Intergeneric bacillus y3522 and application thereof in the production of tremella oligosaccharide
By using Bacillus interstitial Y3522 to produce enzymes and then performing post-processing and enzymatic hydrolysis, the problems of high energy consumption, instability, and high cost in the production of Tremella fuciformis polysaccharides have been solved, achieving efficient preparation of Tremella fuciformis oligosaccharides and expanding their application range.
Patent Information
- Application Number
- CN202510079704.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-18
- Publication Date
- 2026-01-02
- Estimated Expiration
- 2045-01-18
AI Technical Summary
Existing technologies for the production of Tremella polysaccharides suffer from problems such as high energy consumption, instability, high cost, and high risk, and there are few reports on methods for the enzymatic degradation of Tremella polysaccharides by microorganisms.
The enzyme was produced by fermentation using Bacillus subtilis Y3522 (Mesobacillus sp. CGMCC No. 32779). The oligosaccharide was prepared from Tremella fuciformis through fermentation, post-treatment and enzymatic hydrolysis. The specific steps included fermentation culture, centrifugation, filtration, enzymatic hydrolysis and purification.
It achieves efficient, environmentally friendly, and controllable degradation of the molecular weight of Tremella fuciformis polysaccharides to below 20kDa, expanding the application range of Tremella fuciformis oligosaccharides and making them suitable for the food, pharmaceutical, health product, and cosmetic fields.
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Figure CN119752732B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to an interspecies Bacillus spores Y3522 and its application in the production of tremella oligosaccharide, belonging to the field of food, cosmetics, and pharmaceutical biotechnology. BACKGROUND
[0002] Tremella fuciformis Berk. Tremella fuciformis Also known as tremella, white fungus, and snow fungus, it is a kind of 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 is tremella polysaccharide, which can account for more than 65% of the dry weight of tremella. Tremella polysaccharide is an acidic heteropolysaccharide, and its main chain is a mannose connected by α-1,3-glycosidic bond, and the side chain is composed of glucuronic acid, fucose, galactose, and xylose. Studies have shown that tremella polysaccharide has the effects of anti-tumor, antioxidant, intestinal flora regulation, skin moisturizing, blood glucose and lipid regulation, and immune enhancement, and has attracted much attention in recent years.
[0003] Tremella polysaccharide usually has a very large molecular weight (up to several million daltons), low solubility, and high viscosity after dissolution, which greatly limits its biological activity and application scope. Therefore, degrading tremella polysaccharide to prepare tremella oligosaccharide with small molecular weight and good water solubility can significantly improve its biological activity and effectively expand its application scope, which has important application value. In the current reports, the methods for preparing tremella oligosaccharide mainly include physical and chemical methods, which usually have the disadvantages of high energy consumption, instability, and high cost, and are not suitable for large-scale production. For example, the chemical degradation method by hydrogen peroxide can easily destroy the structure of monosaccharide, thereby reducing the biological activity of tremella polysaccharide, but hydrogen peroxide is a strong oxidizing agent, which has potential risks during transportation and production; for example, the physical method of high-pressure homogenization needs to use a high-pressure homogenizer to repeatedly homogenize tremella polysaccharide to reduce the molecular weight of tremella polysaccharide, but the production efficiency is low and the energy consumption is high, and the degree of degradation of tremella polysaccharide is limited, resulting in great limitations in production. In recent years, microbial enzyme method has gradually become the trend of industrial production of degrading polysaccharide to prepare oligosaccharide due to its environmental protection, high efficiency, controllability, and sustainability. However, so far, there have been few reports on the method of degrading tremella polysaccharide to produce oligosaccharide by microbial enzyme method. Therefore, isolating and screening strains that can efficiently degrade tremella polysaccharide and obtaining high-activity tremella polysaccharide-degrading enzyme can effectively promote the industrial production of tremella oligosaccharide and expand its application scope. SUMMARY
[0004] The application aims to provide an intergeneric Bacillus Y3522 and its application in the production of tremella oligosaccharide, so that the method for industrially producing tremella oligosaccharide is more environmentally friendly, efficient, controllable and sustainable, thereby solving the problems of high energy consumption, instability, high cost and high risk in the current industrial production of tremella oligosaccharide.
[0005] To solve the above technical problems, the technical solutions adopted by the application are as follows:
[0006] An intergeneric Bacillus Y3522, which is classified and named as Bacillus sp. Mesobacillus , has a preservation number of CGMCC No. 32779, is preserved in the China General Microbiological Culture Collection Center, and is preserved on November 25, 2024 at an address of No. 3, Beichen West Road, Chaoyang District, Beijing.
[0007] An application of the intergeneric Bacillus Y3522 in the production of tremella oligosaccharide, comprising: fermentation for enzyme production, post-treatment, and enzymolysis.
[0008] The fermentation for enzyme production is performed by activating and culturing the intergeneric Bacillus Y3522, when OD 600 the optical density reaches 1.0-1.5, inoculating into a fermentation medium, and performing fermentation culture to obtain a fermentation liquor.
[0009] In the fermentation for enzyme production, the inoculation amount when inoculating into the fermentation medium is 1-10%.
[0010] The fermentation culture is performed at a temperature of 30-40 DEG C, an aeration amount of 5-15 L / min, a rotation speed of 100-300 rpm, and a fermentation time of 24-48 h.
[0011] The fermentation medium comprises tremella polysaccharide 5-15 g / L, beef extract 5-20 g / L, yeast extract 5-20 g / L, peptone 5-20 g / L, dipotassium hydrogen phosphate 1-10 g / L, sodium chloride 1-10 g / L, ammonium sulfate 1-10 g / L, and magnesium sulfate 0.05-0.2 g / L, and a solvent is distilled water, and a pH value is 6.8-7.5.
[0012] In the post-treatment, the fermentation liquor is centrifuged to obtain a crude enzyme liquor.
[0013] In the post-treatment, the centrifugal speed is 8000-9000 rpm, and the time is 14-16 min.
[0014] Preferably, in the post-treatment, an ultrafiltration membrane with a filtration precision of 10 kDa is used to filter the crude enzyme liquor, so that a purified enzyme liquor can be obtained.
[0015] The enzyme hydrolysis is carried out by adding the crude enzyme solution into the tremella polysaccharide solution, and an enzyme hydrolysis solution is obtained; the enzyme hydrolysis solution is inactivated and purified, and low-molecular tremella polysaccharide is obtained.
[0016] In the enzyme hydrolysis, the concentration of the tremella polysaccharide solution is 0.5-0.6%.
[0017] Preferably, the pH of the tremella polysaccharide solution is 7.5.
[0018] Preferably, the pH of the tremella polysaccharide solution is 7.5.
[0019] The temperature of the enzyme hydrolysis reaction is 35-40℃.
[0020] The time of the enzyme hydrolysis reaction is 30-120min.
[0021] Preferably, the temperature of the enzyme hydrolysis reaction is 35℃.
[0022] The temperature of the inactivation is 95-98℃, and the time is 30-40min.
[0023] In the purification, the enzyme hydrolysis solution after inactivation is diluted once, centrifuged once, and the supernatant is collected, alcohol precipitation is carried out, and the operation of alcohol precipitation, secondary centrifugation and dilution is repeated 2-3 times.
[0024] In the purification, the dilution ratio of the first dilution is 2-5 times.
[0025] The centrifugal speed of the first centrifugation is 10000-11000rpm, and the time is 20-25min.
[0026] In the alcohol precipitation, 4-5 times of ethanol is added, and the treatment is carried out at 3-5℃ for 3-3.5h.
[0027] The centrifugal speed of the second centrifugation is 6000-7000rpm, and the time is 10-12min.
[0028] In the second dilution, the same volume of deionized water is added for dilution.
[0029] Compared with the prior art, the present application has the following beneficial effects:
[0030] The present application provides an intergeneric bacillus Y3522 and its application in tremella oligosaccharide production, which is the first time to use intergeneric bacillus Y3522 to produce tremella polysaccharase, and can be used for the production of low-molecular tremella polysaccharide, and can degrade tremella polysaccharide with a molecular weight of 1000-4000kDa into tremella oligosaccharide with a molecular weight of less than 20kDa, which has a wide application prospect in the fields of food, medicine, health care products and cosmetics. BRIEF DESCRIPTION OF DRAWINGS
[0031] Figure 1 The polysaccharide degradation rate of 40 strains of the experimental group in Example 1;
[0032] Figure 2 The standard curve graph of Example 1 with the content of glucuronic acid as the abscissa and the OD 530 as the ordinate;
[0033] Figure 3 The polysaccharase activity of 10 strains of the experimental group in Example 1;
[0034] Figure 4 The morphological results of strain Y3522 in Example 2;
[0035] wherein, Figure 4 (a) is the colony morphology of strain Y3522 on trypticase soy agar medium; Figure 4 (b) is the optical microscope photograph of strain Y3522;
[0036] Figure 5 The phylogenetic tree of strain Y3522 and related strains based on 16S rRNA gene in Example 2;
[0037] Figure 6 The results of polysaccharase activity at different temperatures in Example 4;
[0038] Figure 7 The results of polysaccharase activity at different pH in Example 4;
[0039] Figure 8 The GPC graph of the enzymatic product under different enzymolysis time in Example 5;
[0040] wherein, Figure 8 (a) is the GPC graph of the enzymatic product when the enzymolysis time is 30 min; Figure 8 (b) is the GPC graph of the enzymatic product when the enzymolysis time is 60 min; Figure 8 (c) is the GPC graph of the enzymatic product when the enzymolysis time is 90 min; Figure 8 (d) is the GPC graph of the enzymatic product when the enzymolysis time is 120 min. DETAILED DESCRIPTION
[0041] The present application will be further described in connection with the accompanying drawings and specific embodiments, but the scope of protection of the present application is not limited thereto.
[0042] Example 1 Isolation and screening of strain Y3522
[0043] 1. Pretreatment of tremella sample
[0044] Collect several rotten Tremella fuciformis samples from Tremella fuciformis production areas, take 10 g of the Tremella fuciformis sample into 90 mL of sterile normal saline, transfer to a soybean milk machine for crushing and mixing, to obtain a sample suspension; take 10 mL of the sample suspension into a 250 mL flask, add 100 mL of 1% Tremella fuciformis polysaccharide culture medium, and enrich culture under the condition of a temperature of 35°C and a rotation speed of 150 rpm for 3 days, to obtain an enriched culture;
[0045] The components of the 1% Tremella fuciformis polysaccharide enrichment culture medium are: Tremella fuciformis polysaccharide 10 g / L, yeast powder 10 g / L, proteose peptone 5 g / L, potassium phosphate dibasic 2 g / L, sodium chloride 5 g / L, magnesium sulfate 0.2 g / L;
[0046] The molecular weight of the Tremella fuciformis polysaccharide is 1250 kDa;
[0047] 2. Preliminary screening: Tremella fuciformis polysaccharide degradation experiment
[0048] Dilute the enriched culture with sterile normal saline by 10 times in gradient, and take 200 μL of each of the dilutions of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 dilutions and spread on the 1% Tremella fuciformis polysaccharide culture medium, and culture at a temperature of 35°C for 5 days, and then observe the growth of the strains, and perform 3 times of separation and purification on the strains with different colony morphologies until pure strains are obtained; a total of 40 strains are obtained, and the 40 strains are numbered as Y301-Y304, T301-T305, Y3501-Y3523, and T351-T358, respectively;
[0049] The 1% Tremella fuciformis polysaccharide culture medium is the same as the 1% Tremella fuciformis polysaccharide culture medium used in the first step of preliminary screening;
[0050] As an experimental group, inoculate all the strains obtained by separation and purification into 2% Tremella fuciformis polysaccharide culture medium according to an inoculation amount of 5% for Tremella fuciformis polysaccharide degradation experiments, and as a control group, replace the bacterial liquid with an equal volume of normal saline and add it into the 2% Tremella fuciformis polysaccharide culture medium;
[0051] The components of the 2% Tremella fuciformis polysaccharide culture medium are: Tremella fuciformis polysaccharide 20 g / L, yeast powder 10 g / L, proteose peptone 5 g / L, potassium phosphate dibasic 2 g / L, sodium chloride 5 g / L, magnesium sulfate 0.2 g / L;
[0052] After culturing for 48h at a temperature of 35℃ and a rotation speed of 220rpm, the viscosity of the culture medium in the experimental group and the control group was measured using a viscometer to obtain the viscosity V1 of the experimental group and the viscosity V0 of the control group, and the degradation rate (DR) of tremella polysaccharide was calculated according to the following formula:
[0053] The higher the degradation rate, the higher the enzyme activity of the tremella polysaccharase produced by the microorganism.
[0054] After calculating the degradation rate of tremella polysaccharide of the 40 strains in the experimental group, the statistical results are shown in Table 1. Figure 1 From Table 1, it can be seen that the degradation rates of strains Y301, Y302, Y304, T301, Y353, Y354, Y357, Y358, Y3521 and Y3522 are higher, all greater than 20%, so the above 10 strains with a degradation rate of tremella polysaccharide greater than 20% are selected for subsequent experiments. Figure 1
[0055] 3. Rescreening: enzyme activity determination
[0056] (1) Preparation of crude enzyme solution: 10 strains selected by preliminary screening were inoculated into rescreening fermentation medium at an inoculation amount of 5%, and fermented at a temperature of 35℃ and a rotation speed of 180rpm for 36h to obtain fermentation broth, then the fermentation broth was centrifuged at 5000rpm for 10min to remove bacteria and insoluble components, and the supernatant was the crude enzyme solution;
[0057] The composition of the rescreening fermentation medium is: tremella polysaccharide 10g / L, glucose 5g / L, yeast powder 10g / L, peptone 5g / L, beef extract 5g / L, potassium phosphate dibasic 2g / L, sodium chloride 5g / L, magnesium sulfate 0.2g / L.
[0058] (2) Determination of tremella polysaccharase activity
[0059] a. Establishment of tremella polysaccharase activity determination method: Tremella polysaccharide is composed of mannose, glucuronic acid, xylose, etc., among which glucuronic acid has reducing property, and accordingly a method for determining tremella polysaccharase by DNS method is established, and the tremella polysaccharase activity is defined as 1mg of glucuronic acid generated by hydrolysis of 1L of fermentation broth per hour under the conditions of a temperature of 35℃ and a pH of 7.0, which is 1 enzyme activity unit, symbolized as U, expressed in milligrams per liter per hour (mg / L·h).
[0060] b. Determination of tremella polysaccharase activity:
[0061] The experimental group was that 1.8 mL of tremella polysaccharide solution with a concentration of 0.5% (w / v) was added into a test tube, the crude enzyme solution obtained in step (1) was diluted 5 times to obtain a diluted enzyme solution, 200 μL of the diluted enzyme solution was added into the tremella polysaccharide solution, and the mixture was uniformly mixed, reacted for 1 h under the condition of a temperature of 35°C and a pH of 7.0, 2 mL of DNS reagent was added and uniformly mixed, heated in boiling water for 5 min, cooled to room temperature, and the absorbance at 530 nm was measured;
[0062] The blank group was that 1.8 mL of tremella polysaccharide solution with a concentration of 0.5% (w / v) was added into a test tube, 2 mL of DNS reagent was added, the crude enzyme solution obtained in step (1) was diluted 5 times to obtain a diluted enzyme solution, and then 200 μL of the diluted enzyme solution was added, uniformly mixed, heated in boiling water for 5 min, cooled to room temperature, and the absorbance at 530 nm was measured;
[0063] c. Preparation of a standard curve: different amounts of glucose uronic acid standard solution with a concentration of 500 mg / L and phosphate buffer were mixed with DNS reagent according to the following table to react, and the absorbance (OD 530 ) at 530 nm of the reaction solution after reaction was recorded, as shown in the following table:
[0064]
[0065] According to the data in the above table, the content of glucose uronic acid was taken as the abscissa, and the OD 530 was taken as the ordinate, a standard curve graph was drawn, the obtained standard curve graph is shown in Figure 2 , and the linear regression equation y=4.0289x-0.2089, R 2 =0.9992 was obtained according to Figure 2 .
[0066] According to the linear regression equation, the tremella polysaccharase activity of 10 strains of the experimental group, specifically strains Y301, Y302, Y304, T301, Y353, Y354, Y357, Y358, Y3521, and Y3522, was calculated, and the results are shown in Figure 3 . It can be seen from Figure 3 that the tremella polysaccharide degradation rate and the tremella polysaccharase activity of strain Y3522 are the highest.
[0067] Example 2 Morphological and molecular biological identification of strain Y3522
[0068] The colony morphology of strain Y3522 screened in Example 1 on trypticase soy agar medium (TSA) is as shown in Figure 4(a) as shown, the colony morphology is round, slightly raised, the colony diameter is 2-6 mm, beige, the surface is moist, smooth, shiny, and easy to pick up. The morphology of strain Y3522 under ordinary optical microscope is shown in Figure 4 (b) as shown, which is long rod-shaped.
[0069] The genomic DNA of the strain was extracted and separated by using a bacterial DNA extraction kit, and the 16S rRNA gene fragment of the strain Y3522 was amplified by PCR using the bacterial universal primer upstream primer 27F (the sequence is shown in SEQ ID NO. 1) and downstream primer 1492R (the sequence is shown in SEQ ID NO. 2). The PCR amplification reaction system is: 1 μL of each of the upstream and downstream primers, 12.5 μL of Green Taq Mix, 1 μL of DNA, 9.5 μL of ddH2O. The PCR amplification reaction conditions are: 95℃ pre-denaturation for 10 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 90 s, a total of 35 cycles, 72℃ extension for 15 min, and 4℃ incubation. After the PCR amplification product is purified, gel electrophoresis experiment is carried out, and the PCR product with normal amplification fragment length is sent to Goldengene Biotechnology Co., Ltd. for sequencing.
[0070] After sequencing, the length of the 16S rRNA gene fragment of the Paenibacillus sp. Y3522 is 1442 bp, and the sequencing result is shown in SEQ ID NO. 3. The 16S rRNA gene sequence is compared in the NCBI database, and the result shows that the 16S rRNA gene sequence of the strain Y3522 has high homology with the 16S rRNA gene sequence of the registered Paenibacillus sp. in the NCBI database (JQ684234.1 99.86%, OL944330.1 99.86%, DQ314538.1 99.79%, MT163452.1 99.72%). According to the comprehensive analysis of the similarity and integrity value, the standard strain sequence with the closest genetic relationship with the identified strain is selected, the phylogenetic tree based on the NJ (Neighbor-Joining) method is drawn by using the Mega software, and the confidence test is carried out for 1000 times, and the phylogenetic tree is shown in Mesobacillus . Figure 5
[0071] The strain Y3522 screened in the application is classified and named as Mesobacillus Paenibacillus sp., and the preservation number is CGMCC No. 32779, which is preserved in the General Microbial Center of China Microbial Culture Collection Management Committee, the preservation date is November 25, 2024, and the preservation address is No. 1, Beichen West Road, Chaoyang District, Beijing.
[0072] Example 3 Preparation of tremella polysaccharide-degrading enzyme by using the strain Y3522
[0073] 1. Fermentation enzyme production
[0074] Take strain Y3522 for activation culture, when OD 600 When reaching 1.0, inoculate into fermentation medium with 5% (v / v) inoculation amount, under the condition of temperature 35℃, ventilation amount 10L / min, rotation speed 150rpm, after 36h fermentation, fermentation liquor is obtained;
[0075] The composition of the fermentation medium is: tremella polysaccharide 15g / L, beef extract 10g / L, yeast extract 10g / L, peptone 10g / L, potassium phosphate dibasic 5g / L, sodium chloride 5g / L, ammonium sulfate 5g / L, magnesium sulfate 0.1g / L, the solvent is distilled water, and the pH value is 7.5.
[0076] 2. Post-processing
[0077] Centrifuge the fermentation liquor at a centrifugal speed of 8000rpm for 15min to remove the bacteria and insoluble components in the fermentation liquor, take the supernatant to obtain the crude enzyme liquor.
[0078] Example 4: Basic property exploration of enzyme
[0079] 1. Optimum reaction temperature exploration of enzyme
[0080] Prepare phosphoric acid buffer with pH value of 7.5, and then prepare tremella polysaccharide (molecular weight of 1250kDa) solution with concentration of 0.5% (w / v) using the phosphoric acid buffer;
[0081] The enzyme activity determination steps of the experimental group are: take 6 test tubes, add 1.8mL tremella polysaccharide solution into the test tubes, dilute the crude enzyme liquor prepared in Example 3 by 5 times, take 200μL and add, mix well, and place in water bath at 25℃, 30℃, 35℃, 40℃, 45℃ and 50℃ respectively for 1h, add 2mL DNS reagent and mix well, heat in boiling water for 5min, then take out and cool to room temperature, and use spectrophotometer to measure the absorbance at 530nm;
[0082] The enzyme activity determination steps of the blank group are: take 1 test tube, add 1.8mL tremella polysaccharide solution into the test tube, add 2mL DNS reagent, dilute the crude enzyme liquor prepared in Example 3 by 5 times, take 200μL and add, mix well, heat in boiling water for 5min, cool to room temperature, and use spectrophotometer to measure the absorbance at 530nm;
[0083] According to the absorbance at 530nm and the calculation method of tremella polysaccharide enzyme activity in Example 1, the tremella polysaccharide enzyme activity is calculated, and the results of tremella polysaccharide enzyme activity corresponding to different reaction temperatures are shown in Figure 6 ,Figure 6 It can be seen that the optimum temperature of strain Y3522 is 35℃.
[0084] 2. Optimal reaction pH of enzyme
[0085] First, prepare the phosphoric acid buffer with pH of 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and then prepare the tremella polysaccharide (molecular weight of 1250 kDa) solution with a concentration of 0.5% (w / v) using the phosphoric acid buffer with different pH.
[0086] The steps for measuring the enzyme activity of the experimental group are as follows: take 7 test tubes, add 1.8 mL of different pH tremella polysaccharide solution to each test tube, dilute the crude enzyme solution prepared in Example 3 by 5 times, and then add 200 μL of the diluted solution to each test tube, mix well, and react in a 35℃ water bath for 1 h. Then add 2 mL of DNS reagent, 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.
[0087] The steps for measuring the enzyme activity of the blank group are as follows: take 7 test tubes, add 1.8 mL of different pH tremella polysaccharide solution to each test tube, add 2 mL of DNS reagent, dilute the crude enzyme solution prepared in Example 3 by 5 times, and then add 200 μL of the diluted solution to each test tube, mix well, heat in boiling water for 5 min, cool to room temperature, and measure the absorbance at 530 nm using a spectrophotometer.
[0088] According to the absorbance at 530 nm and the calculation method of tremella polysaccharide enzyme activity in Example 1, the tremella polysaccharide enzyme activity corresponding to the different pH tremella polysaccharide solutions is calculated, and the results are shown in Table 1. Figure 7 From Table 1, it can be seen that the optimum pH of strain Y3522 is 7.5. Figure 7
[0089] Example 5: Preparation of tremella oligosaccharide using tremella polysaccharide-degrading enzyme produced by strain Y3522
[0090] Weigh the tremella polysaccharide and put it into water, prepare a 0.5% (w / v) tremella polysaccharide (molecular weight of 3000 kDa) solution using a pH 7.5 phosphoric acid buffer, add 10% of the crude enzyme solution prepared in Example 3 to the tremella polysaccharide solution, and enzymatically hydrolyze at a temperature of 35℃ for 4 h. Every 30 min during the enzymatic hydrolysis process, take 5 mL of the reaction solution, inactivate and purify it, and then use it for GPC detection of molecular weight.
[0091] The purification steps of the enzymatic hydrolysate are as follows: dilute the enzymatic hydrolysate by 3 times, centrifuge at 10000 rpm for 20 min to remove the precipitate and collect the supernatant; add 4 times the volume of ethanol, treat at 4℃ for 3 h, centrifuge at 6000 rpm for 10 min to obtain the alcohol precipitation product, and then add an equal volume of deionized water, dissolve thoroughly, and repeat the above operation twice.
[0092] The molecular weight (Mw) of the tremella polysaccharide was determined by high performance liquid chromatography-gel permeation chromatography (GPC) using an Ohpak SB-806 HQ2.0 300 chromatographic column, a differential detector (RI), 0.1M NaNO3 as the mobile phase at a flow rate of 0.5mL / min, and a column temperature of 25℃.
[0093] The GPC graphs of the enzymatic products with different enzymolysis times are shown in Figure 8 As shown in Figure 8 It can be seen that the molecular weight of the tremella polysaccharide decreases with the extension of the enzymolysis time. When the enzymolysis time is 30min, the tremella polysaccharide substrate with a molecular weight of 3000kDa can be degraded to a tremella polysaccharide with a molecular weight of about 900kDa, as shown in Figure 8 (a); when the enzymolysis time is 60min, the tremella polysaccharide substrate with a molecular weight of 3000kDa can be degraded to a tremella polysaccharide with a molecular weight of about 300kDa, as shown in Figure 8 (b); when the enzymolysis time is 90min, the tremella polysaccharide substrate with a molecular weight of 3000kDa can be degraded to a tremella polysaccharide with a molecular weight of about 80kDa, as shown in Figure 8 (c); when the enzymolysis time is 120min, the tremella polysaccharide substrate with a molecular weight of 3000kDa can be degraded to a tremella polysaccharide with a molecular weight of less than 20kDa, as shown in Figure 8 (d).
[0094] Therefore, tremella polysaccharides or tremella oligosaccharides with different molecular weights can be obtained by controlling the enzymolysis time.
[0095] In summary, the tremella polysaccharase is prepared by using the strain Y3522, and different molecular weight tremella polysaccharides or tremella oligosaccharides are prepared by controlling the enzymolysis time. The prepared tremella polysaccharase can degrade the tremella polysaccharide with a molecular weight of 3000kDa to less than 20kDa within 2h. The optimal temperature of the prepared tremella polysaccharase is 35℃, and the optimal pH is 7.5.
Claims
1. A genus of *Bacillus mesenchyme* ( Mesobacillus sp. Y3522, characterized in that, The preservation number of the intergeneric Bacillus Y3522 is CGMCC No. 32779, preserved in the China General Microbiological Culture Collection Center on November 25, 2024, and located at No. 3, Beichen West Road, Haidian District, Beijing.
2. The use of the interspecies Bacillus Y3522 of claim 1 in the production of tremella oligosaccharides, characterized in that, Comprise: Fermentation enzyme production, post-treatment, enzymatic hydrolysis.
3. The use of the intergeneric Bacillus Y3522 according to claim 2 in the production of tremella oligosaccharides, characterized in that, The fermentation enzyme, take intergeneric Bacillus Y3522 to carry on the activation culture, when OD 600 When reaching 1.0-1.5, inoculate into the fermentation medium, carry on the fermentation culture, obtain the fermentation liquor.
4. The use of the intergeneric Bacillus Y3522 according to claim 3 in the production of tremella oligosaccharides, characterized in that, In the fermentation enzyme production, the inoculation amount when inoculated into the fermentation medium is 1-10%; The fermentation temperature is 30-40℃, the ventilation amount is 5-15L / min, the rotation speed is 100-300rpm, and the fermentation time is 24-48h; The components of the fermentation medium are: tremella polysaccharide 5-15g / L, beef extract 5-20g / L, yeast extract 5-20g / L, peptone 5-20g / L, dipotassium hydrogen phosphate 1-10g / L, sodium chloride 1-10g / L, ammonium sulfate 1-10g / L, magnesium sulfate 0.05-0.2g / L, the solvent is distilled water, and the pH value is 6.8-7.
5.
5. The use of the intergeneric Bacillus Y3522 according to claim 2 in the production of tremella oligosaccharides, characterized in that, In the post-treatment, the fermentation broth is centrifuged, and the supernatant is taken to obtain the crude enzyme solution.
6. The use of the intergeneric Bacillus Y3522 according to claim 5 in the production of tremella oligosaccharides, characterized in that, In the post-treatment, the centrifugal speed is 8000-9000rpm, and the time is 14-16min.
7. The use of the intergeneric Bacillus Y3522 according to claim 2 in the production of tremella oligosaccharides, characterized in that, In the enzymatic hydrolysis, the crude enzyme solution is added to the tremella polysaccharide solution to perform enzymatic hydrolysis reaction, to obtain an enzymatic hydrolysis solution, and the enzymatic hydrolysis solution is inactivated, purified, to obtain low molecular tremella polysaccharide.
8. Use of the intergeneric Bacillus Y3522 according to claim 7 in the production of tremella oligosaccharides, characterized in that, In the enzymatic hydrolysis, 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 in the tremella polysaccharide solution is 5-15%; The temperature of the enzymatic hydrolysis reaction is 35-40℃; The time of the enzymatic hydrolysis reaction is 30-120min; The inactivation temperature is 95-98℃, and the time is 30-40min.
9. The use of the intergeneric Bacillus Y3522 according to claim 7 in the production of tremella oligosaccharides, characterized in that, In the purification, after the inactivated enzymatic hydrolysis solution is diluted once, it is centrifuged once, the supernatant is collected, alcohol precipitation is performed, and the operations of alcohol precipitation, secondary centrifugation and secondary dilution are repeated 2-3 times.
10. The use of the intergeneric Bacillus Y3522 according to claim 9 in the production of tremella oligosaccharides, characterized in that, In the purification, the dilution multiple of the first dilution is 2-5 times; The centrifugal speed of the first centrifugation is 10000-11000rpm, and the time is 20-25min; In the alcohol precipitation, 4-5 times the volume of ethanol is added, and the treatment is performed at 3-5℃ for 3-3.5h; The centrifugal speed of the second centrifugation is 6000-7000rpm, and the time is 10-12min; In the secondary dilution, the same volume of deionized water is added for dilution.