CN114874942A - Bacillus cereus producing protease and application thereof in yeast for making hard liquor - Google Patents
Bacillus cereus producing protease and application thereof in yeast for making hard liquor Download PDFInfo
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Images
Classifications
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- 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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- Engineering & Computer Science (AREA)
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The invention provides a strain of bacillus cereus producing proteaseBacillus cereusSAU-Q1 and application thereof, belonging to the technical field of microorganism. A strain of bacillus cereus producing protease is obtained by separating white spirit Daqu, and is preserved in China general microbiological culture collection center at the preservation place of No. 1 Hospital, Xilu, North Chen, of the Chaoyang district, Beijing, with the preservation number of CGMCC No.24721 and the preservation date of 2022 years, 4 months and 19 days. The activity of the strain SAU-Q1 in a solid culture medium for producing protease is 1733.16U/g; the survival rates of the enzyme bacillus cereus SAU-Q1 can still reach 71.31%, 30.21% and 22.14% respectively at 90 ℃, pH4.0 and 16% sodium chloride concentration. When the strain SAU-Q1 is applied to an enhanced experiment of the Daqu liquor, the activity of the test histone enzyme is improved by 10%, and the flavor of the Daqu liquor is stronger. Strain SAU-Q1 in white spiritThe yeast for making hard liquor and vinegar yeast for making edible vinegar have good application potential.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a bacillus cereus producing protease separated from white spirit yeast and application thereof.
Background
The liquor is a distilled liquor prepared by saccharifying and fermenting grains under the combined action of distiller's yeast and various bacteria, and then carrying out later-stage distillation, storage and other process flows; in the distiller's yeast, the microorganism species content is rich, and different microorganisms can generate metabolites through different biochemical actions, thereby influencing the quality and taste of the white spirit; the protease plays a role in decomposing protein in the raw materials in the process of brewing the white spirit; the decomposition mechanism is mainly 3: firstly, macromolecular protein is directly degraded into micromolecular amino acid, the generated amino acid can be utilized by yeast growth metabolism, and energy can be saved for other growth; secondly, hydrolyzing the shells of the raw materials to expose the shells, and using the microorganisms to improve the yield of the white spirit; and thirdly, the wine is indirectly rich in unique fragrance, protease catalyzes the decomposition of protein, amino acid is decomposed, the amino acid participates in Maillard reaction, a series of substances with special fragrance such as pyrazines and furans are generated, and fragrance substances can be generated under the catalysis of microorganisms and enzyme.
In the process of brewing the white spirit, the participation of microorganisms is extremely important, wherein bacillus is an important protease-producing bacterium, and the protease-producing capability of the bacillus is extremely strong; moreover, the genus can have good environmental tolerance and can bear higher alcohol concentration; 6 strains of high-protease-producing bacteria screened from the high-temperature Daqu by Zhu national star and the like are all bacillus; king Jing and so on regard sauce flavor Daqu as the sample, 1 high protease's genus that isolate from it, also is bacillus; thus the genus that gives rise to proteases in yeast should be bacillus; patent CN201510180738.5 discloses protease-producing bacillus, the protease activity after optimization is 294.44U/mL; the invention patent CN202111620797.1 discloses a bacillus amyloliquefaciens for high yield of protease, the protease activity reaches 1390.6U/mL; the WangJing cell is separated from Maotai-flavor Daqu to obtain Bacillus licheniformis FBKL1.0199, and neutral protease reaches 3925.80U/g under laboratory conditions; the protease activity in the screening process of the bacillus cereus disclosed by the invention reaches 1733.16U/g, and meanwhile, the bacillus cereus disclosed by the invention is applied to a yeast fermentation test, and the protease activity in yeast is higher, and the bacillus cereus and bacillus licheniformis belong to different genera and have larger characteristic difference when being detected; as the bacillus can form spores and has stronger resistance in natural environment than other microorganisms (high temperature resistance, low water resistance and the like), the application of the strain SAU-Q1 in liquor Daqu and vinegar Daqu has huge potential.
Disclosure of Invention
The invention mainly aims to screen and obtain a strain of bacillus cereus producing protease from the liquor yeast and explore the application of the bacillus cereus producing protease.
The purpose of the invention is realized by the following technical scheme.
The invention provides a strain of bacillus cereus producing proteaseBacillus cereusSAU-Q1, which has been deposited in China general microbiological culture Collection center on 19 th month at 2022 by the general culture Collection management Committee of microorganisms at the location of No. 1 Xilu-Beijing university Hospital, Chaoyang, with the collection number of CGMCC number 24721.
The bacillus cereus is separated from the white spirit yeast, and pure bacteria are obtained after primary screening, secondary screening and purification, and have strong protease production capacity.
The culture characteristics of the bacillus cereus SAU-Q1 are as follows: the surface is rough, flat and not transparent, the color is dirty white, the edge color is milky white, the shape of a single colony is nearly circular, the surface has no luster, and the texture is soft.
The morphological characteristics of the bacillus cereus SAU-Q1 under a microscope are as follows: oval or near-round, gram-positive bacteria.
The bacillus cereus SAU-Q1 tolerance test indicates that: SAU-Q1 is resistant to normal growth at 80 deg.C, pH4.0, and 16% sodium chloride.
The bacillus cereus SAU-Q1 is applied to Daqu fermentation, protease activity of the Daqu of the test group white spirit is improved by 10%, and the Daqu fragrance of the Daqu of the test group white spirit is stronger.
Drawings
FIG. 1 is a transparent circle of the 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 all conventional methods unless otherwise specified; the reagents and biomaterials, if not specifically mentioned, are commercially available; the percentages are all by mass unless otherwise specified.
The following examples further illustrate the practice of the invention but are not intended to be limiting.
The media formulations in the examples below were as follows:
skim milk culture medium: a (sodium chloride 10.0g, yeast extract 5.0g, tryptone 10.0g, pH adjusted to 7.0); b-20 g of skimmed milk powder. After the two are distributed, performing damp-heat sterilization, and performing sterilization at 115 ℃ for 20 min; taking out and cooling after sterilization, and uniformly mixing according to the proportion of one part A and 9 parts B for later use;
beef extract peptone solid medium: 10.0 g/L of peptone, 5.0 g/L of sodium chloride, 3.0 g/L of beef extract, 15-20 g/L of nutrient agar and a proper amount of natamycin, and finally adding distilled water, adjusting the pH to 7.0-7.2, boiling, bottling, sealing and sterilizing at 121 ℃ for 20 min;
beef extract peptone liquid medium: 5.0 g/L of sodium chloride, 10.0 g/L of peptone, 3.0 g/L of beef extract and a proper amount of natamycin are added with distilled water and stirred uniformly, and finally, the distilled water is added, the pH is adjusted to 7.0-7.2, the mixture is boiled first, bottled, sealed and sterilized at 121 ℃ for 20 min;
solid medium: the distilled water and the bran are respectively filled into a triangular flask according to the amount of 20.0g and 20mL, and then the triangular flask is sealed and sterilized for 20min at the temperature of 121 ℃ for standby.
Example 1: screening strains producing protease in the white spirit yeast.
(1) 20.0g of Daqu is weighed and put into a mortar under the aseptic condition for sufficient grinding. Preparing sufficient sterile water and a sterilized triangular flask, adding 90ml of sterilized normal saline into the triangular flask, adding 10.0g of ground Daqu fine powder, placing the triangular flask in a shaking incubator, adjusting the rotation speed to 200 r/min, adjusting the temperature to 37 ℃, taking out the triangular flask after culturing for 30min, placing the triangular flask in a water bath kettle at 85 ℃, carrying out water bath for 10 min, filtering, and collecting filtrate for later use; the filtrate was then diluted in a gradient of 10 in a sterile environment -2 、10 -3 、10 -4 、10 -5 、10 -6 Is of equal concentration and is selected from 10 -4 、10 -5 、10 -6 Performing coating culture; three concentrations of each 0.5 mL were plated on beef extract peptone solid media, each requiring 3 replicates. The flat plate is placed upside down and kept still at the constant temperature of 37 ℃ for 24 hours, and is taken out for observation for many times during the period; the strain with different morphology is picked up and inoculated on a new culture medium during the culture completion period, so as to purify the strain.
(2) Primary screening of protease-producing strains: preparing skim milk culture medium plates, performing spot-connection on each plate for three times, separating and purifying the obtained strains, then standing upside down, and culturing at constant temperature of 37 ℃ for 20 hours. The plate was removed and the morphology of each strain and the ratio of the transparent circle to the single colony were recorded.
(3) Re-screening of protease-producing strains: simulating the production and fermentation process, firstly activating the primarily screened strain, then selecting a ring of single colony to be inoculated into a liquid culture medium, then placing the liquid culture medium into a shaking table incubator, adjusting the rotating speed to 200 r/min, and adjusting the temperature to 37 ℃; after 24 hours of culture, adding 10 percent of the bacterial suspension into a triangular flask filled with a solid culture medium, and placing the flask at 35 ℃ for standing culture for 3 days; then adding 10g of the fermented product and 90ml of distilled water into a triangular flask, standing in a water bath kettle at 40 ℃ for 1h, uniformly stirring at the frequency of 15 min/time, and slowly filtering by using circular filter paper to obtain a crude enzyme solution; preparing a phosphate buffer solution with the pH of 7.2 for measuring the activity of the neutral protease; protease activity was measured by the Folin phenol method, which was performed according to the commercial industry Standard SB/T10317-1999 "protease Activity assay".
The ratio of clearing circle to single colony for strain SAU-Q1 was measured to be 2.474 (FIG. 1), and the protease activity of SAU-Q1 strain was 1733.16U/g.
(4) Identification of the strain SAU-Q1:
and (3) morphological identification: streaking the strain SAU-Q1 on a beef extract peptone plate, and culturing at 37 ℃ for 1 day to observe the morphological characteristics of bacterial colonies; simultaneously performing gram staining; the colony and cell morphology of the strain SAU-Q1 are shown in FIG. 2.
And (3) molecular identification: the SAU-F4 strain is subjected to line drawing and inoculated on a PDA plate for activation, the strain is cultured for 24h at the constant temperature of 28 ℃, a 16S rDNA full-length sequence is amplified by adopting a fungus direct amplification kit of Hippon Biotechnology Limited company and using 16S rDNA general primers 27F and 1492R, a PCR product is tested to be qualified and then sent to Chengdu Hippon Biotechnology Limited company for sequencing, the obtained sequencing result is compared with an NCBI database, the strain with the nearest species relation with the tested strain is selected, and a phylogenetic tree is constructed.
The results showed (see FIG. 3) that strain SAU-Q1 has 99.59% homology with Bacillus cereus and that strain SAU-Q1 was identified as Bacillus cereus, in combination with morphological featuresBacillus cereusThe strain SAU-Q1 has been preserved in China general microbiological culture Collection center (CGMCC) on 19.4.2022 at the collection site of No. 1 Xilu-Beijing university Hokko Kogyo-Chao with the collection number of CGMCC number 24721.
Example 2: tolerance of protease-producing Bacillus cereus SAU-Q1.
(1) High temperature resistance test
Culturing strain in sterile LB medium for 48h, centrifuging at 3500r/min for 30min, collecting supernatant, diluting to 10% -4 Taking the last two gradients as one group, three groups in total, and respectively placing at 80 deg.CWater bath at 90 deg.C and 100 deg.C for 30min, cooling with flowing water, respectively inoculating to culture medium with water bath at room temperature as control, culturing in 37 deg.C bacteria incubator for 24 hr, and calculating survival rate by plate counting method.
The high temperature tolerance test result shows that (Table 1), the survival rate of the enzyme Bacillus cereus SAU-Q1 at 90 ℃ can still reach 71.31%.
(2) Acid tolerance test
Sterile LB liquid medium, adjusting its pH value to pH3.0, pH4.0, pH5.0, sucking appropriate amount of strain SAU-Q1 suspension, inoculating into culture medium with different pH values to make its initial concentration about 1 × 10 6 cfu/mL; after the culture is finished, putting the mixture into a shaking incubator at 37 ℃, adjusting the rotating speed to 120r/min, and culturing for 1 h. And taking out, spreading 200 mu L of the suspension on solid culture media, culturing for 24h under the same condition, accurately counting the number of single colonies on a plate, and calculating the survival rate of the single colonies.
The results of the acid tolerance test (Table 2) show that the survival rate of the enzyme Bacillus cereus SAU-Q1 at pH4.0 can still reach 30.21%.
(3) Salt resistance test
The sterile LB liquid culture medium is subpackaged into triangular flasks, the mass concentration of sodium chloride in each triangular flask is adjusted to be 12%, 14% and 16%, respectively, the bacterial suspension of the strain SAU-Q1 is respectively inoculated into triangular flasks with different salt concentrations, and the initial concentration is about 1 × 10 6 Placing cfu/mL in a shaking incubator, adjusting the rotating speed to 120r/min and the temperature to 37 ℃; and taking out after culturing for 1h, taking 200 mu L of the culture medium from each triangular flask, coating the culture medium on a solid culture medium, culturing for 24h under the same condition, accurately counting the number of single colonies on a plate, and calculating the survival rate of the single colonies.
The results of the salt tolerance test show (Table 3) that the survival rate of the enzyme Bacillus cereus SAU-Q1 can reach 22.14% under the condition of 16% sodium chloride concentration.
Example 3 strain SAU-Q1 laboratory simulation of Daqu fermentation.
Pulverizing semen Tritici Aestivi, mixing with water to water content of about 38%The bacterial suspension (strain SAU-Q11.0X 10) was divided to contain 1% (v/w) 6 CFU/mL), pressing the soaked wheat flour tightly into rectangular parallelepiped blocks (wrapped yeast) each weighing about 4.5 kg, with no inoculation of SAU-F4 as blank control; then, stacking layers of the yeast blocks in a fermentation chamber (constant temperature and humidity incubator) for culturing for about 12 days, wherein the temperature and the humidity in the culture process are as follows: the relative humidity of the yeast is more than 90% at 42 ℃ for 0-4 days, the relative humidity of the yeast is more than 90% at 55-60 ℃ for 5-13 days, the relative humidity of the yeast is less than 80% at 45-50 ℃ for 14-24 days, the moisture of the yeast is less than 14% at room temperature for 25-35 days, the protease activity is detected, and the quality of the yeast is analyzed through sensory analysis.
Compared with a blank control group, the protease activity of the liquor Daqu of the test group enhanced by the SAU-Q1 is improved by 10%, the yeast flavor of the liquor Daqu of the test group is stronger, the quality of the liquor Daqu is improved by the bacillus cereus SAU-Q1, and the bacillus cereus SAU-Q1 has certain application potential in the fermentation of the liquor Daqu and vinegar Daqu.
The above examples are intended to illustrate embodiments of the invention only and not to limit the scope of the invention, and modifications and variations of the above description will occur to those skilled in the art, but all such modifications and variations are intended to be within the scope of the invention as defined in the appended claims.
Sequence listing
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Claims (4)
1. Bacillus cereus producing proteaseBacillus cereusSAU-Q1 and application thereof, characterized in that: bacillus cereus preserved in China general microbiological culture collection center with preservation number of CGMCC number 24721Bacillus cereus。
2. A protease producing bacillus cereus according to claim 1, wherein: the bacillus cereus is separated from the white spirit yeast and is applied to fermentation of the white spirit yeast.
3. Use of bacillus cereus according to claim 2, characterized in that: bacillus cereusBacillus cereusThe SAU-Q1 was inoculated into a solid medium at an inoculum size of 1% (v/m), and incubated at a constant temperature of 35 ℃ for 3 days with a protease activity of 1733.16U/g.
4. Use of bacillus cereus according to claim 2, characterized in that: bacillus cereusBacillus cereusWhen the SAU-Q1 is inoculated to the Daqu liquor, compared with a blank control group of the non-inoculated strain SAU-F4, the protease activity in the Daqu liquor of the test group is improved by 10%, and the Daqu liquor of the test group has stronger liquor fragrance.
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