Method for rapidly enriching functional bacteria group with high yield of caproic acid in pit mud and application thereof

Abstract

The application discloses a method for rapidly enriching a high-capacity hexanoic acid functional bacterial group in pit mud and application thereof, and belongs to the technical field of liquor brewing microorganisms. The application uses old pit mud with better quality which is tested and screened as seed mud, and adopts a specific culture medium to perform step-by-step expansion culture on the selected high-quality pit mud. The obtained bacterial group is mainly composed of Clostridium caproicum (85%), and the bacterial group can produce more than 10 g / L of hexanoic acid and less than 2 g / L of butyric acid. The mixed bacterial group obtained by the method can be used for pit mud culture and repair, increase of the main bacterial group for producing hexanoic acid in the pit mud, promotion of pit mud aging, prevention of pit mud aging and hardening, and effective improvement of the quality of raw liquor. The mixed bacterial group can also be used for yellow water fermentation of by-products of Luzhou-flavor liquor, high-capacity hexanoic acid production, and further production of high-quality seasoning liquor.

Description

Technical Field

[0001] This invention relates to a method and application for rapidly enriching high-yield hexanoic acid-producing functional bacteria in cellar mud, belonging to the field of microbial technology in Baijiu brewing. Background Technology

[0002] Strong-aroma baijiu is the largest baijiu variety in my country, accounting for about 60% of the market. It is characterized by its "rich cellar aroma, smooth and sweet taste, and mellow and harmonious body." Mud-cellar brewing is a unique production method for strong-aroma baijiu, and the quality of the cellar mud is a core element affecting the flavor and quality of the baijiu. Among these factors, the metabolic activity of caproic acid-producing functional bacteria (especially *Acroxoproteobacterium*) is a precursor to the synthesis of the key flavor component ethyl hexanoate, directly influencing the typical style and sensory quality of strong-aroma baijiu. Currently, the cultivation and optimization of traditional cellar mud mainly relies on natural aging, which is time-consuming and inefficient, making it difficult to quickly establish a dominant functional microbial community. Significant bottlenecks exist, particularly in the enrichment efficiency, activity, and stability of high-caproic acid-producing bacteria, severely restricting the rapid construction of high-quality cellars and the large-scale release of production capacity.

[0003] At present, the industry generally uses artificial microbial agents to strengthen cellar mud, but there are the following disadvantages or deficiencies: (1) Most microbial agents use single or simple mixed microbial groups, which cannot simulate the complex micro-ecological structure in mature cellar mud and make it difficult to establish a stable and efficient acid production system; (2) A large number of microbial strains are difficult to colonize stably in the complex cellar mud environment, and the actual production effect is difficult to achieve the expected results; (3) Some introduced microbial groups are prone to producing excessive butyric acid, which not only reduces the efficiency of hexanoic acid synthesis, but also causes butyric acid off-flavor in the wine, destroying the original wine flavor; (4) A large amount of yellow water by-products are generated during the brewing process. Traditional treatment methods are mainly discharge or low-value utilization, resulting in resource waste and environmental pressure; at the same time, yellow water is rich in a large amount of usable carbon sources, but current microbial agents are difficult to efficiently and directionally convert it into hexanoic acid for the production of high-quality flavored wine, so as to realize the high-value utilization of resources.

[0004] There are already reports on utilizing yellow water to enhance the functional flora producing hexanoic acid. For example:

[0005] The method for enriching functional microbial communities in pit mud, as described in publication number CN116286452A, involves inoculating pit mud suspension into a culture medium containing 15% (v / v) yellow water, adding fortified Daqu (a type of starter culture), and then conducting multiple rounds of anaerobic culture for expansion. The numbers of *Methanococcus sanguinalis* and *Clostridium* significantly increased, and the abundance of caproic acid-producing bacteria (*Caproiciproducens*) increased, promoting the synthesis of butyric and caproic acids. Although the abundance of caproic acid-producing bacteria increased, it did not reach over 80%; the utilization rate of the yellow water was limited, and the specific value of caproic acid was not clearly defined.

[0006] A method for rapidly enriching functional microbial communities that do not produce off-odor substances in pit mud, as described in publication number CN111088189B, and its application, involves inoculating pit mud into a composite culture medium composed of yellow water (20%-50%) and MM medium, followed by stepwise expansion. The method enriches microbial communities dominated by *Caproiciproducens* (over 50%), which do not produce the off-odor substance 4-methylphenol, and the caproic acid yield is 4.14–4.67 g / L. However, the abundance of caproic acid-producing bacteria is only around 50%, requiring further improvement; the low proportion of yellow water may limit substrate utilization efficiency, resulting in a low caproic acid yield.

[0007] The method for producing hexanoic acid by fermenting baijiu lees through enrichment of hexanoic acid-producing bacteria in pit mud, as described in publication number CN111440838A, involves acclimating the pit mud by continuously adding a mixture of yellow water or baijiu lees, enriching the hexanoic acid-producing bacteria, and then fermenting to produce hexanoic acid. The hexanoic acid concentration reaches 600-750 mg COD / g VS, achieving the resource utilization of baijiu lees. However, the acclimation period is relatively long (20-30 days), the abundance of hexanoic acid-producing bacteria is not clearly quantified, and the direct utilization efficiency of yellow water is not high.

[0008] A method for preparing a functional bacterial solution, functional bacteria, and preparation method for pit mud, disclosed in publication number CN112029682A, involves diluting yellow water and mixing it with pit mud, followed by multiple rounds of anaerobic culture to enrich acid-producing bacteria. The hexanoic acid content increased 21.95 times, lactic acid significantly decreased, and the selectivity of the bacterial community for acid production was enhanced. However, the bacterial community structure was complex, with insufficient targeted enrichment of the genus *Caproiciproducens*, whose abundance did not reach 80%; and lactic acid in the yellow water was not completely converted.

[0009] A method for preparing a compound functional bacterial liquid for strong-aroma baijiu, disclosed in publication number CN113684156A, involves preparing a culture medium using yellow water and fermentation lees, inoculating it with a high-yielding caproic acid-producing bacteria strain, and then using the expanded culture for fermentation pit maintenance. This increases the ethyl caproate content in the base liquor by 40 mg / 100 ml and accelerates the aging process in new fermentation pits. However, the process is complex, requiring multiple expansion cultures; the abundance of caproic acid-producing bacteria is not clearly defined, and the utilization efficiency of the yellow water is limited.

[0010] While existing technologies can utilize yellow water to enrich caproic acid-producing bacteria, they generally suffer from insufficient abundance of caproic acid-producing bacteria (mostly below 80%), low utilization rate of yellow water, and long fermentation cycles. Therefore, it is necessary to develop a new method to increase the abundance of caproic acid-producing bacteria (Caproiciproducens) to over 80% by optimizing the culture medium composition and enhancing the targeted enrichment of bacteria, thereby achieving efficient conversion of yellow water and significantly improving caproic acid yield and pit mud quality. Summary of the Invention

[0011] To address the aforementioned problems, this invention proposes a method and its application for rapidly enriching high-caproic acid-producing functional microbial communities in pit mud. Based on optimized seed mud, this method constructs a step-by-step domestication and screening system using the simplest culture medium. Within a short period, it efficiently enriches a mixed microbial community with caproic acid production as its core, exhibiting ecological stability and directional metabolic capabilities. This enhances the metabolic flux and selectivity of caproic acid synthesis while effectively avoiding the problem of excessive butyric acid production. This method has broad application prospects and can be widely used in the rapid cultivation and aging remediation of pit mud, driving the effective utilization of yellow water by-products and the efficient production of caproic acid.

[0012] To address the shortcomings of existing technologies, this invention provides a method and application for the rapid enrichment of functional microorganisms in pit mud that produce high levels of hexanoic acid. This method can rapidly enrich functional microorganisms in pit mud that produce beneficial flavor substances and precursors such as hexanoic acid, as well as small amounts of butyric acid. It eliminates the need to isolate and purify individual functional microorganisms from the pit mud, thus avoiding contamination. The obtained mixed microbial community represents the main microbial community in the pit mud that produces beneficial flavor substances, and this community produces high levels of hexanoic acid and small amounts of butyric acid.

[0013] The method for rapidly enriching high-caproic acid-producing functional bacteria in pit mud according to the present invention mainly involves: inoculating the pit mud into LCGM medium and culturing it for one generation, and then culturing it for three generations in LY medium with different contents of yellow water.

[0014] In one implementation, the method includes the following steps:

[0015] (1) After inoculating the pit mud into LCGM medium for cultivation, the first round of enriched bacterial solution F1 was prepared;

[0016] (2) The first round of enriched bacterial solution F1 was inoculated into LY enrichment medium with 8-10% (v / v) yellow water and cultured to prepare the second round of enriched bacterial solution F2;

[0017] (3) The second round of enriched bacterial solution F2 was inoculated into LY enrichment medium containing 15-20% (v / v) yellow water and cultured to prepare the third round of enriched bacterial solution F3.

[0018] (4) The third round of enriched bacterial solution F3 was inoculated into LY enrichment medium with 35-40% (v / v) yellow water and cultured to prepare the fourth round of enriched bacterial solution F4.

[0019] (5) The fourth round of enriched bacterial solution F4 was inoculated into LCGM medium and cultured to prepare functional bacterial groups.

[0020] The LY enrichment medium contains, in g / L: 8-10g glucose, 3-5g yeast extract, 4-5g peptone, and 5-6g sodium acetate.

[0021] In one embodiment, the pit mud used for inoculation is selected as seed pit mud: the pit mud is 30-35 years old, the pH of the pit mud is 6-7, and the pit mud with hexanoic acid and lactic acid content is selected by measuring the hexanoic acid content of the pit mud and comprehensively selecting pit mud with hexanoic acid content greater than 5g / Kg and lactic acid content less than 20g / Kg.

[0022] In one embodiment, the yellow liquid is a byproduct produced during the fermentation of strong-aroma baijiu, mainly containing ethanol, lactic acid, and other trace flavor components.

[0023] In one embodiment, the LCGM culture medium composition (g / L) is as follows: glucose 10-12, tryptone 8-10, yeast extract 8-10, ammonium sulfate 1-2, sodium dihydrogen phosphate 0.5-1, dipotassium hydrogen phosphate 0.5-1, magnesium sulfate heptahydrate 0.05-0.1, ferrous sulfate 0.015-0.02, manganese sulfate 0.01-0.02, cobalt chloride 0.01-0.02, calcium chloride 0.01-0.02, pH 6.7.

[0024] In one embodiment, in step (1), the amount of inoculated mud is 5-8% (m / v) by mass-volume ratio.

[0025] In one embodiment, in step (1), the amount of inoculated mud is 5% (m / v) by mass-volume ratio.

[0026] In one embodiment, in step (2), the inoculation amount of the first round of enriched bacterial solution F1 is 8-10%, and the culture conditions are anaerobic culture at 35-37℃ for 5-6 days.

[0027] In one embodiment, in step (3), the inoculation amount of the second round of enriched bacterial solution F2 is 8-10%, and the culture conditions are anaerobic culture at 35-37℃ for 5-6 days.

[0028] In one embodiment, in step (4), the inoculation amount of the third round of enriched bacterial solution F3 is 8-10%, and the culture conditions are anaerobic culture at 35-37℃ for 30-35 days.

[0029] In one embodiment, in step (5), the inoculation amount of the fourth round of enriched bacterial solution F4 is 8-10%, and the culture conditions are anaerobic culture at 35-37℃ for 5-6 days.

[0030] In one embodiment, the yellow water contains 70-80 g / L of lactic acid, 5-7 g / L of acetic acid, and has a pH of 3-4.

[0031] The present invention also provides a high-caproic acid-producing functional bacterial group prepared by the above method.

[0032] This invention also provides a method for preparing hexanoic acid using a high-production hexanoic acid functional bacterial community, the method comprising the following steps:

[0033] (1) The high hexanoic acid-producing functional bacterial group prepared above was inoculated into the seed culture medium and gradually expanded to the third seed culture. The culture conditions for each seed culture were: anaerobic culture at 35-37℃ for 36-60h, and the inoculation amount was 10-12% (v / v).

[0034] (2) Inoculate the prepared seed liquid into the fermentation medium at an inoculation rate of 10-12% (v / v) and culture it under anaerobic conditions at 35-37℃ for 5-7 days.

[0035] In one embodiment, the seed culture medium composition (g / L) is: glucose 10-12, acetic acid 3-5, tryptone 8-10, yeast extract 8-10, ammonium sulfate 1-2, sodium dihydrogen phosphate 0.5-1, dipotassium hydrogen phosphate 0.5-1, magnesium sulfate heptahydrate 0.05-0.1, ferrous sulfate 0.015-0.02, manganese sulfate 0.01-0.02, cobalt chloride 0.01-0.02, calcium chloride 0.01-0.02, pH 6.7;

[0036] In one embodiment, the fermentation medium composition (g / L) is: lactic acid 28-30; yeast extract 3-5; peptone 4-5; sodium acetate 3-5.

[0037] The present invention also provides a method for preparing hexanoic acid using yellow water, the method comprising the following steps:

[0038] (1) The above-mentioned high-caproic acid-producing functional bacteria were inoculated into the seed culture medium and gradually expanded to the third-level seed liquid to prepare the seed liquid. The culture conditions for each level of seed liquid were: anaerobic culture at 35-37℃ for 36-60h, and the inoculation amount was 10-12% (v / v).

[0039] (2) The prepared seed liquid was inoculated into the fermentation medium containing yellow water at an inoculation rate of 10-12% (v / v) and cultured under anaerobic conditions at 35-37℃ for 5-6 days to prepare hexanoic acid.

[0040] In one embodiment, the seed culture medium composition (g / L) is: glucose 10-12, acetic acid 3-5, tryptone 8-10, yeast extract 8-10, ammonium sulfate 1-2, sodium dihydrogen phosphate 0.5-1, dipotassium hydrogen phosphate 0.5-1, magnesium sulfate heptahydrate 0.05-0.1, ferrous sulfate 0.015-0.02, manganese sulfate 0.01-0.02, cobalt chloride 0.01-0.02, calcium chloride 0.01-0.02, pH 6.7;

[0041] In one embodiment, the fermentation medium composition (g / L) is: lactic acid 28-30; yeast extract 3-5; peptone 4-5; sodium acetate 3-5;

[0042] In one embodiment, the amount of yellow water added is 25-30% (v / v), and the yellow water contains 70-80 g / L of lactic acid, 5-7 g / L of acetic acid, and has a pH of 3-4.

[0043] This invention also provides the application of the above method in improving the passage stability of high-caproic acid-producing functional bacterial groups.

[0044] Using the above-described gradual cultivation method, the main microbial community obtained from the pit mud consisted of functional bacteria that utilize glucose to produce small amounts of acetic acid and butyric acid, as well as a high amount of hexanoic acid. Metabolite testing of the fermentation broth from gradual subculturing showed that the main product of the fermentation broth was hexanoic acid, and the concentration of hexanoic acid gradually increased during the scale-up cultivation process. This indicates that the obtained microbial community was a functional community that produced high levels of hexanoic acid and small amounts of acetic acid and butyric acid.

[0045] The present invention also provides an application of the functional microbial community enriched from cellar mud in the baijiu brewing system, that is, a method for brewing strong-aroma baijiu using the above-mentioned microbial community.

[0046] The aforementioned method for brewing and producing baijiu includes the application of the aforementioned functional microbial communities during the baijiu fermentation process.

[0047] In one embodiment, the functional microbial community is applied in the grain fermentation process of a strong-aroma baijiu system without cellar mud.

[0048] In one embodiment, the method for brewing strong-aroma baijiu includes adding functional bacterial liquid for co-fermentation during the mixing of fermented grains.

[0049] This invention also provides a method for cultivating or maintaining artificial pit mud, comprising: adding a certain amount of functional bacterial solution enriched using the method of rapidly enriching high-caproic acid-producing functional bacteria in pit mud according to this invention during the cultivation or maintenance of artificial pit mud. This method, by increasing the caproic acid-producing bacteria in the pit mud, promotes the conversion of lactic acid in the pit mud and mash into caproic acid, generating other beneficial flavor substances, and also promotes the aging of the pit mud, preventing it from becoming aging and compacted.

[0050] Beneficial effects:

[0051] 1) The main microorganisms of the functional microbial community of pit mud enriched by the method of this invention are Caproiciproducens. High-throughput sequencing results show that the relative abundance of these Caproiciproducens is over 80%, and these Caproiciproducens can completely convert lactic acid in yellow water into caproic acid. Compared with strains, the obtained microbial community has strong anti-interference ability and produces a rich variety of beneficial flavor substances (such as esters, alcohols, acids, etc.).

[0052] 2) It enriches the important functional microbial community in the pit mud, requiring a small initial inoculation amount of pit mud, thus reducing damage to high-quality pits.

[0053] 3) High-quality functional microbial communities of pit mud can be obtained in a short period of time. Through domestication in various culture media, functional microbial communities with high hexanoic acid production can be obtained, which can be further used for industrial applications.

[0054] The functional microbial community of pit mud obtained by this method can be used for the cultivation of artificial pit mud, as well as for daily pit mud maintenance. It can increase the main functional microbial community of hexanoic acid production in the pit, promote the aging of pit mud, and prevent pit mud aging and compaction. Attached Figure Description

[0055] Figure 1 Enriched bacterial community composition at the genus level.

[0056] Figure 2 GC-MS image of bacterial community utilizing yellow water products. Detailed Implementation

[0057] The present invention will now be described in detail with reference to specific embodiments.

[0058] The yellow liquid mentioned in this invention is a byproduct produced during the fermentation of strong-aroma baijiu, mainly containing ethanol, lactic acid, and other trace flavor components. Specific indicators of the yellow liquid are shown in Table 1.

[0059] Table 1: Yellow Water Parameters

[0060]

[0061]

[0062] The culture media involved in the following examples are as follows:

[0063] LCGM medium (g / L): tryptone 8-10; yeast extract 8-10; ammonium sulfate 1-2; sodium dihydrogen phosphate 0.5-1; dipotassium hydrogen phosphate 0.5-1; magnesium sulfate heptahydrate 0.05-0.1; ferrous sulfate 0.015-0.02; manganese sulfate 0.01-0.02; cobalt chloride 0.01-0.02; calcium chloride 0.01-0.02; pH 6.7; autoclave at 115℃ for 30 min.

[0064] LY medium (g / L): Yellow water 10%-40%; glucose 8-10; yeast extract 3-5; peptone 4-5; sodium acetate 5-6.

[0065] Treatment method for yellow water in culture medium: Take yellow water, a by-product of winery production, let it stand for at least 30 minutes, and dilute it with water at a volume ratio of 30% to 40%. After adjusting the pH of the diluted yellow water to 3.5-3.8, deoxygenate it.

[0066] The following examples illustrate the method for treating undiluted yellow water:

[0067] Take the yellow water, a byproduct of winery production, let it stand for at least 30 minutes, then directly adjust its pH to 3.5-3.8 and deoxygenate it for use in subsequent experiments.

[0068] The detection methods involved in the following embodiments:

[0069] Detection of hexanoic acid, acetic acid, butyric acid, and valeric acid:

[0070] Qualitative and quantitative analysis was performed using gas chromatography (GC). A gas chromatograph equipped with a flame ionization detector (FID) and an HP-FFAP capillary column (or equivalent) was used. After high-speed centrifugation, the supernatant of the sample was collected, filtered through a 0.22 μm microporous membrane, and a standard curve was plotted using organic acid standards (such as hexanoic acid, acetic acid, etc.). Quantification was performed using the external standard method.

[0071] Detection of microbial abundance:

[0072] The absolute abundance of total bacteria and specific functional groups (such as caproic acid bacteria) in samples was determined using real-time quantitative PCR (qPCR). After extracting total DNA from the samples, universal primers for bacteria (338F / 806R targeting the 16S rRNA gene) and primers for caproic acid bacteria-specific functional genes were used for amplification. Gene copy numbers were calculated based on standard curves to characterize microbial abundance.

[0073] Microbial diversity index:

[0074] Analysis was performed using high-throughput sequencing of the 16S rRNA genes of the microbial community in the samples. Bioinformatics software such as QIIME2 or Mothur was used to process the sequencing data, including quality filtering, noise reduction, generation of operational taxonomic units (OTUs) or amplified sequence variants (ASVs), and Alpha diversity indices, such as the Chao1 index (richness estimate), Shannon index (diversity index), and Simpson index (diversity index), were calculated based on the processed data.

[0075] Methods for detecting metabolic kinetics under lactate conditions:

[0076] The target bacterial group or sample was inoculated into a basal culture medium with lactic acid as the sole or main carbon source and cultured in batches or continuously under anaerobic conditions. Samples were taken periodically to detect changes in lactic acid concentration (using high-performance liquid chromatography, HPLC) and the concentration of metabolites (such as hexanoic acid and acetic acid, using GC-Fid). Kinetic parameters such as the specific consumption rate of lactic acid and the specific formation rate of hexanoic acid were calculated by plotting time curves of substrate consumption and product formation.

[0077] A method for detecting the metabolic kinetics of microbial communities producing hexanoic acid from yellow water, a byproduct of brewing, under different conditions:

[0078] Equal amounts of bacterial cultures were inoculated into yellow water culture media with different treatment conditions (such as different dilution ratios, pH, and the addition of different nutrients) and cultured under strictly anaerobic conditions. Samples were taken periodically, and the concentrations of key metabolites such as hexanoic acid, acetic acid, and butyric acid were determined by GC. By analyzing the hexanoic acid concentration change curve over time, kinetic parameters such as the maximum hexanoic acid concentration and the maximum specific formation rate were calculated, and the effects of different conditions on metabolic kinetics were compared.

[0079] Example 1: Method for Enriching Functional Microbial Communities in Pit Mud

[0080] 1. Selection of pit mud samples:

[0081] The selected pit mud is of high quality and aged 30-35 years, with a pit bottom depth of 1-3 cm. 0.4-0.5 kg of fresh pit mud is collected using anaerobic sampling bags.

[0082] 2. Approximately 500g of fresh pit mud was collected using anaerobic sampling bags. Pit mud with a pH of 6.0-7.0 was selected, and mud with high hexanoic acid content and low lactic acid content was chosen as the seed pit mud for subsequent use. The parameters of the pit mud are shown in Table 2 below:

[0083] Table 2: Pit Mud Parameters

[0084]

[0085] 3. Prepare the culture medium:

[0086] LY medium (g / L): 10% (v / v) yellow water / 20% (v / v) yellow water / 40% (v / v) yellow water; 10g glucose; 5g yeast extract; 5g peptone; 5g sodium acetate.

[0087] Treatment method for yellow water in culture medium: Take yellow water, a by-product of winery production, let it stand for at least 30 minutes, and dilute it with water at a volume ratio of 30% to 40%. Adjust the pH of the diluted yellow water to 3.5-3.8 and then deoxygenate it. Add the deoxygenated yellow water to the culture medium at volume ratios of 10% (v / v), 20% (v / v), and 40% (v / v), respectively.

[0088] LCGM medium (g / L): glucose 10, tryptone 10; yeast extract 10; ammonium sulfate 2; sodium dihydrogen phosphate 0.5; dipotassium hydrogen phosphate 1; magnesium sulfate heptahydrate 0.1; ferrous sulfate 0.015; manganese sulfate 0.01; cobalt chloride 0.01; calcium chloride 0.01, pH 6.7.

[0089] 4. Microbial community enrichment process

[0090] First round of enrichment: The selected pit mud from step 2 was added to LCGM medium at an inoculum of 5% (m / v, g / 100ml) and anaerobic cultured at 35℃ for 5 days to prepare the first round of enriched bacterial solution F1.

[0091] Second round of enrichment: The first round of enriched bacterial solution F1 was inoculated into the LY medium containing 10% (v / v) yellow water prepared in step 3 at an inoculation amount of 10% (v / v) and cultured anaerobically at 35℃ for 5 days to prepare the second round of enriched bacterial solution F2.

[0092] Third round of enrichment: The second round of enriched bacterial solution F2 was inoculated into the LY medium containing 20% ​​(v / v) yellow water prepared in step 3 at an inoculation rate of 10% (v / v). After anaerobic culture at 35℃ for 5 days, the third round of enriched bacterial solution F3 was obtained.

[0093] Fourth round of enrichment: The third round of enriched bacterial solution F3 was inoculated into the LY medium containing 40% (v / v) yellow water prepared in step 3 at an inoculation rate of 10% (v / v). After being cultured in an anaerobic workstation at 35℃ for 30 days, the fourth round of enriched bacterial solution F4 was obtained.

[0094] Fifth round of enrichment: The bacterial culture F4 enriched in the fourth round was inoculated into LCGM medium at an inoculation rate of 10% (v / v) and cultured in an anaerobic workstation at 35℃ for 5 days to prepare functional bacterial groups, which were named functional bacterial groups L1.

[0095] 5. Experimental Results

[0096] The genus-level bacterial community structure in the enriched functional bacterial communities (first-round enriched bacterial solution F1, second-round enriched bacterial solution F2, third-round enriched bacterial solution F3, fourth-round enriched bacterial solution F4, and functional bacterial community L1) was analyzed, and the results are shown in Table 3.

[0097] Table 3: Changes in microbial abundance (%) during enrichment and domestication process

[0098]

[0099] The results showed that the main component of the obtained microbial community was Caproiciproducens, with an abundance of 85.8% (Table 3). Caproiciproducens is the main microbial genus that produces caproic acid in the cellar mud of strong-aroma baijiu.

[0100] Comparative Example 1:

[0101] In this comparative embodiment, only the following modifications were made compared to Embodiment 1, while the other steps and parameters remained the same as in Embodiment 1;

[0102] 1. The influence of culture medium

[0103] Control A: Same as Example 1, except that the following culture medium was used when preparing the first round of enriched bacterial solution F1:

[0104] The following ingredients were added: 10% yellow water (v / v), 15 g / L ethanol, 10 g / L yeast extract, 10 g / L peptone, and 5 g / L sodium acetate; cultured for 12 days; other steps were performed according to the method in Example 1 to enrich the microbial community A.

[0105] Control B: Same as Example 1, except that the following culture medium was used when preparing the first round of enriched bacterial solution F1:

[0106] 10% yellow water (v / v), 15 g / L lactic acid, 10 g / L yeast extract, 10 g / L peptone, and 5 g / L sodium acetate were added and cultured for 12 days. After enrichment according to the method in Example 1, bacterial community B was obtained.

[0107] Control C: In the first round of enrichment culture of functional bacterial solution, glucose was removed and 15 g / L of undiluted yellow water raw material was added. After culturing for 12 days, bacterial group C was obtained.

[0108] 2. The impact of the enrichment process

[0109] (1) The effect of enrichment steps

[0110] Control D: The specific steps are the same as in Example 1, except that the second round of enrichment is removed; after enrichment in other steps according to the method of Example 1, bacterial community D is obtained.

[0111] Control E: The specific details are the same as in Example 1, except that the second round of enrichment is removed and the time for the third round of enrichment is extended to 8 days; after enrichment in other steps according to the method of Example 1, bacterial community E is obtained.

[0112] (2) The effect of yellow water concentration in a single step

[0113] Control F: The specific details are the same as in Example 1, except that the concentration of yellow water in the second round of enrichment is adjusted to 5% (v / v); after enrichment in other steps according to the method of Example 1, bacterial community F is obtained.

[0114] Control G: The specific details are the same as in Example 1, except that the concentration of yellow water in the third enrichment round is adjusted to 30% (v / v); after enrichment in other steps according to the method of Example 1, bacterial community G is obtained.

[0115] Control H: The specific details are the same as in Example 1, except that the concentration of yellow water in the fourth round of enrichment is adjusted to 50% (v / v); after enrichment in other steps according to the method of Example 1, bacterial community H is obtained.

[0116] (3) The effect of enrichment time

[0117] Control I: The specific steps are the same as in Example 1, except that the anaerobic culture time for the second round of enrichment is adjusted to 8 days; after enrichment in other steps according to the method of Example 1, bacterial community I is obtained.

[0118] Control J: The specific details are the same as in Example 1, except that the anaerobic culture time for the third round of enrichment is adjusted to 8 days; after enrichment in other steps according to the method of Example 1, bacterial community J is obtained.

[0119] Control K: The specific steps are the same as in Example 1, except that the anaerobic culture time for the fourth round of enrichment is adjusted to 25 days; after enrichment in other steps according to the method of Example 1, the bacterial community K is obtained.

[0120] (4) The impact of changes in yellow water concentration in multiple steps

[0121] Comparison L: Specifically the same as Example 1, except that the concentration of the concentrated yellow water in the second round of enrichment is adjusted to 20% (v / v), the concentration of the concentrated yellow water in the third round of enrichment is adjusted to 20% (v / v), and the concentration of the concentrated yellow water in the fourth round of enrichment is adjusted to 20% (v / v).

[0122] Comparison M: Specifically the same as Example 1, except that the concentration of the concentrated yellow water in the second round of enrichment is adjusted to 10% (v / v), the concentration of the concentrated yellow water in the third round of enrichment is adjusted to 20% (v / v), and the concentration of the concentrated yellow water in the fourth round of enrichment is adjusted to 30% (v / v).

[0123] 3. Results of different experiments:

[0124] The results are shown in Table 4:

[0125] Table 4: Microbial and metabolic kinetics of caproic acid-producing bacteria communities obtained from different groups after fermentation under different conditions.

[0126]

[0127] As can be seen from the data in Table 4:

[0128] (1) There were significant differences in bacterial composition and metabolic performance between the control groups and Example 1. With the optimization of the enrichment process, the abundance of Caproiciproducens showed a significant upward trend, reaching the highest of 85.8% in Example 1, while the abundance of the control groups was generally low, with Control A (ethanol carbon source) at only 52.3%, indicating that glucose as a carbon source is more conducive to the enrichment of the target bacteria.

[0129] (2) The diversity of the microbial community (Shannon index) was negatively correlated with the abundance of the target bacteria. The Shannon index of Example 1 was the lowest (0.95), indicating the highest purity of the microbial community. The diversity of Control D (removed from the second round of enrichment) was the highest (2.01), proving that multi-round gradient enrichment is crucial for eliminating contaminating bacteria.

[0130] (3) In terms of metabolic performance, hexanoic acid production was directly related to the abundance of Caproiciproducens. Example 1 showed the highest production (3.33 g / L) and the best production intensity (0.28 g / L / d), while control A (ethanol carbon source) showed the lowest production (1.97 g / L) and a decline in hexanoic acid production in the later stages of fermentation, which may be related to the inhibition of ethanol metabolism. The byproduct profile showed that butyric acid and acetic acid were the main byproducts in all groups, but the lactic acid group (control B) had the lowest total amount of byproducts, while the yellow water group (control C) showed a more complex byproduct profile.

[0131] (4) The adjustment of the yellow water concentration gradient has a significant impact on the results. The control M (10%-20%-30% gradient) is close to the effect of Example 1, but the fourth round of 40% yellow water (Example 1) can more thoroughly eliminate contaminating bacteria. Although the extension of the cultivation time (such as 25 days for control K) increases the yield, it needs to be combined with the optimization of the yellow water concentration to achieve the best results.

[0132] In summary, the strategy of five rounds of enrichment, gradient increase of yellow water concentration (10%-40%) and glucose carbon source adopted in Example 1 showed the best results in terms of bacterial community purity, stability and metabolic performance.

[0133] Example 2: Microbial Community Stability

[0134] The functional bacterial group L1 obtained in Example 1 was activated and a seed culture was prepared using LCGM-1 medium. The specific composition of the medium was the same as that of LCGM, except that the carbon source was adjusted to a composite carbon source consisting of 10 g / L glucose and 5 g / L acetic acid.

[0135] The specific steps are as follows:

[0136] 1. Prepare the culture medium:

[0137] LCGM-1 medium (g / L): glucose 10g / L, acetic acid 5g / L, tryptone 10g; yeast extract 10g; ammonium sulfate 2g; sodium dihydrogen phosphate 0.5g; dipotassium hydrogen phosphate 1g; magnesium sulfate heptahydrate 0.05g; ferrous sulfate 0.015g; manganese sulfate 0.01g; cobalt chloride 0.01g; calcium chloride 0.01g; pH 6.7.

[0138] 2. Subculture of the microbial community

[0139] (1) The functional bacterial group L1 obtained in Example 1 was inoculated into LCGM-1 medium at an inoculation rate of 10% (v / v) and cultured at 35°C in an anaerobic workstation for 60 h to prepare an activation solution; the prepared activation solution was inoculated into 20 mL LCGM-1 medium at an inoculation rate of 10% (v / v) and cultured at 35°C under anaerobic conditions for 36 h to prepare the seed culture of L1 bacterial group.

[0140] (2) The L1 seed culture prepared in step (1) was inoculated into LCGM-1 medium at an inoculation rate of 10% (v / v). The culture was carried out under anaerobic conditions at 35℃ for 36 h to obtain a secondary seed culture.

[0141] (3) The secondary seed solution from step (2) was inoculated into LCGM-1 medium at an inoculation rate of 10% (v / v) and cultured under anaerobic conditions at 35℃ for 36 h to obtain the tertiary seed solution.

[0142] (4) First round of culture: The tertiary seed culture from step (3) was inoculated into LCGM-1 medium at an inoculation rate of 10% (v / v) and cultured for 5 days, with samples taken daily.

[0143] (5) Second round of culture: The fermentation broth from step (4) on day 5 of fermentation was inoculated into LCGM-1 medium at an inoculation rate of 10% (v / v) and cultured for 5 days, with samples taken daily.

[0144] (6) Third round of culture: The fermentation broth from step (5) on day 5 of fermentation was inoculated into LCGM-1 medium at an inoculation rate of 10% (v / v) and cultured for 5 days, with samples taken daily.

[0145] (7) Fourth round of culture: The fermentation broth from the 5th day of fermentation in step (6) was inoculated into LCGM-1 medium at an inoculation rate of 10% (v / v) and cultured for 5 days.

[0146] 3. Experimental Results

[0147] The content of short-chain fatty acids in the bacterial culture obtained after each round of cultivation in step 2 was detected, and the results are shown in Tables 5 and 6.

[0148] Table 5: Stability (Metabolic) of the L1 Microbial Community

[0149]

[0150] Table 6: Abundance of microbial variations (species) in L1 community stability

[0151]

[0152] The results are shown in Tables 5 and 6 below. This functional microbial community exhibited multi-level stability during four consecutive rounds of fermentation.

[0153] At the metabolic level, the concentration of the key product hexanoic acid remained highly stable (4.44±0.26 g / L, RSD=5.86%), while the fluctuations of propionic acid and valeric acid were extremely low (RSD≤3.96%), the pH difference was only 0.05 units (RSD=0.41%), and the repeatability error of cell growth (OD600) was only 2.34%. At the community structure level, although the core strain Ruminococcaceae bacterium CPB6 experienced a third round of brief fluctuations (55.91%), it quickly recovered to 71.38% and remained dominant. The key functional bacterium uncultured g_Caproiciproducens achieved a dynamic balance of abundance through self-regulation (5.22%→26.95%→5.18%), while the auxiliary bacteria unclassified f_Lachnospiraceae (abundance range 17.16-21.09%) and Ruminococcaceae bacterium CPC-11 (fluctuation range <2%) provides stable redundancy support.

[0154] Example 3: Optimization of Acid-Producing Fermentation Conditions of Functional Bacterial Liquid

[0155] The functional bacterial group L1 obtained in Example 1 was activated and a seed culture was prepared using LCGM-1 medium. The specific composition of the medium was the same as that of LCGM, with a composite carbon source consisting of 10 g / L glucose and 5 g / L acetic acid.

[0156] The specific steps are as follows:

[0157] 1. Prepare the culture medium:

[0158] LY-1 medium (g / L): lactic acid 28-30; yeast extract 5; peptone 5; sodium acetate 5.

[0159] LCGM-1 medium (g / L): glucose 10g / L, acetic acid 5g / L, tryptone 10g; yeast extract 10g; ammonium sulfate 2g; sodium dihydrogen phosphate 0.5g; dipotassium hydrogen phosphate 1g; magnesium sulfate heptahydrate 0.05g; ferrous sulfate 0.015g; manganese sulfate 0.01g; cobalt chloride 0.01g; calcium chloride 0.01g; pH 6.7.

[0160] 2. Subculture of the microbial community

[0161] (1) The functional bacterial group L1 obtained in Example 1 was inoculated into LCGM-1 medium at an inoculation rate of 10% (v / v) and cultured at 35°C in an anaerobic workstation for 60 h to prepare an activation solution; the prepared activation solution was inoculated into 20 mL LCGM-1 medium at an inoculation rate of 10% (v / v) and cultured at 35°C under anaerobic conditions for 36 h to prepare the seed culture of L1 bacterial group.

[0162] (2) The L1 seed culture prepared in step (1) was inoculated into LCGM-1 medium at an inoculation rate of 10% (v / v). The culture was carried out under anaerobic conditions at 35℃ for 36 h to obtain a secondary seed culture.

[0163] (3) The secondary seed solution from step (2) was inoculated into LCGM-1 medium at an inoculation rate of 10% (v / v) and cultured under anaerobic conditions at 35℃ for 36 h to obtain the tertiary seed solution.

[0164] (4) Inoculate the tertiary seed culture from step (3) into the LY-1 medium containing 30 g / L lactic acid prepared in step 1 at an inoculation rate of 10% (v / v) and culture it under anaerobic conditions at 35°C for 5 days.

[0165] 3. Experimental Results

[0166] The metabolic kinetics of the bacterial culture obtained after step 2 culture under lactic acid conditions were detected, and the results are shown in Table 7.

[0167] Table 7: Metabolic kinetics of L1 gut microbiota under lactic acid conditions

[0168]

[0169] The results show:

[0170] The fermentation process exhibits significant metabolic dynamics, as shown in Table 6:

[0171] In the initial stage (0-1d), lactic acid was rapidly consumed (from 28.14 g / L to 21.62 g / L) and mainly converted into acetic acid (carbon flux ratio 39.6%). As fermentation progressed (1-5d), the metabolic pathway underwent a systematic remodeling, characterized by continuous degradation of lactic acid (final residue 1.42 g / L) and a continuously enhanced hexanoic acid synthesis capacity (from 1.98 g / L to 9.78 g / L). Its carbon flux ratio increased from 23.4% to 62.9%, becoming the absolutely dominant pathway.

[0172] Meanwhile, the carbon flux ratio of acetic acid decreased from 39.6% to 8.1%, while the proportions of medium-chain acids such as propionic acid / butyric acid / valeric acid remained relatively stable. This metabolic shift was accompanied by a continuous improvement in carbon utilization efficiency (from 28.7% to 68.7%), and increased microbial growth (OD). 600 It showed a strong positive correlation with hexanoic acid production, and its activity was restored after the pH dropped to 5.88 and eventually stabilized at 7.00.

[0173] Example 4: High production of hexanoic acid by L1 bacterial group using yellow water under laboratory conditions

[0174] Hexanoic acid was produced by fermentation using the optimized functional bacterial culture from Example 3. The functional bacterial group L1 obtained in Example 1 was activated and a seed culture was prepared using LCGM-1 medium. The specific composition of the medium was the same as that of LCGM, with a composite carbon source consisting of 10 g / L glucose and 5 g / L acetic acid.

[0175] The specific steps are as follows:

[0176] 1. Prepare the culture medium:

[0177] LY-2 medium (g / L): 30% yellow water (v / v); glucose 10, yeast extract 5; peptone 5; sodium acetate 5.

[0178] LCGM-1 medium (g / L): glucose 10, acetic acid 5, tryptone 10; yeast extract 10; ammonium sulfate 2; sodium dihydrogen phosphate 0.5; dipotassium hydrogen phosphate 1; magnesium sulfate heptahydrate 0.05; ferrous sulfate 0.015; manganese sulfate 0.01; cobalt chloride 0.01; calcium chloride 0.01; pH 6.7.

[0179] 2. Subculture of the microbial community

[0180] (1) Inoculate the functional bacterial group L1 into the LCGM-1 medium prepared in step 1 at an inoculation rate of 10% (v / v) and culture it at 35℃ in an anaerobic workstation for 60h to prepare an activation solution; inoculate the prepared activation solution into 20mL LCGM-1 medium at an inoculation rate of 10% (v / v) and culture it at 35℃ in an anaerobic condition for 36h to prepare the seed culture of the L1 bacterial group.

[0181] (2) The L1 seed culture prepared in step (1) was inoculated into LCGM-1 medium at an inoculation rate of 10% (v / v). The culture was carried out under anaerobic conditions at 35℃ for 36 h to obtain a secondary seed culture.

[0182] (3) The secondary seed solution from step (2) was inoculated into LCGM-1 medium at an inoculation rate of 10% (v / v) and cultured under anaerobic conditions at 35℃ for 36 h to obtain the tertiary seed solution.

[0183] (4) Inoculate the tertiary seed culture from step (3) into the LY-2 medium containing 30% yellow water prepared in step 1 at an inoculation rate of 10% (v / v), and culture at 35℃ under anaerobic conditions for 13 days; on the 7th day of fermentation, add 200mL of undiluted yellow water to the culture system. During the fermentation process, the operating conditions are anaerobic, 37℃, static culture, and the system volume is 2L.

[0184] 3. Experimental Results

[0185] The metabolic kinetics of the bacterial culture obtained after step 2 were tested under different conditions in which the bacterial community produced hexanoic acid using yellow water, a byproduct of brewing. The results are shown in Table 8.

[0186] The results show:

[0187] Under laboratory conditions, as shown in Table 8, the fermentation process exhibits typical metabolic dynamics:

[0188] In the initial stage (0h), the lactic acid concentration was 20.70±0.82g / L, which was rapidly consumed to 3.34±1.19g / L after 48 hours. At the same time, the hexanoic acid production reached 6.82±0.16g / L. When the lactic acid was completely depleted at 96h, the hexanoic acid production increased to 14.51±0.42g / L, and finally stabilized at 15.37±0.17g / L at 108h.

[0189] During this process, butyric acid gradually accumulated from 0 g / L to 2.74 ± 0.02 g / L, while propionic acid and valeric acid stabilized at 1.65 ± 0.03 g / L and 1.05 ± 0.00 g / L, respectively. Cell growth (OD)... 600 The pH value showed a continuous upward trend, rising from 0 to 4.05±0.20, while the pH value initially decreased from 6.66±0.10 to 5.91±0.06 before recovering to 6.83±0.05, indicating that the bacterial community has a good environmental adaptability. GC-MS analysis revealed that this bacterial community utilizes yellow water to produce hexanoic acid, which, in addition to acidic compounds, can also produce esters and alcohols. Figure 2 ).

[0190] Example 5: Production of hexanoic acid by bacterial community L1 from yellow water under pilot-scale conditions

[0191] The functional bacterial population obtained in Example 1 was activated and seed culture was prepared using LCGM-1 medium. The specific composition of the medium was the same as that of LCGM, with a composite carbon source consisting of 10 g / L glucose and 5 g / L acetic acid.

[0192] The specific steps are as follows:

[0193] 1. Prepare the culture medium:

[0194] LY-2 medium (g / L): 30% yellow water (v / v); glucose 10, yeast extract 5; peptone 5; sodium acetate 5.

[0195] LCGM-1 medium (g / L): glucose 10g / L, acetic acid 5g / L, tryptone 10g; yeast extract 10g; ammonium sulfate 2g; sodium dihydrogen phosphate 0.5g; dipotassium hydrogen phosphate 1g; magnesium sulfate heptahydrate 0.05g; ferrous sulfate 0.015g; manganese sulfate 0.01g; cobalt chloride 0.01g; calcium chloride 0.01g; pH 6.7.

[0196] 2. Subculture of the microbial community

[0197] (1) The functional bacterial group L1 was inoculated into LCGM medium at an inoculation rate of 10% (v / v) and cultured at 35℃ in an anaerobic workstation for 60h to prepare an activation solution; the prepared activation solution was inoculated into 20mL LCGM-1 medium at an inoculation rate of 10% (v / v) and cultured at 35℃ under anaerobic conditions for 36h to prepare the seed culture of L1 bacterial group.

[0198] (2) The L1 seed liquid prepared in step (1) is inoculated into the LCGM-1 medium described in step (1) at an inoculation amount of 10% (v / v) and cultured under anaerobic conditions at 35℃ for 36 h to obtain a secondary seed liquid.

[0199] (3) The secondary seed culture from step (2) was inoculated into the LCGM-1 medium from step (1) at an inoculation rate of 10% (v / v) and cultured under anaerobic conditions at 35°C for 36 h to obtain the tertiary seed culture.

[0200] (4) The tertiary seed culture from step (3) was inoculated into the LY medium containing 30 g / L lactic acid prepared in step 1 at an inoculation rate of 10% (v / v) and cultured for 108 h. At the 48 h of fermentation, 200 mL of undiluted yellow water was added to the culture system. During the fermentation process, the operating system on the tank was 4 L and the temperature was 35 °C.

[0201] 3. Experimental Results

[0202] The metabolic kinetics of the bacterial culture obtained after step 2 were tested under different conditions in which the bacterial community produced hexanoic acid using yellow water, a byproduct of brewing. The results are shown in Table 8.

[0203] Table 8. Metabolic kinetics of microbial communities producing hexanoic acid from brewing byproduct yellow water under different conditions.

[0204]

[0205]

[0206] The results show:

[0207] Under pilot-scale conditions, as shown in Table 8, although the initial lactate concentration was high (25.72±0.06 g / L), the metabolic trend was highly consistent with that under laboratory conditions.

[0208] The yield of hexanoic acid reached 10.38±0.46 g / L after 48 hours, increased to 16.04±0.27 g / L after 96 hours, and finally stabilized at 16.47±0.19 g / L.

[0209] The accumulation patterns of other products, including butyric acid (2.23±0.04 g / L), propionic acid (1.19±0.02 g / L), and valeric acid (1.06±0.01 g / L), were not significantly different from those under laboratory conditions. However, the highest cell growth (OD600) was only 8.23±0.04, which was about twice that under laboratory conditions, and the pH variation range (6.80±0.19 to 6.93±0.05) was also relatively gradual.

[0210] The high consistency of hexanoic acid production under the two conditions indicates that the fermentation process has good scalability, while the difference in cell growth may be due to stricter environmental control or different mass transfer efficiencies under the pilot-scale conditions.

[0211] Example 6: Preparation of Functional Microbial Agents

[0212] The specific steps are as follows:

[0213] (1) Activation of functional flora L1:

[0214] The L1 bacterial group from Example 1 was inoculated into LCGM-1 medium in an anaerobic chamber at 37°C and cultured for 48-60 hours to obtain a primary seed culture. After activation under the same conditions, the culture was gradually expanded to three generations to obtain a tertiary seed culture.

[0215] (2) Based on an inoculation ratio of 10% (v / v), the tertiary seed culture was inoculated into LY medium containing 50% yellow water and cultured under anaerobic conditions at 37℃ until the logarithmic growth phase, i.e., 36-48 hours, to obtain a bacterial suspension with relatively high activity. The bacterial suspension was centrifuged at 8000 rpm for 10 minutes at 4℃, and the bacterial cells were collected. Then, skim milk was added in an anaerobic chamber according to the bacterial cell content, mixed well, and then freeze-dried under vacuum and thawed to obtain the freeze-dried bacterial biological agent.

[0216] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone 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 enriching a high-caproic acid-producing functional bacterial community, characterized in that, The method includes the following steps: (1) Select the bottom mud of the pit with a year of 30-35 years, pH 6.0-7.0, hexanoic acid >5 g / Kg mud and lactic acid <20 g / Kg mud. Inoculate the mud into LCGM medium at a mass-volume ratio of 5% and anaerobic culture at 35℃ for 5 days to prepare the first round of enriched bacterial solution F1. (2) The first round of enriched bacterial solution F1 was inoculated into LY enrichment medium with 10% v / v added at an inoculation rate of 10% v / v. After anaerobic culture at 35℃ for 5 days, the second round of enriched bacterial solution F2 was prepared. (3) The second round of enriched bacterial solution F2 was inoculated into LY enrichment medium with 20% v / v added at an inoculation rate of 10% v / v. After anaerobic culture at 35℃ for 5 days, the third round of enriched bacterial solution F3 was prepared. (4) The third round of enriched bacterial solution F3 was inoculated into LY enrichment medium with 40% v / v added at an inoculation rate of 10% v / v. After anaerobic culture at 35°C for 30 days, the fourth round of enriched bacterial solution F4 was prepared. (5) The fourth round of enriched bacterial solution F4 was inoculated into LCGM medium at an inoculation rate of 10% v / v and anaerobic cultured at 35℃ for 5 days to prepare functional bacterial groups; The LY enrichment medium consists of the following components (g / L): glucose 8-10, yeast extract 3-5, peptone 4-5, sodium acetate 5-6; the LCGM medium consists of the following components (g / L): glucose 10-12, tryptone 8-10, yeast extract 8-10, ammonium sulfate 1-2, sodium dihydrogen phosphate 0.5-1, dipotassium hydrogen phosphate 0.5-1, magnesium sulfate heptahydrate 0.05-0.1, ferrous sulfate 0.015-0.02, manganese sulfate 0.01-0.02, cobalt chloride 0.01-0.02, calcium chloride 0.01-0.02, pH 6.

7. The yellow water contains 70-80 g / L lactic acid, 5-7 g / L acetic acid, 8-10 g / L propionic acid, 4-6 g / L butyric acid, and 4-6 g / L glucose, with a pH of 3-4.

2. A method for preparing hexanoic acid using a high-production functional bacterial community, characterized in that, The method includes the following steps: (1) A high-caproic acid-producing functional bacterial group was obtained by enriching the bacterial group according to the method of claim 1; (2) The high-caproic acid-producing functional bacteria described in step (1) are inoculated into the seed culture medium and gradually expanded to three-level seed liquid. The culture conditions for each level of seed liquid are: anaerobic culture at 35-37℃ for 36-60 h, and the inoculation amount is 10-12% v / v. (3) Inoculate the prepared seed liquid into the fermentation medium at an inoculation rate of 10-12% v / v and culture it under anaerobic conditions at 35-37℃ for 5-7 days.

3. The method according to claim 2, characterized in that, The seed culture medium composition, in g / L, is as follows: glucose 10-12, acetic acid 3-5, tryptone 8-10, yeast extract 8-10, ammonium sulfate 1-2, sodium dihydrogen phosphate 0.5-1, dipotassium hydrogen phosphate 0.5-1, magnesium sulfate heptahydrate 0.05-0.1, ferrous sulfate 0.015-0.02, manganese sulfate 0.01-0.02, cobalt chloride 0.01-0.02, calcium chloride 0.01-0.02, pH 6.7; The fermentation medium consists of the following components in g / L: lactic acid 28-30, yeast extract 3-5, peptone 4-5, and sodium acetate 3-5.

4. A method for preparing hexanoic acid using yellow water, characterized in that, The method includes the following steps: (1) A high-caproic acid-producing functional bacterial group was obtained by enriching the bacterial group according to the method of claim 1; (2) The high-caproic acid-producing functional bacteria described in step (1) are inoculated into the seed culture medium and gradually expanded to a third-level seed liquid to prepare the seed liquid. The culture conditions for each level of seed liquid are: anaerobic culture at 35-37℃ for 36-60 h, and the inoculation amount is 10-12% v / v. (3) The prepared seed liquid was inoculated into the fermentation medium containing yellow water at an inoculation rate of 10-12% v / v and cultured under anaerobic conditions at 35-37℃ for 5-6 days to prepare hexanoic acid; the amount of yellow water added was 25-30% v / v; the yellow water contained 70-80 g / L of lactic acid, 5-7 g / L of acetic acid, 8-10 g / L of propionic acid, 4-6 g / L of butyric acid, 4-6 g / L of glucose, and pH 3-4.

5. The method according to claim 4, characterized in that, The seed culture medium composition, in g / L, is as follows: glucose 10-12, acetic acid 3-5, tryptone 8-10, yeast extract 8-10, ammonium sulfate 1-2, sodium dihydrogen phosphate 0.5-1, dipotassium hydrogen phosphate 0.5-1, magnesium sulfate heptahydrate 0.05-0.1, ferrous sulfate 0.015-0.02, manganese sulfate 0.01-0.02, cobalt chloride 0.01-0.02, calcium chloride 0.01-0.02, pH 6.7; The fermentation medium consists of the following components in g / L: lactic acid 28-30, yeast extract 3-5, peptone 4-5, and sodium acetate 3-5.

6. The application of the method described in claim 1 in the brewing of strong-aroma baijiu or in the cultivation, repair, or maintenance of artificial cellar mud.

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