Space bacillus subtilis CEGF-TKJ-011 and application thereof

Abstract

The application discloses space bacillus subtilis CEGF-TKJ-011 and application thereof. The space bacillus subtilis CEGF-TKJ-011 is obtained through space mutagenesis, has a preservation number of CCTCC NO: M 2026269, and is classified and named as Bacillus subtilis subsp. subtilis CEGF-TKJ-011. The space strain belongs to Bacillus subtilis subsp. subtilis, and the subspecies is not 'natto bacteria' with a natural secretion natto kinase capacity. However, the strain not only has a natural high secretion natto kinase capacity, but also can utilize non-soy and / or soy substrates to ferment and prepare natto kinase, and can be applied to preparation of natto kinase.

Description

Technical Field

[0001] This invention belongs to the field of microbial preservation and fermentation engineering technology, and more specifically, relates to a space-brood Bacillus subtilis CEGF-TKJ-011 and its applications. Background Technology

[0002] Nattokinase is a serine protease produced by Bacillus subtilis during the fermentation of natto. Nattokinase can directly degrade fibrin, the main scaffold of thrombi, breaking down insoluble fibrin into soluble peptides. Simultaneously, it lowers the level of plasminogen activator inhibitor-1 (PAI-1), thus enhancing the body's own thrombolytic ability in a two-pronged approach. Due to its highly efficient and safe thrombolytic properties and other beneficial characteristics, it has been widely researched and applied in the fields of dietary supplements and pharmaceuticals.

[0003] However, the natural secretor of nattokinase is not the common Bacillus subtilis, but a specific subspecies of Bacillus subtilis natto or its closely related subspecies (Bacillus subtilis var. natto), commonly referred to as "natto bacteria". Examples include strain LNUB236 of Bacillus subtilis natto in patent CN101560478A, strain N14 of Bacillus subtilis natto in patent CN115678793A, and strain ODK-BN1 of Bacillus subtilis natto in patent CN101731706A. However, wild-type Bacillus subtilis or other common Bacillus subtilis subsp. subtilis usually do not have the natural ability to secrete nattokinase. For example, the classic model bacterium Bacillus subtilis 168 (classified as Bacillus subtilis subsp. subtilis), which is commonly used in the laboratory, does not secrete nattokinase.

[0004] Space mutagenesis refers to the treatment of microorganisms in the unique space environment in order to induce genetic variations that are difficult to obtain using conventional methods on Earth. However, the effects of space mutagenesis on strain performance are random, and current research on space-induced Bacillus subtilis is still limited. Summary of the Invention

[0005] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a space-bred Bacillus subtilis CEGF-TKJ-011 strain and its applications. The purpose is to discover a new strain of Bacillus subtilis subsp. subtilis CEGF012 (accession number CCTCC NO: M 20241702) that exhibits high natural nattokinase production capacity through space mutagenesis and screening. This strain has been identified as a new strain of Bacillus subtilis subsp. subtilis and named space-bred Bacillus subtilis CEGF-TKJ-011. It has been deposited with accession number CCTCC NO: M 2026269 and classified as Bacillus subtilis subsp. subtilis CEGF-TKJ-011 (also known as CEGF-TKJ-011 Bacillus subtilis subsp. subtilis), with a deposit date of January 28, 2026. This addresses the shortcomings of existing Bacillus subtilis subsp. subtilis... The technical problem of the low natural nattokinase secretion capacity of the *Subtilis subsp. Subtilis* strain.

[0006] To achieve the above objectives, according to the first aspect of the present invention, a space-bringing Bacillus subtilis CEGF-TKJ-011 is provided, with accession number CCTCC NO: M 2026269, classified as Bacillus subtilis subsp. subtilis CEGF-TKJ-011 (also known as CEGF-TKJ-011 Bacillus subtilis subsp. subtilis), and accession date of January 28, 2026.

[0007] Preferably, the space-bred Bacillus subtilis CEGF-TKJ-011 is obtained by space mutagenesis and screening of Bacillus subtilis subsp. subtilis CEGF012 strain with accession number CCTCC NO: M 20241702, and its 16S rDNA sequence is shown in SEQ ID NO:1.

[0008] According to a second aspect of the present invention, the application of Bacillus subtilis CEGF-TKJ-011 as described in the present invention in the preparation of nattokinase is also provided.

[0009] Preferably, in the application, the space-bred Bacillus subtilis CEGF-TKJ-011 is used to prepare nattokinase, specifically as follows:

[0010] Using Bacillus subtilis CEGF-TKJ-011 as the seed culture, the culture was passaged in a culture medium until the 4th to 10th generations. The supernatant was collected by centrifugation and filtered through a nanofiltration membrane to remove the Bacillus subtilis cells, resulting in a solution containing nattokinase.

[0011] Preferably, in the application, the culture medium is LB medium, and the culture is carried out to the 4th to 8th generation.

[0012] Preferably, in the application, the culture medium is LB medium, and the culture is carried out to the 4th to 6th generation.

[0013] Preferably, in the application, the space-bred Bacillus subtilis CEGF-TKJ-011 is used in the fermentation of Cistanche deserticola to prepare nattokinase, specifically as follows:

[0014] Using the space-borne Bacillus subtilis CEGF-TKJ-011 described in this invention as the fermentation strain, it is inoculated into Cistanche deserticola culture medium for fermentation to obtain a fermentation product containing nattokinase, or after fermentation, the fermentation product is taken and centrifuged to obtain a supernatant containing nattokinase; the Cistanche deserticola culture medium includes Cistanche deserticola and is used as a fermentation substrate.

[0015] Preferably, in the application, the OD of the space-bred Bacillus subtilis CEGF-TKJ-011 bacterial suspension is... 600 The inoculation rate is 1-2, and the inoculation amount is 5%-10% in the Cistanche deserticola culture medium for fermentation.

[0016] Preferably, in the application, the OD of the space-bred Bacillus subtilis CEGF-TKJ-011 bacterial suspension is... 600 The concentration was 1.5, and 6% was inoculated into the Cistanche deserticola culture medium for fermentation.

[0017] According to a third aspect of the present invention, a fermentation product having nattokinase activity is also provided, which is prepared by the following method:

[0018] The *Bacillus subtilis* CEGF-TKJ-011, as described in this invention, is used as the fermentation strain, and the product is obtained through fermentation substrates. The substrates include *Cistanche deserticola* and / or legumes, such as one or more of *Cistanche deserticola*, soybean meal, chickpeas, black beans, and tiger peas. Preferably, the substrates, by weight percentage, comprise 50%–75% *Cistanche deserticola* and 25%–50% legumes.

[0019] In summary, compared with the prior art, the technical solutions conceived in this invention, by obtaining a new strain with the ability to naturally secrete nattokinase through space mutagenesis and screening of the Bacillus subtilis subsp. subtilis CEGF012 strain with accession number CCTCC NO: M 20241702, can achieve the following beneficial effects:

[0020] Bacillus subtilis subsp. subtilis is not a "natto bacterium" with the natural ability to secrete nattokinase. However, the space-bred Bacillus subtilis CEGF-TKJ-011 provided in this invention belongs to Bacillus subtilis subsp. subtilis, which not only has the natural ability to secrete high levels of nattokinase, but can also prepare nattokinase by fermentation using non-soybean substrates such as Cistanche deserticola. The fermented product obtained by fermenting Cistanche deserticola using this space-bred Bacillus subtilis CEGF-TKJ-011 has an nattokinase activity of up to 77736 IU / g. Attached Figure Description

[0021] Figure 1 The effects of fermentation on enzyme production by different ratios of Cistanche deserticola and soybean meal were investigated.

[0022] Figure 2 Comparison of nattokinase activity secreted by Bacillus subtilis subsp. subtilis CEGF012 before and after space-induced mutagenesis.

[0023] Figure 3 This study compares the flavonoid content after fermentation by different Bacillus subtilis strains.

[0024] Figure 4 The content of phenylethanol glycosides after fermentation by different Bacillus subtilis strains.

[0025] Figure 5 The polysaccharide content after fermentation of different Bacillus subtilis strains. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0027] The inventors previously isolated and purified bacterial strains from the roots of poplar forests in the Taklamakan Desert of Xinjiang. From the isolated strains, a strain with high activity and good activity consistency was screened out. The strain was identified as a new strain of Bacillus subtilis by 16S rRNA and named Bacillus subtilis subsp. subtilis CEGF012. It was deposited on July 31, 2024, at the China Center for Type Culture Collection (CCCCC) at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province (Wuhan University Collection Center), with accession number CCTCC NO: M 20241702.

[0028] Furthermore, the *Bacillus subtilis* subsp. *subtilis* CEGF012 strain with accession number CCTCC NO: M 20241702 was carried into space aboard the "Shijian-19" spacecraft for mutagenesis. After mutagenesis, the samples were retrieved and stored at 4°C. Based on criteria such as faster colony growth, larger colonies, and significant differences in color and morphology compared to the originating strain (*Bacillus subtilis* subsp. *subtilis* CEGF012), 247 mutant strains were initially selected. Further screening was conducted, and based on strain performance measurements, 50 space-bred *Bacillus subtilis* strains exhibiting single or combined advantages in growth, enzyme production, or environmental adaptability were selected.

[0029] The re-screened space-induced Bacillus subtilis strains were passaged multiple times in test tubes (e.g., 10 generations). Unstable strains were eliminated during passage. Nattokinase activity and bioactivity were tested in F2, F4, F6, F8, and F10 generations. The performance stability of the space-induced mutant strains after multiple passages was compared, and the space-induced Bacillus subtilis strain with the best performance and most stable enzyme production was selected. This strain, corresponding to number 6, was deposited on January 28, 2026, at the China Center for Type Culture Collection (CTCC), No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province (Wuhan University Collection Center). Its accession number is CCTCC NO: M 2026269, and the deposit date is January 28, 2026. It is classified and named *Bacillus subtilis* subsp. *subtilis* CEGF-TKJ-011 (also known as CEGF-TKJ-011 *Bacillus subtilis*). subsp. subtilis).

[0030] Based on this, the present invention provides a space-bred Bacillus subtilis CEGF-TKJ-011, with accession number CCTCCNO: M 2026269, classified as Bacillus subtilis subsp. subtilis CEGF-TKJ-011 (also known as CEGF-TKJ-011 Bacillus subtilis subsp. subtilis), and accession date of January 28, 2026.

[0031] The space-bred Bacillus subtilis CEGF-TKJ-011 provided by this invention belongs to Bacillus subtilis subsp. subtilis. While Bacillus subtilis subsp. subtilis is not a "natto bacterium" with the natural ability to secrete nattokinase, this space-bred Bacillus subtilis CEGF-TKJ-011 not only possesses the ability to naturally secrete nattokinase with high nattokinase activity, but it can also utilize non-soybean substrates such as Cistanche deserticola to produce high-yield nattokinase. In some embodiments, when the space-bred Bacillus subtilis CEGF-TKJ-011 is passaged to the 4th generation, the naturally secreted nattokinase activity of the strain can reach 4399.0 IU / mL. The fermented product obtained by fermenting Cistanche deserticola using this space-bred Bacillus subtilis CEGF-TKJ-011 has a nattokinase activity of up to 77736 IU / g.

[0032] The space-bred Bacillus subtilis strain CEGF012 with accession number CCTCC NO: M 20241702 was obtained by space mutagenesis and screening aboard the "Shijian-19" spacecraft. Its 16S rDNA sequence is shown in SEQ ID NO:1.

[0033] Furthermore, it has been discovered that the space-bred Bacillus subtilis CEGF-TKJ-011 provided by this invention not only has the ability to naturally secrete nattokinase, but can also prepare nattokinase by fermentation using non-soybean substrates such as Cistanche deserticola.

[0034] In addition, the present invention also provides the application of Bacillus subtilis CEGF-TKJ-011 as described in the present invention in the preparation of nattokinase.

[0035] In some embodiments, the space-borne Bacillus subtilis CEGF-TKJ-011 is used to prepare nattokinase, as follows:

[0036] Using the aforementioned space-bred Bacillus subtilis CEGF-TKJ-011 as the seed culture, the bacteria were passaged in LB medium for 4 to 10 generations. The supernatant was collected by centrifugation and filtered through a 0.22 μm filter membrane to remove the bacterial cells, resulting in a solution containing nattokinase.

[0037] The passage culture is preferably cultured to the 4th to 8th generation, and the supernatant is collected by centrifugation; more preferably cultured to the 4th to 6th generation, and the supernatant is collected by centrifugation; especially cultured to the 4th generation, and the supernatant is collected by centrifugation.

[0038] In some embodiments, the culture was carried out at an inoculum rate of 1% in LB medium until the 4th generation. The supernatant was collected by centrifugation and filtered through a 0.22 μm filter membrane, yielding nattokinase with an enzyme activity of up to 4399.0 IU / mL. The LB medium contained 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride.

[0039] In some embodiments, the space-bred Bacillus subtilis CEGF-TKJ-011 is applied to the fermentation of Cistanche deserticola to prepare nattokinase, as detailed below:

[0040] Using the aforementioned space-brood Bacillus subtilis CEGF-TKJ-011 as the fermentation strain, it was inoculated into Cistanche deserticola culture medium for fermentation to obtain a fermentation product containing nattokinase.

[0041] For example, the bacterial culture of the aforementioned space-brood Bacillus subtilis CEGF-TKJ-011 can be used as a seed culture and inoculated into Cistanche deserticola culture medium for fermentation to obtain a fermentation product containing nattokinase. Alternatively, after fermentation, the fermentation product can be taken and centrifuged to obtain a supernatant containing nattokinase.

[0042] The OD of the space-borne Bacillus subtilis CEGF-TKJ-011 bacterial suspension 600 The inoculum is 1-2, and 5%-10% is inoculated into the Cistanche deserticola culture medium for fermentation. In some embodiments, the OD of the *Bacillus subtilis* CEGF-TKJ-011 bacterial suspension is... 600 The concentration was 1.5, and 6% was inoculated into Cistanche deserticola soybean meal culture medium. Fermentation was carried out at 37℃ for 24 hours to obtain a fermentation product containing nattokinase. The fermentation product was centrifuged, and the supernatant was used to detect the nattokinase activity. The enzyme activity of nattokinase in the fermentation product was found to be up to 77736 IU / g.

[0043] This invention also provides a fermentation product with nattokinase activity, which is prepared according to the following method:

[0044] The *Bacillus subtilis* CEGF-TKJ-011, as described in this invention, is used as the fermentation strain, obtained through a fermentation substrate. The substrate includes *Cistanche deserticola* and / or legumes, wherein the legumes can be selected from one or more of soybean meal, black soybeans, chickpeas, and tiger peas. Preferably, the substrate, by weight percentage, comprises 50%–75% *Cistanche deserticola* and 25%–50% legumes.

[0045] The following are examples.

[0046] Example 1: Screening of space-borne Bacillus subtilis

[0047] 1. Experimental Materials

[0048] 1.1 The starting strain, namely the original strain Bacillus subtilis subsp. subtilis CEGF012, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20241702 and deposit date of July 31, 2024.

[0049] 1.2 Fermentation medium (LB medium): 10g protein extract, 5g yeast extract, 10g NaCl, 800mL water, adjust pH to 7.5 with NaOH, add water to make up to 1000mL, autoclave at 121℃ for 20min.

[0050] 2. Experimental Methods

[0051] 2.1 Space mutagenesis treatment of bacterial strains

[0052] The freshly cultured Bacillus subtilis subsp. subtilis CEGF012 was streaked on a culture medium slant, the cap was tightened, and the sample was sent into space aboard the "Shijian-19" spacecraft for mutagenesis. After space mutagenesis, the sample was retrieved and stored in a 4°C refrigerator for later use.

[0053] 2.2 Isolation and screening of space-bred Bacillus subtilis

[0054] (1) Initial screening: The Bacillus subtilis strain returned from space mutagenesis in section 2.1 was resuspended in phosphate buffer, isolated by the plate dilution method, and plated on LB agar plates and incubated at 37°C for 24 hours. Based on the criteria of faster colony growth, larger colonies, and significant differences in color and morphology from the original strain (Bacillus subtilis subsp. subtilis CEGF012), 247 mutant strains were initially selected. After multiple streaking isolations, the obtained single colonies were numbered 1-247 and stored on nutrient agar slants. After the colonies grew, they were stored at 4°C for later use.

[0055] (2) Secondary screening: The performance of the strains was determined by inoculating a loopful of the initially screened strains into a test tube containing LB medium and activating it for 18 hours. Then, the strains were inoculated at a rate of 3% into a 250 mL Erlenmeyer flask containing 50 mL of fermentation medium (LB medium) and cultured at 37 °C and 200 r / min for 72 hours. After fermentation, the levels of nattokinase, protease, biofilm, and OD were measured. 600 The specific detection methods for nattokinase are as follows:

[0056] Agarose fibrin plate method: Take an agarose fibrin plate and use the stainless steel tube of a hole punch to vertically punch holes in the fibrin plate. Maintain a 15mm distance between holes to prevent the fusion rings from crossing and affecting the measured diameter. Accurately measure 10µL of the sample solution using a pipette and spot it, simultaneously preparing 1-2 parallel samples. Spot the urokinase standard curve series and the sample solution on the same fibrin plate, and accurately label the sample numbers. After spotting, cover the plate and incubate at 37℃ for 18 hours. Immediately after removing the plate, begin measuring the fusion ring diameter.

[0057] The specific detection of protease is as follows:

[0058] Add sterile skim milk powder (final concentration 1-2%, w / v) to LB agar medium cooled to approximately 50°C after sterilization, mix quickly, and pour onto plates. Take 1-2 μL of fresh bacterial suspension (logarithmic growth phase) of the test strain and spot it in the center of the seed plate using a sterile toothpick or micropipette. Invert the plate and incubate at a suitable temperature (e.g., 37°C) for 24-48 hours. Observe whether a clear zone appears around the colony and take a photograph. Measure the diameter of the clear zone (total diameter of the clearly defined area including the colony) and the diameter of the colony (diameter of the bacterial growth area). Calculate the ratio: Hydrolysis zone ratio = Clear zone diameter / Colony diameter.

[0059] The specific detection methods for biofilms are as follows:

[0060] Dilute the overnight cultured bacterial suspension with fresh culture medium to an OD600 of approximately 0.05–0.1. Add 200 μL of the diluted bacterial suspension to the wells of a sterile 96-well polystyrene plate. Set up duplicate wells (at least 3) and a blank control well containing only culture medium. Incubate at a suitable temperature for 24–48 hours. Carefully aspirate or discard any floating bacterial suspension from the wells.

[0061] Gently rinse the well walls three times with 200 μL of 1X PBS to remove any unattached airborne bacteria. Note: Avoid damaging the biofilm at the bottom of the well. Air dry at room temperature or allow to air dry naturally. Add 200 μL of 0.1% crystal violet solution to each well and stain for 15-20 minutes at room temperature. Discard the staining solution and rinse thoroughly with deionized water or PBS until the washings are colorless. Air dry. Add 200 μL of 95% ethanol (or 33% glacial acetic acid solution) to each well and incubate at room temperature for 15-30 minutes to allow the dye to dissolve completely.

[0062] After mixing the solution in each well by pipetting, transfer 100-150 μL to a new 96-well plate (or measure directly in the original plate, but ensure the bottom of the well is clean). Measure the absorbance at OD570 nm or OD590 nm using a microplate reader.

[0063] OD 600 The specific testing procedure is as follows: Add 200 μL of the overnight culture to the wells of a sterile 96-well polystyrene plate. Set up replicates (at least 3 wells) and a blank control well containing only the culture medium. Measure the absorbance at OD600 nm using a microplate reader.

[0064] Based on nattokinase, protease, biomembrane and OD 600 The test data were analyzed, and the original data and plate photos of these strains were manually reviewed following the process of initial screening based on individual indicators and verification of biological characteristics. Strains with high total scores but abnormal indicators (such as extremely high biofilm, strange colony morphology, or suspected contamination) were removed. As a result, 50 space-mutated strains with single or combined advantages in growth, enzyme production, or environmental adaptability were selected, as shown in Table 1.

[0065] Table 1 Results of rescreening of Bacillus subtilis in space

[0066]

[0067]

[0068] In Table 1, “CEGF012” refers to the originating strain Bacillus subtilis subsp. subtilis CEGF012, whose accession number is CCTCC NO: M 20241702.

[0069] (3) Secondary screening: The 50 dominant space-mutated strains selected in Table 1 and the starting strain Bacillus subtilis subsp. subtilis CEGF012 were separately inoculated into a fermentation medium with Cistanche deserticola as the sole carbon and nitrogen source for small-scale fermentation, and the viable cell count was detected at different time points. Preparation of the fermentation medium: 10g of Cistanche deserticola powder was weighed, dissolved in 1L of purified water, and autoclaved at 121℃ for 20min. The powder was inoculated into the fermentation medium at a 6% inoculation rate and incubated on a shaker at 200rpm at 37℃ for 24h. Samples were taken to detect the viable cell count, and the results are shown in Table 2.

[0070] Table 2 Viable cell count after 24 hours of fermentation

[0071]

[0072] The viable cell count of the starting strain, Bacillus subtilis subsp. subtilis CEGF012, after 24 hours of fermentation was 80 × 10⁻⁶. 7 CFU / mL.

[0073] According to the fermentation time of 24 hours, the number of viable bacteria is greater than or equal to 2.5 × 10⁻⁶. 7 The CFU / mL screening of space-induced mutant strains yielded 10 strains, which are identified as space-induced mutant strains numbered 1, 2, 6, 40, 46, 87, 114, 141, 179 and 217.

[0074] (4) Stability test of space Bacillus subtilis strain

[0075] Ten space-bred Bacillus subtilis strains selected after secondary screening, along with the starting strain Bacillus subtilis subsp. subtilis CEGF012, were inoculated from glycerol storage tubes into 3 mL of liquid culture medium and cultured at 37°C for 12–16 hours to obtain the first-generation seed culture.

[0076] Each strain was individually inoculated into test tubes and passaged multiple times to eliminate strains with unstable traits during passage. The specific passage culture process is as follows: Each strain will be treated independently and in parallel as a complete passage cycle.

[0077] First generation inoculation: Vortex the above seed culture to mix well. Using a sterile pipette, inoculate 50 μL (1% inoculation volume, v / v) into tube A containing 5 mL of fresh LB medium (containing 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride). Incubate at 37°C for 24 hours. Take an appropriate amount of the bacterial culture, centrifuge to collect the supernatant, filter through a 0.22 μm filter membrane for determining nattokinase activity, and use the remaining bacterial culture as the "seed culture" for the next generation.

[0078] Repeated subculturing: Take 50 μL of bacterial culture from the freshly cultured tube and inoculate it into another tube (B) containing 5 mL of fresh LB medium. Incubate at 37°C for 24 hours. Take an appropriate amount of bacterial culture, centrifuge to collect the supernatant, filter it through a 0.22 μm filter membrane for determining nattokinase activity, and use the remaining bacterial culture as the "seed culture" for the next generation.

[0079] Repeat this process until the preset number of passages is completed. Ensure that the inoculum size, culture medium volume, and culture conditions are completely consistent each time. At least two independent parallel passages should be set up for each strain (including the starting strain) to assess operational errors and random fluctuations.

[0080] In this embodiment, each strain was cultured to the 10th generation (F10), and the nattokinase activity of the F2, F4, F6, F8, and F10 generations was detected. The performance stability changes of the space-induced Bacillus subtilis strains after multiple passages were compared, and the space-induced mutant strains with the best performance and the most stable enzyme production were screened out. The nattokinase activity of each strain after multiple passages is shown in Table 3.

[0081] Table 3 Results of Nattokinase Activity Assay

[0082]

[0083] In Table 3, “CEGF012” refers to the originating strain Bacillus subtilis subsp. subtilis CEGF012, whose accession number is CCTCC NO: M 20241702.

[0084] Using the nattokinase activity of the starting strain Bacillus subtilis subsp. subtilis CEGF012 as a reference, strains with significantly increased enzyme activity were screened. The results showed that the enzyme activities of space-mutated strains numbered 6 (16S rDNA sequence as shown in SEQ ID NO: 1) and 114 (16S rDNA sequence as shown in SEQ ID NO: 2) were significantly higher than those of the starting strain Bacillus subtilis subsp. subtilis CEGF012. Among the F2, F4, F6, F8 and F10 generations, the F4 generation space-mutated strain had the highest nattokinase activity, and it still had a high enzyme activity (1114.10 IU / mL) even after 10 generations. In particular, the nattokinase activity of the space-mutated strain numbered 6 was significantly increased and the nattokinase activity was high. On January 28, 2026, the space-bred Bacillus subtilis strain corresponding to accession number 6 was deposited at the China Center for Type Culture Collection (Wuhan University Collection Center), No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M2026269 and classification name Bacillus subtilis subsp. subtilis CEGF-TKJ-011 (also known as CEGF-TKJ-011 Bacillus subtilis subsp. subtilis). The deposit date was January 28, 2026.

[0085] Example 2

[0086] Soybean meal and Cistanche deserticola were soaked in pure water until saturated. A fermentation medium (100mL Erlenmeyer flask) was prepared by mixing Cistanche deserticola and soybean meal in a specific ratio. 50mL of solid fermentation medium (30g weight) was used, with Cistanche deserticola comprising 100%, 99%, 90%, 83%, 75%, 50%, and 0% of the total Cistanche deserticola by weight. The medium was autoclaved at 121℃ for 20min. A 6% inoculum of *Bacillus subtilis* seed culture (CEGF-TKJ-011, preservation number CCTCC NO: M 2026269) was inoculated and cultured at 37℃ for 24h. 5g of the fermentation product was weighed and centrifuged at 5000rpm for 5min. The supernatant was used to detect nattokinase activity. The experimental results are as follows: Figure 1 The specific enzyme activity assay data are shown in Table 4.

[0087] Table 4. Enzyme production effect of fermentation with different ratios of Cistanche deserticola and soybean meal.

[0088]

[0089] Table 4 shows that nattokinase was detectable in the fermentation product of this strain using a medium containing 100% Cistanche deserticola (i.e., pure Cistanche deserticola medium), indicating that this space-bred Bacillus subtilis CEGF-TKJ-011 can produce nattokinase using pure Cistanche deserticola medium. Compared to pure Cistanche deserticola medium, the enzyme production capacity of this strain was significantly improved using a fermentation medium combining Cistanche deserticola and soybean meal, especially when the mass ratio of Cistanche deserticola was 50%–75%, and the mass ratio of Cistanche deserticola to soybean meal was 1:1. Compared to pure soybean meal medium, the fermentation medium using a combination of Cistanche deserticola and soybean meal achieved enzyme production effects comparable to or even better than those using pure soybean meal medium. With a mass ratio of Cistanche deserticola to soybean meal of 1:1, the enzyme activity in the Erlenmeyer flask medium was 5667.569 IU / g, representing an improvement of more than three times compared to fermentation using pure soybean meal medium.

[0090] Example 3: Scale-up Fermentation Verification

[0091] Culture medium preparation: Weigh 250g of Cistanche deserticola and 250g of soybean meal, add 1250g of purified water, soak for 2 hours, stir evenly, and sterilize in an autoclave at 121°C for 20 minutes to obtain Cistanche deserticola and soybean meal culture medium, which is used as fermentation culture medium.

[0092] Seed culture preparation: Using the starting strain Bacillus subtilis subsp. subtilis CEGF012 as a control, single colonies of Bacillus subtilis numbered 6 and 114 were picked and inoculated into LB liquid medium, and cultured at 37°C with shaking at 120 rpm for 12-16 hours until the late logarithmic growth phase (OD200). 600 ≈1.5), as seed liquid.

[0093] Inoculation and Fermentation: After the Cistanche deserticola soybean meal culture medium has cooled naturally, inoculate the seed liquid at 6% of its weight and stir well. Place it in an incubator and ferment at 37℃ for 24 hours. Weigh 5g of the fermentation material into a centrifuge tube, centrifuge at 5000rpm for 5min, and take the supernatant to detect nattokinase activity, flavonoid content, phenylethyl glycoside content, and polysaccharide content. The results are as follows: Figures 2 to 5 As shown. Among them. Figure 2 Comparison of nattokinase activity secreted by Bacillus subtilis subsp. subtilis before and after space-induced mutagenesis. Figure 3 To compare the flavonoid content after fermentation by different Bacillus subtilis strains, Figure 4 The content of phenylethanol glycosides after fermentation by different Bacillus subtilis strains, Figure 5 The polysaccharide content after fermentation of different Bacillus subtilis strains.

[0094] Although *Bacillus subtilis* is the natural host of nattokinase, only specific subspecies (varieties) possess this ability. Other common *Bacillus subtilis* strains (such as wild-type) typically do not have the natural secretion capacity; for example, conventional laboratory-grade *Bacillus subtilis* strains (such as WB800 and 168) do not naturally secrete nattokinase. Therefore, not all *Bacillus subtilis* strains can secrete nattokinase. Nattokinase products labeled as "produced by *Bacillus subtilis*" on the market are often genetically engineered products, not naturally secreted nattokinase. In fact, currently only *Bacillus subtilis* natto, or its closely related pathogenic subspecies *Bacillus subtilis* (natto), can naturally secrete nattokinase.

[0095] Depend on Figure 2 The results showed that the starting strain, *Bacillus subtilis* subsp. *subtilis* CEGF012, was identified as *Bacillus subtilis* subsp. *subtilis*, which possesses the ability to secrete nattokinase. Compared to the starting strain *Bacillus subtilis* subsp. *subtilis* CEGF012, the nattokinase activity of the space-maligned *Bacillus subtilis* strains numbered 6 and 114, selected after space mutagenesis, was significantly increased. Specifically, the nattokinase activity in the fermentation product of strain number 6 reached 77736 IU / g (based on the dry weight of the fermentation product). There was no statistically significant difference in nattokinase activity between the space-maligned *Bacillus subtilis* strains numbered 6 and 114. Therefore, the space-maligned *Bacillus subtilis* strains numbered 6 and 114, selected after space mutagenesis, have a significantly superior natural ability to secrete nattokinase. The space-maligned *Bacillus subtilis* strain numbered 6 was identified as belonging to the *Bacillus subtilis* subsp. *subtilis*.

[0096] Depend on Figure 3 The results showed that, compared with the original strain Bacillus subtilis subsp. subtilis CEGF012, the flavonoid content of the space-maligned Bacillus subtilis corresponding to number 114 after space mutagenesis was not statistically different from that of the original strain Bacillus subtilis subsp. subtilis CEGF012. However, the flavonoid content of the space-maligned Bacillus subtilis corresponding to number 6 after space mutagenesis was significantly increased after fermentation.

[0097] Depend on Figure 4The results showed that, compared with the original strain Bacillus subtilis subsp. subtilis CEGF012, the phenylethanol glycoside content of Bacillus subtilis corresponding to space mutagenesis (number 114) after fermentation was not statistically different from that of the original strain Bacillus subtilis subsp. subtilis CEGF012. However, the phenylethanol glycoside content of Bacillus subtilis corresponding to space mutagenesis (number 6) after fermentation was significantly increased.

[0098] Depend on Figure 5 The results showed that, compared with the original strain Bacillus subtilis subsp. subtilis CEGF012, the polysaccharide content of space-bred Bacillus subtilis corresponding to number 114 after fermentation was not statistically different from that of the original strain Bacillus subtilis subsp. subtilis CEGF012. However, the polysaccharide content of space-bred Bacillus subtilis corresponding to number 6, which was screened after space mutagenesis, was significantly reduced after fermentation.

[0099] Flavonoids and phenylethanoid glycosides in Cistanche deserticola often form complexes with polysaccharides or are "encapsulated" within the cell wall by polysaccharides. For example, Cistanche deserticola phenylethanoid glycosides (such as cistanche glycosides) typically have a structure of aglycone (flavonoid / phenylethanoid glycoside) + sugar chain, resulting in poor water solubility and difficulty in release. To promote the dissolution of flavonoids and phenylethanoid glycosides in Cistanche deserticola, the processing method is crucial. The most classic method is water soaking followed by stewing. Prolonged stewing and soaking degrade polysaccharides, promoting the dissolution of active ingredients such as flavonoids and phenylethanoid glycosides. However, prolonged stewing and soaking may lead to the degradation of heat-sensitive components such as flavonoid glycosides due to excessively high temperatures.

[0100] After fermenting Cistanche deserticola with Bacillus subtilis (number 6), it was found that the content of flavonoids and phenylethanoid glycosides in the fermentation supernatant increased significantly, while the content of polysaccharides decreased significantly. It is speculated that Bacillus subtilis (number 6) can act as a polysaccharide degrading agent to degrade polysaccharides, destroy the complex structure formed by flavonoids or phenylethanoid glycosides and polysaccharides in Cistanche deserticola, thereby promoting the release of active ingredients flavonoids and phenylethanoid glycosides and improving their bioavailability.

[0101] The *Bacillus subtilis* strain corresponding to accession number 6 was deposited on January 28, 2026, at the China Center for Type Culture Collection (CCCCC), No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province (Wuhan University Collection Center). Its accession number is CCTCC NO: M 2026269, and its classification name is *Bacillus subtilis subsp. Subtilis* CEGF-TKJ-011 (also known as CEGF-TKJ-011 *Bacillus subtilis subsp. subtilis*). This strain (*Bacillus subtilis subsp. Subtilis* CEGF-TKJ-011) not only possesses the ability to naturally secrete high levels of nattokinase, but also can prepare nattokinase through fermentation using non-soybean and / or soybean substrates, enabling its application in the preparation of nattokinase.

[0102] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A space-bred Bacillus subtilis CEGF-TKJ-011, characterized in that, Its accession number is CCTCC NO: M2026269, its classification name is Bacillus subtilis subsp. subtilis CEGF-TKJ-011, and its accession date is January 28, 2026; The space-bred Bacillus subtilis CEGF-TKJ-011 was obtained by space mutagenesis and screening of Bacillus subtilis subsp. subtilis CEGF012 strain with accession number CCTCC NO: M 20241702. Its 16S rDNA sequence is shown in SEQ ID NO:

1.

2. The application of the space-bred Bacillus subtilis CEGF-TKJ-011 as described in claim 1 in the preparation of nattokinase, characterized in that, This invention is applied to the fermentation of Cistanche deserticola to prepare nattokinase, wherein the substrate for the fermentation of Cistanche deserticola to prepare nattokinase comprises, by mass percentage, 50%~75% Cistanche deserticola and 25%~50% legumes.

3. The application as described in claim 2, characterized in that, The space-bred Bacillus subtilis CEGF-TKJ-011 is applied to the fermentation of Cistanche deserticola to prepare nattokinase, specifically as follows: Using the space-borne Bacillus subtilis CEGF-TKJ-011 as described in claim 1 as the fermentation strain, it is inoculated into Cistanche deserticola culture medium for fermentation to obtain a fermentation product containing nattokinase, or after the fermentation is completed, the fermentation product is taken and centrifuged to obtain a supernatant containing nattokinase.

4. The application as described in claim 3, characterized in that, The OD of the space-borne Bacillus subtilis CEGF-TKJ-011 bacterial suspension 600 The inoculation rate is 1-2, and the inoculation amount is 5%-10% in the Cistanche deserticola culture medium for fermentation.

5. The application as described in claim 4, characterized in that, The OD of the space-borne Bacillus subtilis CEGF-TKJ-011 bacterial suspension 600 The concentration was 1.5, and 6% was inoculated into the Cistanche deserticola culture medium for fermentation.

6. A fermentation product with nattokinase activity, characterized in that, It is prepared according to the following method: The fermentation strain is Bacillus subtilis CEGF-TKJ-011 as described in claim 1, obtained through fermentation substrate; the substrate, by mass percentage, includes 50%~75% Cistanche deserticola and 25%~50% legumes.

Images