Geobacillus habitoformans and application thereof

Abstract

This invention provides a *Bacillus hygroscopicus* strain and its application, relating to the field of microbial fermentation technology. The strain is named *Bacillus hygroscopicus* (…). Bacillus altitudinis Bacillus subtilis (W09), 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 December 26, 2025, with accession number CGMCC No. 37218. This invention provides Bacillus subtilis (W09). Bacillus altitudinis W09 can be used to increase the content of schisandrol A in the fermentation products of Schisandra chinensis vine stems.

Description

Technical Field

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

[0002] Schisandra chinensis ( Schisandra chinensis Schisandra chinensis (Turez.) Bail is the dried, mature fruit of *Schisandra chinensis* or *Schisandra sinensis*, belonging to the Magnoliaceae family. It is one of the authentic medicinal materials of Heilongjiang Province. Schisandra chinensis was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) and has the effects of astringing the lungs and promoting fluid production, tonifying the kidneys and nourishing the heart, and consolidating and strengthening the body. It is mainly used to treat symptoms such as cough and asthma due to lung deficiency, thirst due to fluid depletion, insomnia, and forgetfulness, and is a commonly used tonic in traditional Chinese medicine. Schisandrol A, as one of the main lignan active components in Schisandra chinensis, is an important material basis for its pharmacological effects. Modern pharmacological studies have shown that schisandrarol A has various biological activities, including hepatoprotective, anti-tumor, anti-inflammatory, antioxidant, immune regulation, and cognitive function improvement, and has good development and application prospects.

[0003] However, schisandrin A is mainly derived from Schisandra chinensis fruit, and its supply is insufficient due to limited total resources and the long growth cycle of the plant. In contrast, Schisandra chinensis vines, as a traditionally underutilized non-medicinal part, have relatively abundant resources, but their overall utilization rate is low. Recent systematic analyses of non-medicinal parts of Schisandra chinensis have shown that the vines and fruits have high similarity in chemical composition, both being rich in lignans, polysaccharides, terpenes, and flavonoids, among other active substances, and possess potential development value. Therefore, they are expected to be utilized as a novel plant source for schisandrin A.

[0004] Microbial fermentation technology, as an important means of modernizing and utilizing traditional Chinese medicine (TCM), can improve the cell wall breaking efficiency of plants through microbial metabolism and the action of secreted enzymes, promoting the release and transformation of active ingredients. It may also generate new active metabolites, thereby enhancing the comprehensive utilization rate of TCM resources. Given the large proportion of xylem and the dense cell wall structure in the stems of Schisandra chinensis, microbial fermentation to produce cellulase and related degrading enzymes holds promise for effectively degrading its lignocellulose structure, thereby promoting the release of active ingredients such as schisandrin A.

[0005] Highland Bacillus ( Bacillus altitudinisAs a functional strain with good enzyme-producing ability, it is mainly used in fields such as biological control, environmental remediation, and industrial enzyme fermentation. However, there are no reports on using *Bacillus hygroscopicus* to increase the content of schisandrol A in the fermentation products of *Schisandra chinensis* vine stems. Previous reports indicated that fermentation of *Schisandra chinensis* using *Bacillus subtilis* increased the content of the active ingredient schisandrol A by 37.06%. Compared with the above-mentioned prior art, this invention uses *Bacillus hygroscopicus* as the fermentation strain to ferment the non-medicinal part of *Schisandra chinensis* vine stems, achieving a 59% increase in the content of schisandrol A in the fermented vine stems. Compared with the prior art, the improvement of this invention is about 22 percentage points, achieving a significant performance leap, indicating that *Bacillus hygroscopicus* has good application potential in promoting the release and accumulation of schisandrol A in *Schisandra chinensis* vine stems. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention provides a highland Bacillus strain and its application, which can increase the content of schisandrol A in the fermentation products of Schisandra chinensis vine stems.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0008] This invention provides a strain of Bacillus hygroscopicus, named Bacillus hygroscopicus (B. hygroscopicus). Bacillus altitudinis W09 is 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. The deposit date is December 26, 2025, and the accession number is CGMCC No. 37218.

[0009] This invention provides a bacterial agent, the active ingredient of which is the above-mentioned Bacillus hygroscopicus.

[0010] 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.

[0011] This invention provides the application of Bacillus hygroscopicus in increasing the content of schisandrol A in the fermentation products of Schisandra chinensis vine stems.

[0012] The aforementioned Bacillus hygroscopicus can be used to increase the content of schisandrol A in the fermentation products of Schisandra chinensis vine stems.

[0013] This invention provides the application of the above-mentioned microbial agent in increasing the content of schisandrol A in the fermentation products of Schisandra chinensis vine stems.

[0014] The above-mentioned microbial agents can be used to increase the content of schisandrol A in the fermentation products of Schisandra chinensis vine stems.

[0015] This invention provides a method for increasing the content of schisandrol A in the fermentation product of Schisandra chinensis vine stems, using the above-mentioned Bacillus hygroscopicus or the above-mentioned inoculum to increase the content of schisandrol A in the fermentation product of Schisandra chinensis vine stems.

[0016] The use of Bacillus hygroscopicus of the present invention can increase the content of schisandrol A in the fermentation products of Schisandra chinensis vine stems.

[0017] Further, the method includes the following steps: inoculating Bacillus hygroscopicus or the above-mentioned inoculum into a culture medium containing Schisandra chinensis vine stems, and then fermenting and culturing it.

[0018] Furthermore, the inoculation amount of Bacillus hygroscopicus is 1-11%.

[0019] Furthermore, the fermentation culture temperature was 30℃.

[0020] Furthermore, the fermentation time was 3 days.

[0021] The above technical solution has the following beneficial effects: This invention uses *Bacillus hygroscopicus* WO9 to ferment the stems of *Schisandra chinensis*, and uses liquid chromatography to determine the changes in the content of schisandrol A in the samples before and after fermentation. The results show that after fermentation with *Bacillus hygroscopicus* WO9, the content of schisandrol A in the stems of *Schisandra chinensis* is significantly increased, with an increase rate of up to 59%. *Bacillus hygroscopicus* WO9 has good application potential in promoting the release and accumulation of schisandrol A in the stems of *Schisandra chinensis*, which helps to broaden the raw material sources of schisandrol A and alleviate the problem of insufficient raw material supply caused by limited total resources and long plant growth cycle. Attached Figure Description

[0022] Figure 1 The morphology of strain W09 on LB solid medium;

[0023] Figure 2 Phylogenetic tree of strain W09;

[0024] Figure 3 The figure shows the HPLC chromatogram of schisandrol A in Schisandra chinensis vine stem before fermentation, where 1) represents the chromatographic peak of schisandrol A in Schisandra chinensis vine stem before fermentation.

[0025] Figure 4 The figure shows the HPLC chromatogram of schisandrol A in the fermented Schisandra chinensis stem, where 2) represents the chromatographic peak of schisandrol A in the fermented Schisandra chinensis stem.

[0026] Figure 5 The graph shows the effect of strain W09 on increasing the content of schisandrin A in the fermentation products of Schisandra chinensis vine stems. Detailed Implementation

[0027] 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.

[0028] This invention obtains *Bacillus hygroscopicus* W09 from the roots of *Schisandra chinensis* using an enrichment culture method. After culturing on LB solid medium at 30°C for 48 h, the colonies of this strain are round, white, opaque, regularly shaped, with clear edges, a smooth, moist, and glossy surface, are not sticky, have weak flowability, and have regularly round, wrinkled protrusions in the center. At 30°C, this strain W09 can be used to increase the content of schisandrol A in the fermentation products of *Schisandra chinensis* vine stems, with an increase rate reaching 59%. This invention is the first to propose using *Bacillus hygroscopicus* W09 to increase the content of schisandrol A in the fermentation products of *Schisandra chinensis* vine stems.

[0029] 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.

[0030] 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.

[0031] 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. Reagents used, 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, unless otherwise specified, are all conventional products that can be purchased from legitimate channels. Unless otherwise specified, the solutions of this invention are prepared using water as the solvent.

[0032] The following is a description through specific embodiments.

[0033] Example 1

[0034] The strains were isolated and screened from the roots of Schisandra chinensis, including the following steps:

[0035] (1) Using the tissue separation method, the fresh roots of Schisandra chinensis (collected from Maoershan District, Heilongjiang Province) were rinsed clean under running water, and the water was blotted dry with sterile filter paper. Under sterile conditions, the roots were surface disinfected in the following order: soaked in 75% ethanol for 30-60 s, rinsed with sterile water 3-5 times, soaked in 5% sodium hypochlorite solution for 2 min, rinsed with sterile water 5-7 times, and the water was blotted dry with sterile filter paper. The disinfected Schisandra chinensis roots were cut into 0.5 cm segments with a sterilized knife. The disinfected Schisandra chinensis root samples were obtained by the above method.

[0036] (2) Take the Schisandra chinensis root sample after repeated disinfection 3 times and inoculate it on LB solid medium. Then place it in a 30℃ biochemical incubator and invert it for 2-5 days. Observe the growth of the strain until colonies grow.

[0037] (3) Take the colonies obtained in step (2), and when they grow well, pick the tip of the hyphae and inoculate them into a new LB solid medium. Incubate them upside down in a 30℃ biochemical incubator for 1-2 days for isolation and purification until a single colony is obtained. The strain is named W09 and preserved with glycerol.

[0038] Example 2

[0039] Strawberry strain W09 was streaked onto LB agar and incubated at 30°C for 48 h. The morphological characteristics of the colonies were then observed. Figure 1 As shown, the colonies are round, white and opaque on the front, with regular shape, clear edges, and wrinkled protrusions in the middle.

[0040] The white strain W09 obtained from the screening was sent to Shanghai Lingen Biotechnology Co., Ltd., which performed 16S rRNA gene sequencing using primers 27F / 1492R.

[0041] Primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1);

[0042] Primer 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO.2);

[0043] All the primers mentioned above were provided by Shanghai Lingen Biotechnology Co., Ltd.

[0044] The sequence of the 16S rRNA gene of strain W09 is shown below:

[0045]

[0046] The sequencing results were compared using BLAST on the NCBI website, and a phylogenetic tree was constructed, such as... Figure 2 As shown. Based on morphological observation and phylogenetic tree construction results, strain W09 was identified as belonging to Bacillus hygroscopicus (Bacillus hygroscopicus). Bacillus altitudinis ).

[0047] The strain W09 was named Bacillus hygroscopicus ( ). Bacillus altitudinis W09 is 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 December 26, 2025, with accession number CGMCCNo.37218.

[0048] Example 3

[0049] Using Bacillus hygroscopicus W09 to increase the content of schisandrin A in the fermentation products of Schisandra chinensis vine stems includes the following steps:

[0050] (1) Preparation of fermentation medium: Take 2.5 g of Schisandra chinensis stem powder (passed through a 60-mesh sieve), add water to 75 mL, put it into a 150 mL wide-mouth bottle, seal it with tissue culture sealing film and stopper it, put it in an autoclave at 121℃ for 20 min, and let it cool before use.

[0051] (2) Activation of bacterial strain: Bacillus hygroscopicus W09 was transferred to LB liquid medium and cultured at 30℃ and 140 r / min for 16 h until the enrichment bottle became turbid, thus obtaining a bacterial suspension. The activation of Bacillus hygroscopicus W09 was completed through the above steps.

[0052] (3) Preparation of seed culture: The bacterial suspension obtained in step (2) is inoculated into 50 mL of LB liquid culture medium at an inoculation amount of 5 mL and shaken at 30℃ and 140 r / min to obtain seed culture.

[0053] (4) Fermentation: Fermentation of the OD-dependent cells 600 The seed culture with a concentration of 0.6 was evenly inoculated into the fermentation medium prepared in step (1) at an inoculation rate of 5% (volume percentage). The wide-mouth bottle was sealed with tissue culture sealing film and placed in a shaker at 30℃ and 130 r / min for fermentation for 3 days. The fermented sample was freeze-dried to obtain pure fermented Schisandra chinensis vine stem sample powder.

[0054] Example 4

[0055] The content of schisandrol A in the pure fermented Schisandra chinensis vine stem sample powder obtained in Example 3 and in the Schisandra chinensis vine stem before fermentation was detected, including the following steps:

[0056] (1) Accurately weigh 0.1 g of the powdered Schisandra chinensis stem before fermentation and the powdered Schisandra chinensis stem after pure fermentation, place them in different 10 mL volumetric flasks, add 8 mL of methanol to each, and sonicate for 30 min at 100 W. After cooling to room temperature, dilute to 10 mL with methanol, shake well, filter through a 0.22 μm organic filter membrane, and take the filtrate for later use to prepare the test solution.

[0057] (2) The content of schisandrol A in the test sample solution was determined by liquid chromatography.

[0058] The content of schisandrol A in the test solution was detected using a Waters 2695 high performance liquid chromatograph (HPLC), an Ultimate® XB-C18 column (250 nm × 4.6 nm, 5 μm), and a UV (220 nm) detector. The liquid chromatography detection conditions were set as follows: mobile phase A was water, mobile phase C was acetonitrile, and the gradient elution conditions were as follows: 0-28 min: 51%-4% (A), 49%-96% (C); 28-31 min: 4% (A), 96% (C); 31-35 min: 4%-0% (A), 96%-100% (C); 35-38 min: 0%-51% (A), 100%-49% (C). All percentages are volume percentages. The flow rate was set to 0.5 mL / min, the injection volume was set to 10 μL, the detection wavelength was 220 nm, the column temperature was 30℃, the theoretical plate number was not less than 2000, and the resolution between adjacent peaks was greater than 1.5.

[0059] (3) Plotting the standard curve: Accurately weigh 10 mg of schisandrol A reference standard and place it in a 10 mL volumetric flask. Dilute to the mark with chromatographic grade methanol and shake well to prepare a reference standard solution with a mass concentration of 1 mg / mL. After shaking well, gradient dilute to prepare 7 different concentration reference standard solutions of 0.3 mg / g, 0.2 mg / g, 0.1 mg / g, 0.08 mg / g, 0.05 mg / g, 0.03 mg / g, and 0.01 mg / g. Filter the solutions through a 0.22 μm organic filter membrane and determine the peak area of ​​schisandrol A in the reference standard solution according to the chromatographic conditions in step (2). Plot the standard curve with the concentration of the reference standard solution (X) as the abscissa and the peak area (Y) as the ordinate.

[0060] (4) Substitute the peak area obtained by HPLC in step (2) into the standard curve obtained in step (3) to calculate the content of schisandrol A in the schisandra vine stem before and after fermentation.

[0061] Figure 3 and Figure 4The images show the HPLC chromatograms of schisandrol A in Schisandra chinensis vine stems before and after fermentation. The pre-fermentation sample, after lyophilization, was analyzed using the above method, and the calculated schisandrol A content was 4.31 mg / g (i.e., 4.31 mg of schisandrol A per gram of lyophilized sample). The post-fermentation sample, fermented for 3 days, was lyophilized and analyzed using the above method; the calculated schisandrol A content was 6.85 mg / g (i.e., 6.85 mg of schisandrol A per gram of lyophilized sample). Figure 3 and Figure 4 As can be seen, the chromatographic peak corresponding to schisandrin A in the fermented sample was significantly higher than that before fermentation.

[0062] Example 5

[0063] Pure-culture fermented Schisandra chinensis vine stem sample powder was prepared using the method of Example 3. The fermentation inoculum amounts in step (4) of Example 3 were set to 1%, 3%, 5%, 7%, 9%, and 11%, respectively. The fermented samples with different inoculum amounts were freeze-dried to obtain pure-culture fermented Schisandra chinensis vine stem sample powders with different inoculum amounts. The method for detecting the schisandrol A content in the pure-culture fermented Schisandra chinensis vine stem sample powder was the same as in Example 4, and the increase rate of schisandrol A content in the pure-culture fermented Schisandra chinensis vine stem sample powder was calculated using the following formula:

[0064] Increase rate = (Schisandrol A content in the sample after fermentation - Schisandrol A content in the sample before fermentation) / Schisandrol A content in the sample before fermentation × 100%.

[0065] Experimental results are as follows Figure 5 As shown, from Figure 5 It can be seen that, compared with the sample with an inoculum of 0%, the content of schisandrol A in the fermentation product of Schisandra chinensis vine stems fermented by Bacillus hygroscopicus W09 increased at different inoculum amounts. Among them, the highest increase rate of schisandrol A content in the fermentation product of Schisandra chinensis vine stems by Bacillus hygroscopicus W09 was 59% when the fermentation inoculum amount was 5%.

[0066] 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 highland Bacillus ( Bacillus altitudinis W09, characterized in that, The accession number is CGMCCNo.37218.

2. A microbial agent, characterized in that, The active ingredient is the Bacillus hygroscopicus described in claim 1.

3. The application of the Bacillus hygroscopicus of claim 1 in increasing the content of schisandrol A in the fermentation products of Schisandra chinensis vine stems.

4. The application of the microbial agent according to claim 2 in increasing the content of schisandrol A in the fermentation products of Schisandra chinensis vine stems.

5. A method for increasing the content of schisandrol A in the fermentation products of Schisandra chinensis vine stems, characterized in that, The content of schisandrol A in the fermentation product of Schisandra chinensis vine stem can be increased by using the Bacillus hygroscopicus of claim 1 or the inoculum of claim 2.

6. The method for increasing the content of schisandrol A in the fermentation product of Schisandra chinensis vine stem according to claim 5, characterized in that, The Bacillus hygroscopicus of claim 1 or the inoculum of claim 2 is inoculated into a fermentation medium containing Schisandra chinensis vine stems, and fermented.

7. The method for increasing the content of schisandrol A in the fermentation product of Schisandra chinensis vine stem according to claim 6, characterized in that, The inoculation rate of Bacillus hygroscopicus is 1-11%.

8. The method for increasing the content of schisandrol A in the fermentation product of Schisandra chinensis vine stem according to claim 6, characterized in that, The fermentation culture temperature is 30℃.

9. The method for increasing the content of schisandrol A in the fermentation product of Schisandra chinensis vine stem according to claim 6, characterized in that, The fermentation culture time was 3 days.

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