CN112358998B - Novel strain of Beijing bacillus and application thereof - Google Patents
Novel strain of Beijing bacillus and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/07—Bacillus
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- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G3/00—Preparation of other alcoholic beverages
- C12G3/02—Preparation of other alcoholic beverages by fermentation
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Abstract
The invention discloses a novel strain of Beijing Bacillus and application thereof, wherein the strain is the Beijing Bacillus (Bacillus beijinginensis) and the preservation number of the strain in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC NO. 19202. Experiments prove that the novel strain Bacillus beijing (Bacillus beijingensis) is provided, is separated from white spirit cellar mud, can produce substances with strong aroma and flavor, and can be used for fermenting and brewing wine.
Description
Technical Field
The invention relates to the technical field of brewing microorganisms, and particularly relates to a novel strain of bacillus beijing and application thereof.
Background
The white spirit is one of three distilled spirits in the world, is produced by adopting a unique fermentation technology, has a history of thousands of years, and the whole inner wall of a cellar pool is covered with pre-cultured cellar mud. The fermented raw materials are cooked, mixed, crushed and distilled. Adding saccharification leaven into the steamed raw materials, then putting the steamed raw materials into a cellar for fermentation, taking the fermented materials out of the cellar, and distilling the fermented materials to prepare the white spirit. Microorganisms in pit mud produce various flavor components such as butyric acid, caproic acid and ethyl caproate. Ethyl caproate is considered as a key ingredient affecting the flavor and quality of Luzhou-flavor liquor. In the brewing process, the pit mud microorganisms have important aroma-producing and flavor-enhancing effects, and the quality of the Luzhou-flavor liquor is improved along with the increase of the age of the wine pit. The high quality of the Luzhou-flavor liquor is attributed to the maturation process of pit mud, which results in the balance of microbial community structures and the diversity of pit mud, thereby generating unique flavor.
The strong aromatic Chinese spirits have the flavor characteristics of strong cellar aroma, sweet and mellow taste, harmonious aroma and long aftertaste, are deeply favored by consumers, and the annual sale amount accounts for about 70 percent of the total amount of Chinese spirits. Microorganisms in pit mud can generate various flavor components such as butyric acid, caproic acid and ethyl caproate, and the ethyl caproate is considered as a key component influencing the flavor and the quality of the Luzhou-flavor liquor. The pit mud microbial flora is a main factor influencing the quality of pit mud and white spirit, and is an important microbial source in the brewing process of the white spirit. The microorganisms in the pit mud are various and have very rich species diversity, but most of the microorganisms are still in a difficult culture state. Although the culturability of microorganisms in different environments is different, the culturability is very low, for example, the culturability in seawater is less than 0.1 percent, and the culturability in soil is about 0.3 percent. Only a small proportion of bacteria in a natural habitat can be cultured using conventional bacterial culture methods. At present, microorganisms cultured by using a conventional method are known microorganisms, and no new microorganism species is found.
Disclosure of Invention
Therefore, the invention provides a novel strain of Bacillus beijing and application thereof.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a new bacterial strain, which is Bacillus beijing (Bacillus beijinginensis) and the preservation number of the bacterial strain is CGMCC NO.19202 in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms.
In one embodiment of the invention, the 16S rDNA sequence of the Bacillus beijing is shown in SEQ ID No. 1.
The invention also provides a microbial inoculum, the active ingredient of which is the strain of claim 1.
The application of the strain or the microbial inoculum in the following (A1) or (A2):
(A1) generating a flavor substance;
(A2) a product with a flavour material is prepared.
The application of the strain or the microbial inoculum in the following (B1) or (B2):
(B1) brewing wine;
(B2) and (5) preparing a wine brewing product.
The invention also provides a screening method of the strain, which comprises the following steps: diluting pit mud, coating the pit mud on a lactobacillus selective agar culture medium, carrying out aerobic culture at 37 ℃ for 24h, carrying out streak purification culture at three regions for 3 times to obtain pure culture bacteria, amplifying 16SrDNA by using the pure culture bacteria PCR technology, and carrying out sequence comparison analysis and identification to obtain the Bacillus beijing (Bacillus beijijinensis).
In one embodiment of the invention, the lactobacillus selective agar medium comprises the following raw materials by mass: 10.0g of tryptone, 5.0g of yeast extract powder, 6.0g of monopotassium phosphate, 2.0g of ammonium citrate, 25g of sodium acetate, 0.575g of magnesium sulfate, 0.12g of manganese sulfate, 0.034g of ferric sulfite, 801.0 g of tween, 20g of glucose and 15.0g of agar, no acetic acid is added after sterilization, the volume is fixed to 1L by adding distilled water at 25 ℃.
The invention has the following advantages:
experiments prove that the novel strain of the Bacillus beijing (Bacillus beijingensis) is separated from pit mud, can generate substances with strong fragrance and flavor, and can be used for fermenting and brewing wine.
According to the invention, the lactobacillus selective agar culture medium is adopted firstly, common bacteria are slow to reproduce, and new zoomicrobials difficult to culture grow fast on the same culture dish and are little influenced by conventional microorganisms; meanwhile, after the culture time is prolonged for 36 hours, a single colony is picked, and new microorganisms difficult to culture grow fully for a long time until the colony is visible.
Bacterial preservation description: the Beijing Bacillus (Bacillus beijinginensis) is preserved in the China general microbiological culture Collection center in 2019, 12 months and 16 days, and the preservation address is as follows: the collection number of the microorganism research institute of Chinese academy of sciences, No. 3 Xilu No.1 of Beijing, Chaoyang, and the institute of microbiology, is CGMCC NO. 19202.
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 should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.
FIG. 1 is 16S rDNA sequence and evolutionary tree diagram of Bacillus beijing (Bacillus beijingensis) provided by the present invention;
FIG. 2 is a diagram showing the detection result of menaquinone MK-7 of Bacillus beijing (Bacillus beijingensis) provided by the invention;
FIG. 3 is a diagram showing the results of detecting polar esters of Bacillus beijing (Bacillus beijingensis) according to the present invention.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. 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.
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. 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 identification of Bacillus beijing (Bacillus beijingensis)
Separation of novel strain of Bacillus beijingensis
Step one, preparing a lactobacillus selective agar culture medium: 10.0g of tryptone, 5.0g of yeast extract powder, 6.0g of potassium dihydrogen phosphate III, 2.0g of ammonium citrate, 25g of sodium acetate, 0.575g of magnesium sulfate, 0.12g of manganese sulfate, 0.034g of ferric sulfite, 801.0 g of tween, 20g of glucose and 15.0g of agar, sterilizing, adding no acetic acid, adding distilled water at 25 ℃ and keeping the volume to 1L.
Step two, taking 10g of pit mud, and diluting the pit mud to 10g by using sterile distilled water-5Coating on a prepared lactobacillus selective agar culture medium culture dish, and performing aerobic culture at 28 ℃;
step three, carrying out aerobic culture at 28 ℃ for 36 hours, then selecting a single colony, and carrying out streak purification on three regions for 3 times to obtain pure culture bacteria;
and step four, extracting bacterial DNA, amplifying a 16SrDNA sequence by utilizing a PCR technology, and preliminarily determining a new strain of the Bacillus beijing (Bacillus beijijinensis) through sequence comparison.
Second, identification of Bacillus beijingensis
The new bacteria separated and screened from pit mud are gram-positive bacteria, bacillus, named as: beijing Bacillus (Bacillus beijingensis)
1. The strain of the Beijing Bacillus (Bacillus beijingensis) is subjected to morphological, physiological and biochemical, cytochemical and gene level research. Physiological and biochemical characteristics of Bacillus beijing REN3(Bacillus beijingensis).
The colony morphology of the Beijing Bacillus (Bacillus beijingensis) is round, smooth in surface, transparent and white; gram-positive bacteria, the optimum temperature is 28 ℃, the optimum pH value is 7-8, the sodium chloride concentration can be tolerated to be 1% -3%, the starch can be hydrolyzed, the catalase is negative, the alkaline phosphatase, the esterase (C4), the lipoid esterase (C8), the leucine arylamine, the valine arylamine, the cystine arylamine, the trypsin, the chymotrypsin, the acid phosphatase, the beta-galactosidase and the alpha-glucosidase are positive, the fermentation (glucose) is carried out, and the glucosidase-esculin is hydrolyzed.
2. Detection of cell chemistry characteristics of Bacillus beijing (Bacillus beijingensis)
The cytochemical components of Bacillus beijing (Bacillus beijijinensis) such as fatty acids, quinone type, polar lipids, etc., were detected by GC, HPLC liquid chromatography and TLC thin-layer chromatography (Sasser M. identification of bacterial by gas chromatography of cellular lipids, MIDI Technical Note 101.Newark, DE: MIDIinc; 1990.Minnikin DE, O' Donnell AG, Goodfell M, Alderson G, Athaly M et al. integrated procedure for the extraction of bacterial isocyanates and lipid microorganisms J. Microbiol Methods 1984; 2: 233-.
Fatty acid component of Beijing Bacillus (Bacillus beijingensis) cell: saturated fatty acids: c16:0, Antesio-C15:0, Antesio-C17:0, Iso-C14:0, Iso-C15:0, Iso-C16:0, Iso-C17: 0;
the cellular fatty acid composition is shown in Table 1
TABLE 1
As shown in figure 2, in the detection of the quinone component in the cell respiration of the strain Bacillus beijing (Bacillus beijingensis), the main advantage of the strain is quinone menaquinone MK-7, and quinone compounds are a class of oxidation active substances which are widely existed in natural products, antitumor drugs, in-vivo biochemical metabolites, environmental pollutants or polycyclic aromatic hydrocarbon metabolism. Each bacterium has a main quinone component, and the difference in the kinds and amounts of quinones in a microbial population reflects the diversity of the population composition, and the quinone spectrum has been widely used in environmental microorganisms as an index of the diversity of the bacterial population composition.
The detection of the polar ester of the Beijing Bacillus (Bacillus beijingensis) comprises the following specific steps: polar lipid extraction: 100mg of lyophilized cells of Bacillus beijing were suspended in 9.5mL of chloroform: methanol: 0.3% NaCl (2.5:5: 2). The thallus solution is placed in a water bath at 80 ℃ for 15 min. After cooling, the filter paper was filtered into a 50mL centrifuge tube, 2.5mL chloroform and 2.5mL 0.3% NaCl were added, and centrifuged at 4000rpm for 5 min. The lower chloroform phase was carefully separated into a clean rotary evaporator-dedicated flask and the chloroform removed by rotary evaporation under reduced pressure on the rotary evaporator, the temperature of the water bath not exceeding 40 ℃. 250 mu.L of chlorine anti-methanol (2:1, v/v) is added and transferred to a brown screw sample bottle, and the bottle is placed in a refrigerator at 4 ℃ for storage and testing.
TLC analysis of polar lipids: a10 cm × 10cm Silica gel plate (Merck 25TLC aluminum sheets 20cm × 20cm Silica gel 60F254) was activated in an oven at 110 deg.C for 1 hour, and then cooled. Pipette 2 μ L of total lipid sample onto TLC plate, and spot 3 times.
And (3) placing the TLC thin plate into a first chromatographic cylinder for layer development, wherein the first spreading agent is chloroform, methanol and water (65:25:4, v/v), taking out the thin plate after the solvent is spread to the top, drying the thin plate by blowing, placing the thin plate into a second chromatographic cylinder, and the second spreading agent is chloroform: methanol: acetic acid: water (80: 12: 15: 4, V/V), ascending in a direction perpendicular to the first direction, spreading the solvent to the top, taking out the thin plate for drying, spraying phosphomolybdic acid color developing agent to the TLC plate until the TLC plate is completely wet, heating at 100 ℃ for 5-8min, displaying clear spots, immediately scanning the TLC plate on a scanning instrument, and recording the result. As shown in fig. 3, bacillus beijing contains 4 polar esters, DPG: diphosphatidylglycerol, diphosphatidylglycerol; PG: phosphatidylglycerol, phosphonoglycolic, glycophospholipidid; PE: phosphatidylethanolamine, phosphatidyl ethanolamines; AL: an amino polar ester; PI: phosphatidylinositol, phasetdylinosotol; phospholipids (phospholipids) belong to polar lipids (polar lipids), which together with proteins, sugars, etc. form cell membranes and play an important role in substance transport, metabolism and maintenance of normal osmotic pressure. The phospholipidic components of different genera of bacteria are different, and are one of the important characteristics for identifying the genus, and are indispensable classification indicators in chemical classification projects.
3. 16S rDNA sequence and evolutionary tree of Bacillus beijing (Bacillus beijingensis)
Bacillus beijing (Bacillus beijingensis), the strain is subjected to whole genome sequencing by Shanghai Meiji biological medicine science and technology Limited to obtain a whole genome sequence. The phylogenetic tree is shown in figure 1.
The measured REN3T 16S rRNA sequence is compared with model strains in NCBI (https:// www.ncbi.nlm.nih.gov /), 24 model strain 16S rRNA sequences are selected according to similarity to construct a phylogenetic tree, as shown in FIG. 1, the relative distance between Bacillus beijing (Bacillus beijingensis) REN3T and the zoogloea is similar, and the phylogenetic tree is an independent branch to determine that the phylogenetic tree is the zoogloea, and the similar strain is Bacillus formianis strain CV53 according to the similarity (98.66%). Then, the REN3T strain is sent to Shanghai Meiji biological medicine science and technology limited to perform whole genome sequencing, so as to obtain a whole genome sequence. At the same time, the whole genome of a similar strain is downloaded from NCBI, the whole genome of REN3T is compared with the whole genome of a similar strain Bacillus formarnis strain CV53 at DSMZ (http:// ggdc.dsmz.de/home.php), and genomic DNA-DNA homology hybridization (DDH): DNA Homology (DDH) is 18.20% [ 16.1-20.6% ], DDH is less than 70%, and thus it was preliminarily determined as a new species.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> Beijing university of Industrial and commercial
Novel <120> Beijing bacillus strain and application thereof
<130> GG19719818A
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1412
<212> DNA
<213> Artificial Sequence
<400> 1
ttaggcggct ggccccaaaa aggttacccc accgacttcg ggtgttacaa actctcgtgg 60
tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc tgatccgcga 120
ttactagcga ttccggcttc atgcaggcga gttgcagcct gcaatccgaa ctgagaatgg 180
ttttatggga ttggcttcac ctcgcggctt cgctgccctt tgttccatcc attgtagcac 240
gtgtgtagcc caggtcataa ggggcatgat gatttgacgt catccccacc ttcctccggt 300
ttgtcaccgg cagtcacctt agagtgccca actgaatgct ggcaactaag atcaagggtt 360
gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca accatgcacc 420
acctgtcact ctgtcccccg aaggggaacg ccctatctct aggggtggca gaggatgtca 480
agacctggta aggttcttcg cgttgcttcg aattaaacca catgctccac cgcttgtgcg 540
ggcccccgtc aattcctttg agtttcagcc ttgcggccgt actccccagg cggagtgctt 600
aatgcgtttg ctgcagcact aaagggcgga aaccctctaa cacttagcac tcatcgttta 660
cggcgtggac taccagggta tctaatcctg ttcgctcccc acgctttcgc gcctcagcgt 720
cagttacaga ccagagagcc gccttcgcca ctggtgttcc tccacatctc tacgcatttc 780
accgctacac gtggaattcg ctctcctctt ctgcactcaa gtcccccagt ttccaatgac 840
cctccacggt tgagccgtgg gctttcacat cagacttaaa ggaccgcctg cgcgcgcttt 900
acgcccaata attccggaca acgcttgcca cctacgtatt accgcggctg ctggcacgta 960
gttagccgtg gctttctggt caggtaccgt caaggtgccg gcagtgactc cggcacttgt 1020
tcttccctga caacagagct ttacgacccg aaggccttca tcgctcacgc ggcgttgctc 1080
cgtcagactt tcgtccattg cggaagattc cctactgctg cctcccgtag gagtctgggc 1140
cgtgtctcag tcccagtgtg gccgatcacc ctctcaggtc ggctacgcat cgttgccttg 1200
gtgagccatt acctcaccaa ctagctaatg cgccgcgggc ccatctgtaa gtgacagccg 1260
aaaccgtctt tcagctctcc ctcatgcgag ggaaagggtt atccggtatt agccccggtt 1320
tcccggagtt atcccagtct tacaggcagg ttgcccacgt gttactcacc cgtccgccgc 1380
tgacttcagg gagcaagctc ccttctgtcc gc 1412
Claims (4)
1. The preservation number of the Bacillus beijing (Bacillus beijingensis) in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms is CGMCC NO. 19202.
2. The strain of claim 1,
the 16S rDNA sequence of the Beijing bacillus is shown in SEQ ID No. 1.
3. A bacterial agent characterized in that the active ingredient thereof is the strain according to claim 1.
4. Use of the strain of claim 1 or the microbial agent of claim 3 in (B1) or (B2) as follows:
(B1) brewing wine;
(B2) and (5) preparing a wine brewing product.
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