Bacillus velezensis CZ-1 for specifically and efficiently degrading astragalus polysaccharide and application of bacillus velezensis CZ-1

Abstract

The invention discloses a bacillus velezensis for efficiently degrading astragalus polysaccharide from fish intestinal tracts and application of the bacillus velezensis, and belongs to the technical field of microorganisms. The strain is named as Bacillus velezensis CZ-1, and is preserved in the China Center for Type Culture Collection on April 15, 2025, and the preservation number is CCTCC (China Center for Type Culture Collection) NO: M20251128. The strain has efficient degradation capacity on astragalus polysaccharide, when the astragalus polysaccharide is 2.5 g / L and bacillus velezensis is inoculated according to the inoculation rate of 5%, the degradation rate in 12 h is larger than or equal to 55.60%, the degradation rate in 24 h is larger than or equal to 74.10%, the degradation rate in 48 h is larger than or equal to 87.70%, and a main product is oligosaccharide with the polymerization degree of 2 and 3. The invention provides an efficient and safe microbial solution for resource utilization of astragalus polysaccharide.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Bacillus velezensis CZ-1 capable of specifically and efficiently degrading astragalus polysaccharide and an application thereof. Background Art

[0002] Astragalus membranaceus, also known as Huangqi in Chinese, is a plant of the genus Astragalus in the Leguminosae family. Its main active ingredients include polysaccharides, flavonoids, and astragalosides. Astragalus polysaccharides (APS) are among the most abundant and bioactive components, exhibiting immunomodulatory, antioxidant, anticancer, anti-aging, and anti-inflammatory properties. APS has a wide molecular weight range (10-2000 kDa) and poor water solubility, resulting in low direct utilization efficiency and limited intestinal absorption. Therefore, reducing the degree of polymerization of APS by appropriate methods can not only enhance its bioactivity but also improve its utilization, which is of great significance for its application.

[0003] Common methods for degrading polysaccharides include physical degradation, chemical degradation, and biodegradation. Physical degradation, primarily through ultrasound, microwaves, and hydrothermal processes, is simple and efficient, but can easily damage the natural structure of polysaccharides and incompletely degrades them. Chemical degradation, primarily through acid / base hydrolysis and oxidation reactions, can rapidly break carbon chains, but the products are uncontrollable and pollute the environment. Biodegradation, through enzymatic reactions or enzymes secreted by bacterial strains, offers advantages such as mild conditions and environmental friendliness. However, due to the complex structure of polysaccharides, the selection of efficient strains and enzyme systems remains a technical challenge.

[0004] Currently, there are few reports on microbial strains that can degrade APS. Developing efficient APS-degrading strains is crucial for improving the resource utilization of Astragalus membranaceus. Bacillus velezensis, a new species of the genus Bacillus, possesses probiotic properties and carries numerous biosynthetic gene clusters, potentially producing diverse metabolites. However, its ability to degrade APS has not been reported. Summary of the Invention

[0005] The purpose of the present invention is to provide a strain of Bacillus velezensis CZ-1 that can specifically and efficiently degrade astragalus polysaccharides. The strain is isolated from the intestinal contents of crucian carp and has a deposit number of CCTCC NO: M20251128.

[0006] Another object of the present application is to provide the use of Bacillus Velezii CZ-1 in degrading astragalus polysaccharides.

[0007] In order to achieve the above object, the present invention adopts the following technical measures:

[0008] The applicant isolated and screened a bacterium, CZ-1, from the intestinal contents of crucian carp, which has the ability to efficiently degrade astragalus polysaccharides. The applicant identified the bacterium as Bacillus velezensis. The strain was deposited with the China Center for Type Culture Collection (CCTCC) on May 20, 2025, with CCTCC NO: M20251128 and taxonomic name: Bacillus velezensis CZ-1. The address is Wuhan University, China.

[0009] Bacillus velezensis CZ-1 was cultured on LB agar plate medium at 37°C for 18 h to form round, milky white colonies that were relatively round with rough, irregular, and raised edges.

[0010] The protection scope of the present invention also includes:

[0011] The fermentation broth of Bacillus Velez CZ-1 contains live bacteria of Bacillus Velez CZ-1.

[0012] A compound, wherein the active ingredient of the compound contains Bacillus velezensis CZ-1.

[0013] Application of Bacillus velez CZ-1, Bacillus velez CZ-1 fermentation broth and / or a compound thereof in degrading polysaccharides.

[0014] Application of Bacillus velez CZ-1, Bacillus velez CZ-1 fermentation broth and / or its compound in preparing microbial agent for degrading polysaccharides

[0015] In the above application, preferably, the polysaccharide comprises: astragalus polysaccharide, locust bean gum, guar gum, tuckahoe polysaccharide, polygonatum polysaccharide, laminarin, beech xylan and / or wheat arabinoxylan.

[0016] Application of Bacillus velez CZ-1, Bacillus velez CZ-1 fermentation broth and / or a compound thereof in the preparation of astragalus polysaccharide oligosaccharides.

[0017] The above-mentioned application preferably comprises the step of inoculating Bacillus velez CZ-1, Bacillus velez CZ-1 fermentation broth and / or a combination thereof into a SM liquid culture medium containing astragalus polysaccharide as the sole carbon source.

[0018] In the above application, preferably, the astragalus polysaccharide oligosaccharide includes oligosaccharides with a degree of polymerization of 2 and 3;

[0019] In the above application, preferably, the inoculation amount is 1% to 10%.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] The present invention discloses for the first time a strain of Bacillus velezensis CZ-1, which has the ability to degrade various polysaccharides, especially has a specific and efficient degradation ability for astragalus polysaccharide, and is a supplement to the astragalus polysaccharide degradation strain resources.

[0022] The Bacillus Velez CZ-1 provided by the present invention has a high degradation rate for astragalus polysaccharide. When inoculated into SM culture medium containing astragalus polysaccharide as the sole carbon source, the Bacillus Velez CZ-1 can efficiently degrade astragalus polysaccharide in the sample. When 2.5g of astragalus polysaccharide is inoculated with Bacillus Velez CZ-1 at a 5% inoculation rate, the degradation rate is ≥55.60% after 12 hours of inoculation, ≥74.10% after 24 hours of inoculation, and ≥87.70% after 48 hours of inoculation, showing good application potential.

[0023] The Bacillus velezensis CZ-1 provided by the present invention can degrade astragalus polysaccharide into astragalus oligosaccharides containing disaccharides and trisaccharides, and the fermentation liquid containing the astragalus oligosaccharides has antioxidant activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Colony morphology of Bacillus velezensis CZ-1 strain

[0025] Figure 2 This is a Gram-stained microscopic morphology of Bacillus velezensis CZ-1 strain (1000×).

[0026] Figure 3 This is the phylogenetic tree of Bacillus velezensis CZ-1 strain.

[0027] Figure 4 To determine the process parameters of astragalus polysaccharide fermentation by Bacillus velezensis CZ-1;

[0028] Among them: A is the growth curve of Bacillus velezensis CZ-1 strain during the fermentation process, B is the change curve of the pH value of the fermentation liquid, C is the change curve of the total sugar content of the fermentation liquid, and D is the change curve of the reducing sugar content of the fermentation liquid.

[0029] Figure 5 This is a thin layer analysis diagram (A) and polymerization degree change diagram (B) of the fermentation broth of astragalus polysaccharide fermented by Bacillus velezensis CZ-1 at different time points provided in the examples of this application. DETAILED DESCRIPTION

[0030] Example 1:

[0031] Isolation, Purification and Identification of Bacillus Velezii Strain CZ-1

[0032] 1) Sample collection: The applicant collected crucian carp from Derun Aquatic Products Cooperative in Xishui County, Hubei Province in June 2024, and obtained the crucian carp intestines and their contents.

[0033] 2) Isolation of the strain: Place crucian carp intestinal tissue in a grinder and grind with 1 mL of sterile PBS. In a clean bench, aspirate 1 mL of the supernatant and serially dilute it with sterile PBS. From each dilution series, spread 100 μL onto a GAM solid culture plate and incubate in a constant temperature incubator at 37°C for 24 h.

[0034] 3) Strain Purification: Single colonies of varying color and morphology were selected from GAM solid medium within a clean bench. These colonies were inoculated into GAM liquid medium for enrichment, then streaked again. Separation and purification were performed stepwise until a single strain was obtained. The diluted suspension of the single strain was then plated onto selective solid medium (SM) supplemented with astragalus polysaccharide (2.5 mg / mL) as the sole carbon source and incubated at 37°C for 24 hours to obtain colonies capable of metabolizing astragalus polysaccharide.

[0035] Each 200 mL of the SM liquid culture medium includes the following components: KCl 0.9 g, NaCl 0.9 g, NaHCO 3 0.3 g, KH 2 PO 4 0.1 g, K 2 HPO 4 0.1 g, NH 4 Cl 0.06 g, MgSO 4 0.04 g, 200 μL of alkaline trace elements and 200 μL of acidic trace elements; wherein,

[0036] The alkaline trace elements include Na2SeO3 0.1mM, Na2WO4 0.1mM, Na2MoO4 0.1mM, and NaOH 10mM; the acidic trace elements include FeCl2 7.5mM, H3BO4 1mM, ZnCl2 0.5mM, CuCl2 0.1mM, MnCl2 0.5mM, CoCl2 0.5mM, NiCl2 0.1mM, and HCl 50mM.

[0037] SM solid medium is prepared by adding agar to SM liquid medium with a final concentration of 20 g / L.

[0038] Identification of strain microscopy: Pick colonies and make three-zone lines on LB solid medium. After culturing at 37℃ for 24h, observe the following Figure 1 The colony morphology is milky white, relatively round, with rough, irregular and raised edges.

[0039] Gram staining microscopic morphological identification: Use an optical microscope to observe the morphology of bacteria after Gram staining. Figure 2 It can be seen that the bacteria are blue-purple, are Gram-positive bacteria, and are short rod-shaped.

[0040] Bacterial 16S rDNA sequencing identification: Use the extraction kit to extract and sequence the strain DNA, submit the sequencing results to the NCBI database for homology comparison, and draw a phylogenetic tree, such as Figure 3 From the developmental tree, it can be preliminarily judged that the strain has the highest similarity with Bacillus velezensis strain FZB42 of the Bacillaceae family and Bacillus bacteria, so it is named Bacillus velezensis CZ-1. The strain was deposited in the China Center for Type Culture Collection on May 20, 2025, with the deposit number CCTCC NO: M20251128, classification name: Bacillus velezensis CZ-1, address: Wuhan University, Wuhan, China.

[0041] Example 2:

[0042] Application of Bacillus Velez CZ-1 in polysaccharide degradation:

[0043] In this example, the applicant investigated the degradation ability of Bacillus velezensis CZ-1 on various polysaccharides, including astragalus polysaccharide, locust bean gum, guar gum, xanthan gum, tuckahoe polysaccharide, polygonatum polysaccharide, laminarin, beech xylan and wheat arabinoxylan.

[0044] The specific steps are as follows:

[0045] Preparation of Bacillus Velez CZ-1 seed solution:

[0046] The inoculation amount of Bacillus velez CZ-1 was 1%, and Bacillus velez CZ-1 was cultured in LB liquid medium at 37°C and 200 rpm until the plateau phase (the plateau phase was reached after 24 h), and the OD 600 The bacterial suspension was diluted to 1.50, the LB medium was removed by centrifugation, and the suspension was resuspended in SM medium without carbon source to an OD of 600 is 1.50.

[0047] Prepare 20 mL of SM liquid culture medium containing 2.5 mg / mL polysaccharide (astragalus polysaccharide, locust bean gum, guar gum, xanthan gum, tuckahoe polysaccharide, polygonatum polysaccharide, laminarin, beech xylan or wheat arabinoxylan), inoculate the above-prepared Bacillus Velezii liquid at an inoculum size of 5%, and culture at 37°C, 200 rpm, and pH 7. The total sugar content of the samples at 0 h and different fermentation time points was determined using the phenol-sulfuric acid method.

[0048] Polysaccharide degradation rate = (initial total sugar - total sugar at different fermentation time points) / initial total sugar * 100%.

[0049] The degradation results of different polysaccharides by Bacillus Velez CZ-1 are shown in Table 1:

[0050] Table 1

[0051]

[0052] As can be seen from Table 1, the Bacillus Velez CZ-1 described in the present application has high activity against astragalus polysaccharide, beech xylan and wheat arabinoxylan, among which the degradation rate of astragalus polysaccharide is the highest.

[0053] Example 3:

[0054] Method for preparing oligosaccharides by fermenting astragalus polysaccharide with Bacillus velez CZ-1

[0055] 1) The inoculation amount of Bacillus velez CZ-1 was 1%, and Bacillus velez CZ-1 was cultured in LB liquid medium at 37°C and 200 rpm until the plateau phase, and the OD 600 The bacterial suspension was diluted to 1.50, the LB medium was removed by centrifugation, and the suspension was resuspended in SM medium without carbon source to an OD of 600 The bacterial suspension was inoculated at a ratio of 5% into 60 mL of SM medium containing astragalus polysaccharide (2.5 mg / mL) as the sole carbon source for fermentation. Three parallel experiments were performed for each group. The control group consisted of SM medium without inoculation of Bacillus velezensis CZ-1 and containing astragalus polysaccharide (2.5 mg / mL) as the sole carbon source.

[0056] 2) Regularly monitor the culture medium OD during fermentation 600 , pH, total sugar, and reducing sugar changes. The results are as follows Figure 4 shown. Figure 4 A shows the OD of Bacillus Velez CZ-1 within 24 hours after inoculation. 600 The value increased from 0.29 ± 0.02 to 0.62 ± 0.01; Figure 4 Middle B shows that after inoculation with Bacillus Velez CZ-1, the pH value of the fermentation broth of Bacillus Velez decreased from 7.84±0.17 to 6.64±0.09 within 24 hours. As the fermentation time prolonged, the pH value remained around 6.6. Figure 4Middle C shows that after inoculation with Bacillus Velez CZ-1, the concentration of total carbohydrates in the fermentation broth of the fermentation group decreased from 2.26±0.05 mg / mL to 0.59±0.02 mg / mL within 24 hours, and dropped to a minimum of 0.28±0.02 mg / mL within 48 hours; Figure 4 D in the middle shows that after inoculation with Bacillus Velez CZ-1, the concentration of reducing sugar in the fermentation broth of the fermentation group increased from 0.05±0.003 mg / mL to 0.63±0.001 mg / mL within 12 h.

[0057] 3) During the fermentation process, the fermentation broth was collected regularly, deproteinized, dialyzed, concentrated, and then freeze-dried to obtain astragalus oligosaccharides, which were then analyzed by thin-layer chromatography. Figure 5 Figure A shows a thin layer chromatogram of a mixed standard of fiber and sugar (column 10), and fermentation broth samples at 0h, 3h, 6h, 12h, 24h, 36h, 48h, 60h and 72h (columns 1-9). Figure 5 As shown in Figure A, as the fermentation time progresses, the color of the astragalus polysaccharide spots in the fermentation broth gradually becomes lighter, indicating that its content gradually decreases, and the main spots produced correspond to oligosaccharides with a degree of polymerization of 2 and 3. Figure 5 Middle B shows the HPLC chromatograms of the fiber sugar mixed standard, mannose mixed standard and fermentation broth samples at 0h, 12h, 24h, 36h, 48h, 60h and 72h. It can be seen from the figure that Bacillus Velezii CZ-1 degrades astragalus polysaccharide mainly to produce oligosaccharides with a degree of polymerization of 2 and 3.

[0058] In the thin layer chromatography analysis, a mixed solution of n-butanol: isopropanol: acetic acid: water = 7:5:2:2 (v / v) was used as a developing solvent, and a 10% sulfuric acid ethanol solution (v / v) was used as a color developer.

[0059] Among them, the HPLC analysis conditions include: detector, evaporative light scattering detector: gain 100, gas 25 psi, drift tube 80°C, nebulizer heating, power level 95%; chromatographic column, XAmide column (4.6×150 mm, 5 μm); mobile phase A, acetonitrile 0.02 mol / L; mobile phase B, 100 mM ammonium formate (pH=3.2) (B); elution conditions: isocratic elution, 40 min; flow rate, 1.0 mL / min; column temperature, 30°C; injection volume, 10 μL.

[0060] The present application example applies Bacillus velez to the degradation of astragalus polysaccharide for the first time and has the advantages of being green, efficient, low-cost, and easy to operate, and is suitable for large-scale industrial production. In view of this, the use of Bacillus velez to degrade astragalus polysaccharide to prepare oligosaccharides has broad application prospects.

[0061] The above is only a preferred specific implementation method of the present application, but the scope of protection of the present application is not limited thereto. Any changes or replacements that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed in this application should be covered by the scope of protection of the present application.

Claims

1. A strain of Bacillus velezensis that can specifically and efficiently degrade Astragalus polysaccharides ( Bacillus velezensis ) CZ-1, the deposit number of the Bacillus Velez is CCTCC NO: M20251128.

2. The fermentation broth of Bacillus Velez CZ-1 according to claim 1, wherein the fermentation broth contains live bacteria of Bacillus Velez CZ-1.

3. A compound, wherein the active ingredient of the compound contains Bacillus Velez CZ-1.

4. Use of the Bacillus Velez CZ-1 according to claim 1, the fermentation broth of the Bacillus Velez CZ-1 according to claim 2, and / or the compound according to claim 3 in degrading polysaccharides.

5. Use of the Bacillus Velez CZ-1 according to claim 1, the fermentation broth of the Bacillus Velez CZ-1 according to claim 2, and / or the compound according to claim 3 in the preparation of a microbial agent for degrading polysaccharides.

6. The use according to claim 4 or 5, wherein the polysaccharide comprises: Astragalus polysaccharide, locust bean gum, guar gum, tuckahoe polysaccharide, polygonatum polysaccharide, laminarin, beech xylan and / or wheat arabinoxylan.

7. Use of the Bacillus Velez CZ-1 according to claim 1, the fermentation broth of the Bacillus Velez CZ-1 according to claim 2, and / or the compound according to claim 3 in the preparation of astragalus polysaccharide oligosaccharides.

8. The use according to claim 7, wherein the use process comprises inoculating Bacillus Velez CZ-1, Bacillus Velez CZ-1 fermentation broth and / or a combination thereof into SM liquid culture medium containing astragalus polysaccharide as the sole carbon source.

9. The astragalus polysaccharide oligosaccharide includes oligosaccharides with a degree of polymerization of 2 and 3.

10. The use according to claim 8, wherein the inoculation amount is 1% to 10%.

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