WO2024131919A1 - Paenibacillus and application thereof in preparation of tremella fuciformis oligosaccharide - Google Patents

Abstract

Provided are a paenibacillus and an application thereof in the preparation of a tremella fuciformis oligosaccharide. The deposit number of the paenibacillus is CGMCC No: 25534. Further provided are a method for preparing a tremella fuciformis polysaccharide degradative enzyme, and a preparation method for a tremella fuciformis oligosaccharide. The paenibacillus can produce a high-activity tremella fuciformis polysaccharide degradative enzyme under the induction of tremella fuciformis or a tremella fuciformis polysaccharide. Tremella fuciformis and a tremella fuciformis polysaccharide can be efficiently degraded, a generated tremella fuciformis oligosaccharide is stable in quality and high in yield. The degradation process is mild and controllable, the energy consumption is low, and the operation is simple and suitable for large-scale production, with significant economic benefits.

Description

A kind of Paenibacillus and its application in preparing Tremella oligosaccharide

This application claims the priority of Chinese Patent Application No. 2022116595702 filed on December 22, 2022. This application cites the entire text of the above Chinese Patent Application.

Technical Field

The invention relates to the field of microbial fermentation, in particular to a Paenibacillus sp. and application thereof in the preparation of Tremella oligosaccharide.

Background technique

The main active ingredient of Tremella is Tremella polysaccharide. The main chain of Tremella polysaccharide is polymannose connected by α-1,3-glycosidic bonds, and the side chains contain glucose, xylose, fructose, arabinose and glucuronic acid. Tremella polysaccharide has been reported to have moisturizing, immunomodulatory, anti-liver cancer cell proliferation, hypoglycemic, wound healing, antioxidant, anti-radiation, anti-photoaging, anti-virus and nerve cell protection activities, among which the immunomodulatory effect has received the most attention. Tremella polysaccharide has a large molecular weight (ranging from tens of thousands of Daltons to millions of Daltons), low solubility, and high viscosity after dissolution. The characteristic of excessive molecular weight hinders the absorption of Tremella polysaccharide by the esophagus, limiting its scope of application. Therefore, on the premise of retaining the physiological activity of Tremella polysaccharide, the degradation of Tremella polysaccharide to prepare Tremella oligosaccharides with small molecular weight and good water solubility has important application value.

The process of preparing oligosaccharides by degrading polysaccharides is mainly divided into physical method, chemical method and enzymatic method. Among them, the physical method breaks the polysaccharide chain by high shear, electrolysis, high-pressure homogenization and other methods; the chemical method degrades the polysaccharide chain by using chemical reagents such as acid and hydrogen peroxide. The physical or chemical degradation process randomly breaks the polysaccharide chain, and there are problems of poor production controllability and unstable product quality. 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 is selective in hydrolyzing the chemical bonds of the substrate, and the enzymatic hydrolysis process is precisely controllable. However, there is no literature report on the specific degradation of Tremella polysaccharides by microorganisms or microbial enzymes. Therefore, screening Tremella polysaccharide-degrading strains and developing highly active Tremella polysaccharide-degrading enzymes are of great significance for the industrial production of Tremella oligosaccharides.

Summary of the invention

In order to solve the problems of poor production controllability and unstable product quality caused by random interruption of polysaccharide chains in the process of degrading Tremella polysaccharides in the prior art, the present invention provides a Bacillus sporogenes and its application in the preparation of Tremella oligosaccharides, so that the degradation process is mild and controllable, with low energy consumption, simple operation, suitable for large-scale production, and significant economic benefits.

In order to solve the above technical problems, the first aspect of the present invention provides a Paenibacillus sp. strain, and the preservation number of the Paenibacillus sp. is CGMCC No: 25534.

The colony morphology of the Bacillus strain with the accession number of CGMCC No: 25534 (hereinafter referred to as "strain WSK-2") on the modified TSB solid medium is: round flat colonies with complete edges, slightly raised and smooth surface, translucent, light gray, shiny, and no obvious odor. Under the microscope, it is rod-shaped, Gram-positive, about 1 to 6 microns. It grows aerobically, with the optimal growth pH of 6.0 to 7.0 and the optimal growth temperature of 28°C to 35°C. The strain can produce Tremella polysaccharide degrading enzyme in a culture environment containing Tremella polysaccharide or Tremella. Strain WSK-2 was sequenced and compared with 16S rRNA gene, and it is a Bacillus strain.

In order to solve the above technical problems, the second aspect of the present invention provides a method for preparing Tremella polysaccharide degrading enzyme, the method comprising:

The Paenibacillus as described in the first aspect is fermented to obtain a fermentation broth, and the fermentation broth is centrifuged or filtered to obtain a crude enzyme solution of Tremella polysaccharide degrading enzyme.

In some preferred embodiments, the fermentation culture medium contains the following ingredients in a mass volume ratio: 0.1% to 2.0% tremella or tremella polysaccharide, 0.1% to 2.0% yeast extract, 0.1% to 2.0% soy peptone, 0.1% to 2.0% beef extract, 0.1% to 1.0% dipotassium hydrogen phosphate, 0.1% to 1.0% sodium chloride, 0.1% to 1.0% ammonium sulfate, 0.005% to 0.05% calcium chloride, 0.01% to 0.1% magnesium sulfate, 0.01% to 0.05% ferric citrate, 0.1% to 1.0% cysteine and 0.1% to 1.0% arginine.

In some more preferred embodiments, the fermentation medium is: 2.0% Tremella powder, 1.0% yeast extract, 1.0% soy 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% semi- cystine and 0.1% arginine.

In some embodiments, the fermentation conditions include: the inoculation amount of the Paenibacillus is 1% to 10%, the temperature is 28 to 32° C., and sufficient stirring and ventilation are performed.

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

In some embodiments, the crude enzyme solution is obtained by centrifuging the fermentation broth at 5000 rpm for 10 minutes, and then filtering the fermentation broth using filter cloth, filter paper or a 0.22 μm filter membrane to obtain the crude enzyme solution.

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

In some more preferred embodiments, the molecular pore size of the ultrafiltration membrane is 10 KDa or above.

In order to solve the above technical problems, the third aspect of the present invention provides a crude enzyme solution for degrading Tremella polysaccharide, wherein the crude enzyme solution is prepared by the method described in the second aspect of the present invention.

In order to solve the above technical problems, the fourth aspect of the present invention provides a method for preparing Tremella oligosaccharides, which comprises using the Bacillus as described in the first aspect of the present invention or the crude enzyme solution as described in the third aspect of the present invention to degrade Tremella polysaccharides to obtain a degradation solution containing Tremella oligosaccharides.

In the present invention, the Tremella oligosaccharide refers to a degradation product of Tremella polysaccharide with a molecular weight ranging from 1 to 10 KDa.

In some preferred embodiments, the preparation method is: mixing the crude enzyme solution with a solution containing Tremella polysaccharide or Tremella, and performing enzymolysis at 30-55°C.

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.

In some further more 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.

In some embodiments, the preparation method further comprises removing impurities from the degradation liquid to obtain a Tremella oligosaccharide solution.

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

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

In some embodiments, the preparation method further comprises: concentrating the Tremella oligosaccharide solution to a solid content of 10% to 20% by mass volume, and then spray drying to obtain Tremella oligosaccharide powder.

In order to solve the above technical problems, the fifth aspect of the present invention provides a use of the Paenibacillus as described in the first aspect of the present invention and the crude enzyme solution as described in the third aspect of the present invention in the preparation of Tremella oligosaccharides.

On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

The reagents and raw materials used in the present invention are commercially available.

The positive and progressive effects of the present invention are:

(1) The Paenibacillus sp. strain CGMCC No. 25534 provided by the present invention can produce highly active Tremella polysaccharide-degrading enzyme under the induction of Tremella or Tremella polysaccharide. The strain is easy to culture, has stable properties, and is suitable for industrial production.

(2) The Tremella polysaccharide-degrading enzyme produced by Paenibacillus sp. strain WSK-2 can efficiently degrade Tremella and Tremella polysaccharide 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.

(3) The Tremella oligosaccharide product prepared by the technology provided by the present invention has a minimum molecular weight of 1.0-1.2 KDa, and the Tremella oligosaccharide produced has stable quality, high yield and stable quality. The production process for preparing Tremella polysaccharide degrading enzyme and Tremella oligosaccharide of the present invention has low energy consumption, simple operation, precise and controllable degradation process, is suitable for large-scale production, and has significant economic benefits.

Biomaterial Deposit Information

The Paenibacillus sp. strain WSK-2 of the present invention has been It was deposited in the China General Microbiological Culture Collection (CGMCC) on June 15, 2011. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, the deposit number is: CGMCC No: 25534, the culture name is WSK-2, and the classification name is Paenibacillus sp. strain.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A and Figure 1B show the colony morphology and cell morphology under microscope of strain Paenibacillus sp. strain WSK-2; Figure 1A is a colony photograph of strain Paenibacillus sp. strain WSK-2; Figure 1B is a magnified microscopic photograph of strain Paenibacillus sp. strain WSK-2, with a scale of 5 μm.

Figure 2 shows the effect of pH on the activity of Tremella polysaccharide-degrading enzyme in Paenibacillus sp. strain WSK-2.

Figure 3 shows the effect of temperature on the activity of Tremella polysaccharide-degrading enzyme in Paenibacillus sp. strain WSK-2.

Figure 4 shows the molecular weight distribution of Tremella oligosaccharides produced by Paenibacillus sp. strain WSK-2 Tremella polysaccharide degrading enzyme. The horizontal axis represents the retention time of substances with different molecular weights in the gel chromatography column, and the vertical axis represents the abundance signal of substances with different molecular weights. a is the molecular weight distribution of 0.5% Tremella polysaccharide solution; b is the molecular weight distribution of Tremella polysaccharide degradation products; c is the molecular weight distribution of Tremella oligosaccharide products. The analysis conditions are: Shimadzu high performance liquid chromatography system, TSK-GEL G2000SWXL gel chromatography column as the analytical column, 0.1mol/L sodium nitrate as the mobile phase, flow rate 0.5ml/min, and differential refractometer.

Detailed ways

The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

The following examples are provided to further illustrate the present invention, but the present invention is not limited to the following examples.

The enzyme activity was determined using the DNS method, and the specific operation was as follows:

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

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

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

(1) Enrichment and isolation of Tremella fuciformis-degrading strains

Collect several portions of humus and soil from naturally grown Tremella fuciformis and its planting area, add appropriate amounts of physiological saline, shake for 10 minutes, and obtain a sample suspension. Take 1 ml of each sample suspension and add it to 100 ml of enrichment medium, and culture it at 30 ° C and 100 rpm for 10 days. The composition of the enrichment medium is (w/v): 1% Tremella fuciformis polysaccharide, 0.5% yeast extract, 0.5% soy peptone, 0.5% ammonium sulfate, 0.2% dipotassium hydrogen phosphate, 0.5% sodium chloride, 0.01% calcium chloride and 0.01% magnesium sulfate, sterilized at 121 ° C for 30 minutes before use.

The viscosity of the culture medium containing 1% Tremella polysaccharide is very high. After 10 days of enrichment, the enrichment with significantly reduced viscosity is selected. After appropriate dilution, it is spread on a sterile solid culture plate. The solid culture plate has the same composition as the enrichment medium, except that 1.5% agar powder is added. After static culture at 30°C for 7 days, strains with different colony morphology are inoculated into test tubes containing 4ml enrichment medium and cultured at 30°C and 100rpm for 2 days. Observe whether the viscosity of the culture medium in the test tube decreases. The strains with Tremella polysaccharide degradation effect are transferred to shake flasks containing 50ml enrichment medium respectively. After culturing for 2 days at 30°C and 100rpm, the viscosity of the culture medium in each shake flask is measured using a viscometer. The lower the viscosity after fermentation, the higher the enzyme activity of the microorganism in degrading Tremella polysaccharide.

After repeated enrichment and screening, the present invention discovered the strain WSK-2 that can efficiently degrade Tremella polysaccharide. The colony morphology of strain WSK-2 on the modified TSA medium is: round flat colonies with complete edges, slightly raised and smooth surface, translucent, light gray, shiny, and no obvious odor. Under a microscope, strain WSK-2 cells are rod-shaped, Gram-positive, and about 1 to 6 microns (see Figure 1A and Figure 1B). The formula of the modified TSA solid medium is (w/v): 2% soybean powder papain digest, 0.25% dipotassium hydrogen phosphate, 0.5% sodium chloride, 0.25% glucose, 0.5% Tremella polysaccharide, and 1.5% agar powder.

(2) Molecular biological identification of strain WSK-2.

The strain WSK-2 was cultured in the above enrichment medium, and the bacterial precipitate was collected by centrifugation at 5000 rpm for 5 minutes. The DNA of the bacterial cells was extracted using a commercial DNA extraction kit, and the 16S rRNA gene sequence of the strain was determined. By searching and comparing on NCBI, the strain was found to be a Paenibacillus sp. strain, which was deposited under the accession number CGMCC No: 25534.

Example 2: Preparation and property determination of Paenibacillus sp. strain WSK-2 Tremella polysaccharide degrading enzyme

(1) Preparation of Tremella polysaccharide degrading enzyme

Fermentation medium:

0.1% ~ 2.0% Tremella or Tremella polysaccharide, 0.1% ~ 2.0% yeast extract, 0.1% ~ 2.0% soy 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.

The culture medium components used in this example are (w/v):

2.0% Tremella powder or 1.0% Tremella polysaccharide, 1.0% yeast extract, 1.0% soy 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.

Dissolve the above culture medium components in water, sterilize at 121°C for 30 minutes, and cool to 30°C. Inoculate the strain WSK-2 seed solution into the above cooled culture medium at a volume ratio of 1% to 10%, and culture at 28 to 32°C with sufficient stirring and ventilation for 24 to 48 hours. Produce Tremella polysaccharide degrading enzyme.

Preparation of Tremella polysaccharide degrading enzyme:

The fermentation liquid is centrifuged at 5000 rpm for 10 minutes, or filtered using filter cloth, filter paper and 0.22 μm filter membrane to remove the bacterial cells and insoluble components in the fermentation liquid, and the resulting supernatant is the crude enzyme liquid. The crude enzyme liquid can be stored at 4°C for more than one month after adding potassium sorbate preservative, or can be frozen after adding 10% glycerol.

The crude enzyme solution can be further concentrated and purified by membrane filtration technology. The membrane pore size used is 10KDa. The permeate side is discarded and the concentrated side is the purified enzyme solution.

(2) Determination of the optimal reaction pH of Tremella polysaccharide-degrading enzyme

Prepare buffer:

Buffer solutions of pH 4.0, pH 5.0, and pH 6.0 were prepared with 0.1 mol/L citric acid-sodium hydrogen phosphate buffer pair;

Prepare pH 7.0 and pH 8.0 buffers with 0.1 mol/L Tris-HCl;

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

0.5% Tremella polysaccharide was dissolved in the above buffer solutions.

Determination of relative enzyme activity:

Take 900μl of the above substrate solutions with different pH values and add 100μl of appropriately diluted crude enzyme solution. React at 35℃ for 1 hour, then add 1ml DNS reagent, heat in boiling water for 10 minutes, cool to room temperature, and measure the absorbance at 540nm with a cuvette. Each pH gradient test group uses the crude enzyme solution inactivated by boiling for 5 minutes as a negative control. The relative enzyme activity of each pH gradient is calculated with the enzyme activity of the test group with the largest absorbance as 100%, and 3 parallel samples are set for each test group. The results are shown in Figure 2. The results show that the activity of the enzyme is inhibited under both acidic and alkaline conditions, and the optimal reaction pH range is 6.0-7.0.

(3) Determination of the optimal reaction temperature of Tremella polysaccharide-degrading enzyme

Prepare pH 7.0 buffer with 0.1 mol/L Tris-HCl, add 0.5% Tremella polysaccharide as substrate solution. Take 900 μl of substrate solution and add 100 μl of enzyme solution diluted with water, react for 1 hour at 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, then add 1 ml of DNS reagent, heat in boiling water for 10 minutes, cool to room temperature, and measure absorbance at 540 nm with a cuvette. The crude enzyme solution inactivated by boiling for 5 minutes was used as negative control in each temperature gradient test group. The relative enzyme activity of each temperature gradient was calculated with the enzyme activity of the test group with the largest absorbance as 100%, and 3 parallel samples were set for each test group. The results are shown in Figure 3.

The results showed that the enzyme had a certain degree of heat resistance, and the optimal reaction temperature range was 45℃～50℃.

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

(1) Degradation of Tremella polysaccharide by Tremella polysaccharidase

Take one portion of the purified enzyme solution of the Tremella polysaccharide degrading enzyme described in Example 2 and mix it with 9 portions of 2% (w/v) Tremella polysaccharide or Tremella solution, and fully stir for 20 hours at 45°C. After the enzymolysis is complete, heat to 100°C and incubate for 30 minutes to deactivate the enzyme. The enzymolysis solution is cooled to 70°C, 1% (w/v) activated carbon is added, and stirring is continued for 1 hour. Then, red diatomaceous earth is used as a filter aid, and the mixed solution is filtered with filter paper to remove activated carbon and insoluble matter. The obtained filtrate is colorless and clarified, and then by means of concentration, its solid content reaches 10% to 20% (w/v), and spray-dried Tremella oligosaccharide powder is obtained.

(2) Analysis of degradation products

The substrate solution, the enzymatic hydrolyzate during the degradation process, and the solution prepared from Tremella oligosaccharide powder were taken for molecular weight analysis. The analysis conditions were: Shimadzu HPLC system, TSK-GEL G2000SWXL gel chromatography column, 0.1 mol/L sodium nitrate as mobile phase, flow rate 0.5 ml/min, and differential refractometer.

The analysis results are shown in Figure 4. It can be seen that under the action of the enzyme, the high molecular weight Tremella polysaccharide is degraded, and the final product is mainly oligosaccharides. The oligosaccharides characteristically include three molecular weight ranges of 1.0KDa to 1.2KDa, 2.0KDa to 2.2KDa and 2.9KDa to 3.1KDa.

The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of implementation of the present invention. Therefore, any changes or modifications made according to the claims and description of the present invention should fall within the scope of the patent of the present invention.

Claims (10)

A Bacillus sp., characterized in that the preservation number of the Bacillus sp. is CGMCC No: 25534. A method for preparing Tremella polysaccharide degrading enzyme, characterized in that the method comprises: fermenting the Paenibacillus according to claim 1 to obtain a fermentation liquid, and centrifuging and/or filtering the fermentation liquid to obtain a crude enzyme liquid of Tremella polysaccharide degrading enzyme. The method according to claim 2, characterized in that the fermentation medium contains the following components in a mass volume ratio: 0.1% to 2.0% tremella or tremella polysaccharide, 0.1% to 2.0% yeast extract, 0.1% to 2.0% soy peptone, 0.1% to 2.0% beef extract, 0.1% to 1.0% dipotassium hydrogen phosphate, 0.1% to 1.0% sodium chloride, 0.1% to 1.0% ammonium sulfate, 0.005% to 0.05% calcium chloride, 0.01% to 0.1% magnesium sulfate, 0.01% to 0.05% ferric citrate, 0.1% to 1.0% cysteine and 0.1% to 1.0% arginine; Preferably, the fermentation medium is: 2.0% tremella powder, 1.0% yeast extract, 1.0% soy 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. The method according to claim 2 or 3, characterized in that the fermentation conditions include: the inoculation amount of the Paenibacillus is 1% to 10%, the temperature is 28 to 32°C, and it is fully stirred and aerated; Preferably, the fermentation time is 24 to 48 hours. The method according to any one of claims 2 to 4, characterized in that the conditions for obtaining the crude enzyme solution are: centrifuging the fermentation broth at 5000 rpm for 10 minutes, and then filtering using filter cloth, filter paper or a 0.22 μm filter membrane to obtain the crude enzyme solution; Preferably, the crude enzyme solution can be filtered through an ultrafiltration membrane to obtain an enzyme concentrate, or can be subjected to ammonium sulfate precipitation, centrifugation, desalting and freeze-drying to obtain a solid enzyme preparation; More preferably, the molecular pore size of the ultrafiltration membrane is 10 KDa or above. A crude enzyme solution for degrading Tremella polysaccharide, characterized in that the crude enzyme solution is prepared by the method according to any one of claims 2 to 5. A method for preparing Tremella oligosaccharides, characterized in that the preparation method comprises using the Paenibacillus according to claim 1 or the crude enzyme solution according to claim 6 to degrade Tremella polysaccharides to obtain a degradation solution containing Tremella oligosaccharides; Preferably, the preparation method is: mixing the crude enzyme solution with a solution containing Tremella polysaccharide or Tremella, and performing enzymolysis at 30-55° C.; More preferably, the ratio of the crude enzyme 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; Further more preferably, the ratio of the crude enzyme to the Tremella polysaccharide or Tremella solution is 1:9, the concentration of the solution is 2%, the ratio is a volume ratio, and the concentration is a mass-to-volume ratio. The preparation method according to claim 7, characterized in that the preparation method further comprises removing impurities from the degradation liquid to obtain a Tremella oligosaccharide solution; Preferably, the impurity removal includes one or more of enzyme or bacteria inactivation, activated carbon adsorption, red diatomaceous earth filtration and filter paper filtration; More preferably, the inactivation temperature is 100° C., the time is 10 to 30 minutes; and the added amount of the activated carbon is 1% by mass volume. The preparation method according to claim 7 or 8 is characterized in that the preparation method further comprises: concentrating the solid content of the Tremella fuciformis oligosaccharide solution to a mass volume ratio of 10% to 20%, and spray drying to obtain Tremella fuciformis oligosaccharide powder. A use of the Paenibacillus as claimed in claim 1 or the crude enzyme solution as claimed in claim 6 in the preparation of Tremella oligosaccharides.