the caproic acid production potential of cellar mud and provide a key path to overcome the bottlenecks of existing technologies in terms of cycle, cost and equipment. It is an important direction for realizing the efficient utilization of functional microbial resources in baijiu. Summary of the Invention

[0006] To address the insufficient selectivity of existing enrichment methods for ethanol-utilizing caproic acid-producing bacteria, this invention proposes a rapid and efficient method for enriching this functional microbial community. Using pit mud as raw material, this enrichment method first reduces redundant species in the native microorganisms through heat treatment, lowering niche competition. Then, using ethanol as a specific guiding factor, several rounds of transfer are used to directionally enrich ethanol-utilizing caproic acid-producing bacteria, with *Clostridium kluyveri* as the main functional bacterium. The resulting mixed microbial community not only exhibits strong caproic acid synthesis capacity but also has a stable community structure and reproducible functions. This enrichment method is simple to operate, has a short cycle time, and low equipment requirements (no need for sealed anaerobic tanks or continuous feeding systems), and possesses good potential for process scale-up. It is particularly suitable for the enhanced preparation and application of functional microbial agents in the brewing process of baijiu (Chinese liquor).

[0007] To achieve the above objectives, the present invention adopts the following technical solution: a method for rapidly enriching caproic acid bacteria from baijiu cellar mud, comprising the following steps: (1) adding physiological saline to baijiu cellar mud, mixing well to obtain cellar mud suspension; (2) treating the cellar mud suspension obtained in step (1) in a constant temperature water bath, inoculating and transferring it to a saline bottle containing specialized ES medium, and incubating at a constant temperature, this batch is regarded as the first generation culture; (3) taking the fermentation liquid obtained in step (2) and placing it in a test tube, treating it in a constant temperature water bath, and then inoculating it into a saline bottle containing specialized ES medium, and incubating at a constant temperature, this batch is regarded as the second generation culture; (4) repeating step (3), subculturing to the third to fourth generation to obtain a stable caproic acid-producing microbial community.

[0008] Preferably, the pH of the baijiu cellar mud in step (1) is 4.8-6.8, the humus content is 8-12wt%, the water content is 50-70wt%, and the available phosphorus is 400-800mg / kg.

[0009] Preferably, the weight ratio of the liquor cellar mud to physiological saline in step (1) is 1:8-10.

[0010] Preferably, the concentration of the physiological saline is 8-10 g / L.

[0011] Preferably, the mixing in step (1) is as follows: add physiological saline to the baijiu cellar mud, then add glass beads, and mix with a glass rod to obtain a cellar mud suspension.

[0012] Preferably, the glass beads have a diameter of 0.2-0.4 cm, and the weight ratio of the glass beads to the liquor cellar mud is 1:35-45.

[0013] Preferably, the preparation method of the specialized ES culture medium in step (2) or (3) is as follows: 0.8-1.2g of yeast powder, 4-7g of peptone, 4-6g of sodium acetate, 0.15-0.35g of magnesium sulfate heptahydrate, 0.3-0.5g of dipotassium hydrogen phosphate, 0.4-0.8g of ammonium sulfate, 0.3-0.8g of L-cysteine ​​hydrochloride, and 970-980mL of distilled water are mixed, with a pH of 6.8-6.9. After sterilization and cooling to room temperature, 20-30mL of anhydrous ethanol is added.

[0014] Preferably, the volume ratio of the inoculation mud suspension / fermentation broth to the specialized ES culture medium in step (2) or (3) is 3-6%.

[0015] Preferably, the constant temperature water bath in step (2) or (3) is 70-80℃ and the time is 10-15min.

[0016] Preferably, the constant temperature culture in step (2) or (3) is 35-38℃ and the time is 5-10 days.

[0017] The positive and beneficial effects of this invention are as follows: 1. The cellar mud of this invention is fine, soft, and elastic. It can be formed into a ball by hand and easily crumbles when rubbed, without any gritty feel. This indicates that it has a good granular structure and porosity, which is conducive to the retention of moisture and nutrients and can form a stable anaerobic microenvironment, providing ideal conditions for the colonization and metabolism of strictly anaerobic functional microorganisms such as caproic acid bacteria and butyric acid bacteria. In addition, there are no white crystals (such as calcium lactate, sodium acetate, etc.) in the cellar mud, indicating that there has been no serious aging or salt accumulation, and the ionic environment is balanced, effectively avoiding the inhibition of functional bacteria. In terms of sensory aspects, the cellar mud presents a rich and pure complex cellar aroma with a slight fruity ester aroma, without any off-flavors or putrid odors. This directly reflects the coordinated generation of organic acids such as caproic acid, butyric acid, and acetic acid and their corresponding esters (such as ethyl hexanoate and ethyl butyrate), which is an important indicator of the activity of acid-producing functional bacteria such as caproic acid bacteria. This macro-evaluation standard based on appearance and odor helps operators quickly and initially screen out relatively high-quality cellar mud on-site, laying the foundation for further refined selection through physicochemical indicators and nutritional components. In terms of physicochemical properties, the cellar mud has a pH of 4.8-6.8, falling within the weakly acidic range, which matches the optimal growth pH (usually 6.0-7.0) of caproic acid bacteria (such as Clostridium kluyveri) in baijiu cellars. This promotes caproic acid synthesis while effectively inhibiting the excessive proliferation of other bacteria. Humus, as a natural organic colloid with a mass content of 8-12%, can chelate metal ions, buffer pH fluctuations, adsorb and slowly release nutrients, and also serve as a long-term carbon source to support long-term continuous fermentation, maintaining the stability of the cellar mud's micro-ecosystem. Simultaneously, the available phosphorus content of 400-800 mg / kg provides ample guarantee for microbial synthesis of key components such as nucleic acids, ATP, and coenzymes, indicating that the cellar mud is rich in nutrients, sufficient to support the vigorous growth and metabolic activity of a high-density functional microbial community.

[0018] 2. In this invention, a microbial suspension is extracted from the baijiu cellar mud using a water-soluble method and then inoculated into a liquid culture medium. This allows for more uniform dispersion of microorganisms in the suspension, avoiding the uneven inoculation problem caused by direct inoculation of cellar mud blocks. The cellar mud suspension / fermentation liquid is then treated in a constant-temperature water bath at 70-80℃ for 10-15 minutes. This constant-temperature water bath heat treatment acts as a strong selective pressure, significantly reshaping the composition of the cellar mud microbial community. During the treatment, non-spore-forming bacteria that are intolerant to high temperatures are eliminated, reducing microbial redundancy in the cellar mud microbial community. The relative abundance of heat-resistant and sporulating microorganisms (such as Clostridium kluyveri) increases significantly.

[0019] 3. This invention uses a specialized ES medium, which, unlike traditional ES medium, uses a milder L-cysteine ​​hydrochloride as the antioxidant. Simultaneously, anhydrous ethanol is added as an enrichment guide factor to target the substrate feeding preferences of Clostridium caproate, and this is added after the medium has cooled to avoid ethanol evaporation caused by the high temperature of the sterilized medium. Using ethanol as a guide factor in this invention offers advantages such as high selectivity, a clear metabolic pathway, simple operation, and good reproducibility. It effectively drives Clostridium caproate to synthesize caproate using ethanol and acetic acid, while inhibiting the growth of non-target bacteria. This method is suitable for targeted enrichment, resulting in a stable caproate-producing microbial community with high caproate concentration. It solves the problem that "traditional methods use complex yellow water as a composite carbon source and growth factor, which suffers from unstable composition, significant batch-to-batch variations, and the potential introduction of contaminating bacteria or unknown inhibitors, affecting the controllability and reproducibility of enrichment."

[0020] 4. This invention utilizes the characteristics of caproic acid-producing bacteria, employing a liquid static submerged culture at a temperature of 35-38℃ for 5-10 days. This allows the microorganisms to be evenly dispersed in the culture medium, facilitating biomass counting. Furthermore, the static submerged culture provides a favorable anaerobic environment, maximizing caproic acid production. Since the microbial cells inevitably settle at the bottom of the fermentation vessel, and caproic acid-producing bacteria are strictly anaerobic, the transfer process involves slowly mixing the cells using a pipette. The saline bottle should be equipped with a one-way valve cap to prevent gas generation during cultivation from opening the cap and contaminating the culture medium, thus avoiding contact with oxygen.

[0021] 5. This invention optimizes the microbial community structure through heat treatment and, combined with specific guiding factors, can directionally enrich caproic acid-producing microorganisms. Caproic acid-producing microbial communities obtained from Baijiu cellar mud are passaged to the third or fourth generation. The experimental cycle is 21-28 days. The operation method is simple, the operation cycle is short, and the maximum cumulative biomass OD during the fermentation cycle is [missing information]. 600 The peak value was 0.9, and the cumulative concentration of hexanoic acid in the fermentation broth reached as high as 5.62 g / L, which significantly increased the hexanoic acid content in the liquor and can be used for subsequent expansion culture and large-scale baijiu enhancement culture. Attached Figure Description

[0022] Figure 1 shows the colorimetric effect of the enriched hexanoic acid bacteria in Example 1 of the present invention; Figure 2 shows the changes in biomass and the content of major volatile acids (acetic acid, butyric acid, hexanoic acid) in Example 1 of the present invention during the complete fermentation cycle; Figure 3 shows the changes in β diversity in different rounds of Example 1 of the present invention. Detailed Implementation

[0023] The present invention will be further described below with reference to some specific embodiments.

[0024] Example 1 A method for rapidly enriching caproic acid bacteria from baijiu cellar mud, comprising the following steps: (1) Obtaining strong-aroma baijiu cellar mud, the cellar mud is dark brown and has no white crystals; the texture is delicate, soft and elastic, can be kneaded into a ball by hand and easily dispersed by rubbing, without a gritty feel; it has a rich and pure compound cellar aroma, slightly fruity ester aroma, and no off-flavors or putrid odors; the pH is 5.0, the humic content is 11wt%, the water content is 60wt%, and the available phosphorus is 720mg / kg, indicating that it is in a good active state; (2) Adding physiological saline to the strong-aroma baijiu cellar mud, the weight ratio of strong-aroma baijiu cellar mud to physiological saline is 1:9, the concentration of physiological saline is 9g / L, placed in a 250mL conical flask, adding 3-6 glass beads and then carefully mixing with a glass rod to avoid violent shaking as much as possible, the diameter of the glass beads is 0.2-0.4cm, to obtain a cellar mud suspension; (3) The step ( 2) The obtained pit mud suspension was heated in a constant temperature water bath at 75℃ for 10 minutes. After cooling to room temperature, the treated pit mud suspension was slowly mixed by pipetting with a 5mL pipette. It was then transferred to a 100mL saline bottle containing specialized ES medium at an inoculation ratio of 5% (V / V). The bottle was then incubated in a liquid static deep layer at 37℃ for 7 days, with three replicates. This batch was considered the first generation culture. The saline bottle used should be equipped with a one-way valve cap to prevent gas generation during the culture process from opening the cap and contaminating the medium. Specialized ES medium was prepared by mixing 1g yeast powder, 5g peptone, 5g sodium acetate, 0.2g magnesium sulfate heptahydrate, 0.4g dipotassium hydrogen phosphate, 0.5g ammonium sulfate, 0.5g L-cysteine ​​hydrochloride, and 980mL distilled water. The pH was 6.8-6.9. After sterilization, the mixture was placed in a clean bench and cooled to room temperature before adding 20mL of anhydrous ethanol.

[0025] (4) The content of hexanoic acid was determined by the copper sulfate-hexanoic acid colorimetric method. The culture medium with the best colorimetric effect was used for subculturing. After slowly mixing with a 5 mL pipette, 30 mL of fermentation broth was placed in three test tubes (10 mL each). The test tubes were then placed in a 75°C constant temperature water bath for 10 min. After cooling to room temperature, the culture medium was slowly mixed with a 5 mL pipette. Then, the culture medium was inoculated into three 100 mL saline bottles containing specialized ES culture medium (the same as in step (3)) at an inoculation ratio of 5% (V / V). Liquid culture in a constant temperature incubator at 7℃ for 7 days is considered second-generation culture. The saline bottle used should be equipped with a one-way valve cap to prevent gas generation during culture from opening the cap and contaminating the culture medium. The steps for detecting hexanoic acid content by the copper sulfate-hexanoic acid colorimetric method are as follows: Take 5 mL of fermentation broth into a colorimetric tube, add 1 mL of 2% copper sulfate solution and 1 mL of petroleum ether, mix thoroughly, and let it stand to separate into layers. When the hexanoic acid content in the fermentation broth reaches the detection line, the petroleum ether extract layer will show blue or blue-green color. The darker the color (the better the colorimetric effect), the higher the hexanoic acid content.

[0026] Repeat step (4) until the fourth generation of culture to obtain a stable caproic acid-producing microbial community.

[0027] Example 2 The method for rapidly enriching caproic acid bacteria from the cellar mud of Baijiu in this example is basically the same as that in Example 1. The similarities will not be repeated. The differences are: the pH of the strong aroma type Baijiu cellar mud in step (1) is 5.2, the humus content is 10wt%, the water content is 70wt%, and the available phosphorus is 600mg / kg.

[0028] Example 3 The method for rapidly enriching hexanoic acid functional bacteria from the mud of a liquor cellar in this example is basically the same as that in Example 1. The similarities will not be repeated. The difference is that the constant temperature water bath temperature in steps (3) and (4) is 80°C.

[0029] Comparative Example 1: The method for rapidly enriching caproic acid bacteria from the mud of a liquor cellar in this embodiment is basically the same as that in Example 1. The similarities will not be repeated. The difference is that the culture was repeated up to the seventh generation.

[0030] Comparative Example 2 The method for rapidly enriching hexanoic acid functional bacteria from the baijiu cellar mud in this example is basically the same as that in Example 1. The similarities will not be repeated. The differences are: in steps (3) and (4), the amount of anhydrous ethanol in the specialized ES culture medium is 10 mL and the amount of distilled water is 990 mL.

[0031] Comparative Example 3 The method for rapidly enriching caproic acid bacteria from the cellar mud of baijiu is basically the same as that in Example 1. The similarities will not be repeated. The difference is that the pH of the strong-aroma baijiu cellar mud in step (1) is 3.5.

[0032] Comparative Example 4 The method for rapidly enriching caproic acid bacteria from the cellar mud of strong-aroma baijiu is basically the same as that in Example 1. The similarities will not be repeated. The difference is that the constant temperature water bath temperature in steps (3) and (4) is 60°C.

[0033] The comparative example 5 is basically the same as the comparative example 1. The similarities will not be repeated. The difference is that the special ES medium mentioned in steps (3)-(5) is replaced with glucose medium. The specific composition is as follows: glucose medium (g / L): glucose 10g, peptone 5g, dipotassium hydrogen phosphate 0.5g, yeast powder 5g, ammonium sulfate 1g, ferrous sulfate 0.0075g, magnesium sulfate heptahydrate 0.05g, calcium chloride 0.005g, manganese sulfate monohydrate 0.005g, zinc sulfate 0.001g, cobalt chloride 0.001g, potassium dihydrogen phosphate 0.25g, distilled water 1000mL, pH adjusted to 7 before sterilization.

[0034] The comparative example 6 uses a method that is basically the same as that of example 1. The similarities will not be repeated. The differences are as follows: the specialized ES medium mentioned in steps (3)-(5) is replaced with CPB6 medium. The specific composition is as follows: CPB6 medium (g / L): ammonium chloride 0.25g, magnesium sulfate heptahydrate 0.2g, potassium dihydrogen phosphate 0.23g, dipotassium hydrogen phosphate 0.31g, sodium chloride 0.8g, L-cysteine ​​hydrochloride 0.25g, lactic acid 30g, vitamin solution 1mL, trace element nutrient solution 1mL, distilled water 1000mL, sterilized; wherein, vitamin solution (g / L): 2.0g of thymine triacetic acid, 1.0g of manganese sulfate monohydrate. 0g, ferrous ammonium sulfate hexahydrate 0.8g, cobalt chloride hexahydrate 0.2g, zinc sulfate heptahydrate 0.0002g, copper chloride dihydrate 0.02g, nickel chloride hexahydrate 0.02g, sodium molybdate dihydrate 0.02g, sodium selenate dihydrate 0.02g, sodium tungstate 0.02g, distilled water 1000mL, sterilized; Trace element nutrient solution (mg / L): vitamin B12 100mg, para-aminobenzoic acid 80mg, D-biotin 20mg, nicotinic acid 200mg, calcium pantothenate 100mg, pyridoxine hydrochloride 300mg, thiamine hydrochloride 200mg, distilled water 1000mL, adjust pH to 6~6.5 before sterilization.

[0035] The comparative example 7 is basically the same as the comparative example 1. The similarities will not be repeated. The difference is that the special ES medium mentioned in steps (3)-(5) is replaced with the traditional ES medium. The specific composition is as follows: traditional ES medium: 1g yeast powder, 5g peptone, 5g sodium acetate, 0.2g magnesium sulfate heptahydrate, 0.4g dipotassium hydrogen phosphate, 0.5g ammonium sulfate, 0.5g ferrous sulfate, 980mL distilled water. After sterilization, it is placed in a clean bench and cooled to room temperature before adding 20mL anhydrous ethanol.

[0036] The method of Comparative Example 8 is basically the same as that of Example 1. The similarities will not be repeated. The differences are as follows: the constant temperature water bath treatment is omitted in steps (3) and (4). Specifically, in step (3), the pit mud suspension obtained in step (2) is transferred to a 100mL saline bottle containing specialized ES medium at an inoculation ratio of 5% (V / V). The mixture is then placed in a 37℃ constant temperature incubator for 7 days for liquid static deep culture. Three replicates are set up. This batch is regarded as the first generation culture. The saline bottle used should be equipped with a one-way gas valve cap to prevent gas generation during the culture process. (4) Use the copper sulfate-hexanoic acid colorimetric method to detect the hexanoic acid content, take the culture medium with the best colorimetric effect for subculture, and then inoculate into three 100mL saline bottles containing specialized ES culture medium at an inoculation ratio of 5% (V / V). Incubate in a 37℃ constant temperature incubator for 7 days in a liquid static deep culture. This batch is considered as the second generation culture. The saline bottles used should be equipped with one-way gas valve caps to prevent gas generation during the culture process from opening the bottle caps and contaminating the culture medium; (5) Repeat step (4) until the seventh generation culture.

[0037] The comparative method of Comparative Example 9 is basically the same as that of Example 1. The similarities will not be repeated. The differences are as follows: Step (1) The cellar mud is replaced with cellar bottom mud of high-quality baijiu that has been selected for more than 10 years and can stably produce baijiu that meets the standard of GB / T10781.1-2021 strong aroma baijiu. The cellar mud is dark brown with a little white crystals; the texture is slightly rough with a little particle and a little cellar aroma.

[0038] Comparative Example 10 The method of this comparative example is basically the same as that of Example 1. The similarities will not be repeated. The differences are as follows: Step (2) add physiological saline to the strong aroma type baijiu cellar mud. The weight ratio of strong aroma type baijiu cellar mud to physiological saline is 1:9. The concentration of physiological saline is 9g / L. Place it in a 250mL conical flask and mix it carefully with a glass rod, avoiding violent shaking as much as possible, to obtain cellar mud suspension.

[0039] The content of hexanoic acid obtained by the enrichment methods of Examples 1-3 and Comparative Examples 1-10 of the present invention was detected, and the detection results are shown in Table 1 and Figure 1 below.

[0040] Table 1 Results of hexanoic acid content detection

[0041] As shown in Table 1, Example 1, employing the complete screening and enrichment method described in this invention, achieved a peak yield (5.62 g / L) in the 4th generation (G4). Example 2 relaxed the initial sludge screening criteria, resulting in slightly lower initial microbial diversity or functional potential compared to Example 1. Its hexanoic acid yield in the G1 generation and subsequent generations was also slightly lower than that of Example 1. Example 3 used a higher water bath pretreatment temperature, applying stronger environmental selection pressure. While this treatment helped eliminate non-tolerant bacteria, it also caused sublethal damage to some functional bacteria, resulting in a lower hexanoic acid yield in the G1 generation, and the yield tended to stabilize between the G3 and G4 generations.

[0042] Comparative Example 1 and Example 1 showed similar performance in the first four generations (G1–G4) of culture, both exhibiting the expected trend of steadily increasing hexanoic acid production. However, starting from the fifth generation, although Comparative Example 1 showed a further narrowing of intragroup differences in the G5–G7 stages, hexanoic acid production failed to continue to increase, instead stagnating or even slightly declining. This indicates that after G4, continuous heat treatment for passage has entered a marginal effect stage—the contribution of heat treatment to the enrichment of hexanoic acid-producing bacteria has significantly weakened, and the damage it causes to cells may have outweighed the selective advantage, thus limiting further increases in production. After three to four generations of enrichment, the enrichment content of hexanoic acid-producing functional bacteria in this invention already meets the requirements.

[0043] Although the hexanoic acid yield in Comparative Example 2 showed a similar growth trend to that in Example 1, its maximum yield was lower than that in Example 1. This was mainly due to the lack of ethanol in the culture medium—a key electron donor in the hexanoic acid synthesis pathway. The absence of ethanol led to insufficient substrate supply, thus limiting the production of hexanoic acid. The hexanoic acid yield in Comparative Example 3 did not show a clear pattern. This was because the acidity of the selected pit mud was too high, which not only resulted in a low initial relative abundance of hexanoic acid bacteria, making rapid enrichment difficult, but also the presence of a large number of lactic acid bacteria in the high-acid pit mud further inhibited the accumulation of hexanoic acid. Comparative Example 4 used a milder heat treatment method with a constant water bath temperature of 60°C. Although this resulted in a continuous and slow increase in hexanoic acid yield, the intra-group variation was consistently greater than that in Example 1, and the yield was generally lower than that in Example 1 at the same generation number. This indicates that while the weaker selection pressure alleviated the "niche depletion" of the community and preserved more microbial species, it also intensified interspecific competition, making it difficult for functional bacteria (such as hexanoic acid-producing bacteria) to dominate, thus reducing enrichment efficiency.

[0044] The culture media used in Comparative Examples 5 and 6 failed to effectively support the enrichment of hexanoic acid-producing bacteria in this system, and the overall performance was poor. Comparative Example 7 showed acceptable results, but it was not the optimal choice. Comparative Example 8 completely omitted constant temperature water bath heat treatment and relied solely on specialized ES medium for multiple rounds of culture. Its hexanoic acid yield showed a fluctuating upward trend, but the intra-group differences were the most significant among all groups, reflecting that in the absence of directional selection pressure, the assembly of the microbial community was dominated by a random process, resulting in unstable fermentation performance and limiting its application value. Comparative Example 9 lowered the screening criteria for pit mud. Although it was able to obtain hexanoic acid-producing bacteria in four rounds of enrichment, its initial (G1) and peak hexanoic acid yields were lower than those in Example 1, indicating that the quality of the raw pit mud has a decisive influence on the upper limit of enrichment. Comparative Example 10 dispersed the pit mud in physiological saline by stirring with a glass rod without adding glass beads, which failed to effectively release or activate functional bacteria, resulting in a low maximum hexanoic acid yield. Although the yield plateau was not reached within four generations, the increase was limited, and the enrichment time was too long, which affected rapid enrichment.

[0045] As shown in Figure 1, the petroleum ether extraction layer of sample G1 in Example 1 is almost colorless, exhibiting a slightly emulsified, translucent appearance, indicating a low hexanoic acid content. The extraction layer of sample G2 shows a lighter green color, indicating that the hexanoic acid concentration has reached a detectable level. Samples G3 and G4, however, show a distinct deep blue-green petroleum ether layer, indicating that hexanoic acid production increases further with further enrichment. Therefore, this invention demonstrates optimal overall performance in terms of enrichment efficiency, yield level, community stability, and operational economy. The combination of its heat treatment intensity and the sludge screening standards achieves efficient and robust enrichment of hexanoic acid-producing functional microbial communities.

[0046] As shown in Figure 2, after culturing the G4 generation of Example 1 for 7 days, it was cultured for another 3 days. During the 10-day fermentation process, the biomass of the G4 community began to increase significantly on day 4, reaching a peak (0.9) on day 7, and then tended to stabilize, indicating that the cells entered the logarithmic growth phase and entered the stationary phase around day 7. Correspondingly, the concentrations of hexanoic acid and butyric acid accumulated rapidly from day 4 to day 7, with hexanoic acid reaching its highest value (2.0 g / L) on day 7, slightly higher than butyric acid (1.5 g / L). Thereafter, both maintained relatively stable levels, indicating that their synthesis mainly occurred during the vigorous growth phase, which is consistent with the characteristics of typical growth-coupled metabolites. Acetic acid, as one of the initial carbon sources, remained at a high level in the early stages of fermentation (days 0-4), with an average concentration of 2.64 g / L. It was gradually consumed as the cells grew and acid production progressed. From day 5 onwards, the acetic acid concentration decreased significantly, reaching an average of 1.68 g / L by day 7. This consumption trend was highly synchronized with the peak synthesis of hexanoic acid and butyric acid, indicating that acetic acid is an important precursor for the synthesis of short-chain fatty acids and exhibits a clear metabolic coupling relationship. Furthermore, the initial high concentration of acetic acid stemmed from the specialized ES medium formulation, providing ample substrate support for subsequent acid production. The biomass growth and hexanoic acid accumulation curves rose almost parallel from day 4 to day 7, further validating the ability of this bacterial community to efficiently synthesize hexanoic acid using ethanol as a carbon source through a growth-coupled pathway.

[0047] The microbial composition during multiple rounds of enrichment culture in Example 1 was detected using high-throughput sequencing technology. The results are shown in Table 2.

[0048] Table 2. Microbial composition of the four enrichment rounds in Example 1

[0049] As shown in Table 2, during the four rounds of enrichment culture in Example 1, the overall relative abundance of *Clostridium sensu stricto 12* in the pit mud microbial community significantly increased. Simultaneously, the relative abundance of *Clostridium kluyveri* within this genus increased from an initial 1.96% to a maximum of 58.47%, becoming the most significant contributor to the increased abundance within this genus. In contrast, the relative abundance of other members of the *Clostridium sensu stricto 12* genus significantly decreased from 23.49% to 0.81%. These results demonstrate that the enrichment method of this invention exhibits high selectivity for *Clostridium kluyveri*, enabling targeted enrichment while effectively inhibiting the proliferation of other species within the same genus, thus achieving precise and targeted regulation of the functional microbial community structure.

[0050] To further clarify the enrichment effect of Example 1, this invention used high-throughput sequencing technology to analyze the dynamic changes of the microbial community in the four generations of enriched samples (G1 to G4), as shown in Figure 3. Figure 3 shows that, based on PCoA (principal coordinate analysis) combined with the PERMANOVA test, there was a highly significant difference in β-diversity among samples G1-G4 (p<0.01), indicating that the community structure underwent significant succession with increasing enrichment generations. However, the difference in β-diversity between G3 and G4 was not significant (p=0.3), indicating that the community structure tended to stabilize after the third generation. The PCoA plot shows that samples G1 and G2 clustered in the second and third quadrants, respectively, while samples G3 and G4 highly overlapped, concentrated in the boundary region between the first and fourth quadrants. This distribution pattern clearly reflects that the heat treatment enrichment method adopted in this invention can rapidly and effectively reshape the microbial community structure. Furthermore, the high overlap between G3 and G4 reflects the marginal effect in the enrichment process, meaning that further enrichment is unlikely to cause significant changes in the community. This provides a clear biological signal for accurately determining the experimental endpoint, effectively avoiding time waste and potential cell function damage caused by over-treatment. In addition, the intra-group dispersion of each sample group significantly decreases with increasing enrichment generations, indicating that although there is some community heterogeneity among the initial samples, after three to four generations of enrichment, this method can stably obtain structurally highly similar hexanoic acid-producing functional bacterial communities, demonstrating good reproducibility and process robustness.

[0051] The application experiment was conducted at a winery in Henan Province. After obtaining stable caproic acid-producing bacteria using the enrichment method described in Example 1 of this invention, the bacteria were progressively expanded to a three-stage seed culture. The culture conditions for each stage of the seed culture were: specialized ES medium, anaerobic culture at 37°C for 5 days, and an inoculum size of 10% (v / v). The expanded three-stage seed culture was evenly sprinkled onto the upper layer of mash in the fermentation pit and immediately sealed with mud. The ratio of mash to seed culture was 1000 kg mash: 5 L seed culture. After 40 days of fermentation, the liquor was distilled and ethyl caproic acid was tested. The traditional strong-aroma baijiu production process without the addition of seed culture was used as a comparison. The test results are shown in Table 3 below.

[0052] Table 3. Detection results of ethyl hexanoate content in Baijiu (Chinese liquor)

[0053] As shown in Table 3, the functional microorganisms producing hexanoic acid obtained by the method of the present invention (under the conditions described in Example 1) can effectively colonize in the actual Baijiu brewing system and significantly improve the synthesis level of ethyl hexanoate, thereby improving the flavor and quality of Baijiu. This further verifies the practicality, scalability and industrial application value of the enrichment method of the present invention. Even under different cellar backgrounds (such as 81#, 51# and 87#), the content of ethyl hexanoate can still be stably increased, demonstrating good process stability.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Any other modifications or equivalent substitutions made by those skilled in the art to the technical solutions of the present invention, as long as they do not depart from the spirit and scope of the technical solutions of the present invention, should be covered within the scope of the claims of the present invention.

Claims

1. A method for rapidly enriching caproic acid bacteria from liquor cellar mud, characterized in that, Includes the following steps: (1) Add physiological saline to the strong-aroma baijiu cellar mud, mix well, and obtain cellar mud suspension; (2) Treat the cellar mud suspension obtained in step (1) in a constant temperature water bath, inoculate and transfer it to a saline bottle containing a special ES medium, and incubate at a constant temperature. This batch is considered as the first generation of culture; (3) Take the fermentation liquid obtained in step (2) and place it in a test tube, treat it in a constant temperature water bath, and then inoculate it into a saline bottle containing a special ES medium, and incubate at a constant temperature. This batch is considered as the second generation of culture; (4) Repeat step (3) and subculture to the third to fourth generation to obtain a stable hexanoic acid-producing microbial community.

2. The method for rapidly enriching caproic acid bacteria from liquor cellar mud according to claim 1, characterized in that, The pH of the baijiu cellar mud in step (1) is 4.8-6.8, the humus content is 8-12wt%, the water content is 50-70wt%, and the available phosphorus is 400-800mg / kg.

3. The method for rapidly enriching caproic acid bacteria from liquor cellar mud according to claim 1, characterized in that, The weight ratio of the baijiu cellar mud to physiological saline in step (1) is 1:8-10.

4. The method for rapidly enriching caproic acid bacteria from liquor cellar mud according to claim 3, characterized in that, The concentration of the saline solution is 8-10 g / L.

5. The method for rapidly enriching caproic acid bacteria from liquor cellar mud according to claim 1, characterized in that, The mixing in step (1) is as follows: add physiological saline to the baijiu cellar mud, then add glass beads, and mix with a glass rod to obtain a cellar mud suspension.

6. The method for rapidly enriching caproic acid bacteria from liquor cellar mud according to claim 5, characterized in that, The glass beads have a diameter of 0.2-0.4 cm, and the weight ratio of the glass beads to the liquor cellar mud is 1:35-45.

7. The method for rapidly enriching caproic acid bacteria from liquor cellar mud according to claim 1, characterized in that, The preparation method of the specialized ES culture medium described in step (2) or (3) is as follows: Mix 0.8-1.2g of yeast powder, 4-7g of peptone, 4-6g of sodium acetate, 0.15-0.35g of magnesium sulfate heptahydrate, 0.3-0.5g of dipotassium hydrogen phosphate, 0.4-0.8g of ammonium sulfate, 0.3-0.8g of L-cysteine ​​hydrochloride, and 970-980mL of distilled water. The pH should be 6.8-6.

9. After sterilization, let it cool to room temperature and then add 20-30mL of anhydrous ethanol.

8. The method for rapidly enriching caproic acid bacteria from liquor cellar mud according to claim 1, characterized in that, The volume ratio of the inoculated pit mud suspension / fermentation broth to the specialized ES culture medium in step (2) or (3) is 3-6%.

9. The method for rapidly enriching caproic acid bacteria from liquor cellar mud according to claim 1, characterized in that, The constant temperature water bath temperature in step (2) or (3) is 70-80℃ and the time is 10-15min.

10. The method for rapidly enriching caproic acid bacteria from liquor cellar mud according to claim 1, characterized in that, The constant temperature culture in step (2) or (3) is 35-38℃, and the time is 5-10 days.

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