Bacillus amyloliquefaciens and application thereof

Abstract

The application provides a bacillus amyloliquefaciens and application thereof, and relates to the field of microbial fermentation. Bacillus amyloliquefaciens The strain is named bacillus amyloliquefaciens (B2), is preserved in the China General Microbiological Culture Collection Center, 3, Xili Beichen, Chaoyang District, Beijing, China, and has a preservation date of July 30, 2025 and a preservation number of CGMCC NO.35462. Bacillus amyloliquefaciens The bacillus amyloliquefaciens (B2) provided by the application can be used for degrading calcium oxalate needle crystals in Banxia Qu.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a Bacillus amyloliquefaciens and its applications. Background Technology

[0002] Pinellia Pinellia ternate (Thunb) Breit. Pinellia ternata is the dried tuber of a plant belonging to the genus Pinellia in the Araceae family. Its medicinal use was first recorded in the *Fifty-Two Prescriptions*. The raw form is toxic and irritating to mucous membranes, and is listed as an inferior medicine in the *Shennong's Classic of Materia Medica*. Ingestion of raw or fresh Pinellia ternata can cause swelling of the lips, tongue, and throat, and in severe cases, even death. Studies have shown that calcium oxalate needle crystals are one of the primary irritant components of raw Pinellia ternata. These crystals are chemically stable (insoluble in water and organic solvents) and heat-resistant, making their crystal structure difficult to destroy using conventional decoction processes. Therefore, it is often used clinically in compound prescriptions or as a processed product. Pinellia ternata fermentation is a traditional compound fermentation preparation made by naturally fermenting Pinellia ternata or its processed form with various medicinal materials such as alum, flour, ginger juice, and Shenqu (a type of fermented medicinal herb) or its raw material.

[0003] Currently, the toxicity of processed Pinellia ternata is judged solely by observing the morphology of calcium oxalate needle crystals (such as length, diameter, and tip sharpness) under a microscope. This is essentially a qualitative or semi-quantitative subjective judgment, lacking quantifiable "morphology-toxicity" correspondence standards. Therefore, it suffers from significant subjective errors, inability to quantify content, and poor sample representativeness. Bacillus amyloliquefaciens is mainly used for biological control, plant growth promotion, and pollutant degradation. There are no reports of using Bacillus amyloliquefaciens to degrade calcium oxalate needle crystals in processed Pinellia ternata. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides a Bacillus amyloliquefaciens strain and its application, which is suitable for survival in an environment where calcium oxalate is the main carbon source and can degrade calcium oxalate.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0006] This invention provides a strain of Bacillus amyloliquefaciens, named Bacillus amyloliquefaciens (B. amyloliquefaciens). Bacillus amyloliquefaciens B2, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, on July 30, 2025, with accession number CGMCC NO.35462.

[0007] The application utilizes calcium oxalate directional culture medium to screen functional bacteria degrading calcium oxalate, and determines the bacteria degrading calcium oxalate needle crystals in pinellia ternate through content determination and calcium oxalate needle crystal morphology screening, and changes fermentation conditions according to the properties of the bacteria, so that the toxicity is reduced to the maximum extent, the production cost of enterprises is reduced, the loss is reduced, and the production benefit is improved. It is verified through experiments that the bacillus amyloliquefaciens B2 provided by the application can be used for degrading calcium oxalate needle crystals in pinellia ternate at normal temperature conditions, and the degradation rate of calcium oxalate needle crystals can reach 60%-65%.

[0008] The application provides a kind of bacterial agent, including the above-mentioned bacillus amyloliquefaciens and / or the fermentation liquor of the above-mentioned bacillus amyloliquefaciens.

[0009] The dosage form of the bacterial agent is not particularly limited, and can be a solid preparation, a liquid preparation, or other types of dosage forms in the art. The bacterial agent can be added with excipients or other ingredients according to the use requirements.

[0010] The application provides a fermentation method of the above-mentioned bacillus amyloliquefaciens strain, including the following steps: inoculating the above-mentioned bacillus amyloliquefaciens into a pinellia ternate fermentation system and carrying out fermentation culture.

[0011] The application provides the application of bacillus amyloliquefaciens in degrading calcium oxalate needle crystals.

[0012] The above-mentioned bacillus amyloliquefaciens strain can be used for degrading calcium oxalate needle crystals in pinellia ternate.

[0013] The application provides the application of the above-mentioned bacterial agent in degrading calcium oxalate needle crystals.

[0014] The above-mentioned bacterial agent can be used for degrading calcium oxalate needle crystals in pinellia ternate.

[0015] The application provides a method for degrading calcium oxalate needle crystals, including the following steps: using the above-mentioned bacillus amyloliquefaciens or the above-mentioned bacterial agent to degrade calcium oxalate needle crystals.

[0016] The bacillus amyloliquefaciens of the application is used to degrade calcium oxalate needle crystals in pinellia ternate.

[0017] Further, the following steps are included: inoculating the bacillus amyloliquefaciens or the above-mentioned bacterial agent into pinellia ternate and carrying out fermentation culture.

[0018] Further, the inoculation amount is 3%.

[0019] Further, the fermentation culture temperature is 30°C.

[0020] Further, the fermentation culture humidity is 85%.

[0021] Further, the culture time is 72 h.

[0022] The invention prepares pinellia tuber powder and detects the removal rate of calcium oxalate needle crystals in the pinellia tuber powder by Bacillus amyloliquefaciens B2. The detection shows that the removal rate of calcium oxalate needle crystals by Bacillus amyloliquefaciens B2 can reach 62.01%. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The result of the strain B2 cultured on LB solid plate.

[0024] Figure 2 The phylogenetic tree.

[0025] Figure 3 The effect diagram of the degradation rate of calcium oxalate by the strain B2.

[0026] Figure 4 The effect diagram of the damage of calcium oxalate needle crystals by the strain B2. DETAILED DESCRIPTION

[0027] The principles and features of the invention are described below in combination with the drawings, and the examples are only used to explain the invention and not to limit the scope of the invention.

[0028] The Bacillus amyloliquefaciens B2 is obtained from the natural pinellia tuber powder fermentation sample through enrichment culture, step-by-step screening and other methods. The strain colony is regular round, the diameter is about 0.5 mm, the edge is slightly wavy, the color is milky white, the surface is smooth with slight wrinkles, and the strain B2 can be used for degrading calcium oxalate needle crystals in the pinellia tuber powder at 30℃ for 48 h. The degradation rate of calcium oxalate needle crystals can reach 60%-65%. The invention first proposes to use Bacillus amyloliquefaciens B2 to degrade the toxic component calcium oxalate in the traditional Chinese medicine pinellia tuber powder.

[0029] The formula of the calcium oxalate solid culture medium includes 1% of calcium oxalate, 0.01% of yeast extract, 0.005% of K2HPO4, 0.005% of MgSO4·7H2O and 1.5% of agar, which are prepared with water, and the pH value is 7.0, and the sterilization is performed at 121℃ for 30 min. The percentages are all mass percentages.

[0030] The formula of the LB liquid culture medium includes 10 g of tryptone, 5 g of yeast extract and 10 g of sodium chloride per 1000 mL of water, the pH value is adjusted to 7.2-7.4, and the sterilization is performed at 121℃ for 20 min.

[0031] The formula of the LB solid culture medium includes 10 g of tryptone, 5 g of yeast extract and 10 g of sodium chloride per 1000 mL of water, 15 g of agar, the pH value is adjusted to 7.2-7.4, and the sterilization is performed at 121℃ for 20 min.

[0032] The Qingbanxia, Liushenqu, and white alum were purchased from Bozhou Chinese Herbal Medicine Market in Anhui. The ginger was purchased from Daoli Market in Harbin. The flour was purchased from Jinshahe Group.

[0033] Unless otherwise specified, the specific techniques or conditions in the examples were performed according to the conventional methods or the techniques or conditions described in the literature in the field, or according to the product instructions. The experimental materials used in the present application, unless otherwise specified, were conventional products that can be purchased through regular channels or prepared according to conventional methods in the field. The instruments used in the present application, unless otherwise specified, were conventional instruments that can be purchased through regular channels.

[0034] The solutions of the present application were prepared with water as the solvent, unless otherwise specified.

[0035] The following will be described through specific examples.

[0036] Example 1

[0037] The strain was enriched and screened from natural Banxiaqu fermentation, including the following steps:

[0038] The fresh sample of naturally fermented Banxiaqu was prepared, and the specific preparation process was as follows:

[0039] The ginger juice was prepared, including the following steps: fresh ginger was cut into pieces, and an appropriate amount of water was added to squeeze the juice. According to the conversion of 1 g of ginger to 1 mL of ginger juice, 1 g / mL of ginger juice was prepared.

[0040] Take 160 g of Qingbanxia, 10 g of white alum, and grind them into fine powder. Mix 32 g of flour and 5 g of Liushenqu. Add 20 mL of ginger juice to 60-80 mL of water, and mix the above components. Knead into a ball (hold it together, and it will scatter when touched).

[0041] (2) The Banxiaqu prepared in step (1) was naturally fermented at 30°C and 85% humidity.

[0042] (3) Take 1 mg of fresh sample at different fermentation time points of 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h in a sterile environment and add to a 150 mL conical flask containing 9 mL of sterile water and 2 mm glass shaking beads. Shake at 140 r / min for about 20 min to make the bacteria fully diffuse. Use a 1 mL sterile pipette to draw 1 mL of bacterial suspension from the conical flask and add it to a sterile tube containing 9 mL of sterile water. 10 -1 fold bacterial suspension was obtained. According to the method, 10 -2 , 10 -4 , 10 -6 , 10 -8 fold dilution of bacterial suspension was prepared.

[0043] (4) 100 μL of 10 -2 , 10 -4 , 10 -6 , 10 -8 bacterial suspension was taken with a sterile pipette and spread evenly on the surface of the prepared calcium oxalate solid medium (3 replicates for each dilution) using a sterile metal spreader. The relevant dilution marks were made on the plate. Then the plate was placed in a 30°C biochemical incubator for 2-5 d until colonies grew. The growth of the strain was observed.

[0044] (5) The white single colony in step (4) was purified on an LB solid medium plate and inverted in a constant temperature incubator at 30°C for 1-2 d to obtain a pure strain, which was named B2 and preserved in glycerol.

[0045] Example 2

[0046] The strain B2 was streak inoculated on an LB solid medium plate and cultured at 30°C for 24 h. The morphological characteristics of the colony were observed. As shown in Table 1, the colony was round with a slightly wavy edge; milky white, opaque, smooth surface with slight wrinkles. Figure 1

[0047] The white strain B2 obtained by screening was sent to Shanghai Ling'en Biological Technology Co., Ltd. The 16S rRNA gene sequencing was performed by Shanghai Ling'en Biological Technology Co., Ltd. using primers 27F / 1492R.

[0048] Primer 27F: AGRGTTYGATYMTGGCTCAG (SEQ ID NO. 1);

[0049] Primer 1492R: RGYTACCTTGTTACGACTT (SEQ ID NO. 2);

[0050] The above primers were provided by Shanghai Ling'en Biological Technology Co., Ltd.

[0051] The sequence of the 16S rRNA gene of the strain B2 is as follows:

[0052] ​

[0053] The sequencing results were compared using BLAST on the NCBI website, and a phylogenetic tree was constructed, such as... Figure 2 As shown, strain B2 was identified as belonging to Bacillus amyloliquefaciens (Bacillus). Bacillus amyloliquefaciens ).

[0054] Strain B2 was named *Bacillus amyloliquefaciens*. Bacillus amyloliquefaciens B2, deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, on July 30, 2025, with accession number CGMCCNo. 35462.

[0055] Example 3

[0056] The use of Bacillus amyloliquefaciens B2 to degrade calcium oxalate needle crystals in Pinellia ternata includes the following steps:

[0057] (1) Fermentation material formula: According to the following ratio, 160 g of Pinellia ternata, 10 g of alum, 32 g of flour, 5 g of Shenqu (a type of fermented wheat starter), and 20 mL of ginger juice. The preparation method of the ginger juice includes the following steps: cut fresh ginger into pieces, add an appropriate amount of water and juice it, and calculate that 1 g of fresh ginger makes 1 mL of ginger juice, so as to make 1 g / mL of ginger juice.

[0058] (2) Preparation of fermentation starter for Pinellia ternata: Take Pinellia ternata, flour, alum and shenqu and mix them evenly. Add 20 mL of ginger juice to water to 60~80 mL. Mix the above components together and make them into a state that can be formed into a ball when squeezed and can be easily broken when touched. Distribute them into 250 mL wide-mouth bottles, seal them with kraft paper and stopper them. Sterilize them in an autoclave at 121℃ for 20 min. Let them cool and use them for later use.

[0059] (3) Activation of bacterial strain: The preserved Bacillus amyloliquefaciens B2 was transferred to LB liquid medium and shaken at 30℃ and 140 r / min until the enrichment bottle became turbid, thus obtaining a bacterial suspension. The activation of Bacillus amyloliquefaciens was completed through the above steps.

[0060] (4) Preparation of seed culture: The above bacterial suspension was inoculated into 50 mL of LB liquid medium at an inoculation rate of 5 mL and shaken at 30℃ and 140 r / min to obtain seed culture.

[0061] (5) Fermentation: Fermentation of the OD-dependent cells 600The seed liquid with OD600=0.8 is inoculated into the sterilized fermentation starter of Banxia koji at an inoculation amount of 3%, that is, the ratio of the volume (mL) of the seed liquid to the mass (g) of the fermentation starter of Banxia koji is 3%; the mouth of the conical flask is sealed with a tissue culture sealing film, and the conical flask is placed in a biochemical incubator with a temperature of 30°C and a humidity of 85% for fermentation. The fermented Banxia koji is taken at 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h, respectively; the fermented sample is placed in a 40°C electric heating air drying oven for low-temperature drying for 4 h to obtain fermented Banxia koji sample powder taken at different time periods, which is ready for use.

[0062] Example 4

[0063] The fermented samples taken at different time points in Example 3 are processed, including the following steps:

[0064] (1) 0.2 g of the fermented Banxia koji sample powder taken at different time periods in Example 3 is placed in a 10 mL EP tube, 3 mL of ultrapure water is added, and after shaking, 10 min of 100 W ultrasonic treatment is performed; then the EP tube is placed in a 65°C water bath for heating and oscillation (140 r / min, 15 min), and centrifugation is performed at a speed of 4000 r / min for 5 min; the supernatant is discarded, and 2 mL of hot ultrapure water is added to the residue for repeated washing for 3 times; after each washing, the mixing and centrifugation operation is performed.

[0065] (2) 0.4 mL of 6 M HCl (hydrochloric acid) and 3 mL of ultrapure water are added to the residue and mixed, heated and stirred in a 75 o C water bath for 25 min, and the centrifugation operation is performed under the conditions of step (1) above to obtain the supernatant and residue; the supernatant is placed in a 10 mL volumetric flask;

[0066] The residue is added with 2 mL of 0.1 M HCl, heated and stirred in a 75 o C water bath for 25 min, and the centrifugation operation is performed under the conditions of step (1) above to obtain the supernatant and residue; the supernatant is placed in a 10 mL volumetric flask;

[0067] (3) Degradation rate of calcium oxalate was determined by liquid chromatography. A Waters 2695 high-performance liquid chromatograph, a CE553-C18-AQ (250nm×4.6nm, 5μm) column, and a UV (210 nm) detector were used to detect the calcium oxalate content. The liquid chromatography detection conditions were set as follows: mobile phase was 0.2% (v / v) H3PO4-H2O solution, flow rate was 0.5 mL / min, injection volume was 10 μL, column temperature was 30℃, theoretical plate number was not less than 2000, and resolution between adjacent peaks was greater than 1.5.

[0068] The degradation rate is calculated as follows: Degradation rate = (Calcium oxalate content in the sample before fermentation - Calcium oxalate content in the sample after fermentation) / Calcium oxalate content in the sample before fermentation × 100%.

[0069] Experimental results are as follows Figure 3 As shown, from Figure 3 It can be seen that, compared with the control group at 0 h, the B2 strain of Pinellia ternata fermented by pure culture degraded calcium oxalate to a certain extent at different time periods. Among them, the degradation rate of calcium oxalate by the B2 strain was the highest at 72 h, reaching 62.01%.

[0070] The microstructure of calcium oxalate needle crystals in the dried Pinellia ternata powder before and after fermentation was observed using a scanning electron microscope (SEM). After depositing a metal film in a high-vacuum ion sputtering apparatus, the image was imaged at 10 kV resolution. Figure 4 The study observed the needle crystal destruction effect under the conditions of 30℃, 85% humidity, 3% inoculum, a ginger juice to water volume ratio of 1:3, and a fermentation time of 72 h. It can be seen that Bacillus amyloliquefaciens B2 has a good degradation effect on calcium oxalate needle crystals in Pinellia ternata. The needle crystals exhibit a fractured morphology with relatively flat and rounded fracture surfaces.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A Bacillus amyloliquefaciens (B2) characterized in that Bacillus amyloliquefaciens the genome of said Bacillus amyloliquefaciens (B2) comprises a mutation in the gene encoding the protein of SEQ ID NO: 1, and , and the preservation number is CGMCC NO. 35462.

2. An inoculant characterized in that, The effective component is the Bacillus amyloliquefaciens in claim 1.

3. The Bacillus amyloliquefaciens in claim 1 is used for degrading calcium oxalate.

4. The bacterial agent in claim 2 is used for degrading calcium oxalate.

5. A method of degrading calcium oxalate, characterized by, The Bacillus amyloliquefaciens in claim 1 or the bacterial agent in claim 2 is used for degrading calcium oxalate.

6. The method of degrading calcium oxalate of claim 5, wherein, The Bacillus amyloliquefaciens in claim 1 or the bacterial agent in claim 2 is inoculated into the fermented material of pinellia pedatisecta, and then fermented and cultured.

7. The method of degrading calcium oxalate of claim 6, wherein, The inoculation amount of the Bacillus amyloliquefaciens is 3%.

8. The method of degrading calcium oxalate according to claim 6 or 7, wherein, The temperature of the fermentation and culture is 30℃, and / or the humidity of the fermentation and culture is 85%.

9. The method of degrading calcium oxalate according to claim 6 or 7, wherein, The time of the fermentation and culture is 72 h.

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