A novel microbial species derived from cellar mud and its application in the efficient synthesis of butyric acid.

Abstract

This invention belongs to the field of microbial technology, specifically relating to the identification of a new species of the bacterium *Clostridium cellarense* and its application in the efficient synthesis of butyric acid from the metabolism of acetic acid and ethanol. The *Clostridium cellarense* BJN0013 disclosed in this invention possesses the ability to metabolize acetic acid and ethanol to produce high levels of butyric acid, reaching a yield of 31.11 ± 0.94 g / L, which is the highest known yield of butyric acid synthesized by *Clostridium* microorganisms. The high-quality new microbial species resource provided by this invention offers a preferred microbial resource for functional microorganisms that produce high-flavor compounds in the baijiu (Chinese liquor) brewing process, laying a foundation for improving the flavor and quality of baijiu.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a new species of microorganism derived from pit mud, Clostridium cellarense BJN0013, and its application in the production of short-chain fatty acid butyric acid. Background Technology

[0002] Ethanol and water account for approximately 98% of the total mass of baijiu (Chinese white liquor), while flavor compounds comprise about 2%, representing trace components that play a decisive role in its quality. Currently, there are over 3000 such trace components, including esters, alcohols, acids, aldehydes, ketones, heterocyclic compounds, and carbonyl compounds. Among them, acids such as short-chain fatty acids effectively reduce the spiciness and bitterness of baijiu, increasing its smoothness and significantly impacting its taste. An appropriate proportion of organic acids in baijiu can make the liquor fuller-bodied, more harmonious, and have a longer aftertaste, while also promoting the aging of new liquor. Studies have reported that Clostridium perfringens in the cellar mud of strong-aroma baijiu exhibits high potential for the metabolic synthesis of acids such as butyric acid and hexanoic acid. Butyric acid, in particular, has been shown to promote digestion, enhance intestinal function, alleviate obesity, and inhibit inflammation after ingestion. Therefore, research on the role of cellar mud microorganisms in the synthesis of acids during baijiu production and the stability of product quality is of great significance. However, due to the strictly anaerobic environment and harsh conditions required for cultivating microorganisms in fermentation pit mud, a large number of unisolated microorganisms still exist, hindering the stability of baijiu fermentation production. Therefore, screening functional microorganisms in fermentation pit mud capable of synthesizing short-chain fatty acids, enriching microbial resources, rationally regulating the fermentation process, and promoting the stability of in-situ metabolic synthesis of acidic substances are of great significance.

[0003] Therefore, the purpose of this invention is to provide a new high-quality microbial species derived from cellar mud, Clostridium cellarense BJN0013, and its application in the efficient synthesis of butyric acid from the metabolism of acetic acid and ethanol, thereby promoting the production of beneficial acidic flavor substances in baijiu and providing a preferred microbial resource for the synthesis of acidic substances during the baijiu fermentation process. Summary of the Invention

[0004] The purpose of this invention is to provide a new strain of microorganisms in pit mud and its application in the production of short-chain fatty acid butyric acid.

[0005] This invention is achieved through the following technical solution:

[0006] This invention discloses a novel microbial species of baijiu (Chinese liquor) *Clostridium cellarense* BJN0013 and its application in the high-yield production of short-chain fatty acid butyric acid from the metabolism of acetic acid and ethanol. The novel species underwent whole-genome sequencing, including 16S rRNA gene sequencing and genome-wide comparison. The results showed that the sequence similarity values ​​of this strain and its most similar strain, based on 16S rRNA, average nucleotide identity (ANI) at the genome level, and DNA hybridization (DDH) at the genome level, were all below the threshold for novel microbial species. This indicates that the strain is a novel species of the genus *Clostridium*.

[0007] This invention also discloses the application of Clostridium cellarense BJN0013 in the synthesis of short-chain fatty acid butyric acid from acetic acid and ethanol under fermentation culture conditions.

[0008] Compared with the prior art, the present invention has the following beneficial technical effects:

[0009] This invention utilizes microbial technology to isolate and purify a strain of *Clostridium cellarense* BJN0013, which has been identified as a novel microbial species. Under specific fermentation conditions, *Clostridium cellarense* BJN0013 provided by this invention exhibits the ability to efficiently synthesize butyric acid from acetic acid and ethanol, making it the most efficient wild-type microbial strain for butyric acid production from fermentation pit mud to date. The identification of this novel microbial species as a *Clostridium* species originating from baijiu (Chinese liquor) has resulted in its naming as *Clostridium cellarense*, with the serial number BJN0013. This provides fundamental data and microbial resources for the high-yield butyric acid production from the conversion of acetic acid and ethanol by microbial strains derived from fermentation pit mud.

[0010] The novel species Clostridium cellarense BJN0013 described in this invention has a highly efficient ability to metabolize and synthesize short-chain fatty acids, providing an optimal resource for improving the flavor and quality of baijiu (Chinese liquor).

[0011] Preservation instructions:

[0012] The novel Clostridium cellarense of this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46148 and deposit date of October 15, 2024. Attached Figure Description

[0013] Figure 1 A phylogenetic tree was constructed using the neighbor-joining method (MEGA 11.0) based on the 16S rRNA sequences of Clostridium cellarense BJN0013 and the closest type strains in the Ezbiocloud database.

[0014] Figure 2 Comparison of the genome sequences of Clostridium cellarense BJN0013 with five most closely related microbial strains (average nucleotide identity (ANI) was calculated using Ortho ANI, and DNA-DNA hybridization (DDH) was performed using The Type (Strain) Genome Server (TYGS)).

[0015] Figure 3 Liquid chromatography chromatogram of butyric acid synthesized by Clostridium cellarense BJN0013

[0016] Figure 4 Fermentation metabolic characteristics of Clostridium cellarense BJN0013 under basal culture medium supplemented with sodium acetate and ethanol Detailed Implementation

[0017] The present invention will be described in detail with reference to specific embodiments, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. In the following embodiments of the present invention, unless otherwise specified, all reagents used are commercially available, and all methods involved are conventional methods.

[0018] Example 1: Phylogenetic analysis of 16S rRNA from Clostridium cellarense BJN0013

[0019] Based on the whole-genome sequencing results, the 16S rRNA (1511 bp) gene sequence of C. cellarense BJN0013 was extracted and submitted to EzBioCloud for preliminary identification. The 16S rRNA gene sequences of the newly isolated strain and similar strains were compared using ClustalW in MEGA 11.0 software. A phylogenetic tree was constructed using the neighbor-joining method, and the expansion support was obtained by performing 1000 iterations on the generated topology tree.

[0020] The 16S rRNA (1511 bp) gene sequence of C. cellarense BJN0013 was extracted and submitted to the EzBioCloud database for comparison. The results showed that C. cellarense BJN0013 is similar to Clostridium kluyveri DSM555. T The 16S rRNA gene sequence had the highest similarity at 98.24%, followed by Clostridium aromativorans WLY-B-L2. T (94.78%), Clostridium luticellarii DSM 29923 T (94.22%). The similarity values ​​were all below the threshold used to define new microbial species (98.67%). Therefore, *Clostridium kluyveri* DSM555 T The strain was selected as the closest phylogenetic reference microorganism for comparative analysis of its physiological and biochemical characteristics. Neighbor-joining phylogenetic analysis of the 16S rRNA gene sequence showed that the strain formed a single-origin evolutionary branch. Figure 1 ).

[0021] Example 2: Whole genome alignment of Clostridium cellarense BJN0013

[0022] ANI and DDH are primarily used to assess phylogenetic relationships between species at the whole-genome level. The DDH of *C. cellarense* BJN0013 and the reference strain was calculated using The Type (Strain) Genome Server (TYGS), and the ANI of *C. cellarense* BJN0013 and the reference strain was calculated using OrthoANI software.

[0023] The complete genome sequence of strain BJN0013 was submitted to TYGS for DDH value calculation compared with the model strain. Results showed that the strain was similar to Clostridium kluyveri DSM 555. T The strain had the highest DDH value, at 41.80%. Secondly, it was similar to *Irregularibacter muris* DSM 28593. T , Pelotomaculum isophthalicum JI T , Clostridium luticellarii DSM 29923 T , Clostridium aromativorans WLY-B-L2 TThe DDH levels were 39.80%, 36.80%, 22.40%, and 21.80%, respectively, far below the DDH species classification threshold of 70%. Similar to DDH, the strain was associated with *Clostridium kluyveri* DSM 555. T , Irregularibacter muris DSM 28593 T , Pelotomaculum isophthalicum JI T , Clostridium luticellarii DSM 29923 T , Clostridium aromativorans WLY-B-L2 T The ANI values ​​were 90.09%, 72.78%, 78.47%, 75.91%, and 76.30%, respectively, which are far below the recommended species delineation point of 95%. In conclusion, the genome-wide comparison results show that the ANI and DDH values ​​significantly support the identification of C. cellarense BJN0013 as a new microbial species of the genus Clostridium.

[0024] Example 3: Determination of the ability of Clostridium cellarense BJN0013 to produce butyric acid and ethanol under fermentation culture conditions.

[0025] Sodium ethanolacetate fermentation medium: KH2PO4 0.03-0.3g, K2HPO4 0.03-0.3g, CaCl2 0.001-0.01g, Fe2(SO4)3·3H2O 0.001-0.01g, (NH4)2SO4 0.1-0.5g, MgSO4·7H2O 0.02-0.2g, sodium acetate 3-25g, yeast extract 1-10g, calcium carbonate 2-10g, sodium resazurin 0.02-0.2mg, ethanol 0.5-2% (v / v), L-cysteine ​​0.001-0.005g, distilled water 1L.

[0026] The chromatographic column was a Bio Rad Aminex HPX-87H. Detection conditions: mobile phase was 0.006M H2SO4 solution, flow rate was 0.9 mL / min, column temperature was 65℃, injection volume was 10 μL, detector was a differential refractive index detector, and detection time was 20 min.

[0027] The results confirmed that the novel microbial species C. cellarense BJN0013 provided by this invention has the ability to efficiently synthesize butyric acid from acetic acid and ethanol (e.g., Figure 3 As shown), the yield was 31.11 ± 0.94 g / L (as shown). Figure 4 (As shown).

Claims

1. A new microbial species, Clostridium cellarense BJN0013, characterized in that, The strain exhibits the characteristic of metabolizing acetic acid and ethanol with high butyric acid production. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46148.

2. The novel microbial species Clostridium cellarense BJN0013 as described in claim 1, characterized in that, The strain was identified as a novel microbial species of the genus Clostridium by 16S rRNA gene sequence alignment and whole-genome-level assessment of interspecies relationships.

3. The novel microbial species Clostridium cellarense BJN0013 as described in claim 1 has a butyric acid yield of 31.11 ± 0.94 g / L.

4. The novel microbial species Clostridium cellarense BJN0013 as described in claim 1, with a high butyric acid production culture medium of sodium ethanolacetate: KH2PO4 0.03-0.3g, K2HPO4 0.03-0.3g, CaCl2 0.001-0.01g, Fe2(So4)3·3H2O 0.001-0.01g, (NH4)2SO4 0.1-0.5g, MgSO4·7H2O 0.02-0.2g, sodium acetate 3-25g, yeast extract 1-10g, calcium carbonate 2-10g, sodium resazurin 0.02-0.2mg, ethanol 0.5-2% (v / v), L-cysteine ​​0.001-0.005g, distilled water 1L, culture conditions 30-40℃, strictly anaerobic.

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