CN112725235A - Novel strain of clostridium and application thereof - Google Patents

Abstract

The invention belongs to the technical field of brewing microorganisms, and particularly relates to a novel clostridium strain and application thereof. Aiming at the problem that the existing functional microorganisms capable of improving the aroma in the white spirit fermentation process are few in variety, the invention provides a new strain of clostridium, and the preservation number is CGMCC NO. 19455. The novel strain can simultaneously produce butyric acid, ethyl valerate, ethyl acetate and 2-pentanone with high yield, provides a novel strain for aroma production in liquor brewing, enriches resources of brewing microorganisms, and has good application value.

Description

Novel strain of clostridium and application thereof

Technical Field

The invention belongs to the technical field of brewing microorganisms, and particularly relates to a novel clostridium strain and application thereof.

Background

Chinese liquor is known as one of six distilled liquors in the world due to unique taste and aroma, and strong-flavor liquor plays a significant role. Since ancient times, the 'good liquor is produced in the old cellar', and the cellar mud has an important influence on the quality and flavor of the strong aromatic Chinese spirits. Acid generated by the metabolism of pit mud bacteria is an important substance in the strong aromatic white spirit, and plays roles in generating fragrance, assisting fragrance, reducing stimulation of a spirit body and buffering balance in the spirit. The clostridium can produce caproic acid, and is an important precursor for synthesizing ethyl caproate which is a main aroma-producing substance of the strong aromatic white spirit. In the production of white spirit, the white spirit is strengthened in pit mud by an artificial pit mud method.

It is known that the basic components of wine are consistent, mainly ethanol and water, accounting for about 98% of the total amount, and the rest 2% of the components are the chromatographic skeleton components and trace aroma substances forming the unique style of white spirit. At present, 470 kinds of aroma components in white spirit are detected, and mainly comprise esters, organic acids, alcohols, aldehyde ketone compounds, aromatic compounds and the like. Their content, although very small, determines the style and characteristics of white spirits.

Few functional microorganisms capable of producing fragrance are found at present, and the technology for flavoring the white wine by utilizing microbial fermentation is yet to be further developed.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: the existing functional microorganisms capable of improving the aroma in the white spirit fermentation process are few in types and need to be developed.

The technical scheme for solving the technical problems comprises the following steps: a novel strain of Clostridium is provided. The new clostridium strain has a preservation number of CGMCC NO. 19455. The preservation time is 3 months and 6 days in 2020, the preservation place is the China general microbiological culture Collection center, and the address is microbial research institute of China academy of sciences No. 3, West Lu No.1 Hospital, North Cheng, the south China area, Beijing, zip code: 100101.

wherein, the nucleotide sequence of the 16S rDNA of the new clostridium strain is shown as SEQ ID NO:1 is shown.

SEQ ID NO 1 16S rDNA nucleotide sequence of novel Clostridium species

TCAGGACGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGGGCGAAGAAGCTCCTTCGGGAGAATCTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTCAAAGAGGGGGATAGCCTCCCGAAAGGGAGATTAATACCGCATGACAAATATAATCCGCATGGATTATATTTTAAAGGAGAAATCCGCTTTGAGATGGACCCGCGGCGCATTAGCTAGTTGGCAGGGTAACGGCCTACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGAACGGCCACATTGGAACTGAGAGACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATTGTAAAGCTCTGTCATCTGGGACGATAATGACGGTACCAGATGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTACTGGGCGTAAAGGGTGCGCAGGCGGACATTTAAGTGAGATGTGAAATACCCGGGCTTAACCCGGGCAGTGCATTTCAAACTGGGTGTCTGGAGTGCAGGAGAGGAGAACGGAATTCCTAGTGTAGCGGTGAAATGCGTAGAGATTAGGAAGAACACCAGTGGCGAAGGCGGTTCTCTGGACTGTAACTGACGCTGAGGCACGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTACTAGGTGTAGGAGGTATCGACCCCTTCTGTGCCGCAGTAAACACAATAAGTACTCCGCCTGGGAAGTACGATCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTGGACTTGACATCCCCTGCATATCTCAGAGATGAGAGAAGCCCTTCGGGGCAGGGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTAGGTTAAGTCCTGCAACGAGCGCAACCCCTGTTGTTAGTTGCTAACAGTAAGATGAGCACTCTAACGAGACTGCCGCGGTTAACGCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCCAGGGCAACACACGTGCTACAATGGGCAGAACAGAGAGAAGCAAGGCCGCGAGGTGGAGCGAACCTCAAAAACTGTTCCCAGTTCGGATTGCAGGCTGAAACCCGCCTGCATGAAGCTGGAGTTGCTAGTAATCGCGAATCAGCATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGCCGGCAACACCCGAAGTCCGTAGTCTAACGAAAGAGGACGCGGCCGAAGGTGGGGTTGGTGATTGGGGTG。

Wherein, the cytological characteristics of the new clostridium strain are as follows: the thallus is rod-shaped, and the bacterial colony is light yellow and round, which shows smooth and moist and neat edge.

Wherein, the biological characteristics of the new clostridium strain are as follows: can utilize salicin, D-xylose, L-arabinose, gelatin, D-melezitose, D-raffinose, D-sorbitol, L-rhamnose or D-trehalose; tryptophan, urea, D-glucose, D-mannitol, D-lactose, D-sucrose, D-maltose, ferric esculetin citrate, glycerol, D-cellobiose or D-mannose are not available; the hydrogen peroxide test was negative.

Wherein the new Clostridium species produces ethyl valerate and 2-pentanone simultaneously.

Wherein, the new Clostridium bacterial simultaneously produces butyric acid, ethyl valerate, ethyl acetate and 2-pentanone.

Wherein the ethyl valerate producing ability of the new Clostridium bacterial strain reaches 6.37 ppm.

Wherein, the 2-pentanone producing capability of the new clostridium strain reaches 3.16 ppm.

Wherein, the butyric acid producing capability of the new clostridium strain reaches 588 ppm.

Wherein the ethyl acetate producing ability of the new Clostridium bacteria strain reaches 2.62 ppm.

The invention also provides application of the new clostridium strain in liquor brewing.

The invention has the beneficial effects that:

the method screens and obtains a new clostridium strain with the preservation number of CGMCC NO.19455 from the strong aromatic Chinese liquor pit mud, the clostridium strain capable of producing ethyl valerate and 2-pentanone is screened and obtained for the first time, and meanwhile, the clostridium strain can produce butyric acid and ethyl acetate while producing ethyl valerate and 2-pentanone, so that flavor substances in the Chinese liquor are enriched, and the liquor quality of the Chinese liquor can be improved in brewing of the strong aromatic Chinese liquor. The invention provides a new microorganism for producing aroma of strong aromatic Chinese spirits, and has wide application space.

The novel clostridium strain is CGMCC (China general microbiological culture Collection center) in 3-6.3-2020, with the preservation number as follows: CGMCC NO. 19455. The preservation address is the microbiological research institute of China academy of sciences, No. 3, Xilu No.1, Beijing, Chaoyang, Beijing. Classified and named Clostridium sp.

Drawings

FIG. 1 shows the morphology of the new strain Clostridium sp.according to the present invention;

FIG. 2 is a diagram showing the 16S rDNA phylogenetic tree of the new strain Clostridium;

FIG. 3 shows the HS-SPME peak of fermentation broth of new strain Clostridium bacteria;

FIG. 4 shows a graph of the HS-SPME peak of DSM29923 fermentation broth;

FIG. 5 shows the HS-SPME peak of a blank fermentation broth.

Detailed Description

The invention provides a new strain of clostridium strain, which is obtained by screening strong-flavor liquor cellar mud of Yibin wuliangye GmbH and is named as WLY-B-L2. The novel strain is preserved in 6 days 3 months in 2020, the preservation number is CGMCC NO.19455, the preservation place is the China general microbiological culture Collection center, the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beicheng, and the address is: 100101.

the new strain of clostridium WLY-B-L2 has the butyric acid production capacity of less than or equal to 588ppm, the ethyl valerate production capacity of less than or equal to 6.37ppm, the ethyl acetate production capacity of less than or equal to 2.62ppm and the 2-pentanone production capacity of less than or equal to 3.16 ppm.

The clostridium WLY-B-L2 is a new strain, supplements new precious strain resources, and has high application value.

The following examples are intended to illustrate specific embodiments of the present invention without limiting the scope of the invention to the examples.

Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

The isolation medium composition used in the examples included: dissolving 20 parts of glucose, 1 part of yeast extract and 20 parts of calcium carbonate in 1000 parts of distilled water, mixing, and adjusting the pH value to 6.5-7.0 by using NaOH.

The composition of the RCM medium used in the examples included: 10 parts of peptone, 10 parts of beef extract, 3 parts of yeast powder, 5 parts of glucose, 1 part of soluble starch, 5 parts of sodium chloride, 3 parts of sodium acetate, 0.5 part of L-cysteine hydrochloride, 1000ml of distilled water and NaOH for adjusting the pH value to 6.5-7.0.

The slant and plate used in the examples were prepared by adding 2% agar powder to the corresponding medium.

EXAMPLE 1 isolation, screening and characterization of the New Strain, Clostridium WLY-B-L2

(I) separation and screening

A pit mud sample taken from a brewing workshop of Yibin wuliangye Co., Ltd is put into a triangular flask containing glass beads and sterile normal saline in an amount of 10%, vibrated for a certain time, subjected to water bath at 80 ℃ for 20min, and then kept stand for later use. Then taking supernatant for gradient dilution, taking 10^-2-10^-6Coating the diluent on a separation culture medium plate, placing the plate in an anaerobic box, standing and culturing for 15 days at 37 ℃, selecting bacterial colonies on the plate, streaking, culturing, repeating for three times until a purified strain is obtained by separation, and storing on a slant. The strain was named WLY-B-L2.

(II) Classification and identification

1. Morphological characteristics of the Strain

The WLY-B-L2 strain obtained by separation is gram-negative bacteria and rod-shaped, and the bacterial colony on the RCM culture medium is beige, has a wet and smooth surface and is neat in edge.

2. Physiological and biochemical characteristics

The WLY-B-L2 strain obtained by separation can utilize at least one of salicin, D-xylose, L-arabinose, gelatin, D-melezitose, D-raffinose, D-sorbitol, L-rhamnose and D-trehalose. Tryptophan, urea, D-glucose, D-mannitol, D-lactose, D-sucrose, D-maltose, ferric citrate esculetin, glycerol, D-cellobiose, and D-mannose cannot be used. The hydrogen peroxide test was negative.

3. 16S rDNA identification

Inoculating the strain WLY-B-L2 into RCM culture medium, standing and culturing at 37 ℃ for 7 days, taking 1mL of bacterial liquid, centrifugally collecting thalli, extracting genomic DNA by using a bacterial genome extraction kit, and preserving at-20 ℃ for later use.

Amplification of bacterial genome 16S rDNA using primers 27F and 1492R, 27F: AGTTTGATCMTGGCTCAG (SEQ ID NO:2), 1492R: GGTTACCTTGTTACGACTT (SEQ ID NO: 3).

Amplification system (25. mu.L total): 10 XPCR buffer 2.5. mu.L, dNTP (2.5mmol/L each) 1. mu.L, DNA 10ng, primer F (10. mu. mol/L) 0.5. mu.L, primer R (10. mu. mol/L) 0.5. mu.L, double distilled water to 25. mu.L. The amplification procedure is as follows: 4min at 94 ℃; 30 cycles of 94 ℃ for 45s, 55 ℃ for 45s and 72 ℃ for 60 s; the reaction was stopped at 72 ℃ for 10min and 4 ℃.

16S rDNA sequencing: purifying PCR amplification products by using a gel recovery kit; the sequencing work of the purified product is completed by Shanghai Biotechnology Limited, and the 16S rDNA sequence of the strain WLY-B-L2 obtained by the determination result is shown as SEQ ID NO. 1.

And (3) construction of a phylogenetic tree: the BLASTn similarity analysis of the determined 16S rDNA gene sequences of the bacteria with GenBank database respectively shows that the similarity of Clostridium lyticellarii FW431(NR 145907) with the closest relationship to the experimental strains is 97.42% and less than 98.65%. Selecting a standard strain which is relatively close to the genetic relationship of the experimental strain, performing sequence comparison by using Clustalw software, performing phylogenetic analysis by using MEGA 7 software, and constructing a phylogenetic tree, as shown in figure 2.

4. Whole genome dDDH assay

The clostridium WLY-B-L2 and the model strain DSM29923 (purchased from German collection center for culture of microorganisms) are sent to China center for culture Collection of microorganisms (CICC) for genome-wide dDDH analysis. The results showed a dDDH value of 28.1%, less than 70% with model strain DSM 29923.

The strain WLY-B-L2 is identified as a new strain of Clostridium (Clostridium sp.) by integrating the analysis of thallus morphology, physiological and biochemical characteristics, 16S rDNA gene sequence and complete gene dDDH. The bacterial strain WLY-B-L3 is preserved in China general microbiological culture Collection center (CGMCC for short, address: No. 3 of West Lu No.1 of Beijing city Kogyo Beichen, institute of microbiology, China academy of sciences, postal code 100101) at 3.6.2020, and the preservation number is CGMCC NO. 19455.

EXAMPLE 2 determination of flavor production by the New Strain Clostridium WLY-B-L2

The above strains were inoculated in RCM medium at an inoculum size of 5% and cultured in an anaerobic cassette at 37 ℃ for 30 days.

(I) flavor component

1. The detection method comprises the following steps: centrifuging the fermentation liquor for 10min at 5000r/min, filtering the supernatant with 0.22 μm filter membrane to obtain 2ml supernatant, and measuring volatile flavor components by headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS).

(1) HS-SPME condition: 2mL of the filtered fermentation broth was taken in a 20mL headspace bottle and 10. mu.L of internal standard (4-octanol) was added. Equilibrating at 60 deg.C for 15min, inserting needle 1cm away from liquid surface, adsorbing for 40min, and analyzing for 3min at sample inlet.

(2) GC conditions were as follows: TG-WAXMS (30.0m multiplied by 0.25mm multiplied by 0.25 mu m), the injection inlet temperature is 250 ℃, the flow rate of carrier gas (He) is 1mL/min, and the sample injection is carried out in a non-split mode; gas chromatography temperature program: the initial column temperature was 40 deg.C, held for 5min, ramped to 230 deg.C at 4 deg.C/min, held for 15 min.

(3) MS conditions: mass spectrum EI source, electron bombardment energy 70 eV; the ion source temperature is 200 ℃, the quadrupole rod temperature is 150 ℃, and the mass number scanning range is 35-350 amu.

The semi-quantitative analysis was performed by peak area normalization using 4-octanol (0.5ppm) as an internal standard. The peak area of each compound was calculated using a Selected Ion (SIM), and the concentration of each compound was calculated as the ratio of the peak area of the internal standard substance.

2. As a result: in RCM medium, Clostridium sp.WLY-B-L2 produces the main flavors of ethyl valerate, ethyl acetate and 2-pentanone.

(1) HS-SPME Peak plot

The peak of the WLY-B-L2 fermentation broth is shown in FIG. 3, the peak of the DSM29923 fermentation broth is shown in FIG. 4, and the peak of the blank fermentation broth is shown in FIG. 5.

(2) The flavour information is shown in table 1.

TABLE 1 flavor information table (ppm) of New Strain, Clostridium and model Strain

Note: "-" indicates no detection.

As can be seen from Table 1, the flavor substances mainly produced by the Clostridium new bacterial strain WLY-B-L2 are ethyl valerate, ethyl acetate and 2-pentanone, and the flavor substances are 6.37ppm, 2.62ppm and 3.16ppm respectively, which are all important flavor substances in white spirit. Whereas model strain DSM29923 produces very little flavour. From this, it was found that the two bacteria were significantly different.

(II) organic acids

1. Detection method

Taking 0.1mL of the fermentation liquid, diluting the fermentation liquid to 1mL by using 0.1% phosphoric acid aqueous solution, uniformly oscillating the fermentation liquid in a 2mL centrifuge tube, and filtering the fermentation liquid by using a 0.2-micrometer filter membrane to obtain a sample to be detected.

The content of the main organic acids in the fermentation broth was determined by HPLC. The chromatographic procedure was: the column temperature is 30 ℃; mobile phase: a is methanol; b is 0.1% phosphoric acid aqueous solution. Gradient elution: 0 min: 100% of B; 8 min: 100% of B; 25 min: 10% of B; 28.5 min: 10% of B; 29 min: 100% of B; 35 min: 100% of B; stopping: 35 min; flow rate: 1.0 mL/min. Detection wavelength: 214.8 nm.

2. Results

HPLC (high Performance liquid chromatography) measurement of the main organic acid contents of fermentation liquids of the new strain, namely Clostridium clostridia WLY-B-L2 and the model strain DSM29923 is shown in Table 2.

TABLE 2 information table (ppm) of main organic acids of new strain Clostridium and model strain

Name (R)	Blank space	WLY-B-L2	DSM29923
				Lactic acid	733±3	-	494±2
Acetic acid	1598±6	1555±5	3032±9
				Butyric acid	505±2	1093±1	338±1
Hexanoic acid	-	-	-

Note: "-" indicates no detection.

As can be seen from Table 2, the new strain Clostridium WLY-B-L2 produces predominantly butyric acid up to 588ppm, while the model strain DSM29923 produces predominantly acetic acid up to 1432 ppm. From this, it was found that the two bacteria were significantly different. The examples show that the strain WLY-B-L2 obtained by screening the invention is different from the model strain in main fragrant substances and organic acids, and the two belong to different species.

Sequence listing

<110> Yibin wuliangye GmbH

<120> novel strain of Clostridium and application thereof

<130> A210024K (preface)

<141> 2021-01-20

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Claims (9)

1. The novel clostridium strain is characterized in that: the preservation number of the new strain is CGMCC NO. 19455.

2. The novel Clostridium species of claim 1, wherein: the 16S rDNA nucleotide sequence of the new strain is shown as SEQ ID NO:1 is shown.

3. The novel Clostridium species of claim 1, wherein: the new clostridium strain produces ethyl valerate and 2-pentanone simultaneously.

4. The novel Clostridium species of claim 1, wherein: the new clostridium strain simultaneously produces butyric acid, ethyl valerate, ethyl acetate and 2-pentanone.

5. The novel Clostridium species of claim 1, wherein: the ethyl valerate producing capacity of the new clostridium bacterial reaches 6.37 ppm.

6. The novel Clostridium species of claim 1, wherein: the 2-pentanone producing capacity of the new clostridium strain reaches 3.16 ppm.

7. The novel Clostridium species of claim 1, wherein: the butyric acid production capacity of the new strain reaches 588 ppm.

8. The novel Clostridium species of claim 1, wherein: the ethyl acetate producing capacity of the new strain reaches 2.62 ppm.

9. Use of a novel Clostridium species of any one of claims 1-8 in brewing white spirit.

Images