Pit mud-sourced microbial new species and application thereof in efficient synthesis of butyric acid

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to identification of a new species of Clostridium cell and application of the new species in high-efficiency synthesis of butyric acid by metabolizing acetic acid and ethanol. The Clostridium cell BJN0013 disclosed by the invention has the capability of metabolizing acetic acid and ethanol to produce butyric acid at high yield, and the yield can reach 31.11 + / -0.94 g / L, which is the highest yield of butyric acid synthesized by metabolism of known Clostridium microorganisms at present. According to the novel high-quality microorganism resource provided by the invention, an optimal microorganism resource is provided for functional microorganisms with high yield of flavor substances in the white spirit brewing process, and a strain foundation is laid for improving the flavor and quality of the white spirit.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a new microorganism species Clostridium cellarense BJN0013 derived from pit mud and application thereof in the production of short-chain fatty acid butyrate. Background Art

[0002] The content of ethanol and water in liquor accounts for about 98% of the total mass, and flavor substances account for about 2% of the total mass. They are trace components in liquor and play a decisive role in the quality of liquor. At present, there are more than 3,000 trace components, including esters, alcohols, acids, aldehydes and ketones, heterocyclic compounds, carbonyl compounds, etc. Among them, acid substances such as short-chain fatty acids can effectively reduce the spiciness and bitterness of liquor, increase the refreshingness of liquor, and have a very important influence on the taste. The appropriate proportion of organic acids in liquor can make the body fuller, more coordinated, and have a long aftertaste, and is conducive to promoting the aging of new wine. Studies have reported that Clostridium in the mud of Luzhou-flavor liquor cellars showed efficient metabolic synthesis potential of acid substances such as butyric acid and caproic acid. Among them, butyric acid has been shown to promote digestion, enhance intestinal function, improve obesity, and inhibit inflammation after being ingested into the body. Therefore, it is of great significance to study the synthesis of acid substances in the production process of liquor and the stability of product quality by cellar mud microorganisms. However, due to the strict anaerobic environment and harsh conditions of cellar mud microorganisms, there are still a large number of unisolated microorganisms, which restrict the stability of liquor fermentation production. Therefore, it is of great significance to screen functional microorganisms with the ability to synthesize short-chain fatty acids in cellar mud, enrich microbial resources, rationally regulate the fermentation process, and promote the improvement of the in situ metabolic synthesis stability of acid substances.

[0003] Therefore, the purpose of the present invention is to provide a high-quality new species of cellar mud-derived microorganism Clostridium cellarense BJN0013 and its application in metabolizing acetic acid and ethanol to efficiently synthesize butyric acid, promote the production of beneficial acid flavor substances in liquor, and provide a preferred microbial resource for the synthesis of acid substances in the liquor fermentation process. Summary of the invention

[0004] The purpose of the present invention is to provide a new cellar mud microorganism species and its application in the production of short-chain fatty acid butyrate.

[0005] The present invention is achieved through the following technical solutions:

[0006] The present invention discloses a new species of liquor microorganism Clostridium cellarense BJN0013 and its application in metabolizing acetic acid and ethanol to produce short-chain fatty acid butyrate. The new species is subjected to whole genome sequencing, and 16S rRNA gene sequence and whole genome level comparison are performed. The results show that the sequence similarity values ​​of the strain and the most similar strain are all lower than the threshold limit of the new microorganism species through 16S rRNA, average nucleotide identity (Average Nucleotide Identity, AND) at the genome level, and DNA DNA hybridization (DNA DNA hybridization, DDH), and the results show that the strain is a new species of microorganism of the genus Clostridium.

[0007] The invention also discloses an application of Clostridium cellarense BJN0013 in metabolizing acetic acid and ethanol to synthesize short-chain fatty acid butyrate under fermentation culture conditions.

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

[0009] The present invention utilizes microbial technology to separate and purify a strain of Clostridium cellarense BJN0013, which is identified as a new microbial species. The Clostridium cellarense BJN0013 provided by the present invention has the ability to metabolize acetic acid and ethanol to efficiently synthesize butyric acid under specific fermentation conditions, and is the most efficient wild-type strain for producing butyric acid from cellar mud. The identification of the new microbial species resource is a Clostridium microorganism from liquor source, named Clostridium cellarense, numbered BJN0013, which provides basic data and microbial resources for the conversion of acetic acid and ethanol to high-yield butyric acid by strains from cellar mud.

[0010] The new species Clostridium cellarense BJN0013 described in the present invention has the ability to efficiently metabolize and synthesize short-chain fatty acids, and provides an optimal resource for improving the flavor and quality of liquor.

[0011] Storage Instructions:

[0012] The new Clostridium microorganism (Clostridium cellarense) of the present invention is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, the storage location is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the storage number is CGMCC No.46148, and the storage date is October 15, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The 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 model strain in the Ezbiocloud database.

[0014] Figure 2 Comparison of the genome sequences of Clostridium cellarense BJN0013 and the 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 chromatogram of butyric acid synthesis catalyzed by Clostridium cellarense BJN0013

[0016] Figure 4 Fermentation metabolic characteristics of Clostridium cellarense BJN0013 in minimal medium supplemented with sodium acetate and ethanol DETAILED DESCRIPTION

[0017] The present invention is described in detail in conjunction with specific embodiments, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. In the following examples of the present invention, unless otherwise specified, the reagents used can be purchased on the market, and unless otherwise specified, the methods involved are all conventional methods.

[0018] Example 1 16S rRNA phylogenetic analysis of Clostridium cellarense BJN0013

[0019] According to 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. ClustalW in MEGA 11.0 software was used to align the sequences of the 16S rRNA gene of the newly isolated strain and similar strains. The phylogenetic tree was constructed using the neighbor-joining method, and the generated topological tree was repeated 1000 times to obtain the bootstrap support rate.

[0020] The 16S rRNA (1511 bp) gene sequence of C. cellarense BJN0013 was submitted to the EzBioCloud database for comparison. The results showed that C. cellarense BJN0013 and Clostridium kluyveri DSM555 T The 16S rRNA gene sequence similarity of 1.477 mmol / l to 1.317 mmol / l was the highest, 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 for defining a new species of microorganism (98.67%). Therefore, Clostridium kluyveri DSM555 T The closest related reference microorganisms were selected for comparative analysis of physiological and biochemical characteristics. The results of neighbor-joining phylogenetic analysis of 16S rRNA gene sequences showed that the strains formed a single-source evolutionary branch ( Figure 1 ).

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

[0022] ANI and DDH are mainly used to evaluate the relationship 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 whole genome sequence of strain BJN0013 was submitted to TYGS for calculation of DDH value with the model strain. The results showed that the strain was similar to Clostridium kluyveri DSM 555. T The DDH value of the strain was the highest, at 41.80%. T , Pelotomaculum isophthalicum JI T , Clostridium luticellarii DSM 29923 T , Clostridium aromativorans WLY-B-L2 TThe DDH of the strains were 39.80%, 36.80%, 22.40%, and 21.80%, respectively, which were far below the DDH species classification threshold of 70%. T , Irregularibacter muris DSM 28593 T , Pelotomaculum isophthalicum JI T , Clostridium uliginosum DSM12992 T , Clostridiumluticellarii DSM 29923 T , Clostridium aromativorans WLY-B-L2 T The ANI values ​​of the three strains were 90.09%, 72.78%, 78.47%, 75.91% and 76.30%, respectively, which were far lower than the recommended species demarcation point of 95%. In conclusion, the ANI and DDH values ​​of the whole genome comparison results significantly supported that C. cellarense BJN0013 was a new species of Clostridium.

[0024] Example 3 Determination of the ability of Clostridium cellarense BJN0013 to produce butyrate and ethanol under fermentation conditions

[0025] Ethanol sodium acetate 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 powder 1-10g, calcium carbonate 2-10g, resazurin sodium 0.02-0.2mg, ethanol 0.5-2% (v / v), L-cysteine ​​0.001-0.005g, distilled water 1L.

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

[0027] The results confirmed that the new microbial species C. cellarense BJN0013 provided by the present invention has the ability to metabolize acetic acid and ethanol to efficiently synthesize butyric acid (such as Figure 3The yield was 31.11±0.94 g / L (as shown in Figure 4 shown).

Claims

1. A new microbial species Clostridium cellarense, numbered BJN0013, characterized by: The strain has the characteristics of metabolizing acetic acid and ethanol to produce high butyric acid.

2. The new microbial species Clostridium cellarense BJN0013 according to claim 1, characterized in that: The strain was compared with 16S rRNA gene sequences and the relationship between species was evaluated at the whole genome level. The results showed that the strain was a new species of Clostridium.

3. The Clostridium cellarense BJN0013 as claimed in claim 1, which has the ability to metabolize acetic acid and ethanol to produce butyric acid in high yield, with a yield of 31.11±0.94 g / L.

4. The Clostridium cellarense BJN0013 as claimed in claim 1, wherein the culture medium for efficiently metabolizing and synthesizing butyric acid is ethanol sodium acetate 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, resazurin sodium 0.02-0.2mg, ethanol 0.5-2% (v / v), L-cysteine ​​0.001-0.005g, distilled water 1L), the culture conditions are 30-40°C, and the culture is strictly anaerobic.

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