Brevibacillus parabrevis and application thereof in tissue isolation

Abstract

The invention relates to brevibacillus parabrevis and application thereof in tissue isolation, and belongs to the technical field of biology. The brevibacillus parabrevis JN-6 disclosed by the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC NO.29951, and the preservation date is March 7, 2024. The brevibacillus parabrevis JN-6 disclosed by the invention has the advantages that the preservation number is CGMCC NO.29951; the brevibacillus parabrevis provided by the invention has collagen degradation capability, and the produced collagenase can be used for adipose tissue separation and is further cultured into adipose-derived stem cells. When the produced collagenase is used for digestion and separation, the number of obtained cells can be 6.17 * 10 < 6 > / mL, the adipose-derived stem cells can be effectively differentiated, and the method has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a strain of Bacillus parabrevis and application thereof in tissue separation. Background Art

[0002] Collagen is the most abundant protein in animals and a major structural protein in the extracellular matrix. It plays a supporting and connecting role in animal tissues and is widely distributed in the tendons, bones, ligaments, skin, blood vessels and other connective tissues of mammals, as well as the skin, scales, bones and other tissues of aquatic animals. These collagens mainly exist in the form of insoluble fibrous proteins, making collagen an insurmountable obstacle in tissue dissociation. The full and reasonable use of collagen degrading enzymes to ensure the complete separation of tissues and to protect the integrity of cells to the greatest extent is of great significance in tissue dissociation.

[0003] Enzyme digestion is widely used in tissue separation. Its basic principle is to use enzymes to destroy collagen fibers and elastic fibers between tissues, hydrolyze the proteins and mucopolysaccharides in the tight connection structure of tissue cells, and disperse the solid tissue into single cells. Collagenase is one of the commonly used enzymes for separating cells and tissues, and is mainly used to hydrolyze the collagen component in connective tissue.

[0004] At present, collagenase used for tissue separation mainly comes from microorganisms. People have obtained collagenase from microorganisms such as Bacillus subtilis, Bacillus cereus, Actinomycetes, and Pseudoalteromonas. However, some strains require special induction conditions when producing enzymes, and there is a lack of strains that can secrete efficient collagenase and then degrade collagen for tissue separation. Therefore, it is particularly important to screen a strain with efficient collagenase activity for tissue separation. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a Bacillus parabrevis and its application. The strain belongs to a new species of the genus Bacillus parabrevis, can secrete collagenase, and can be used for separation of adipose tissue and acquisition of stem cells.

[0006] The first object of the present invention is to provide a Bacillus parabrevis, characterized in that the taxonomic name of the Bacillus parabrevis is Brevibacillus parabrevis JN-6, which is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, a deposit number of CGMCC NO.29951, and a deposit date of March 7, 2024.

[0007] In one embodiment of the present invention, the Bacillus parabrevis is isolated from a soil sample mixed with waste such as fish skin and fish scales in a fishery.

[0008] In one embodiment of the present invention, the 16S rDNA gene sequence of Bacillus parabrevis is shown as SEQ ID NO.1.

[0009] The second object of the present invention is to provide a microbial agent, which contains the living cells of Brevibacillus parabrevis or its fermentation liquid, or contains freeze-dried powder or inactivated bacterial cells of Brevibacillus parabrevis.

[0010] In one embodiment of the present invention, the amount of Bacillus parabrevis added to the microbial agent is not less than 6.1×10 7 CFU / mL.

[0011] The third object of the present invention is to provide a use of the Bacillus parabrevis or the microbial agent in the preparation of a drug for targeted reduction of adipose tissue amount, reduction and / or elimination of lipoma.

[0012] The fourth object of the present invention is to provide a use of the Bacillus parabrevis or the microbial agent in the preparation of collagenase or chemicals containing collagenase.

[0013] In one embodiment of the present invention, the preparation method of collagenase specifically comprises the following steps: inoculating Bacillus parabrevis or microbial agent into a fermentation medium for fermentation culture, and obtaining a supernatant, i.e., collagenase solution, by centrifugation, and measuring the relative enzyme activity to be 9.4 U / mL.

[0014] In one embodiment of the present invention, the fermentation medium comprises: sucrose 1.5%-2.5%, yeast powder 0.1%-0.2%, peptone 0.5%-1.5%, Na 2 HPO 4 ·2H 2 O 0.05%-0.1%, K 2 HPO 4 ·3H 2 O 0.2%-0.3%, CaCl 2 0.001%-0.005%, pH 7.0-7.2.

[0015] In one embodiment of the present invention, the fermentation culture conditions are: the shaking speed is 200rpm-220rpm, the culture temperature is 36°C-40°C, and the culture time is 16h-24h.

[0016] In one embodiment of the present invention, the centrifugal speed is 7200r / min-8000r / min, and the time is 5min-15min.

[0017] The fifth object of the present invention is to provide a use of the Bacillus parabrevis or the microbial agent in the preparation of daily chemical products, cosmetics, skin care products, medical supplies, biomaterials, hair care products or in the separation of adipose tissue, cell separation, and acquisition of stem cells.

[0018] In one embodiment of the present invention, the cell separation is separation of adipose stem cells by digestion.

[0019] In one embodiment of the present invention, the method for digesting and isolating adipose stem cells specifically comprises the following steps: adding collagenase solution to the sheared fat particles in a digestive tube according to a fat volume to collagenase solution volume ratio of 1: (1-3), then adding lactated Ringer buffer, digesting in a metal bath and centrifuging; fully resuspending the precipitate in a washing solution (PBS: P / S ratio is 100: 1) and filtering through a cell filter and centrifuging, and discarding the supernatant; resuspending the precipitate in a low-glucose complete medium, and counting with a hemocytometer to obtain a stem cell number of 6.17×10 6 / mL.

[0020] In one embodiment of the present invention, the temperature of the metal bath digestion is 36°C-38°C, the rotation speed is 200rpm-400rpm, and the time is 50min-1h.

[0021] In one embodiment of the present invention, the centrifugal force of the centrifugation is 790 rcf-820 rcf, and the time is 10 min-15 min.

[0022] The sixth object of the present invention is to provide a collagen hydrolyzing agent, characterized in that the collagen hydrolyzing agent contains the Bacillus parabrevis or the microbial agent.

[0023] The seventh object of the present invention is to provide a method for hydrolyzing collagen, comprising the following steps: adding the Bacillus parabrevis, the microbial agent or the collagen hydrolyzing agent to a hydrolysis system containing collagen for reaction.

[0024] In one embodiment of the present invention, the amount of Bacillus parabrevicaulis added is not less than 6.1×10 7 CFU / mL.

[0025] The eighth object of the present invention is to provide an application of the Bacillus parabrevis, the microbial agent or the collagen hydrolyzing agent for hydrolyzing protein in the fields of biomedicine, food processing, leather industry, drug delivery, environmental protection, cosmetics, and medical treatment.

[0026] The technical solution of the present invention has the following advantages over the prior art:

[0027] The Bacillus parabrevis of the present invention has the ability to degrade collagen, and the collagenase produced can be used to separate adipose tissue and further cultured into adipose stem cells. The number of cells obtained by digestion and separation using the produced collagenase is 6.17×10 6 / mL, which can effectively differentiate adipose stem cells and has good application prospects.

[0028] Deposit of biological materials:

[0029] A strain of Bacillus parabrevis, taxonomically named Brevibacillus parabrevis JN-6, has been deposited in the General Microbiology Center of China Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC NO.29951, and the deposit date is March 7, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0031] Figure 1 is the standard curve of glycine of the present invention;

[0032] Figure 2 It is the gelatin hydrolysis circle of the primary screening strain in Example 1 of the present invention;

[0033] Figure 3 The gelatin flat plate hydrolysis circle in Example 1 of the present invention;

[0034] Figure 4 This is the polyacrylamide gel electrophoresis diagram of strain JN-6 in Example 1 of the present invention;

[0035] Figure 5 The colony morphology of strain JN-6 in Example 3 of the present invention;

[0036] Figure 6 The microscopic examination results of strain JN-6 in Example 3 of the present invention are shown;

[0037] Figure 7 is the phylogenetic tree of strain JN-6 in Example 3 of the present invention;

[0038] Figure 8 The results of cowhide hydrolysis of the supernatant of strain JN-6 in Example 4 of the present invention;

[0039] Fig. 9 is the degradation rate of cowhide of the supernatant of strain JN-6 in Example 4 of the present invention;

[0040] Fig.10 The adipose stem cells separated and cultured from adipose tissue in Example 4 of the present invention;

[0041] Fig.11 The effect of reaction temperature on collagenase produced by strain JN-6 in Example 6 of the present invention;

[0042] Fig.12 The effect of the reaction pH on the collagenase produced by the strain JN-6 in Example 6 of the present invention;

[0043] Fig.13 This is the effect of metal ions on collagenase produced by strain JN-6 in Example 6 of the present invention. DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. It should be understood that the specific embodiments are only used to explain the present invention, but the embodiments are not intended to limit the present invention.

[0045] In the present invention, unless otherwise explained, technical and scientific terms used in the present invention have the same meanings as commonly understood by those skilled in the art to which the present invention belongs.

[0046] In the present invention, unless otherwise stated, the experimental methods used in the embodiments of the present invention are conventional methods unless otherwise stated, and the materials, reagents, etc. used are all commercially available unless otherwise stated.

[0047] In the present invention, unless otherwise specified, the culture medium involved in the embodiments of the present invention is as follows:

[0048] Enrichment medium: gelatin 0.1%, NaCl 0.5%, peptone 0.5%, pH 7.2-7.5, sterile.

[0049] Gelatin screening medium: Gelatin 2%, NaCl 0.01%, peptone 0.5%, KH 2 PO 4 0.05%,MgSO 4 7H 2O 0.02%, agar 1.5%, pH 7.2-7.5;

[0050] Seed culture medium: beef extract 0.5%, NaCl 0.5%, peptone 1%, pH 7.2-7.4;

[0051] Fermentation medium: sucrose 2%, yeast powder 0.2%, peptone 0.5%, Na 2 HPO 4 ·2H 2 O 0.05%, K 2 HPO 4 ·3H 2 O 0.3%, CaCl 2 0.001%, pH 7.0-7.2;

[0052] Low-glucose complete medium: 44.5 mL DMEM low-glucose medium + 5 mL FBS + 0.5 mL P / S.

[0053] In the present invention, unless otherwise specified, the glycine standard curve ( Figure 1 ) includes the following steps: thoroughly mix 1 mL of glycine solution of different concentrations (0.056 μmol / mL, 0.122 μmol / mL, 0.199 μmol / mL, 0.287 μmol / mL, 0.389 μmol / mL, 0.47 μmol / mL) with 1 mL of acetic acid buffer (2 mol / L), add 1 mL of ninhydrin colorimetric solution, mix thoroughly, cover the mouth of the test tube, boil in 80°C water bath for 20 min, cool completely in an ice bath, add 3 mL of 60% ethanol to dilute, mix thoroughly, and compare colors at 570 nm.

[0054] In the present invention, unless otherwise specified, the collagenase activity U involved in the embodiments of the present invention is defined as: at 37°C and pH 7.5, 1 mL of enzyme solution hydrolyzes collagen within 5 hours to produce an amount equivalent to 1 μmol of glycine, which is 1 enzyme activity unit.

[0055] Example 1 Screening and Isolation of Bacteria

[0056] (1) Sample collection: Soil samples were collected from the fish skin sediments in the fishing grounds, placed in an ice box and brought back to the laboratory.

[0057] (2) Enrichment culture: Take 1 g of sample, soak it in 10 mL of sterile saline, shake it well, take 1 mL of the suspension and add it to the enrichment medium, and culture it in a shaking incubator at 37°C and 200 rpm for 2 days to obtain an enriched bacterial solution.

[0058] (3) Screening and isolation of strains: Use physiological saline to perform gradient dilutions on the enriched bacterial solution. Take 100 μL of 10 -6 -10 -8 The dilution was spread on a gelatin screening plate and cultured at 37°C for 48 h. The number of single colonies on the plate was observed. If the number was > 300, the dilution was continued to be graded to 10. -11 Then, take 100 μL of 10 -9 -10 -11 The diluted solution was spread on the plate and incubated at 37°C for 48 hours. At this time, a single colony was generated on the gelatin screening plate. The strain that formed a transparent hydrolysis circle on the plate was selected and spotted on two identical gelatin screening medium in parallel. After incubation at 37°C for 48 hours, the size of the hydrolysis circle was observed and the strain was selected for the next screening. Figure 2 As shown;

[0059] In the clean bench, use a pipette to pick up the colonies with obvious hydrolysis circles on the plate, inoculate them into the seed culture medium, and culture them in a shaking incubator at 37°C and 220 rpm for 12 h. After the culture is completed, take 2% of the bacterial solution of the seed culture medium and transfer it to the rescreened fermentation medium, and culture it in a shaking incubator at 37°C and 220 rpm for 24 h.

[0060] The supernatant of the bacterial culture fluid obtained in step (3) was centrifuged and the collagenase activity was measured to screen out a strain with good collagenase activity, which was named JN-6.

[0061] (4) Gelatin hydrolysis: A single colony of JN-6 was selected and inoculated onto a gelatin screening medium plate. After culturing at 37°C for 2 days, the plate was coated with 35% trichloroacetic acid. The hydrolysis zone of the gelatin plate was as follows: Figure 3 As shown. Figure 3 It can be seen that JN-6 forms a hydrolysis zone (1.1 cm) and is able to hydrolyze gelatin.

[0062] (5) SDS-PAGE: The supernatant obtained in step (3) was subjected to gel electrophoresis. The results were as follows: Figure 4 As shown. Figure 4 It can be seen that the supernatant has a single protein band.

[0063] Example 2 Determination of collagenase activity produced by strains

[0064] (1) Preparation of strain fermentation supernatant: streak frozen JN-6 on a gelatin screening medium plate, culture at 37°C for 24 h, pick a single colony and inoculate it into a seed medium after recovery of activity, culture it in a shaking incubator at 37°C and 220 rpm for 12 h, inoculate it into a rescreening fermentation medium at an inoculum size of 2% (v / v), and culture it in a shaking incubator at 37°C and 220 rpm for 24 h; take the rescreening fermentation culture medium into a centrifuge tube, centrifuge it at 8000 r / min for 10 min, take the fermentation supernatant, and measure the collagenase activity U.

[0065] (2) Definition and determination method of collagenase activity

[0066] Experimental group: Type I collagen was used as substrate (2 mg / mL); the reaction system was: 250 μL substrate solution, 200 μL Tris-HCl (0.1 mol / L, pH 7.5, containing 50 mmol / L CaCl 2 ), 250 μL of the fermentation supernatant of the strain (2 mg / mL), react at 37°C for 5 h, and add an equal volume of 10% trichloroacetic acid to terminate the reaction;

[0067] Control group: add an equal volume (i.e. 700 μL) of 10% trichloroacetic acid stop solution as control;

[0068] Add 500 μL of the above sample, 500 μL of acetic acid buffer, and 500 μL of ninhydrin colorimetric solution to the test tube in sequence, heat in an 80°C water bath for 20 min, and after the ninhydrin colorimetric reaction, ice bath until completely cooled, add 1500 μL of 60% ethanol, centrifuge at 12000 rpm for 1 min, take 200 μL to an ELISA plate, and compare the color at 570 nm. The absorbance value obtained is used to calculate the collagenase activity U according to the glycine standard curve. The collagenase activity of the screened strain JN-6 is 9.4 U / mL.

[0069] Example 3 Identification of strain JN-6

[0070] (1) Morphological characteristics: The colony morphology of strain JN-6 is as follows Figure 5 As shown in the figure, after culturing strain JN-6 on gelatin medium plate for 2 days, uniform colonies appeared, and it can be observed that the colonies were milky white, smooth, and had neat edges. Figure 6 As shown. Figure 6 It can be seen that strain JN-6 is slender rod-shaped and Gram-negative.

[0071] (2) Physiological and biochemical characteristics: This bacterium is a Gram-negative, aerobic bacterium that has the ability to hydrolyze casein and gelatin, has no amylase activity, and is catalase positive.

[0072] (3) Sequence amplification and identification of 16S rDNA of strains: After a single colony of the selected strain was selected and added to LB (10 mL) for culture, the genome was extracted using a genome extraction kit, amplified and sequenced using universal primers. The amplified 16S rDNA sequence was subjected to homology analysis with the 16S rDNA sequences of all standard strains in the database. The results showed that the sequences with higher homology all belonged to the genus Parabrevibacillus. Strains closely related to the genus Brevibacillus were selected for phylogenetic analysis with the strains isolated in Example 1, and a phylogenetic tree was constructed using MEGA-X, as shown in FIG. Figure 7 shown.

[0073] The 16S rDNA sequence (SEQ ID NO.1) is as follows:

[0074]

[0075] According to the results of 16S rDNA sequence comparison and combined with the biological characteristics of the strain, the strain was identified as Brevibacillus parabrevis. Brevibacillus parabrevis JN-6 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with a deposit number of CGMCC NO.29951 and a deposit date of March 7, 2024.

[0076] Example 4 Evaluation of the titer of collagenase produced by strains

[0077] Cowhide digestion experiment: The selected strain JN-6 was selected and inoculated into the seed culture medium, and grown at 37°C and 220rpm for 12 hours to obtain the seed solution; the seed solution was inoculated into the rescreened fermentation culture medium at a 2% (v / v) inoculation amount, and cultured at 37°C and 220rpm for 24 hours to obtain the fermentation liquid; the fermentation liquid was centrifuged at 8000r / min and 15min to obtain the supernatant; the obtained supernatant was subjected to the cowhide digestion experiment, and was specifically divided into an experimental group and a control group:

[0078] Experimental group: 25 mL of supernatant was added to fresh cowhide and digested at 37°C and 220 rpm for 1-6 days to obtain cowhide digestion solution;

[0079] Control group: 25 mL of supernatant was boiled in water for 10 min, then added to fresh cowhide and digested for 1-6 days under the same conditions to obtain cowhide digestion solution;

[0080] The results of cowhide hydrolysis are as follows Figure 8 As shown. Figure 8 It can be seen that after 4 days of digestion, the cowhide in the experimental group was partially hydrolyzed and became thinner and softer overall compared with the control group.

[0081] The weight of cowhide after enzyme digestion is Fig. 9 As shown in Figure 2, with the extension of enzyme digestion time, the weight of cowhide decreased significantly.

[0082] (2) Isolation and digestion of adipose stem cells: After mice were killed by dislocation, the inguinal fat of the mice was isolated and removed using surgical instruments and placed in a washing solution for immersion and rinsing. Ten mice were grouped into one group and washed three times respectively. Sterile forceps were used to place the isolated fat in a dish lid and clean ophthalmic surgical scissors were used to cut the fat tissue into a paste of about 1 mm. 3The fat particles were placed in 15 mL digestive tubes, and the supernatant obtained in step (1) was added to each tube at a ratio of 1:1 by fat volume. The tubes were sealed with sealing film and mixed for 2-3 times. The tightly sealed digestive tubes were placed in a metal bath and digested at 37°C and 300 rpm for 40 min-2 h. The tubes were centrifuged at 800 rcf for 10 min. After centrifugation, the solution in the tubes was divided into three layers, the top layer was white fat, the middle layer was a clear solution, and the bottom layer was a red and white precipitate (SVF) and unlysed tissue blocks. The upper two layers of material were sucked off with a Pasteur pipette, and the bottom precipitate was retained. The bottom precipitate was washed with a washing solution (50 mL PBS + 0.5 mL After being fully resuspended, the cells were filtered through a 40 μm cell strainer and transferred to a 15 mL centrifuge tube. The cells were centrifuged at 800 rcf for 10 min and the supernatant was discarded. After washing, the cells were resuspended in 1 mL of low-glucose complete medium, mixed and 10 μL was transferred to a hemocytometer for counting. The mouse adipose stem cells were isolated and digested using the collagenase produced by the screened strain.

[0083] According to the above method, commercially available collagenase (the concentration of commercially available collagenase is 2 mg / mL enzyme solution, and the enzyme activity of commercially available collagenase is greater than 125 U / mg) was used as a comparison. The specific results are shown in Table 1:

[0084] Table 1

[0085] name Digestion time (min) Digestion status Cell number Blank control 120min Undigested <![CDATA[3.38×10 3 / mL]]> Commercially available collagenase 120min Digested to pulp <![CDATA[2.23×10 7 / mL]]> Screening collagenase 120min Partial digestion <![CDATA[6.17×10 6 / mL]]>

[0086] As can be seen from Table 1, since excessive digestion can cause damage to cells, the digestion time was limited to 120 min. The number of cells obtained during the digestion of adipose stem cells by the screened strain producing collagenase was approximately 6.17 × 10 6 / mL; although it was lower than the 2.23×10 7 / mL was different, but the experimental effect was still better than that of the blank control group. After a period of culture, the obtained cells grew into the morphology of adipose stem cells. The results are as follows Fig.10 shown.

[0087] Example 5 Collagenase-related substrate spectrum produced by strains

[0088] The enzyme produced by strain JN-6 was used to conduct relevant degradation experiments to preliminarily explore the substrate spectrum of the enzyme, which specifically includes the following steps:

[0089] (1) Type I collagen: Type I collagen was used as a substrate, and the collagenase activity was determined according to the collagenase activity determination method in Example 2;

[0090] (2) PZ peptide: PZ peptide was used as a substrate and reacted with the supernatant (prepared according to the method of step (1) of Example 2), and the absorbance at 320 nm was measured;

[0091] (3) Gelatin: According to the gelatin hydrolysis method (4) in Example 1: a single colony of JN-6 was inoculated on a gelatin screening medium plate, cultured at 37°C for 2 days, and then coated with 35% trichloroacetic acid to observe whether a hydrolysis zone was formed;

[0092] (4) Sheep skin, fish skin, wool: Add the substrate to the supernatant (prepared according to the method of step (1) of Example 2), incubate at 37° C. for 5 days, then take out and observe whether the wool or other substrate is degraded;

[0093] (5) Casein, nitrilase, papain, bovine serum albumin, lipase: Casein, nitrilase, papain, bovine serum albumin, and lipase were incubated with the supernatant (prepared according to the method of step (1) of Example 2, and the supernatant inactivated at 100° C. for 10 min was used as a control) at a ratio of 1:1 for 5 h, and then subjected to gel electrophoresis. The proteins were separated according to their molecular weight. The original bands of the degraded proteins would become lighter or disappear, and smaller molecular weight bands would appear on the gel, indicating that they had been degraded. The bands were observed to see if they were degraded.

[0094] The substrate spectrum is shown in Table 2:

[0095] Table 2

[0096] Protein substrate Molecular weight (KDa) Can it be degraded? Collagen Type I 100-130 + PZ-peptide / + gelatin 30-40 + sheepskin / + Casein 20-25 + Fish skin / + Nitrilase 35 - Papain 23 - wool / - BSA 66 + Lipase 30-70 -

[0097] As can be seen from Table 2, the supernatant of JN-6 can degrade type I collagen, PZ-peptide, gelatin, sheepskin, casein, fish skin and bovine serum albumin, but cannot degrade nitrilase, papain, wool and lipase.

[0098] Example 6 Enzymatic properties of collagenase produced by strain JN-6

[0099] Preparation of enzyme solution: streak the frozen strain JN-6 on a gelatin screening medium plate, culture it at 37°C for 24 hours, pick a single colony and inoculate it into a seed medium after the activity is restored, culture it in a shaking incubator at 37°C and 220rpm for 12 hours, inoculate it into a re-screening fermentation medium at an inoculum size of 2% (v / v), culture it in a shaking incubator at 37°C and 220rpm for 24 hours to obtain a fermentation culture medium; take the fermentation culture medium into a centrifuge tube, centrifuge it at 8000r / min for 10 minutes, and then take the supernatant to obtain an enzyme solution.

[0100] Enzyme activity detection method: Type I collagen was used as substrate (2 mg / mL); the reaction system was: 250 μL of type I collagen substrate solution (2 mg / mL), 200 μL Tris-HCl (0.1 mol / L, pH 7.5, containing 50 mmol / L CaCl2 ), 250 μL of strain supernatant, react at 37°C for 5 h, and add an equal volume of 10% trichloroacetic acid to terminate the reaction.

[0101] The enzymatic performance analysis of the enzyme solution is as follows:

[0102] (1) The effect of reaction temperature on the collagenase produced by strain JN-6: The enzyme solution was reacted with 4 mg / mL collagen substrate in a shaker at different temperatures (the temperatures in the enzyme activity detection method were adjusted to 25°C, 30°C, 37°C, 40°C, and 45°C, respectively) for 5 h; the enzyme activity measured at the optimal reaction temperature was defined as 100% relative enzyme activity. The results are shown in Table 1. Fig.11 As shown. Fig.11 It can be seen that the optimum temperature of collagenase produced by strain JN-6 is 37°C.

[0103] (2) The effect of reaction pH on the collagenase produced by strain JN-6: The enzyme solution was reacted with 4 mg / mL collagen substrate under different pH conditions (the pH in the enzyme activity detection method was adjusted to 3, 4, 5, 6, 7, 7.5, 8, 9, and 10, respectively) for 5 h. The enzyme activity measured at the optimal reaction pH was defined as 100% relative enzyme activity. The results are shown in Table 1. Fig.12 As shown. Fig.12 It can be seen that the optimum pH of collagenase produced by strain JN-6 is 7.

[0104] (3) The effect of metal ions on the collagenase produced by strain JN-6: The enzyme solution was mixed with 4 mg / mL collagen substrate in 5 mmol / L different metal ion solutions (metal ions Mn 2+ 、Na + , Li 2+ 、Zn 2+ Mg 2+ , Cu 2+ , Ca 2+ ) for 5 h; the enzyme activity measured without metal ions was defined as 100% relative enzyme activity. Fig.13 As shown. Fig.13 It can be seen that the collagenase produced by strain JN-6 is 2+ In the presence of , the relative enzyme activity can reach 170%.

[0105] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A Brevibacillus parasporus, characterized in that The taxonomic name of the Bacillus parabrevis is Brevibacillus parabrevis JN-6, which is deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit number being CGMCC NO.29951, and the deposit date being March 7, 2024.

2. A microbial agent, characterized in that: The microbial agent contains the living cells of Brevibacillus parasporus or its fermentation liquid as claimed in claim 1, or contains freeze-dried powder or inactivated bacterial cells of Brevibacillus parasporus.

3. The microbial agent according to claim 2, characterized in that: The amount of Bacillus parabrevis added in the microbial agent is not less than 6.1×10 7 CFU / mL.

4. Use of the Bacillus parabrevis according to claim 1 or the microbial agent according to any one of claims 2 to 3 in the preparation of a drug for targeted reduction of adipose tissue amount, reduction and / or elimination of lipoma.

5. Use of the Bacillus parabrevis according to claim 1 or the microbial agent according to any one of claims 2 to 3 in the preparation of collagenase or chemicals containing collagenase.

6. Use of the Bacillus parabrevis according to claim 1 or the microbial agent according to any one of claims 2 to 3 in the preparation of daily chemical products, cosmetics, skin care products, medical supplies, biomaterials, hair care products, or in the separation of adipose tissue, cell separation, and acquisition of stem cells.

7. A collagen hydrolyzing agent, characterized in that The collagen hydrolyzing agent contains the Bacillus parabrevis according to claim 1 or the microbial agent according to any one of claims 2-3.

8. A method for hydrolyzing collagen, characterized in that: The method comprises the following steps: adding the bacillus parabrevis according to claim 1, the microbial agent according to any one of claims 2 to 3, or the collagen hydrolyzing agent according to claim 7 to a hydrolysis system containing collagen for reaction.

9. The method for hydrolyzing collagen according to claim 8, characterized in that: The amount of Bacillus parabrevis added is not less than 6.1×10 7 CFU / mL.

10. Use of the Bacillus parabrevis according to claim 1, the microbial agent according to any one of claims 2-3, or the collagen hydrolyzing agent according to claim 7 in hydrolyzing proteins in the fields of biomedicine, food processing, leather industry, drug delivery, environmental protection, cosmetics, and medical treatment.

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