CN114874942B - Bacillus cereus for producing protease and application of bacillus cereus in Daqu - Google Patents
Bacillus cereus for producing protease and application of bacillus cereus in Daqu Download PDFInfo
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- 239000004365 Protease Substances 0.000 title claims abstract description 36
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- 238000000855 fermentation Methods 0.000 claims description 9
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- 239000002054 inoculum Substances 0.000 claims 1
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- 239000000796 flavoring agent Substances 0.000 abstract description 4
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- 238000002474 experimental method Methods 0.000 abstract description 2
- 235000019634 flavors Nutrition 0.000 abstract description 2
- 108010033040 Histones Proteins 0.000 abstract 1
- 238000005728 strengthening Methods 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 241000193830 Bacillus <bacterium> Species 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 7
- 238000012258 culturing Methods 0.000 description 7
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- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 108090000145 Bacillolysin Proteins 0.000 description 2
- 241000194108 Bacillus licheniformis Species 0.000 description 2
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- OHCQJHSOBUTRHG-KGGHGJDLSA-N FORSKOLIN Chemical compound O=C([C@@]12O)C[C@](C)(C=C)O[C@]1(C)[C@@H](OC(=O)C)[C@@H](O)[C@@H]1[C@]2(C)[C@@H](O)CCC1(C)C OHCQJHSOBUTRHG-KGGHGJDLSA-N 0.000 description 2
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- NCXMLFZGDNKEPB-FFPOYIOWSA-N natamycin Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C[C@@H](C)OC(=O)/C=C/[C@H]2O[C@@H]2C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 NCXMLFZGDNKEPB-FFPOYIOWSA-N 0.000 description 2
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- SUZLHDUTVMZSEV-UHFFFAOYSA-N Deoxycoleonol Natural products C12C(=O)CC(C)(C=C)OC2(C)C(OC(=O)C)C(O)C2C1(C)C(O)CCC2(C)C SUZLHDUTVMZSEV-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention provides bacillus cereus Bacillus cereus SAU-Q1 for producing protease and application thereof, belonging to the technical field of microorganisms. The bacillus cereus producing protease is separated from the white spirit Daqu and is preserved in China general microbiological culture Collection center, the preservation place is North Star Xiyu No. 1 institute in the Korean region of Beijing city, the preservation number is CGMCC No.24721, and the preservation date is 2022, 4 months and 19 days. The activity of the strain SAU-Q1 in the solid culture medium for producing protease is 1733.16U/g; the survival rate of the bacillus cereus SAU-Q1 can still reach 71.31%, 30.21% and 22.14% respectively at 90 ℃, pH 4.0 and 16% sodium chloride concentration. The strain SAU-Q1 is applied to the strengthening experiment of the white spirit Daqu, the experimental histone enzyme activity is improved by 10%, and the yeast flavor of the experimental group white spirit Daqu is stronger. The strain SAU-Q1 has good application potential in the Daqu of white spirit and vinegar yeast.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to protease-producing bacillus cereus separated from white spirit Daqu and application thereof.
Background
The distilled spirit is prepared by saccharification and fermentation of grains under the combined action of distiller's yeast and various bacteria, and the process flows of later distillation, storage and the like; in distiller's yeast, the microorganism species content is rich, different microorganisms can produce metabolites through different biochemical actions, so that the quality and the taste of the white wine are affected; the protease plays a role in decomposing protein in raw materials in the process of brewing white spirit; the decomposition mechanism is mainly 3: firstly, macromolecular proteins are directly degraded into micromolecular amino acids, and the generated amino acids can be utilized by yeast growth metabolism, and can save energy for other growth; secondly, the shells of the raw materials are hydrolyzed to be exposed, and microorganisms are utilized, so that the white spirit yield is improved; thirdly, the wine is indirectly rich in unique fragrance, protease catalyzes the decomposition of protein, amino acid is decomposed to participate in Maillard reaction, a series of substances with special fragrance such as pyrazines, furans and the like are produced, and fragrance substances can be produced under the catalysis of microorganisms and enzymes.
In the white spirit brewing process, the participation of microorganisms and the importance of the microorganisms are that bacillus is a very important genus for producing protease, and the protease production capability of the bacillus is very strong; moreover, the genus bacteria can have good environmental tolerance and can bear higher alcohol concentration; zhu Guoxing and other 6 strains of high-yield protease selected from high-temperature Daqu are bacillus; wang and the like take Maotai-flavor Daqu as a sample, and 1 strain of high protease bacteria obtained by separation is also bacillus; thus the genus of the main protease in Daqu should be bacillus; patent CN201510180738.5 discloses bacillus producing protease, and the protease activity is 294.44U/mL after optimization; the invention patent CN202111620797.1 discloses a bacillus amyloliquefaciens strain with high protease yield, and the protease activity reaches 1390.6U/mL; wang separating Bacillus licheniformis FBKL1.0199 from Maotai-flavor Daqu, and allowing the neutral protease to reach 3925.80U/g under laboratory conditions; the protease activity of the bacillus cereus screening process disclosed by the invention reaches 1733.16U/g, and meanwhile, the bacillus cereus disclosed by the invention is applied to a Daqu fermentation test, so that the protease activity in Daqu is detected to be higher, and the bacillus cereus and the bacillus licheniformis are of different genus and have larger characteristic difference; as bacillus can form spores, the resistance in natural environment is stronger than that of other microorganisms (high temperature resistance, low moisture resistance and the like), and the application of the strain SAU-Q1 in white spirit Daqu and edible vinegar Daqu has great potential.
Disclosure of Invention
The invention mainly aims to screen and obtain a protease-producing bacillus cereus from the white spirit Daqu and explore the application of the protease-producing bacillus cereus.
The aim of the invention is achieved by the following technical scheme.
The invention provides a protease-producing bacillus cereus Bacillus cereus SAU-Q1 which is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 4 months and 19 days in 2022, wherein the preservation place is North Star Xiyu No. 1 institute of Kogyo in Beijing city, and the preservation number is CGMCC No.24721.
The bacillus cereus is separated from the Daqu of white spirit, and pure bacteria are obtained after preliminary screening, secondary screening and purification, so that the bacillus cereus has stronger protease production capability.
The bacillus cereus SAU-Q1 culture characteristics are as follows: the surface is rough and smooth and opaque, the color is dirty white, the edge color is milky white, the shape of a single colony is nearly round, the surface is not glossy, and the texture is soft.
The bacillus cereus SAU-Q1 is characterized by the following bacterial forms under a microscope: oval or near circular, gram positive bacteria.
The bacillus cereus SAU-Q1 tolerance test shows that: SAU-Q1 is resistant to normal growth at 80℃and pH 4.0 at 16% sodium chloride.
The bacillus cereus SAU-Q1 is applied to Daqu fermentation, the protease activity of the Daqu of the test group white spirit is improved by 10%, and the flavor of the Daqu of the test group white spirit is stronger.
Drawings
FIG. 1 is a transparent circle of strain SAU-Q1.
FIG. 2 is a colony and microscopic cell morphology of strain SAU-Q1.
FIG. 3 is a phylogenetic tree of strain SAU-Q1.
Detailed Description
The test methods described in the following examples are conventional methods unless otherwise specified; the reagents and biological materials, unless otherwise specified, are commercially available; the percentages are mass percentages unless otherwise indicated.
The following examples further illustrate the practice of the invention, but are not limited thereto.
The following media formulations were used in the examples:
Skim milk medium: a (sodium chloride 10.0g, yeast extract 5.0g, tryptone 10.0g, pH adjusted to 7.0); b-skimmed milk powder 20g. After the two are distributed, carrying out damp-heat sterilization, and killing for 20min at 115 ℃; taking out and cooling after sterilization is finished, and uniformly mixing according to the proportion of one part of A and 9 parts of B for standby;
Beef extract peptone solid medium: 10.0g/L of peptone, 5.0g/L of sodium chloride, 3.0g/L of beef extract, 15-20 g/L of nutrient agar and a proper amount of natamycin, finally, adding distilled water, regulating the pH to 7.0-7.2, boiling, bottling, sealing and sterilizing at 121 ℃ for 20min;
Beef extract peptone liquid medium: 5.0g/L of sodium chloride, 10.0g/L of peptone, 3.0g/L of beef extract and a proper amount of natamycin are added with distilled water and stirred uniformly, finally distilled water is added, the pH is regulated to 7.0-7.2, and the mixture is boiled, bottled, sealed and sterilized at 121 ℃ for 20min;
Solid medium: distilled water and bran are respectively put into triangular bottles according to the amounts of 20.0g and 20mL, and then are sealed and sterilized for 20min at 121 ℃ for standby.
Example 1: screening protease-producing strains in koji.
(1) Weighing 20.0g of Daqu, and grinding in a mortar under aseptic condition. Preparing a sufficient amount of sterile water and a sterilizing triangular flask, adding 90ml of sterilized normal saline into the triangular flask, adding 10.0g of ground Daqu fine powder, placing the mixture into a shaking incubator, adjusting the rotating speed to 200r/min, adjusting the temperature to 37 ℃, culturing for 30min, taking out, placing into a water bath kettle at 85 ℃, carrying out water bath for 10min, filtering, and collecting filtrate for later use; then in a sterile environment, the filtrate is diluted to 10 -2、10-3、10-4、10-5、10-6 equal concentration in a gradient way, and 10 -4、10-5、10-6 is selected for coating culture; each of the three concentrations was applied to a beef extract peptone solid culture in an amount of 0.5mL, each of which was subjected to 3 parallel experiments. The flat plate is placed upside down and is kept at the constant temperature of 37 ℃ for culturing for 24 hours, taking out for observation for multiple times during the period; the strains with different forms are selected and inoculated on a new culture medium during the period of culture completion, so as to purify the strain.
(2) Primary screening of protease-producing strains: preparing skim milk culture medium plates, inoculating three separated and purified strains on each plate, and standing at 37 ℃ for 20h. The plates were removed and the morphology of each strain and the ratio of transparent circles to single colonies were noted.
(3) Rescreening of protease-producing strains: according to the production fermentation process, firstly, the strain screened initially is activated, then a loop of single colony is selected and connected into a liquid culture medium, then the liquid culture medium is placed in a shaking incubator, the rotating speed is regulated to be 200r/min, and the temperature is regulated to be 37 ℃; culturing for 24 hr, adding 10% bacterial suspension into triangular flask containing solid culture medium, and standing at 35deg.C for 3d; then adding 10g of fermented product, 90ml of distilled water and a triangular flask, standing for 1h in a water bath at 40 ℃, uniformly stirring at the frequency of 15 min/time, and slowly filtering by using circular filter paper to obtain crude enzyme liquid; phosphate buffer pH 7.2 was prepared for determination of neutral protease activity; the protease activity was determined using the forskolin phenol method, with reference to the commercial industry standard SB/T10317-1999 "protease activity assay".
The SAU-Q1 strain had a protease activity 1733.16U/g, measured as a transparent circle to single colony ratio of 2.474 (FIG. 1).
(4) Identification of Strain SAU-Q1:
morphological identification: streaking the strain SAU-Q1 on a beef extract peptone plate, and culturing at 37 ℃ for 1 day to observe colony morphological characteristics; simultaneously carrying out gram staining; bacterial colony and cell morphology of strain SAU-Q1 are shown in FIG. 2.
Molecular identification: the SAU-Q1 strain is inoculated on a PDA flat plate for activation, the temperature is constant for 24 hours at 28 ℃, a fungus direct amplification kit of the Qingkei catalpa Xixi biotechnology Co., ltd is adopted, the full-length sequence of the 16S rDNA is amplified by using the universal primers 27F and 1492R of the 16S rDNA, the PCR product is sent to the Chengdu Qingqike catalpa biotechnology Co., ltd for sequencing after being checked to be qualified, the sequencing result is compared with NCBI database, and the strain with the closest relation with the tested strain species is selected to construct a phylogenetic tree.
The results show (see FIG. 3) that the homology of the strain SAU-Q1 and the Bacillus cereus is 99.59%, the strain SAU-Q1 is identified as Bacillus cereus by combining morphological characteristics, the strain SAU-Q1 is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) on the 4 th month 19 of 2022, and the preservation place is the Games of North Star West way 1 of the Korean area of Beijing and the preservation number is CGMCC No.24721.
Example 2: tolerance of protease-producing bacillus cereus SAU-Q1.
(1) High temperature tolerance test
Culturing strain in sterile LB culture medium for 48 hr, centrifuging at 3500r/min for 30min, taking out supernatant, diluting to 10 -4, taking the last two gradients as one group, respectively placing into water baths at 80deg.C, 90deg.C and 100deg.C for 30min, cooling with flowing water, respectively inoculating into culture medium with water bath at room temperature as control, placing into a bacteria incubator at 37deg.C, culturing for 24 hr, and calculating survival rate by plate counting method.
The results of the high temperature tolerance test show (Table 1) that the survival rate of the enzyme bacillus cereus SAU-Q1 at 90 ℃ can still reach 71.31%.
(2) Acid tolerance test
The pH value of the sterile LB liquid culture medium is adjusted to be pH3.0, pH4.0 and pH5.0, a proper amount of bacterial suspension of the strain SAU-Q1 is absorbed, and the bacterial suspension is respectively connected into culture mediums with different pH values, so that the initial concentration is about 1 multiplied by 10 6 cfu/mL; after completion, the mixture is placed in a shaking incubator at 37 ℃ and the rotating speed is regulated to 120r/min, and the culture is carried out for 1h. Taking out, coating 200 mu L of each on the solid culture medium, culturing for 24 hours under the same condition, accurately counting the number of single colonies on the flat plate, and calculating the survival rate.
The results of the acid tolerance test show (Table 2) that the survival rate of the enzyme bacillus cereus SAU-Q1 at pH 4.0 can still reach 30.21%.
(3) Salt tolerance test
Sub-packaging sterile LB liquid culture medium into triangular flasks, adjusting the mass concentration of sodium chloride in each triangular flask to be 12%, 14% and 16% respectively, tapping SAU-Q1 bacterial suspension of the strain into triangular flasks with different salt concentrations, enabling the initial concentration to be about 1X 10 6 cfu/mL, placing the strain into a shaking incubator, adjusting the rotating speed to 120r/min, and adjusting the temperature to 37 ℃; after 1h of culture, 200 mu L of the culture medium is taken out from each triangular flask and coated on a solid culture medium, and the culture is carried out for 24h under the same condition, so that the number of single colonies on a flat plate is accurately counted, and the survival rate of the single colonies is calculated.
The salt tolerance test results show (Table 3) that the survival rate of the enzyme bacillus cereus SAU-Q1 at the concentration of 16% sodium chloride can reach 22.14%.
Example 3 Strain SAU-Q1 laboratory simulated Daqu fermentation.
Crushing raw material wheat, mixing the crushed wheat with water to make the water content of the crushed wheat be about 38%, wherein the water content comprises 1% (v/w) of bacterial suspension (strain SAU-Q1.0X10 6 CFU/mL), tightly pressing the soaked wheat flour into a rectangular curved block (bag Bao Qu), and taking a non-inoculated SAU-Q1 as a blank control group, wherein each block weighs about 4.5 kg; then, the yeast blocks are stacked layer by layer and cultured in a fermentation chamber (constant temperature and humidity incubator) for about 12 days, and the temperature and humidity during the culture process are as follows: the relative humidity at 42 ℃ is higher than 90% in 0-4 days, the relative humidity at 55-60 ℃ is higher than 90% in 5-13 days, the relative humidity at 45-50 ℃ is lower than 80% in 14-24 days, the moisture of the yeast at room temperature is lower than 14% in 25-35 days, the protease activity is detected, and the yeast quality is analyzed by adopting sensory analysis.
Compared with a blank control group, the protease activity of the SAU-Q1 reinforced test group white spirit yeast is improved by 10%, the yeast fragrance of the test group white spirit yeast is stronger, the bacillus cereus SAU-Q1 improves the quality of the white spirit yeast, and the method has certain application potential in the fermentation of the white spirit yeast and vinegar yeast.
The above examples are intended to illustrate embodiments of the invention and not to limit the scope of the invention, and modifications or variations may be made to the above description by those skilled in the art, all of which fall within the scope of the invention as defined in the appended claims.
Sequence listing
<110> Sichuan university of agriculture
<120> Bacillus cereus for producing protease and application thereof in Daqu
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<170> SIPOSequenceListing 1.0
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gacactgagg cgcgaaagcg tggggagcaa acaggattag ataccctggt agtccacgcc 780
gtaaacgatg agtgctaagt gttagagggt ttccgccctt tagtgctgaa gttaacgcat 840
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ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc 960
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cgttctcagt tcggattgta ggctgcaact cgcctacatg aagctggaat cgctagtaat 1320
cgcggatcag catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 1380
cacgagagtt tgtaacaccc gaagtcggtg gggtaacctt ttggaaccag ccgcctaagg 1440
tgggacagat gattggggtg atcaaaagaa gggg 1474
Claims (4)
1. A protease-producing Bacillus cereus SAU-Q1 is characterized in that: the bacillus cereus SAU-Q1 is preserved in China general microbiological culture collection center with a preservation number of CGMCC No. 24721.
2. The use of protease-producing bacillus cereus in the fermentation of white spirit Daqu according to claim 1, characterized in that: the bacillus cereus is separated from the white spirit Daqu and is applied to fermentation of the white spirit Daqu.
3. The use of bacillus cereus as claimed in claim 2, wherein: bacillus cereus SAU-Q1 is inoculated into a solid culture medium according to an inoculum size of 1% (v/m), and is subjected to stationary culture at a constant temperature of 35 ℃ for 3 days, wherein protease activity is 1733.16U/g.
4. The use of bacillus cereus as claimed in claim 2, wherein: inoculating bacillus cereus SAU-Q1 into the white spirit Daqu, comparing with a blank control group without inoculating strain SAU-F4, wherein protease activity in the white spirit Daqu of the test group is improved by 10%, and the yeast fragrance of the white spirit Daqu of the test group is stronger.
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| CN116904376A (en) * | 2023-09-13 | 2023-10-20 | 广州大学 | High-salt-tolerance bacillus cereus strain, microbial agent and application thereof |
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| CN102517238B (en) * | 2011-12-30 | 2014-02-26 | 山东宝来利来生物工程股份有限公司 | Acid-producing bacillus cereus and application thereof |
| CN103525718B (en) * | 2013-07-18 | 2015-04-08 | 河南科技大学 | Bacillus cereus and probiotics powder thereof as well as preparation and application of probiotics powder |
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| RU2723411C2 (en) * | 2018-07-13 | 2020-06-11 | Рашит Накипович Кадыров | Bacillus cereus strain rcam04578, a biologically active compounds producer having probiotic properties |
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| CN110656063B (en) * | 2019-10-21 | 2020-09-25 | 黄山学院 | Strain for producing high-temperature-resistant protease |
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| CN116904376A (en) * | 2023-09-13 | 2023-10-20 | 广州大学 | High-salt-tolerance bacillus cereus strain, microbial agent and application thereof |
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