A β1,3-glucan and its preparation method

Abstract

The present invention discloses a β1,3-glucan and a preparation method thereof. Firstly, a multi-enzyme system is used to enzymatically hydrolyze lentinan, and microwave is used during the enzymatic hydrolysis process to accelerate the polysaccharide enzymatic hydrolysis process. Then, Saccharomyces cerevisiae is used for fermentation to purify and enrich β-glucan while increasing the polysaccharide yield. The reaction conditions of the multi-enzyme system and the strain fermentation system are optimized to obtain the optimal conditions for the synergistic conversion of lentinan by enzymatic hydrolysis and fermentation, which can significantly improve the extraction rate of polysaccharide, shorten the extraction cycle, and at the same time, the prepared β-1,3-glucan has strong antioxidant activity and remarkable antibacterial effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological fermentation, and particularly relates to a β1,3-glucan and a preparation method thereof. Background Art

[0002] β-glucan is a type of macromolecular polysaccharide widely present in microorganisms, plants, and animals. The main chain structure is connected by β-1,3-glycosidic bonds. Due to its excellent antioxidant, immunological, and anticancer effects, it is applied in aspects such as daily chemicals, medicine, and agriculture. However, there are currently few mature research results. Therefore, how to efficiently prepare β-1,3-glucan and explore its biological activity has become a research focus and key point.

[0003] Currently, the biological method for degrading β-glucan mainly uses enzymatic hydrolysis. Commonly, β-glucanase is used to degrade long-chain macromolecular β-glucan into small-molecule short-chain polysaccharides, thereby increasing water solubility and improving its biological activity. Moreover, the antitumor and antioxidant activities of glucan with good water solubility after enzymatic hydrolysis are also significantly enhanced. However, the substances remaining after extracting the polysaccharide will be discarded without being utilized, resulting in waste of raw materials.

[0004] The microbial fermentation method is a type of biotransformation method, which refers to using intact microbial cells as biocatalysts, capable of improving the production efficiency of active polysaccharides. Carbon sources, nitrogen sources, inorganic salts, etc. in the raw materials can be utilized by the growth of microorganisms, but microorganisms cannot directly utilize polysaccharides, thereby achieving the purpose of purifying, separating, and enriching polysaccharides.

[0005] Lentinula edodes is a well-known traditional edible mushroom in China and was the first to be artificially domesticated and cultivated in the world. Lentinula edodes is rich in nutrients and has a delicious taste, and is regarded as the "king of mushrooms". Lentinan is an effective active ingredient extracted from high-quality Lentinula edodes fruit bodies. The active ingredient in lentinan is branched β1,3-glucan, which has good immune activation and antitumor activities. However, there are few reports on the related research of the synergistic transformation of lentinan by polysaccharide enzymatic hydrolysis and microbial fermentation. Summary of the Invention

[0006] The purpose of this part is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Simplifications or omissions may be made in this part, as well as in the abstract and title of the present application, to avoid obscuring the purpose of this part, the abstract, and the title. However, such simplifications or omissions cannot be used to limit the scope of the present invention.

[0007] In view of the above and / or problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a preparation method for β1,3-glucan.

[0009] To solve the above technical problems, the present invention provides the following technical solutions:

[0010] Pre-treat the lentinan, enzymatically hydrolyze the polysaccharide using a multi-enzyme system, ferment the enzymatic hydrolysate, and separate and purify the product.

[0011] As a preferred embodiment of the present invention, wherein: the pre-treatment of lentinan includes soaking lentinus edodes in water, grinding it into a slurry, centrifuging and drying to obtain lentinan powder; washing the obtained powder with distilled water and centrifuging again to collect the precipitate; repeating the above step and then adding distilled water to obtain a suspension, adjusting the pH to 6.5 and then filtering and drying again to obtain the lentinan to be enzymatically hydrolyzed.

[0012] As a preferred embodiment of the present invention, wherein: the enzymatic hydrolysis of the polysaccharide using a multi-enzyme system includes,

[0013] Pass the lentinan to be enzymatically hydrolyzed through a 100-mesh sieve, add distilled water and stir evenly to obtain an enzymatic hydrolysis solution, and place it in a constant temperature water bath;

[0014] First add the activated cellulase and papain to the enzymatic hydrolysis solution to react to obtain a mixed enzymatic hydrolysis solution A;

[0015] Then add the activated β-glucanase to the mixed enzymatic hydrolysis solution A and react in a microwave oven;

[0016] After the reaction, filter while it is hot, take the precipitate, wash it, and freeze-dry it to obtain the enzymatic hydrolysate for standby.

[0017] As a preferred embodiment of the present invention, wherein: the mass ratio of cellulase, papain, and β-glucanase is 1:1:1, and based on the total mass of lentinan, the addition amount of each enzyme is 0.5% - 2% of lentinan.

[0018] As a preferred embodiment of the present invention, wherein: the reaction in the microwave oven, wherein the reaction time is 20 min and the microwave power is 700 W.

[0019] As a preferred embodiment of the present invention, wherein: the fermentation of the enzymatic hydrolysate includes,

[0020] Dissolve the enzymatic hydrolysate in distilled water, mix and shake to completely dissolve the enzymatic hydrolysate, add it to the liquid medium and adjust the pH to 6.5; inoculate the strain for fermentation into the above solution for fermentation, and obtain a fermentation broth after 36 h of fermentation.

[0021] As a preferred embodiment of the present invention, wherein: the strain for fermentation is Saccharomyces cerevisiae, and the inoculation amount is 2% - 4%.

[0022] As a preferred embodiment of the present invention, the liquid culture medium is prepared from tryptone, glucose, NaCl, agar, and distilled water.

[0023] As a preferred embodiment of the present invention, the separation and purification of the product includes

[0024] Centrifuge the above fermentation broth to obtain the supernatant, wash it with ethanol, refrigerate it overnight, centrifuge it again, wash it with ethanol again, refrigerate it overnight and centrifuge it again. Finally, a white precipitate is obtained, which is β-1,3-glucan.

[0025] Another object of the present invention is to overcome the deficiencies in the prior art and provide a β1,3-glucan prepared by an enzymatic hydrolysis fermentation method.

[0026] Advantages of the present invention:

[0027] The present invention first uses a multi-enzyme system to enzymatically hydrolyze lentinan. During the enzymatic hydrolysis process, microwave is used for the reaction. The microwave causes changes in the internal structure of cells, accelerates the transfer of lentinan from the outer layer of cells to the enzymatic hydrolysis solution, and accelerates the polysaccharide enzymatic hydrolysis process. Then, Saccharomyces cerevisiae is used for fermentation. While increasing the polysaccharide yield, the purification and enrichment of β-glucan are achieved. The reaction conditions of the multi-enzyme system and the strain fermentation system are optimized, and the optimal conditions for the synergistic conversion of lentinan by enzymatic hydrolysis and fermentation are obtained, which can significantly improve the extraction rate of polysaccharides, shorten the extraction cycle, and at the same time, the prepared β-1,3-glucan has strong antioxidant activity and significant antibacterial effect. Detailed implementation manners

[0028] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following detailed description of the specific implementation manners of the present invention will be given in combination with the embodiments of the specification.

[0029] In the following description, many specific details are set forth in order to fully understand the present invention. However, the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar generalizations without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0030] Secondly, the so-called "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that can be included in at least one implementation manner of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment that mutually excludes other embodiments.

[0031] The present invention uses the Congo red method to determine the purity of the product;

[0032] The present invention uses the spectrophotometry method to determine the antioxidant activity of the product;

[0033] The present invention uses the filter paper method to measure the inhibitory effect of the product on Staphylococcus aureus respectively.

[0034] Raw materials used in the present invention:

[0035] Lentinus edodes: Anhui Yanzhifang E-commerce Co., Ltd., commercially available; cellulase (enzyme activity 11000u / g), papain (enzyme activity 800000u / g), pectinase (enzyme activity 50000u / g), neutral protease (enzyme activity 50000u / g): Hefei Shengrun Bioproducts Co., Ltd., commercially available; β-glucanase (enzyme activity 40000u / g): Hubei Taiduoyuan Bioengineering Co., Ltd., commercially available; Saccharomyces cerevisiae: Beijing Wanjia Shouhua Biotechnology Co., Ltd., strain number BWCC59811; other raw materials involved in the present invention, without special instructions, are all commercially available.

[0036] Example 1

[0037] Soak Lentinus edodes in water and grind it into slurry, and obtain Lentinus edodes powder after centrifugation and drying; wash the obtained powder 5 times with distilled water and then centrifuge again to collect the precipitate; repeat the above steps and then add distilled water according to the ratio of 1:8 to obtain a suspension, and adjust the pH to 6.5 with citric acid-sodium hydroxide-hydrochloric acid buffer solution; the suspension is filtered and dried again to obtain the Lentinus edodes polysaccharide to be enzymolyzed.

[0038] Weigh 100g of the Lentinus edodes polysaccharide to be enzymolyzed, sieve it through a 100-mesh sieve, add an appropriate amount of distilled water and stir evenly to obtain the solution to be enzymolyzed, place it in a 50°C constant temperature water bath, weigh 1g of cellulase and 1g of papain, dissolve them in distilled water at 55°C and activate for 5 minutes, and after activation, place them in a 55°C constant temperature water bath with the solution to be enzymolyzed and react for 10 minutes to obtain the mixed enzymolysis solution A;

[0039] Weigh 1g of β-glucanase again, dissolve it in distilled water at 45°C and activate for 5 minutes, after activation, mix it with the enzymolysis solution A, fully mix it and then place it in a microwave oven, and continue to react for 20 minutes under the condition of a power of 700W. After the reaction, filter while it is hot, take the precipitate and wash it; obtain the enzymolysis product after freeze-drying.

[0040] Weigh 10g of tryptone, 5g of glucose, 10g of NaCl, 20g of agar, add 1000mil of distilled water to prepare a liquid medium; dissolve the enzymolysis product in sterile water, mix and shake to make the enzymolysis product completely dissolve, add it to the liquid medium and adjust the pH to 6.5; inoculate 3% of the activated Saccharomyces cerevisiae into the above solution for shake flask fermentation, and obtain the fermentation broth after 36h of fermentation.

[0041] Centrifuge the obtained fermentation broth, take the supernatant, wash it with 75% ethanol with a volume twice that of the supernatant, centrifuge again after refrigerating overnight, take the supernatant, wash it with 75% ethanol with a volume six times that of the supernatant, refrigerate overnight again, and centrifuge to obtain a white precipitate, which is the β-1,3-glucan product.

[0042] Example 2

[0043] Soak the Lentinus edodes in water and grind it into a slurry, and obtain Lentinus edodes powder after centrifugation and drying; wash the obtained powder 5 times with distilled water and then centrifuge again to collect the precipitate; repeat the above step and then obtain a suspension by adding distilled water in a ratio of 1:8, and adjust the pH to 6.5 with a citric acid-sodium hydroxide-hydrochloric acid buffer solution; filter and dry the suspension again to obtain the Lentinus edodes polysaccharide to be enzymolyzed.

[0044] Weigh 100 g of the Lentinus edodes polysaccharide to be enzymolyzed, sieve it through a 100-mesh sieve, add an appropriate amount of distilled water and stir evenly to obtain a solution to be enzymolyzed, place it in a 50 °C constant temperature water bath, weigh 1 g of cellulase and 1 g of papain, dissolve them in distilled water at 55 °C and activate for 5 min, and react with the solution to be enzymolyzed in a 55 °C constant temperature water bath for 10 min to obtain a mixed enzymolysis solution A;

[0045] Weigh 1 g of β-glucanase again, dissolve it in distilled water at 45 °C and activate for 5 min, mix it with the enzymolysis solution A after activation, and place it in a microwave oven after thorough mixing, and continue to react for 20 min under the conditions of powers of 500 W, 600 W, 700 W, and 800 W respectively. After the reaction is completed, filter while it is hot, take the precipitate and wash it; obtain the enzymolysis product after freeze-drying.

[0046] Weigh 10 g of tryptone, 5 g of glucose, 10 g of NaCl, 20 g of agar, and add 1000 mil of distilled water to prepare a liquid medium; dissolve the enzymolysis product in sterile water, mix and shake to completely dissolve the enzymolysis product, add it to the liquid medium and adjust the pH to 6.5; inoculate 3% of the activated Saccharomyces cerevisiae into the above solution for shake flask fermentation, and obtain a fermentation broth after 36 h of fermentation.

[0047] Centrifuge the obtained fermentation broth, take the supernatant, wash it with 75% ethanol with a volume twice that of the supernatant, centrifuge again after refrigerating overnight, take the supernatant, wash it with 75% ethanol with a volume six times that of the supernatant, refrigerate overnight again, and centrifuge to obtain a white precipitate, which is the β-1,3-glucan product.

[0048] Measure the purity, antioxidant activity (free radical scavenging rate), and antibacterial property of the product, and the results are shown in Table 1:

[0049] Table 1 Purity and properties of β-1,3-glucan prepared under different microwave powers

[0050]

[0051] As can be seen from Table 1, with the increase of microwave power, the yield of lentinan increases, that is, the purity of the final product increases. However, when the power exceeds 700W, the polysaccharide yield not only does not increase but decreases. This is because the increase of microwave power can accelerate the molecular movement speed and promote the transfer of lentinan from the outer cells to the solution. But when the power is too high, the polysaccharide is easily carbonized, resulting in a lower final yield.

[0052] Example 3

[0053] Soak the lentinus edodes in water and grind it into slurry. After centrifugation and drying, lentinus edodes powder is obtained; wash the obtained powder with distilled water 5 times and then centrifuge again to collect the precipitate; repeat the above steps and then add distilled water in a ratio of 1:8 to obtain a suspension, and adjust the pH to 6.5 with citric acid - sodium hydroxide - hydrochloric acid buffer solution; filter and dry the suspension again to obtain the lentinan to be enzymolyzed.

[0054] Weigh 100g of the lentinan to be enzymolyzed, pass it through a 100 - mesh sieve, add an appropriate amount of distilled water and stir evenly to obtain the enzymolysis solution to be treated. Place it in a 50°C constant - temperature water bath. Weigh 1g of cellulase and 1g of papain, dissolve them in distilled water at 55°C and activate for 5min. After activation, place them in the 55°C constant - temperature water bath with the enzymolysis solution to be treated and react for 10min to obtain the mixed enzymolysis solution A.

[0055] Weigh 1g of β - glucanase again, dissolve it in distilled water at 45°C and activate for 5min. After activation, mix it with the enzymolysis solution A. After full mixing, place it in a microwave oven and continue to react for 20min under the condition of 700W power. After the reaction, filter while it is hot, take the precipitate and wash it; after freeze - drying, the enzymolysis product is obtained.

[0056] Weigh 10g of tryptone, 5g of glucose, 10g of NaCl, 20g of agar, and add 1000ml of distilled water to prepare a liquid medium; dissolve the enzymolysis product in sterile water, mix and shake to make the enzymolysis product completely dissolve, and add it to the liquid medium to adjust the pH to 6.0, 6.5, 7.0, and 7.5 respectively; inoculate 3% of the activated Saccharomyces cerevisiae into the above solution for shake - flask fermentation, and after 36h of fermentation, the fermentation broth is obtained.

[0057] Centrifuge the obtained fermentation broth, take the supernatant, wash it with 75% ethanol with a volume twice that of the supernatant, refrigerate overnight and then centrifuge again, take the supernatant, wash it with 75% ethanol with a volume six times that of the supernatant, refrigerate overnight again, and centrifuge to obtain a white precipitate, which is the β - 1,3 - glucan product.

[0058] Measure the purity, antioxidant activity (free radical scavenging rate), and antibacterial property of the product. The results are shown in Table 2:

[0059] Table 2 Purity and properties of β-1,3-glucan prepared at different pH values of fermentation broth

[0060]

[0061] As can be seen from Table 2, the polysaccharide yield is affected to a certain extent by the initial pH of the fermentation broth. When the pH of the fermentation broth is 6.5, the polysaccharide purity is the highest. It can be determined that in this invention, 6.5 is the optimal pH for fermentation.

[0062] Example 4

[0063] Soak Lentinula edodes in water and grind it into slurry. After centrifugation and drying, obtain Lentinula edodes powder; wash the obtained powder 5 times with distilled water and then centrifuge again to collect the precipitate; repeat the above step and then obtain a suspension by adding distilled water in a ratio of 1:8, and adjust the pH to 6.5 with citric acid-sodium hydroxide-hydrochloric acid buffer; filter and dry the suspension again to obtain the Lentinula edodes polysaccharide to be enzymolyzed.

[0064] Weigh 100 g of the Lentinula edodes polysaccharide to be enzymolyzed, pass it through a 100-mesh sieve, add an appropriate amount of distilled water and stir evenly to obtain the solution to be enzymolyzed. Place it in a 50 °C constant temperature water bath. Weigh 1 g of cellulase and 1 g of papain, dissolve them in distilled water at 55 °C and activate for 5 min. After activation, place them in the 55 °C constant temperature water bath with the solution to be enzymolyzed and react for 10 min to obtain the mixed enzymolysis solution A;

[0065] Weigh 1 g of β-glucanase again, dissolve it in distilled water at 45 °C and activate for 5 min. After activation, mix it with the enzymolysis solution A, mix well and then place it in a microwave oven, and continue to react for 20 min under the condition of a power of 700 W. After the reaction, filter while it is hot and take the precipitate for washing; obtain the enzymolysis product after freeze-drying.

[0066] Weigh 10 g of tryptone, 5 g of glucose, 10 g of NaCl, 20 g of agar, add 1000 mil of distilled water to prepare a liquid medium; dissolve the enzymolysis product in sterile water, mix and shake to make the enzymolysis product completely dissolve, add it to the liquid medium and adjust the pH to 6.5; inoculate 2.0%, 2.5%, 3.0%, 3.5% of the activated Saccharomyces cerevisiae into the above solution respectively for shake flask fermentation, and obtain the fermentation broth after 36 h of fermentation.

[0067] Centrifuge the obtained fermentation broth, take the supernatant, wash it with 75% ethanol with a volume twice that of the supernatant, centrifuge again after refrigerating overnight, take the supernatant, wash it with 75% ethanol with a volume six times that of the supernatant, refrigerate again overnight, and centrifuge to obtain a white precipitate, which is the β-1,3-glucan product.

[0068] Measure the purity, antioxidant activity (free radical scavenging rate), and antibacterial property of the product, and the results are shown in Table 3:

[0069] Table 3 Purity and properties of β-1,3-glucan prepared with different inoculation amounts of fermentation strains

[0070]

[0071] As can be seen from Table 3, the inoculation amount of yeast in the fermentation process is optimized in the present invention. When the inoculation amount is 3%, the product purity is the highest. When the inoculation amount continues to increase, the polysaccharide yield decreases instead.

[0072] Example 5

[0073] Soak the Lentinula edodes in water and grind it into slurry. After centrifugation and drying, obtain Lentinula edodes powder; wash the obtained powder with distilled water 5 times and then centrifuge again to collect the precipitate; repeat the above step and then add distilled water in a ratio of 1:8 to obtain a suspension, and adjust the pH to 6.5 with citric acid-sodium hydroxide-hydrochloric acid buffer; filter and dry the suspension again to obtain the Lentinula edodes polysaccharide to be enzymolyzed.

[0074] Weigh 100 g of the Lentinula edodes polysaccharide to be enzymolyzed, pass it through a 100-mesh sieve, add an appropriate amount of distilled water and stir evenly to obtain an enzymolysis solution to be treated. Place it in a 50 °C constant temperature water bath. Weigh 1 g of cellulase and 1 g of papain, dissolve them in distilled water at 55 °C and activate for 5 min. After activation, place them in the enzymolysis solution to be treated in a 55 °C constant temperature water bath and react for 10 min to obtain a mixed enzymolysis solution A;

[0075] Weigh 1 g of β-glucanase again, dissolve it in distilled water at 45 °C and activate for 5 min. After activation, mix it with enzymolysis solution A, mix well and then place it in a microwave oven. Under the condition of a power of 700 W, continue to react for 20 min. After the reaction, filter while it is hot and take the precipitate for washing; obtain the enzymolysis product after freeze-drying.

[0076] Weigh 10 g of tryptone, 5 g of glucose, 10 g of NaCl, 20 g of agar, add 1000 mil of distilled water to prepare a liquid medium; dissolve the enzymolysis product in sterile water, mix and shake to make the enzymolysis product completely dissolve, add it to the liquid medium and adjust the pH to 6.5; inoculate 3% of the activated Saccharomyces cerevisiae into the above solution for shake flask fermentation, and obtain fermentation broth after fermenting for 24, 30, 36, and 42 h respectively.

[0077] Centrifuge the obtained fermentation broth, take the supernatant, wash it with 75% ethanol with a volume twice that of the supernatant, centrifuge again after refrigerating overnight, take the supernatant, wash it with 75% ethanol with a volume six times that of the supernatant, refrigerate overnight again, and centrifuge to obtain a white precipitate, which is the β-1,3-glucan product.

[0078] Measure the purity, antioxidant activity (free radical scavenging rate), and antibacterial property of the product. The results are shown in Table 4:

[0079] Table 4 Purity and properties of β-1,3-glucan prepared at different fermentation times

[0080]

[0081] By adjusting the fermentation time in the present invention, it is found that the product yield is the highest and the antioxidant activity is the strongest when fermenting for 36 h. Therefore, the optimal fermentation time of the present invention is determined to be 36 h

[0082] Comparative Example 1

[0083] Soak the Lentinus edodes in water and grind it into slurry. After centrifugation and drying, obtain Lentinus edodes powder; wash the obtained powder with distilled water 5 times and then centrifuge again to collect the precipitate; repeat the above steps and then add distilled water according to a ratio of 1:8 to obtain a suspension, and adjust the pH to 6.5 with citric acid-sodium hydroxide-hydrochloric acid buffer; filter and dry the suspension again to obtain the Lentinus edodes polysaccharide to be enzymolyzed

[0084] Weigh 100 g of the Lentinus edodes polysaccharide to be enzymolyzed, pass it through a 100-mesh sieve, add an appropriate amount of distilled water and stir evenly to obtain an enzymolysis solution to be enzymolyzed. Place it in a 50 °C constant temperature water bath. Weigh 1 g of cellulase pectinase and 1 g of neutral protease, dissolve them in distilled water at 55 °C and activate for 5 min. After activation, place them in a 55 °C constant temperature water bath with the enzymolysis solution to be enzymolyzed and react for 10 min to obtain a mixed enzymolysis solution A

[0085] Weigh 1 g of β-glucanase again, dissolve it in distilled water at 45 °C and activate for 5 min. After activation, mix it with the enzymolysis solution A, fully mix it and then place it in a microwave oven, and continue to react for 20 min under the condition of a power of 700 W. After the reaction, filter while it is hot, take the precipitate and wash it; after freeze-drying, obtain the enzymolysis product

[0086] Weigh 10 g of tryptone, 5 g of glucose, 10 g of NaCl, 20 g of agar, add 1000 mil of distilled water to prepare a liquid medium; dissolve the enzymolysis product in sterile water, mix and shake to make the enzymolysis product completely dissolve, add it to the liquid medium and adjust the pH to 6.5; inoculate 3% of the activated Saccharomyces cerevisiae into the above solution for shake flask fermentation, and obtain a fermentation broth after 36 h of fermentation

[0087] Centrifuge the obtained fermentation broth, take the supernatant, wash it with 75% ethanol with a volume twice that of the supernatant, centrifuge again after refrigerating overnight, take the supernatant, wash it with 75% ethanol with a volume six times that of the supernatant, refrigerate overnight again, and centrifuge to obtain a white precipitate, which is the β-1,3-glucan product

[0088] Comparative Example 2

[0089] Soak the shiitake mushrooms in water and then grind them into a slurry. After centrifugation and drying, obtain shiitake mushroom powder. Wash the obtained powder 5 times with distilled water and then centrifuge again to collect the precipitate. Repeat the previous step and then add distilled water in a ratio of 1:8 to obtain a suspension. Adjust the pH to 6.5 with a citric acid-sodium hydroxide-hydrochloric acid buffer solution. Filter and dry the suspension again to obtain the shiitake mushroom polysaccharide to be enzymatically hydrolyzed.

[0090] Weigh 100 g of the shiitake mushroom polysaccharide to be enzymatically hydrolyzed, sieve it through a 100-mesh sieve, add an appropriate amount of distilled water and stir evenly to obtain the solution to be enzymatically hydrolyzed. Place it in a 50 °C constant temperature water bath. Weigh 1 g of cellulase and 1 g of papain, dissolve them in distilled water at 55 °C and activate for 5 min. After activation, place them in the 55 °C constant temperature water bath with the solution to be enzymatically hydrolyzed and react for 10 min to obtain the mixed enzymatically hydrolyzed solution A.

[0091] Weigh 1 g of β-glucanase again, dissolve it in distilled water at 45 °C and activate for 5 min. After activation, mix it with the enzymatically hydrolyzed solution A. After thorough mixing, place it in a microwave oven and continue to react for 20 min under the condition of a power of 700 W. After the reaction, filter while it is hot and wash the precipitate. After freeze-drying, obtain the enzymatically hydrolyzed product.

[0092] Weigh 10 g of tryptone, 5 g of glucose, 10 g of NaCl, 20 g of agar, add 1000 ml of distilled water to prepare a liquid medium. Dissolve the enzymatically hydrolyzed product in sterile water, mix and shake until the enzymatically hydrolyzed product is completely dissolved, add it to the liquid medium and adjust the pH to 6.5. Inoculate 3% of the activated actinomycetes into the above solution for shake flask fermentation. After 36 h of fermentation, obtain the fermentation broth.

[0093] Centrifuge the obtained fermentation broth, take the supernatant, wash it with 75% ethanol with a volume twice that of the supernatant, centrifuge again after refrigerating overnight, take the supernatant, wash it with 75% ethanol with a volume six times that of the supernatant, refrigerate overnight again, and centrifuge to obtain a white precipitate, which is the β-1,3-glucan product.

[0094] Comparative Example 3

[0095] Soak the shiitake mushrooms in water and then grind them into a slurry. After centrifugation and drying, obtain shiitake mushroom powder. Wash the obtained powder 5 times with distilled water and then centrifuge again to collect the precipitate. Repeat the previous step and then add distilled water in a ratio of 1:8 to obtain a suspension. Adjust the pH to 6.5 with a citric acid-sodium hydroxide-hydrochloric acid buffer solution. Filter and dry the suspension again to obtain the shiitake mushroom polysaccharide to be enzymatically hydrolyzed.

[0096] Weigh 100 g of the shiitake mushroom polysaccharide to be enzymatically hydrolyzed, sieve it through a 100-mesh sieve, add an appropriate amount of distilled water and stir evenly to obtain the solution to be enzymatically hydrolyzed. Place it in a 50 °C constant temperature water bath. Weigh 1 g of cellulase and 1 g of papain, dissolve them in distilled water at 55 °C and activate for 5 min. After activation, place them in the 55 °C constant temperature water bath with the solution to be enzymatically hydrolyzed and react for 10 min to obtain the mixed enzymatically hydrolyzed solution A.

[0097] Weigh 1 g of β-glucanase again and dissolve it in distilled water at 45 °C for 5 min of activation. After activation, mix it with enzymatic hydrolysate A. After thorough mixing, heat it in a boiling water bath for 20 min. After the reaction is completed, filter it while it is hot, and take the precipitate for washing; after freeze-drying, the enzymatic hydrolysis product is obtained.

[0098] Weigh 10 g of tryptone, 5 g of glucose, 10 g of NaCl, 20 g of agar, and add 1000 mil of distilled water to prepare a liquid medium; dissolve the enzymatic hydrolysis product in sterile water, mix and shake to completely dissolve the enzymatic hydrolysis product, and add it to the liquid medium to adjust the pH to 6.5; inoculate 3% of the activated Saccharomyces cerevisiae into the above solution for shake flask fermentation, and the fermentation broth is obtained after 36 h of fermentation.

[0099] Centrifuge the obtained fermentation broth, take the supernatant, wash it with 75% ethanol with a volume twice that of the supernatant, centrifuge it again after refrigerating overnight, take the supernatant, wash it with 75% ethanol with a volume six times that of the supernatant, refrigerate it overnight again, and centrifuge to obtain a white precipitate, which is the β-1,3-glucan product.

[0100] Determine the purity, antioxidant activity (free radical scavenging rate), and antibacterial property of the product. The results are shown in Table 3:

[0101] Table 5 Purity and properties of β-1,3-glucan under different preparation processes

[0102]

[0103]

[0104] The present invention first uses a multi-enzyme system to enzymatically hydrolyze lentinan, selects cellulase, papain, and β-glucanase to form a multi-enzyme catalytic system, first uses cellulase and papain to act on the cell wall of Lentinus edodes, then adds β-glucanase, and simultaneously optimizes the temperature, time, and enzyme addition amount in the multi-enzyme reaction system; the present invention uses microwave for the reaction during the enzymatic hydrolysis process. Since microwave can cause changes in the internal structure of cells, it accelerates the transfer of lentinan from the outer layer of cells to the enzymatic hydrolysate, accelerating the polysaccharide enzymatic hydrolysis process; then uses Saccharomyces cerevisiae for fermentation, purifying and enriching β-glucan while increasing the polysaccharide yield, obtaining the optimal conditions for the synergistic transformation of lentinan by enzymatic hydrolysis and fermentation, which can significantly improve the extraction rate of polysaccharide, shorten the extraction cycle, and at the same time, the prepared β-1,3-glucan has strong antioxidant activity and significant antibacterial effect.

[0105] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing β-1,3-glucan, characterized in that: including pretreating lentinan, enzymatically hydrolyzing the polysaccharide using a multi-enzyme system, fermenting the enzymatic hydrolysate, and separating and purifying the product The pretreated lentinan is obtained by soaking lentinus edodes in water, grinding it into a slurry, centrifuging and drying to obtain lentinan powder; washing the obtained powder with distilled water and centrifuging again to collect the precipitate; repeating the above steps and then adding distilled water to obtain a suspension, adjusting the pH to 6.5 and then filtering and drying again to obtain the lentinan to be enzymatically hydrolyzed The enzymatic hydrolysis of the polysaccharide using a multi-enzyme system is to pass the lentinan to be enzymatically hydrolyzed through a 100-mesh sieve, add distilled water and stir evenly to obtain an enzymatic hydrolysis solution, and place it in a constant temperature water bath; first add the activated cellulase and papain to the enzymatic hydrolysis solution to react to obtain a mixed enzymatic hydrolysis solution A. Among them, based on the total mass of lentinan, the addition amount of each enzyme is 0.5% - 2% of lentinan; then add the activated β-glucanase to the mixed enzymatic hydrolysis solution A and place it in a microwave oven for reaction, the reaction time is 20 min, and the microwave power is 700 W; after the reaction, filter while it is hot, take the precipitate, wash it, and freeze-dry it to obtain the enzymatic hydrolysis product for standby The mass ratio of the cellulase, papain, and β-glucanase is 1:1:1 The fermentation of the enzymatic hydrolysis product is to dissolve the enzymatic hydrolysis product in distilled water, mix and shake to completely dissolve the enzymatic hydrolysis product, add it to a liquid medium and adjust the pH to 6.5; inoculate the strain for fermentation into the above solution for fermentation. The strain for fermentation is Saccharomyces cerevisiae, the inoculation amount is 3%, and the fermentation broth is obtained after 36 h of fermentation 2. The preparation method of β-1,3-glucan according to claim 1, characterized in that, The separation and purification of the product includes Centrifuge the above fermentation broth to obtain the supernatant, wash it with ethanol, refrigerate it overnight and centrifuge again, wash it with ethanol again, refrigerate it overnight and centrifuge, and finally obtain a white precipitate, which is β-1,3-glucan

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