Serratia marcescens Y3521 capable of degrading tremella polysaccharide as well as culture method and application of serratia marcescens Y3521

Abstract

The invention discloses serratia marcescens Y3521 capable of degrading tremella polysaccharide and a culture method and application thereof, and belongs to the biotechnical field of food, cosmetics and drugs, the classification name of the serratia marcescens Y3521 is serratia marcescens sp., the serratia marcescens Y3521 is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.32778, the preservation date is November 25, 2024, and the preservation number is CGMCC No.32778. The preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the serratia marcescens Y3521 disclosed by the invention can be used for preparing low-molecular tremella polysaccharide, is high in tremella polysaccharide specificity and can be applied to industrial production.

Description

Technical Field

[0001] The invention relates to Serratia Y3521 capable of degrading Tremella polysaccharide and a culture method and application thereof, belonging to the field of food, cosmetics and pharmaceutical biotechnology. Background Art

[0002] Tremella Tremella fuciformis ), also known as Tremella fuciformis, white fungus, snow fungus, etc., is given the laudatory title of "the crown of fungi". It is an edible fungus with economic value, rich in polysaccharides, proteins, fats, crude fiber and a small amount of vitamins, and has rich nutritional value. The main active ingredient of Tremella fuciformis is Tremella polysaccharide, which can account for more than 65% of the dry weight of Tremella fuciformis. Tremella polysaccharide is an acidic heteropolysaccharide, the main chain of which is mannan connected by α-1,3-glycosidic bonds, and the side chain is composed of glucuronic acid, fucose, galactose and xylose. Studies have shown that Tremella polysaccharide has anti-tumor, antioxidant, intestinal flora regulation, skin moisturizing, blood sugar and blood lipid regulation, and immunity enhancement effects, so it has attracted much attention in recent years.

[0003] Tremella polysaccharides usually have a large molecular weight (up to several million Daltons), low solubility, and high viscosity after dissolution, which greatly limits the development of their biological activity and application scope. Therefore, degrading Tremella polysaccharides to prepare Tremella oligosaccharides with small molecular weight and good water solubility can significantly improve their biological activity and effectively expand their application scope, which has important application value. In current reports, the methods for preparing Tremella oligosaccharides mainly include physical degradation, chemical degradation and enzyme degradation. Physical degradation methods include heating, mechanical shearing, ultrasound and high-pressure homogenization. These physical degradation methods are usually simple processes, but usually have low production efficiency, serious losses and high energy consumption, and the degree of degradation of Tremella polysaccharides is limited, and ultra-low molecular weight Tremella polysaccharides cannot be obtained. Chemical degradation methods mainly include alkaline hydrolysis, acid hydrolysis and oxidative degradation. Chemical degradation methods are usually low-cost and easy to mass produce, but they are easy to destroy the monosaccharide structure, thereby reducing the biological activity of Tremella polysaccharides, and strong acids, strong bases and strong oxidants are potentially dangerous during transportation and production.

[0004] In recent years, microbial enzyme method has gradually become the trend of degrading polysaccharides to prepare oligosaccharides in industrial production due to its advantages of environmental protection, high efficiency, controllability and sustainability. Enzymatic hydrolysis has strong specificity, mild reaction conditions, and the structure of polysaccharides has not changed. It can obtain Tremella polysaccharides of different molecular weights by controlling different degradation times. It is an ideal method for preparing low molecular weight Tremella polysaccharides. At present, the sources of Tremella polysaccharide enzymes on the market are very limited and the price is relatively expensive, which greatly limits its application. The Tremella polysaccharide enzyme microorganisms reported in the literature include Paenibacillus, and so far, there is no information about Serratia marcescens ( Serrati asp. ) has never been reported. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, the present invention provides a Serratia Y3521 for degrading Tremella polysaccharide and a culture method and application thereof, which can be used in the preparation of low-molecular Tremella polysaccharide, has high specificity for Tremella polysaccharide, and can be applied to industrial production.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: A Serratia marcescens Y3521 capable of degrading Tremella fuciformis polysaccharide, classified and named Serratia marcescens Serratia sp. , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.32778, the deposit date is November 25, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] A method for culturing the aforementioned Serratia Y3521, comprising: seed culture, fermentation culture, and post-treatment; The seed culture comprises inoculating the activated Serratia Y3521 strain into a liquid seed culture medium, and culturing the culture medium on a shaking table for 15-24 hours at a temperature of 30-40° C. and a rotation speed of 150-300 rpm to obtain a seed solution; The seed culture medium comprises: 10-20 g / L Tremella polysaccharide, 5-20 g / L yeast extract, 5-20 g / L peptone, 1-10 g / L dipotassium hydrogen phosphate, 0.05-0.2 g / L magnesium sulfate, the solvent is distilled water, and the pH value is 6.0-8.0; The fermentation culture comprises inoculating the seed liquid into a liquid fermentation medium, and expanding the culture for 18-36 hours at a temperature of 30-40° C. and a rotation speed of 150-300 rpm to obtain a fermentation liquid; The liquid fermentation medium comprises: 10-20 g / L Tremella polysaccharide, 5-20 g / L yeast extract, 5-20 g / L peptone, 1-10 g / L dipotassium hydrogen phosphate, 0.05-0.2 g / L magnesium sulfate, the solvent is distilled water, and the pH value is 6.0-8.0; The post-treatment comprises centrifuging the fermentation broth and collecting the supernatant to obtain a crude enzyme solution; In the post-treatment, the centrifugal speed is 8000-9000 rpm and the time is 14-16 min; Preferably, in the post-treatment, the crude enzyme solution is filtered using an ultrafiltration membrane with a filtration accuracy of 10 kDa to obtain a purified enzyme solution.

[0008] An application of the aforementioned Serratia Y3521, adding a crude enzyme solution to a Tremella polysaccharide solution, performing an enzymatic hydrolysis reaction to obtain an enzymatic hydrolyzate, and inactivating and purifying the enzymatic hydrolyzate to obtain a low-molecular Tremella polysaccharide; The concentration of the Tremella polysaccharide solution is 0.5-0.6%; The pH of the Tremella polysaccharide solution is 7.0-7.5; Preferably, the pH of the Tremella polysaccharide solution is 7.0; The volume fraction of the crude enzyme solution to the Tremella polysaccharide solution is 5-15%; The temperature of the enzymatic hydrolysis reaction is 35-40°C; The enzymatic reaction time is 30-180min; Preferably, the temperature of the enzymatic hydrolysis reaction is 35°C; The inactivation temperature is 95-98°C and the time is 30-40min; The purification comprises diluting the inactivated enzymatic hydrolysate once, centrifuging once, collecting the supernatant, performing alcohol precipitation, performing a second centrifugation, performing a second dilution, and repeating the alcohol precipitation, second centrifugation, and dilution operations 2-3 times; In the purification, the dilution multiple of the primary dilution is 2-5 times; The centrifugal speed of the first centrifugation is 10000-11000 rpm, and the time is 20-25 min; The alcohol precipitation is performed by adding 4-5 times the volume of ethanol and treating at 3-5°C for 3-3.5h; The secondary centrifugation has a centrifugal speed of 6000-7000 rpm and a time of 10-12 min; For the secondary dilution, an equal volume of deionized water is added for dilution.

[0009] Compared with the prior art, the present invention has the following beneficial effects: The invention prepares tremella polysaccharidase by utilizing Serratia Y3521 for the first time, and uses the enzyme for preparing low-molecular tremella polysaccharide. The tremella polysaccharide with a molecular weight of 1000-2000 kDa can be degraded to less than 10 kDa, has high specificity for tremella polysaccharide, can be applied to industrial production, thereby improving the yield and stability of the tremella polysaccharidase, and has broad application prospects in the fields of food, medicine, health products, cosmetics, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is the degradation rate of Tremella polysaccharide of 40 strains in the experimental group of Example 1; Figure 2 In Example 1, the glucuronic acid content is taken as the horizontal axis and the OD 530 A standard curve graph is plotted for the ordinate; Figure 3 is the Tremella polysaccharidase activity of the 10 strains in the experimental group of Example 1; Figure 4 The morphological results of strain Y3521 in Example 2; in, Figure 4 (a) Colony morphology of strain Y3521 on tryptone soy agar medium; Figure 4 (b) is an optical microscope photo of strain Y3521; Figure 5 The phylogenetic tree of strain Y3521 and related strains based on 16S rRNA gene in Example 2; Figure 6 The results of Tremella polysaccharide enzyme activity at different pH values ​​in Example 4; Figure 7 The results of Tremella polysaccharide enzyme activity at different temperatures in Example 4; Figure 8 The GPC diagram of the enzymatic hydrolysis products at different enzymatic hydrolysis times in Example 5; Fig. 9 This is the GPC diagram of the enzymatic hydrolysis product after 3 hours of enzymatic hydrolysis in Example 5. DETAILED DESCRIPTION

[0011] The present invention will be further described below in conjunction with the accompanying drawings and specific implementation methods, but the protection scope of the present invention is not limited thereto.

[0012] Example 1 Enrichment and screening of strain Y3521 1. Processing Tremella samples Collect several portions of rotten Tremella from Tremella production areas, weigh 10g Tremella sample and put it into 90mL sterile physiological saline, transfer it to a soybean milk machine for grinding and mixing, and obtain a sample suspension. Take 10mL of sample suspension in a 250mL triangular flask, add 100mL of 1% Tremella polysaccharide enrichment medium, and enrich and culture it at a temperature of 35℃ and a rotation speed of 150rpm for 3 days; The composition of the 1% Tremella polysaccharide enrichment medium is: Tremella polysaccharide 10g / L, yeast powder 10g / L, peptone 5g / L, dipotassium hydrogen phosphate 2g / L, sodium chloride 5g / L, magnesium sulfate 0.2g / L; The molecular weight of the Tremella polysaccharide is 1250 kDa.

[0013] 2. Preliminary screening: Tremella polysaccharide degradation experiment After the sample was pretreated, the sample bacterial suspension was diluted 10 times with sterile saline, and the dilutions of 10 -1 , 10 -2 , 10 -3 , 10-4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 200 μL of each dilution was spread on a 1% Tremella polysaccharide enriched medium, and after culturing for 5 days at a temperature of 35°C, the growth of the strains was observed, and the strains with different colony morphologies were separated and purified three times until pure strains were obtained, and a total of 40 strains were obtained, and the 40 strains were numbered as Y301-Y304, T301-T305, Y3501-Y3523, and T351-T358, respectively; the strains were inoculated into a 2% Tremella polysaccharide medium at an inoculation rate of 5% to carry out a Tremella polysaccharide degradation experiment as the test group, and an equal volume of physiological saline was used instead of the bacterial solution as the control group; The composition of the 1% Tremella polysaccharide enrichment medium is: Tremella polysaccharide 10g / L, yeast powder 10g / L, peptone 5g / L, dipotassium hydrogen phosphate 2g / L, sodium chloride 5g / L, magnesium sulfate 0.2g / L; The composition of the 2% Tremella polysaccharide culture medium is: Tremella polysaccharide 20g / L, yeast powder 10g / L, peptone 5g / L, dipotassium hydrogen phosphate 2g / L, sodium chloride 5g / L, magnesium sulfate 0.2g / L; After culturing for 48 h at a temperature of 35 °C and a rotation speed of 220 rpm, the viscosity of the culture medium in the shake flasks of the experimental group and the control group was measured using a viscometer to obtain the viscosity of the experimental group. V 1 and control group viscosity V 0, the degradation rate (DR) of Tremella fuciformis polysaccharide was calculated according to the following formula. The higher the degradation rate after fermentation, the higher the enzyme activity of Tremella fuciformis polysaccharide produced by the microorganism.

[0014]

[0015] The degradation rates of Tremella polysaccharide of 40 strains in the experimental group were calculated and statistically analyzed. The statistical results are shown in Figure 1 ,from Figure 1 It can be seen that the degradation rates of Tremella fuciformis polysaccharides of strains Y301, Y302, Y304, T301, Y353, Y354, Y357, Y358, Y3521 and Y3522 were relatively high, all greater than 20%. Therefore, the above 10 strains with Tremella fuciformis polysaccharide degradation rates greater than 20% were selected for subsequent experiments.

[0016] 3. Secondary screening: enzyme activity determination (1) Preparation of crude enzyme solution: The 10 strains screened out in the primary screening were inoculated into the secondary screening fermentation medium at an inoculation rate of 5%, and the fermentation culture was carried out at a temperature of 35°C and a rotation speed of 180 rpm for 36 h to obtain a fermentation liquid. The fermentation liquid was then centrifuged at 5000 rpm for 10 min to remove the bacteria and insoluble components. The supernatant was the crude enzyme solution.

[0017] The components of the rescreened fermentation medium are: 10 g / L tremella polysaccharide, 5 g / L glucose, 10 g / L yeast powder, 5 g / L peptone, 5 g / L beef extract, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium chloride, and 0.2 g / L magnesium sulfate.

[0018] (2) Determination of Tremella polysaccharide enzyme activity a. Establish a method for determining the activity of Tremella polysaccharidase: Tremella polysaccharide is composed of mannan, glucuronic acid, xylose, etc., among which glucuronic acid has reducing property. Based on this, a method for determining Tremella polysaccharidase by DNS method was established. The activity of Tremella polysaccharidase is defined as 1 L fermentation liquid hydrolyzing Tremella polysaccharide to produce 1 mg of glucuronic acid in 1 h under the conditions of temperature of 35°C and pH of 7.0, which is 1 enzyme activity unit, with the symbol U, expressed in milligrams per liter per hour (mg / L·h).

[0019] b. Determination of Tremella polysaccharide enzyme activity The experimental group was prepared by adding 1.8 mL of 0.5% (w / v) Tremella polysaccharide solution into a test tube, diluting the crude enzyme solution prepared in step (1) by 5 times to obtain a diluted enzyme solution, adding 200 μL of the diluted enzyme solution to the Tremella polysaccharide solution, mixing well, reacting for 1 h at 35 °C and pH 7.0, adding 2 mL of DNS reagent, mixing well, heating in boiling water for 5 min, cooling to room temperature, and measuring the absorbance at 530 nm; The blank group was prepared by adding 1.8 mL of 0.5% (w / v) Tremella polysaccharide solution into a test tube, adding 2 mL of DNS reagent, and diluting the crude enzyme solution prepared in step (1) by 5 times to obtain the diluted enzyme solution. Then, 200 μL of the diluted enzyme solution was added, mixed evenly, heated in boiling water for 5 min, cooled to room temperature, and the absorbance at 530 nm was measured; c. Prepare the standard curve: Mix different amounts of 500 mg / L glucuronic acid standard solution and phosphate buffer with DNS reagent according to the table below, and record the absorbance of the reaction solution at 530 nm (OD 530 ), as shown in the following table:

[0020] According to the data in the table above, the glucuronic acid content is taken as the horizontal axis and the OD 530As the vertical axis, draw the standard curve graph, and the obtained standard curve graph is shown in Figure 2 As shown, according to Figure 2 The linear regression equation obtained is y=4.0289x-0.2089, R 2 =0.9992.

[0021] According to the above linear regression equation, the Tremella polysaccharidase activity of the 10 strains in the experimental group, specifically strains Y301, Y302, Y304, T301, Y353, Y354, Y357, Y358, Y3521, and Y3522, was calculated. The results are shown in Figure 3 .Depend on Figure 3 It can be seen that the degradation rate of Tremella polysaccharide and Tremella polysaccharase activity of strain Y3521 are both high, and the degradation rate of Tremella polysaccharide is 66.04%.

[0022] Example 2 Morphology and molecular biological identification of strain Y3521 The colony morphology of strain Y3521 screened in Example 1 on tryptone soy agar (TSA) medium is as follows: Figure 4 As shown in (a), the colony is round, with a red center and a white, slightly raised edge. The colony diameter is 1-3 mm, and the surface is moist, smooth, and easy to pick up. Figure 4 As shown in (b), it is spherical or short rod-shaped. The strain can grow on a culture medium with Tremella polysaccharide as the sole carbon source.

[0023] The genomic DNA of the isolated strain was extracted using a bacterial DNA extraction kit, and the 16S rRNA gene fragment of strain Y3521 was PCR amplified using the bacterial universal primers upstream primer 27F (sequence shown in SEQ ID NO.1) and downstream primer 1492R (sequence shown in SEQ ID NO.2). The PCR amplification reaction system was: 1 μL of upstream and downstream primers, 12.5 μL of Green Taq Mix, 1 μL of DNA, and 9.5 μL of ddH2O. The PCR amplification reaction conditions were: 95°C pre-denaturation for 10 min, 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 90 s, a total of 35 cycles, 72°C extension for 15 min, and 4°C insulation. The PCR amplification products were purified and subjected to gel electrophoresis experiments. The PCR products with normal PCR amplification fragment length were sent to Jinweizhi Biotechnology Co., Ltd. for sequencing.

[0024] After sequencing, the length of the 16S rRNA gene fragment of Serratia marcescens Y3521 was 1471bp, and the sequencing results are shown in SEQ ID NO.3. The 16S rRNA gene sequence was compared in the NCBI database, and the results showed that the 16S rRNA gene sequence of the strain of the present invention had a high homology with the 16S rRNA gene sequence of some Serratia marcescens registered in the NCBI database (AJ550467.1 97.02%, OP810732.1 97.02%, OP810789.1 97.02%, CP081470.1 96.95%). The similarity and integrity values ​​were comprehensively analyzed, and the standard strain sequence with the closest relationship to the identified strain was selected. The phylogenetic evolutionary tree based on the NJ (Neighbor-Joining) method and passed 1000 confidence tests was drawn using Mega software, see Figure 5 shown.

[0025] The strain Y3521 screened by the present invention is classified and named as Serratia Serratia sp. , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.32778, the deposit date is November 25, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0026] Example 3 Preparation of Tremella polysaccharide degrading enzyme using strain Y3521 1. After activating strain Y3521, pick a single colony and inoculate it into liquid seed culture medium. Incubate it in a shaking incubator at 35°C and 200 rpm for 18 hours to obtain seed liquid. The liquid seed culture comprises the following ingredients: 15 g / L tremella polysaccharide, 10 g / L yeast extract, 10 g / L peptone, 4 g / L dipotassium hydrogen phosphate, and 0.2 g / L magnesium sulfate; the solvent is distilled water; and the pH value is 7.0.

[0027] 2. Inoculate the seed liquid into the fermentation medium at an inoculum rate of 5%, and culture at a temperature of 35°C and a rotation speed of 200 rpm for 24 hours to obtain a fermentation liquid; The fermentation medium comprises the following components: 10 g / L tremella polysaccharide, 10 g / L yeast extract, 10 g / L peptone, 4 g / L dipotassium hydrogen phosphate, and 0.1 g / L magnesium sulfate; the solvent is distilled water; and the pH value is 7.0.

[0028] 3. Centrifuge the fermentation broth at 8000 rpm for 15 min to remove the bacteria and insoluble components, collect the supernatant and obtain the crude enzyme solution.

[0029] Example 4 Basic properties of enzymes 1. Study on the optimal reaction pH of enzyme First, phosphate buffers with pH values ​​of 5, 5.5, 6, 6.5, 7, 7.5, and 8 were prepared, and then 0.5% (w / v) solutions of Tremella polysaccharide (molecular weight 1250 kDa) with different pH values ​​were prepared using phosphate buffers with different pH values; The steps for determining the enzyme activity of the experimental group are as follows: take 7 test tubes, add 1.8 mL of Tremella polysaccharide solution of different pH values ​​to each test tube, dilute the crude enzyme solution obtained in Example 3 by 5 times to obtain a diluted crude enzyme solution, add 200 μL of the diluted crude enzyme solution to each Tremella polysaccharide solution, mix well, react in a 35°C water bath for 1 h, add 2 mL of DNS reagent and mix well, heat in boiling water for 5 min, take out and cool to room temperature, and measure the absorbance at 530 nm using a spectrophotometer; The steps of enzyme activity determination of the blank group are as follows: take 7 test tubes, add 1.8 mL of Tremella polysaccharide solution of different pH values ​​to each test tube, add 2 mL of DNS reagent, dilute the crude enzyme solution obtained in Example 3 by 5 times to obtain a diluted crude enzyme solution, add 200 μL of the diluted crude enzyme solution, mix well, heat in boiling water for 5 min, cool to room temperature, and measure the absorbance at 530 nm with a spectrophotometer; The activity of Tremella polysaccharide was calculated according to the absorbance at 530 nm and the calculation method of Tremella polysaccharide activity in Example 1. The results of Tremella polysaccharide activity corresponding to Tremella polysaccharide solutions with different pH values ​​were shown in Table 1. Figure 6 ,Depend on Figure 6 It can be seen that the most suitable pH for strain Y3521 is 7.0.

[0030] 2. Study on the optimal reaction temperature of enzyme A phosphate buffer with a pH value of 7.0 was prepared, and then a 0.5% (w / v) solution of Tremella polysaccharide (molecular weight 1250 kDa) was prepared using the phosphate buffer; The steps for determining the enzyme activity of the experimental group are as follows: take 6 test tubes, add 1.8 mL of the above-mentioned Tremella polysaccharide solution into the test tubes, dilute the crude enzyme solution obtained in Example 3 by 5 times to obtain a diluted crude enzyme solution, add 200 μL of the diluted crude enzyme solution to the Tremella polysaccharide solution respectively, mix well, place in a water bath at 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C for reaction for 1 h, add 2 mL of DNS reagent and mix well, heat in boiling water for 5 min, take out and cool to room temperature, and measure the absorbance at 530 nm using a spectrophotometer; The steps of enzyme activity determination of the blank group are as follows: take a test tube, add 1.8 mL of the above Tremella polysaccharide solution into the test tube, add 2 mL of DNS reagent, dilute the crude enzyme solution obtained in Example 3 by 5 times to obtain a diluted crude enzyme solution, add 200 μL of the diluted crude enzyme solution to the Tremella polysaccharide solution respectively, mix well, heat in boiling water for 5 min, cool to room temperature, and measure the absorbance at 530 nm with a spectrophotometer; The activity of Tremella polysaccharidase was calculated according to the absorbance at 530 nm and the calculation method of Tremella polysaccharidase activity in Example 1. The results of Tremella polysaccharidase activity corresponding to different reaction temperatures were shown in Table 1. Figure 7 ,Depend on Figure 7 It can be seen that the most suitable temperature for strain Y3521 is 35°C.

[0031] Example 5 Preparation of Tremella oligosaccharides using Tremella polysaccharide degrading enzyme prepared by strain Y3521 Take 100 mL of a 0.5% (w / v) solution of Tremella polysaccharide (molecular weight 1250 kDa) prepared in phosphate buffer with a pH value of 7.0 in Example 4 as a substrate, add the crude enzyme solution prepared in Example 3 at a volume fraction of 10%, react in a 35°C water bath for 3 h, take 5 mL of the reaction solution every 30 min, inactivate and purify it, and then use it for GPC detection of molecular weight.

[0032] The steps for purifying the enzymatic hydrolysate are as follows: dilute the enzymatic hydrolysate 3 times, centrifuge at 10,000 rpm for 20 minutes, remove the precipitate and collect the supernatant; add 4 times the volume of ethanol, treat at 4°C for 3 hours, centrifuge at 6,000 rpm for 10 minutes to obtain the alcohol precipitate product, add an equal volume of deionized water, and repeat the above operation twice after it is fully dissolved.

[0033] High performance liquid chromatography-gel permeation chromatography (GPC) was used to determine the molecular weight (Mw) of Tremella polysaccharide using an Ohpak SB-806 HQ2.0 300 column. A differential detector (RI) was used, and 0.1 M NaNO3 was set at a flow rate of 0.5 mL / min as the mobile phase. The column temperature was 25 °C.

[0034] The GPC diagrams of the enzymatic hydrolysis products at different enzymatic hydrolysis times are shown in Figure 8 As shown by Figure 8 It can be seen that the molecular weight of Tremella polysaccharide decreases with the extension of enzymatic hydrolysis time. The enzyme solution can degrade the Tremella polysaccharide substrate with a molecular weight of 1250 kDa into a low molecular weight Tremella polysaccharide with a molecular weight of about 200 kDa after enzymatic hydrolysis for 30 min. The GPC graph of Tremella oligosaccharide after enzymatic hydrolysis for 3 h is shown in FIG. Fig. 9 As shown by Fig. 9It can be seen that the enzyme solution can continue to degrade into Tremella oligosaccharides with a molecular weight below 10 kDa. Figure 8 and Fig. 9 It can be seen that by controlling the enzymatic hydrolysis time, tremella polysaccharides or tremella oligosaccharides of different molecular weights can be obtained.

[0035] In summary, the present invention uses strain Y3521 to prepare Tremella polysaccharidase, and the prepared Tremella polysaccharidase can prepare Tremella polysaccharides or Tremella oligosaccharides of different molecular weights by controlling the reaction time, and can degrade Tremella polysaccharides with a molecular weight of 1250 kDa to less than 10 kDa within 3 hours. The prepared Tremella polysaccharidase has an optimum pH of 7.0 and an optimum temperature of 35°C, and has high specificity for Tremella polysaccharides.

Claims

1. A Serratia marcescens Y3521 that degrades Tremella polysaccharides, characterized in that: Serratia Serratia sp. , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCCNo.32778, the deposit date is November 25, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A method for culturing Serratia Y3521 according to claim 1, characterized in that: include: Seed culture, fermentation culture, and post-processing.

3. The method for cultivating Serratia Y3521 according to claim 2, characterized in that: The seed culture comprises inoculating the activated Serratia Y3521 strain into a liquid seed culture medium, and culturing the culture medium on a shaking table for 15-24 hours at a temperature of 30-40° C. and a rotation speed of 150-300 rpm to obtain a seed solution; The seed culture medium comprises the following components: 10-20 g / L tremella polysaccharide, 5-20 g / L yeast extract, 5-20 g / L peptone, 1-10 g / L dipotassium hydrogen phosphate, and 0.05-0.2 g / L magnesium sulfate; the solvent is distilled water; and the pH value is 6.0-8.

0.

4. The method for cultivating Serratia Y3521 according to claim 2, characterized in that: The fermentation culture comprises inoculating the seed liquid into a liquid fermentation medium, and expanding the culture for 18-36 hours at a temperature of 30-40° C. and a rotation speed of 150-300 rpm to obtain a fermentation liquid; The liquid fermentation medium comprises the following components: 10-20 g / L tremella polysaccharide, 5-20 g / L yeast extract, 5-20 g / L peptone, 1-10 g / L dipotassium hydrogen phosphate, and 0.05-0.2 g / L magnesium sulfate; the solvent is distilled water; and the pH value is 6.0-8.

0.

5. The method for cultivating Serratia Y3521 according to claim 2, characterized in that: The post-treatment comprises centrifuging the fermentation broth and collecting the supernatant to obtain a crude enzyme solution; In the post-treatment, the centrifugal speed is 8000-9000 rpm and the time is 14-16 min.

6. A use of the Serratia Y3521 according to claim 1, characterized in that: The crude enzyme solution obtained by fermentation of the Serratia Y3521 is added to the Tremella polysaccharide solution to carry out enzymolysis reaction to obtain an enzymolysis solution, and the enzymolysis solution is inactivated and purified to obtain low-molecular Tremella polysaccharide.

7. The use of Serratia marcescens Y3521 according to claim 6, characterized in that: The concentration of the Tremella polysaccharide solution is 0.5-0.6%; The pH of the Tremella polysaccharide solution is 7.0-7.5; The volume fraction of the crude enzyme solution to the Tremella polysaccharide solution is 5-15%; The temperature of the enzymatic hydrolysis reaction is 35-40°C; The enzymatic reaction time is 30-180min; The inactivation temperature is 95-98°C and the time is 30-40 minutes.

8. The use of Serratia marcescens Y3521 according to claim 6, characterized in that: The purification comprises diluting the inactivated enzymatic hydrolysate once, centrifuging once, collecting the supernatant, performing alcohol precipitation, performing a second centrifugation, performing a second dilution, and repeating the alcohol precipitation, second centrifugation, and dilution operations 2-3 times; In the purification, the dilution multiple of the primary dilution is 2-5 times; The centrifugal speed of the first centrifugation is 10000-11000 rpm, and the time is 20-25 min; The alcohol precipitation is performed by adding 4-5 times the volume of ethanol and treating at 3-5°C for 3-3.5h; The secondary centrifugation has a centrifugal speed of 6000-7000 rpm and a time of 10-12 min; For the secondary dilution, an equal volume of deionized water is added for dilution.

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