A bacillus subtilis TN, a live bacteria preparation, a preparation method and application thereof

Abstract

The present application relates to a bacillus subtilis TN, a live bacterial preparation and a preparation method and application thereof, and belongs to the technical field of microorganisms. The bacillus subtilis TN was preserved in the Guangdong Provincial Microbial Culture Collection Center on March 8, 2023, and the strain preservation number is GDMCC No: 63246, and the preservation address is No. 100, Martyr Road, Guangzhou City, Guangdong Province. The bacillus subtilis TN and the live bacterial preparation provided in the present application can effectively avoid the problem of secondary infection of a burn wound caused by an unsuitable bandaging therapy, or the problem of dysbacteriosis and drug resistance caused by the abuse of biological preparations such as antibiotics when the bacillus subtilis TN and the live bacterial preparation are applied to treat a burn wound, and have the effects of anti-inflammation, antioxidant, inhibition of melanin production and moisturizing, are beneficial to promoting wound recovery and preventing scarring, and through compounding with a nutrient solution, the problems of difficulty in survival of the bacillus subtilis when applied to a wound and poor film-forming hemostatic effect are effectively solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms, in particular to a bacillus subtilis TN, a live bacteria preparation and a preparation method and application thereof. BACKGROUND

[0002] Burn is a kind of tissue damage caused by heat material such as flame, electric energy, chemical substance and radiation, mainly showing skin tissue damage, and can damage subcutaneous or submucosal tissue such as muscle, bone, joint and even internal organs in severe cases.

[0003] Since there are a large number of damaged and inactivated tissues on the burn wound, it is a good place for bacteria to breed. If not treated in time and correctly after burn, it can lead to systemic invasive infection, which not only causes local skin damage, decay, swelling and pain, blood stasis and stagnation, and damage of heat toxin to yin fluid, but also causes imbalance of zang-fu yin and yang and disorder of qi and blood, and even endangers human life. With the rapid development of modern industry, burn caused by various types of heat energy is increasing day by day, and burn has become one of the common and serious traumatic diseases which seriously harm human beings and are extremely painful.

[0004] In related technologies, modern medicine mainly uses bandaging therapy and wound medication to treat burn wounds. The bandaging therapy is a common method for treating burn wounds with dressings, which can reduce the adverse effects of external stimulation on the wound. However, improper bandaging methods such as too tight bandaging often lead to secondary infection of the wound, causing gangrene, putrid meat and other conditions, and further causing secondary infection of the burn wound, affecting the recovery of the burn wound. The wound medication mainly uses biological agents such as silver sulfadiazine and antibiotics. Although silver sulfadiazine has good antibacterial effect, it can cause the side effect of deepening the burn wound, causing the problem of scarring of the burn wound. When biological agents such as antibiotics are used for treatment, although the effect is good, it is easy to cause bacterial flora imbalance and drug resistance due to the abuse of antibiotics.

[0005] Therefore, there is an urgent need for a biological agent with antibacterial and bacteriostatic effects and capable of reducing the pain of burn wounds. When applied to the treatment of burn wounds, it can effectively avoid the problem of secondary infection of burn wounds caused by improper bandaging therapy, or the problem of bacterial flora imbalance and drug resistance caused by the abuse of biological agents such as antibiotics, and has the effects of anti-inflammatory, antioxidant, inhibition of melanin production and moisturizing, which is beneficial to promote wound recovery and prevent scarring. SUMMARY

[0006] To overcome the problems existing in related technologies, this application provides Bacillus subtilis TN, a live bacterial preparation, its preparation method, and its application. When applied to the treatment of burn wounds, Bacillus subtilis TN and the live bacterial preparation can effectively avoid the problem of secondary infection of burn wounds caused by inappropriate bandaging therapy, or the problem of dysbiosis and drug resistance caused by the abuse of antibiotics and other biological agents. It also has anti-inflammatory, antioxidant, melanin inhibition, and moisturizing effects, which are beneficial to promoting wound healing and preventing scarring.

[0007] One of the purposes of this application is to provide a Bacillus subtilis TN strain, which was deposited on March 8, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 63246, and the deposit address is No. 100, Xianlie Middle Road, Guangzhou, Guangdong Province.

[0008] The second objective of this application is to provide a live Bacillus subtilis preparation, comprising the aforementioned Bacillus subtilis TN, its physiological metabolites, and nutrient solution.

[0009] In a preferred embodiment of the present invention, the physiological metabolites include natto polysaccharide and natto protease.

[0010] The third objective of this application is to provide a method for preparing a live Bacillus subtilis preparation, which mainly includes the following steps:

[0011] Bacillus subtilis TN was placed in LB liquid medium and cultured in a shaker at 37°C for 24 hours to obtain Bacillus subtilis bacterial suspension.

[0012] The bacterial solution was mixed with the nutrient solution and cultured in a 37°C incubator for 20 hours. After filtration, the live Bacillus subtilis preparation was obtained.

[0013] In a preferred embodiment of the present invention, the preparation of the nutrient solution includes the following steps:

[0014] After washing the soybeans, soak them thoroughly and then dissolve them in soy peptone and agar.

[0015] After dissolving, steam or boil for 1 hour, then cool to obtain the nutrient solution.

[0016] In a preferred embodiment of the present invention, the preparation of Bacillus subtilis TN includes the following steps:

[0017] The preparation of the Bacillus subtilis TN includes the following steps:

[0018] Rice straw is pretreated to obtain disinfected rice straw;

[0019] Disinfected rice straw was ground into a sample suspension, which was then spread on LB solid medium for culture to generate colonies.

[0020] The newly grown colonies were isolated and purified using the streak plate method.

[0021] The purified strain was stored in a preservation solution to obtain Bacillus subtilis TN.

[0022] In a preferred embodiment of the present invention, the pretreatment of rice straw includes:

[0023] Cut rice straw into 3-5cm pieces with sterilized scissors, soak in 2% sodium hypochlorite for 2 minutes, then soak in 70% alcohol for 2 minutes, and then wash several times with sterile water.

[0024] In a preferred embodiment of the present invention, the step of grinding rice straw into a sample suspension includes:

[0025] Cut off 1cm from both ends of the disinfected rice straw, add sterile water and quartz sand, grind thoroughly with a grinding rod, and then dilute in a 10-fold gradient to obtain concentrations of 10... －4 10 －5 and 10 －6 The sample suspension.

[0026] In a preferred embodiment of the present invention, the preservation solution comprises the following components: 20% glycerol.

[0027] The fourth objective of this application is to provide an application of a live Bacillus subtilis preparation, specifically for the treatment of burn wounds.

[0028] Compared with the prior art, the technical solution provided in this application may include the following beneficial effects:

[0029] (1) The Bacillus subtilis TN provided in this application can metabolize and secrete substances such as natto polysaccharide and natto protease. The natto protease can decompose fibrin coagulation in inflamed areas and has the effect of removing gangrene, necrotic tissue and debris from the wound surface. It effectively solves the problem that endogenous collagenase is insufficient to remove all necrotic tissue due to limited production in cases of non-physiological necrotic tissue caused by burns. Natto protease can hydrolyze and remove the necrotic self-collagen at the base of deep second-degree wounds, promote epithelial growth and accelerate the healing of deep second-degree wounds. Natto polysaccharide has anti-inflammatory, antioxidant, melanin-inhibiting and moisturizing effects on burn wounds, which is conducive to promoting the rapid recovery of burn wounds and effectively preventing scarring.

[0030] (2) The Bacillus subtilis TN and Bacillus subtilis live bacteria preparation provided in this application are biological agents with antibacterial and bacteriostatic effects, and can also relieve the pain of burn wounds. When applied to the treatment of burn wounds, it can effectively avoid the problem of secondary infection of burn wounds caused by inappropriate bandaging therapy, or the problem of dysbiosis and drug resistance caused by the abuse of antibiotics and other biological agents. It is also simple and convenient to use, and the preparation can be directly applied to the burn wound.

[0031] (3) The Bacillus subtilis live bacterial preparation provided by the present invention uses a mixture of Bacillus subtilis bacterial liquid and nutrient solution. On the wound surface, the nutrients in the Bacillus subtilis live bacterial preparation and the exudate of the burn wound are used as nutrients to rapidly proliferate and form dominant bacteria, and quickly form a bacterial film, thereby forming a biological barrier on the wound surface to inhibit the reproduction of other bacteria, effectively protecting the burn wound and preventing infection. In addition, after fermentation, Bacillus subtilis produces a large amount of viscous substance, which helps wound healing and prevents the wound from being stimulated by the outside world. At the same time, the Bacillus subtilis live bacterial preparation can accelerate the reproduction of beneficial strains for wound recovery, so that the burn wound can quickly stop bleeding and reduce inflammation, effectively reduce the patient's pain, and accelerate the healing of the burn wound.

[0032] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description

[0033] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments thereof in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments thereof.

[0034] Figure 1 This is a colony diagram of the TN strain activated by the streak plate method, as shown in the embodiments of this application, after being cultured on LB solid medium for 24 hours;

[0035] Figure 2 This is a screening diagram of Bacillus subtilis strains that produce protease, as shown in the embodiments of this application;

[0036] Figure 3 This is a schematic diagram of an initial burn as shown in an embodiment of this application;

[0037] Figure 4 This is a schematic diagram illustrating a burn 8 hours after the application of a live Bacillus subtilis preparation;

[0038] Figure 5 This is a schematic diagram of a wound surface shown in an embodiment of this application;

[0039] Figure 6This is a schematic diagram showing the wound surface after 8 hours of applying a live Bacillus subtilis preparation, as illustrated in the embodiments of this application. Detailed Implementation

[0040] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of protection of the present invention. Unless otherwise specified, the experimental materials, reagents, instruments, etc., used in the embodiments of the present invention are commercially available; unless otherwise specified, all technical means in the embodiments of the present invention are conventional means well known to those skilled in the art.

[0041] Currently, modern medicine primarily treats burn wounds with bandaging and medication. However, inappropriate bandaging methods, such as overly tight bandaging, can lead to secondary infections, causing gangrene and necrotic tissue, thus hindering wound healing. While antibiotics and other biological agents are effective, their overuse can easily lead to dysbiosis and antibiotic resistance.

[0042] To address the aforementioned issues, this application provides Bacillus subtilis TN, a live bacterial preparation, its preparation method, and its application. This effectively avoids secondary infection of burn wounds caused by inappropriate bandaging therapy, or dysbiosis and drug resistance caused by the overuse of antibiotics and other biological agents. Furthermore, it has anti-inflammatory, antioxidant, melanin-inhibiting, and moisturizing effects, which are beneficial for promoting wound healing and preventing scarring.

[0043] The technical solutions of the embodiments of this application are described in detail below with reference to the accompanying drawings.

[0044] Example 1: Obtaining the TN strain

[0045] 1. Strains are isolated, purified, and preserved.

[0046] Strains TN were isolated and purified from rice straw. The specific isolation and purification steps are as follows:

[0047] The rice straw is pretreated as follows: First, the rice straw is cut into 3-5 cm pieces using sterilized scissors. Then, the surface dust is washed off with sterile water to obtain clean rice straw. The cleaned rice straw is then soaked in a 2% sodium hypochlorite solution for 2 minutes, rinsed once with sterile water, and then soaked in 70% alcohol for 2 minutes. It is then rinsed with sterile water. Preferably, it is rinsed 8 times with sterile water, each time for 5 minutes. The sterile water from the last rinse is spread onto LB solid medium for incubation at 37℃ for 24-48 hours. After 24 hours of incubation, the bacterial growth on the LB solid medium can be observed. If no bacteria grow on the LB solid medium after 24 hours, it indicates that the rice straw has been thoroughly disinfected, and the disinfected rice straw is obtained.

[0048] Cut off 1cm from both ends of the disinfected rice straw to obtain the middle section. Place the middle section of rice straw in a mortar, add sterile water and quartz sand, and grind thoroughly with a grinding stick to obtain a grinding liquid. It is important to note that the grinding operation must be carried out in a sterile environment, that is, the mortar, quartz sand, and grinding stick must all be sterilized in advance.

[0049] Take 1 mL of the above-mentioned homogenate and add it to 9 mL of sterile water. Mix thoroughly, and then continue to dilute with sterile water in a 10-fold serial dilution series to obtain concentrations of 10... －4 10 －5 and 10 －6 200 μL of the sample suspension with a concentration of 10 was taken respectively. -4 10 -5 and 10 -6 The sample suspension was evenly spread on LB solid medium that had been sterilized at 121°C for 20 min and cultured to form colonies.

[0050] The newly generated colonies are isolated and purified by the streak plate method, which specifically includes: picking the newly generated colonies by the streak plate method and purifying them on LB solid medium, and repeatedly subculturing them until the colonies have the same color, shape, size, texture and transparency.

[0051] It should be noted that the LB solid medium comprises 9-12 g / L peptone, 4-6 g / L yeast extract, 9-12 g / L NaCl, and 14-18 g / L sucrose. Preferably, it comprises 9 g / L peptone, 6 g / L yeast extract, 9 g / L sodium chloride (NaCl), and 16 g agar powder. The pH value is 7.0. The conditions for subculturing with LB solid medium are a temperature of 20-45℃ and a pH value of 6.0-10.0. Preferably, the temperature is 37℃ and the pH value is 7.0.

[0052] Finally, its morphology was further observed by simple staining (carbohydrate fuchsin staining) and microscopic examination (oil immersion), with uniform length, consistent width, and uniform staining as the purification criteria for the strain. Three to four loops of the purified strain were picked up using a sterilized inoculation loop and preserved in a preservation solution (20% glycerol), and stored at -20℃ and -80℃ to obtain Bacillus subtilis TN (abbreviated as: strain TN).

[0053] Bacillus subtilis, also known as natto bacteria, belongs to the Bacillus subtilis subspecies. The Bacillus subtilis TN provided in this application is further subdivided into Bacillus subtilis. Since Bacillus subtilis can produce nattokinase, an alkaline serine protease that can hydrolyze protease plates to form clear zones, effective strains can be efficiently selected based on this characteristic. Specifically, the process involves sterilizing skim milk and other components of the skim milk culture medium separately. After cooling to 45℃-50℃, the skim milk and other components are mixed evenly, poured into plates, and placed in a clean bench to fully dry the surface moisture of the skim milk plates. Using an inoculation loop, pick up activated antagonistic bacterial colonies and inoculate them in the center of a skim milk agar plate. Then, invert the plate and incubate it in a 28°C incubator. After 2 days of incubation, observe whether a clear zone appears around the colonies growing in the skim milk plate. If a clear zone appears, it indicates that protease is produced; otherwise, it indicates that no protease is produced. Select the strain with strong protease production ability as the effective strain, which is strain TN. Extract DNA from strain TN.

[0054] Example 2: 16S rDNA analysis of strain TN.

[0055] DNA was extracted from strain TN, isolated and purified in Example 1. 16S rDNA was amplified using universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG) and 1492R (5'-TACCTTGTTACGACTT), and detected using agarose gel electrophoresis. The PCR amplification product was sent to a testing company for sequencing to obtain the 16S rRNA gene sequence of strain TN. The 16S rRNA gene sequence was entered into GenBank for BLAST alignment to preliminarily determine the genus and species position of strain TN in taxonomy. The results showed that strain TN of this invention has 99.93% similarity to the Bacillus subtilis type strain. Therefore, strain TN of this invention should be classified as Bacillus subtilis, and more specifically, as Bacillus natto within Bacillus subtilis.

[0056] The 16S rDNA gene sequence determination results are as follows:

[0057]

[0058] The Bacillus subtilis TN described in this invention was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 8, 2023, with the strain accession number GDMCC No: 63246 and the deposit address being No. 100, Xianlie Middle Road, Guangzhou, Guangdong Province.

[0059] Example 3: Preparation of live Bacillus subtilis preparation

[0060] Preparation of Bacillus subtilis bacterial culture: 1 LLB liquid culture medium was dispensed into an Erlenmeyer flask, sterilized at 121°C for 30 min, cooled, and then the Bacillus subtilis strain obtained in Example 1 was added. The flask was then incubated at 37°C in a shaker for 24 hours to obtain the Bacillus subtilis bacterial culture. The LB liquid culture medium comprised 8-10 g / L peptone, 5-7 g / L yeast extract, and 8-10 g / L NaCl; preferably, it contained 9 g / L peptone, 6 g / L yeast extract, and 9 g / L sodium chloride (NaCl); the pH was 7.0.

[0061] Preparation of nutrient solution: Take 60g of plump soybeans, wash them 4-5 times, and soak them thoroughly in pure water until the soybeans absorb water and swell until the surface is smooth, wrinkle-free, the skin is easy to peel off, and the soybeans are easy to break when pinched, with the cross-section thoroughly soaked and without a hard core. Pour off the water to remove impurities, and then add water to 1L. Add 7g of soybean peptone and 5g of agar to dissolve. After dissolving, pour the solution into a tray and steam or boil for 1 hour. After cooling, the nutrient solution is obtained. The nutrient solution can be dispensed into conical flasks, stoppered, and sealed. After sterilization at 121℃ for 30 minutes, remove, cool, and seal for storage.

[0062] The above-mentioned Bacillus subtilis bacterial suspension was mixed with nutrient solution and poured into a sterilized tray. It was then incubated in a 37°C incubator for 20 hours, allowing the strains in the Bacillus subtilis bacterial suspension to produce a large amount of bacterial film and viscous metabolites such as natto protease and natto polysaccharide. The soybean particles were then filtered out using a 100-mesh filter and dispensed into sterilized containers to obtain a live Bacillus subtilis preparation.

[0063] Example 4: Application of Bacillus subtilis live bacterial preparation

[0064] like Figures 3-4 As shown, after applying live Bacillus subtilis preparation directly to the burn wound surface, no significant improvement was observed in the burn wounds without the application of live Bacillus subtilis preparation after 8 hours. Figures 5-6 As shown, after 8 hours, the burn wound treated with the live Bacillus subtilis preparation turned black and began to scab. In terms of wound healing speed, the live Bacillus subtilis preparation has a good effect on accelerating the growth of new granulation tissue in burn wounds and promoting burn tissue healing.

[0065] Regarding the apparatus in the above embodiments, the specific manner in which each module performs its operation has been described in detail in the embodiments related to the method, and will not be elaborated further here.

[0066] The solution of this application has been described in detail above with reference to the accompanying drawings. In the above embodiments, the descriptions of each embodiment have different emphases; parts not described in detail in a certain embodiment can be referred to in the relevant descriptions of other embodiments. Those skilled in the art should also understand that the actions and modules involved in the specification are not necessarily essential to this application. Furthermore, it is understood that the steps in the method of this application embodiment can be adjusted, combined, and deleted according to actual needs, and the modules in the device of this application embodiment can be combined, divided, and deleted according to actual needs.

[0067] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A Bacillus subtilis TN strain, characterized in that: It was deposited on March 8, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with the accession number GDMCC 63246 and the deposit address at No. 100, Xianlie Middle Road, Guangzhou, Guangdong Province.

2. A live Bacillus subtilis preparation, characterized in that: Includes Bacillus subtilis TN as described in claim 1, its physiological metabolites, and nutrient solution.

3. A method for preparing a live Bacillus subtilis preparation, characterized in that: The preparation of the Bacillus subtilis live bacterial preparation as described in claim 2 mainly includes the following steps: Bacillus subtilis TN was placed in LB liquid medium and cultured in a shaker at 37°C for 24 hours to obtain Bacillus subtilis bacterial suspension. The bacterial solution was mixed with the nutrient solution and cultured in a 37°C incubator for 20 hours. After filtration, the live Bacillus subtilis preparation was obtained.

4. The method for preparing the live Bacillus subtilis preparation as described in claim 3, characterized in that: The preparation of the nutrient solution includes the following steps: After washing the soybeans, soak them thoroughly and then dissolve them in soy peptone and agar. After dissolving, steam or boil for 1 hour, then cool to obtain the nutrient solution.

5. The method for preparing the live Bacillus subtilis preparation as described in claim 3, characterized in that: The preparation of the Bacillus subtilis TN includes the following steps: Rice straw is pretreated to obtain disinfected rice straw; Disinfected rice straw was ground into a sample suspension, which was then spread on LB solid medium for culture to generate colonies. The newly grown colonies were isolated and purified using the streak plate method. The purified strain was stored in a preservation solution to obtain Bacillus subtilis TN.

6. The method for preparing the live Bacillus subtilis preparation as described in claim 5, characterized in that: The pretreatment of rice straw includes: Cut rice straw into 3-5cm pieces with sterilized scissors, soak in 2% sodium hypochlorite for 2 minutes, then soak in 70% alcohol for 2 minutes, and then wash several times with sterile water.

7. The method for preparing the live Bacillus subtilis preparation as described in claim 5, characterized in that: The process of grinding disinfected rice straw into a sample suspension includes: Cut off 1 cm from both ends of the disinfected rice straw, add sterile water and quartz sand, grind thoroughly with a grinding rod, and dilute in a 10-fold gradient to obtain the following dilution gradients: －4 10 －5 and 10 －6 The sample suspension.

8. The method for preparing the live Bacillus subtilis preparation as described in claim 5, characterized in that: The preservation solution comprises the following components: 20% glycerol.

9. The application of a live Bacillus subtilis preparation, characterized in that, The application of the live Bacillus subtilis preparation as described in claim 2 in the preparation of burn medications.

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