Paenibacillus sp. and application thereof in preparing tremella oligosaccharide

Abstract

The application provides a kind of Paenibacillus and its application in preparing tremella oligosaccharide, the preservation number of the Paenibacillus is CGMCC No:25534.A method for preparing tremella polysaccharide degrading enzyme and a method for preparing tremella oligosaccharide are also disclosed.The Paenibacillus of the application can produce high-activity tremella polysaccharide degrading enzyme under the induction of tremella or tremella polysaccharide, can efficiently degrade tremella and tremella polysaccharide, the quality of the produced tremella oligosaccharide is stable, the yield is high, and the degradation process is mild and controllable, the energy consumption is low, the operation is simple, is suitable for large-scale production, has significant economic benefits.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial fermentation, and particularly to a Paenibacillus sp. and its application in preparing tremella oligosaccharide. BACKGROUND

[0002] The main active ingredient of tremella is tremella polysaccharide. The main chain of tremella polysaccharide is α-1, 3-glycosidic bond connected poly-mannose, and the side chain contains glucose, xylose, fructose, arabinose and glucuronic acid. Tremella polysaccharide is reported to have the activities of moisturizing, immunomodulation, anti-hepatoma cell proliferation, blood sugar reduction, wound healing promotion, antioxidant, anti-radiation, anti-photoaging, anti-virus and nerve cell protection, among which the immunomodulation is most concerned. Tremella polysaccharide has a large molecular weight (from tens of thousands of daltons to several million daltons) and low solubility, and has a very high viscosity after being dissolved. The characteristics of too large molecular weight hinder the absorption of tremella polysaccharide by the esophagus, and limit its application scope. Therefore, under the premise of retaining the physiological activity of tremella polysaccharide, it has important application value to degrade tremella polysaccharide and prepare tremella oligosaccharide with small molecular weight and good water solubility.

[0003] The process for preparing oligosaccharide by degrading polysaccharide mainly includes physical method, chemical method and enzymatic method. Among them, the physical method breaks the polysaccharide chain through high shear, electrolysis, high pressure homogenization and the like; the chemical method degrades the polysaccharide chain by using chemical reagents such as acid and hydrogen peroxide. The physical or chemical degradation process is a random breaking of the polysaccharide chain, and there are problems of poor production controllability and unstable product quality. The enzymatic degradation is a modern green production process with mild reaction conditions, high production efficiency, low energy consumption and environmental friendliness. At the same time, the enzyme has selectivity in hydrolyzing chemical bonds of substrates, and the enzymatic degradation process is accurately controllable. However, there is no literature report on the specific degradation of tremella polysaccharide by microorganisms or microbial enzymes. Therefore, it is of great significance to screen tremella polysaccharide degrading strains and develop high-activity tremella polysaccharide degrading enzymes for the industrialized production of tremella oligosaccharide. SUMMARY

[0004] In order to solve the problems of poor production controllability and unstable product quality caused by random breaking of polysaccharide chain in the process of degrading tremella polysaccharide in the prior art, the present application provides a Paenibacillus sp. and its application in preparing tremella oligosaccharide, so that the degradation process is mild and controllable, the energy consumption is low, the operation is simple, it is suitable for large-scale production, and has significant economic benefits.

[0005] In order to solve the above technical problems, the present application provides a Paenibacillus sp. (Paenibacillus sp. strain) in a first aspect, and the preservation number of the Paenibacillus sp. is CGMCC No: 25534.

[0006] The colony morphology of *Bacillus* strain WSK-2 (CGMCC No:25534) on modified TSB solid medium is as follows: round, flat colonies with intact edges, slightly raised and smooth surfaces, translucent, light gray, glossy, and odorless. Under a microscope, they appear as rod-shaped, Gram-positive colonies, approximately 1–6 micrometers in size. They are aerobic, with an optimal growth pH of 6.0–7.0 and an optimal growth temperature of 28–35°C. This strain can produce *Tremella fuciformis* polysaccharide-degrading enzymes in cultures containing *Tremella fuciformis* polysaccharide or *Tremella fuciformis* polysaccharide. 16S rRNA gene sequencing of strain WSK-2 confirms it as a *Bacillus* strain.

[0007] To address the aforementioned technical problems, a second aspect of the present invention provides a method for preparing a tremella polysaccharide-degrading enzyme, the method comprising:

[0008] Fermenting the Bacillus-like bacteria as described in the first aspect yields a fermentation broth. The fermentation broth is then centrifuged or filtered to obtain a crude enzyme solution of Tremella polysaccharide degrading enzyme.

[0009] In some preferred embodiments, the fermentation medium comprises the following components in the following mass-volume ratios: 0.1%–2.0% *Tremella fuciformis* or *Tremella fuciformis* polysaccharide, 0.1%–2.0% yeast extract, 0.1%–2.0% soybean peptone, 0.1%–2.0% beef extract, 0.1%–1.0% dipotassium hydrogen phosphate, 0.1%–1.0% sodium chloride, 0.1%–1.0% ammonium sulfate, 0.005%–0.05% calcium chloride, 0.01%–0.1% magnesium sulfate, 0.01%–0.05% ferric citrate, 0.1%–1.0% cysteine, and 0.1%–1.0% arginine.

[0010] In some preferred embodiments, the fermentation medium comprises: 2.0% tremella powder, 1.0% yeast extract, 1.0% soybean peptone, 1.0% beef extract, 0.5% dipotassium hydrogen phosphate, 0.5% sodium chloride, 0.5% ammonium sulfate, 0.005% calcium chloride, 0.01% magnesium sulfate, 0.01% ferric citrate, 0.1% cysteine, and 0.1% arginine.

[0011] In some embodiments, the fermentation conditions include: an inoculum of 1% to 10% of the Bacillus subtilis, a temperature of 28 to 32°C, thorough stirring, and aeration.

[0012] In some preferred embodiments, the fermentation time is 24 to 48 hours.

[0013] In some embodiments, the conditions for obtaining the crude enzyme solution are as follows: the fermentation broth is centrifuged at 5000 rpm for 10 minutes, and then filtered using a filter cloth, filter paper, or a 0.22 μm filter membrane to obtain the crude enzyme solution.

[0014] In some preferred embodiments, the crude enzyme solution can be filtered through an ultrafiltration membrane to obtain an enzyme concentrate, or obtained as a solid enzyme preparation by ammonium sulfate precipitation, centrifugation, desalting, and freeze-drying.

[0015] In some preferred embodiments, the molecular pore size of the ultrafiltration membrane is 10 kDa or above.

[0016] To address the aforementioned technical problems, a third aspect of the present invention provides a crude enzyme solution for degrading Tremella polysaccharides, wherein the crude enzyme solution is prepared by the method described in the second aspect of the present invention.

[0017] To address the aforementioned technical problems, a fourth aspect of the present invention provides a method for preparing Tremella oligosaccharides, wherein the method involves using Bacillus subtilis as described in the first aspect of the present invention or crude enzyme solution as described in the third aspect of the present invention to degrade Tremella polysaccharides, thereby obtaining a degradation solution containing Tremella oligosaccharides.

[0018] In this invention, the tremella oligosaccharide refers to the degradation products of tremella polysaccharide with a molecular weight range of 1 to 10 kDa.

[0019] In some preferred embodiments, the preparation method is as follows: the crude enzyme solution is mixed with a solution containing tremella polysaccharide or tremella, and enzymatically hydrolyzed at 30-55°C.

[0020] In some more preferred embodiments, the ratio of the crude enzyme solution to the solution containing Tremella polysaccharide or Tremella is 1:(1-20), the concentration of the solution is 0.2%-2%, the ratio is a volume ratio, and the concentration is a mass-volume ratio.

[0021] In some further preferred embodiments, the ratio of the crude enzyme solution to the solution containing Tremella polysaccharide or Tremella is 1:9, the concentration of the solution is 2%, the ratio is a volume ratio, and the concentration is a mass-volume ratio.

[0022] In some embodiments, the preparation method further includes removing impurities from the degradation solution to obtain a Tremella oligosaccharide solution.

[0023] In some preferred embodiments, the impurity removal includes one or more of the following: enzyme or bacteria inactivation, activated carbon adsorption, red diatomaceous earth filtration, and filter paper filtration.

[0024] In some preferred embodiments, the inactivation temperature is 100°C and the time is 10 to 30 minutes; the amount of activated carbon added is 1% by mass-volume ratio.

[0025] In some embodiments, the preparation method further includes: concentrating the solid content of the Tremella oligosaccharide solution to a mass-volume ratio of 10% to 20%, and then spray drying it to obtain Tremella oligosaccharide powder.

[0026] To address the aforementioned technical problems, the fifth aspect of this invention provides the application of Bacillus subtilis as described in the first aspect of this invention and crude enzyme solution as described in the third aspect of this invention in the preparation of Tremella fuciformis oligosaccharides.

[0027] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0028] The reagents and raw materials used in this invention are all commercially available.

[0029] The positive and progressive effects of this invention are as follows:

[0030] (1) The Paenibacillus sp. strain CGMCC No:25534 provided by this invention can produce highly active tremella polysaccharide-degrading enzymes under the induction of tremella or tremella polysaccharide. This strain is easy to culture, has stable characteristics, and is suitable for industrial production.

[0031] (2) The tremella polysaccharide degrading enzyme produced by Paenibacillus sp. strain WSK-2 can efficiently degrade tremella and tremella polysaccharides within 12 to 24 hours without producing monosaccharides. The degradation process is mild and controllable, with low energy consumption and no need to add additional chemical reagents.

[0032] (3) The Tremella oligosaccharide products prepared using the technology provided by this invention have a minimum molecular weight of 1.0–1.2 kDa. The produced Tremella oligosaccharides are of stable quality, with high yield and consistent quality. The production process for preparing Tremella polysaccharide degrading enzymes and Tremella oligosaccharides described in this invention has low energy consumption, is simple to operate, and the degradation process is precise and controllable, making it suitable for large-scale production and yielding significant economic benefits.

[0033] Biological material preservation information

[0034] The Paenibacillus sp. strain of this invention, WSK-2, was deposited on August 15, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No:25534. The culture name is WSK-2, and the classification name is Paenibacillus sp. strain. Attached Figure Description

[0035] Figure 1A and Figure 1BThe colony morphology and microscopic cell morphology of strain Paenibacillus sp. strain WSK-2 are shown; among which... Figure 1A A colony photograph of strain Paenibacillus sp. strain WSK-2; Figure 1B This is a magnified micrograph of strain Paenibacillus sp. strain WSK-2, with a scale bar of 5 μm.

[0036] Figure 2 The activity of the polysaccharide-degrading enzyme in Paenibacillus sp. strain WSK-2 was affected by pH.

[0037] Figure 3 The activity of the Paenibacillus sp. strain WSK-2 polysaccharide degrading enzyme is affected by temperature.

[0038] Figure 4 The molecular weight distribution of *Tremella fuciformis* oligosaccharides produced using *Paenibacillus sp. strain* WSK-2, a polysaccharide-degrading enzyme, was determined. The horizontal axis represents the retention time of different molecular weight substances in the gel chromatography column, and the vertical axis represents the abundance signal of different molecular weight substances. a represents the molecular weight distribution of a 0.5% *Tremella fuciformis* polysaccharide solution; b represents the molecular weight distribution of the *Tremella fuciformis* polysaccharide degradation product; and c represents the molecular weight distribution of the *Tremella fuciformis* oligosaccharide product. Analytical conditions were as follows: Shimadzu high-performance liquid chromatography system, using a TSK-GEL G2000SWXL gel chromatography column, with 0.1 mol / L sodium nitrate as the mobile phase, a flow rate of 0.5 mL / min, and a differential refractive index detector. Detailed Implementation

[0039] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0040] The following embodiments are further illustrations of the present invention, but the present invention is not limited to the following embodiments.

[0041] In all examples, the DNS method was used to determine enzyme activity, and the specific procedures are as follows:

[0042] (1) Preparation of DNS reagent. Dissolve 6.3g of 3,5-dinitrosalicylic acid in 300ml of distilled water, and dissolve 21g of sodium hydroxide in 400ml of distilled water. Then slowly add the two solutions dropwise while stirring constantly. Next, add 182g of potassium sodium tartrate tetrahydrate, 5g of phenol, and 5g of anhydrous sodium sulfite in sequence, stirring constantly in a warm water bath until the solution is clear and transparent. Finally, dilute to 1000ml with distilled water, store in a brown bottle, and let stand for 5-7 days before use.

[0043] (2) Enzyme activity assay. Mix 100 μl of appropriately diluted enzyme solution with 900 μl of 0.5% (w / v) Tremella fuciformis polysaccharide solution and react at a specific temperature and pH for 1 hour. Add 1 ml of DNS reagent, mix well, heat in boiling water for 10 minutes, cool to room temperature, and measure the absorbance at 540 nm using a cuvette. Higher enzyme activity results in higher absorbance.

[0044] Example 1: Isolation and identification of strain Paenibacillus sp. strain WSK-2

[0045] (1) Enrichment and isolation of Tremella fuciformis degrading strains

[0046] Several samples of naturally grown Tremella fuciformis and its planting area's humus and soil were collected. Appropriate amounts of physiological saline were added to each sample, and the mixture was shaken for 10 minutes to obtain sample suspensions. 1 ml of each sample suspension was added to 100 ml of enrichment medium and incubated at 30℃ and 100 rpm for 10 days. The enrichment medium consisted of (w / v) 1% Tremella fuciformis polysaccharide, 0.5% yeast extract, 0.5% soybean peptone, 0.5% ammonium sulfate, 0.2% dipotassium hydrogen phosphate, 0.5% sodium chloride, 0.01% calcium chloride, and 0.01% magnesium sulfate, and was sterilized at 121℃ for 30 minutes before use.

[0047] The culture medium containing 1% Tremella fuciformis polysaccharide had a high viscosity. After 10 days of enrichment, the enriched samples with significantly reduced viscosity were selected. These were appropriately diluted and spread onto sterile solid culture plates. The solid culture plates had the same composition as the enrichment medium, except for the addition of 1.5% agar powder. After static incubation at 30℃ for 7 days, strains with different colony morphologies were inoculated into test tubes containing 4 ml of enrichment medium and incubated at 30℃ and 100 rpm for 2 days. The viscosity of the medium in the test tubes was observed to see if it decreased. Strains exhibiting Tremella fuciformis polysaccharide degradation effects were transferred to shake flasks containing 50 ml of enrichment medium and incubated at 30℃ and 100 rpm for 2 days. The viscosity of the medium in each shake flask was then measured using a viscometer. The lower the viscosity after fermentation, the higher the enzyme activity of the microorganism in degrading Tremella fuciformis polysaccharide.

[0048] Through repeated enrichment and screening, this invention discovered the strain WSK-2, which can efficiently degrade Tremella fuciformis polysaccharides. On modified TSA medium, the colony morphology of strain WSK-2 is as follows: round, flat colonies with intact edges, slightly raised and smooth surfaces, translucent, light gray, glossy, and without a noticeable odor. Under a microscope, the cells of strain WSK-2 are rod-shaped, Gram-positive, and approximately 1–6 micrometers in size (see...). Figure 1A and Figure 1B The modified TSA solid culture medium formula is as follows (w / v): 2% soybean flour papain digest, 0.25% dipotassium hydrogen phosphate, 0.5% sodium chloride, 0.25% glucose, 0.5% tremella polysaccharide, and 1.5% agar powder.

[0049] (2) Molecular biological identification of strain WSK-2.

[0050] Strain WSK-2 was cultured in the enrichment medium described above, and the bacterial pellet was collected by centrifugation at 5000 rpm for 5 minutes. DNA was extracted from the bacterial cells using a commercially available DNA extraction kit, and the 16S rRNA gene sequence of this strain was determined. A search and comparison on NCBI identified this strain as a Paenibacillus sp. strain, which has been deposited with the accession number CGMCC No:25534.

[0051] Example 2: Preparation and property determination of Paenibacillus sp. strain WSK-2 tremella polysaccharide degrading enzyme

[0052] (1) Preparation of Tremella polysaccharide degrading enzyme

[0053] Fermentation medium:

[0054] 0.1%–2.0% Tremella fuciformis or Tremella fuciformis polysaccharide, 0.1%–2.0% yeast extract, 0.1%–2.0% soybean peptone, 0.1%–2.0% beef extract, 0.1%–1.0% dipotassium hydrogen phosphate, 0.1%–1.0% sodium chloride, 0.1%–1.0% ammonium sulfate, 0.005%–0.05% calcium chloride, 0.01%–0.1% magnesium sulfate, 0.01%–0.05% ferric citrate, 0.1%–1.0% cysteine ​​and 0.1%–1.0% arginine.

[0055] The culture medium composition used in this embodiment is (w / v):

[0056] 2.0% Tremella powder or 1.0% Tremella polysaccharide, 1.0% yeast extract, 1.0% soybean peptone, 1.0% beef extract, 0.5% dipotassium hydrogen phosphate, 0.5% sodium chloride, 0.2% ammonium sulfate, 0.005% calcium chloride, 0.01% magnesium sulfate, 0.01% ferric citrate, 0.1% cysteine ​​and 0.1% arginine.

[0057] Dissolve the above-mentioned culture medium components in water, sterilize at 121°C for 30 minutes, and cool to 30°C. Inoculate the WSK-2 seed culture at a volume ratio of 1% to 10% into the cooled culture medium, and culture at 28 to 32°C with thorough stirring and aeration for 24 to 48 hours. The fermentation broth will then produce tremella polysaccharide degrading enzymes.

[0058] Preparation of Tremella polysaccharide degrading enzyme:

[0059] Centrifuge the fermentation broth at 5000 rpm for 10 minutes, or filter the broth using filter cloth, filter paper, and a 0.22 μm filter membrane to remove bacterial cells and insoluble components. The resulting supernatant is the crude enzyme solution. This crude enzyme solution, after adding potassium sorbate preservative, can be stored at 4°C for more than one month, or it can be frozen after adding 10% glycerol.

[0060] The crude enzyme solution can be further concentrated and purified using membrane filtration technology. The membrane used has a molecular pore size of 10 kDa. The permeate side is discarded, and the concentrated side is the purified enzyme solution.

[0061] (2) Determination of the optimal reaction pH of Tremella polysaccharide degrading enzyme

[0062] Prepare the buffer solution:

[0063] Buffer solutions with pH 4.0, pH 5.0, and pH 6.0 were prepared using a 0.1 mol / L citrate-disodium hydrogen phosphate buffer pair.

[0064] Prepare buffer solutions with pH 7.0 and pH 8.0 using 0.1 mol / L Tris-HCl;

[0065] Buffer solutions with pH 9.0, pH 10.0, and pH 11.0 were prepared using 0.1 mol / L glycine-sodium hydroxide.

[0066] Dissolve 0.5% Tremella polysaccharide in each of the above buffer solutions.

[0067] Determination of relative enzyme activity:

[0068] Take 900 μl of substrate solutions at different pH values ​​and add 100 μl of appropriately diluted crude enzyme solution to each solution. React at 35℃ for 1 hour, then add 1 ml of DNS reagent, heat in boiling water for 10 minutes, cool to room temperature, and measure the absorbance at 540 nm using a cuvette. For each pH gradient experimental group, use crude enzyme solution inactivated by boiling for 5 minutes as a negative control. Calculate the relative enzyme activity for each pH gradient using the enzyme activity of the experimental group with the highest absorbance as 100%. Three replicates were set up for each experimental group. Results are as follows: Figure 2 The results showed that the enzyme activity was inhibited under both acidic and alkaline conditions, with the optimal reaction pH range being 6.0–7.0.

[0069] (3) Determination of the optimal reaction temperature of Tremella polysaccharide degrading enzyme

[0070] A pH 7.0 buffer solution was prepared using 0.1 mol / L Tris-HCl, and 0.5% Tremella fuciformis polysaccharide was added as the substrate solution. 900 μl of the substrate solution was added to 100 μl of enzyme solution appropriately diluted with water, and the mixture was reacted at 30℃, 35℃, 40℃, 45℃, 50℃, and 55℃ for 1 hour each. Then, 1 ml of DNS reagent was added, and the mixture was heated in boiling water for 10 minutes. After cooling to room temperature, the absorbance was measured at 540 nm using a cuvette. Crude enzyme solution inactivated by boiling for 5 minutes was used as a negative control for each temperature gradient experimental group. The relative enzyme activity of each temperature gradient was calculated with the enzyme activity of the experimental group with the highest absorbance as 100%. Three replicates were set up for each experimental group. The results are as follows: Figure 3 .

[0071] The results showed that the enzyme has certain heat resistance, and the optimal reaction temperature range is 45℃~50℃.

[0072] Example 3: Production of Tremella oligosaccharides using Paenibacillus sp. strain WSK-2 tremella polysaccharide degrading enzyme

[0073] (1) Degradation of Tremella polysaccharide using Tremella polysaccharide enzyme

[0074] One part of the purified enzyme solution of the Tremella fuciformis polysaccharide degrading enzyme described in Example 2 was mixed with nine parts of 2% (w / v) Tremella fuciformis polysaccharide or Tremella fuciformis solution, and stirred thoroughly at 45°C for 20 hours. After enzymatic hydrolysis, the temperature was raised to 100°C and kept at that temperature for 30 minutes to inactivate the enzyme. The hydrolysate was cooled to 70°C, and 1% (w / v) activated carbon was added, and stirring was continued for 1 hour. Then, using red diatomaceous earth as a filter aid, the mixed solution was filtered through filter paper to remove activated carbon and insoluble matter. The resulting filtrate was colorless and clear, and then concentrated to achieve a solid content of 10%–20% (w / v). After spray drying, Tremella fuciformis oligosaccharide powder was obtained.

[0075] (2) Degradation product analysis

[0076] Molecular weight analysis was performed on the substrate solution, the enzymatic hydrolysate from the degradation process, and the solution prepared from Tremella fuciformis oligosaccharide powder. The analytical conditions were as follows: Shimadzu high-performance liquid chromatography system, TSK-GEL G2000SWXL gel chromatography column, 0.1 mol / L sodium nitrate as mobile phase, flow rate 0.5 mL / min, and differential refractive index detector.

[0077] Analysis results as follows Figure 4 It is evident that under the action of enzymes, high molecular weight Tremella polysaccharides undergo degradation, with the final products mainly being oligosaccharides. These oligosaccharides characteristically include three molecular weight ranges: 1.0 kDa–1.2 kDa, 2.0 kDa–2.2 kDa, and 2.9 kDa–3.1 kDa.

[0078] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Therefore, any changes or modifications made in accordance with the claims and specification of the present invention should fall within the scope of the present invention.

Claims

1. A type of Bacillus-like bacterium, characterized in that, The preservation number of the Bacillus subtilis is CGMCC No: 25534.

2. A method for preparing a polysaccharide-degrading enzyme from Tremella fuciformis, characterized in that, The method includes: fermenting the Bacillus subtilis as described in claim 1 to obtain a fermentation broth, and centrifuging and / or filtering the fermentation broth to obtain a crude enzyme solution of Tremella fuciformis polysaccharide degrading enzyme.

3. The method as described in claim 2, characterized in that, The fermentation medium contains the following components in the following mass-volume ratios: 0.1%~2.0% Tremella fuciformis or Tremella fuciformis polysaccharide, 0.1%~2.0% yeast extract, 0.1%~2.0% soybean peptone, 0.1%~2.0% beef extract, 0.1%~1.0% dipotassium hydrogen phosphate, 0.1%~1.0% sodium chloride, 0.1%~1.0% ammonium sulfate, 0.005%~0.05% calcium chloride, 0.01%~0.1% magnesium sulfate, 0.01%~0.05% ferric citrate, 0.1%~1.0% cysteine, and 0.1%~1.0% arginine.

4. The method as described in claim 3, characterized in that, The fermentation medium consisted of: 2.0% tremella powder, 1.0% yeast extract, 1.0% soybean peptone, 1.0% beef extract, 0.5% dipotassium hydrogen phosphate, 0.5% sodium chloride, 0.5% ammonium sulfate, 0.005% calcium chloride, 0.01% magnesium sulfate, 0.01% ferric citrate, 0.1% cysteine, and 0.1% arginine.

5. The method as described in claim 2, characterized in that, The fermentation conditions include: an inoculum of 1% to 10% of the Bacillus subtilis, a temperature of 28 to 32°C, thorough stirring, and aeration.

6. The method as described in claim 5, characterized in that, The fermentation time is 24-48 hours.

7. The method according to any one of claims 2 to 6, characterized in that, The conditions for obtaining the crude enzyme solution are as follows: the fermentation broth is centrifuged at 5000 rpm for 10 minutes, and then filtered using filter cloth, filter paper or a 0.22 μm filter membrane to obtain the crude enzyme solution.

8. The method as described in claim 7, characterized in that, The crude enzyme solution is filtered through an ultrafiltration membrane to obtain an enzyme concentrate, or it is precipitated with ammonium sulfate, centrifuged, desalted, and freeze-dried to obtain a solid enzyme preparation.

9. The method as described in claim 8, characterized in that, The ultrafiltration membrane has a molecular pore size of 10 kDa.

10. A method for preparing Tremella oligosaccharides, characterized in that, The preparation method involves using the Bacillus subtilis described in claim 1 to degrade Tremella polysaccharides to obtain a degradation solution containing Tremella oligosaccharides.

11. The preparation method according to claim 10, characterized in that, The preparation method further includes removing impurities from the degradation solution to obtain a Tremella oligosaccharide solution.

12. The preparation method according to claim 11, characterized in that, The impurity removal includes one or more of the following: enzyme or bacteria inactivation, activated carbon adsorption, red diatomaceous earth filtration aid, and filter paper filtration.

13. The preparation method according to claim 12, characterized in that, The inactivation temperature is 100℃ and the time is 10~30 minutes; the amount of activated carbon added is 1% by mass volume.

14. The preparation method according to any one of claims 11 to 13, characterized in that, The preparation method further includes: concentrating the solid content of the tremella oligosaccharide solution to a mass-volume ratio of 10% to 20%, and then spray drying to obtain tremella oligosaccharide powder.

15. The use of the Bacillus subtilis as described in claim 1 in the preparation of Tremella fuciformis oligosaccharides.

Images