Chryseobacterium sp. s1-14 and application thereof in enzymolysis of high molecular polysaccharide with α-1,3-glycosidic bond
The α-1,3-glycosidase enzymatic hydrolysis method prepared by Chlorella S1-14 solves the problem of efficient degradation of α-1,3-glycosidic polysaccharides, and realizes the degradation of polysaccharide molecular weight to below 20kDa under mild conditions, which is suitable for industrial production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANDONG FOCUSFREDA BIOTECH CO LTD
- Filing Date
- 2026-04-10
- Publication Date
- 2026-06-23
AI Technical Summary
Existing technologies are inefficient at degrading polysaccharides linked by α-1,3-glycosidic bonds. Traditional methods are inefficient, costly, or may damage the polysaccharide structure and pose safety risks.
α-1,3-glycosidase was prepared using Chlorella S1-14. The macromolecular polysaccharide was degraded into low-molecular-weight polysaccharide by enzymatic hydrolysis under mild conditions. The crude enzyme solution was prepared by steps such as strain activation, seed culture, fermentation culture and post-treatment. The low-molecular-weight polysaccharide was then obtained by enzymatic hydrolysis, inactivation and purification.
It enables the degradation of polysaccharide molecular weight from 1000kDa to below 20kDa under mild conditions, improving enzymatic hydrolysis efficiency and stability, and making it suitable for industrial production.
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Figure CN122256196A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a Chlorella S1-14 strain and its application in enzymatic hydrolysis of α-1,3-glycosidic bonds in high molecular weight polysaccharides, belonging to the fields of food, cosmetics, and pharmaceutical biotechnology. Background Technology
[0002] Polysaccharides are high-molecular-weight carbohydrates composed of at least 10 monosaccharides linked by glycosidic bonds. They are widely distributed in nature and typically play crucial roles in plant and animal cells. Currently, many bioactive polysaccharides have been discovered and utilized, such as hyaluronic acid, chondroitin sulfate, sodium alginate, lentinan (from shiitake mushrooms), and tremella fuciformis (from silver ear fungus). Polysaccharides typically have large molecular weights, low solubility, and high viscosity after dissolution. This characteristic hinders their ability to cross multiple cell membrane barriers to exert biological activity and significantly limits their applications. Therefore, degrading polysaccharides to prepare oligosaccharides with smaller molecular weights and better water solubility can significantly improve their bioactivity and effectively expand their applications, which has significant application value.
[0003] Large polysaccharides are formed by monosaccharides linked by glycosidic bonds, including α-1,3-glycosidic bonds, α-1,4-glycosidic bonds, β-1,3-glycosidic bonds, and β-1,4-glycosidic bonds. Current reports indicate that polysaccharide degradation methods mainly include physical degradation, chemical degradation, and enzymatic degradation. Physical degradation methods include heating, mechanical shearing, ultrasound, and high-pressure homogenization. These methods are generally simple, but often suffer from low production efficiency, significant losses, and high energy consumption. Furthermore, they have limited degradation potential for Tremella fuciformis polysaccharides, failing to yield ultra-low molecular weight Tremella fuciformis polysaccharides. Chemical degradation methods mainly include alkaline hydrolysis, acid hydrolysis, and oxidative degradation. Chemical degradation methods are generally lower in cost and easier for large-scale production, but they easily damage the monosaccharide structure, thus reducing the bioactivity of Tremella fuciformis polysaccharides. Additionally, strong acids, strong bases, and strong oxidants pose potential hazards during transportation and production.
[0004] In recent years, microbial enzymatic methods have gradually become a trend in industrial production for degrading polysaccharides to prepare oligosaccharides due to their advantages such as environmental friendliness, high efficiency, controllability, and sustainability. Currently, microbial mass production of enzymes that hydrolyze some common glycosidic bonds has been achieved, such as cellulase and β-glucosidase for hydrolyzing β-1,4-glycosidic bonds; α-amylase for hydrolyzing α-1,4-glycosidic bonds; and isoamylase for hydrolyzing α-1,6-glycosidic bonds. However, research on α-1,3-glycosidic bond hydrolases is scarce. Therefore, screening strains with high production of α-1,3-glycosidic bond hydrolases is of great significance for the degradation of polysaccharides linked by α-1,3-glycosidic bonds. Summary of the Invention
[0005] To overcome the shortcomings of existing technologies, this invention provides a Chlorella S1-14 strain and its application in the enzymatic hydrolysis of α-1,3-glycosidic bonds in high molecular weight polysaccharides. It can be used to degrade high molecular weight polysaccharides containing α-1,3-glycosidic bonds, with advantages such as high degradation rate, mild reaction conditions, and low molecular weight of the degradation products, and can be applied to industrial production.
[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A type of *Cyclocarya* S1-14, classified as *Cyclocarya*. Chryseobacterium sp., deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37778, deposited on February 10, 2026, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
[0007] An application of the aforementioned Chlorella S1-14 in the enzymatic hydrolysis of α-1,3-glycosidic bond high molecular weight polysaccharides, wherein the method of application includes: preparing crude enzyme solution and enzymatic hydrolysis; The preparation of crude enzyme solution includes: strain activation, seed culture, fermentation culture, and post-treatment; The strain was activated by streaking a solution of Chlorella S1-14 onto an activation plate and incubating it statically at 29-31°C for 24-25 hours to obtain the activated strain. The activated plate consists of: glucose 9.8-10.2 g / L, yeast extract 4.8-5.2 g / L, peptone 4.8-5.2 g / L, dipotassium hydrogen phosphate 4.8-5.2 g / L, sodium chloride 4.8-5.2 g / L, agar 14.5-15.5 g / L, distilled water as solvent, and a pH of 7.4-7.6. The seed culture involves inoculating the activated strain into the seed culture medium at an inoculation rate of 4.8-5.2%, and then culturing it on a shaker for 15-16 hours at a temperature of 28-32℃ and a rotation speed of 210-230 rpm to obtain the seed liquid. The seed culture medium consists of: 4.8-5.2 g / L Tremella polysaccharide, 4.8-5.2 g / L glucose, 9.8-10.2 g / L yeast extract, 4.8-5.2 g / L peptone, and 4.8-5.2 g / L sodium chloride, with distilled water as the solvent and a pH of 7.4-7.6. The fermentation culture involves inoculating the seed culture into the fermentation medium at an inoculation rate of 4.8-5.2%, and fermenting for 24-25 hours at a temperature of 28-32℃ and a rotation speed of 230-250 rpm to obtain the fermentation broth. The fermentation medium consists of: 9.8-10.2 g / L of Tremella fuciformis polysaccharide, 4.8-5.2 g / L of yeast extract, 9.8-10.2 g / L of peptone, 4.8-5.2 g / L of dipotassium hydrogen phosphate, 4.8-5.2 g / L of sodium chloride, and 0.9-1.1 g / L of magnesium sulfate. The solvent is distilled water, and the pH value is 6.8-7.2. The post-processing involves centrifuging the fermentation broth, collecting the supernatant, and obtaining a crude enzyme solution. The centrifugation speed is 8000-8500 rpm, and the centrifugation time is 15-20 min. The enzymatic hydrolysis involves adding crude enzyme solution to an α-1,3-glycosidic bond high molecular weight polysaccharide solution, carrying out the enzymatic hydrolysis reaction, and then inactivating and purifying the enzymatic hydrolysate to obtain a low molecular weight polysaccharide. In the enzymatic hydrolysis, the concentration of the α-1,3-glycosidic bond high molecular weight polysaccharide solution is 18-22 g / L; The volume ratio of the α-1,3-glycosidic polymeric polysaccharide solution to the crude enzyme solution was 1:1-6. The enzymatic hydrolysis reaction is carried out at a temperature of 38-42℃ for 2-2.5 hours. The inactivation temperature during the inactivation process is 88-92℃, and the inactivation time is 20-25 min. The purification method is as follows: dilute the inactivated enzyme hydrolysate by 2.8-3.2 times, centrifuge at 10000-11000 rpm for 20-25 min, take the supernatant, add 3.8-4.2 times the volume of ethanol, treat at 3-5℃ for 3-3.5 h, centrifuge at 8000-9000 rpm for 10-15 min to obtain the alcohol precipitation product, add an equal volume of deionized water, dissolve, and repeat the above operation twice.
[0008] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes Chlorella S1-14 to prepare α-1,3-glycosidase, which is then used in the preparation of low molecular weight polysaccharides. Under mild conditions, it can degrade polysaccharides with an average molecular weight of 1000 kDa to below 20 kDa, making it applicable to industrial production. This improves the yield and stability of α-1,3-glycosidase and has broad application prospects in the fields of food, pharmaceuticals, health products, and cosmetics. Attached Figure Description
[0009] Figure 1 The morphological results of strain S1-14 in Example 2; In the picture, Figure 1 (a) Colony morphology of strain S1-14 on tryptone soybean agar medium; Figure 1 (b) is an optical microscope image of strain S1-14; Figure 2 This is a phylogenetic tree of strain S1-14 and closely related strains in Example 2 based on the 16S rRNA gene. Detailed Implementation
[0010] 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.
[0011] Example 1: Enrichment and Screening of Strains S1-14 (1) Screening of α-1,3-glycosidase-producing strains The strains were screened using polysaccharides with α-1,3-glycosidic bonds as the main chain and α-glycosidic bonds as the sole carbon source. In this experiment, Tremella fuciformis polysaccharide was used. Tremella fuciformis polysaccharide is an acidic heteropolysaccharide obtained from the fruiting body of Tremella fuciformis. Its main chain structure consists of mannan linked by α-1,3-glycosidic bonds, and its side chains are composed of glucuronic acid and xylose.
[0012] The specific screening process is as follows: Take 10 mL of wastewater sample and add it to 90 mL of sterile physiological saline. Place the sample in an incubator at 30°C and mix at 180 rpm for 30 min to obtain a bacterial suspension. Perform 10-fold serial dilutions of the bacterial suspension with sterile physiological saline, and collect samples from different dilution levels (10... -1 -10 -10 100 μL of each diluted solution was spread onto a Tremella fuciformis polysaccharide solid screening plate (GS1). After incubation at 30℃ for 3 days, strains with different colony morphologies were isolated and purified three times until pure strains were obtained. The purified strains were inoculated into Tremella fuciformis polysaccharide screening medium S1 and incubated at 30℃ and 240 rpm for 48 h. The ability of the strains to produce α-1,3-glycosidase was characterized by the decrease in viscosity of the screening medium (degradation rate). The control group was replaced with an equal volume of physiological saline. The degradation rate was calculated using the formula: Degradation rate (%) = ( V 0- V 1) / V 0×100%. Among them... V 0 and V 1 represents the viscosity of the culture medium in the shake flasks of the control group and the experimental group, respectively. The higher the degradation rate, the higher the activity of microbial Tremella polysaccharide enzyme.
[0013] The components of the Tremella polysaccharide screening medium S1 are: Tremella polysaccharide 10g / L, peptone 10g / L, dipotassium hydrogen phosphate 4g / L, sodium chloride 5g / L, and magnesium sulfate 0.5g / L; The polysaccharide was prepared by adding 20 g / L of agar to the solid screening plate S1, so that the molecular weight of the polysaccharide was 1000 kDa.
[0014] Based on colony characteristics such as morphology and color, 30 pure bacterial strains were picked from the Tremella fuciformis polysaccharide solid selection plate (GS1) and numbered from strain S1-1 to strain S1-30. The degradation ability of these 30 strains (S1-1 to S1-30) on α-1,3-glycoside was detected using Tremella fuciformis polysaccharide selection medium S1. The results are shown in Table 1. Table 1. Degradation capacity of strains for α-1,3-glycosides
[0015] Strains S1-2, S1-7, S1-14, S1-16, S1-17, S1-23, S1-27, and S1-30 with a polysaccharide degradation rate greater than 30% were selected for further enzyme activity assays.
[0016] (2) Enzyme activity assay The enzyme activity was tested as follows: Single colonies were picked from the Tremella fuciformis polysaccharide solid screening plate (GS1) and inoculated into the fermentation medium. Fermentation was carried out at 30℃ and 220 r / min for 48 h. 1 mL of the bacterial culture was collected to determine the Tremella fuciformis polysaccharide enzyme activity, with each treatment repeated three times. The DNS method was used to determine the enzyme activity. One unit of enzyme activity (U / mL) was defined as the amount of reducing sugar produced by the hydrolysis of Tremella fuciformis polysaccharide by 1 mL of fermentation broth in 1 h at 30℃ and pH 7.0, which was 1 μg of enzyme activity.
[0017] The enzyme activities of strains S1-2, S1-7, S1-14, S1-16, S1-17, S1-23, S1-27, and S1-30 were determined using the methods described above, and the results are shown in Table 2. Strain S1-14 exhibited both high degradation rate and high enzyme activity, indicating that strain S1-14 has a strong ability to produce α-1,3-glycosidase.
[0018] Table 2 Enzyme Activities of Strains
[0019] Example 2: Morphological and molecular biological identification of strain S1-14 The colony morphology of strain S1-14 screened in Example 1 on tryptone soybean agar (TSA) medium is as follows. Figure 1 As shown in (a), the colonies are round, golden yellow in color, slightly raised, and 1-3 mm in diameter. The surface is moist, smooth, and easily picked up. The morphology of strain S1-14 under a regular optical microscope is shown in the optical microscope photograph. Figure 1 As shown in (b), it is short rod-shaped. This strain can grow on a medium with Tremella fuciformis polysaccharide as the sole carbon source.
[0020] Strain S1-14 was sent to Genewiz Biotechnology Co., Ltd. for 16S rRNA sequencing identification. Sequencing revealed that the 16S rRNA gene fragment of strain S1-14 was 1400 bp in length, as shown in SEQ ID NO.1. The 16S rRNA gene sequence was compared with the EzBioCloud database. The results showed that the 16S rRNA gene sequence of the strain of this invention had high homology with the 16S rRNA gene sequences of *Chlorella vulgaris* registered in the database, with 99.21% similarity to LUVZ01000011, 98.85% similarity to EU034659, and 98.29% similarity to BAVL01000024. Based on a comprehensive analysis of similarity and integrity values, the sequence of the standard strain most closely related to the identified strain was selected. A phylogenetic tree based on the Neighbor-Joining (NJ) method and passing 1000 confidence tests was constructed using Mega software, as shown in [see image]. Figure 2 As shown.
[0021] The strain S1-14 screened in this invention is classified and named *Cryptobacter chrysogenum*. Chryseobacterium sp., deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37778, deposited on February 10, 2026, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
[0022] Example 3: Preparation of α-1,3-glycosidase using strain S1-14 1. Using a sterile inoculation loop, take a sample of Chlorella S1-14 bacterial suspension from a cryopreservation tube, streak it on an activation plate, and incubate it statically in an incubator at 30℃ for 24 hours to form a single colony, thus obtaining the activated strain; The activated plate consists of: 10 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, 5 g / L dipotassium hydrogen phosphate, 5 g / L sodium chloride, 15 g / L agar, and distilled water as the solvent, with a pH of 7.5. 2. Take the activated strain and inoculate it into the seed culture medium at an inoculation rate of 5%. Incubate it in a shaker at a temperature of 30℃ and a rotation speed of 220 rpm for 15 hours to obtain the seed liquid. The seed culture medium consists of: 5 g / L Tremella polysaccharide, 5 g / L glucose, 10 g / L yeast extract, 5 g / L peptone, and 5 g / L sodium chloride, with distilled water as the solvent and a pH of 7.5. 3. Inoculate the seed culture at a rate of 5% into the fermentation medium and ferment for 24 hours at a temperature of 30℃ and a rotation speed of 240 rpm to obtain the fermentation broth; The fermentation medium consists of: 10 g / L Tremella polysaccharide, 5 g / L yeast extract, 10 g / L peptone, 5 g / L dipotassium hydrogen phosphate, 5 g / L sodium chloride, 1 g / L magnesium sulfate, and distilled water as the solvent, with a pH of 7.0. 4. Centrifuge the fermentation broth at 8000 rpm for 15 min to remove the cells and insoluble components, collect the supernatant to obtain the crude enzyme solution.
[0023] Example 4: Preparation of Tremella oligosaccharides using α-1,3-glycosidase prepared from strain S1-14 200g of Tremella fuciformis polysaccharide (molecular weight 1000kDa) was weighed and added to 10L of pure water to prepare a sugar solution with a concentration of 20g / L. The solution was reacted according to the crude enzyme solution:sugar solution volume ratio of 1:1-6 prepared in Example 3, in a 40℃ water bath for 2h, followed by complete inactivation at 90℃ for 20min, and finally purification. The molecular weight was determined by high-performance liquid chromatography-gel permeation chromatography (GPC). An Ohpak SB-806 HQ2.0300 column was used, with a differential detector (RI), a mobile phase of 0.1M NaNO3 at a flow rate of 0.5mL / min, and a column temperature of 25℃.
[0024] The purification steps for the enzymatic hydrolysate are as follows: dilute the inactivated enzymatic hydrolysate 3 times, centrifuge at 10000 rpm for 20 min, remove the precipitate and take the supernatant; add 4 times the volume of ethanol, treat at 4℃ for 3 h, centrifuge at 8000 rpm for 10 min to obtain the alcohol precipitation product, add an equal volume of deionized water, dissolve fully and repeat the above operation twice.
[0025] The molecular weights of polysaccharides after reacting with crude enzyme solution and sugar solution at different volume ratios are shown in Table 3. According to the data in Table 3, the average molecular weight of polysaccharides can be effectively reduced to below 20 kDa within the range of crude enzyme solution to sugar solution ratio of 1:(1-3).
[0026] Table 3. Molecular weight of polysaccharides after reaction with different ratios of crude enzyme solution to sugar solution.
[0027] Comparative Example 1: Preparation of Tremella Oligosaccharides by Acid Method Weigh 200g of Tremella polysaccharide (molecular weight 1000kDa) and add it to 10L of pure water to make a sugar solution with a concentration of 20g / L. Adjust the pH of the solution to 2 with 1mol / L hydrochloric acid and stir and hydrolyze for 3h at room temperature. After the viscosity of the solution is significantly reduced, it is purified and the average molecular weight is determined to be 96.9kDa by GPC.
[0028] Comparative Example 2: Preparation of Tremella oligosaccharides by hydrogen peroxide method Weigh 200g of Tremella polysaccharide (molecular weight 1000kDa) and add it to 10L of pure water to prepare a sugar solution with a concentration of 20g / L. Add 22.67g of 30% hydrogen peroxide and 35.22g of ascorbic acid, and stir the reaction at 45℃ for 3h. Stop the reaction after the solution viscosity decreases significantly. Add catalase to remove residual hydrogen peroxide, and purify according to the enzymatic hydrolysate purification steps in Example 4. The average molecular weight was determined to be 57.8kDa by GPC.
[0029] Comparative Example 3: Preparation of Tremella oligosaccharides by the Fenton electro-method Weigh 200g of Tremella fuciformis polysaccharide (molecular weight 1000kDa) and add it to 10L of pure water to prepare a sugar solution with a concentration of 20g / L. Place this solution into a 24L electrolytic cell. Weigh 99.4g of sodium sulfate and dissolve it in the polysaccharide solution. Adjust the pH to 3.0 with 1M hydrochloric acid. Use a stainless steel mesh as the anode and porous graphite as the cathode. The electrode size is 30cm × 30cm, the electrode spacing is 5cm, and the current density is 12mA / cm². 2 Oxygen was introduced at a flow rate of 0.6 L / min, and the stirrer was set at 240 rpm. Electrolysis was carried out at room temperature for 2 hours. Immediately after the reaction was complete, the power supply was turned off, and the aeration and stirring were stopped. The pH of the electrolyte was neutralized to 7.0 with 1M sodium hydroxide solution to terminate the Fenton reaction. After purification, the average molecular weight was determined by GPC to be 31.1 kDa.
[0030] The average molecular weights of the Tremella oligosaccharides prepared in Example 4 and Comparative Examples 1-3 were statistically analyzed. Example 4 used a crude enzyme solution to sugar solution ratio of 1:1. The statistical results are shown in Table 4. Table 4. Average molecular weight of Tremella fuciformis oligosaccharides prepared in Example 4 and Comparative Examples 1-3
[0031] The results above show that, compared with traditional degradation methods such as acid degradation, hydrogen peroxide degradation, and electro-Fenton degradation, this invention can degrade Tremella polysaccharides into smaller molecule polysaccharides with lower molecular weight, thus having better industrial application value.
[0032] In summary, this invention provides a novel strain of *C. chrysogenum* S1-14 (CGMCC No. 37778) and utilizes its generated α-1,3-glycosidase to construct a highly efficient process for degrading high-molecular-weight polysaccharides. This strain can significantly and mildly degrade polysaccharides containing α-1,3-glycosidic bonds, such as *Tremella fuciformis* polysaccharide, from 1000 kDa to below 20 kDa, with a degradation effect superior to traditional physical and chemical methods. This invention has the advantages of high efficiency, controllability, and environmental friendliness, providing a new biotechnological approach for the preparation of highly active oligosaccharides, and has broad industrial application prospects in the food, cosmetics, and pharmaceutical fields.
Claims
1. A type of *Chlorella* S1-14, classified and named *Chlorella*. Chryseobacterium sp., deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37778, deposited on February 10, 2026, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
2. The application of *Chlorella vulgaris* S1-14 as described in claim 1 in the enzymatic hydrolysis of α-1,3-glycosidic bonds in high molecular weight polysaccharides, characterized in that... The method of application includes: preparing crude enzyme solution and enzymatic hydrolysis.
3. The application of *Chlorella vulgaris* S1-14 according to claim 2 in the enzymatic hydrolysis of α-1,3-glycosidic bonds in high molecular weight polysaccharides, characterized in that... The preparation of crude enzyme solution includes: strain activation, seed culture, fermentation culture, and post-treatment.
4. The application of *Chlorella vulgaris* S1-14 according to claim 3 in the enzymatic hydrolysis of α-1,3-glycosidic bonds in high molecular weight polysaccharides, characterized in that... The strain was activated by streaking a solution of Chlorella S1-14 onto an activation plate and incubating it statically at 29-31°C for 24-25 hours to obtain the activated strain. The activated plate consists of: glucose 9.8-10.2 g / L, yeast extract 4.8-5.2 g / L, peptone 4.8-5.2 g / L, dipotassium hydrogen phosphate 4.8-5.2 g / L, sodium chloride 4.8-5.2 g / L, agar 14.5-15.5 g / L, distilled water as solvent, and a pH of 7.4-7.
6.
5. The application of *Chlorella vulgaris* S1-14 according to claim 3 in the enzymatic hydrolysis of α-1,3-glycosidic bonds in high molecular weight polysaccharides, characterized in that... The seed culture involves inoculating the activated strain into the seed culture medium at an inoculation rate of 4.8-5.2%, and then culturing it on a shaker for 15-16 hours at a temperature of 28-32℃ and a rotation speed of 210-230 rpm to obtain the seed liquid. The seed culture medium consists of: 4.8-5.2 g / L Tremella polysaccharide, 4.8-5.2 g / L glucose, 9.8-10.2 g / L yeast extract, 4.8-5.2 g / L peptone, and 4.8-5.2 g / L sodium chloride, with distilled water as the solvent and a pH of 7.4-7.
6.
6. The application of *Chlorella vulgaris* S1-14 according to claim 3 in the enzymatic hydrolysis of α-1,3-glycosidic bonds in high molecular weight polysaccharides, characterized in that... The fermentation culture involves inoculating the seed culture into the fermentation medium at an inoculation rate of 4.8-5.2%, and fermenting for 24-25 hours at a temperature of 28-32℃ and a rotation speed of 230-250 rpm to obtain the fermentation broth. The fermentation medium consists of: 9.8-10.2 g / L of Tremella polysaccharide, 4.8-5.2 g / L of yeast extract, 9.8-10.2 g / L of peptone, 4.8-5.2 g / L of dipotassium hydrogen phosphate, 4.8-5.2 g / L of sodium chloride, and 0.9-1.1 g / L of magnesium sulfate. The solvent is distilled water, and the pH value is 6.8-7.
2.
7. The application of *Chlorella vulgaris* S1-14 according to claim 3 in the enzymatic hydrolysis of α-1,3-glycosidic bonds in high molecular weight polysaccharides, characterized in that... The post-processing involves centrifuging the fermentation broth, collecting the supernatant, and obtaining a crude enzyme solution. The centrifugation speed is 8000-8500 rpm, and the centrifugation time is 15-20 min.
8. The application of *Chlorella vulgaris* S1-14 according to claim 2 in the enzymatic hydrolysis of α-1,3-glycosidic bonds in high molecular weight polysaccharides, characterized in that... The enzymatic hydrolysis involves adding crude enzyme solution to a solution of α-1,3-glycosidic high molecular weight polysaccharide, performing an enzymatic hydrolysis reaction, and then inactivating and purifying the hydrolysate to obtain low molecular weight polysaccharide.
9. The application of *Chlorella vulgaris* S1-14 according to claim 8 in the enzymatic hydrolysis of α-1,3-glycosidic bonds in high molecular weight polysaccharides, characterized in that... In the enzymatic hydrolysis, the concentration of the α-1,3-glycosidic bond high molecular weight polysaccharide solution is 18-22 g / L; The volume ratio of the α-1,3-glycosidic polymeric polysaccharide solution to the crude enzyme solution was 1:1-6. The enzymatic hydrolysis reaction is carried out at a temperature of 38-42℃ for 2-2.5 hours. The inactivation temperature during the inactivation process is 88-92℃, and the inactivation time is 20-25 min. The purification method is as follows: dilute the inactivated enzyme hydrolysate by 2.8-3.2 times, centrifuge at 10000-11000 rpm for 20-25 min, take the supernatant, add 3.8-4.2 times the volume of ethanol, treat at 3-5℃ for 3-3.5 h, centrifuge at 8000-9000 rpm for 10-15 min to obtain the alcohol precipitation product, add an equal volume of deionized water, dissolve, and repeat the above operation twice.