CN112251386B

CN112251386B - Bacillus licheniformis Scu-01 and application thereof

Info

Publication number: CN112251386B
Application number: CN202011195454.0A
Authority: CN
Inventors: 周荣清; 唐秋香; 黄钧
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2020-10-30
Filing date: 2020-10-30
Publication date: 2021-08-20
Anticipated expiration: 2040-10-30
Also published as: CN112251386A

Abstract

The invention discloses bacillus licheniformis Scu-01 and application thereof, and belongs to the technical field of microbial food processing. The bacillus licheniformis Scu-01 disclosed by the invention can obviously improve the contents of volatile substances including tetramethylpyrazine, acetoin and 2, 3-butanediol in a small koji, is stable in genetic property, and has a good development and application prospect.

Description

Bacillus licheniformis Scu-01 and application thereof

Technical Field

The invention relates to the technical field of microbial food processing, in particular to a bacillus licheniformis Scu-01 strain and application thereof.

Background

The tetramethylpyrazine is an important flavor substance in a white spirit fermentation product, has the fragrance of baking, hazelnut, peanut and cocoa, and the acetoin and the 2, 3-butanediol are important components forming the import fragrance, mellow and sweet taste and lasting aftertaste of the famous and high-quality white spirit. The research proves that the generation of the components is closely related to the bacillus. The currently reported bacillus strains for fermenting white spirit only relate to the improvement of a single component for producing tetramethylpyrazine, the flavor harmony of the white spirit is not fully considered, the defects of single strain function, insufficient comprehensive performance and the like exist, and the quality of the white spirit is reduced because the strains are not stable during passage.

Therefore, the problem to be solved by the technical personnel in the field is to provide the bacillus licheniformis Scu-01 and the application thereof.

Disclosure of Invention

In view of the above, the invention provides a bacillus licheniformis Scu-01 and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

bacillus licheniformis (Scu-01) strain with the preservation number of CCTCC NO: m2018220, already preserved in China center for type culture Collection, CCTCC for short, addresses China, Wuhan university, and has a preservation date of 19.04.2018, and is classified and named as Bacillus Scu-01 and Bacillus sp.scu-01.

Further, the application of the bacillus licheniformis Scu-01 in fermentation wine brewing.

Further, the application of the Bacillus licheniformis Scu-01 in preparing the Xiaoqu.

Further, the application of the bacillus licheniformis Scu-01 in preparing the Xiaoqu comprises the following specific operations: will OD₆₀₀The bacillus licheniformis Scu-01 suspension of 2.4-3.0 is inoculated on the wheat material in an inoculation amount of 6% -8% (V/V).

Further, the application of the bacillus licheniformis Scu-01 in high yield of tetramethylpyrazine, 2, 3-butanediol and acetoin.

According to the technical scheme, compared with the prior art, the invention discloses and provides the bacillus licheniformis Scu-01 and the application thereof, the bacillus licheniformis Scu-01 can obviously improve the content of the volatile substances of the minor koji, namely tetramethylpyrazine, acetoin and 2, 3-butanediol, and has stable genetic property and good development and application prospects.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a diagram showing the colony morphology of Bacillus licheniformis Scu-01 cultured in a nutrient broth solid medium;

FIG. 2 is an electron microscope scanning image of Bacillus licheniformis Scu-01 of the present invention;

FIG. 3 is a phylogenetic tree of Bacillus licheniformis Scu-01 of the present invention;

FIG. 4 is a drawing showing the advantageous components of Bacillus licheniformis Scu-01 of the present invention compared with a standard strain;

FIG. 5 shows the generation stability of fermentation products (acids, alcohols, etc.) of Bacillus licheniformis Scu-01 of the present invention;

FIG. 6 shows the passage stability of fermentation products (acetoin, 2, 3-butanediol, tetramethylpyrazine) of Bacillus licheniformis Scu-01 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

EXAMPLE 1 isolation and screening of the strains

Taking Maotaizhen maotai Maotai-flavor Daqu as a raw material, selecting a nutrient broth solid culture medium (10 g of peptone, 3g of beef extract, 5g of sodium chloride, 1000ml of deionized water and 20g of agar), respectively taking 2g of yeast powder of each part of the maotai-flavor Daqu, scattering the yeast powder in a 150ml triangular flask filled with sterile glass beads and 45ml of physiological saline, carrying out water bath treatment at 80 ℃ for 30min to obtain spore suspension, diluting and coating the spore suspension on a flat plate according to a series, carrying out constant temperature culture at 37 ℃, selecting colonies, carrying out flat plate streaking to obtain 120 single colonies, inoculating the single colonies to wheat (the wheat is purchased from Chengdu Xiandao Jian powder Co., Ltd.) to measure volatile components, screening out a strain which can obviously increase the variety of the volatile components of the Xiaoqu and improve the content of main volatile components, and naming the strain as Bacillus licheniformis Scu-01, hereinafter, it will be abbreviated as "Scu-01".

Example 2 Strain identification

(1) Morphological characteristics of the strain: the bacillus licheniformis Scu-01 is gram-positive bacteria, is not easy to pick up when being irregularly shaped, white, opaque, convex, dry, creased and fringed in a nutrient broth solid culture medium and firmly attached to the culture medium, and a plurality of single bacterial colonies are obtained by diluting and coating the Scu-01 strain, as shown in figure 1. The strains are observed by a scanning electron microscope, and the results are shown in figure 2, and the thalli are rod-shaped under the electron microscope, have the sizes of 0.40-0.68 mu m multiplied by 1.12-2.50 mu m and are arranged singly or in pairs.

(2) Physicochemical properties of the strain: oxygen demand, contact enzyme activity, oxidase activity, gelatin hydrolysis activity, starch hydrolysis activity and cellulose hydrolysis activity, for which reference is made to Bergey's Manual of bacteriological identification. The results show that the bacillus licheniformis Scu-01: glucose, fructose, sucrose, mannose, maltose and inositol are fermented positively, rhamnose, lactose, sorbitol and mannitol are fermented negatively, cyclodextrin, dextrin and arbutin are fermented variably, a V-P test and a catalase reaction are positive, and an oxidase reaction is negative. The highest salinity tolerance can reach 12 percent, and the pH range is 5.0-9.0.

(3)16S rRNA gene sequencing and evolutionary tree construction

Extraction of DNA of the Scu-01 Strain: after culturing the Scu-01 strain at 37 ℃ for 24h at 180r/min, extracting the total DNA of the lysate according to the operation manual of the UNIQ-10 column type bacterial genome DNA extraction kit, and storing the extracted DNA at-20 ℃ for later use.

PCR primers: bacterial universal primers 27F and 1492R.

27F：5’-AGAGTTTGATCMTGGCTCAG-3’；SEQ ID NO.1；

1492R：5’-TACGGYTACCTTGTTACGACTT-3’；SEQ ID NO.2；

And (3) PCR reaction conditions: pre-denaturation at 94 deg.C for 3min, denaturation at 95 deg.C for 30s, and annealing at 52 deg.C for 30 s; extending for 1min and 45s at 72 ℃, circulating for 35 times, and finally preserving the heat for 10min at 72 ℃. After the reaction is finished, taking a proper amount of PCR sample, mixing the PCR sample with the dye uniformly, and detecting the mixture by 1% agarose gel electrophoresis.

16S rRNA sequence obtained: the PCR product was sequenced by Shanghai Bioengineering Co., Ltd. After the sequencing is finished, the 16S rRNA sequence (SEQ ID NO.3) of Scu-01 is submitted to NCBI for on-line alignment service to search similarity sequences, and a phylogenetic tree is constructed through software MEGA, and the result is shown in figure 3.

16S rRNA sequence of Scu-01:

TGCAAGTCGAGCGGACCGACGGGAGCTTGCTCCCTTAGGTCAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGATTGAACCGCATGGTTCAATCATAAAAGGTGGCTTTTAGCTACCACTTGCAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTGGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGACAACCCTAGAGATAGGGCTTCCCCTTGGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAAAGGGCAGCGAAGCCGCGAGGCTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTGGAGCCAGCCGCCGAA；SEQ ID NO.3。

example 3 application of Scu-01 Strain

Activating and culturing the strain Scu-01, namely inoculating the strain into a nutrient broth liquid culture medium (10 g of peptone, 3g of beef extract, 5g of sodium chloride and 1000ml of deionized water), carrying out shake culture at the temperature of 37 ℃ at 150r/min for 24h, and then carrying out OD (OD) culture₆₀₀The bacterial suspension 2.4 was inoculated to wheat (purchased from Chengdu Xiongjian powder Co., Ltd.) at an inoculation amount of 6% (V/V), incubated at 37 ℃ for 14 days, and the volatile components contained in the wheat were measured by gas chromatography-mass spectrometry.

Two standard strains (Bacillus licheniformis ATCC 14580) were used simultaneously^TPurchased from the culture collection of the institute for Industrial microorganisms, Shanghai, hereinafter referred to as ATCC 14580T; bacillus paranichnformis KACC 18426T, supplied by Korea Agricultural Culture Collection (KACC), hereinafter abbreviated as KACC 18426T) were subjected to the same operation, and the difference in the content of the dominant component was compared.

The gas chromatograph-mass spectrometer used: trace GC Ultra-DSQ II gas chromatography-single-shot quadrupole mass spectrometer, Thermo Fisher Electron Inc. USA. Adopting ethanol to leach Xiaoqu wheat material, manually feeding 1 mu l of liquid, and carrying out chromatographic conditions: the column type was HP-Innowax (30 m.times.250 nm. times.0.25 um film, J & W, USA). Maintaining at 40 deg.C for 5min, heating to 120 deg.C at 6 deg.C/min, and heating to 230 deg.C at 8 deg.C/min for 20 min. The injection inlet temperature is 250 ℃, high-purity helium gas is used as carrier gas (the flow rate is 1ml/min), and the split ratio is 10: 1.

Mass spectrum conditions: the ionization mode EI has electron intensity of 70eV, ion source temperature of 230 ℃, conversion line temperature of 250 ℃ and chromatographic scanning range of m/z of 40-400 amu.

The fermentation was carried out under the same conditions with the same starting concentration of Scu-01 and the standard strain, and the results are shown in FIG. 4; when the culture on the Xiaoqumai material is finished, the contents of acetoin, 2, 3-butanediol, 2,3,5, 6-tetramethylpyrazine and 1, 4-dimethylpiperidine are respectively 52.08mg/kg, 48.51mg/kg, 16.07mg/kg and 37.63 mg/kg. The results show that the content of the acetoin, the 2, 3-butanediol, the 1, 4-dimethylpiperidine and the 2,3,5, 6-tetramethylpyrazine generated by the Scu-01 is higher than that of the two standard strains, so that the capability of generating high-quality components in the white spirit fermentation by the Scu-01 is higher than that of the two standard strains in the experiment, and the application prospect is good.

The content of the target metabolite of the Scu-01 isolate is detected by monthly passage to the ninth generation, and the result is shown in FIGS. 5-6; the results show that the contents of acetoin, 2, 3-butanediol and 2,3,5, 6-tetramethylpyrazine are stable, and the contents of acid, pyrazine, ketone and other volatile components are relatively stable; therefore, the genetic property of the Scu-01 is stable.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

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<120> bacillus licheniformis Scu-01 strain and application thereof

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tgcaagtcga gcggaccgac gggagcttgc tcccttaggt cagcggcgga cgggtgagta 60

acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg 120

atgcttgatt gaaccgcatg gttcaatcat aaaaggtggc ttttagctac cacttgcaga 180

tggacccgcg gcgcattagc tagttggtga ggtaacggct caccaaggcg acgatgcgta 240

gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg 300

aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag 360

tgatgaaggt tttcggatcg taaaactctg ttgttaggga agaacaagta ccgttcgaat 420

agggcggcac cttgacggta cctaaccaga aagccacggc taactacgtg ccagcagccg 480

cggtaatacg taggtggcaa gcgttgtccg gaattattgg gcgtaaagcg cgcgcaggcg 540

gtttcttaag tctgatgtga aagcccccgg ctcaaccggg gagggtcatt ggaaactggg 600

gaacttgagt gcagaagagg agagtggaat tccacgtgta gcggtgaaat gcgtagagat 660

gtggaggaac accagtggcg aaggcgactc tctggtctgt aactgacgct gaggcgcgaa 720

agcgtgggga gcgaacagga ttagataccc ctggtagtcc acgccgtaaa cgatgagtgc 780

taagtgttag agggtttccg ccctttagtg ctgcagcaaa cgcattaagc actccgcctg 840

gggagtacgg tcgcaagact gaaactcaaa ggaattgacg ggggcccgca caagcggtgg 900

agcatgtggt ttaattggaa gcaacgcgaa gaaccttacc aggtcttgac atcttctgac 960

aaccctagag atagggcttc cccttggggg gcagagtgac aggtggtgca tggttgtcgt 1020

cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tgatcttagt 1080

tgccagcatt cagttgggca ctctaaggtg actgccggtg acaaaccgga ggaaggtggg 1140

gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtgcta caatgggcag 1200

aacaaagggc agcgaagccg cgaggctaag ccaatcccac aaatctgttc tcagttcgga 1260

tcgcagtctg caactcgact gcgtgaagct ggaatcgcta gtaatcgcgg atcagcatgc 1320

cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccacga gagtttgtaa 1380

cacccgaagt cggtgaggta acctttggag ccagccgccg aa 1422

Claims

1. A strain of Bacillus licheniformis (Scu-01) is characterized in that the preservation number is CCTCC NO: m2018220.

2. The use of a bacillus licheniformis Scu-01 according to claim 1 in fermentation brewing.

3. The use of a Bacillus licheniformis Scu-01 strain according to claim 1 for making a koji.

4. The use of a Bacillus licheniformis Scu-01 strain according to claim 3 for making a koji₆₀₀The bacillus licheniformis Scu-01 suspension of 2.4-3.0 is inoculated on the wheat material in an inoculation amount of 6% -8% (V/V).

5. The application of the bacillus licheniformis Scu-01 disclosed in claim 1 in high yield of tetramethylpyrazine, 2, 3-butanediol and acetoin.