CN115197983A

CN115197983A - Beta 1,3 glucan and preparation method thereof

Info

Publication number: CN115197983A
Application number: CN202210747384.8A
Authority: CN
Inventors: 曲丽君; 张见; 李萍; 邢军; 李夏瑛; 曹礼船; 包万军
Original assignee: Tianjin Tianlongjiangda Biotechnology Co ltd
Current assignee: Tianjin Tianlongjiangda Biotechnology Co ltd
Priority date: 2022-06-28
Filing date: 2022-06-28
Publication date: 2022-10-18

Abstract

The invention discloses beta 1,3 glucan and a preparation method thereof, and the method comprises the steps of firstly carrying out enzymolysis on lentinan by adopting a multienzyme system, and accelerating the enzymolysis process of the lentinan by utilizing microwave to carry out reaction in the enzymolysis process; and the saccharomyces cerevisiae is used for fermentation, so that the purification and enrichment of beta-glucan are realized while the yield of the polysaccharide is improved, the reaction conditions of a multienzyme system and a strain fermentation system are optimized, the optimal conditions for the synergistic conversion of lentinan by enzymolysis and fermentation are obtained, the extraction rate of the polysaccharide can be obviously improved, the extraction period is shortened, and the prepared beta-1, 3 glucan has strong antioxidant activity and remarkable bacteriostatic effect.

Description

Beta 1,3 glucan and preparation method thereof

Technical Field

The invention belongs to the technical field of biological fermentation, and particularly relates to beta 1,3 glucan and a preparation method thereof.

Background

Beta-glucan is a kind of macromolecular polysaccharide widely existing in microorganisms, plants and animals, and the main chain structure is connected by beta-1, 3-glycosidic bond, so that the beta-glucan is applied to the aspects of daily chemicals, medicines, agriculture and the like due to superior oxidation resistance, immunocompetence and anticancer effect, but the current mature research results are less. Therefore, how to efficiently prepare beta-1, 3-saccharide and explore its biological activity become a hot spot and focus of research.

At present, the beta-glucan degraded by a biological method is mainly degraded into micromolecular short-chain polysaccharide by using an enzymolysis method and commonly using beta-glucanase to degrade long-chain macromolecular beta-glucan, so that the water solubility is increased, the biological activity of the beta-glucan is improved, the anti-tumor activity and the anti-oxidation activity of the glucan with better water solubility are obviously enhanced after enzymolysis, but substances remained after the polysaccharide is extracted cannot be utilized and discarded, and the waste of raw materials is caused.

The microbial fermentation method is one of the biotransformation methods, and means that the complete microbial cells are used as a biocatalyst, so that the production efficiency of active polysaccharide can be improved, carbon sources, nitrogen sources, inorganic salts and the like in raw materials can be utilized by the growth of microorganisms, but the microorganisms cannot directly utilize the polysaccharide, and the aims of purifying, separating and enriching the polysaccharide can be achieved.

The mushroom is a traditional famous edible mushroom in China, and is originally artificially domesticated and cultivated in the world. The mushroom is rich in nutrition and delicious in taste, and is regarded as the king of mushroom. Lentinan is an effective active component extracted from high-quality lentinus edodes fruiting bodies, and the active component in the lentinan is beta 1,3 glucan with branches, so that the lentinan has good immune activation and anti-tumor activity. However, there are few reports on the related researches on the synergistic conversion of lentinan by enzymolysis and microbial fermentation.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

The present invention has been made keeping in mind the above problems and/or problems occurring in the prior art.

Therefore, the present invention aims to overcome the defects in the prior art and provide a preparation method of beta 1,3 glucan.

In order to solve the technical problems, the invention provides the following technical scheme:

pretreating lentinan, performing enzymolysis on the lentinan by using a multienzyme system, fermenting an enzymolysis product, and separating and purifying the product.

As a preferable aspect of the present invention, wherein: the pretreatment of lentinan comprises the steps of soaking lentinus edodes in water, grinding into slurry, and centrifugally drying to obtain lentinus edodes powder; washing the obtained powder with distilled water, centrifuging again, and collecting precipitate; and repeating the previous step, adding distilled water to obtain a suspension, adjusting the pH to 6.5, and performing suction filtration and drying again to obtain the lentinan to be subjected to enzymolysis.

As a preferable aspect of the present invention, wherein: the polysaccharide is subjected to enzymolysis by utilizing a multienzyme system, comprising,

taking lentinan to be subjected to enzymolysis, sieving with a 100-mesh sieve, adding distilled water, stirring uniformly to obtain a solution to be subjected to enzymolysis, and placing in a constant-temperature water bath kettle;

firstly adding activated cellulase and papain into a solution to be subjected to enzymolysis to react to obtain a mixed enzymolysis solution A;

adding the activated beta-glucanase into the mixed enzymatic hydrolysate A, and placing the mixed enzymatic hydrolysate A in a microwave oven for reaction;

and after the reaction is finished, carrying out suction filtration while the reaction is hot, taking the precipitate, washing, and freeze-drying to obtain an enzymolysis product for later use.

As a preferable aspect of the present invention, wherein: the mass ratio of the cellulase to the papain to the beta-glucanase is 1:1:1, wherein the addition amount of each enzyme is 0.5-2% of the lentinan by mass of the total lentinan.

As a preferable aspect of the present invention, wherein: the mixture is placed in a microwave oven for reaction, wherein the reaction time is 20min, and the power of the microwave oven is 700W.

As a preferable aspect of the present invention, wherein: the fermentation enzymolysis product comprises the following components,

dissolving an enzymolysis product in distilled water, mixing and shaking to completely dissolve the enzymolysis product, adding the enzymolysis product into a liquid culture medium, and adjusting the pH value to 6.5; inoculating the strain for fermentation into the solution for fermentation for 36h to obtain fermentation liquor.

As a preferable aspect of the present invention, wherein: the strain for fermentation is saccharomyces cerevisiae, and the inoculation amount is 2-4%.

As a preferable aspect of the present invention, wherein: the liquid culture medium is prepared from tryptone, glucose, naCl, agar and distilled water.

As a preferable aspect of the present invention, wherein: the separation and purification of the product comprises the following steps,

and centrifuging the fermentation liquor to obtain supernatant, adding ethanol for washing, refrigerating overnight, centrifuging again, adding ethanol for washing again, refrigerating overnight, centrifuging again, and finally obtaining white precipitate, namely the beta-1, 3 glucan.

It is a further object of the present invention to overcome the deficiencies of the prior art and to provide a beta 1,3 glucan produced by enzymatic fermentation.

The invention has the beneficial effects that:

according to the invention, firstly, a multi-enzyme system is adopted to carry out enzymolysis on lentinan, microwaves are utilized to carry out reaction in the enzymolysis process, the microwaves cause the change of the internal structure of cells, the transfer of the lentinan from outer-layer cells to enzymolysis liquid is accelerated, and the enzymolysis process of the lentinan is accelerated; and the saccharomyces cerevisiae is used for fermentation, so that the purification and enrichment of beta-glucan are realized while the yield of the polysaccharide is improved, the reaction conditions of a multienzyme system and a strain fermentation system are optimized, the optimal conditions for the synergistic conversion of lentinan by enzymolysis and fermentation are obtained, the extraction rate of the polysaccharide can be obviously improved, the extraction period is shortened, and the prepared beta-1, 3 glucan has strong antioxidant activity and remarkable bacteriostatic effect.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced otherwise than as specifically described herein, and it will be appreciated by those skilled in the art that the present invention may be practiced without departing from the spirit and scope of the present invention and that the present invention is not limited by the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

The purity of the product is determined by adopting a Congo red method;

the invention adopts a spectrophotometry method to determine the antioxidant activity of the product;

the invention adopts a filter paper method to respectively determine the inhibition effect of the product on staphylococcus aureus.

The raw materials used in the invention are as follows:

mushroom: e-commerce, inc., of Fang of Anhui Yan, commonly available; cellulase (enzyme activity is 11000u/g, papain (enzyme activity is 800000 u/g), pectinase (enzyme activity is 50000 u/g) and neutral protease (enzyme activity is 50000 u/g), hefeihuang biological product limited company which is sold in the common market, beta-glucanase (enzyme activity is 40000 u/g), hubei Tai multisource bioengineering limited company which is sold in the common market, saccharomyces cerevisiae, beijing Wanjia first biological technology limited company, the strain number is BWCC59811, and other related raw materials in the invention are sold in the common market without special description.

Example 1

Soaking Lentinus Edodes in water, grinding into slurry, and centrifuging and drying to obtain Lentinus Edodes powder; washing the obtained powder with distilled water for 5 times, centrifuging again, and collecting precipitate; repeating the previous step and then performing the following steps of 1:8, adding distilled water to obtain a suspension, and adjusting the pH to 6.5 by using a citric acid-sodium hydroxide-hydrochloric acid buffer solution; and carrying out suction filtration and drying on the suspension again to obtain the lentinan to be subjected to enzymolysis.

Weighing 100g of lentinan to be subjected to enzymolysis, sieving with a 100-mesh sieve, adding an appropriate amount of distilled water, uniformly stirring to obtain a solution to be subjected to enzymolysis, placing in a 50-DEG C constant-temperature water bath, weighing 1g of cellulase and 1g of papain, dissolving in 55-DEG C distilled water, activating for 5min, and placing the activated solution and the solution to be subjected to enzymolysis in a 55-DEG C constant-temperature water bath for reaction for 10min to obtain a mixed enzymolysis solution A;

weighing 1g of beta-glucanase, dissolving the beta-glucanase in distilled water at 45 ℃ for activation for 5min, mixing the activated beta-glucanase with the enzymolysis liquid A, fully mixing the beta-glucanase and the enzymolysis liquid A, placing the mixture in a microwave oven, continuously reacting for 20min under the condition that the power is 700W, carrying out suction filtration while the mixture is hot after the reaction is finished, and taking the precipitate for washing; freeze drying to obtain enzymolysis product.

Weighing 10g of tryptone, 5g of glucose, 10g of NaCl, 20g of agar and 1000mil of distilled water to prepare a liquid culture medium; dissolving the enzymolysis product in sterile water, mixing and shaking to completely dissolve the enzymolysis product, and adding into a liquid culture medium to adjust the pH to 6.5; inoculating 3% of activated saccharomyces cerevisiae into the solution for shake flask fermentation, and obtaining fermentation liquor after fermentation for 36 hours.

And centrifuging the obtained fermentation liquor, taking supernatant, washing with 75% ethanol in a volume ratio of 2 times of that of the supernatant, refrigerating overnight, centrifuging again, taking supernatant, washing with 75% ethanol in a volume ratio of 6 times of that of the supernatant, refrigerating again overnight, and centrifuging to obtain white precipitate, namely the beta-1, 3 glucan product.

Example 2

weighing 1g of beta-glucanase, dissolving the beta-glucanase in distilled water at 45 ℃ for activation for 5min, mixing the beta-glucanase with the enzymolysis liquid A after activation, placing the mixture in a microwave oven after full mixing, continuously reacting for 20min under the conditions of the power of 500W, 600W, 700W and 800W respectively, carrying out suction filtration while the solution is hot after the reaction is finished, and taking and washing the precipitate; freeze drying to obtain enzymolysis product.

The purity, antioxidant activity (free radical scavenging rate) and bacteriostatic activity of the product were determined, and the results are shown in table 1:

TABLE 1 purity and Properties of beta-1, 3-glucans prepared at different microwave powers

As can be seen from table 1, with the increase of the microwave power, the yield of lentinan is increased, i.e. the purity of the final product is increased, but when the power exceeds 700W, the yield of lentinan is not increased or decreased, because the increase of the microwave power can accelerate the movement speed of molecules, accelerate the transfer of lentinan from outer cells to the solution, but the power is too high, the lentinan is easily carbonized, and the final yield is not high.

Example 3

Weighing 100g of lentinan to be subjected to enzymolysis, sieving with a 100-mesh sieve, adding an appropriate amount of distilled water, uniformly stirring to obtain a solution to be subjected to enzymolysis, placing in a 50-DEG C constant-temperature water bath, weighing 1g of cellulase and 1g of papain, dissolving in 55-DEG C distilled water, activating for 5min, placing in a 55-DEG C constant-temperature water bath after activation, and reacting with the solution to be subjected to enzymolysis for 10min to obtain a mixed enzymatic hydrolysate A;

weighing 1g of beta-glucanase, dissolving the beta-glucanase in distilled water at 45 ℃ for activation for 5min, mixing the beta-glucanase with the enzymolysis liquid A after activation, placing the mixture in a microwave oven after full mixing, continuing to react for 20min under the condition that the power is 700W, carrying out suction filtration while the solution is hot after the reaction is finished, and taking the precipitate for washing; freeze drying to obtain enzymolysis product.

Weighing 10g of tryptone, 5g of glucose, 10g of NaCl, 20g of agar and 1000mil of distilled water to prepare a liquid culture medium; dissolving the enzymolysis product in sterile water, mixing and shaking to completely dissolve the enzymolysis product, adding into liquid culture medium, and adjusting pH to 6.0, 6.5, 7.0, and 7.5; inoculating 3% of activated saccharomyces cerevisiae into the solution for shake flask fermentation, and obtaining fermentation liquor after fermentation for 36 hours.

And centrifuging the obtained fermentation liquor, taking supernatant, washing with 75% ethanol in a volume ratio of 2 times that of the supernatant, refrigerating overnight, centrifuging again, taking supernatant, washing with 75% ethanol in a volume ratio of 6 times that of the supernatant, refrigerating overnight again, and centrifuging to obtain white precipitate, namely the beta-1, 3 glucan product.

The purity, antioxidant activity (free radical scavenging rate), and bacteriostatic activity of the product were determined, and the results are shown in table 2:

TABLE 2 purity and Properties of beta-1, 3 glucans prepared at different pH of fermentation broths

As can be seen from Table 2, the polysaccharide yield is influenced to some extent by the initial pH of the fermentation broth, and when the pH of the fermentation broth is 6.5, the purity of the polysaccharide is the highest, and it can be determined that 6.5 is the optimum pH for fermentation in the present invention.

Example 4

Weighing 10g of tryptone, 5g of glucose, 10g of NaCl, 20g of agar and 1000mil of distilled water to prepare a liquid culture medium; dissolving an enzymolysis product in sterile water, mixing and shaking to completely dissolve the enzymolysis product, and adding the enzymolysis product into a liquid culture medium to adjust the pH value to 6.5; respectively inoculating 2.0%, 2.5%, 3.0% and 3.5% of activated Saccharomyces cerevisiae, performing shake flask fermentation in the above solution, and fermenting for 36h to obtain fermentation liquid.

The purity, antioxidant activity (free radical scavenging rate) and bacteriostatic activity of the product were determined, and the results are shown in table 3:

TABLE 3 purity and Properties of beta-1, 3 glucan prepared with different inoculum sizes of ferments

As can be seen from Table 3, the present invention prefers the inoculation amount of yeast in the fermentation process, and when the inoculation amount is 3%, the product purity is the highest, and when the inoculation amount is continuously increased, the polysaccharide yield is rather reduced.

Example 5

Weighing 10g of tryptone, 5g of glucose, 10g of NaCl, 20g of agar and 1000mil of distilled water to prepare a liquid culture medium; dissolving an enzymolysis product in sterile water, mixing and shaking to completely dissolve the enzymolysis product, and adding the enzymolysis product into a liquid culture medium to adjust the pH value to 6.5; inoculating 3% activated saccharomyces cerevisiae into the solution for shake flask fermentation, and respectively fermenting for 24 h, 30 h, 36h and 42h to obtain fermentation liquor.

The purity, antioxidant activity (free radical scavenging rate), bacteriostatic activity of the product were determined and the results are shown in table 4:

TABLE 4 purity and Properties of beta-1, 3 glucans prepared at different fermentation times

According to the invention, by adjusting the fermentation time, the highest product yield is found when the fermentation is carried out for 36 hours, and the antioxidant activity is strongest, so that the optimal fermentation time is determined to be 36 hours

Comparative example 1

Weighing 100g of lentinan to be subjected to enzymolysis, sieving with a 100-mesh sieve, adding an appropriate amount of distilled water, uniformly stirring to obtain a solution to be subjected to enzymolysis, placing in a 50-DEG C constant-temperature water bath, weighing 1g of cellulose pectinase and 1g of neutral protease, dissolving in 55-DEG C distilled water, activating for 5min, and placing in a 55-DEG C constant-temperature water bath with the solution to be subjected to enzymolysis for reaction for 10min after activation to obtain a mixed enzymatic hydrolysate A;

Weighing 10g of tryptone, 5g of glucose, 10g of NaCl, 20g of agar and 1000mil of distilled water to prepare a liquid culture medium; dissolving the enzymolysis product in sterile water, mixing and shaking to completely dissolve the enzymolysis product, and adding into a liquid culture medium to adjust the pH to 6.5; inoculating 3% activated Saccharomyces cerevisiae, performing shake flask fermentation in the above solution for 36 hr to obtain fermentation broth

Comparative example 2

Weighing 10g of tryptone, 5g of glucose, 10g of NaCl, 20g of agar and 1000mil of distilled water to prepare a liquid culture medium; dissolving the enzymolysis product in sterile water, mixing and shaking to completely dissolve the enzymolysis product, and adding into a liquid culture medium to adjust the pH to 6.5; inoculating 3% of activated actinomycetes into the solution for shake flask fermentation, and fermenting for 36h to obtain fermentation liquor.

Comparative example 3

weighing 1g of beta-glucanase, dissolving in distilled water at 45 ℃, activating for 5min, mixing with the enzymolysis liquid A after activation, heating in a boiling water bath for 20min after full mixing, carrying out suction filtration when the reaction is finished, and taking the precipitate for washing; freeze drying to obtain enzymolysis product.

TABLE 5 purity and Properties of beta-1, 3-Glucan for different preparation Processes

According to the invention, a multi-enzyme system is adopted to carry out enzymolysis on lentinan, a multi-enzyme catalytic system consisting of cellulase, papain and beta-glucanase is selected, the cellulase and the papain act on cell walls of lentinus edodes, and then the beta-glucanase is added, so that the temperature, the time and the enzyme adding amount in the multi-enzyme reaction system are optimized; according to the invention, microwave is utilized to carry out reaction in the enzymolysis process, and the change of the internal structure of the cell can be caused by the microwave, so that the transfer of lentinan from outer-layer cells to enzymolysis liquid is accelerated, and the enzymolysis process of the lentinan is accelerated; and the saccharomyces cerevisiae is used for fermentation, so that the purification and enrichment of beta-glucan are realized while the yield of the polysaccharide is improved, the optimal condition for converting lentinan by enzymolysis and fermentation in a synergistic manner is obtained, the extraction rate of the polysaccharide can be obviously improved, the extraction period is shortened, and the prepared beta-1, 3 glucan has strong antioxidant activity and remarkable bacteriostatic effect.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims

1. A method for preparing beta-1, 3 glucan, which is characterized by comprising the following steps: comprises the steps of (a) preparing a substrate,

2. The method of producing β -1,3 glucan according to claim 1, wherein: the pretreatment of lentinan comprises the steps of soaking lentinus edodes in water, grinding into slurry, and carrying out centrifugal drying to obtain lentinus edodes powder; washing the obtained powder with distilled water, centrifuging again, and collecting precipitate; and repeating the previous step, adding distilled water to obtain a suspension, adjusting the pH to 6.5, and performing suction filtration and drying again to obtain the lentinan to be subjected to enzymolysis.

3. The method of producing β -1,3 glucan according to claim 1, wherein: the polysaccharide is subjected to enzymolysis by utilizing a multienzyme system, which comprises,

taking lentinan to be subjected to enzymolysis, sieving with a 100-mesh sieve, adding distilled water, stirring to obtain a solution to be subjected to enzymolysis, and placing in a constant-temperature water bath kettle;

adding activated cellulase and papain into a solution to be subjected to enzymolysis for reaction to obtain a mixed enzymolysis solution A;

4. A process for the preparation of β -1,3 glucan according to claim 3, wherein: the mass ratio of the cellulase to the papain to the beta-glucanase is 1:1:1, wherein the addition amount of each enzyme is 0.5-2% of the lentinan by mass total amount of the lentinan.

5. A process for the preparation of β -1,3 glucan according to claim 3, wherein: and placing the mixture in a microwave oven for reaction, wherein the reaction time is 20min, and the power of the microwave oven is 700W.

6. The method of producing β -1,3 glucan according to claim 1, wherein: the fermentation enzymolysis product comprises the following components,

dissolving an enzymolysis product in distilled water, mixing and shaking to completely dissolve the enzymolysis product, adding the enzymolysis product into a liquid culture medium, and adjusting the pH value to 6.5; inoculating a strain for fermentation into the solution for fermentation, and fermenting for 36h to obtain a fermentation liquid.

7. The method for producing beta-1, 3 glucan according to claim 6, wherein: the strain for fermentation is saccharomyces cerevisiae, and the inoculation amount is 2-4%.

8. The method for producing beta-1, 3 glucan according to claim 6, wherein: the liquid culture medium is prepared from tryptone, glucose, naCl, agar and distilled water.

9. The method for producing beta-1, 3 glucan according to claim 1, wherein the separation and purification of the product comprises,

10. A product obtained by the method for producing β -1,3 glucan according to any one of claims 1 to 9.