Bacillus amyloliquefaciens and application thereof

Abstract

The invention provides bacillus amyloliquefaciens and application thereof, and relates to the field of microbial fermentation. The name of the strain is bacillus amyloliquefaciens B2, the strain is preserved in the China General Microbiological Culture Collection Center, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, China, the preservation date is July 30, 2025, and the preservation number is CGMCC NO.35462. The invention further discloses a preparation method of the bacillus amyloliquefaciens strain. The bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B2 provided by the invention can be used for degrading the calcium oxalate needle crystal in the fermented pinellia tuber.

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: 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.

[0006] This invention utilizes a calcium oxalate-directed culture medium to screen functional bacteria that degrade calcium oxalate. The degrading bacteria for calcium oxalate needle crystals in Pinellia ternata are determined through both content determination and calcium oxalate needle crystal morphology screening. Fermentation conditions are adjusted according to the characteristics of the bacterial strains to minimize toxicity, thereby reducing production costs, minimizing losses, and improving production efficiency. Experimental verification shows that the Bacillus amyloliquefaciens B2 provided by this invention can degrade calcium oxalate needle crystals in Pinellia ternata under room temperature conditions, achieving a degradation rate of 60%-65%.

[0007] The present invention provides a microbial agent comprising the above-mentioned Bacillus amyloliquefaciens and / or the fermentation broth of the above-mentioned Bacillus amyloliquefaciens.

[0008] This invention does not impose any particular limitation on the dosage form of the microbial agent; it can be a solid dosage form, a liquid dosage form, or other types of dosage forms in the art. This invention can add excipients or other components to the microbial agent according to usage requirements.

[0009] The present invention provides a fermentation method for the above-mentioned Bacillus amyloliquefaciens strain, comprising the following steps: inoculating the above-mentioned Bacillus amyloliquefaciens into a Pinellia ternata fermentation system and fermenting and culturing it.

[0010] This invention provides the application of Bacillus amyloliquefaciens in the degradation of calcium oxalate needle crystals.

[0011] The aforementioned Bacillus amyloliquefaciens strain can be used to degrade calcium oxalate needle crystals in Pinellia ternata.

[0012] This invention provides the application of the above-mentioned microbial agent in the degradation of calcium oxalate needle crystals.

[0013] The above-mentioned microbial agents can be used to degrade calcium oxalate needle crystals in Pinellia ternata.

[0014] The present invention provides a method for degrading calcium oxalate needle crystals, comprising the following steps: using the above-mentioned Bacillus amyloliquefaciens or the above-mentioned bacterial agent to degrade calcium oxalate needle crystals.

[0015] The present invention utilizes Bacillus amyloliquefaciens to degrade calcium oxalate needle crystals in Pinellia ternata.

[0016] Further, the method includes the following steps: inoculating Bacillus amyloliquefaciens or the above-mentioned bacterial agent into Pinellia ternata and fermenting it.

[0017] Furthermore, the vaccination rate is 3%.

[0018] Furthermore, the fermentation culture temperature was 30℃.

[0019] Furthermore, the humidity during fermentation was 85%.

[0020] Furthermore, the incubation time is 72 hours.

[0021] This invention prepares Pinellia ternata curd and tests the removal rate of calcium oxalate needle crystals in Pinellia ternata curd by Bacillus amyloliquefaciens B2. The test results show that the removal rate of calcium oxalate needle crystals by Bacillus amyloliquefaciens B2 provided by this invention can reach 62.01%. Attached Figure Description

[0022] Figure 1 The results are for strain B2 cultured on LB solid plates.

[0023] Figure 2 This is a phylogenetic tree.

[0024] Figure 3 The graph shows the effect of bacteria B2 on the degradation rate of calcium oxalate.

[0025] Figure 4 This image shows the effect of bacteria B2 on the destruction of calcium oxalate needle crystals. Detailed Implementation

[0026] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0027] This invention obtains *Bacillus amyloliquefaciens* B2 from naturally fermented *Pinellia ternata* samples through enrichment culture and stepwise screening. When cultured at 30°C for 48 h on a solid medium containing 1% (w / w) calcium oxalate, the colonies of this strain are regularly round, approximately 0.5 mm in diameter, with slightly wavy edges; they are milky white, opaque, and have a smooth surface with slight wrinkles. At 30°C, strain B2 can degrade calcium oxalate needle crystals in *Pinellia ternata*, achieving a degradation rate of 60%-65%. This invention is the first to propose the use of *Bacillus amyloliquefaciens* B2 to degrade calcium oxalate, a toxic component of the traditional Chinese medicine *Pinellia ternata*.

[0028] The calcium oxalate solid culture medium formula includes: 1% calcium oxalate, 0.01% yeast extract, 0.005% K₂HPO₄, 0.005% MgSO₄·7H₂O, and 1.5% agar, prepared with water, pH 7.0, and sterilized at 121℃ for 30 min. All percentages mentioned above are by mass.

[0029] The LB liquid culture medium formula includes: 10 g tryptone, 5 g yeast extract, and 10 g sodium chloride per 1000 mL of water, with the pH adjusted to 7.2-7.4, and sterilized at 121℃ for 20 min.

[0030] The LB solid medium formula includes: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and 15 g agar per 1000 mL of water, with the pH adjusted to 7.2-7.4, and sterilized at 121℃ for 20 min.

[0031] Pinellia ternata, Shenqu (a type of fermented medicinal herb), and alum were all purchased from the Bozhou Traditional Chinese Medicine Market in Anhui Province. Ginger was purchased from the Daoli Vegetable Market in Harbin. Flour was purchased from Jinshahe Group.

[0032] Unless otherwise specified, all techniques or conditions used in the embodiments are conventional methods or performed according to techniques or conditions described in the literature in this field, or according to product instructions. Experimental materials used in this invention, unless otherwise specified, are all conventional products that can be purchased from legitimate channels or prepared according to conventional methods in this field. Instruments used in this invention, unless otherwise specified, are all conventional instruments that can be purchased from legitimate channels.

[0033] Unless otherwise specified, all solutions of this invention are prepared using water as the solvent.

[0034] The following is a description through specific embodiments.

[0035] Example 1 Enrichment and screening of strains from natural Pinellia ternata fermentation includes the following steps: The specific preparation process for naturally fermented fresh samples of Pinellia ternata is as follows: The preparation of ginger juice includes the following steps: cut fresh ginger into pieces, add an appropriate amount of water and juice it, and convert 1 g of ginger into 1 mL of ginger juice to make 1 g / mL ginger juice.

[0036] Take 160 g of Pinellia ternata and 10 g of alum, grind them into fine powder, mix them with 32 g of flour and 5 g of Shenqu (medicated leaven), add 20 mL of ginger juice to water to make 60-80 mL, mix the above components well, and knead into a ball (it can be formed into a ball when squeezed, but will crumble when touched).

[0037] (2) The prepared Pinellia ternata in step (1) is naturally fermented at 30°C and 85% humidity.

[0038] (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 respectively, and add it to a 150 mL Erlenmeyer flask containing 9 mL of sterile water and 2 mm glass shaking beads in a sterile environment. Shake at 140 r / min for about 20 min to allow the bacteria to spread fully. Use a 1 mL sterile pipette to draw 1 mL of bacterial suspension from the Erlenmeyer flask and add it to a sterile tube containing 9 mL of sterile water to obtain 10 -1 The bacterial suspension was prepared into 10 batches according to the method. -2 10 -4 10 -6 10 -8 Bacterial suspensions of equal dilution.

[0039] (4) Use a sterile pipette to draw 100 μL of 10 -2 10-4 10 -6 10 -8 The bacterial suspension was prepared on calcium oxalate solid medium (each dilution was repeated 3 times). Using a sterile metal spreader, the suspension was gently and evenly spread on the surface of the calcium oxalate solid medium, and the corresponding dilutions were marked on the plates. The plates were then incubated in a 30°C biochemical incubator for 2-5 days until colonies appeared. The growth of the bacterial strain was observed.

[0040] (5) Take the white single colony from step (4) and purify it on an LB solid medium plate. Invert the plate and incubate at 30°C for 1-2 days to obtain pure bacteria. Name it B2 and preserve it in glycerol.

[0041] Example 2 Strawberry strain B2 was streaked onto LB agar plates and incubated at 30°C for 24 h. The morphological characteristics of the colonies were then observed. Figure 1 As shown, the colonies are round with slightly wavy edges; they are milky white, opaque, and have a smooth surface with slight wrinkles.

[0042] The white strain B2 obtained from the screening was sent to Shanghai Lingen Biotechnology Co., Ltd., which performed 16S rRNA gene sequencing using primers 27F / 1492R.

[0043] Primer 27F: AGRGTTYGATYMTGGCTCAG (SEQ ID NO.1); Primer 1492R: RGYTACCTTGTTACGACTT (SEQ ID NO.2); All the primers mentioned above were provided by Shanghai Lingen Biotechnology Co., Ltd.

[0044] The sequence of the 16S rRNA gene of strain B2 is shown below:

[0045] 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 ).

[0046] 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.

[0047] Example 3 The use of Bacillus amyloliquefaciens B2 to degrade calcium oxalate needle crystals in Pinellia ternata includes the following steps: (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.

[0048] (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.

[0049] (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.

[0050] (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.

[0051] (5) Fermentation: Fermentation of the OD-dependent cells 600The seed liquid of 0.8 g was uniformly inoculated into the sterilized fermentation material of Pinellia ternata at an inoculation rate of 3%. The fermentation material of Pinellia ternata was prepared by the method in step (2) above. The 3% inoculation rate refers to the ratio of the volume of the seed liquid (mL) to the mass (g) of the fermentation material of Pinellia ternata being 3%. The mouth of the conical flask was sealed with tissue culture sealing film and placed in a biochemical incubator at a temperature of 30℃ and a humidity of 85% for fermentation. The fermented Pinellia ternata samples were collected at 0 h, 12 h, 24 h, 36 h, 48 h, 60 h and 72 h, respectively. The fermentation samples were placed in a 40℃ electric heating drying oven and dried at a low temperature for 4 h to obtain fermented Pinellia ternata sample powders taken at different time periods for later use.

[0052] Example 4 The fermentation samples from different sampling time points in Example 3 were processed, including the following steps: (1) Take 0.2 g of fermented Pinellia ternata sample powder taken at different time periods in Example 3, place it in a 10 mL EP tube, add 3 mL of ultrapure water, shake well and then perform ultrasonic treatment for 10 min at 100 W. Then place the EP tube in a 65℃ water bath and heat and shake (140 r / min, 15 min), centrifuge at 4000 r / min for 5 min, discard the supernatant, add 2 mL of hot ultrapure water to the residue and wash repeatedly 3 times. After each washing, perform a mixing and centrifugation operation.

[0053] (2) Take the residue, add 0.4 mL of 6 M HCl (hydrochloric acid) and 3 mL of ultrapure water, mix well, and heat at 75°C. o Heat and stir in a water bath for 25 min, then centrifuge according to the conditions of step (1) above to obtain supernatant and residue; place the supernatant in a 10 mL volumetric flask; Add the residue to 2 mL of 0.1 M HCl and saline at 75°C. o Heat and stir in a water bath for 25 minutes, then centrifuge according to the conditions of step (1) above to obtain supernatant and residue. Repeat the process twice, combine the supernatants from the three processes into a volumetric flask, and add ultrapure water to make up to the final volume.

[0054] (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.

[0055] 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%.

[0056] 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%.

[0057] 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.

[0058] 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 type of Bacillus amyloliquefaciens, characterized in that... The strain is Bacillus amyloliquefaciens (Bacillus). Bacillus amyloliquefaciens B2, with accession number CGMCC NO.35462.

2. A microbial agent, characterized in that, The main active ingredient is Bacillus amyloliquefaciens as described in claim 1.

3. The application of Bacillus amyloliquefaciens according to claim 1 in the degradation of calcium oxalate.

4. The application of the microbial agent according to claim 2 in the degradation of calcium oxalate.

5. A method for degrading calcium oxalate, characterized in that, Calcium oxalate is degraded using the Bacillus amyloliquefaciens described in claim 1 or the bacterial agent described in claim 2.

6. The method for degrading calcium oxalate according to claim 5, characterized in that, The Bacillus amyloliquefaciens of claim 1 or the inoculum of claim 2 is inoculated into the fermentation material of Pinellia ternata and fermented.

7. The method for degrading calcium oxalate according to claim 6, characterized in that, The vaccination rate was 3%.

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

9. The method for degrading calcium oxalate according to claim 6 or 7, characterized in that, The fermentation time was 72 h.

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