Serratia marcescens Y3521 capable of degrading tremella polysaccharide, culture method and application thereof
The preparation of Tremella polysaccharide enzyme by Serratia Y3521 solved the problems of low solubility and insufficient enzyme source of Tremella polysaccharide, and realized the industrial production and widespread application of low molecular weight Tremella polysaccharide.
Patent Information
- Application Number
- CN202510079705.5
- 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
The low solubility and high viscosity of Tremella polysaccharide in existing technologies limit its biological activity and application range. Furthermore, the availability of Tremella polysaccharide enzymes on the market is limited and the price is expensive, making it difficult to achieve industrial production.
A polysaccharide enzyme for Tremella fuciformis was prepared using Serratia marcescens Y3521. The purified enzyme solution was obtained through seed culture, fermentation culture, and post-treatment. This enzyme solution was then applied to the enzymatic hydrolysis reaction of Tremella fuciformis polysaccharide solution. The hydrolysis conditions were controlled to prepare low-molecular-weight Tremella fuciformis polysaccharide.
This method enables the degradation of high molecular weight Tremella fuciformis polysaccharides into low molecular weight Tremella fuciformis polysaccharides, thereby improving the bioactivity and application range of Tremella fuciformis polysaccharides, making them suitable for the food, pharmaceutical, health product, and cosmetic fields.
Smart Images
Figure CN119776226B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to a Serratia Y3521 degrading tremella polysaccharide and its culture method and application, belonging to the field of food, cosmetics, drugs biotechnology. BACKGROUND
[0002] Tremella fuciformis Berk. Tremella fuciformis ), also known as tremella, white fungus, snow ear, is a kind of edible fungus with economic value, rich in polysaccharide, protein, fat, 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, whose main chain is 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 blood lipid regulation and immune enhancement, so it 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 low molecular weight tremella oligosaccharide with 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 degradation method, chemical degradation method and enzymatic degradation method. Physical degradation methods include heating, mechanical shearing, ultrasonic and high pressure homogenization, etc. These physical degradation methods usually have a simple process, but usually have low production efficiency, serious loss and high energy consumption, and the degree of degradation of tremella polysaccharide is limited, and it is difficult to obtain ultra-low molecular weight tremella polysaccharide. Chemical degradation methods mainly include alkaline hydrolysis, acid hydrolysis and oxidation degradation methods, etc. Chemical degradation methods usually have low cost and are easy to produce in large scale, but the structure of monosaccharide is easily destroyed, thereby reducing the biological activity of tremella polysaccharide, and strong acids, strong bases and strong oxidants have potential dangers in transportation and production process.
[0004] In recent years, microbial enzyme method has gradually become the trend of degrading polysaccharide to prepare oligosaccharide in industrial production due to its environmental protection, high efficiency, controllability and sustainability. Enzymatic hydrolysis has strong specificity, mild reaction conditions, and the structure of polysaccharide does not change, so it is an ideal method for preparing low molecular weight tremella polysaccharide by controlling different degradation time to obtain tremella polysaccharide with different molecular weights. At present, the source of tremella polysaccharide enzyme on the market is very limited, and the price is relatively high, which to a large extent limits its application. The microorganism reported in the literature for tremella polysaccharide enzyme is Paenibacillus, but so far, there has been no report on tremella polysaccharide enzyme from Serratia. Serratia sp. ). SUMMARY
[0005] In order to overcome the deficiencies in the prior art, the present application provides a Serratia Y3521 capable of degrading tremella polysaccharide, 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] To solve the above technical problems, the technical scheme adopted by the present application is as follows:
[0007] A Serratia Y3521 capable of degrading tremella polysaccharide, classified and named as Serratia Serratia sp. , preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC No.32778, a preservation date of November 25, 2024, and an address of No.3, Beichen West Road, Chaoyang District, Beijing.
[0008] A culture method of the aforementioned Serratia Y3521, comprising: seed culture, fermentation culture, and post-treatment.
[0009] The seed culture is inoculating the activated Serratia Y3521 strain into a liquid seed culture medium, and culturing in a shaking bed at a temperature of 30-40 DEG C and a rotation speed of 150-300 rpm for 15-24 h to obtain a seed liquid.
[0010] The components of the seed culture medium are: tremella polysaccharide 10-20 g / L, yeast extract 5-20 g / L, peptone 5-20 g / L, dipotassium hydrogen phosphate 1-10 g / L, magnesium sulfate 0.05-0.2 g / L, solvent is distilled water, and pH value is 6.0-8.0.
[0011] The fermentation culture is inoculating the seed liquid into a liquid fermentation culture medium, and culturing at a temperature of 30-40 DEG C and a rotation speed of 150-300 rpm for 18-36 h to obtain a fermentation liquid.
[0012] The components of the liquid fermentation culture medium are: tremella polysaccharide 10-20 g / L, yeast extract 5-20 g / L, peptone 5-20 g / L, dipotassium hydrogen phosphate 1-10 g / L, magnesium sulfate 0.05-0.2 g / L, solvent is distilled water, and pH value is 6.0-8.0.
[0013] The post-treatment is centrifuging the fermentation liquid to collect the supernatant to obtain a crude enzyme liquid.
[0014] In the post-treatment, the centrifugal speed of the centrifugation is 8000-9000 rpm, and the time is 14-16 min.
[0015] Preferably, in the post-treatment, the crude enzyme solution is filtered by using an ultrafiltration membrane with a filtering precision of 10 kDa, and the purified enzyme solution can be obtained.
[0016] The application of the aforementioned Serratia marcescans Y3521, adding the crude enzyme solution into the tremella polysaccharide solution, performing the enzymatic hydrolysis reaction, obtaining the enzymatic hydrolysis solution, inactivating and purifying the enzymatic hydrolysis solution, and obtaining the low-molecular tremella polysaccharide;
[0017] The concentration of the tremella polysaccharide solution is 0.5-0.6%;
[0018] The pH of the tremella polysaccharide solution is 7.0-7.5;
[0019] Preferably, the pH of the tremella polysaccharide solution is 7.0;
[0020] The volume fraction of the crude enzyme solution in the tremella polysaccharide solution is 5-15%;
[0021] The temperature of the enzymatic hydrolysis reaction is 35-40 DEG C;
[0022] The time of the enzymatic hydrolysis reaction is 30-180 min;
[0023] Preferably, the temperature of the enzymatic hydrolysis reaction is 35 DEG C;
[0024] The temperature of the inactivation is 95-98 DEG C, and the time is 30-40 min;
[0025] In the purification, after the enzymatic hydrolysis solution after inactivation is diluted once, centrifuged once, the supernatant is collected, alcohol precipitation is performed, the operation of secondary centrifugation and secondary dilution is repeated 2-3 times;
[0026] In the purification, the dilution multiple of the first dilution is 2-5 times;
[0027] The centrifugal speed of the first centrifugation is 10000-11000 rpm, and the time is 20-25 min;
[0028] In the alcohol precipitation, 4-5 times of ethanol is added, and the treatment is performed at 3-5 DEG C for 3-3.5 h;
[0029] The centrifugal speed of the second centrifugation is 6000-7000 rpm, and the time is 10-12 min;
[0030] In the second dilution, the same volume of deionized water is added for dilution.
[0031] Compared with the prior art, the application has the following beneficial effects:
[0032] This invention utilizes Serratia marcescens Y3521 to prepare Tremella polysaccharide enzyme for the first time, and uses it to prepare low molecular weight Tremella polysaccharides. It can degrade Tremella polysaccharides with a molecular weight of 1000-2000kDa to below 10kDa, exhibiting high specificity for Tremella polysaccharides. It can be applied to industrial production, thereby improving the yield and stability of Tremella polysaccharide enzyme, and has broad application prospects in the fields of food, pharmaceuticals, health products, and cosmetics. Attached Figure Description
[0033] Figure 1 The degradation rate of Tremella polysaccharides of the 40 strains in the experimental group in Example 1;
[0034] Figure 2 In Example 1, glucuronic acid content was used as the x-axis and OD was used as the y-axis. 530 A standard curve plotted with the vertical axis as the ordinate;
[0035] Figure 3 The polysaccharide enzyme activity of 10 strains in the experimental group of Example 1;
[0036] Figure 4 The morphological results of strain Y3521 in Example 2;
[0037] in, Figure 4 (a) Colony morphology of strain Y3521 on tryptone soybean agar medium; Figure 4 (b) is an optical microscope image of strain Y3521;
[0038] Figure 5 This is a phylogenetic tree of strain Y3521 and closely related strains in Example 2 based on the 16S rRNA gene;
[0039] Figure 6 The results of the activity of Tremella polysaccharide enzymes at different pH values in Example 4;
[0040] Figure 7 The results of the activity of Tremella polysaccharide enzymes at different temperatures in Example 4;
[0041] Figure 8 GPC diagrams of the enzymatic hydrolysis products at different hydrolysis times in Example 5;
[0042] Figure 9 This is a GPC diagram of the enzymatic hydrolysis product after 3 hours of enzymatic hydrolysis in Example 5. Detailed Implementation
[0043] The present invention will now be further described with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto.
[0044] Example 1: Enrichment and Screening of Strain Y3521
[0045] 1. Processing Tremella sample
[0046] Collect several rotten Tremella samples from Tremella producing areas, take 10g of Tremella sample into 90mL of sterile normal saline, transfer to a soybean milk machine for crushing and mixing evenly to obtain a sample suspension. Take 10mL of the sample suspension into a 250mL flask, add 100mL of 1% Tremella polysaccharide enrichment medium, and enrich culture under the condition of a temperature of 35℃ and a rotation speed of 150rpm for 3 days;
[0047] The composition of the 1% Tremella polysaccharide enrichment medium is: Tremella polysaccharide 10g / L, yeast powder 10g / L, peptone 5g / L, potassium phosphate dibasic 2g / L, sodium chloride 5g / L, magnesium sulfate 0.2g / L;
[0048] The molecular weight of the Tremella polysaccharide is 1250kDa.
[0049] 2. Preliminary screening: Tremella polysaccharide degradation experiment
[0050] After the sample is pretreated, 10-fold gradient dilution is performed on the sample bacterial suspension with sterile normal saline, and 200μL 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 are coated on the 1% Tremella polysaccharide enrichment medium, and after 5d of culture under the condition of a temperature of 35℃, the growth of the strains is observed, and strains with different colony morphologies are subjected to 3 times of separation and purification until pure strains are obtained, a total of 40 strains are obtained, which are numbered as Y301-Y304, T301-T305, Y3501-Y3523, and T351-T358, respectively; the strains are inoculated into 2% Tremella polysaccharide medium according to an inoculation amount of 5% for a Tremella polysaccharide degradation experiment as a test group, and an equal volume of normal saline is used to replace the bacterial liquid as a control group.
[0051] The composition of the 1% Tremella polysaccharide enrichment medium is: Tremella polysaccharide 10g / L, yeast powder 10g / L, peptone 5g / L, potassium phosphate dibasic 2g / L, sodium chloride 5g / L, magnesium sulfate 0.2g / L;
[0052] The composition of the 2% Tremella polysaccharide medium is: Tremella polysaccharide 20g / L, yeast powder 10g / L, peptone 5g / L, potassium phosphate dibasic 2g / L, sodium chloride 5g / L, magnesium sulfate 0.2g / L;
[0053] After 48h of cultivation at 35℃ and 220rpm, the viscosity of the medium in the shake flasks of the experimental and control groups was measured using a viscometer, and the viscosity of the experimental group was V 1 and the viscosity of the control group V 0. The degradation rate (DR) of tremella polysaccharide was calculated according to the following formula: the higher the degradation rate after fermentation, the higher the enzyme activity of tremella polysaccharase produced by the microorganism.
[0054]
[0055] After calculating the degradation rate of tremella polysaccharide of the 40 strains in the experimental group, the statistical results are shown in Figure 1 From Figure 1 It can be seen that the degradation rates of tremella polysaccharide of strains Y301, Y302, Y304, T301, Y353, Y354, Y357, Y358, Y3521, and Y3522 are higher, all greater than 20%. Therefore, the above 10 strains with a tremella polysaccharide degradation rate greater than 20% were selected for subsequent experiments.
[0056] 3. Rescreening: enzyme activity determination
[0057] (1) Preparation of crude enzyme solution: 10 strains selected by preliminary screening were inoculated in the rescreening fermentation medium at an inoculation amount of 5%, and fermented at 35℃ and 180rpm for 36h to obtain fermentation broth. Then, the fermentation broth was centrifuged at 5000rpm for 10min to remove the bacterial bodies and insoluble components, and the supernatant was the crude enzyme solution.
[0058] The composition of the rescreening fermentation medium is as follows: 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.
[0059] (2) Determination of tremella polysaccharase activity
[0060] a. Establishment of method for determining tremella polysaccharase activity: Tremella polysaccharide is composed of mannose, glucuronic acid, xylose, etc., among which glucuronic acid has reducing property. Therefore, a method for determining tremella polysaccharase by DNS method was established. The tremella polysaccharase activity was defined as 1 enzyme activity unit (U) for 1mg of glucuronic acid generated by hydrolysis of 1L of fermentation broth per hour at 35℃ and pH 7.0, expressed in milligrams per liter per hour (mg / L·h).
[0061] b. Determination of tremella polysaccharase activity
[0062] 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 at a temperature of 35°C and a pH of 7.0 for 1 h, 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;
[0063] 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;
[0064] c. Preparation of 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, and the absorbance (OD 530 ) of the reaction solution after reaction at 530 nm was recorded, as shown in the following table:
[0065]
[0066] 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, and a standard curve was drawn, as shown in Figure 2 , and the linear regression equation obtained according to Figure 2 was y=4.0289x-0.2089, R 2 =0.9992.
[0067] 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 tremella polysaccharase activity of strain Y3521 were both high, and the degradation rate of tremella polysaccharide was 66.04%.
[0068] Example 2 Morphological and molecular biological identification of strain Y3521
[0069] The colony morphology of strain Y3521 screened in Example 1 on trypticase soy agar medium (TSA) is shown in Figure 4(a) as shown, the colony morphology is round, the colony center is red, the edge is white, slightly raised, the colony diameter is 1-3 mm, the surface is moist and smooth, and the colony is easy to pick up. The morphology of strain Y3521 under a general optical microscope is as shown in Figure 4 (b) as shown, which is spherical or short rod-shaped. The strain can grow on a culture medium with tremella polysaccharide as the only carbon source.
[0070] 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 Y3521 was subjected to PCR amplification by 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, and 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, a gel electrophoresis experiment is performed, and the PCR product with a normal length of the PCR amplification fragment is sent to a company for sequencing.
[0071] After sequencing, the 16S rRNA gene fragment of the Serratia Y3521 has a length of 1471 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 of the present application has a high homology with the 16S rRNA gene sequence of the Serratia 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 value are comprehensively analyzed, the standard strain sequence with the closest genetic relationship to the identified strain is selected, the Mega software is used to draw a phylogenetic tree based on the NJ (Neighbor-Joining) method and through 1000 times confidence test, and the phylogenetic tree is shown in Figure 5 .
[0072] The strain Y3521 screened in the present application is classified and named as Serratia Serratia sp. , which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 32778, the preservation date is November 25, 2024, and the preservation address is No. 1, Xibaheyi, Beijing City, Chaoyang District.
[0073] Example 3 Preparation of tremella polysaccharide-degrading enzyme using strain Y3521
[0074] 1. After activating the strain Y3521, a single colony was inoculated into a liquid seed culture medium, and the seed liquid was prepared by incubating at a temperature of 35°C and a rotation speed of 200 rpm for 18 h;
[0075] The components of the liquid seed culture were: tremella polysaccharide 15 g / L, yeast extract 10 g / L, peptone 10 g / L, potassium phosphate dibasic 4 g / L, magnesium sulfate 0.2 g / L, solvent was distilled water, and the pH value was 7.0.
[0076] 2. The seed liquid was inoculated into a fermentation medium at an inoculation amount of 5%, and the fermentation liquid was prepared by incubating at a temperature of 35°C and a rotation speed of 200 rpm for 24 h;
[0077] The components of the fermentation medium were: tremella polysaccharide 10 g / L, yeast extract 10 g / L, peptone 10 g / L, potassium phosphate dibasic 4 g / L, magnesium sulfate 0.1 g / L, solvent was distilled water, and the pH value was 7.0.
[0078] 3. The fermentation liquid was centrifuged at 8000 rpm for 15 min to remove the bacterial bodies and insoluble components, and the supernatant was collected to obtain the crude enzyme liquid.
[0079] Example 4 Exploration of the basic properties of the enzyme
[0080] 1. Exploration of the optimal reaction pH of the enzyme
[0081] First, prepare phosphate buffer solutions with pH values of 5, 5.5, 6, 6.5, 7, 7.5, and 8, then prepare tremella polysaccharide (molecular weight 1250 kDa) solutions with a concentration of 0.5% (w / v) using different pH phosphate buffer solutions;
[0082] The steps for measuring the enzyme activity of the experimental group were as follows: 7 test tubes were taken, 1.8 mL of different pH tremella polysaccharide solutions were added to each test tube, the crude enzyme liquid obtained in Example 3 was diluted 5 times to obtain a diluted crude enzyme liquid, 200 μL of the diluted crude enzyme liquid was added to each tremella polysaccharide solution, mixed well, and reacted in a 35°C water bath for 1 h, then 2 mL of DNS reagent was added and mixed well, heated in boiling water for 5 min, then cooled to room temperature, and the absorbance at 530 nm was measured using a spectrophotometer;
[0083] The enzyme activity determination steps of the blank group were as follows: 7 test tubes were taken, 1.8 mL of tremella polysaccharide solution with different pH values was added into each test tube, 2 mL of DNS reagent was added, the crude enzyme solution obtained in Example 3 was diluted 5 times to obtain a diluted crude enzyme solution, 200 μL of the diluted crude enzyme solution was added, and the mixture was uniformly mixed, heated in boiling water for 5 min, cooled to room temperature, and the absorbance at 530 nm was determined by spectrophotometry;
[0084] According to the absorbance at 530 nm and the calculation method of tremella polysaccharase activity in Example 1, the tremella polysaccharase activity was calculated, and the results of the corresponding tremella polysaccharase activity of the tremella polysaccharide solution with different pH values were as follows: Figure 6 According to the absorbance at 530 nm and the calculation method of tremella polysaccharase activity in Example 1, the tremella polysaccharase activity was calculated, and the results of the corresponding tremella polysaccharase activity of the tremella polysaccharide solution with different pH values were as follows: Figure 6 It can be seen that the optimum pH of the strain Y3521 is 7.0.
[0085] 2. Optimum reaction temperature of enzyme
[0086] The phosphate buffer with a pH value of 7.0 was prepared, and then the phosphate buffer was used to prepare a tremella polysaccharide (molecular weight: 1250 kDa) solution with a concentration of 0.5% (w / v);
[0087] The enzyme activity determination steps of the experimental group were as follows: 6 test tubes were taken, 1.8 mL of the above-mentioned tremella polysaccharide solution was added into each test tube, the crude enzyme solution obtained in Example 3 was diluted 5 times to obtain a diluted crude enzyme solution, 200 μL of the diluted crude enzyme solution was added into the tremella polysaccharide solution, the mixture was uniformly mixed, and was placed in a water bath at 25℃, 30℃, 35℃, 40℃, 45℃ or 50℃ for 1 h, 2 mL of DNS reagent was added and uniformly mixed, and after being heated in boiling water for 5 min, the mixture was taken out and cooled to room temperature, and the absorbance at 530 nm was determined by spectrophotometry;
[0088] The enzyme activity determination steps of the blank group were as follows: 7 test tubes were taken, 1.8 mL of tremella polysaccharide solution with different pH values was added into each test tube, 2 mL of DNS reagent was added, the crude enzyme solution obtained in Example 3 was diluted 5 times to obtain a diluted crude enzyme solution, 200 μL of the diluted crude enzyme solution was added, and the mixture was uniformly mixed, heated in boiling water for 5 min, cooled to room temperature, and the absorbance at 530 nm was determined by spectrophotometry;
[0089] According to the absorbance at 530 nm and the calculation method of tremella polysaccharase activity in Example 1, the tremella polysaccharase activity was calculated, and the results of the corresponding tremella polysaccharase activity of the tremella polysaccharide solution with different pH values were as follows: Figure 7 According to the absorbance at 530 nm and the calculation method of tremella polysaccharase activity in Example 1, the tremella polysaccharase activity was calculated, and the results of the corresponding tremella polysaccharase activity of the tremella polysaccharide solution with different pH values were as follows: Figure 7 It can be seen that the optimum temperature of the strain Y3521 is 35℃.
[0090] Example 5: Preparation of tremella oligosaccharide by using tremella polysaccharide-degrading enzyme prepared by the strain Y3521
[0091] Take 100 mL of the 0.5% (w / v) tremella polysaccharide (molecular weight 1250 kDa) solution prepared in Example 4 using the phosphate buffer with a pH value of 7.0 as a substrate, add the crude enzyme solution prepared in Example 3 at a volume fraction of 10%, and react in a 35℃ water bath for 3h. Take 5 mL of the reaction solution every 30 min, inactivate and purify, and then use for GPC detection of the molecular weight.
[0092] The purification step of the enzyme solution is as follows: dilute the enzyme solution 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 3h, centrifuge at 6000 rpm for 10 min to obtain the alcohol precipitation product, add an equal volume of deionized water, fully dissolve, and then repeat the above operation twice.
[0093] The molecular weight (Mw) of the tremella polysaccharide is 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, a flow rate of 0.5mL / min as the mobile phase, and a column temperature of 25℃.
[0094] The GPC chart of the enzyme hydrolysis product at different enzyme hydrolysis times is shown in Figure 8 As can be seen from Figure 8 It can be seen that the molecular weight of the tremella polysaccharide decreases with the extension of the enzyme hydrolysis time, and the enzyme solution can degrade the tremella polysaccharide substrate with a molecular weight of 1250 kDa to low molecular weight tremella oligosaccharide with a molecular weight of about 200 kDa when the enzyme hydrolysis time is 30 min. The GPC chart of the tremella oligosaccharide after enzyme hydrolysis for 3h is shown in Figure 9 As can be seen from Figure 9 It can be seen that the enzyme solution can continue to degrade to tremella oligosaccharide with a molecular weight of less than 10 kDa. As can be seen from Figure 8 and Figure 9 It can be seen that different molecular weight tremella polysaccharides or tremella oligosaccharides can be obtained by controlling the enzyme hydrolysis time.
[0095] In summary, the tremella polysaccharase prepared by the strain Y3521 in the present application can prepare different molecular weight tremella polysaccharides or tremella oligosaccharides by controlling the reaction time, and can degrade the tremella polysaccharide with a molecular weight of 1250 kDa to less than 10 kDa within 3h. The optimal pH of the prepared tremella polysaccharase is 7.0, the optimal temperature is 35℃, and the specificity for tremella polysaccharide is high.
Claims
1. A type of Serratia marcescens that degrades polysaccharides from Tremella fuciformis (Serratia marcescens) Serratia sp. Y3521, characterized in that, The Serratia sp. Y3521 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 32778, the preservation date is November 25, 2024, and the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing.
2. The culture method of Serratia marcescens Y3521 according to claim 1, characterized by, It comprises: Seed culture, fermentation culture, post-treatment.
3. The culture method of Serratia y 3521 according to claim 2, characterized by, The seed culture is inoculated into a liquid seed culture medium after the Serratia sp. Y3521 strain is activated, and the seed liquid is obtained by shaking culture at a temperature of 30-40 DEG C and a rotation speed of 150-300 rpm for 15-24 h. The composition of the seed culture medium is: tremella polysaccharide 10-20 g / L, yeast extract 5-20 g / L, peptone 5-20 g / L, dipotassium hydrogen phosphate 1-10 g / L, magnesium sulfate 0.05-0.2 g / L, and the solvent is distilled water, and the pH value is 6.0-8.
0.
4. The culture method of Serratia y 3521 according to claim 2, characterized by, The fermentation culture is inoculated into a liquid fermentation culture medium, and the fermentation liquid is obtained by expanding culture at a temperature of 30-40 DEG C and a rotation speed of 150-300 rpm for 18-36 h. The composition of the liquid fermentation culture medium is: tremella polysaccharide 10-20 g / L, yeast extract 5-20 g / L, peptone 5-20 g / L, dipotassium hydrogen phosphate 1-10 g / L, magnesium sulfate 0.05-0.2 g / L, and the solvent is distilled water, and the pH value is 6.0-8.
0.
5. The culture method of Serratia y 3521 according to claim 2, characterized by, The fermentation liquid is centrifuged to collect the supernatant to obtain the crude enzyme liquid. In the post-treatment, the centrifugal speed of the centrifugation is 8000-9000 rpm, and the time is 14-16 min.
6. The use of the Serratia sp. Y3521 according to claim 1, characterized in that The crude enzyme liquid obtained by fermentation of the Serratia sp. Y3521 is added to the tremella polysaccharide solution to perform enzymatic reaction to obtain an enzymatic hydrolysis liquid, and the enzymatic hydrolysis liquid is inactivated, purified to obtain low molecular tremella polysaccharide.
7. The use of Serratia y 3521 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 liquid in the tremella polysaccharide solution is 5-15%; The temperature of the enzymatic reaction is 35-40 DEG C; The time of the enzymatic reaction is 30-180 min; The temperature of the inactivation is 95-98 DEG C, and the time is 30-40 min.
8. The use of Serratia y 3521 according to claim 6, characterized in that The inactivated enzymatic hydrolysis liquid is diluted once, 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. In the purification, the dilution multiple of the first 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 added with 4-5 times the volume of ethanol, and treated at 3-5 DEG C for 3-3.5 h; The centrifugal speed of the second centrifugation is 6000-7000 rpm, and the time is 10-12 min; The secondary dilution is diluted with an equal volume of deionized water.