A caproic acid composite bacterial population, a bacterial solution and application thereof

Abstract

The present application relates to caproic acid bacteria technical field, specifically relates to a kind of compound caproic acid bacteria group, bacteria liquid and its application, the compound caproic acid bacteria group in it includes caproic acid bacteria Caproicibacterium argilliputei XB1, butyric acid bacteria Clostridium tyrobutyricum DS1, lactic acid bacteria Ligilactobacillus acidipiscis YHS1A and 1 "movable caproic acid bacteria group". The strain and flora of the compound caproic acid bacteria group are all derived from Luzhou-flavor liquor fermentation system, and can still convert lactic acid into caproic acid or butyric acid and other characteristic flavor components of Luzhou-flavor liquor under the condition of tolerating high-concentration lactic acid (90 g / L ‑1 ) or high-concentration ethanol (60 g / L ‑1 ) close to in-situ fermentation system. Therefore, the compound caproic acid bacteria group can be used for reverse addition in Luzhou-flavor liquor fermentation process, to significantly improve the yield of caproic acid in fermentation system by "increasing caproic acid and reducing lactic acid".

Description

Technical Field

[0001] This invention relates to the field of caproic acid bacteria technology, and in particular to a complex caproic acid bacteria group, bacterial solution, and its application. Background Technology

[0002] The current national standard for strong-aroma baijiu (GB / T 10781.1-2021) defines hexanoic acid and ethyl hexanoate as characteristic flavor compounds, and uses their content in the liquor as one of the grading indicators for "superior grade" and "first grade" liquor. During the fermentation process of strong-aroma baijiu, ethyl hexanoate is formed by the esterification of its precursor, hexanoic acid, and ethanol. Therefore, increasing the hexanoic acid synthesis by hexanoic acid-producing bacteria (referred to as "hexanoic acid bacteria") is crucial for improving the fermentation quality of strong-aroma baijiu.

[0003] Recent studies have shown that caproic acid bacteria in the fermentation system of strong-aroma baijiu mainly reside in the surface layer of the bottom mud, and the tendency and tolerance of caproic acid bacteria to the "high acid / alcohol" in-situ fermentation system are essential prerequisites for their efficient synthesis of caproic acid. However, research on existing caproic acid bacteria such as Caproiciproducens, Caproicibacterium, Clostridium, and Caproicibacter has revealed that these bacteria migrate from the bottom mud to the solid-liquid mixture of mash and yellow water during fermentation. This indicates that the actual main fermentation process in strong-aroma baijiu involves the production of high-concentration lactic acid (up to 90 g / L). -1 High concentrations of ethanol (up to 60 g / L) -1 This is particularly relevant in solid-liquid fermentation systems of mash and yellow water with low pH values ​​(referred to as "high acid / alcohol"), rather than in fermentation systems using bottom pit mud. Therefore, while hexanoic acid strains screened or constructed using existing technologies may exhibit good performance under standard laboratory conditions for baijiu fermentation, they often demonstrate poor tropism and tolerance in simulated or real baijiu brewing environments due to insufficient environmental adaptability. This results in severely suppressed metabolic activity and low hexanoic acid synthesis efficiency, failing to meet the stability and efficiency requirements of industrial production. Summary of the Invention

[0004] In view of this, the purpose of this invention is to propose a composite hexanoic acid bacterial community, bacterial solution and its application, in order to solve the problem that existing bacterial strains have poor tropism and tolerance due to insufficient environmental adaptability, resulting in severely suppressed metabolic activity and low hexanoic acid synthesis efficiency, which cannot meet the stability and efficiency requirements of industrial production.

[0005] To achieve the above objectives, the present invention provides a complex caproic acid bacterial community, characterized in that the complex caproic acid bacterial community comprises at least one of the following: caproic acid strain Caproicibacterium argilliputei XB1 and butyric acid strain Clostridium tyrobutyricum DS1 or lactic acid strain Ligilactobacillus acidipiscis YHS1A, with a mass percentage >50%.

[0006] Meanwhile, the present invention also provides a complex hexanoic acid bacteria group, including the aforementioned complex hexanoic acid bacteria group.

[0007] Finally, this invention provides an application of a complex hexanoic acid bacteria group for the synthesis of butyric acid and hexanoic acid during the fermentation process of strong-aroma baijiu.

[0008] The beneficial effects of the present invention are: (1) It provides a complex hexanoic acid bacteria group with synergistic hexanoic acid synthesis, which is more advantageous in growth and hexanoic acid synthesis than single bacteria in the actual strong aroma baijiu fermentation system;

[0009] (2) The core dominant strains in the compound caproic acid bacteria community are caproic acid strain Caproicibacterium argilliputei XB1, butyric acid strain Clostridium tyrobutyricum DS1, and lactic acid bacteria Ligilactobacillus acidipiscis YHS1A. The non-dominant strains are "motile caproic acid bacteria" that are tolerant to "high acid / alcohol" in situ enriched from caproic acid bacteria solution. The "motile caproic acid bacteria" are "motile" and can produce short-chain fatty acids such as acetic acid and butyric acid. This can help the bacteria overcome the gravitational potential difference and resistance to move to the upper mash-yellow water system for metabolism. To a certain extent, this can alleviate the heterogeneity of the fermentation results and provide acetic acid, an electron acceptor for the synthesis of butyric acid or caproic acid, for caproic acid synthesis.

[0010] (3) The strains in the compound hexanoic acid bacteria group generally have "high acid / alcohol" tolerance and can metabolize in the in situ yellow water system with up to 90 g / L lactic acid or 60 g / L ethanol. Attached Figure Description

[0011] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only for this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0012] Figure 1This is a SEM image of the compound hexanoic acid bacteria group in an embodiment of the present invention;

[0013] Among them, A is Caproicibacterium argilliputei XB1, B is Clostridium tyrobutyricum DS1, and C is Ligilactobacillus acidipiscis YHS1A;

[0014] Figure 2 This invention relates to Caproicibacterium argilliputei XB1 and its most closely related strain Caproicibacterium argilliputei ZCY20-5. T Comparative genomics analysis diagram based on hexanoic acid synthase gene;

[0015] Figure 3 This is a comparative genomics analysis diagram based on the hexanoate synthase gene between Clostridium tyrobutyricum DS1 and strain Clostridium tyrobutyricum Cirm BIA 2237 in this invention.

[0016] Figure 4 This is a schematic diagram of the genes involved in hexanoic acid biosynthesis in the Ligilactobacillus acidipiscis YHS1A genome, as described in an embodiment of the present invention.

[0017] Figure 5 Gradient temperature growth (OD) of Caproicibacterium argilliputei XB1 in this embodiment of the invention. 600 )picture;

[0018] Figure 6 This is a growth and metabolic characteristic diagram of Caproicibacterium argilliputei XB1 under different conditions according to an embodiment of the present invention;

[0019] Where A is OD 600 B represents pH, C represents potential carbon source substrates (glucose, lactic acid, ethanol), D represents acetic acid, E represents butyric acid, and F represents hexanoic acid.

[0020] Figure 7 This is a growth and metabolic characteristic diagram of Clostridium tyrobutyricum DS1 under different conditions according to an embodiment of the present invention;

[0021] Where A is OD 600B is glucose, C is lactic acid, D is ethanol, E is pH, F is acetic acid, G is butyric acid, and H is hexanoic acid;

[0022] Figure 8 This is a graph showing the growth of Ligilactobacillus acidipiscis YHS1A under gradient temperatures and different pH values ​​in an embodiment of the present invention.

[0023] Figure 9 This is a graph showing the growth and metabolic characteristics of Ligilactobacillus acidipiscis YHS1A under different conditions according to an embodiment of the present invention.

[0024] Where A is OD 600 B is pH, C is glucose, D is ethanol, E is lactic acid, F is acetic acid, G is butyric acid, and H is hexanoic acid;

[0025] Figure 10 This is a volcano diagram of differentially expressed genes (DEGs) of Ligilactobacillus acidipiscis YHS1A at different time points during fermentation, as described in this embodiment of the invention.

[0026] Among them, A is 0 hours vs 2 hours, B is 0 hours vs 4 hours, C is 0 hours vs 8 hours, D is 2 hours vs 4 hours, and E is 4 hours vs 8 hours.

[0027] F is a key enzyme gene involved in fatty acid biosynthesis (FAB) and rare branched-chain keto acid (RBO) metabolic pathways. The graph shows the fold change of its FPKM value after logarithmic transformation.

[0028] Figure 11 This is a growth and metabolic characteristic diagram of Caproicibacterium argilliputei XB1 and Clostridium tyrobutyricum DS1 under different conditions during 6 consecutive batch fermentations according to embodiments of the present invention.

[0029] Where A is OD 600 B is pH, C is glucose, D is lactic acid, E is acetic acid, F is butyric acid, and G is hexanoic acid;

[0030] Figure 12 This is a graph showing the growth and metabolic characteristics of Caproicibacterium argilliputei XB1 and Ligilactobacillus acidipiscis YHS1A under different conditions during six consecutive batch fermentations according to embodiments of the present invention.

[0031] Where, A is OD 600 B is pH, C is glucose, D is lactic acid, E is acetic acid, F is butyric acid, and G is hexanoic acid;

[0032] Figure 13 The following are simulation diagrams and actual device diagrams for screening "mobile caproic acid bacteria groups" from caproic acid bacteria solution enriched in strong-aroma baijiu, as an embodiment of the present invention.

[0033] In this diagram, A is a simulation image and B is a real-world installation image.

[0034] Figure 14 This is a diagram of the bacterial community structure at the genus and species level based on metagenomic sequencing of the "mobile hexanoic acid bacteria" in this embodiment of the invention.

[0035] Where A represents the genus and B represents the species;

[0036] Figure 15 for Figure 8 Metabolic characteristics of the "mobile caproic acid bacteria" screened in the study during growth in a culture medium containing gradient concentrations of lactic acid as the sole carbon source;

[0037] Where A is OD 600 B represents pH, C represents lactic acid, D represents acetic acid, E represents butyric acid, and F represents hexanoic acid.

[0038] Figure 16 for Figure 8 The "mobile caproic acid bacteria" screened in the presence of gradient concentrations of ethanol and 15 g L -1 Metabolic characteristics of growth in culture media with lactic acid carbon source under different conditions;

[0039] Where A is ethanol, B is OD600, C is lactic acid, D is pH value, E is acetic acid, F is butyric acid, and G is hexanoic acid (G).

[0040] Figure 17 This is a structural diagram of the prokaryotic community in the "mobile hexanoic acid bacteria" of the present invention, which is tolerant to high concentrations of "lactic acid medium" (CTL) and high concentrations of ethanol "ethanol-lactic acid medium" (CTE). Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0042] The fermentation system of strong-aroma baijiu primarily uses glucose, ethanol, or lactic acid as carbon sources to first synthesize pyruvate. Then, acetyl-CoA enters a carbon chain elongation reaction, sequentially synthesizing butyric acid and hexanoic acid. Subsequently, hexanoic acid esterifies with ethanol in the fermentation system to form ethyl hexanoate. This fermentation process occurs in the mash-wax water system. However, the hexanoic acid bacteria in the strong-aroma baijiu fermentation system mainly reside in the surface layer of the bottom mud. This paper addresses the issue that while the hexanoic acid bacteria in the strong-aroma baijiu fermentation system are mainly found in the bottom mud, the actual hexanoic acid synthesis environment is the upper layer of the mash-wax water fermentation system, characterized by "high acid / alcohol" (high concentration of lactic acid and high concentration of ethanol).

[0043] As one possible implementation, Embodiment 1 of the present invention provides a "high acid / alcohol" tolerant caproic acid bacterial community, namely a complex caproic acid bacterial community. This complex caproic acid bacterial community consists of a "motile caproic acid bacterial community" of non-dominant bacteria that assist in the synthesis of caproic acid and are tolerant to "high acid / alcohol" and contains at least one of the following core functional strains: caproic acid strain *Caproicibacterium argilliputei* XB1, butyric acid strain *Clostridium tyrobutyricum* DS1, or lactic acid strain *Ligilactobacillus acidipiscis* YHS1A. The mass percentage of the caproic acid strain *Caproicibacterium argilliputei* XB1 is greater than 50%.

[0044] Currently, three bacterial strains, *Caproicibacterium argilliputei* XB1, *Clostridium tyrobutyricum* DS1, and *Ligilactobacillus acidipiscis* YHS1A, were deposited on November 14, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Their accession numbers are GDMCC No. 67345, GDMCC No. 67347, and GDMCC No. 67348, respectively. These three strains were isolated from enriched caproic acid bacteria broth in a strong-aroma baijiu fermentation system and obtained using a serial dilution and streak plate method in an anaerobic workstation.

[0045] Blasten alignment analysis of the 16S rRNA gene revealed the following:

[0046] The strain Caproicibacterium argilliputei XB1 showed the highest 16S rRNA gene sequence identity with the existing model strains in the NCBI rRNA database, namely Caproicibacterium argilliputei ZCY20-5 (Accession No. CP135996.1), with an identity of 100.00%. Analysis of its whole genome sequence against those of related strains in the NCBI database using ANI (average nucleotide identity), AAI (average amino-acid identity), and POCP (percentage of conserved proteins) confirmed that this strain is a subspecies of Caproicibacterium argilliputei.

[0047] The strain Clostridium tyrobutyricum DS1 showed the highest 16S rRNA gene sequence identity with existing model strains in the NCBI rRNA database, namely Clostridium tyrobutyricum strain ATCC25755 (Accession No. NR_044718.2), with an identity of 99.34%. Analysis of its whole genome sequence with those of related strains in the NCBI database using ANI (average nucleotide identity), AAI (average amino-acid identity), and POCP (percentage of conserved proteins) confirmed that this strain is a subspecies of Clostridium tyrobutyricum.

[0048] The strain Ligilactobacillus acidipiscis YHS1A showed the highest 16S rRNA gene sequence identity with the existing model strains in the NCBI rRNA database, namely Ligilactobacillus acidipiscis NBRC102163 (Accession No. NR_112693.1), with an identity of 99.93%. Analysis of its whole genome sequence with those of related strains in the NCBI database using ANI (average nucleotide identity), AAI (average amino-acid identity), and POCP (percentage of conserved proteins) confirmed that this strain is a subspecies of Ligilactobacillus acidipiscis.

[0049] Comparative genomic analysis of the hexanoic acid synthase gene sequences of the three strains and their related strains showed differences. The 16S rRNA genes of *Caproicibacterium argilliputei* XB1, *Clostridium tyrobutyricum* DS1, and *Ligilactobacillus acidipiscis* YHS1A have NCBI accession numbers PQ870405.1, PV111091, and PQ626723.1, respectively; their whole genome sequences have NCBI accession numbers CP174369.1, CP180725, and CP173417.1, respectively. Furthermore, strain *Clostridium tyrobutyricum* DS1 has an additional plasmid compared to its homologous strains; its plasmid sequence has NCBI accession number CP180726.

[0050] The culture media used for isolation and performance testing are as follows: The isolation medium used for Caproicibacterium argilliputei XB1, Clostridium tyrobutyricum DS1, and Ligilactobacillus acidipiscis YHS1A is a medium with the natural components of "yellow water" from the fermentation system of strong-aroma baijiu as the main component. Specifically, the liquid culture medium consists of fresh yellow water diluted 5 times with deionized water to achieve a lactic acid concentration suitable for the growth of caproic acid bacteria, a sufficiently low ethanol concentration, and the addition of 5 g L / L... -1Glucose was added, and the pH was adjusted to 6.5, the optimal pH for the growth of caproic acid bacteria, using 3 mol / L NaOH solution. The solid plate culture medium was prepared by adding 2% agar to the liquid medium.

[0051] The fermentation medium used (L) -1 The culture medium consisted of: 10 g tryptone, 10 g yeast extract, 2 g ammonium sulfate, 1 g sodium hydrogen phosphate, 0.5 g dipotassium hydrogen phosphate, 0.1 g magnesium sulfate heptahydrate, 0.015 g ferrous sulfate heptahydrate, 0.01 g manganese sulfate monohydrate, 0.01 g calcium chloride, 0.002 g cobalt chloride, 0.002 g zinc sulfate, 15 g L / D-lactic acid, 5 g glucose, 9.72 mL ethanol, and 2 mL of "yellow water" (a type of fermented liquid used in the in-situ fermentation of strong-aroma baijiu). The medium was filtered through a 0.22 μm filter before being added. Based on the carbon source composition of the "yellow water" in the in-situ fermentation liquid system of strong-aroma baijiu, residual glucose and biodegradable lactic acid were added, and ethanol was used as the selective pressure to verify the hexanoic acid synthesis by hexanoic acid bacteria under ethanol-tolerant conditions.

[0052] The "lactic acid medium" used for the gradient lactate concentrations was based on the above-mentioned "fermentation medium," but with glucose and ethanol removed, and lactate concentrations of 15, 30, 60, and 90 g / L replaced. -1 This study was used to examine the effect of high concentrations of lactic acid on hexanoic acid synthesis by a complex hexanoic acid-producing bacterial community.

[0053] The gradient ethanol concentration "ethanol-lactic acid medium" used is based on the "fermentation medium" with glucose and ethanol removed, and the ethanol concentrations replaced with 10, 20, 40, and 60 g / L. -1 This study was used to examine the effect of high concentrations of ethanol on the synthesis of hexanoic acid in bacterial communities.

[0054] The highest lactate concentration (90 g / L) in the "lactic acid medium" used -1 The highest ethanol concentration (60 g / L) in "ethanol-lactic acid medium" and -1 It is slightly higher than the concentration of the corresponding component in the "yellow water" of the in-situ fermentation system of strong-aroma baijiu.

[0055] Caproicibacterium argilliputei XB1, Clostridium tyrobutyricum DS1, and Ligilactobacillus acidipiscis YHS1A have a wide range of usable carbon sources. Besides glucose and lactic acid in the culture medium, their available carbon sources include various sugars such as fructose, trehalose, galactose, cellobiose, dextrin, maltose, mannose, sorbitol, sucrose, melibiose, and melitriose, as well as various acids such as α-butanone, α-ketovalerate, malic acid, β-hydroxybutyric acid, and asparagine. Most of these components are metabolic products within the bacteria.

[0056] Specifically, the compound hexanoic acid bacteria group provided in this embodiment is isolated, identified and cultured using the following method.

[0057] Caproicibacterium argilliputei XB1, Clostridium tyrobutyricum DS1, and Ligilactobacillus acidipiscis YHS1A were all isolated from enriched caproic acid bacteria broth in a strong-aroma baijiu fermentation system, obtained through serial dilution and streak plating in an anaerobic workstation. Therefore, obtaining these three strains required first enriching them in the fermentation system, then continuously purifying them using a separation and purification medium, and finally identifying and culturing them to determine their growth and metabolic characteristics.

[0058] The isolation of Caproicibacterium argilliputei XB1 was achieved during the preparation of caproic acid bacteria solution. It was found that a strain related to Caproicibacterium argilliputei ZCY20-5T was dominant in the bacterial solution, and therefore it was isolated as the core functional strain for preparing caproic acid bacteria solution.

[0059] (1) Isolation of three strains

[0060] Enrichment of in-situ caproic acid bacteria solution: The by-product "yellow water" after the fermentation of strong-aroma baijiu is diluted tenfold to obtain diluted yellow water. The diluted yellow water is mixed with the surface cellar mud at a volume-to-mass ratio of 10:1. After 3 days of cultivation in an anaerobic workstation with anaerobic gas H2:CO2:N2 = 1:1:4, it is used as a starter culture. The diluted yellow water is then used for continuous cultivation and enrichment to obtain the enriched caproic acid bacteria solution.

[0061] Strain isolation: The same components as the fermentation medium (L -1The fermentation medium consisted of: 10 g tryptone, 10 g yeast extract, 2 g ammonium sulfate, 1 g sodium hydrogen phosphate, 0.5 g dipotassium hydrogen phosphate, 0.1 g magnesium sulfate heptahydrate, 0.015 g ferrous sulfate heptahydrate, 0.01 g manganese sulfate monohydrate, 0.01 g calcium chloride, 0.002 g cobalt chloride, 0.002 g zinc sulfate, 15 g L / D-lactic acid, 5 g glucose, 9.72 mL ethanol, and 2 mL yellow water, all filtered through a 0.22 μm filter membrane before being added. This fermentation medium was used as the separation medium, and in an anaerobic workstation (incubator), it was serially diluted 5 times (i.e., 10⁻⁶) using the gradient dilution plate method. -1 10 -2 10 -3 10 -4 and 10 -5 The last three serially diluted bacterial solutions were selected and inoculated onto solid agar plates using the plate spread method. The plates were then inverted and anaerobically incubated at 34°C until colonies appeared. Dominant colonies were selected for three consecutive rounds of purification and identification.

[0062] (2) Identification: First, the bacteria were observed by colony morphology, color and scanning electron microscopy. Then, the full-length 16S rRNA gene was amplified by PCR and sequenced. Finally, the whole genome was sequenced to identify the bacteria.

[0063] (3) Culture of strains: The isolated strains were cultured in a fermentation medium with pH 6.5 and physicochemical parameters, including hexanoic acid, were measured at fixed time intervals until the strains reached the stationary phase.

[0064] Specifically, the characteristics and identification of the compound hexanoic acid bacteria group provided in this embodiment are as follows:

[0065] (1) Colony morphology of the three strains: Caproicibacterium argilliputei XB1 colonies are round with neat edges, milky white raised, moist and viscous; Clostridium tyrobutyricum DS1 colonies are round with irregular edges, slightly yellow, no raised, and flat; Ligilactobacillus acidipiscis YHS1A colonies are round with neat edges and milky white.

[0066] (2) Cell morphology of the three strains: Caproicibacterium argilliputei XB1, Clostridium tyrobutyricum DS1, and Ligilactobacillus acidipiscis YHS1A were all Gram-positive as determined by Gram staining; as shown by scanning electron microscopy results... Figure 1As shown, all three strains were solitary, with no chain-like or aggregated phenomena observed. Among the three, strain *Caproicibacterium argilliputei* XB1 was the largest, a long rod-shaped cell approximately 1 × 5 μm in size, with a thick capsule visible on its surface, indicating adhesion, but no flagella were observed. Strain *Clostridium tyrobutyricum* DS1 was medium-sized, a short rod-shaped cell approximately 1.5 × 5 μm in size, with a smooth surface, a thin capsule or extracellular biofilm layer, and asymmetrical long flagella as a locomotor organ, confirming its motility. Strain *Ligilactobacillus acidipiscis* YHS1A was the smallest, a short rod-shaped cell approximately 1 × 2 μm in size, with pili-like components on its surface.

[0067] (3) Biolog carbon source spectrum analysis of the three strains: The carbon source spectrum results of the three strains were obtained by Biolog AN MicroPlates™ combined with anaerobic identification analysis as follows. Among them, the colored carbon source indicates that the strain can utilize the carbon source.

[0068] The carbon source spectrum of Caproicibacterium argilliputei XB1 is shown in Table 1:

[0069] Table 1. Spectra of Caproicibacterium argilliputei XB1 using different carbon sources

[0070]

[0071] The carbon source spectrum of Clostridium tyrobutyricum DS1 is shown in Table 2.

[0072] Table 2. Spectra of Clostridium tyrobutyricum DS1 using different carbon sources

[0073] ③ The carbon source spectrum of strain Ligilactobacillus acidipiscis YHS1A is shown in Table 3.

[0074] Table 3. Spectra of Ligilactobacillus acidipiscis YHS1A using different carbon sources.

[0075] (4) Molecular biological identification characteristics

[0076] ①The 16S rRNA gene sequence of *Caproicibacterium argilliputei* XB1 has been submitted to the NCBI database, accession number PQ870405.1. Blasten alignment analysis in the NCBI database showed that the 16S rRNA gene sequence of *Caproicibacterium argilliputei* XB1 has the highest identity (100.00%) with the type strain *Caproicibacterium argilliputei* ZCY20-5 (Accession No. CP135996.1). Further whole-genome sequencing was performed, and the sequenced data was submitted to the NCBI database, accession number CP174369.1. By analyzing its whole genome sequence and the sequences of related strains using ANI (average nucleotide identity), AAI (average amino-acid identity), and POCP (percentage of conserved proteins), as shown in Table 4, the AAI value between Caproicibacterium argilliputei XB1 and its closest relative, Caproicibacterium argilliputei ZCY20-5, was far below the species-level threshold of 95.00%. Furthermore, based on the genus-level thresholds of AAI (74.00%) and POCP (50.00%), the strain was further identified as a subspecies of Caproicibacterium argilliputei; it does not belong to the same genus as the other homologous strains. Based on our previous research, metagenomic sequencing revealed that Caproicibacterium argilliputei was the most abundant strain in the in-situ enriched caproic acid-producing bacterial culture. Therefore, in this embodiment, it was identified as one of the core functional strains in the complex caproic acid-producing bacterial community responsible for caproic acid synthesis.

[0077] Table 4. Analysis of ANI, AAI, and POCP values ​​of Caproicibacterium argilliputei XB1 genome and related strains.

[0078]

[0079] To analyze the differences in hexanoate synthase genes between strain Caproicibacterium argilliputei XB1 and its closest relative, Caproicibacterium argilliputei ZCY20-5, comparative genomic analysis of hexanoate synthase in their whole genomes was performed. The results are as follows: Figure 2 As shown, this indicates that strain Caproicibacterium argilliputei XB1 differs from previously published type strains, with a difference in the number of tes genes in its hexanoic acid synthase gene (sequence differences are not shown).

[0080] ②The 16S rRNA gene sequence of *Clostridium tyrobutyricum* DS1 has been submitted to the NCBI database, accession number PV111091. Blasten alignment analysis in the NCBI database showed that the 16S rRNA gene sequence of strain *Clostridium tyrobutyricum* DS1 has the highest identity (99.34%) with the type strain *Clostridium tyrobutyricum* strain ATCC 25755 (Accession No. NR_044718.2). Further whole-genome sequencing was performed, and the sequenced data was submitted to the NCBI database, accession number CP180725. ANI (average nucleotide identity), AAI (average amino-acid identity), and POCP (percentage of conserved proteins) analyses were performed between the whole-genome sequence and the sequences of related strains, as shown in Table 5. Although Blanne analysis of the 16S rRNA gene sequence showed that strain Clostridium tyrobutyricum DS1 is most closely related to strain Clostridium tyrobutyricum strain ATCC 25755, the whole-genome sequencing analysis results shown in Table 5 indicate that strain Clostridium tyrobutyricum DS1 is most closely related to strain Clostridium tyrobutyricum Cirm BIA 2237. The AAI value between the two strains is far below the species-level threshold of 95.00%. Furthermore, based on the genus-level thresholds of AAI value (74.00%) and POCP value (50.00%), strain Clostridium tyrobutyricum DS1 was further identified as a subspecies of Clostridium tyrobutyricum. Based on our previous research, metagenomic sequencing revealed that the relative abundance of Clostridium tyrobutyricum DS1, a relative relative of the in situ enriched caproic acid bacteria, was second only to the absolutely dominant Caproicibacterium argilliputei, and that both strains synergistically enhanced caproic acid production (described later). Therefore, in this embodiment, Clostridium tyrobutyricum DS1 is used as one of the core functional strains in the complex caproic acid bacteria community for caproic acid synthesis.

[0081] Table 5. Analysis of ANI, AAI, and POCP values ​​of C. tyrobutyricum DS1 genome and related strains

[0082]

[0083] To analyze the differences in butyrate synthase genes between strain Clostridium tyrobutyricum DS1 and its closest relative Clostridium tyrobutyricum Cirm BIA 2237, comparative genomic analysis of hexanoate synthase in their whole genomes was performed. The results are as follows: Figure 3 As shown, there is a significant difference in the distribution order of the butyrate synthase gene between the two strains (sequence differences are not shown). Furthermore, strain Clostridium tyrobutyricum DS1 has an additional plasmid (NCBI accession number CP180726) compared to its homologous strains, none of which have plasmids. This indicates that strain Clostridium tyrobutyricum DS1 in this example differs from the related model strains.

[0084] ③ The 16S rRNA gene sequence of *Ligilactobacillus acidipiscis* YHS1A has been submitted to the NCBI database, accession number PQ626723.1. Blasten alignment analysis in the NCBI database showed that the 16S rRNA gene sequence of strain *Ligilactobacillus acidipiscis* YHS1A has the highest identity (99.93%) with the type strain *Ligilactobacillus acidipiscis* NBRC 102163 (Accession No. NR_112693.1). Further whole-genome sequencing was performed, and the sequenced data was submitted to the NCBI database, accession number CP173417.1. ANI (average nucleotide identity), AAI (average amino-acid identity), and POCP (percentage of conserved proteins) analyses were performed between the whole-genome sequence and the sequences of related strains, as shown in Table 6.

[0085] Table 6. Analysis of ANI, AAI, and POCP values ​​of Ligilactobacillus acidipiscis YHS1A genome and related strains.

[0086] Although Blanne analysis of the 16S rRNA gene sequence showed that strain *Ligilactobacillus acidipiscis* YHS1A was most closely related to strain *Ligilactobacillus acidipiscis* NBRC 102163, the whole-genome sequencing analysis results shown in Table 6 indicated that strain *Ligilactobacillus acidipiscis* YHS1A was most closely related to strain *Lactobacillus acidipiscis* ACA-DC 1533. The AAI value between strain *Ligilactobacillus acidipiscis* YHS1A and its most closely related strain *Lactobacillus acidipiscis* ACA-DC 1533 was far below the species-level threshold of 95.00%. Furthermore, based on the genus-level thresholds of AAI value (74.00%) and POCP value (50.00%), the strain was further identified as a subspecies of *Lactobacillus acidipiscis*. Lactobacillus acidipiscis was originally classified under Lactobacillus at the genus level. In 2020, when the current broad genus *Lactobacillus sensu lato* split into 25 new genera, Lactobacillus acidipiscis was reclassified as *Ligilactobacillus acidipiscis*. Therefore, the strain YHS1A in this embodiment is defined as *Ligilactobacillus acidipiscis* YHS1A. Significantly different from other lactic acid bacteria, the genus in this embodiment, because it was isolated from hexanoic acid bacteria culture, possesses the ability to synthesize hexanoic acid. This study further analyzed the function of the hexanoic acid synthase gene in its genome and mapped the metabolic pathways involved in hexanoic acid synthesis in its genome, such as... Figure 4As shown, substrate preparation and hexanoic acid synthesis are performed via RBO (reverse β-oxidation pathway) and FAB (fatty acid biosynthesis pathway). EMP: glycolysis pathway; LDH: lactate dehydrogenase; PDC: pyruvate dehydrogenase complex; ACP: acyl carrier protein; CoA: coenzyme A; PTA: phosphoryltransferase; ACK: acetate kinase; THL: thiolase; KCR: ketoacyl-CoA reductase; HCD: hydroxyacyl-CoA dehydratase; ECR: enoyl-CoA reductase; TES: thioesterase; PTB: phosphorylated butyryltransferase; BUK: butyrate kinase; CAT: butyryl-CoA reductase. A-transferase; ACC: acetyl-CoA carboxylase; MAT: malonyl-CoA; ACP transacylase; ATA: acetyl-transacylase; KAS: β-ketoacyl-ACP synthase II or III; KAR: ketoacyl-ACP synthase; HAD: hydroxyacyl-ACP dehydratase; EAR: enoyl-ACP reductase; H2ase: ferroredoxin hydrogenase; EtfA: electron transport flavoprotein α subunit; EtfB: electron transport flavoprotein β subunit. The locus numbers in parentheses correspond to the gene tags of Ligilactobacillus acidipiscis YHS1A in the GenBank database (accession number: CP173417.1). Enzymes marked with a red cross "×" in the figure indicate that their corresponding genes do not exist in the Ligilactobacillus acidipiscis YHS1A genome. The tag number in parentheses is the locus tag number of the corresponding enzyme gene in the Ligilactobacillus acidipiscis YHS1A genome.

[0087] The results showed that strain Ligilactobacillus acidipiscis YHS1A possesses a metabolic pathway for hexanoic acid synthesis.

[0088] Specifically, this embodiment describes the growth and metabolic characteristics of the three core functional strains in the compound hexanoic acid bacteria group in the fermentation medium.

[0089] To determine whether the functional strains in the complex hexanoic acid bacteria community can perform hexanoic acid synthesis and metabolism under ethanol-tolerant conditions, this study analyzed the growth and metabolic characteristics of the core functional strains in sequence.

[0090] First, the growth (OD600) of the core functional caproic acid-producing strain *Caproicibacterium argilliputei* XB1 in the complex caproic acid bacterial community was analyzed under gradient temperature conditions. Figure 5As shown, the results indicate that Caproicibacterium argilliputei XB1 can grow at temperatures of 15 to 45°C, with a suitable growth temperature range of 20 to 40°C, suggesting that it can grow and metabolize under relatively natural fermentation system conditions during the fermentation of strong-aroma baijiu.

[0091] Secondly, the growth and metabolic characteristics of the core functional caproic acid-producing strain *Caproicibacterium argilliputei* XB1 in the complex caproic acid bacteria community under ethanol-tolerant conditions at 34℃ (the temperature during the later stage of fermentation of strong-aroma baijiu) in the fermentation medium were analyzed. Figure 6 As shown in the figure (error bars represent standard deviation; differences between samples with different letters are significant, p < 0.05, ANOVA, n = 3). The results indicate that the strain *Caproicibacterium argilliputei* XB1 can degrade glucose and lactic acid to synthesize hexanoic acid in a fermentation medium with a simulated carbon source ratio in yellow water, under conditions of ethanol tolerance and non-ethanol degradation. It should be noted that the fermentation medium provided the electron acceptors for hexanoic acid synthesis: acetate and butyrate (provided by sodium acetate and sodium butyrate, respectively). In natural fermentation, these require the assistance of co-existing strains. Therefore, we also analyzed the metabolic characteristics of two other core functional strains under ethanol-tolerant conditions.

[0092] Then, the synergistic functional strain *Clostridium tyrobutyricum* DS1 in the complex hexanoic acid bacteria community was analyzed. Under the same conditions, in the fermentation medium at 34℃ (the temperature during the later stage of fermentation of strong-aroma baijiu), its growth and metabolic characteristics under ethanol-tolerant conditions were examined. Figure 7 As shown in the figure (error bars represent standard deviation; samples with different letters are significantly different, p < 0.05, ANOVA, n = 3). The results indicate that the strain *Clostridium tyrobutyricum* DS1 can degrade glucose and lactic acid to synthesize butyric acid in a fermentation medium with a carbon source ratio simulating that of yellow water, under conditions where it tolerates ethanol without degrading ethanol. This provides butyric acid, the electron acceptor, for hexanoic acid synthesis by hexanoic acid bacteria in the hexanoic acid synthesis metabolism involving a complex hexanoic acid bacterial community.

[0093] Finally, before analyzing the growth and metabolic characteristics of the synergistic strain *Ligilactobacillus acidipiscis* YHS1A in the hexanoic acid complex under ethanol-tolerant conditions, its suitable growth temperature and pH conditions were first determined. Figure 8The image shows the growth of strain *Ligilactobacillus acidipiscis* YHS1A under gradient temperatures and pH values. The results indicate that its optimal growth temperature is 20–45℃; its optimal growth pH is 5.5–9.5, but it can still grow weakly at pH 3–4.5, indicating that it can grow and metabolize under the relatively natural low pH fermentation conditions of strong-aroma baijiu. It should be noted that traditional hexanoic acid strains are intolerant to the "high acid / alcohol" metabolic conditions of the in-situ fermentation system of strong-aroma baijiu, while *Ligilactobacillus acidipiscis* YHS1A, as a lactic acid bacterium that produces butyric acid and trace amounts of hexanoic acid, has a natural tolerance to low pH values. This allows the composite hexanoic acid bacteria group in this embodiment to complete pH synthesis within a wide pH range. Next, the growth and metabolic characteristics of strain *Ligilactobacillus acidipiscis* YHS1A in the same fermentation medium at 34℃ (the temperature in the later stages of strong-aroma baijiu fermentation) are shown below. Figure 9 As shown in the figure (error bars represent standard deviation; samples with different letters are significantly different, p < 0.05, ANOVA, n = 3). The results indicate that although this strain is a lactic acid bacterium in a broad sense, it can indeed synthesize lactic acid, acetic acid, butyric acid, and trace amounts of hexanoic acid under conditions of ethanol tolerance and without ethanol degradation (hexanoic acid may subsequently be degraded via a reversible RBO metabolic pathway due to insufficient carbon source or electron acceptor). The synthesized lactic acid can serve as a carbon source for the core strains Caproicibacterium argilliputei XB1 and Clostridium tyrobutyricum DS1 in the core hexanoic acid bacterium culture; acetic acid can serve as an electron acceptor for butyric acid or hexanoic acid synthesis; and butyric acid can serve as an electron acceptor for hexanoic acid synthesis. This study is the first to discover that acidophilic lactic acid bacteria can synthesize butyric acid and trace amounts of hexanoic acid. Given that this study is the first to discover that the lactic acid strain YHS1A can synthesize fatty acids, we further analyzed its possible metabolic pathways through transcriptomics analysis, providing a theoretical basis for future applications, such as... Figure 10 As shown in the figure. The results indicate that Ligilactobacillus acidipiscis YHS1A can sequentially synthesize butyric acid and hexanoic acid via the FAB (fatty acid biosynthesis) metabolic pathway, and the subsequent degradation of hexanoic acid is carried out via the RBO metabolic pathway.

[0094] In summary, all three core functional strains in the hexanoic acid complex can synthesize fatty acids without degrading ethanol and under ethanol-tolerant conditions. The lack of ethanol degradation indicates that these three strains have no impact on the yield of baijiu production; ethanol tolerance indicates that ethanol does not affect their own metabolism. *Ligilactobacillus acidipiscis* YHS1A primarily degrades glucose to synthesize lactic acid, acetic acid, and butyric acid; *Clostridium tyrobutyricum* DS1 primarily degrades glucose and lactic acid to synthesize butyric acid; and *Caproicibacterium argilliputei* XB1 primarily degrades glucose and lactic acid to synthesize butyric acid and hexanoic acid. Furthermore, the accumulation of electron acceptors acetic acid and butyric acid facilitates the final metabolic chain progression to hexanoic acid. This indicates that the three core strains exhibit synergy and their products are not entirely identical.

[0095] Specifically, this embodiment provides a sequential batch synergistic metabolic characteristic analysis of two synergistic strains and the core hexanoic acid strain Caproicibacterium argilliputei XB1 in a complex hexanoic acid bacterial community.

[0096] To further simulate the batch fermentation mode of strong-aroma baijiu, the synergistic promoting effect of Clostridium tyrobutyricum DS1 and Ligilactobacillus acidipiscis YHS1A on the hexanoic acid synthesis of Caproicibacterium argilliputei XB1 was verified by continuous batch fermentation.

[0097] First, the synergistic metabolic effects of Clostridium tyrobutyricum DS1 (abbreviated as C. tyrobutyricum DS1 in the figure) and strain Caproicibacterium argilliputei XB1 (abbreviated as C. sp. XB1 in the figure) during continuous batch fermentation were analyzed. Figure 11 As shown, the results indicate that during the synergistic continuous fermentation of the two strains, the amount of hexanoic acid synthesized gradually increased with the increase of fermentation batches. Compared with the control pure strain Caproicibacterium argilliputeiXB1, the amount of hexanoic acid synthesized increased by 27.03% to 68.18%. This suggests that the addition of Clostridium tyrobutyricum DS1 strain to the complex hexanoic acid bacterial community helps to increase the yield of hexanoic acid. Since both strains are suitable for growth and metabolism in a weakly acidic to near-neutral physicochemical environment, this indicates that their synergistic metabolism is beneficial for strongly promoting hexanoic acid synthesis within the weakly acidic to near-neutral physicochemical range.

[0098] Then, the synergistic metabolic effects of strains *Ligilactobacillus acidipiscis* YHS1A and *Caproicibacterium argilliputei* XB1 during continuous batch fermentation were analyzed. Figure 12 As shown, the results indicate that during the synergistic continuous fermentation process of the two strains, the hexanoic acid yield was 72.73% higher than that of the single strain *Caproicibacterium argilliputei* XB1 after the first batch of fermentation. Then, with the increase in fermentation batches from 2 to 6, the hexanoic acid synthesis slightly decreased and then stabilized at a low value, showing a decline compared to the single strain *Caproicibacterium argilliputei* XB1. This suggests that, unlike strain *Clostridium tyrobutyricum* DS1, which is favorable for long-term hexanoic acid synthesis, strain *Ligilactobacillus acidipiscis* YHS1A is favorable for rapidly increasing hexanoic acid synthesis. The subsequent decrease in hexanoic acid synthesis is presumably related to the rapid decrease in pH and the synthesis of large amounts of acetic acid and lactic acid. Therefore, in actual use, it is necessary to add a compound of Ligilactobacillus acipiscis YHS1A and Caproicibacterium argilliputei XB1 to each fermentation batch, while a compound of Clostridium tyrobutyricum DS1 and Caproicibacterium argilliputei XB1 needs to be added each time.

[0099] Specifically, this invention provides a screening of "motile hexanoic acid bacteria" and an analysis of their "high acid alcohol" metabolic characteristics in a complex hexanoic acid bacteria community.

[0100] Given that the fermentation of strong-aroma baijiu is solid-state fermentation in the early stage and semi-solid-liquid fermentation in the later stage, the complex caproic acid bacteria added to the surface can sink to the lower layer for metabolism due to gravity during the actual fermentation process. Simultaneously, to enable the complex caproic acid bacteria to overcome gravitational potential difference and resistance and move to the upper mash-yellow water system for metabolism, we further screened for motile caproic acid bacteria from the enriched caproic acid bacteria solution. The specific screening method is as follows... Figure 13As shown in Figure B, the initial enrichment solution of hexanoic acid bacteria is shown in Figure B. CT1-4 represent four segments of the cotton thread from anaerobic bottle B to anaerobic bottle A, and CT5 represents the hexanoic acid bacteria that have completely migrated to anaerobic bottle A. In practice, as shown in the figure, anaerobic bottle B containing the hexanoic acid bacteria solution is placed at a lower position, while another anaerobic bottle A containing sterile screening medium is placed at a higher position. To eliminate interference from the cotton thread's own adsorption, the sterile cotton thread is thoroughly soaked with sterile screening medium beforehand. Then, the cotton thread is used to connect anaerobic bottle A and anaerobic bottle B, maintaining the connection for several days. The mobile hexanoic acid bacteria in sample CT5, which ultimately overcomes the gravitational potential difference and migrates from the lower bottle (B) to the higher bottle (A), are considered part of the composite hexanoic acid bacteria of this invention. The mobility of this hexanoic acid bacteria helps overcome the heterogeneity of hexanoic acid synthesis during the actual fermentation of strong-aroma baijiu. Metagenomic sequencing analysis of the community structure of the mobile hexanoic acid bacteria at the genus (A) and species (B) levels is shown in Figure B. Figure 14 As shown in the figure, Clostridium, Staphylococcus, Sporolactobacillus, and Unclassified Lachnospiraceae are the main mobile groups in the hexanoic acid bacteria community. Among them, the butyric acid-producing bacterium Clostridium tyrobutyricum is absolutely dominant, while traditional hexanoic acid bacteria are present in very small numbers. It is important to note that although Staphylococcus is considered an opportunistic pathogen, some of its species are also common in fermentation systems.

[0101] To ensure that the selected motile caproic acid bacteria can indeed assist the core functional strain Caproic acid bacteria (Caproicibacterium argilliputei XB1) in the actual high-concentration lactic acid and high-concentration ethanol fermentation system of strong-aroma baijiu in caproic acid synthesis and metabolism, it is necessary to test its metabolic capacity under high-concentration lactic acid and high-concentration ethanol conditions. For example... Figure 15 As shown (mean error bars represent standard deviation; significant differences exist between samples without identical lowercase letters, p < 0.05; * indicates significant differences between different samples within the same time period, p < 0.05; n = 3), in fermentation media containing gradient concentrations of lactic acid, motile hexanoic acid bacteria can still perform butyric acid-based anabolic metabolism, providing hexanoic acid as the electron acceptor for hexanoic acid synthesis. This indicates that they are largely unaffected by high lactic acid concentrations during the fermentation of strong-aroma baijiu. Further testing was conducted on the ability of motile hexanoic acid bacteria to increase hexanoic acid and decrease lactic acid under high-concentration ethanol conditions, such as... Figure 16As shown in the figure (error bars represent standard deviation; samples without identical lowercase letters show significant differences, p < 0.05; * indicates significant differences between different samples within the same time period, p < 0.05; n = 3), the results indicate that motile hexanoic acid bacteria can reach up to 60 g / L in the yellow water of a natural reaction system similar to that of strong-aroma baijiu. -1 Butyric acid synthesis can still occur in ethanol, and the amount of hexanoic acid synthesized is increased. Although hexanoic acid is subsequently degraded due to insufficient carbon source, the results indicate that motile hexanoic acid-producing bacteria can synthesize butyric acid normally in high-concentration ethanol, and even promote hexanoic acid synthesis.

[0102] Further analysis of the prokaryotic community structure in motile hexanoic acid bacteria tolerant to high concentrations of lactic acid culture medium (CTL) and high concentrations of ethanol-lactic acid culture medium (CTE) was conducted using 16S rRNA gene amplicon sequencing. Figure 17 As shown, the results indicated that the relative abundance of *Clostridium sensu stricto 12* (mainly *Clostridium tyrobutyricum*) in the bacterial community significantly increased with increasing stress intensity (reaching 93.62% and 81.41% under the highest stress), while the abundance of genera such as *Staphylococcus*, *Unclassified Lachnospiraceae*, and *Sporolactobacillus* decreased accordingly. Furthermore, the most dominant ASV was *Clostridium tyrobutyricum* DS1, one of the core dominant strains of the hexanoic acid-containing bacterial community isolated in this example. This demonstrates that *Clostridium tyrobutyricum* DS1 and strain *Ligilactobacillus acidipiscis* YHS1A in the hexanoic acid-containing bacterial community of this example can continuously metabolize in a high-acid / alcohol metabolic system, assisting in hexanoic acid synthesis.

[0103] Meanwhile, the present invention also provides a complex hexanoic acid bacteria group, including the aforementioned complex hexanoic acid bacteria group.

[0104] Finally, this invention provides an application of a complex hexanoic acid bacteria group for the synthesis of butyric acid and hexanoic acid during the fermentation process of strong-aroma baijiu.

[0105] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention (including the claims) is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in the details for the sake of brevity.

[0106] This invention is intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A complex hexanoic acid bacteria group, characterized in that, The complex hexanoic acid bacteria group contains hexanoic acid strains accounting for >50% by mass. Caproicibacterium argilliputei XB1 and butyric acid strains Clostridium tyrobutyricum DS1 or lactic acid bacteria Ligilactobacillus acidipiscis At least one of YHS1A.

2. The complex hexanoic acid bacteria group according to claim 1, characterized in that, The hexanoic acid strain Caproicibacterium argilliputei XB1 has the accession number GDMCC No: 67345.

3. The complex hexanoic acid bacteria group according to claim 1, characterized in that, The butyric acid strain Clostridium tyrobutyricum The accession number for DS1 is GDMCC No: 67347.

4. The complex hexanoic acid bacteria group according to claim 3, characterized in that, The butyric acid strain Clostridium tyrobutyricum DS1 has one more plasmid than its homologous strains.

5. The complex hexanoic acid bacteria group according to claim 1, characterized in that, The lactic acid strain Ligilactobacillus acidipiscis YHS1A has the accession number GDMCC No: 67348.

6. The complex hexanoic acid bacteria group according to claim 1, characterized in that, The complex caproic acid bacteria group also includes "mobile caproic acid bacteria group" screened from the in-situ enriched caproic acid bacteria solution of strong-aroma baijiu.

7. The complex hexanoic acid bacteria group according to claim 1, characterized in that, The strains in the complex caproic acid bacteria group were all isolated from the enriched caproic acid bacteria liquid of the strong-aroma baijiu fermentation system and obtained by gradient dilution and streak plate method in an anaerobic workstation.

8. A complex hexanoic acid bacteria group, characterized in that, Includes the complex hexanoic acid bacteria group as described in any one of claims 1 to 6.

9. An application of the compound hexanoic acid bacteria group according to claim 7, characterized in that, It is used to synthesize butyric acid and hexanoic acid during the fermentation process of strong-aroma baijiu.

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