Method for preparing high-purity yeast β-glucan

Abstract

A method for preparing a high-purity yeast β-glucan, comprising the following steps: treating a yeast cell wall mixed liquid by adding amylase at a certain temperature, then adjusting the pH, and treating the mixed liquid by adding lipase at a certain temperature; adding an alkali and a nonionic surfactant, carrying out heating and thermal insulation treatment, and cooling to obtain an alkali-treated liquid; washing and separating the alkali-treated liquid, adding water to a precipitate, then adding an acid to adjust the pH to 3-5, carrying out heating and thermal insulation treatment to obtain an acid-treated liquid, and performing separation, washing, and drying on the acid-treated liquid to obtain a yeast β-glucan product; and subjecting the yeast β-glucan to a combined action of a mixed enzyme preparation and ultrasound to obtain an enzymatic hydrolysate, and performing separation treatment on the enzymatic hydrolysate to obtain an insoluble yeast β-glucan and a water-soluble yeast β-glucan, respectively. In the present invention, a high-purity yeast β-glucan product having a content of 90% or more and a water-soluble yeast β-glucan product with biological activity can be prepared, the preparation method is simple, large-scale production can be achieved, and the quality and additional value of the product can be improved.

Description

A method for preparing high-purity yeast beta-glucan Technical Field

[0001] The present invention relates to the technical field of food manufacturing, and in particular to a method for preparing high-purity yeast beta-glucan. Background Art

[0002] Yeast β-glucan, a polysaccharide found in yeast cell walls, has numerous physiological benefits, including immune enhancement, radiation protection, anti-allergy, and intestinal health. The glucans naturally present in yeast cell walls are mostly water-insoluble, accounting for approximately 85%. Once in the intestines, yeast β-glucan is absorbed into the immune system through endocytosis by intestinal epithelial cells, demonstrating its effectiveness in enhancing immunity, improving intestinal function, and protecting against radiation in both animals and humans. Its mechanism of action involves stimulating and activating immune cells such as macrophages and NK cells, leading to the release of cytokines that directly or indirectly kill harmful particles such as tumor cells and antigens. Furthermore, due to its excellent adsorption capacity, it can absorb a large number of toxins in the intestine, enhance intestinal motility, and improve intestinal function. Yeast β-glucan has been approved as a new food ingredient by the US FDA, the EU, China, and other countries and regions, requiring the product to contain at least 70% β-glucan.

[0003] Yeast cell walls are the main source of yeast β-glucan. However, in addition to yeast β-glucan, yeast cell walls also contain a certain amount of mannan, protein and crude fat. In traditional yeast β-glucan preparation methods, common extraction methods such as acid-base method, enzymatic method, ultrasonic method, high-pressure homogenization method, etc. are often difficult to effectively remove the protein and lipid in yeast β-glucan, resulting in a low purity of yeast β-glucan products, generally around 70%.

[0004] To obtain yeast β-glucan products with higher content, methods such as enzymatic methods and organic solvent washing are generally used, but these methods are complex and can lead to residual organic solvents. For example, prior art CN101463373A discloses a method for preparing high-purity yeast immunoreactive β-1,3-glucan. This method involves subjecting the yeast raw material to autolysis pretreatment, followed by enzymatic hydrolysis, centrifugal filtration to collect the precipitate, and then treating it with alkali, followed by acid neutralization, washing, dehydration, drying, and pulverization to obtain the high-purity yeast immunoreactive β-1,3-glucan product. CN101748172A discloses a method for preparing insoluble glucan, which is carried out according to the following steps: 1) suspending a crude glucan product from yeast in 100 mL of a phosphate buffer solution with a pH of 8.0, adding Alcalase protease to hydrolyze the product at 40-60° C., centrifuging, and collecting a precipitate; 2) adding anhydrous ethanol to the precipitate obtained in step 1), stirring the product, and then filtering the product and vacuum drying the product; 3) stirring the product obtained in step 2) in a NaOH solution with a mass fraction concentration of 1%-3% at 60-100° C. for 3-8 hours, and centrifuging the product to collect the product; 4) washing the precipitate obtained by centrifugation after alkali treatment in step 3) with anhydrous ethanol, filtering the product, and vacuum drying the product to obtain an insoluble glucan with a β-1,3-D main chain and a β-1,6-D side chain. CN105925640B discloses a method for preparing yeast β-glucan, comprising the following steps: (1) adding 5-15 parts by weight of brewer's yeast to 30-90 parts by weight of enzyme solution, adjusting the pH to 5-10, inducing autolysis in a water bath at 45-65°C for 10-30 hours, and then inactivating the enzyme in a water bath at 90-100°C for 5-15 minutes to obtain an enzymatic hydrolyzate; (2) centrifuging the enzymatic hydrolyzate and washing with water 2-4 times to obtain a precipitate; (3) adding the precipitate to 10-30 parts by weight of acid solution, acid-hydrolyzing at 65-125°C for 1-5 hours, centrifuging, washing with water 1-3 times to obtain an acid hydrolyzate; and (4) washing the acid hydrolyzate with ethanol 1-3 times, filtering, and drying to obtain the product. However, these existing technologies all have some problems: (1) The extraction method mainly involves simple acid-base treatment, and the final product has a low glucan content; (2) The use of organic solvents and other processes to increase the glucan content is cumbersome and the problem of organic solvent residue is difficult to control; (3) Some processes are not suitable for large-scale production, have poor stability, and low yield.

[0005] Furthermore, yeast β-glucan is poorly soluble in water due to the hydrogen bonding between the multiple hydroxyl groups on the molecular chain, forming a dense triple helix structure. Current applications of yeast β-glucan are primarily focused on animal feed, with commercially available products mostly in the form of tablets and solid beverages, resulting in a relatively narrow product range. Therefore, modification and solubilization of yeast β-glucan are needed to expand its application. Common modification methods include chemical, physical, and biological methods. Conventional chemical methods primarily increase water solubility by introducing hydrophilic groups such as sulfate, carboxylic acid, and phosphate groups into the molecular chain. However, while these methods improve the solubility of yeast β-glucan, they also alter the native conformation of the glucan molecule, thereby affecting its physiological activity. Furthermore, chemical methods are costly and highly polluting. While purely physical methods are relatively low-cost, simple, and low-pollution, they have limited effectiveness in degrading high-molecular-weight chains. Biological methods primarily rely on enzymatic hydrolysis, where enzymes moderately degrade the substrate, reducing its molecular weight and thereby improving its solubility. Degrading higher-molecular-weight polysaccharides to lower molecular weights can significantly enhance their activity. However, the molecular weight range is closely related to the function of glucan. If the molecular weight is too low, it will be difficult for glucan to form an active molecular chain aggregation structure, thus losing its biological activity. Therefore, controlling the degree of degradation is also the key to water-soluble modification. Summary of the Invention

[0006] The purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art and provide a method for preparing high-purity yeast β-glucan by combining multiple enzyme treatments, solubilizer-assisted alkali treatment, and acid treatment. The yeast β-glucan obtained by this method has high purity.

[0007] The present invention is achieved through the following technical solutions:

[0008] A method for preparing high-purity yeast β-glucan comprises the following steps:

[0009] S1. Amylase Treatment: Yeast cell walls are mixed with water to a concentration of 2%-10% by weight. Amylase is added at 60-100°C and incubated for 0.5-5 hours to obtain the first enzymatic hydrolyzate. The weight of amylase should be 0.1%-2% of the milliliters of the mixture. Amylase treatment removes glycogen and increases the permeability of the yeast cell walls, improving subsequent extraction efficiency.

[0010] S2. Lipase Treatment: Adjust the pH of the first hydrolyzate to 5.0-9.0, add lipase at 30-60°C, and incubate for 1-4 hours to produce a second hydrolyzate. The lipase concentration should be 0.05%-1% of the first hydrolyzate. Lipase catalyzes ester hydrolysis or alcoholysis, hydrolyzing residual fats, phospholipids, and other components in the yeast cell walls, thereby facilitating their dissolution and removal.

[0011] S3. Solubilizer-assisted alkali treatment: Add an alkali and a nonionic surfactant to the second enzymatic hydrolysate, heat to 80-100°C, and maintain with constant stirring for 1-4 hours. Then cool to 50-60°C to obtain an alkali-treated solution. The alkali concentration (in grams) should be 1%-4% of the second enzymatic hydrolysate by weight, and the nonionic surfactant should be 0.1%-5% of the second enzymatic hydrolysate by weight. The solubilizer (nonionic surfactant) and alkali treatment together promote the dissolution of lipids, proteins, and other components in the yeast cell wall.

[0012] S4. Separation: The alkali-treated solution is subjected to solid-liquid separation. The precipitate is collected and washed with water. The separation and washing steps are repeated 2-5 times. Yeast glucan is an alkali-insoluble component. Solid-liquid separation can remove dissolved impurities such as proteins, lipids, and polysaccharides.

[0013] S5. Acid treatment: The precipitate obtained in step S4 is mixed with water to form a 3%-8% mixture. Acid is then added to adjust the pH to 3-5. The mixture is heated to 80-90°C and maintained for 0.5-2 hours with constant stirring. The mixture is then cooled to 50-60°C to obtain an acid-treated solution. The acid treatment further dissolves glycogen in the yeast cell walls.

[0014] S6, separation: the acid-treated liquid is subjected to solid-liquid separation, the precipitate is collected and washed with water, and the separation and washing steps are repeated 2-5 times;

[0015] S7. Dry the precipitate obtained in step S6 to obtain a powdered yeast β-glucan product. This yeast β-glucan product can be packaged in a variety of formats. The resulting yeast β-glucan product has a purity exceeding 90%, a glycogen content ≤ 2%, and a β-1,3-glycosidic bond ratio ≥ 80%. Due to the importance of the β-1,3-glycosidic bonded glucan backbone to its immunopotentiating activity, and the essential β-1,3-glycosidic bonded backbone structure, increasing the β-1,3-glycosidic bond ratio is beneficial for improving the efficacy and quality of the yeast β-glucan product.

[0016] Furthermore, the method further includes a water-soluble modification step, the specific steps of which are as follows:

[0017] S8. Add water to the yeast β-glucan obtained in step S7 to form a glucan mixture with a mass concentration of 2%-10%. The pH is adjusted to 4.0-8.0, and then a mixed enzyme preparation is added. The mixture is placed in an ultrasonic thermostat, ultrasonically activated, and heated to 40-75°C and then incubated for 20-100 minutes to obtain an enzymatic hydrolyzate. The weight of the mixed enzyme preparation is 0.1%-0.5% of the milliliters of the glucan mixture. The yeast β-glucan product with a purity of over 90% is subjected to water-soluble modification. The raw material has a low impurity content, the modification method is simple, and other impurities introduced during the modification step are reduced, which greatly simplifies post-processing steps.

[0018] S9. The enzymatic hydrolyzate obtained in step S8 is heated to 100° C., incubated for at least 5 minutes to inactivate the enzyme, cooled to room temperature, and subjected to solid-liquid separation. The separated precipitate and clear liquid are collected. Enzyme inactivation prevents further degradation of the β-glucan by the enzyme, thereby preventing the molecular weight of the β-glucan from being too low and the molecular structure from changing, which could affect its efficacy.

[0019] S10, washing the precipitate obtained in step S9 with water for 2-3 times, and drying to obtain insoluble yeast β-glucan. The insoluble yeast β-glucan can be used in the fields of food, feed, medicine, etc. according to conventional uses.

[0020] S11. Adjust the pH of the clear solution obtained in step S9 to 5.0-8.0 and filter (if necessary, the clear solution may be decolorized with activated carbon). Concentrate the filtered clear solution to 10%-30% of its original volume, add 50%-100% of the concentrated volume of ethanol to precipitate, and dry the precipitated product to obtain water-soluble yeast β-glucan. The water-soluble yeast β-glucan can be used in food, medicine, health products, cosmetics, and other fields, fully utilizing its biological activity and efficacy to further increase its added value.

[0021] Further preferably, the mixed enzyme preparation is a mixture of mannanase and β-glucanase in a mass ratio of 1:(2-5), and the β-glucanase is one or more of β-1,3-glucanase, β-1,4-glucanase, and β-(1,3-1,4)-glucanase, preferably containing at least β-1,3-glucanase.

[0022] β-glucanase belongs to the class of hydrolases. Research has shown that β-glucanase can degrade or break down glucans. β-1,3-1,4-glucanase is a type of endoglycoside hydrolase that can degrade high-molecular-weight glucans. It specifically acts on the β-1,4-glycosidic bond next to the β-1,3-glycosidic bond. β-1,4-glucanase can randomly cleave the β-1,4-glycosidic bond in β-glucans. β-1,3-glucanase is highly specific and can act specifically on the β-1,3-glycosidic bond, randomly cleaving the β-1,3-glycosidic bond in β-glucans, reducing the molecular weight of β-glucans. Mannanase is also a type of hydrolase and can be used in the production of functional oligosaccharides. Yeast β-glucan has a high molecular weight, and the sugar chains on the molecular chain have various structures. The structure and content often vary greatly due to differences in raw materials and preparation methods. Various types of enzymes have specificity. Therefore, the present invention specifically uses a combination of multiple enzymes for treatment, and combines it with an ultrasonic treatment method to improve the effect of enzyme treatment. On the one hand, ultrasound can promote the dispersion of materials and the probability of contact between them, thereby improving the efficiency of enzymatic hydrolysis. On the other hand, the cavitation effect of ultrasound can shear long molecular chains, which is conducive to the degradation of high molecular chains. The advantage of this is that the amount of enzyme preparation can be reduced, reducing costs. At the same time, due to the uniformity of the reaction, the reaction is more controllable and the product molecular weight distribution is narrower, avoiding excessive degradation of glucan molecules due to uneven reaction, thereby affecting the efficacy and quality of the product. Studies have shown that for water-soluble yeast β-glucan, only those with a relative molecular mass of 90,000 or above can form a triple helical structure. Only glucans with a triple helical structure have the immunological activity of polysaccharides.

[0023] Enzyme concentration, enzymatic hydrolysis temperature, pH, time, ultrasonic frequency, power and other conditions are important factors affecting the degradation reaction, which are directly related to the final molecular weight, molecular weight distribution and structure of yeast β-glucan, thereby affecting the yield and biological activity of the final water-soluble product. It is not possible to simply pursue the yield. A high yield often requires a reduction in biological activity. Therefore, it is necessary to strike a balance between the two under certain conditions. Further optimization, the ultrasonic frequency of the ultrasonic constant temperature device is 20-100kHz, and the ultrasonic power is 100-1000W. The filtration in step S11 is performed using diatomaceous earth with a mesh size of 100 or more.

[0024] Furthermore, the amylase in step S1 is α-amylase or β-amylase.

[0025] Further, the lipase in the S2 step is a carboxylesterase or / and a phospholipase. Lipase (Lipase, glycerol ester hydrolase) belongs to the carboxyl ester hydrolase class, can progressively hydrolyze triglyceride into glycerol and fatty acid, and it comprises phosphatase, sterolase and carboxylesterase. Phospholipase, a class of enzyme that can hydrolyze glycerophospholipids existing in organism, comprises phospholipase A1, A2, B, C and D, and they act specifically on each ester bond inside the phospholipid molecule, form different products. The present invention adopts lipase to process yeast cell wall, because the inventor finds in research, the lipid in yeast is mainly lipid, and after research discovery, compared with common protease, saccharifying enzyme, mannanase etc., lipase is more effective to the hydrolysis of this type of lipid.

[0026] Furthermore, the base in step S3 is NaOH, KOH or NaCO3, and the non-ionic surfactant is a polyoxyethylene non-ionic surfactant, such as emulsifier OP series, emulsifier TX series, emulsifier SOPE series, emulsifier MOA series, emulsifier E1300 series, emulsifier O series, Tween series, etc.

[0027] Furthermore, the solid-liquid separation in steps S4 and S6 is performed using a centrifuge or a filtering device.

[0028] Furthermore, the acid in step S5 is hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid or citric acid.

[0029] Furthermore, the drying in step S7 is carried out by spray drying or freeze drying.

[0030] The present invention sequentially uses amylase and lipase to pre-treat yeast cell walls, so that water-soluble polysaccharides and lipid components therein are properly decomposed, which is conducive to subsequent solubilization and extraction. Then, impurity components therein are further dissolved through solubilizer-assisted alkali extraction, and the amount of alkali used is reduced. Then, through acid treatment, separation washing, drying and other steps, a high-purity yeast β-glucan product with a content of more than 90% is prepared, the glycogen content in the product is ≤2%, and the proportion of β-1,3-glycosidic bonds in the product is ≥80%, and the product quality is high. Enzymatic hydrolysis and ultrasound are used together to promote the degradation of long molecular chains of insoluble yeast β-glucan, thereby improving its water solubility and obtaining a water-soluble product. The biological efficacy and biological activity of yeast β-glucan can be fully exerted, and its application field can be expanded. The yeast β-glucan can also be graded and finely processed by this method, thereby further increasing the added value of the product. The overall preparation method is simple, highly operable, has low pollution, and can be produced on a large scale. DETAILED DESCRIPTION

[0031] Example 1

[0032] The specific process flow for producing yeast β-glucan includes: (1) amylase treatment of yeast cell walls; (2) lipase treatment of yeast cell walls; (3) solubilizer-assisted alkaline extraction; (4) solid-liquid separation and washing; (5) acid extraction; (6) solid-liquid separation and washing; and (7) drying.

[0033] Amylase treatment: Yeast cell walls (commercially available, purchased from Angel Yeast Co., Ltd., β-glucan content ≥20%, the same below) were added with water to a concentration of 2% (weight content), and 0.1% (m / v, g / mL) α-amylase (commercially available, purchased from Xiasheng Enzyme Biotechnology Co., Ltd., model FYD2601, enzyme activity 20000u / mL or more, the same below) was added and kept at 60°C for 0.5h.

[0034] Lipase treatment: After amylase treatment, the cell wall milk was adjusted to pH 5.0 and temperature 60°C, and 1% (m / v, g / mL) lipase (commercially available, purchased from Xiasheng Enzyme Biotechnology Co., Ltd., model FYD2261, 100,000 u / mL enzyme activity, the same below) was added and kept warm for 4 hours.

[0035] Solubilizer and alkali treatment: After lipase treatment, add NaOH to the mixed solution to make its content reach 4% (m / v, g / mL), add 0.1% (m / v, g / mL) Tween 80, heat to 100°C for 1 hour, stir continuously, and then cool to 60°C.

[0036] Separation: The mixed liquid after alkali treatment is separated by a stacked disc centrifuge, and water is added to the precipitate to the volume before separation for a second separation. A total of 4 separation and washing operations are performed.

[0037] Acid treatment: Add water to the precipitate after separation and washing in the previous step to a concentration of about 5%, adjust the pH to 3 with hydrochloric acid, heat to about 90°C, keep warm for 0.5h, stir continuously, and then cool to 60°C.

[0038] Separation: The acid-treated mixed liquid was separated by centrifuge, and water was added to the precipitate to the volume before separation for the second separation. A total of 4 washing and separations were performed.

[0039] Drying: Powder spray drying is adopted, and the product is powdery after drying.

[0040] Packaging: Yeast β-glucan products can be packaged in a variety of forms.

[0041] The yeast β-glucan content in the powdered product was tested using the QB / T 4572 standard method, the glycogen content was determined using an enzymatic method, and the β-1,3-glycosidic bond content was determined using nuclear magnetic resonance. The yeast β-glucan content was 91.5%, the glycogen content was 0.8%, and the β-1,3-glycosidic bond content was approximately 88.5%.

[0042] Example 2

[0043] The specific process of yeast β-glucan includes:

[0044] Amylase treatment: Yeast cell walls were diluted with water to a concentration of 10%, and 2% (m / v, g / mL) β-amylase (commercially available from Xiasheng Enzyme Biotechnology Co., Ltd., model FYD2218, 700,000 u / mL enzyme activity, the same below) was added and kept at 100°C for 5 h.

[0045] Lipase treatment: After amylase treatment, the cell wall milk was adjusted to pH 9.0 and temperature 30°C, and 0.05% (m / v, g / mL) phospholipase (commercially available, purchased from Xiasheng Enzyme Biotechnology Co., Ltd., hemolytic phospholipase, model FDG2238, 2900u / mL enzyme activity, the same below) was added and kept warm for 1h.

[0046] Solubilizer and alkali treatment: After lipase treatment, add KOH to the mixture to a concentration of 1% (m / v, g / mL), and then add 5% (m / v, g / mL) Tween 20. Heat to 80°C for 4 h with constant stirring, then cool to 50°C.

[0047] Separation: The mixed liquid after alkali treatment is separated by a stacked disc centrifuge, and water is added to the precipitate to the volume before separation for the second separation, and a total of 4 separation and washing operations are performed.

[0048] Acid treatment: Add water to the precipitate after separation and washing in the previous step to a concentration of about 5%, adjust the pH to 5 with phosphoric acid, heat to about 90°C, keep warm for 2 hours, stir continuously, and then cool to 60°C.

[0049] Separation: The acid-treated mixed liquid was separated by a centrifuge, and the precipitate was added with water to the volume before separation for a second separation, and a total of 4 separation and washing were performed.

[0050] Drying: Freeze drying is used. The dried product is in powder form. The yeast β-glucan content is 92.8%, the glycogen content is 0.7%, and the proportion of β-1,3-glycosidic bonds is about 85.2%.

[0051] Example 3

[0052] The specific process of yeast β-glucan includes:

[0053] Amylase treatment: Yeast cell walls were diluted with water to a concentration of 5%, 1% (m / v, g / mL) β-amylase was added, and the mixture was kept at 90°C for 2 h.

[0054] Lipase treatment: After amylase treatment, the cell wall milk was adjusted to pH 8.0 and temperature 40° C., 0.1% (m / v, g / mL) phospholipase was added, and the temperature was kept for 3.0 h.

[0055] Solubilizer and alkali treatment: After lipase treatment, add NaOH to the mixture to a concentration of 4% (m / v, g / mL), and then add 2% (m / v, g / mL) Tween 80. Heat to 90°C for 3 h with constant stirring, then cool to 60°C.

[0056] Separation: The mixed liquid after alkali treatment is separated by a stacked disc centrifuge, and water is added to the precipitate to the volume before separation for a second separation. A total of 4 separation and washing operations are performed.

[0057] Acid treatment: Add water to the precipitate after separation and washing in the previous step to a concentration of about 5%, adjust the pH to 4 with phosphoric acid, heat to about 90°C, keep warm for 1 hour, stir continuously, and then cool to 60°C.

[0058] Separation: The acid-treated mixed liquid was separated by a centrifuge, and the precipitate was added with water to the volume before separation for a second separation, and a total of 4 separation and washing were performed.

[0059] Drying: Freeze drying is used. The dried product is in powder form. The yeast β-glucan content is 95.7%, the glycogen content is 0.4%, and the proportion of β-1,3-glycosidic bonds is about 83.7%.

[0060] Example 4

[0061] The specific process of yeast β-glucan includes:

[0062] Amylase treatment: Yeast cell walls were diluted with water to a concentration of 5%, 0.5% (m / v, g / mL) β-amylase was added, and the mixture was kept at 90°C for 1 h.

[0063] Lipase treatment: After amylase treatment, the cell wall milk was adjusted to pH 5.0 and temperature 60° C., 0.5% (m / v, g / mL) lipase was added, and the temperature was kept for 4.0 h.

[0064] Solubilizer and alkali treatment: After lipase treatment, add NaOH to the mixture to a concentration of 2% (m / v, g / mL), and then add 1% (m / v, g / mL) Tween 60. Heat to 90°C for 3 h with constant stirring, then cool to 60°C.

[0065] Separation: The mixed liquid after alkali treatment is separated by a stacked disc centrifuge, and water is added to the precipitate to the volume before separation for a second separation, and a total of 2 separation and washing operations are performed.

[0066] Acid treatment: Add water to the precipitate after separation and washing in the previous step to a concentration of about 5%, adjust the pH to 4 with acetic acid, heat to 80°C, keep warm for 2 hours, stir continuously, and then cool to 60°C.

[0067] Separation: The mixed liquid after acid treatment is separated by centrifuge, and the precipitate is separated by adding water to the volume before separation for the second separation, and the separation and washing are carried out twice in total.

[0068] Drying: Spray drying is used. The product is powdery after drying. The yeast β-glucan content is 93.3%, the glycogen content is 0.5%, and the proportion of β-1,3-glycosidic bonds is about 85.6%.

[0069] Comparative Example 1

[0070] The specific process flow for producing yeast β-glucan in this comparative example includes: (1) alkali treatment of yeast cell walls; (2) separation and collection of precipitates; (3) acid extraction; and (4) drying.

[0071] Alkali treatment: Add water to the yeast cell wall to a concentration of 5%, add NaOH to a concentration of 4% (m / v, g / mL), heat to 90°C for 3 hours, stir continuously, and then cool to 60°C.

[0072] Separation: The mixed liquid after alkali treatment is separated by a stacked disc centrifuge, and water is added to the precipitate to the volume before separation for a second separation, and a total of 2 separation and washing operations are performed.

[0073] Acid treatment: Add water to the precipitate after separation and washing in the previous step to a concentration of 5%, adjust the pH to 4 with acetic acid, heat to 80°C, keep warm for 2 hours, stir continuously, and then cool to 60°C.

[0074] Separation: The mixed liquid after acid treatment is separated by centrifuge, and the precipitate is separated by adding water to the volume before separation for the second separation, and the separation and washing are carried out twice in total.

[0075] Drying: Spray drying is used and the product is powdery after drying.

[0076] After testing, the yeast β-glucan content was 72.7% and the glycogen content was 11.8%.

[0077] Comparative Example 2

[0078] The specific process flow for producing yeast β-glucan in this embodiment includes: (1) alkaline treatment of yeast cell walls; (2) separation and collection of precipitates; (3) acid extraction; and (4) drying.

[0079] Alkali treatment: Add water to the yeast cell wall to a concentration of 5%, add NaOH to make its content reach 2% (m / v, g / mL), heat to 90℃ for 3h, stir continuously, and then cool to 60℃.

[0080] Separation: The mixed liquid after alkali treatment is separated by a stacked disc centrifuge, and water is added to the precipitate to the volume before separation for a second separation, and a total of 2 separation and washing operations are performed.

[0081] Acid treatment: The precipitate after separation and washing in the previous step was prepared into a 5% concentration, adjusted to pH 4 with hydrochloric acid, heated to 80°C, kept warm for 2 hours, stirred continuously, and then cooled to 60°C.

[0082] Separation: The mixed liquid after acid treatment is separated by centrifuge, and the precipitate is separated by adding water to the volume before separation for the second separation, and the separation and washing are carried out twice in total.

[0083] Drying: Spray drying is used, and the product is powdered after drying. Testing shows that the yeast β-glucan content is 65.3% and the glycogen content is 14.6%.

[0084] Comparative Example 3

[0085] The specific process flow for producing yeast β-glucan in this embodiment includes: (1) amylase treatment; (2) protease treatment; (3) alkali treatment; (4) separation and collection of precipitates; (3) acid extraction; and (4) drying.

[0086] Amylase treatment: Yeast cell walls were diluted with water to a concentration of 2% (by weight), 0.1% (m / v, g / mL) α-amylase was added, and the mixture was incubated at 60°C for 0.5 h.

[0087] Protease treatment: After amylase treatment, the cell wall milk was adjusted to pH 5.0 and temperature 60°C, and 1% (m / v, g / mL) protease (commercially available, purchased from Xiasheng Enzyme Biotechnology Co., Ltd., model FDG2210, 50,000 u / mL enzyme activity) was added and kept warm for 4 hours.

[0088] Alkali treatment: After protease treatment, add NaOH to the mixed solution to make its content reach 4% (m / v, g / mL), heat to 100°C for 1 hour, stir continuously, and then cool to 60°C.

[0089] Separation: The mixed liquid after alkali treatment is separated by a stacked disc centrifuge, and water is added to the precipitate to the volume before separation for the second separation, and a total of 4 separation and washing operations are performed.

[0090] Acid treatment: The precipitate separated and washed in the previous step was prepared into a 5% concentration, adjusted to pH 3 with hydrochloric acid, heated to about 90°C, kept warm for 0.5h, stirred continuously, and then cooled to 60°C.

[0091] Separation: The mixed liquid after acid treatment is separated by centrifuge, and the precipitate is separated by adding water to the volume before separation for the second separation, and a total of 4 separation and washing are performed.

[0092] Drying: The product is powdered after drying. The yeast β-glucan content is 83.5% and the glycogen content is 8.6%.

[0093] Comparative Example 4

[0094] The specific process flow for producing yeast β-glucan in this embodiment includes: (1) amylase treatment; (2) saccharifying enzyme treatment; (3) alkali treatment; (4) separation and collection of precipitates; (3) acid extraction; and (4) drying.

[0095] Amylase treatment: Yeast cell walls were diluted with water to a concentration of 5%, 1% (m / v, g / mL) β-amylase was added, and the mixture was kept at 90°C for 2 h.

[0096] Saccharifying enzyme treatment: After amylase treatment, the cell wall milk was adjusted to pH 8.0 and temperature 40°C, and 0.1% (m / v, g / mL) glucoamylase (commercially available, purchased from Xiasheng Enzyme Biotechnology Co., Ltd., model FYD2223, 260,000 u / mL enzyme activity) was added and kept warm for 3.0 h.

[0097] Alkali treatment: After the saccharifying enzyme treatment, NaOH was added to the mixture to make its content reach 4% (m / v, g / mL), heated to 90°C for 3 hours with constant stirring, and then cooled to 60°C.

[0098] Separation: The mixed liquid after alkali treatment is separated by a stacked disc centrifuge, and water is added to the precipitate to the volume before separation for the second separation, and a total of 4 separation and washing operations are performed.

[0099] Acid treatment: Add water to the precipitate after separation and washing in the previous step to a concentration of about 5%, adjust the pH to 4 with phosphoric acid, heat to about 90°C, keep warm for 1 hour, stir continuously, and then cool to 60°C.

[0100] Separation: The mixed liquid after acid treatment is separated by centrifuge, and the precipitate is separated by adding water to the volume before separation for the second separation, and a total of 4 separation and washing are performed.

[0101] Drying: Freeze drying is used, and the product is powdered after drying. After testing, the yeast β-glucan content is 86.3% and the glycogen content is 5.5%.

[0102] Example 5

[0103] This example mainly modifies the yeast β-glucan prepared in Example 1 to improve the water solubility of the yeast β-glucan. The specific steps are as follows:

[0104] Ultrasonic enzymatic hydrolysis: The yeast β-glucan obtained in Example 1 (mass m0) was added with water to prepare a glucan mixture with a mass concentration of 5%. The pH was adjusted to about 6.0, and the mixed enzyme preparation was added. The mixture was placed in a laboratory ultrasonic constant temperature water bath (20 L, ultrasonic frequency 35 kHz, ultrasonic power 400 W). Ultrasonic waves were turned on, the temperature was raised to 65° C., and then kept warm for 50 minutes to obtain an enzymatic hydrolyzate. The weight of the mixed enzyme preparation in grams was 0.3% of the milliliters of the glucan mixture. The mixed enzyme preparation is a mixture of mannanase (commercially available, with an enzyme activity of more than 100,000, the same below) and β-glucanase (commercially available, purchased from Xiasheng Enzyme Biotechnology Co., Ltd., model FDG2219, with an enzyme activity of 130,000 u / mL, the same below) in a mass ratio of 1:3. The β-glucanase also contains β-(1,3-1,4)-glucanase, endo-β-1,3-glucanase and endo-β-1,4-glucanase.

[0105] Inactivation separation: After heating the obtained enzymatic hydrolysate to 100°C, keep it warm for 10 minutes, cool it to room temperature, and then perform solid-liquid separation, and collect the separated precipitate and clear liquid.

[0106] Washing and drying: Wash the precipitate with water twice, and then dry it to obtain insoluble yeast β-glucan.

[0107] Concentration and alcohol precipitation: After adjusting the pH of the clear liquid to 6.5, activated carbon is added for decolorization, and then filtered through diatomaceous earth with a mesh size of 100 or above. The filtered clear liquid is concentrated to 20% of the original volume, and 80% of the concentrated volume of ethanol is added for alcohol precipitation. The product after alcohol precipitation is dried to obtain water-soluble yeast β-glucan, and its mass is weighed as m1.

[0108] The yield of the water-soluble yeast β-glucan of the present invention is calculated according to the formula: Yield of water-soluble yeast β-glucan = m1 / m0×100%. The relative molecular mass of the water-soluble yeast β-glucan is sent to a testing agency for determination using gel permeation chromatography (GPC). The test results show that the yield of the water-soluble yeast β-glucan of this example is 45.3%, and the weight average molecular weight of the water-soluble yeast β-glucan is 2.53×10 5 , Mw / Mn is 1.36.

[0109] Example 6

[0110] This example mainly modifies the yeast β-glucan prepared in Example 2 to improve the water solubility of the yeast β-glucan. The specific steps are as follows:

[0111] Ultrasonic enzymatic hydrolysis: Yeast β-glucan (mass m0) obtained in Example 2 was added with water to prepare a 6% glucan mixture. The pH was adjusted to approximately 6.0, and a mixed enzyme preparation was added. The mixture was then placed in an ultrasonic constant-temperature water bath (24 L, ultrasonic frequency 35 kHz, ultrasonic power 600 W). Ultrasonic waves were activated, the temperature was raised to 65°C, and then incubated for 1 hour to obtain an enzymatic hydrolyzate. The weight of the mixed enzyme preparation in grams was 0.4% of the milliliters of the glucan mixture. The mixed enzyme preparation was a mixture of mannanase and β-glucanase in a mass ratio of 1:4.

[0112] Inactivation separation: After heating the obtained enzymatic hydrolysate to 100°C, keep it warm for 10 minutes, cool it to room temperature, and then perform solid-liquid separation, and collect the separated precipitate and clear liquid.

[0113] Washing and drying: Wash the precipitate with water twice, and then dry it to obtain insoluble yeast β-glucan.

[0114] Concentration and alcohol precipitation: After adjusting the pH of the clear liquid to 6.5, the clear liquid is filtered through diatomaceous earth with a mesh size of 100 or above. The filtered clear liquid is concentrated to 15% of the original volume, and ethanol is added at a volume ratio of 1:1 (v / v) to the concentrated liquid to perform alcohol precipitation. The product after alcohol precipitation is dried to obtain water-soluble yeast β-glucan, and its mass is weighed as m1.

[0115] The yield of the water-soluble yeast β-glucan of the present invention is calculated according to the formula: Yield of water-soluble yeast β-glucan = m1 / m0×100%. After testing, the yield of the water-soluble yeast β-glucan of this embodiment is 53.3%, and the weight average molecular weight of the water-soluble yeast β-glucan is 3.56×10 5 , Mw / Mn is 1.28.

[0116] Comparative Example 5

[0117] The rest is the same as Example 5, except that there is no ultrasonic assistance during the enzymatic hydrolysis process. The specific steps are as follows:

[0118] Enzymatic Hydrolysis: Yeast β-glucan (mass m0) obtained in Example 1 was added with water to prepare a 5% glucan mixture. The pH was adjusted to approximately 6.0, and a mixed enzyme preparation was added. The mixture was heated to 65°C and incubated for 50 minutes to obtain an enzymatic hydrolysis solution. The weight of the mixed enzyme preparation was 0.3% of the milliliters of the glucan mixture. The mixed enzyme preparation was a mixture of mannanase and β-glucanase in a mass ratio of 1:3.

[0119] Inactivation separation: After heating the obtained enzymatic hydrolysate to 100°C, keep it warm for 10 minutes, cool it to room temperature, and then perform solid-liquid separation, and collect the separated precipitate and clear liquid.

[0120] Washing and drying: Wash the precipitate with water twice, and then dry it to obtain insoluble yeast β-glucan.

[0121] Concentration and alcohol precipitation: After adjusting the pH of the clear liquid to 6.5, activated carbon is added for decolorization, and then filtered through diatomaceous earth with a mesh size of 100 or above. The filtered clear liquid is concentrated to 20% of the original volume, and 80% of the concentrated volume of ethanol is added for alcohol precipitation. The product after alcohol precipitation is dried to obtain water-soluble yeast β-glucan, and its mass is weighed as m1.

[0122] The yield of the water-soluble yeast β-glucan of the present invention is calculated according to the formula: Yield of water-soluble yeast β-glucan = m1 / m0×100%. After testing, the yield of the water-soluble yeast β-glucan of this embodiment is 30.6%, and the weight average molecular weight of the water-soluble yeast β-glucan is 4.78×10 4 , Mw / Mn is 2.73, and the molecular weight distribution is relatively wide.

[0123] Comparative Example 6

[0124] This example uses ultrasound to treat the yeast β-glucan obtained in Example 2. The specific steps are as follows:

[0125] Ultrasonic treatment: The yeast β-glucan obtained in Example 2 (mass m0) was added with water to prepare a glucan mixture with a mass concentration of 6%. The mixture was placed in an ultrasonic constant temperature water bath (24 L, ultrasonic frequency 35 kHz, ultrasonic power 600 W). Ultrasonication was turned on, the temperature was raised to 65° C., and then kept warm for 1 h to obtain a treated solution.

[0126] Inactivation separation: The treated liquid was heated to 100°C, kept warm for 10 minutes, cooled to room temperature, and then subjected to solid-liquid separation. The precipitate and clear liquid after separation were collected.

[0127] Washing and drying: Wash the precipitate with water twice, and then dry it to obtain insoluble yeast β-glucan.

[0128] Concentration and alcohol precipitation: After adjusting the pH of the clear liquid to 6.5, the clear liquid is filtered through diatomaceous earth with a mesh size of 100 or above. The filtered clear liquid is concentrated to 15% of the original volume. Ethanol is added at a volume ratio of 1:1 (v / v) to the concentrated liquid to carry out alcohol precipitation. The product after alcohol precipitation is dried to obtain water-soluble yeast β-glucan, and its mass is weighed as m1.

[0129] The yield of the water-soluble yeast β-glucan of the present invention is calculated according to the formula: Yield of water-soluble yeast β-glucan = m1 / m0×100%. After testing, the yield of the water-soluble yeast β-glucan of this embodiment is 8.4%. The weight average molecular weight of the water-soluble yeast β-glucan is 3.24×10 5The polydispersity coefficient Mw / Mn is 3.65, indicating that its molecular weight uniformity is poor.

[0130] The above detailed description is a specific description of a feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not depart from the present invention should be included in the patent scope of this case.

Claims

1. A method for preparing high-purity yeast β-glucan, characterized in that: The steps include: S1. Amylase treatment: Yeast cell walls are added with water to form a mixture with a mass concentration of 2%-10%, amylase is added at 60-100°C, and the mixture is kept warm for 0.5-5 hours to obtain a first enzymatic hydrolyzate, wherein the weight of amylase in grams is 0.1%-2% of the weight of the mixture in milliliters; S2. Lipase treatment: adjust the pH of the first hydrolyzate to 5.0-9.0, add lipase at a temperature of 30-60°C, and keep warm for 1-4 hours to obtain a second hydrolyzate, wherein the weight of lipase is 0.05%-1% of the weight of the first hydrolyzate; S3, solubilizer and alkali treatment: add alkali and nonionic surfactant to the second enzymatic hydrolyzate, heat to 80-100°C, keep warm for 1-4 hours under constant stirring, and then cool to 50-60°C to obtain an alkali treatment solution, wherein the weight of the alkali is 1%-4% of the milliliters of the second enzymatic hydrolyzate, and the weight of the nonionic surfactant is 0.1%-5% of the milliliters of the second enzymatic hydrolyzate; S4, separation: solid-liquid separation of the alkali-treated liquid, collecting the precipitate and washing it with water, repeating the separation and washing steps 2-5 times; S5, acid treatment: the precipitate obtained in step S4 is added with water to form a 3%-8% mixed solution, acid is added to adjust the pH to 3-5, heated to 80-90°C, kept warm for 0.5-2h under continuous stirring, and then cooled to 50-60°C to obtain an acid-treated solution; S6, separation: the acid-treated liquid is subjected to solid-liquid separation, the precipitate is collected and washed with water, and the separation and washing steps are repeated 2-5 times; S7, drying the precipitate obtained in step S6 to obtain a powdery yeast β-glucan product; The amylase in step S1 is α-amylase or β-amylase; The lipase in step S2 is phospholipase; The nonionic surfactant in step S3 is a polyoxyethylene nonionic surfactant.

2. The method for preparing high-purity yeast β-glucan according to claim 1, characterized in that: The base in step S3 is NaOH or KOH.

3. The method for preparing high-purity yeast β-glucan according to claim 1, characterized in that: The solid-liquid separation in steps S4 and S6 is performed using a centrifuge or a filtering device.

4. The method for preparing high-purity yeast β-glucan according to claim 1, characterized in that: The acid in step S5 is hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid or citric acid.

5. The method for preparing high-purity yeast β-glucan according to claim 1, characterized in that: The drying in step S7 is carried out by spray drying or freeze drying.

6. A method for preparing water-soluble yeast β-glucan, comprising the following steps: S8, adding water to the yeast β-glucan obtained in step S7 of claim 1 to prepare a glucan mixture with a mass concentration of 2%-10%, adjusting the pH to 4.0-8.0, adding the mixed enzyme preparation, placing it in an ultrasonic thermostat, turning on the ultrasound, heating to 40-75°C and then keeping it warm for 20-100 minutes to obtain an enzymatic hydrolyzate, wherein the weight of the mixed enzyme preparation in grams is 0.1%-0.5% of the milliliters of the glucan mixture; S9, heating the enzymatic hydrolyzate obtained in step S8 to 100° C., keeping the temperature for more than 5 minutes to inactivate the enzyme, cooling it to room temperature, and then performing solid-liquid separation, collecting the separated precipitate and clear liquid; S10, washing the precipitate obtained in step S9 with water 2-3 times, and drying to obtain insoluble yeast β-glucan; S11, adjusting the pH of the clear solution obtained in step S9 to 5.0-8.0 and filtering, concentrating the filtered clear solution to 10%-30% of the original volume, adding 50%-100% of the concentrated volume of ethanol for alcohol precipitation, and drying the product after alcohol precipitation to obtain water-soluble yeast β-glucan; The mixed enzyme preparation is a mixture of mannanase and β-glucanase in a mass ratio of 1:(2-5), and the β-glucanase is one or more of β-1,3-glucanase, β-1,4-glucanase, and β-(1,3-1,4)-glucanase.

7. The method for preparing water-soluble yeast β-glucan according to claim 6, characterized in that: The ultrasonic frequency of the ultrasonic constant temperature device is 20-100 kHz, the ultrasonic power is 100-1000 W, and the filtration in step S11 is performed using diatomaceous earth with a mesh size of 100 or more.