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 increase 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 pit 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, existing caproic acid bacteria, such as... Caproiciproducens, Caproicibacterium, Clostridium, Caproicibacter Fermentation studies have revealed that existing hexanoic acid bacteria migrate from the bottom mud of the fermentation pit to the solid-liquid mixture of mash and yellow water during fermentation. This means that the actual fermentation process of strong-aroma baijiu primarily 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 composite caproic acid bacterial community, wherein the composite caproic acid bacterial community comprises caproic acid strains with a mass percentage >50%. Caproicibacterium argilliputei XB1 and butyric acid strains Clostridium tyrobutyricum DS1 or lactic acid bacteria Ligilactobacillus acidipiscis At least one of YHS1A.

[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; (2) The core dominant strain in the complex hexanoic acid bacteria group is a hexanoic acid strain. Caproicibacterium argilliputei XB1, butyric acid strain Clostridium tyrobutyricum DS1 and lactic acid bacteria Ligilactobacillus acidipiscis YHS1A and the non-dominant bacterial group are "motile hexanoic acid bacteria" that are tolerant to "high acid / alcohol" in situ enriched from hexanoic acid bacteria solution. These "motile hexanoic acid bacteria" are "motile" and can produce short-chain fatty acids such as acetic acid and butyric acid. This helps the bacteria overcome gravitational potential differences and resistance to move to the upper mash-yellow water system for metabolism. To some extent, this can alleviate the heterogeneity of fermentation results, while also providing acetic acid, an electron acceptor for the synthesis of butyric or hexanoic acid, for hexanoic acid synthesis.

[0009] (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

[0010] 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.

[0011] Figure 1 This is a SEM image of the compound hexanoic acid bacteria group in an embodiment of the present invention; Where A is Caproicibacterium argilliputei XB1, B is Clostridium tyrobutyricum DS1, C is Ligilactobacillus acidipiscis YHS1A; Figure 2 This is an embodiment of the present invention. Caproicibacterium argilliputei XB1 and its closest relatives Caproicibacterium argilliputei ZCY20-5 T Comparative genomics analysis diagram based on hexanoic acid synthase gene; Figure 3 This is an embodiment of the present invention.Clostridium tyrobutyricum DS1 and strain Clostridium tyrobutyricum Cirm Comparative genomics analysis diagram of BIA 2237 based on hexanoate synthase gene; Figure 4 This is an embodiment of the present invention. Ligilactobacillus acidipiscis A schematic diagram of genes involved in hexanoic acid biosynthesis in the YHS1A genome; Figure 5 This is an embodiment of the present invention. Caproicibacterium argilliputei Gradient temperature growth of XB1 (OD) 600 )picture; Figure 6 This is an embodiment of the present invention. Caproicibacterium argilliputei Growth and metabolic characteristics of XB1 under different conditions; 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. Figure 7 This is an embodiment of the present invention. Clostridium tyrobutyricum Growth and metabolic characteristics of DS1 under different conditions; Where A is OD 600 B 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; Figure 8 This is an embodiment of the present invention. Ligilactobacillus acidipiscis Figures showing the growth of YHS1A at gradient temperatures and different pH values; Figure 9 This is an embodiment of the present invention. Ligilactobacillus acidipiscis Growth and metabolic characteristics of YHS1A under different conditions; 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; Figure 10 This is an embodiment of the present invention. Ligilactobacillus acidipiscis Volcano plot of differentially expressed genes (DEGs) of YHS1A at different time points during fermentation; 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. 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. Figure 11 This is an embodiment of the present invention. Caproicibacterium argilliputei XB1 and Clostridiumtyrobutyricum Growth and metabolic characteristics of DS1 under different conditions during 6 consecutive batch fermentations; 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; Figure 12 This is an embodiment of the present invention. Caproicibacterium argilliputei XB1 and Ligilactobacillus acidipiscis Growth and metabolic characteristics of YHS1A under different conditions during 6 consecutive batch fermentations. 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; 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. In this diagram, A is a simulation image and B is a real-world installation image. 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. Where A represents the genus and B represents the species; Figure 15 Metabolic characteristics of the selected "mobile caproic acid bacteria" during growth in a culture medium containing gradient concentrations of lactic acid as the sole carbon source; 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. Figure 16 To screen for "motile caproic acid bacteria", they were subjected to a mixture of ethanol at gradient concentrations and 15 g / L. -1 Metabolic characteristics of growth in culture media with lactic acid carbon source under different conditions; 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). 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

[0012] 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.

[0013] 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).

[0014] As one possible implementation method, Embodiment 1 of the present invention provides a "high acid / alcohol" tolerant caproic acid bacterial community, namely a complex caproic acid bacterial community. The complex caproic acid bacterial community consists of a "motile caproic acid bacterial community" of non-dominant bacteria that are "high acid / alcohol" tolerant and assist in the synthesis of caproic acid, and a core functional strain: a caproic acid strain. Caproicibacterium argilliputei XB1 and butyric acid strains Clostridium tyrobutyricum DS1 or lactic acid bacteria Ligilactobacillus acidipiscis At least one component of YHS1A. Specifically, the hexanoic acid strain... Caproicibacterium argilliputei XB1 accounts for more than 50% of the total mass.

[0015] at present Caproicibacterium argilliputei XB1, Clostridium tyrobutyricum DS1 and Ligilactobacillus acidipiscis Three strains of YHS1A were deposited on November 14, 2025, at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The accession numbers are GDMCC No. 67345, GDMCC No. 67347, and GDMCC No. 67348, respectively. The three strains provided in this invention were isolated from the enriched caproic acid bacteria broth of a strong-aroma baijiu fermentation system, obtained through a serial dilution and streak plate method in an anaerobic workstation.

[0016] Blasten alignment analysis of the 16S rRNA gene revealed the following: strain Caproicibacterium argilliputei The bacterial species with the highest 16S rRNA gene sequence identity between XB1 and existing model bacterial species in the NCBI rRNA database is... Caproicibacterium argilliputeiZCY20-5 (Accession No. CP135996.1) has 100.00% identity. 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) confirms that this strain is a species... Caproicibacterium argilliputei Subspecies.

[0017] strain Clostridium tyrobutyricum The bacterial species with the highest 16S rRNA gene sequence identity between DS1 and existing model bacterial species in the NCBI rRNA database is […]. Clostridium tyrobutyricum strain ATCC25755 (Accession No. NR_044718.2) has a 99.34% identity. 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) confirms that this strain is a species of ATCC25755. Clostridium tyrobutyricum Subspecies.

[0018] strain Ligilactobacillus acidipiscis The bacterial species with the highest 16S rRNA gene sequence identity between YHS1A and existing model bacterial species in the NCBI rRNA database is... Ligilactobacillus acidipiscis NBRC102163 (Accession No. NR_112693.1) has a 99.93% identity. 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) confirms that this strain is a species of NBRC102163. Ligilactobacillus acidipiscis Subspecies.

[0019] Genomic analysis comparing the hexanoic acid synthase gene sequences of the three strains with their related strains showed differences. Caproicibacterium argilliputei XB1, Clostridium tyrobutyricum DS1, Ligilactobacillus acidipiscisThe 16S rRNA gene of YHS1A has NCBI sequence accession numbers PQ870405.1, PV111091, and PQ626723.1, respectively; its whole genome sequence has NCBI sequence accession numbers CP174369.1, CP180725, and CP173417.1, respectively; in addition, the strain... Clostridium tyrobutyricum DS1 has an additional plasmid compared to its homologous strains, and its plasmid sequence is accessed by NCBI under the number CP180726.

[0020] The culture media used for separation and performance testing are as follows: Separation Caproicibacterium argilliputei XB1, Clostridium tyrobutyricum DS1 and Ligilactobacillus acidipiscis The isolation medium used in YHS1A is a medium whose main component is the natural components of "yellow water" from the fermentation system of strong-aroma baijiu. Specifically, the liquid 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, while maintaining a sufficiently low ethanol concentration, and adding an additional 5 g / L. -1 Glucose 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.

[0021] 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.

[0022] 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.

[0023] 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. -1This study was used to examine the effect of high concentrations of ethanol on the synthesis of hexanoic acid in bacterial communities.

[0024] 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.

[0025] Caproicibacterium argilliputei XB1, Clostridium tyrobutyricum DS1, Ligilactobacillus acidipiscis YHS1A has a wide range of carbon source applications. Besides glucose and lactic acid in the culture medium, its 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 byproducts in the bacterial cell's metabolic pathways.

[0026] Specifically, the compound hexanoic acid bacteria group provided in this embodiment is isolated, identified and cultured using the following method.

[0027] Caproicibacterium argilliputei XB1, Clostridium tyrobutyricum DS1, Ligilactobacillus acidipiscis YHS1A strains 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 the 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.

[0028] for Caproicibacterium argilliputei The isolation of XB1 was discovered during the preparation of the hexanoic acid bacteria suspension, where it was found to be related to the strain. Caproicibacterium argilliputei The strain related to ZCY20-5T was absolutely dominant in the bacterial solution, so it was isolated as the core functional strain for preparing hexanoic acid bacterial solution.

[0029] (1) Isolation of three strains 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.

[0030] 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.

[0031] (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.

[0032] (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.

[0033] Specifically, the characteristics and identification of the compound hexanoic acid bacteria group provided in this embodiment are as follows: (1) Colony morphology of the three strains: Caproicibacterium argilliputei XB1 colonies are round with neat edges, milky white, raised, moist, and viscous; strain Clostridium tyrobutyricum DS1 colonies are round with irregular edges, slightly yellow, flat, and without any raised areas; strain Ligilactobacillus acidipiscis YHS1A colonies are round with neat edges and are milky white.

[0034] (2) Cell morphology of the three strains: Caproicibacterium argilliputei XB1, Clostridium tyrobutyricum DS1, Ligilactobacillus acidipiscis YHS1A was Gram-positive as determined by Gram staining; scanning electron microscopy results showed... Figure 1 As shown, all three strains were solitary, with no chain or aggregated phenomena observed. Comparing the three strains, the... Caproicibacterium argilliputei XB1 is the largest, a long rod-shaped cell approximately 1×5 μm in size. A thick capsule is visible on the cell surface, indicating its adhesive function; no flagella were observed.Clostridium tyrobutyricum DS1 is medium-sized, short rod-shaped, approximately 1.5 × 5 μm in size. The bacterial cell surface is smooth, with a thin capsule or extracellular biofilm layer, and asymmetrical long flagella as locomotor organs; it has been identified as motile. Ligilactobacillus acidipiscis YHS1A is the smallest, short rod-shaped, about 1×2 μm in size, and has a fimbria-like structure on its surface.

[0035] (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.

[0036] ① Caproicibacterium argilliputei The carbon source spectrum of XB1 is shown in Table 1: Table 1 Caproicibacterium argilliputei XB1 utilizes spectra from different carbon sources

[0037] ② Clostridium tyrobutyricum The carbon source spectrum of DS1 is shown in Table 2.

[0038] Table 2 Clostridium tyrobutyricum DS1 utilizes spectra from different carbon sources

[0039] ③ strain Ligilactobacillus acidipiscis The carbon source spectrum of YHS1A is shown in Table 3.

[0040] Table 3 Ligilactobacillus acidipiscis YHS1A utilizes spectra from different carbon sources

[0041] (4) Molecular biological identification characteristics ① Caproicibacterium argilliputei The 16S rRNA gene sequence of XB1 has been submitted to the NCBI database, accession number PQ870405.1. Blasten alignment analysis in the NCBI database shows that... Caproicibacterium argilliputei XB1 and type strains Caproicibacterium argilliputeiThe 16S rRNA gene sequence of ZCY20-5 (Accession No. CP135996.1) showed the highest identity (100.00%). Further whole-genome sequencing was performed, and the sequenced data was submitted to the NCBI database (accession number CP174369.1). 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 4. Caproicibacterium argillipute i XB1 and its closest relatives Caproicibacterium clay pit The AAI values ​​of ZCY20-5 were far below the species-level threshold of 95.00%. Furthermore, by using the AAI value (74.00%) and POCP value (50.00%), which are at the genus-level threshold, the strain was further identified as a species. Caproicibacterium clay pit It is a subspecies; and does not belong to the same genus as other homologous species. Based on our previous research, metagenomic sequencing revealed that the in situ enriched hexanoic acid bacteria solution contained... Caproicibacterium argilliputei The relative abundance of this strain is absolutely dominant, therefore, in this embodiment, it is identified as one of the core functional strains in the complex hexanoic acid bacteria group used for hexanoic acid synthesis.

[0042] Table 4. Analysis of ANI, AAI, and POCP values ​​of Caproicibacterium argilliputei XB1 genome and related strains.

[0043] To analyze the strains Caproicibacterium argilliputei XB1 and its closest relatives Caproicibacterium argilliputei Differences in hexanoate synthase genes between ZCY20-5 were analyzed through comparative genomic analysis of hexanoate synthases across their entire genomes. The results are as follows: Figure 2 As shown. This indicates the strain. Caproicibacterium argilliputei XB1 differs from previously published type strains in that the number of tes genes in its hexanoic acid synthase gene differs (sequence differences are not shown).

[0044] ② Clostridium tyrobutyricum The 16S rRNA gene sequence of DS1 has been submitted to the NCBI database, accession number PV111091. Blasten alignment analysis in the NCBI database confirms that the strain... Clostridium tyrobutyric DS1 and type strain Clostridium tyrobutyricum strainThe 16S rRNA gene sequence of ATCC 25755 (Accession No. NR_044718.2) showed the highest identity (99.34%). 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 revealed the strain... Clostridium tyrobutyricum DS1 and strain Clostridium tyrobutyricum strain ATCC 25755 is the most closely related strain, while the whole-genome sequencing analysis results shown in Table 5 indicate that the strain... Clostridium tyrobutyricum DS1 and strain Clostridium tyrobutyricum Cirm BIA 2237 is the most closely related strain. The AAI value between the two is far below the species-level threshold of 95.00%. Furthermore, the strain was further identified using the AAI value (74.00%) and POCP value (50.00%) at the genus-level threshold. Clostridium tyrobutyricum DS1 is a bacterial strain Clostridium tyrobutyricum Subspecies. Based on our previous research, metagenomic sequencing revealed that the in situ enriched hexanoic acid bacteria solution contained... Clostridium tyrobutyricum The relative abundance of DS1-related strains was second only to the absolutely dominant strain. Caproicibacterium argilliputei The subdominant strains were identified, and it was determined that both had a synergistic effect in increasing hexanoic acid production (described later). Therefore, this example will... Clostridium tyrobutyricum DS1 is used as one of the core functional strains in the complex hexanoic acid bacteria group for hexanoic acid synthesis.

[0045] Table 5 C. tyrobutyricum Analysis of ANI, AAI, and POCP values ​​of DS1 genome and related strains

[0046] To analyze the strains Clostridium tyrobutyricum DS1 and its closest relatives Clostridium tyrobutyricum Cirm Differences in butyrate synthase genes between BIA 2237 were analyzed by comparative genomic analysis of hexanoate synthase in their whole genomes. The results are as follows: Figure 3 As shown, there are significant differences in the distribution order of the butyrate synthase gene between the two strains (sequence differences are not shown). Furthermore, the strains... Clostridium tyrobutyricumDS1 has one more plasmid (NCBI accession number CP180726) than its homologous strains, none of which have plasmids. This indicates that the strains in this example... Clostridium tyrobutyricum DS1 differs from the related type strain.

[0047] ③ Ligilactobacillus acidipiscis The 16S rRNA gene sequence of YHS1A has been submitted to the NCBI database, accession number PQ626723.1. Blasten alignment analysis in the NCBI database shows that the strain... Ligilactobacillus acidipiscis YHS1A and model strain Ligilactobacillus acidipiscis The 16S rRNA gene sequence of NBRC 102163 (Accession No. NR_112693.1) showed the highest identity (99.93%). 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.

[0048] Table 6 Ligilactobacillus acidipiscis Analysis of ANI, AAI, and POCP values ​​of YHS1A genome and related strains

[0049] Although Blasten analysis of the 16S rRNA gene sequence revealed that the strain... Ligilactobacillus sour fish YHS1A and strain Ligilactobacillus acidipiscis NBRC 102163 is the most closely related strain, while the whole-genome sequencing analysis results shown in Table 6 indicate that the strain... Ligilactobacillus acidipiscis YHS1A and strain Lactobacillus acidipiscis The strain is most closely related to ACA-DC 1533. Ligilactobacillus acidipiscis YHS1A and its closest related strains Lactobacillus acidipiscis The AAI values ​​of ACA-DC 1533 were far below the species-level threshold of 95.00%. Furthermore, the strain was further identified as a species by using the AAI value (74.00%) and POCP value (50.00%), which are at the genus-level threshold. Lactobacillus acidipiscis Subspecies. Lactobacillus acidipiscis Originally classified at the genus level Lactobacillus In 2020, the current broad definition of Lactobacillus (…) Lactobacillus sensu lato When it splits into 25 new genera, Lactobacillus acidipiscis Classified asLigilactobacillus acidipiscis Therefore, strain YHS1A in this embodiment is defined as... Ligilactobacillus acidipiscis YHS1A. Significantly different from other lactic acid bacteria, the genus in this embodiment of the invention, because it was isolated from hexanoic acid bacteria culture, possesses the ability to synthesize hexanoic acid. This study further analyzed the function of hexanoic acid synthase genes in its genome and mapped the metabolic pathways involved in hexanoic acid synthesis in its genome, such as... Figure 4 As 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 those in the GenBank database (accession number: CP173417.1). Ligilactobacillus acidipiscis The gene tag for YHS1A. Enzymes marked with a red cross "×" in the diagram indicate the corresponding gene in... Ligilactobacillus acidipiscis The YHS1A genome does not exist. The tag number in parentheses is... Ligilactobacillus acidipiscis The locus tag number of the corresponding enzyme gene in the YHS1A genome.

[0050] The results showed that the strain Ligilactobacillus acidipiscis YHS1A possesses a metabolic pathway for hexanoic acid synthesis in vivo.

[0051] 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.

[0052] 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.

[0053] First, we analyzed the core functional caproic acid strains in the complex caproic acid bacterial community. Caproicibacterium argilliputei The growth of XB1 under gradient temperature (OD600), such asFigure 5 As shown, the results indicate that Caproicibacterium argilliputei XB1 can grow at temperatures of 15 to 45°C, with an optimal growth temperature range of 20 to 40°C, indicating that it can grow and metabolize under relatively natural fermentation conditions during the fermentation of strong-aroma baijiu.

[0054] Secondly, the core functional caproic acid strains in the complex caproic acid bacterial community were analyzed. Caproicibacterium argilliputei The growth and metabolic characteristics of XB1 under ethanol-tolerant conditions in fermentation medium at 34℃ (the temperature during the later stage of fermentation of strong-aroma baijiu), such as... Figure 6 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... Caproicibacterium argilliputei XB1 can synthesize hexanoic acid by degrading glucose and lactic acid in a fermentation medium with a carbon source ratio simulating that of yellow water, under conditions that are ethanol-tolerant and do not degrade ethanol. It should be noted that the fermentation medium provides 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.

[0055] Then, the synergistic functional strains in the complex hexanoic acid bacteria were analyzed. Clostridium tyrobutyricum DS1, under the same conditions, in the fermentation medium at 34℃ (the temperature in the later stage of fermentation of strong-aroma baijiu), exhibits growth and metabolic characteristics tolerant to ethanol, such as... 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 is tolerant to ethanol and does not degrade ethanol. This provides butyric acid, an electron acceptor, for hexanoic acid synthesis by hexanoic acid bacteria in the hexanoic acid synthesis metabolism involving the complex hexanoic acid bacteria community.

[0056] Finally, the synergistic functional strains in the complex hexanoic acid bacteria community were analyzed. Ligilactobacillus acidipiscis Before determining the growth and metabolic characteristics of YHS1A under ethanol-tolerant conditions, its suitable growth temperature and pH conditions must be identified. For example... Figure 8 The strain shown is Ligilactobacillus acidipiscisThe growth of YHS1A single strain under gradient temperature and pH conditions showed that its optimal growth temperature was 20-45℃; its optimal growth pH was 5.5-9.5, but it could still grow weakly at pH 3-4.5, indicating that it can grow and metabolize under the relatively natural low pH fermentation system conditions in the fermentation process 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 capable of producing butyric acid and trace amounts of hexanoic acid, possesses a natural tolerance to low pH values. This allows the composite hexanoic acid bacteria group in this embodiment to complete pH synthesis within a relatively wide pH range. Next, the strain... Ligilactobacillus acidipiscis The growth and metabolic characteristics of YHS1A in the same fermentation medium at 34℃ (the temperature during the later stage of fermentation of strong-aroma baijiu) are as follows: Figure 9 As shown in the figure (error bars represent standard deviation; significant differences exist between samples with different letters, 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 the core strain in the core hexanoic acid bacterium culture. Caproicibacterium argilliputei XB1 and Clostridium tyrobutyricum Acetic acid, the carbon source for DS1, can serve as an electron acceptor for the synthesis of butyric acid or hexanoic acid, with butyric acid acting as the 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 subsequently further analyzed its possible metabolic pathways through transcriptomics analysis, providing a theoretical basis for future applications, such as... Figure 10 As shown. The results indicate that, Ligilactobacillus acidipiscis YHS1A can be synthesized sequentially into butyric acid and hexanoic acid via the FAB (fatty acid biosynthesis) metabolic pathway. The subsequent degradation of hexanoic acid is carried out via the RBO metabolic pathway.

[0057] In summary, all three core functional strains in the compound caproic acid bacteria group can synthesize fatty acids without degrading ethanol and under ethanol-tolerant conditions. The absence 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; Caproicibacterium argilliputeiXB1 primarily degrades glucose and lactic acid to synthesize butyric acid and hexanoic acid. Furthermore, the accumulation of electron acceptors acetic acid and butyric acid favors the final metabolic pathway towards hexanoic acid. This indicates that the three core strains exhibit synergy and their products are not entirely identical.

[0058] Specifically, this embodiment provides a complex hexanoic acid bacterial community containing two synergistic strains and a core hexanoic acid strain. Caproicibacterium argilliputei Continuous batch synergistic metabolic characterization of XB1.

[0059] To further simulate the batch fermentation method of strong-aroma baijiu, the synergistic strains were verified through continuous batch fermentation. Clostridium tyrobutyricum DS1 and Ligilactobacillus acidipiscis YHS1A Caproicibacterium argilliputei XB1 synergistically promotes the synthesis of hexanoic acid.

[0060] First, analysis Clostridium tyrobutyricum DS1 (abbreviated in the figure) C. tyrobutyricum DS1 and strain Caproicibacterium argilliputei XB1 (abbreviated in the figure) C. sp. The synergistic metabolic role of XB1 in continuous batch fermentation processes. For example... 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 argilliputei Compared to XB1, it increased hexanoic acid synthesis by 27.03% to 68.18%. This indicates that in the complex hexanoic acid bacterial community, Clostridium tyrobutyricum The addition of strain DS1 helped increase the yield of hexanoic acid. Since both strains are adapted to growth and metabolism in weakly acidic to near-neutral physicochemical environments, this indicates that their synergistic metabolism strongly promotes hexanoic acid synthesis within this range.

[0061] Then, the strains were analyzed. Ligilactobacillus acidipiscis YHS1A and strain Caproicibacterium argilliputei The synergistic metabolic role of XB1 in continuous batch fermentation processes. For example... Figure 12 As shown, the results indicate that during the synergistic continuous fermentation process of the two strains, the hexanoic acid yield was higher than that after the first batch of fermentation. Caproicibacterium argilliputei The XB1 single strain showed a 72.73% increase; then, with 2-6 fermentation batches, the amount of hexanoic acid synthesized decreased slightly with increasing batch numbers, and then stabilized at a low value, similar to... Caproicibacterium argilliputei The decrease was observed compared to the single strain XB1. Therefore, it can be concluded that the decrease was related to the strain. Clostridium tyrobutyricum DS1 is beneficial for the long-term synthesis of hexanoic acid, and different strains are involved. Ligilactobacillus acidipiscisYHS1A is beneficial for rapidly increasing hexanoic acid synthesis. The subsequent decrease in hexanoic acid synthesis is presumably related to the rapid drop in pH and the synthesis of large amounts of acetic acid and lactic acid. Therefore, in practical applications, it is necessary to add YHS1A to each fermentation batch. Ligilactobacillus acidipiscis YHS1A bacteria and Caproicibacterium argilliputei XB1 contains a dual-strain complex, while Clostridium tyrobutyricum DS1 and Caproicibacterium argilliputei The XB1 dual-strain compound bacteria needs to be added each time.

[0062] 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.

[0063] 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 13 As 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 It is the main mobile group in the hexanoic acid bacteria community, including butyric acid-producing bacteria. Clostridium tyrobutyricum It is overwhelmingly dominant, with very little traditional caproic acid bacteria present. It is important to note that although... Staphylococcus They are considered conditionally pathogenic bacteria, but some of their species are also common in fermentation systems.

[0064] To ensure that the selected motile caproic acid bacteria can indeed assist the core functional strains in the complex caproic acid bacteria community in the actual high-concentration lactic acid and high-concentration ethanol fermentation system of strong-aroma baijiu. Caproicibacterium argilliputei XB1 undergoes hexanoic acid synthesis metabolism, and its metabolic capacity under conditions of high concentrations of lactic acid and high concentrations of ethanol needs to be tested separately. For example... Figure 15 As shown (the error bars represent the standard deviation; samples without identical lowercase letters are significantly different), p < 0.05; * indicates a significant difference 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 carry out anabolic metabolism mainly based on butyric acid synthesis, 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. Then, the ability of motile hexanoic acid bacteria to increase hexanoic acid and decrease lactic acid under high-concentration ethanol conditions was further examined, such as... Figure 16 As shown in the figure (error bars represent standard deviation; samples without identical lowercase letters are significantly different), p < 0.05; * indicates a significant difference between different samples within the same time period. p < 0.05; n = 3. ), the results showed that motile hexanoic acid bacteria reached a concentration as high as 60 g / L in the yellow water of the natural reaction system, which is 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.

[0065] 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 indicate that the bacterial community... Clostridium sensu stricto 12 (mainly butyric acid bacteria) Clostridium tyrobutyricum The relative abundance of ) increased significantly with increasing stress intensity (reaching 93.62% and 81.41% under the highest stress), while Staphylococcus , Unclassified Lachnospiraceae and Sporolactobacillus The abundance of certain genera decreases accordingly. Furthermore, the most dominant ASV is one of the core dominant strains of the hexanoic acid-containing complex isolated in this embodiment. Clostridium tyrobutyricum DS1. This proves that the complex hexanoic acid bacteria in this embodiment... Clostridium tyrobutyricum DS1 and strain Ligilactobacillus acidipiscisYHS1A can be continuously metabolized in high acid / alcohol metabolic systems, assisting in the synthesis of hexanoic acid.

[0066] Meanwhile, the present invention also provides a complex hexanoic acid bacteria group, including the aforementioned complex hexanoic acid bacteria group.

[0067] 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.

[0068] 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.

[0069] 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 caproic acid bacteria group includes caproic acid strain XB1 and lactic acid strain YHS1A. The caproic acid strain XB1 is classified as Caproicibacterium argilliputei XB1, with accession number GDMCC No: 67345, and the mass percentage of the caproic acid strain XB1 is >50%. The lactic acid strain YHS1A is classified as Ligilactobacillus acidipiscis YHS1A, with accession number GDMCC No: 67348.

2. An application of the compound hexanoic acid bacteria group according to claim 1, 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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