CN117925477B - Bacillus subtilis SP-8-5 and application thereof - Google Patents
Bacillus subtilis SP-8-5 and application thereof Download PDFInfo
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Abstract
The invention discloses a bacillus subtilis SP-8-5, which is preserved in China Center for Type Culture Collection (CCTCC) in 2023, 3 and 06 days, wherein the preservation number is CCTCC NO: m2023254. The invention has the advantages that: the bacillus subtilis SP-8-5 high-yield fermented soybean plasmin is a potential excellent strain for industrially producing the fermented soybean plasmin, provides a new direction for thrombus treatment, has a good antibacterial effect on bacteria and fungi, and has potential application and development prospects.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to bacillus subtilis for efficiently producing plasmin from fermented soybeans and application thereof.
Background
Thrombosis is one of ten diseases which harm human beings, and brings great trouble to the life of people. A serine proteinase which is produced by bacillus subtilis in the fermentation process of fermented soya beans is called fermented soya bean plasmin, has obvious thrombolysis effect and can be used for preventing and treating thrombolytic diseases. Compared with other plasmin, the fermented soybean plasmin has many advantages such as strong thrombolytic ability, high activity, good safety, no internal hemorrhage, durable efficacy, long half-life in vivo, small molecular weight and direct absorption through digestive tract.
Plasmin produced by microorganisms has the characteristics of short period, low cost and the like, and has great application potential. Therefore, the strain for efficiently producing the fermented soybean plasmin is screened, and the thrombolytic drug or the functional health-care food is developed by utilizing microorganisms, so that the strain has important significance for the life of human beings.
Disclosure of Invention
The invention aims to solve the technical problem of providing bacillus subtilis for efficiently producing plasmin from fermented soya beans.
The invention provides a bacillus subtilis SP-8-5, wherein the bacillus subtilis SP-8-5 (Bacillus subtilis SP-8-5) is preserved in China Center for Type Culture Collection (CCTCC) in 3-month 06 of 2023, and the preservation number is CCTCC NO: m2023254.
The invention provides a microbial inoculum comprising bacillus subtilis SP-8-5.
The invention provides an application of bacillus subtilis SP-8-5 or a microbial inoculum or a fermentation product in preparing thrombolytic or bacteriostatic medicines.
The invention has the beneficial effects that:
The bacillus subtilis SP-8-5 screened from the traditional fermented black beans in Yunnan is a potential excellent strain for industrially producing the black bean plasmin, provides a new direction for thrombus treatment, has a good antibacterial effect on bacteria and fungi, and has potential application and development prospects.
Drawings
FIG. 1 is a colony morphology of Bacillus subtilis SP-8-5;
FIG. 2 is a phylogenetic tree of Bacillus subtilis SP-8-5.
Detailed Description
The following describes the embodiments of the present invention further with reference to the drawings. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The invention provides a bacillus subtilis SP-8-5, which is separated from traditional fermented black beans in Yunnan, wherein the bacillus subtilis SP-8-5 (Bacillus subtilis SP-8-5) is preserved in China Center for Type Culture Collection (CCTCC) on 3-month 06 of 2023, and the preservation number is CCTCC NO: m2023254.
The invention provides a microbial inoculum comprising bacillus subtilis SP-8-5.
The invention provides an application of bacillus subtilis SP-8-5 or a microbial inoculum or a fermentation product in preparing thrombolytic or bacteriostatic medicines.
The following examples are provided to illustrate a strain of Bacillus subtilis SP-8-5 and its use in detail, but they should not be construed as limiting the scope of the invention.
Example 1
Separation, purification, identification and preservation of bacillus subtilis SP-8-5
1. Isolation and purification of strains
The special geographical environment and various humane advantages of Yunnan province, the special traditional fermented black beans resources of Yunnan are created, and the traditional fermented black beans represented by Ethernet black beans, watery black beans, fenugreek black beans, hani black beans, spiced black beans, black bean strips and the like are formed. The unique traditional fermented soybeans are stored with microorganism resources such as lactobacillus, bacillus subtilis and the like.
The bacillus subtilis SP-8-5 is isolated from Yunnan traditional fermented soybeans, and the method comprises the following steps: sterilizing the test tube containing 5mLNB liquid culture medium with 121 deg.C high pressure steam for 20min, quickly inoculating 3-5 fermented soybean samples into the test tube, immediately plugging the rubber plug, and culturing the test tube at 37 deg.C for 24h at 150r/min. Absorbing 100 mu L of the bacterial liquid which is cultured for 24 hours and uniformly mixed in a sterile operation table, adding the bacterial liquid into 900 mu L of sterile physiological saline which is packaged for gradient dilution, then taking 100 mu L of 10 5,106,107 diluent which is uniformly coated on NB solid culture medium, making three times of parallelism on each gradient, placing the coated solid culture medium in a 37 ℃ water-proof constant temperature incubator for culturing for 24 hours, after single bacterial colonies grow out, picking the single bacterial colonies in the sterile toothpick by using the sterilized toothpick, streaking the single bacterial colonies on the NB solid culture medium, placing the sterile toothpick in the 37 ℃ water-proof constant temperature incubator for culturing for 24 hours, and repeatedly picking single bacterial colony streaking for 2-3 times by using the toothpick until the single bacterial colonies grow out and are purified. Single colonies are selected according to the morphological characteristics of the colonies, such as morphology, color, size, whether the colonies are raised, transparency, whether edges are regular, and the like. Single colonies were inoculated into 5mL of sterilized NB liquid medium, cultured at 37℃for 48h at 150r/min, and then stored with 50% glycerol to-80℃for further use.
The NB liquid medium and NB solid medium were purchased from Canon Cryptographic microorganism Co., ltd.
2. Morphological identification of strains
Gram staining and microscopic examination prove that the strain is gram positive bacteria, spores are positioned in the center of bacteria, the surface of a bacterial colony is rough and opaque, milky white, and wrinkled walls are often formed when the bacterial colony grows on a solid culture medium, and the bacterial colony is opaque and glossy. The colony morphology of bacillus subtilis SP-8-5 is shown in FIG. 1.
3. Molecular biology identification of strains
The strain was inoculated in a liquid medium of NB, placed in a constant temperature incubator at 37℃for activation culture, and then genomic DNA of the strain was extracted (DNA extraction was performed using the kit QIAamp genomic DNA AND RNA KITS), and the 16S rRNA gene of the isolated strain was subjected to PCR amplification using bacterial 16S rRNA gene universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGACTTAACCCCAATCGC-3'). The reaction system: 2X TAQPCR MASTER Mix 10. Mu.L, 0.5. Mu. L, DNA each of the upstream and downstream primers 1. Mu. L, ddH2O 8. Mu.L; the reaction procedure: 95 ℃ for 5min;94℃1min,55-58℃1min,72℃90s,30 cycles; and at 72℃for 10min. The PCR product of the 16S rRNA of the strain isolated and cultured in the sample is sent to a company limited by biological engineering (Shanghai) to carry out gene sequence determination, then the detected 16S rRNA gene sequence is subjected to Blast comparison in GenBank of NCBI database, the bacterial standard strain with the highest similarity with the isolated strain is selected, the strain type is determined, and a phylogenetic tree is constructed by using MEGA 7.0 software to construct the phylogenetic tree. The bacillus subtilis SP-8-5 phylogenetic tree is shown in figure 2.
Identification result: through morphological identification and molecular biological identification, the 16S rRNA gene sequence is compared, a phylogenetic tree is constructed, and SP-8-5 is identified as bacillus subtilis.
4. Preservation of strains
Bacillus subtilis SP-8-5 (Bacillus subtilis SP-8-5) is preserved in China Center for Type Culture Collection (CCTCC) for type number of collection (CCTCC NO: m2023254; the preservation address is: eight paths of Lopa nationality mountain in Wuhan city of Hubei province; contact phone: 027-68754052.
5. Experiment of liquefaction of gelatin
The SP-8-5 isolated and preserved from fermented soybeans and other isolated strains were rapidly thawed, 20. Mu.L of each was added to NB medium containing 17% gelatin, incubated at 35℃for 48 hours, and then placed in a refrigerator at 4℃for 20 minutes to see if gelatin in the test tube coagulated. If gelatin is liquefied, the positive strain is determined. NB medium composition of 17% gelatin: 170g of gelatin and 1000mL of water are added into 18g of NB medium, and the mixture is uniformly mixed, and sterilized at 121 ℃ for 15min.
The experiment shows that the bacillus subtilis SP-8-5 is a positive strain, which shows that the strain is metabolically active and can produce protease. In addition, there are 11 gelatin strains tested positive.
Example 2
(1) Primary screening of strain producing fermented soya bean plasmin
20. Mu.L of SP-8-5 and 11 other gelatin positive strains were inoculated into 5mL of sterilized fresh NB medium, incubated at 37℃in a shaker at 150r/min for 24h, followed by shaking and mixing, 20. Mu.L of the strain was inoculated into 5mL of fibrin-induced medium (purchased from Guangdong Kai microorganism technology Co., ltd.) at 150r/min for 48h at 37℃and 20. Mu.L of the strain was inoculated into an Erlenmeyer flask containing 20mL of NB medium at 150r/min for 48h at 37 ℃. Then 1mL of culture solution is taken, 5000r/min is centrifuged for 30min, 10 mu L of culture supernatant is taken and added into a 0.3% fibrin agarose plate hole (5-6 holes are evenly punched on the plate), the plate is placed in a constant temperature box for culturing for 48h at 37 ℃, the diameters of fibrinolytic circles are respectively measured for 24h and 48h, and the diameters of the fibrinolytic circles approximately represent the enzyme activity of the fermented soybean plasmin.
The 0.3% fibrin agarose plate: 3.0g of fibrin was dissolved in 500mL of sterilized phosphate buffer (concentration: 50mmol/L, pH 7.4) and stirred overnight; 10g of agarose was then dissolved in 500mL of sterile phosphate buffer (50 mmol/L, pH 7.4) and mixed well to prepare plates, 25mL each, after solidification and perforation (pore size 6.0 mm) after cooling the agarose solution to 70 ℃.
Experimental results: 12 positive strains of gelatin test separated from fermented soya beans only have 8 strains with proteolytic loops, and the other strains have no proteolytic loops. The results of the measured diameter of the proteolytic loop of 8 strains showing the proteolytic loop are shown in the following table after culturing for 24 hours and 48 hours:
It can be seen that the diameter of the proteolytic loop of SP-8-5 is much larger than that of other strains, indicating that the enzymatic activity of Bacillus subtilis SP-8-5 is optimal.
(2) Determination of plasmin Activity of fermented Soy
The method is characterized in that the method is determined by adopting an ninhydrin method, a substrate is changed into a 0.6% fibrin solution, the OD value of a sample enzyme solution is measured at 570nm, and the OD value is converted into the activity of fermented soya bean plasmin. The specific method comprises the following steps:
Inoculating the 8 strains with the soluble protein circles into NB medium respectively, shake culturing for 24h, centrifuging, taking 200 mu L of supernatant, adding 300 mu L of buffer solution containing 0.6% of fibrin, shake culturing for 2h at 37 ℃ and 150r/min, then adding 500 mu L of 0.3mol/L TCA,12000rpm, centrifuging for 10min, taking 100 mu L of supernatant, adding 900 mu L of citric acid buffer solution, 100 mu L of stannic chloride and 2mL of ninhydrin solution, uniformly mixing, boiling for 20min in a water bath, cooling, fixing the volume to 10mL by using Tris-HCl buffer (purchased from Guangdong CycloKy microorganism technology Co., ltd.), taking 500 mu L of mixed solution with the fixed volume, adding 500 mu L of distilled water, diluting for 2 times, and measuring absorbance at 570 nm. After shaking culture for 2h at 37 ℃ with 500 mu L of Tris-HCl buffer as a blank control and 150r/min, subsequent experiments are carried out for calibration, 300 mu L of Tris-HCl buffer+200 mu L of crude enzyme solution (one control is carried out for each sample) is used as an enzyme solution sample control, 300 mu L of 0.3% fibrin solution+200 mu L of Tris-HCl buffer is used as a substrate control, the absorbance value is measured, and the plasmin activity of the enzyme at 37 ℃ and pH7.5 is calculated (final sample OD value=sample OD value-enzyme solution sample control OD value-substrate control OD value; fermented soybean plasmin activity= (AxC)/T, A represents the leucine content in the sample reaction solution, C represents the dilution of the sample and T represents the reaction time of 120 min).
The required reagent formula is as follows:
0.6% fibrin solution: 30.8g of Na2HPO4 and 2.5g of NaH2PO4 are dissolved in 800mL of distilled water, the pH is adjusted to 7.4, 6.0g of fibrin is added, and the volume is fixed to 1L after the fibrin is dissolved; 4.89g of TCA is weighed by 0.3mol/L of TCA, distilled water is added to fix the volume to 100mL, a brown bottle is protected from light, and the mixture is stored in a refrigerator at 4 ℃.
Citric acid buffer: 21.0g of citric acid+250 mL of H2O+1mol/L of NaOH 200mL, adjusting the pH to 5.0, fixing the volume to 500mL, and preserving in a refrigerator at 4 ℃. Tin chloride solution (ready-to-use): 0.2g of stannic chloride is added with 12.4g of citric acid solution for dissolution, and the brown bottle is protected from light and stored in a refrigerator at 4 ℃.
Ninhydrin solution: 1g of ninhydrin, 25mL of ethylene glycol monomethyl ether and 20mL of citric acid buffer solution are fully stirred and uniformly mixed, the volume is fixed to 50mL, the ninhydrin is easy to decompose in light, a brown bottle is protected from light, and the mixture is stored in a refrigerator at 4 ℃.
The results of the calculated fermented soybean plasmin activities of 8 strains are shown in the following table:
Researches show that the bacillus subtilis SP-8-5 has the highest activity of fermented soybean plasmin reaching 13108U/mL, and then SP-4-1, YN9 and SP-5-2. Wherein the enzyme activity of SP-4-1 reaches 9087U/mL, the enzyme activity of YN9 reaches 8904U/mL, the enzyme activity of SP-5-2 reaches 7010U/mL, and the enzyme activity of the rest strains is lower, so that the enzyme activity of SP-8-5 is far higher than that of the rest strains as can be seen from the results.
Example 3
Bacteriostasis test
The bacteriostasis activity of probiotics is measured by oxford cup method: uniformly pouring about 10mL of NB solid culture medium at the bottom of a flat plate, airing, placing an oxford cup in the middle of the flat plate, adding SP-8-5 with the CFU of 10 8 CFU/mL into the oxford cup, adding a proper amount of pathogenic bacteria into the culture medium when the culture medium corresponding to the pathogenic bacteria is cooled to 40 ℃, enabling the final concentration of the pathogenic bacteria to be 10 6 CFU/mL, respectively obtaining staphylococcus aureus (the culture medium is BHI solid culture medium and purchased from Guangdong Cyclo microorganism technology Co., ltd.), escherichia coli (the culture medium is LB solid culture medium and purchased from Guangdong Cyclo microorganism technology Co., ltd.) and candida albicans (the culture medium is sand culture medium and purchased from Guangdong Cyclo microorganism technology Co., ltd.), culturing at a constant temperature for 24 hours at 37 ℃ after the culture medium is cooled and solidifying completely, and measuring the diameter of a bacteriostasis zone, wherein all experiments are repeated for 3 times. The results of measuring the diameter of the inhibition zone of each strain are shown in the following table:
The antibacterial result shows that the bacillus subtilis SP-8-5 has antibacterial effects on three pathogenic bacteria of staphylococcus aureus, escherichia coli and candida albicans, wherein the antibacterial effect on the escherichia coli is strongest, the diameter of a antibacterial circle is 31mm, the bacteria are staphylococcus aureus, the diameter of the antibacterial circle is 27mm, the antibacterial effect of the SP-8-5 on fungi is obvious, and the diameter of the antibacterial circle is more than 15mm.
The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.
Claims (3)
1. The bacillus subtilis SP-8-5 is characterized in that: the bacillus subtilis SP-8-5 (Bacillus subtilis SP-8-5) is preserved in China Center for Type Culture Collection (CCTCC) in 2023, 3 and 06 days, and the preservation number is CCTCC NO: m2023254.
2. A microbial inoculum comprising the bacillus subtilis SP-8-5 according to claim 1.
3. The use of bacillus subtilis SP-8-5 according to claim 1 or the microbial inoculum according to claim 2 for the preparation of thrombolytic or bacteriostatic drugs, wherein the bacteriostatic pathogenic bacteria is escherichia coli, staphylococcus aureus or candida albicans.
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| CN105238720A (en) * | 2015-10-28 | 2016-01-13 | 华中农业大学 | Fibrinolytic enzyme-producing Bacillus subtilis and fermentation method and application thereof |
| CN116806259A (en) * | 2020-11-04 | 2023-09-26 | 艾力格生物科技有限公司 | Propionibacterium acnes recombinant phage, production method and application thereof |
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|---|---|---|---|---|
| CN105238720A (en) * | 2015-10-28 | 2016-01-13 | 华中农业大学 | Fibrinolytic enzyme-producing Bacillus subtilis and fermentation method and application thereof |
| CN116806259A (en) * | 2020-11-04 | 2023-09-26 | 艾力格生物科技有限公司 | Propionibacterium acnes recombinant phage, production method and application thereof |
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