Glucan product as well as preparation method and application thereof

Abstract

The invention relates to the technical field of skin care products, in particular to a glucan product as well as a preparation method and application thereof. According to the invention, the glucan product is obtained by performing enzymolysis on the material containing the glucan through the incision enzyme for specifically treating the 1, 4 glucosidic bonds, and the number ratio of the 1, 4 glucosidic bonds to the 1, 3 glucosidic bonds in the glucan product is 1.8 or less, so that the intramolecular and intermolecular agglomeration of the glucan is improved, the room-temperature stability of the glucan is improved, and the yield of the glucan is increased. And the obtained glucan product can be added in cosmetics in a large amount, has a stronger anti-inflammatory effect, and has an outstanding effect of promoting skin soothing after being added into the cosmetics.

Description

Technical Field

[0001] The present invention relates to the technical field of skin care products, and in particular to a glucan product and a preparation method and application thereof. Background Art

[0002] Due to its polyhydroxy structure and long linear chains, dextran can form hydrogen bonds within and between molecules during storage. This can lead to aggregation and agglomeration of dextran molecules, resulting in product instability and reduced efficacy. This is primarily due to the formation of hydrophobic regions between adjacent 1,4-glycosidic bonds, which can cause dextran precipitation during storage. This characteristic limits the use of dextran in clear, aqueous cosmetics.

[0003] There are some technical solutions in the related art that use chemical modification methods to try to solve the problem of glucan stability. Chemical modification methods include carboxymethylation and phosphorylation of glucan. Carboxymethylated glucan has been approved for use in cosmetics, which can effectively solve the stability problem of glucan. However, the carboxymethylation process requires the use of sodium hydroxide, chloroacetic acid or its sodium salt, and organic solvents such as ethanol or isopropanol. The characteristics of the carboxymethylation reaction cause this method to cause reagent residues, thereby causing product safety issues. In addition, the degree of carboxymethylation must be strictly controlled. When the degree of carboxymethylation is too high, the activity of glucan will be reduced. The same is true for the characteristics of phosphorylation. Chemical modification often leads to a decrease in the activity of glucan, and there is a risk of solvent residue.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] One object of the present invention is to provide a method for preparing a glucan product, which can solve the problem of poor efficacy and stability of such products during long-term storage.

[0006] Another object of the present invention is to provide a glucan product.

[0007] Another object of the present invention is to provide a use of the above-mentioned glucan product in the preparation of pharmaceutical products and / or cosmetics.

[0008] In order to achieve the above-mentioned object of the present invention, the present invention provides a method for preparing a glucan product on the one hand, wherein a material containing glucan is enzymatically hydrolyzed by an endoenzyme that specifically treats 1,4-glycosidic bonds to obtain a glucan product, wherein the ratio of the number of 1,4-glycosidic bonds to 1,3-glycosidic bonds in the glucan product is less than 1.8.

[0009] The glucan prepared by the present invention using an endonuclease that specifically processes 1,4-glycosidic bonds helps to improve the agglomeration within and between glucan molecules and increase its room temperature stability. The obtained glucan product can be added in large quantities to cosmetics and has a stronger anti-inflammatory effect. When added to cosmetics, it has an outstanding skin soothing effect.

[0010] Another aspect of the present invention provides a glucan product prepared by the above-mentioned method for preparing a glucan product, wherein the ratio of 1,4 glycosidic bonds to 1,3 glycosidic bonds in the glucan product is less than 1.8.

[0011] The glucan product of the present invention has good stability and can be added in large quantities in cosmetics. It also has a stronger anti-inflammatory effect and can be added to cosmetics to promote skin soothing effects.

[0012] Another aspect of the present invention provides a use of the above-mentioned glucan product in the preparation of pharmaceutical products and / or cosmetics.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The glucan prepared by the present invention using an endonuclease that specifically processes 1,4-glycosidic bonds helps to improve the agglomeration within and between glucan molecules and increase its room temperature stability. The obtained glucan product can be added in large quantities to cosmetics and has a stronger anti-inflammatory effect. When added to cosmetics, it has an outstanding skin soothing effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 This is a comparative data diagram showing the anti-inflammatory efficacy of flexible glucan samples in cells according to a specific embodiment of the present invention. DETAILED DESCRIPTION

[0017] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. Those who do not specify specific conditions in the embodiments are carried out according to normal conditions or the conditions recommended by the manufacturer. Those whose reagents or instruments are not specified by the manufacturer are conventional products that can be purchased commercially.

[0018] On one hand, the present invention provides a method for preparing a glucan product, comprising enzymatically hydrolyzing a glucan-containing material with an endoenzyme that specifically treats 1,4-glycosidic bonds to obtain a glucan product, wherein the ratio of 1,4-glycosidic bonds to 1,3-glycosidic bonds in the glucan product is less than 1.8.

[0019] The glucan prepared by the present invention using an endonuclease that specifically processes 1,4-glycosidic bonds helps to improve the agglomeration within and between glucan molecules and increase its room temperature stability. The obtained glucan product can be added in large quantities to cosmetics and has a stronger anti-inflammatory effect. When added to cosmetics, it has an outstanding skin soothing effect.

[0020] In a specific embodiment of the present invention, the glucan-containing material is preferably a material from which the cellulose component has been removed. In the presence of cellulose, endocellulase will preferentially react with cellulose, thereby affecting the action of cellulase on glucan.

[0021] In one embodiment of the present invention, the glucan comprises β-glucan, a natural product consisting of linked glucose molecules and found in a variety of plants and fungi. β-glucan possesses numerous important biological and pharmacological activities and is widely used in medicine, healthcare products, food, and other fields.

[0022] In one embodiment of the present invention, the glucans suitable for use in the present invention may be derived from various sources, including cereal β-glucans. All cereal β-glucans are unbranched, linear, non-starch polysaccharides composed of glucose linked by β-1,3 and β-1,4 glycosidic bonds. Therefore, different cereal β-glucans have similar properties, namely, they all experience instability due to hydrogen bonding.

[0023] In a specific embodiment of the present invention, the glucan includes one or more of oat β-glucan, highland barley β-glucan and barley β-glucan.

[0024] In a specific embodiment of the present invention, the endonuclease that specifically processes 1,4-glycosidic bonds refers to an endonuclease that has an enzymatic effect only on 1,4-glycosidic bonds, but has no enzymatic effect on other types of glycosidic bonds in glucan (such as 1,3-glycosidic bonds, etc.). Various commercially available endonucleases that meet the above requirements can be used, including one or more of cellulases (such as Novozymes composite cellulase, C0057 cellulase, etc.), mannanase and glucanase.

[0025] In a specific embodiment of the present invention, the enzymatic hydrolysis time is 30-60 min, and the enzymatic hydrolysis time is preferably sufficient to fully carry out the enzymatic hydrolysis reaction, for example, it can be exemplified as 30 min, 35 min, 40 min, 44 min, 47 min, 50 min, 56 min, and 60 min.

[0026] In a specific embodiment of the present invention, the enzymatic hydrolysis temperature is 50-60°C, preferably the suitable activity temperature of endocellulose enzyme, such as 50°C, 52°C, 55°C, 56°C, 58°C, and 60°C.

[0027] In a specific embodiment of the present invention, the material containing glucan includes an extract containing glucan. The glucan can be obtained from various sources by conventional extraction or extraction, or can be directly purchased.

[0028] In a preferred embodiment of the present invention, the glucan-containing material is obtained by enzymatically extracting a glucan-containing plant material, or is prepared by dissolving a glucan-containing material.

[0029] In a preferred embodiment of the present invention, the glucan-containing material is obtained by enzymatically extracting a glucan-containing plant material, and one or more combinations of various enzymes disclosed in the prior art for enzymatic hydrolysis of the glucan-containing plant material, such as alkaline protease, can be used. Cellulase can also be used in combination to decompose the cellulose contained in the plant material.

[0030] In a preferred embodiment of the present invention, during the enzymatic extraction, the mass ratio of the plant material containing glucan to water is 1:(10-20), preferably 1:15.

[0031] Different mass ratios of plant raw materials containing glucan and water can be used according to the actual equipment and target product concentration requirements. The requirements for glucan enzymatic hydrolysis reaction can be met within the above-mentioned mass ratio range of the glucan-containing material and water. For example, in different embodiments, the mass ratio of the glucan-containing material to water can be exemplified as 1:10, 1:12.5, 1:14, 1:15, 1:16, 1:18, 1:20, etc.

[0032] In a preferred embodiment of the present invention, when dissolving the raw material containing glucan, the mass ratio of the raw material containing glucan to water is 1:(12-70), preferably 1:14.

[0033] According to the actual equipment and target product concentration requirements, the subsequent enzymatic hydrolysis requirements of glucan can be met within the above-mentioned mass ratio range of the raw material containing glucan to water. For example, in different embodiments, the mass ratio of the raw material containing glucan to water can be exemplified as 1:12, 1:12.5, 1:14, 1:18, 1:20, 1:26, 1:34, 1:37, 1:42, 1:50, 1:53, 1:62, 1:69, 1:70, etc.

[0034] In a preferred embodiment of the present invention, the temperature of the enzymatic extraction is 70-90°C, preferably 80-85°C; the temperature is preferably the suitable activity temperature of the selected enzyme, such as alkaline protease, for example, 70-73°C, 74-76°C, 78-80°C, 81-83°C, 88-90°C, etc.

[0035] In a preferred embodiment of the present invention, the pH of the enzymatic extraction is preferably the pH at which the selected enzyme, such as alkaline protease, is active.

[0036] In a preferred embodiment of the present invention, the temperature for dissolving the raw material containing glucan is 70-90°C, preferably 80-85°C; in order to facilitate the dissolution of the raw material containing glucan, there is no strict temperature requirement, for example, it can be 70-73°C, 74-76°C, 78-80°C, 81-83°C, 88-90°C, etc.

[0037] In a preferred embodiment of the present invention, the plant raw material containing glucan includes plants with a relatively high glucan content, such as cereals, preferably one or more of oats, highland barley and barley.

[0038] In a preferred embodiment of the present invention, the plant raw material containing glucan includes a portion containing more glucan components, such as plant bran.

[0039] In a specific embodiment of the present invention, the preparation method further comprises at least one of decolorization, desalting and protein removal. Other corresponding treatment methods can be selected according to actual needs, such as the needs of subsequent formulation applications.

[0040] In a specific embodiment of the present invention, the decolorization is performed 1-3 times, preferably 2 times.

[0041] In a specific embodiment of the present invention, the total amount of the decolorizing agent used for decolorization is 2%-5% of the total mass of the material to be decolorized, and more preferably 2-3%.

[0042] The total amount of decolorant added can be determined according to the color of the obtained product and the decolorization requirements. The use of conventional decolorants does not affect the properties of glucan. For example, in different embodiments, the total amount of decolorant added for decolorization can be exemplified as 2%, 2.1%, 2.4%, 2.9%, 3.15%, 3.6%, 4.2%, 4.74%, 5%, etc., of the total mass of the material to be decolorized.

[0043] In one embodiment of the present invention, the decolorizing agent comprises activated carbon.

[0044] In a specific embodiment of the present invention, the decolorization time is 30-60 minutes each time.

[0045] For example, in different embodiments, the decolorization time for each decolorization step can be illustratively 30 min, 34 min, 39 min, 43 min, 46 min, 52 min, 57 min, 60 min, etc.

[0046] In a specific embodiment of the present invention, the decolorization temperature is 75-85°C.

[0047] The decolorization temperature can be set according to the requirements of the equipment, production line, decolorization efficiency, etc. The decolorization temperature does not affect the properties of the glucan. For example, in different embodiments, the decolorization temperature can be exemplified as 75-77°C, 76-78°C, 78-80°C, 78.5-81°C, 83-85°C, etc.

[0048] In a specific embodiment of the present invention, the decolorization is performed twice. The first time, 3% of the total mass of the material to be decolorized is added as a decolorant, and the material is decolorized at 80-85°C for 30 minutes. The second time, 2% of the total mass of the material to be decolorized as a decolorant activated carbon is added, and the material is decolorized at 80-85°C for 30 minutes.

[0049] In a specific embodiment of the present invention, anion and cation exchange resins are used for desalination.

[0050] In a specific embodiment of the present invention, the material flow rate during the desalination process is 1-3 t / h. More preferably, the material flow rate during the desalination process is 2-3 t / h.

[0051] The material flow rate during the desalination process can be set according to the requirements of the equipment, production line, desalination efficiency, etc. The material flow rate does not affect the properties of the glucan. For example, in different embodiments, the material flow rate during the desalination process can be exemplified by 1-1.3 t / h, 1.2-1.6 t / h, 1.9-2.4 t / h, 2-2.5 t / h, 2.3-2.7 t / h, 2.8-3 t / h, etc.

[0052] By combining isoelectric point analysis with heat treatment, trace amounts of protein in the sample can be destroyed, denatured and precipitated, and then removed by subsequent filtration steps, thereby improving the appearance and stability of the product as needed. The present invention can also use other protein removal processes as alternatives, such as the Sevage method, the TCA method, and the protease method, depending on product and process requirements.

[0053] In a specific embodiment of the present invention, the obtained product is filtered and then compounded to obtain a glucan product.

[0054] In a specific embodiment of the present invention, one or more of hexylene glycol, pentylene glycol, glycerol and PEHG are used as compounding solvents for compounding.

[0055] In a specific embodiment of the present invention, the amount of the compound solvent can be adjusted as needed. In a preferred specific embodiment of the present invention, the obtained glucan is added with glycerol, hexylene glycol and pentylene glycol, and the glycerol content in the obtained product is 10% of the total mass, the hexylene glycol content is 2% of the total mass, and the pentylene glycol content is 2% of the total mass.

[0056] In a specific embodiment of the present invention, sterilization is performed after compounding.

[0057] Another aspect of the present invention provides a glucan product prepared by the above-mentioned method for preparing a glucan product, wherein the ratio of 1,4 glycosidic bonds to 1,3 glycosidic bonds in the glucan product is less than 1.8.

[0058] Generally, the ratio of 1,4 glycosidic bonds to 1,3 glycosidic bonds in glucan is more than 2-3. The applicant has found that controlling the ratio of 1,4 glycosidic bonds to 1,3 glycosidic bonds in the glucan product can effectively improve the stability of the obtained glucan product. The ratio of 1,4 glycosidic bonds to 1,3 glycosidic bonds in the obtained glucan product can be obtained by selecting different plant raw materials / glucan products of different specifications, controlling the amount of the endonuclease that specifically processes the 1,4 glycosidic bonds, and the enzymatic reaction temperature, time and other related conditions. However, as a glucan product, For example, some 1,4 glycosidic bonds should be retained rather than completely hydrolyzed (to meet the definition of glucan), for example, the ratio of 1,4 glycosidic bonds to 1,3 glycosidic bonds in the obtained glucan product is controlled to be above 0.1. For example, in different embodiments, the ratio of 1,4 glycosidic bonds to 1,3 glycosidic bonds in the glucan product can be exemplified by 1.8, 1.75, 1.72, 1.7, 1.6, 1.5, 1.3, 1.2, 1.0, 0.8, 0.5, 0.3, 0.2, 0.1, etc. It can be expected that the smaller the ratio of 1,4 glycosidic bonds to 1,3 glycosidic bonds in the glucan product, the lower the weight average molecular weight of the obtained glucan product.

[0059] The glucan product of the present invention has good stability and can be added in large quantities in cosmetics. It also has a stronger anti-inflammatory effect and can be added to cosmetics to promote skin soothing effects.

[0060] Another aspect of the present invention provides a use of the above-mentioned glucan product in the preparation of pharmaceutical products and / or cosmetics.

[0061] Example 1

[0062] Preparation of enzymatic oat β-glucan, the specific steps are as follows:

[0063] 5 kg of oat bran was added to 75 kg of water (1:15), and NaOH was added to adjust the pH to 10-11. Then, 0.135% of the total mass of oat bran and water was added with alkaline protease (Alcalase (Novozymes)), and the mixture was extracted at 55° C. for 60 min. The temperature was then raised to 85° C. and kept for 2 h to obtain solution A.

[0064] Add citric acid to solution A to adjust the pH to 4.0-4.5, add 0.02% of the total mass of solution A to α-thermostable amylase, add 1% of the total mass of solution A to decolorize (first decolorization) for 1 hour, and then let it stand for 12-18 hours;

[0065] After aging, the supernatant was extracted for a second decolorization, activated carbon was added at 2% of the total mass of the supernatant, and decolorization was carried out at 80-85°C for 30 minutes, and then filtered to obtain solution B;

[0066] Solution B was passed through an anion and cation chromatography column at a flow rate of 3.0 t / h for desalination, and then the pH was adjusted to 4.64 to obtain solution C;

[0067] Solution C was added with 0.04% of the total mass of Solution C of Novozymes composite endocellulose enzyme (Celluclast 1.5L), kept at 50°C for 30 minutes, and then concentrated under reduced pressure to a β-glucan mass fraction of 1%, to obtain Solution D;

[0068] Ethylene glycol and pentylene glycol were added to solution D, and the obtained product contained 2% ethylene glycol and 2% pentylene glycol of the total mass, and then sterilized at 100° C. for 30 minutes to obtain an enzymatically hydrolyzed oat β-glucan.

[0069] Example 2

[0070] Preparation of enzymatic oat β-glucan, the specific steps are as follows:

[0071] To 20 kg of a commercially available oat β-glucan aqueous solution with a β-glucan mass fraction of 1% (provided by Beijing Oriental Miaosen Biotechnology Co., Ltd., NW-01-1008), 0.02% of the total mass of the oat β-glucan aqueous solution was added endocellulase (Sigma COO57), and enzymatic hydrolysis was performed at 55° C. for 1 h to obtain an enzymatically hydrolyzed oat β-glucan.

[0072] Examples 3, 4, and 5

[0073] Preparation of enzymatic oat β-glucan, the specific steps are as follows:

[0074] 20 kg of commercially available oat β-glucan aqueous solution with a β-glucan mass fraction of 1% was added with 0.025% (Example 3), 0.03% (Example 4), and 0.05% (Example 5) of Novozymes composite cellulase (Celluclast 1.5L) of the total mass of the oat β-glucan aqueous solution, respectively, and enzymatic hydrolysis was performed at 60° C. for 30 min to obtain an enzymatically hydrolyzed oat β-glucan.

[0075] Example 6

[0076] Preparation of enzymatically hydrolyzed highland barley β-glucan, the specific steps are as follows:

[0077] 5 kg of crushed highland barley bran was added to 75 kg of water (1:15), and NaOH was added to adjust the pH to 10-11. Then, alkaline protease (Alcalase (Novozymes)) at a concentration of 0.135% of the total mass of highland barley bran and water was added. The mixture was extracted at 55° C. for 60 min, and the temperature was continuously raised to 85° C. and kept warm for 2 h to obtain solution A.

[0078] Add citric acid to solution A to adjust the pH to 4.0-4.5, add 0.02% of the total mass of solution A to α-thermostable amylase, add 1% of the total mass of solution A to decolorize (first decolorization) for 1 hour, and then let it stand for 12-18 hours;

[0079] After aging, the supernatant was extracted for a second decolorization, activated carbon was added at 2% of the total mass of the supernatant, and decolorization was carried out at 80-85°C for 30 minutes, and then filtered to obtain solution B;

[0080] Solution B was passed through an anion and cation chromatography column at a flow rate of 3.0 TPH for desalination, and then the pH was adjusted to 4.64 to obtain solution C;

[0081] Solution C was added with 0.025% of the total mass of Solution C of Novozymes complex cellulase (Celluclast 1.5 L), kept at 50° C. for 30 min, and then concentrated under reduced pressure to a β-glucan mass fraction of 1%, to obtain Solution D;

[0082] Ethylene glycol and pentylene glycol are added to solution D, and the content of ethylene glycol in the obtained product is 2% of the total mass and the content of pentylene glycol is 2% of the total mass. The product is then sterilized at 100° C. for 30 minutes to obtain an enzymatically hydrolyzed highland barley β-glucan.

[0083] Example 7

[0084] Preparation of enzymatically hydrolyzed barley β-glucan, the specific steps are as follows:

[0085] 5 kg of barley bran was added to 75 kg of water (1:15), and NaOH was added to adjust the pH to 10-11. Then, 0.135% of the total mass of barley bran and water was added with alkaline protease (Alcalase (Novozymes)), and the mixture was extracted at 55° C. for 60 min. The temperature was then raised to 85° C. and kept for 2 h to obtain solution A.

[0086] Add citric acid to solution A to adjust the pH to 4.0-4.5, add 0.02% of the total mass of solution A to α-thermostable amylase, add 1% of the total mass of solution A to decolorize (first decolorization) for 1 hour, and then let it stand for 12-18 hours;

[0087] After aging, the supernatant was extracted for a second decolorization, activated carbon was added at 2% of the total mass of the supernatant, and decolorization was carried out at 80-85°C for 30 minutes, and then filtered to obtain solution B;

[0088] Solution B was passed through an anion and cation chromatography column at a flow rate of 3.0 TPH for desalination, and then the pH was adjusted to 4.64 to obtain solution C;

[0089] Solution C was added with 0.005% of the total mass of solution C of Novozymes composite cellulase (Celluclast 1.5L), kept at 50°C for 30 minutes, and then concentrated under reduced pressure to a β-glucan mass fraction of 1% to obtain solution D;

[0090] Ethylene glycol and pentylene glycol were added to solution D, and the obtained product had an ethylene glycol content of 2% and a pentylene glycol content of 2% of the total mass, and then sterilized at 100° C. for 30 min to obtain barley β-glucan.

[0091] Comparative Example 1

[0092] 5 kg of oat bran was added to 75 kg of water (1:15), and NaOH was added to adjust the pH to 10-11. Then, 0.135% of the total mass of oat bran and water was added with alkaline protease (Alcalase (Novozymes)), and the mixture was extracted at 55° C. for 60 min. The temperature was then raised to 85° C. and kept for 2 h to obtain solution A.

[0093] Add citric acid to solution A to adjust the pH to 4.0-4.5, add 0.02% of the total mass of solution A to α-thermostable amylase, add 1% of the total mass of solution A to decolorize (first decolorization) for 1 hour, and then let it stand for 12-18 hours;

[0094] After aging, the supernatant was extracted for a second decolorization, activated carbon was added at 2% of the total mass of the supernatant, and decolorization was carried out at 80-85°C for 30 minutes, and then filtered to obtain solution B;

[0095] Solution B was passed through an anion and cation chromatography column at a flow rate of 3.0 t / h for desalination, and then the pH was adjusted to 4.64 to obtain solution C;

[0096] Solution C was concentrated under reduced pressure until the mass fraction of β-glucan was 1% to obtain solution D;

[0097] Ethylene glycol and pentylene glycol were added to solution D, and the obtained product had an ethylene glycol content of 2% and a pentylene glycol content of 2% of the total mass, and then sterilized at 100° C. for 30 minutes to obtain oat β-glucan.

[0098] Comparative Example 2

[0099] Preparation of oat β-glucan, the specific steps are as follows:

[0100] A cosmetic grade oat β-glucan aqueous solution with a mass fraction of 1% (provided by Beijing Dongfang Miaosen Biotechnology Co., Ltd., NW-01-1008) was purchased from the market as Comparative Example 2.

[0101] Comparative Example 3

[0102] Preparation of highland barley β-glucan, the specific steps are as follows:

[0103] 5 kg of crushed highland barley bran was added to 75 kg of water (1:15), and NaOH was added to adjust the pH to 10-11. Then, alkaline protease (Alcalase (Novozymes)) at a concentration of 0.135% of the total mass of highland barley bran and water was added. The mixture was extracted at 55° C. for 60 min, and the temperature was continuously raised to 85° C. and kept warm for 2 h to obtain solution A.

[0104] Add citric acid to solution A to adjust the pH to 4.0-4.5, add 0.02% of the total mass of solution A to α-thermostable amylase, add 1% of the total mass of solution A to decolorize (first decolorization) for 1 hour, and then let it stand for 12-18 hours;

[0105] After aging, the supernatant was extracted for a second decolorization, activated carbon was added at 2% of the total mass of the supernatant, and decolorization was carried out at 80-85°C for 30 minutes, and then filtered to obtain solution B;

[0106] Solution B was passed through an anion and cation chromatography column at a flow rate of 3.0 TPH for desalination, and then the pH was adjusted to 4.64 to obtain solution C;

[0107] Concentrate under reduced pressure until the mass fraction of β-glucan is 1% to obtain solution D;

[0108] Ethylene glycol and pentylene glycol are added to solution D, and the content of ethylene glycol in the obtained product is 2% of the total mass and the content of pentylene glycol is 2% of the total mass. The product is then sterilized at 100° C. for 30 minutes to obtain an enzymatically hydrolyzed highland barley β-glucan.

[0109] Comparative Example 4

[0110] Preparation of enzymatically hydrolyzed barley β-glucan, the specific steps are as follows:

[0111] 5 kg of barley bran was added to 75 kg of water (1:15), and NaOH was added to adjust the pH to 10-11. Then, 0.135% of the total mass of barley bran and water was added with alkaline protease (Alcalase (Novozymes)), and the mixture was extracted at 55° C. for 60 min. The temperature was then raised to 85° C. and kept for 2 h to obtain solution A.

[0112] Add citric acid to solution A to adjust the pH to 4.0-4.5, add 0.02% of the total mass of solution A to α-thermostable amylase, add 1% of the total mass of solution A to decolorize (first decolorization) for 1 hour, and then let it stand for 12-18 hours;

[0113] After aging, the supernatant was extracted for a second decolorization, activated carbon was added at 2% of the total mass of the supernatant, and decolorization was carried out at 80-85°C for 30 minutes, and then filtered to obtain solution B;

[0114] Solution B was passed through an anion and cation chromatography column at a flow rate of 3.0 TPH for desalination, and then the pH was adjusted to 4.64 to obtain solution C;

[0115] Then, the solution was concentrated under reduced pressure until the mass fraction of β-glucan was 1%, thereby obtaining solution D;

[0116] Ethylene glycol and pentylene glycol were added to solution D, and the obtained product had an ethylene glycol content of 2% and a pentylene glycol content of 2% of the total mass, and then sterilized at 100° C. for 30 min to obtain barley β-glucan.

[0117] Performance Evaluation

[0118] The physical and chemical indicators of the samples prepared in Examples 1-7 and Comparative Examples 1-4 were tested, and the results are shown in Table 1:

[0119] Physical and chemical index testing methods:

[0120] Chroma: A platinum cobalt colorimeter (HI96727) was used for colorimetry. Ultrapure water was first used for zeroing, and then samples of the products of each embodiment / comparative example were added for colorimetry testing.

[0121] Turbidity: An ISO portable turbidimeter (HI98713-01) was used for turbidity detection. A standard turbidity sample was used for calibration before use. Then, samples of the products of each embodiment / comparative example were added to the instrument for turbidity testing.

[0122] Table 1 Physical and chemical index test data

[0123] Serial number Sample name Chroma Turbidity 1 Example 1 67 2.56 2 Example 2 42 7.32 3 Example 3 89 4.89 4 Example 4 23 9.11 5 Example 5 91 6.04 6 Example 6 106 3.77 7 Example 7 34 1.23 8 Comparative Example 1 78 8.50 9 Comparative Example 2 95 5.98 10 Comparative Example 3 140 7.22 11 Comparative Example 4 49 2.17

[0124] The stability of the samples prepared in Examples 1-7 and Comparative Examples 1-4 was observed, and the results are shown in Table 2:

[0125] Stability observation method:

[0126] The samples were placed at room temperature in the dark (25°C ± 2°C, 60% RH ± 10% RH) and observed every 30 days using white light in a small high-definition studio (Chunying). If the samples showed precipitation or gelation, they were recorded as unstable. The number of stable days was then counted and recorded.

[0127] Table 2 Stability observation data table

[0128]

[0129]

[0130] It can be seen from Table 1 and Table 2 that in fact, the glucan prepared by the method of the present application may not have a relatively obvious difference from the glucan of general origin only in terms of chromaticity and turbidity, but the glucan prepared by the method of the present application has a high stability that is unmatched by glucans from other sources in terms of further stability. Compared with Comparative Examples 1-4, Examples 1-7 all passed the freeze-thaw cycle test (15 cycles, 30 days), and the shape of each sample group did not change, and it remained a uniform solution; each group of Comparative Examples 1-4 showed different degrees of precipitation under the freeze-thaw cycle conditions, among which Comparative Examples 1, 2, and 3 showed white to light yellow gel at the bottom, and Comparative Example 4 showed white gel and granular precipitate. (Note: The freeze-thaw cycle is -18°C 24h, 45°C 24h, and one cycle is 2 days. This method is used to accelerate the evaluation of cereal glucan gel stability). It is proved that the present invention can effectively improve the stability of glucan (freeze-thaw and room temperature conditions).

[0131] Structural testing (polysaccharide methylation) was performed on Example 1 and Comparative Example 1. The results are shown in Table 3:

[0132] Methylation method:

[0133] 1. Sample pretreatment for polysaccharide methylation detection

[0134] (1) Take a small amount of sample (3 mg) and dissolve it in 500 μL DMSO.

[0135] (2) Add 1 mg of NaOH and incubate for 30 minutes.

[0136] (3) Add 50 μL of iodomethane solution and react for 1 h.

[0137] (4) Add 1 mL of water and 2 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the water wash three times.

[0138] (5) Aspirate the lower dichloromethane phase and blow dry with nitrogen.

[0139] (6) Add 100 μL of 2M TFA (trifluoroacetic acid) and react at 121°C for 90 min.

[0140] (7) Evaporate to dryness at 30°C.

[0141] (8) Add 50 μL of 2M ammonia water and 50 μL of 1M NaBD4, mix well, and react at room temperature for 2.5 h.

[0142] (9) Add 20 μL of acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250 μL of methanol, and blow dry with nitrogen.

[0143] (10) Add 250 μL of acetic anhydride, vortex mix, and react at 100 °C for 2.5 h.

[0144] (11) Add 1 mL of water and let stand for 10 minutes.

[0145] (12) Add 500 μL of dichloromethane, vortex to mix, centrifuge, discard the aqueous phase, and repeat the water washing three times.

[0146] (13) The lower dichloromethane phase was removed and tested by GC-MS.

[0147] Detection method:

[0148] The analytical instrument used in this experiment was an Agilent Technologies Inc. (CA, USA) 6890A-5977B gas chromatography-mass spectrometer, and the autosampler model was G4567A.

[0149] An Agilent gas chromatograph (Agilent 6890A; Agilent Technologies, USA) was used, using a TG-200 column (30 m × 0.25 mm × 0.25 μm, SGE, Australia). The injection volume was 1 μL, the split ratio was 10:1, and the carrier gas was high-purity helium at a flow rate of 1.5 mL / min. The column oven temperature was initially set at 150°C for 2.0 min, then programmed to 210°C at 2°C / min, held for 3 min, and then to 240°C at 2°C / min, held for 5 min.

[0150] The mass spectrometry system used was an Aiglent quadrupole mass spectrometry system (Agilent 5977B; Agilent Technologies, USA), equipped with an electron impact ionization (EI) source and a MassHunter workstation. Analytes were detected in full scan (SCAN) mode using the EI source temperature of 200°C, the MS quadrupole temperature of 110°C, the ionization energy of 50 eV, the transfer line temperature of 210°C, and the mass scan range (m / z) of 50–350.

[0151] Data Analysis:

[0152] Under the combined action of the mass analyzer's electric and magnetic fields, the fragments are separated by m / z, detected, and recorded, yielding characteristic fragments of polysaccharide methylation. These fragments are then compared against existing databases to confirm their bonding patterns. This project primarily calculates the relative molar weight of the compound by calculating the ratio of the chromatographic peak area to the weight-average molecular weight of the corresponding derivative, thereby approximating the relative molar ratio of the various glycosidic bonds in the sample.

[0153] Table 3 Polysaccharide methylation structure detection data table

[0154]

[0155]

[0156] The results show that both Example 1 and Comparative Example 1 are cereal β-glucans linked by β-1,3 glycosidic bonds and β-1,4 glycosidic bonds, wherein the molar ratio of β-1,4 glycosidic bonds to β-1,3 glycosidic bonds in Comparative Example 1 is 2.58, and the molar ratio of β-1,4 glycosidic bonds to β-1,3 glycosidic bonds in Example 1 is 1.72, which proves that the method of the present invention can effectively reduce the molar ratio of β-1,4 glycosidic bonds to β-1,3 glycosidic bonds in β-glucan, and combined with the stability test results, it can be seen that this can effectively improve the stability of the β-glucan sample.

[0157] The cellular anti-inflammatory efficacy of the enzymatically hydrolyzed cereal glucans obtained in Examples 1, 6, 7 and Comparative Examples 1, 3, 4 was characterized.

[0158] RAW264.7 cells were cultured at a cell density of 3*10 5 Cells were inoculated into 24-well plates and incubated in a CO2 cell culture incubator overnight. The next day, samples of the test examples / comparative example products were dissolved in high-glucose DMEM medium (gibio, C11965500BT), and the cells were treated with 0.25% (mass fraction) of the test solution of different examples and comparative example products, respectively, and incubated for 24 hours. On the third day, 10 ng / mL of LPS (lipopolysaccharide) was added to induce inflammation and incubated for 24 hours. On the fourth day, the supernatant of each group was tested for inflammatory factors according to the TNF-α ELISA kit.

[0159] The experimental results are as follows Figure 1 As shown, TNF-α is a pro-inflammatory cytokine secreted by immune cells (such as macrophages and T cells). It plays a key regulatory role in inflammatory responses and is closely related to skin irritation and sensitivity. Highland barley, oat, and barley glucans treated with this patented process significantly enhance their ability to scavenge TNF-α, demonstrating that this patented process can enhance the anti-inflammatory and irritation-relieving effects of cereal glucans.

[0160] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a glucan product, characterized in that: The glucan-containing material is enzymatically hydrolyzed by an endoenzyme that specifically treats 1,4-glycosidic bonds to obtain a glucan product, wherein the ratio of 1,4-glycosidic bonds to 1,3-glycosidic bonds in the glucan product is less than 1.

8.

2. The method for preparing a glucan product according to claim 1, wherein: The glucan includes β-glucan; Preferably, the glucan comprises cereal beta-glucan; Preferably, the glucan comprises one or more of oat β-glucan, highland barley β-glucan and barley β-glucan; Preferably, the endoenzyme specifically processing 1,4-glycosidic bonds includes one or more of endocellulase, mannanase and glucanase; Preferably, the enzymatic hydrolysis time is 30-60 min; Preferably, the enzymatic hydrolysis temperature is 50-60°C.

3. The method for preparing a glucan product according to claim 1, wherein: The glucan-containing material is obtained by enzymatically extracting a glucan-containing plant material, or by dissolving a glucan-containing material; Preferably, the enzymatic extraction is performed using alkaline protease; Preferably, during the enzymatic extraction, the mass ratio of the plant material containing glucan to water is 1:(10-20), preferably 1:15; Preferably, when dissolving the raw material containing glucan, the mass ratio of the raw material containing glucan to water is 1:(12-70), preferably 1:14; Preferably, the temperature of the enzymatic extraction is 70-90°C, preferably 80-85°C; Preferably, the temperature for dissolving the raw material containing glucan is 70-90°C, preferably 80-85°C.

4. The method for preparing a glucan product according to claim 3, wherein: The plant material containing glucan includes cereals, preferably one or more of oats, highland barley and barley; Preferably, the plant material containing glucan comprises plant bran.

5. The method for preparing a glucan product according to claim 3, wherein: The preparation method further comprises at least one of decolorization, desalting and protein removal.

6. The method for preparing a glucan product according to claim 5, characterized in that: The number of decolorization is 1-3 times, preferably 2 times; Preferably, the total amount of the decolorizing agent added for decolorization is 2%-5% of the total mass of the material to be decolorized, more preferably 2-3%; Preferably, the decolorizing agent comprises activated carbon; Preferably, the decolorization time is 30-60 minutes per decolorization; Preferably, the temperature of each decolorization step is 75-85°C; Further preferably, the decolorization is performed twice, the first time 3% of the total mass of the material to be decolorized is added as a decolorant, and the temperature is 80-85° C. for 30 minutes, and the second time 2% of the total mass of the material to be decolorized as a decolorant activated carbon is added, and the temperature is 80-85° C. for 30 minutes; Preferably, desalination is performed using anion and cation exchange resins; Preferably, the material flow rate during the desalination process is 1-3 t / h, and further preferably, the material flow rate during the desalination process is 2-3 t / h.

7. The method for preparing a glucan product according to claim 5 or 6, characterized in that: The obtained product is filtered and then compounded to obtain a glucan product; Preferably, one or more of hexanediol, pentanediol, glycerol and PEHG are used as compounding solvents for compounding; Preferably, sterilization is performed after compounding.

8. A glucan product, characterized in that: The glucan product is prepared by the preparation method of any one of claims 1 to 7, wherein the ratio of 1,4 glycosidic bonds to 1,3 glycosidic bonds in the glucan product is less than 1.

8.

9. A glucan product prepared by the method for preparing a glucan product according to any one of claims 1 to 7, or use of the glucan product according to claim 8 in the preparation of pharmaceutical products and / or cosmetics.

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