A method for preparing a branched-chain-rich yeast beta-glucan and use thereof

Abstract

The application discloses a branched yeast beta-glucan preparation method and application thereof, relates to the technical field of yeast beta-glucan, and aims to solve the problems of poor dispersibility and low bioavailability of natural yeast beta-glucan caused by the problems of too large molecular weight and tight triple helix structure, and overcome the defects of random damage to the glucan skeleton structure caused by the existing strong alkali or oxidation degradation process. The method comprises the following steps: raw material pretreatment and gradient extraction; multi-enzyme synergistic controllable enzymolysis, directional regulation of molecular weight and branch degree; ultrasonic-microjet synergistic physical modification, improvement of dispersibility; selective grading and purification. The yeast beta-glucan prepared by the method has a weight average molecular weight of 100-200 kDa, and the branch degree of the beta-1,6 branch is maintained at 10-23%, so that the unification of high dispersibility, excellent processing adaptability and significant immune regulation activity is realized, and the yeast beta-glucan is suitable for various foods such as powder, beverage and soft candy.

Description

Technical Field

[0001] This invention relates to the field of yeast β-glucan technology, specifically to a method for preparing richly branched yeast β-glucan and its application. Background Technology

[0002] Yeast β-glucan is a structural polysaccharide found in the inner layer of yeast cell walls. Its chemical structure is primarily composed of a main chain linked by β-1,3-glycosidic bonds, with side chains linked by β-1,6-glycosidic bonds. Modern immunological research indicates that yeast β-glucan is a highly effective biological response modifier. It can activate the host immune system by specifically binding to Dectin-1 and CR3 receptors on the surfaces of macrophages, neutrophils, and dendritic cells, exerting anti-infective, anti-tumor, lipid-regulating, and wound-healing effects. However, the industrial application of natural yeast β-glucan faces a severe structure-efficacy contradiction:

[0003] (1) Low bioavailability due to high molecular weight and high branching: Natural β-glucan has a very large molecular weight, with a weight average molecular weight of 500-2000 kDa and a branching degree of β-1,6 branches of 20-30%. The molecular chains form a tight triple helix structure through a large number of hydrogen bonds. This large and dense molecular conformation makes it difficult for the body to cross the intestinal barrier and be effectively absorbed and utilized, resulting in extremely low bioavailability. Most unmodified β-glucan can only have limited contact with immune cells in the intestine and is difficult to enter the blood circulation system to exert a systemic immunomodulatory effect, thus limiting the expression of its physiological activity at the source.

[0004] (2) Bottlenecks in processing performance and product form caused by structural characteristics: The aforementioned macromolecular characteristics also make it extremely difficult to disperse in water. This physical property not only further hinders its absorption in the digestive tract, but also directly leads to the limitation of application forms. In liquid products, it will quickly precipitate and separate into layers; in semi-solid foods, such as jellies and gummies, it will cause the system to become turbid.

[0005] (3) Limitations of existing processes: The mainstream preparation method in industry is acid-base extraction. Although it can remove proteins and mannans, it usually involves long-term treatment with strong alkali at high temperatures. These harsh conditions can cause random breakage of polysaccharide chains and alkaline peeling reactions, resulting in the loss of a large number of branched structures. Although the purity is improved, a large number of β-1,6 branched structures are lost, and the main chain undergoes random breakage, thereby destroying its inherent active conformation, resulting in a low yield and generating a large amount of high-salt and high-alkali wastewater, which puts great pressure on environmental protection.

[0006] Based on research on yeast β-glucan, existing technology CN120989185A discloses a method for preparing yeast β-glucan derived from Saccharomyces cerevisiae. While this patent proposes an environmentally friendly approach that avoids the use of organic solvents and employs a complex enzyme for impurity removal, its core process still relies on high-pressure homogenization followed by high-temperature, strong-alkali extraction. This method is essentially a way to purify insoluble cell walls, and its product is insoluble yeast β-glucan powder. This patent does not address the application pain point of poor dispersibility, nor does it achieve control over molecular weight and the branching degree of β-1,6 branches.

[0007] Therefore, developing a preparation process that is green and mild throughout the entire process, free of strong acids and bases and organic solvents, can precisely control the molecular weight to the optimal activity range, retain a moderate branched structure to maintain stability, and significantly improve dispersibility and dispersion stability is a key challenge for realizing the high-value application of yeast β-glucan. Summary of the Invention

[0008] The primary objective of this invention is to provide a method for preparing highly branched yeast β-glucan. This method integrates the specificity of enzymatic shearing with the solubilizing effect of physical field modification to prepare a highly active and highly dispersible product with a weight-average molecular weight of 100-200 kDa and a β-1,6 branching degree of 10-23%.

[0009] The specific technical solution is as follows: A method for preparing branched yeast β-glucan includes crude extraction, complex enzymatic hydrolysis, physical modification, fractional purification, and drying. The crude extraction is characterized by using an enzyme-assisted activation-extraction combined with a segmented gradient water extraction method; the complex enzymatic hydrolysis utilizes the synergistic effect of β-1,3-glucan endonuclease, β-1,6-glucan endonuclease, and β-glucosidase with specific enzyme activity ratios for functional differentiation, wherein the β-1,3-glucan endonuclease performs restrictive random cleavage of the backbone, adjusting the weight-average molecular weight to 100-200 kDa. The β-1,6-glucan endonuclease selectively prunes excessively dense or long side chains, controlling the branching degree of β-1,6 branches to 10-23%; the β-glucosidase hydrolyzes the glucose residues at the non-reducing ends of β-glucan to reduce the polydispersity coefficient; the process of the complex enzymatic hydrolysis is quantitatively controlled by real-time monitoring of the system viscosity; the physical modification is carried out by a combination of ultrasonic dispersion and high-pressure microfluidic homogenization; the fractional purification uses programmed cooling to achieve molecular weight difference sedimentation fractionation, and is purified by membrane separation.

[0010] Furthermore, the method for preparing branched yeast β-glucan includes the following steps: S1: Crude extraction of food-grade brewing yeast powder is carried out in sequence, including activation autolysis treatment, segmented water extraction treatment, enzymatic hydrolysis and salting out impurities, and finally, crude β-glucan is obtained by solid-liquid separation and washing. S2: Prepare a suspension with a solid content of 2-8% (w / w) using pure water with crude β-glucan obtained in step S1. Disperse the suspension fully by high-speed shearing, adjust the pH of the system to 5.5-6.5, control the temperature at 40-50°C, and add the compound enzyme solution to carry out the enzymatic hydrolysis reaction. Monitor the viscosity of the reaction system in real time during the reaction to adjust the enzymatic hydrolysis time and temperature. When the viscosity of the system drops to 5-20% of the initial viscosity value, rapidly raise the temperature to 85-95°C and maintain it for 10-20 minutes to inactivate the enzyme and obtain the enzymatically hydrolyzed solution. S3: The enzymatic hydrolysis solution obtained in S2 is physically modified by first performing ultrasonic dispersion treatment and then high-pressure microfluidic homogenization treatment to break up the aggregates and build a stable dispersion structure, thus obtaining the physically modified solution. S4: The physically modified liquid obtained in S3 is subjected to programmed cooling and fractionation precipitation to separate and collect the upper liquid phase rich in the target component; the upper liquid phase is purified by membrane separation, concentrated and spray-dried to obtain yeast β-glucan product with a weight average molecular weight of 100-200kDa and a branching degree of 10-23% for β-1,6 branches.

[0011] Further, the specific method for the activation autolysis treatment described in step S1 is as follows: dilute the food-grade brewer's yeast powder with pure water to a solid content of 10-20% (w / w), adjust the pH of the system to 5.0-6.0, and stir at 45-50°C for 12-24 hours to autolyze; add 0.01-0.1 wt% of papain to assist autolysis; after autolysis, rapidly heat to 85-95°C and keep warm for 10-15 minutes to inactivate endogenous enzymes, and collect the yeast precipitate by centrifugation.

[0012] Furthermore, the segmented water extraction process described in step S1 involves first performing a low-temperature primary water extraction, followed by a medium-temperature secondary water extraction. The specific method for the low-temperature primary water extraction is as follows: the yeast precipitate is prepared into a suspension with a solid content of 8.0-12.0% (w / w) using pure water; the pH of the system is adjusted to 6.0-7.0; and the mixture is subjected to ultrasound at 45-55°C, with the ultrasound frequency set to 20-40 kHz and the ultrasound power density to 0.1-0.3 W / cm³. 2 After stirring and extracting for 30-60 minutes, centrifuge to separate and collect the precipitate; the specific method of the medium-temperature secondary water extraction is as follows: the precipitate after the primary water extraction is prepared again with pure water to form a suspension with a solid content of 10-20% (w / w), the pH is adjusted to 7.0-8.0, the temperature is raised to 70-80°C, and the mixture is stirred and extracted for 1-2 hours.

[0013] Furthermore, the method for adjusting the pH of the system is achieved by adding food-grade citric acid or food-grade sodium bicarbonate.

[0014] Furthermore, the specific method for enzymatic hydrolysis and salting out impurities described in step S1 is as follows: Cool the suspension after segmented water extraction to 45-55°C, add pure water to dilute to a solid content of 6% (w / w), stir thoroughly, add 0.05-0.5 wt% of food-grade neutral protease based on the dry weight of the substrate, hydrolyze for 2-6 hours, then add 3-5 wt% sodium chloride for salting out, so that the dissolved proteins and nucleic acids flocculate and precipitate.

[0015] Further, the complex enzyme solution in step S2 is prepared by mixing β-1,3-glucan endonuclease, β-1,6-glucan endonuclease and β-glucosidase in a ratio of (10-50):(1-5):(0.5-2) activity units; the amount of the complex enzyme solution added is 500-4000 U / g substrate dry weight.

[0016] Furthermore, the ultrasonic dispersion treatment conditions in step S3 are as follows: ultrasonic frequency is 20-40kHz, and the total applied energy density is 50-200J / mL; the high-pressure micro-jet homogenization treatment conditions are as follows: homogenization pressure is set at 100-180MPa, the treatment is repeated 3-5 times, and the temperature of the liquid is kept below 60°C throughout the process.

[0017] Further, the specific method for programmed cooling and fractional precipitation in step S4 is as follows: at an initial temperature of 50-70°C, the feed solution is cooled to 10-30°C at a programmed rate of 0.1-1.0°C / min, and the upper liquid phase is obtained by separation; the specific method for membrane separation and purification is as follows: the upper liquid phase is separated by a membrane module with a molecular weight cutoff of 10kDa to remove inorganic salts, small molecule sugars and peptide impurities; the spray drying conditions are as follows: the inlet air temperature is 160-190°C, and the outlet air temperature is 80-95°C.

[0018] Furthermore, the yeast β-glucan is used in solid beverages, candies, gel foods, plant-based beverages, or dairy products as a solubilizing agent, texture modifier, thickener, or stabilizer.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) Synergistic optimization of structure and function: This invention locks the molecular weight at 100-200 kDa through multi-enzyme synergy and physical modification, and controls the branching degree of β-1,6 branches at 10-23%. β-glucan with these structural parameters has near-true solution processing performance, significantly retains the inherent high immunomodulatory activity of β-glucan, and its binding ability to the key immune receptor Dectin-1 is much higher than that of traditional alkaline degradation products.

[0020] (2) Excellent dispersibility and dispersion stability: The micro-aggregate state of the material is changed by ultrasonic-microjets. The prepared dry powder can be quickly dispersed in cold water, and forms a uniform colloid after rehydration. It is not easy to separate into layers after standing.

[0021] (3) Safety: The entire process uses only water, enzyme preparations and conventional food processing aids, completely avoiding the high-temperature cooking of sodium hydroxide and alcohol precipitation process. There is no risk of organic solvent residue, making it low-carbon and environmentally friendly. Attached Figure Description

[0022] Figure 1 This is a flowchart of a method for preparing branched yeast β-glucan according to the present invention; Figure 2 This is a schematic diagram illustrating the principle of the present invention for the synergistic modification of the β-1,3 main chain and β-1,6 side chain of crude β-glucan through complex enzymatic hydrolysis. Figure 3 This is a comparison chart of the weight-average molecular weight and the branching degree of β-1,6 branches in various embodiments and comparative examples of the present invention. Figure 4 This is a comparison chart of the relative immune activity and dispersion stability index of various embodiments and comparative examples of the present invention. Detailed Implementation

[0023] The following embodiments further explain and illustrate the technical solutions of the present invention. It should be specifically noted that each specific embodiment is a concretization and explanation of the technical solution and should not be considered as a limitation on the scope of protection of the present invention. Those skilled in the art still have the right to modify the technical solutions of these embodiments and make equivalent substitutions for some or all of the technical features, and these modifications or substitutions do not change the essence of the corresponding technical solutions, nor do they cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions described in the present invention.

[0024] As attached Figure 1 The diagram shows a flowchart of a method for preparing branched yeast β-glucan according to the present invention, which specifically includes the following steps: 1. Mild pretreatment and segmented gradient water extraction This invention employs a multi-step, gentle extraction strategy aimed at effectively separating β-glucan while minimizing chemical damage to its β-1,3-main chain and β-1,6-branch: (1) Activation autolysis: Dilute food-grade brewer's yeast powder with pure water to a solid content of 10-20% (w / w). Adjust the pH of the system to 5.0-6.0. In a water bath at 45-50°C, stir and autolyze for 12-24 hours. Add 0.01-0.1 wt% of papain to assist autolysis. After autolysis, rapidly heat to 85-95°C and hold for 10-15 minutes to inactivate endogenous enzymes. Centrifuge and collect the autolyzed yeast precipitate.

[0025] (2) Low-temperature primary water extraction: Prepare a suspension with a solid content of 8.0-12.0% (w / w) using pure water, and adjust the pH to 6.0-7.0. At 45-55°C, turn on the ultrasonic processor, set the frequency to 20-40kHz, and the power density to 0.1-0.3W / cm³. 2 After stirring and extracting for 30-60 minutes, centrifuge to separate the supernatant and remove most of the mannan, then collect the precipitate.

[0026] (3) Medium-temperature secondary water extraction: The precipitate after the primary water extraction is prepared again with pure water to form a suspension with a solid content of 10-20% (w / w), the pH is adjusted to 7.0-8.0, the temperature is raised to 70-80°C, and the mixture is stirred for 1-2 hours to promote the dissolution of residual impurities such as proteins and nucleic acids, and to provide conditions for the subsequent enzymatic impurity removal steps.

[0027] (4) Enzymatic purification: Cool the suspension after secondary water extraction to 45-55°C, add pure water to dilute to a solid content of 6% (w / w), and stir thoroughly to ensure homogeneity. Add 0.05-0.5 wt% of food-grade neutral protease based on the dry weight of the substrate, and perform enzymatic hydrolysis at this temperature for 2-6 hours. After enzymatic hydrolysis, add 3-5 wt% sodium chloride (NaCl) for salting out, causing the dissolved proteins and nucleic acids to flocculate and precipitate. Centrifuge to separate the precipitate, collect the precipitate, and wash repeatedly with deionized water to obtain crude β-glucan.

[0028] 2. Multi-enzyme synergistic controllable enzymatic hydrolysis system The crude β-glucan obtained in step 1 was prepared into a suspension with a solid content of 2-8% (w / w) using pure water. High-speed shearing was used to fully disperse the suspension, and the pH was adjusted to 5.5-6.5. The temperature was controlled at 40-50°C. A compound enzyme solution was added to initiate the enzymatic hydrolysis reaction. The amount of compound enzyme solution added was 500-4000 U / g dry weight of the substrate. An online rotational viscometer was used to monitor the viscosity of the reaction system in real time. When the system viscosity dropped to 5-20% of the initial viscosity value, the enzyme activity was completely inactivated by rapidly raising the system temperature to 85-95°C and maintaining it for 10-20 minutes, thus precisely freezing the structural state of the β-glucan. (See attached image) Figure 2 The diagram shown illustrates the principle of synergistic modification of crude β-glucan by complex enzymatic hydrolysis.

[0029] The complex enzyme solution is prepared by mixing β-1,3-glucan endonuclease, β-1,6-glucan endonuclease and β-glucosidase in a ratio of activity units (U) of (10-50):(1-5):(0.5-2).

[0030] The β-1,3-glucan endonuclease acts by randomly cleaving the main chain, rapidly reducing the weight-average molecular weight. The β-1,6-glucan endonuclease selectively prunes overly long or dense side chains, but due to steric hindrance, it does not remove all branches, thus controlling the branching degree of β-1,6 branches to 10-23%. This range ensures molecular dispersion, preventing excessive aggregation, while retaining the side chain fingerprint required for Dectin-1 recognition. The β-glucosidase acts by hydrolyzing the glucose residues at the non-reducing ends of β-glucan, further reducing the polydispersity index (PDI) of the product and improving the uniformity of the dextran molecular structure.

[0031] 3. Ultrasonic-microfluidic synergistic and gentle physical modification Although the molecular weight of dextran decreases after enzymatic hydrolysis, intermolecular hydrogen bonds can still lead to retroprecipitation. This invention employs purely physical methods for structural remodeling, with the specific steps as follows: (1) Ultrasonic pretreatment: The enzyme-inactivated liquid is pumped into a flow-through high-intensity ultrasonic reactor or treated with a probe-type ultrasonic processor. The ultrasonic frequency is 20-40kHz, and the total energy density applied is controlled at 50-200J / mL.

[0032] (2) Micro-jet homogenization: The ultrasonically treated liquid is immediately pumped into a micro-jet homogenizer. The homogenization pressure is set at 100-180 MPa, and the process is repeated 3-5 times. The temperature of the liquid is controlled to not exceed 60°C throughout the process by using a cooling jacket.

[0033] 4. Fractional purification and drying (1) Temperature-controlled fractionation: The feed solution is fractionated by programmed cooling to separate and collect the target components. Specifically, the physically modified feed solution is pumped into a separation tank with a temperature-controlled jacket. At an initial temperature of 50-70°C, the temperature is slowly reduced to a final temperature of 10-30°C at a programmed rate of 0.1-1.0°C / min. During this process, some high-molecular-weight, insufficiently modified impurities will precipitate. The upper liquid phase is collected, which is the fractionated solution rich in the target product.

[0034] (2) Purification and concentration: The fractionated solution is pumped into the ultrafiltration system for purification and concentration. The molecular weight cutoff of the ultrafiltration membrane is preferably 10 kDa. Impurities such as inorganic salts, small molecule sugars and peptides are effectively removed through percolation. Then, the solution is concentrated to achieve a solid content of 10-20% (w / w).

[0035] (3) Drying: The concentrate is spray-dried, with the inlet air temperature controlled at 160-190°C and the outlet air temperature controlled at 80-95°C, and finally the finished powder is obtained.

[0036] In the above preparation process, the required pH range for each step is achieved by adding food-grade citric acid or food-grade sodium bicarbonate.

[0037] Table 1. Reagents used in the following examples and comparative examples.

[0038] Example 1 (1) Pretreatment and extraction: Take 10 kg of food-grade brewer's yeast powder and mix it with pure water to prepare a yeast milk with a solid content of 15% (w / w). Adjust the pH of the system to 5.5 with food-grade citric acid, add 0.05 wt% papain, and autolyze at 48°C for 18 hours. After autolysis, rapidly heat to 90°C to inactivate the enzyme for 10 minutes. Centrifuge and discard the autolysis supernatant. Dilute the obtained precipitate with pure water to make the solid content 10% (w / w). Adjust the pH of the system to 6.5 with food-grade sodium bicarbonate, and turn on the sonication at 50°C with a frequency of 30 kHz and a power density of 0.2 W / cm³. 2 Extraction was performed for 45 minutes with assisted extraction. The supernatant was discarded by centrifugation, and the resulting precipitate was diluted with water to a solids content of 15% (w / w). The pH of the system was adjusted to 7.5 with food-grade sodium bicarbonate, and extraction was carried out at 75°C for 1.5 hours. The suspension was cooled to 50°C, diluted with pure water to a solids content of 6% (w / w), and thoroughly stirred to ensure homogeneity. 0.3 wt% food-grade neutral protease was added, and enzymatic hydrolysis was performed at this temperature for 4 hours. After enzymatic hydrolysis, 4 wt% NaCl was added, and the mixture was allowed to settle. The precipitate was collected by centrifugation and washed three times with pure water to obtain crude β-glucan.

[0039] (2) Complex enzymatic hydrolysis: The crude β-glucan was prepared into a 6% (w / w) suspension, and the suspension was fully dispersed by high-speed shearing. The pH of the system was adjusted to 6.0 with food-grade citric acid, and the temperature was raised to 45°C. The complex enzyme solution was added at a rate of 2000 U / g of substrate dry weight, and the activity ratio of the three enzymes was β-1,3-glucan endonuclease: β-1,6-glucan endonuclease: β-glucosidase = 25:2.5:1. The reaction was started, and the viscosity was monitored online. After 3.5 hours, the viscosity dropped to 15% of the initial value. The temperature was immediately raised to 90°C and maintained for 15 minutes to inactivate the enzymes.

[0040] (3) Physical modification: The enzyme-inactivated solution was passed through a pipeline ultrasonic processor with an ultrasonic frequency of 30 kHz and an energy density of 100 J / mL. It was then pumped into a microfluidic homogenizer and homogenized 4 times at a homogenization pressure of 150 MPa, while controlling the temperature of the solution to not exceed 60°C.

[0041] (4) Fractional purification and drying: The physically modified liquid was pumped into a separator and cooled from 60°C to 20°C at a rate of 0.5°C / min, and the upper and middle supernatant was collected. The supernatant was purified by an ultrafiltration membrane system with a molecular weight cutoff of 10 kDa and concentrated to a solid content of 15% (w / w). The concentrate was spray-dried, with the inlet air temperature controlled at 180°C and the outlet air temperature controlled at 90°C, finally yielding the finished powder.

[0042] Example 2 (1) Pretreatment and extraction: Take 10 kg of food-grade brewer's yeast powder and mix it with pure water to prepare a yeast milk with a solid content of 20% (w / w). Adjust the pH of the system to 6.0 with food-grade citric acid, add 0.1 wt% papain, and autolyze at 50°C for 24 hours. After autolysis, rapidly heat to 95°C to inactivate the enzyme for 15 minutes. Centrifuge and discard the autolysis supernatant. Dilute the obtained precipitate with pure water to make the solid content 12% (w / w). Adjust the pH of the system to 7.0 with food-grade sodium bicarbonate, and turn on the sonication at 55°C with a frequency of 40 kHz and a power density of 0.3 W / cm³. 2 Extraction was performed for 60 minutes with assisted extraction. The supernatant was discarded by centrifugation. The resulting precipitate was diluted with water to a solid content of 20% (w / w). The pH of the system was adjusted to 8.0 with food-grade sodium bicarbonate. Extraction was carried out at 80°C for 2 hours. The suspension was cooled to 55°C and diluted with pure water to a solid content of 6% (w / w). The system was thoroughly stirred to ensure homogeneity. 0.5 wt% food-grade neutral protease was added, and enzymatic hydrolysis was performed at this temperature for 6 hours. After enzymatic hydrolysis, 5 wt% NaCl was added, and the mixture was allowed to settle. Centrifugation was performed, the precipitate was collected, and the mixture was washed three times with pure water to obtain crude β-glucan.

[0043] (2) Complex enzymatic hydrolysis: The crude β-glucan was prepared into an 8% (w / w) suspension, and the suspension was fully dispersed by high-speed shearing. The pH of the system was adjusted to 6.5 with food-grade citric acid, and the temperature was raised to 45°C. The complex enzyme solution was added at a rate of 500 U / g of substrate dry weight, and the activity ratio of the three enzymes was β-1,3-glucan endonuclease: β-1,6-glucan endonuclease: β-glucosidase = 10:1:0.5. The reaction was started, and the viscosity was monitored online. After 2 hours, when the viscosity dropped to 20% of the initial value, the temperature was immediately raised to 95°C and maintained for 20 minutes to inactivate the enzymes.

[0044] (3) Physical modification: The enzyme-inactivated solution was passed through a pipeline ultrasonic processor with an ultrasonic frequency of 40 kHz and an energy density of 200 J / mL. It was then pumped into a microfluidic homogenizer and homogenized 4 times at a homogenization pressure of 180 MPa, while controlling the temperature of the solution to not exceed 60°C.

[0045] (4) Fractional purification and drying: The physically modified liquid was pumped into a separator and cooled from 70°C to 30°C at a rate of 1.0°C / min, and the clear liquid in the middle and upper parts was collected. The clear liquid was purified by an ultrafiltration membrane system with a molecular weight cutoff of 10 kDa and concentrated to a solid content of 20% (w / w). The concentrate was spray-dried, with the inlet air temperature controlled at 190°C and the outlet air temperature controlled at 95°C, finally obtaining the finished powder.

[0046] Example 3 (1) Pretreatment and extraction: Take 10 kg of food-grade brewer's yeast powder and add pure water to prepare a yeast milk with a solid content of 10% (w / w). Adjust the pH of the system to 5.0 with food-grade citric acid, add 0.01 wt% papain, and autolyze at 45°C for 12 hours. After autolysis, rapidly heat to 85°C to inactivate the enzyme for 10 minutes. Centrifuge and discard the autolysis supernatant. Dilute the obtained precipitate with pure water to make the solid content 8% (w / w). Adjust the pH of the system to 6.0 with food-grade sodium bicarbonate, and turn on the sonication at 45°C with a frequency of 20 kHz and a power density of 0.1 W / cm³. 2 Extraction was assisted for 30 minutes, followed by centrifugation to discard the supernatant. The resulting precipitate was diluted with water to a solid content of 10% (w / w). The pH of the system was adjusted to 7.0 with food-grade sodium bicarbonate. Extraction was carried out at 70°C for 1 hour. The suspension was cooled to 45°C, diluted with pure water to a solid content of 6% (w / w), and thoroughly stirred to ensure homogeneity. 0.05 wt% food-grade neutral protease was added, and enzymatic hydrolysis was performed at this temperature for 2 hours. After enzymatic hydrolysis, 3 wt% NaCl was added, and the mixture was allowed to settle. Centrifugation was performed, the precipitate was collected, and the mixture was washed three times with pure water to obtain crude β-glucan.

[0047] (2) Complex enzymatic hydrolysis: The crude β-glucan was prepared into a 2% (w / w) suspension, and the suspension was fully dispersed by high-speed shearing. The pH of the system was adjusted to 5.5 with food-grade citric acid, and the temperature was raised to 40°C. The complex enzyme solution was added at a rate of 4000 U / g of substrate dry weight, and the activity ratio of the three enzymes was β-1,3-glucan endonuclease: β-1,6-glucan endonuclease: β-glucosidase = 50:5:1. The reaction was started, and the viscosity was monitored online. After 5 hours, when the viscosity dropped to 5% of the initial value, the temperature was immediately raised to 85°C and maintained for 10 minutes to inactivate the enzymes.

[0048] (3) Physical modification: The enzyme-inactivated solution was passed through a pipeline ultrasonic processor with an ultrasonic frequency of 20 kHz and an energy density of 50 J / mL. It was then pumped into a microfluidic homogenizer and homogenized 4 times at a homogenization pressure of 100 MPa, while controlling the temperature of the solution to not exceed 60°C.

[0049] (4) Fractional purification and drying: The physically modified liquid was pumped into a separator and cooled from 50°C to 10°C at a rate of 0.1°C / min. The clear liquid in the middle and upper part was collected. The clear liquid was purified by an ultrafiltration membrane system with a molecular weight cutoff of 10 kDa and concentrated to a solid content of 10% (w / w). The concentrate was spray-dried with the inlet air temperature controlled at 160°C and the outlet air temperature controlled at 80°C to finally obtain the finished powder.

[0050] Comparative Example 1 Traditional alkaline extraction was used. Food-grade brewer's yeast powder from the same batch as in the example was taken, mixed with water, and pre-hydrated to form a homogeneous suspension. This suspension was then homogenized under high pressure to enhance cell wall disruption. Food-grade sodium hydroxide was added to adjust the pH to 13, and alkaline extraction was carried out at 95°C with stirring for 4 hours. This allowed the yeast cell walls to fully lyse under strong alkaline and high-temperature conditions, releasing soluble impurities. After extraction, the sample was centrifuged, and the insoluble crude β-glucan precipitate was collected and repeatedly washed with pure water until the washings were neutral. The precipitate was reconstituted, and alkaline protease and lipase were added. Enzymatic hydrolysis was performed at 55°C for 6 hours to further remove residual protein and lipid impurities. After enzymatic hydrolysis, the precipitate was centrifuged, washed with water, and spray-dried to obtain the traditional process β-glucan powder.

[0051] Comparative Example 2 Similar to Example 1, except that the ultrasonic and microfluidic treatments are omitted, the liquid material after enzymatic hydrolysis is directly subjected to ultrafiltration and drying to obtain the product powder.

[0052] Comparative Example 3 Same as Example 1, but only β-1,3-glucanase was added during enzymatic hydrolysis, excluding β-1,6-glucanase and β-glucosidase. The hydrolysis time was also 3.5 hours, yielding the product powder.

[0053] Analysis and testing methods: 1. Molecular weight determination Sample preparation: Accurately weigh 10 mg of dried sample and dissolve it in 10 mL of 0.1 mol / L NaNO3 solution, containing 0.02% NaN3. Stir and dissolve at 4°C for 24 hours. Filter through a 0.22 μm filter membrane.

[0054] Instrumentation and Columns: Size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS) system was used. This system mainly consists of a high-performance liquid chromatography (HPLC) unit, a multi-angle laser light scattering detector (MALLS), and a differential refractive index detector (DRI) connected in series. Shodex OHpak SB-805 HQ and SB-804 HQ columns were used in series.

[0055] Detection procedure: The mobile phase is 0.1 mol / L NaNO3, containing 0.02% NaN3; the flow rate is 0.5 mL / min; the column temperature is 40°C; and the injection volume is 100 μL.

[0056] Data processing: Using Wyatt ASTRA software, the weight-average molecular weight Mw (kDa) and number-average molecular weight Mn (kDa) were calculated based on the Zimm plot method; the polydispersity index PDI was calculated as Mw / Mn.

[0057] 2. Determination of branching degree by carbon nuclear magnetic resonance spectroscopy Sample preparation: Weigh 50 mg of dry sample, dissolve it in 1 mL of D2O, dissolve it completely at 80°C, freeze dry it, and then dissolve it again in D2O to replace the exchangeable protons. Finally, dissolve it in 1.0 mL of D2O, take the supernatant, and transfer it into an NMR tube.

[0058] Instrument: Nuclear magnetic resonance spectrometer.

[0059] Test method: Quantitative heteronuclear single quantum coherent two-dimensional nuclear magnetic resonance spectra (qHSQC) were acquired using a nuclear magnetic resonance spectrometer, and sufficient scans were performed at 70-80°C to improve the signal-to-noise ratio.

[0060] Data processing: Based on the cross peaks corresponding to the β-1,3 main chain C-3-H and the β-1,6 side chain C-3-H in the qHSQC diagram, the branch I is obtained by integration. br With non-branched I unbr And calculate the branching degree: DB=[I br / (I br +I unbr )]×100%.

[0061] 3. Dispersion stability test Sample preparation: Accurately weigh 0.40 g of sample and add it to 19.60 g of deionized water at 25°C to form a 2% (w / w) dispersion.

[0062] Test procedure: Stir at 300 rpm for 30 minutes using a magnetic stirrer. Transfer 10 mL of the dispersion to a centrifuge tube and centrifuge at 3000 g for 10 minutes.

[0063] Data processing: A certain volume of the upper layer liquid was taken. The total sugar content (C) was determined using the phenol-sulfuric acid method. sup Meanwhile, an equal volume of the original dispersion that had not been centrifuged was taken, and its total sugar content (C) was determined. total Dispersion stability index (%) = (C sup / C total The index is calculated as (1) × 100%. This index reflects the ability of a dispersion to remain homogeneous under centrifugal force. A higher index indicates less sedimentation of β-glucan colloidal particles and a more stable dispersion system.

[0064] 4. Immunological activity test Cell model: HEK293 cells that stably express the human Dectin-1 receptor were used as model cells.

[0065] Sample preparation: Each β-glucan sample was labeled with fluorescein isothiocyanate (FITC) to prepare the sample solution to be tested.

[0066] Detection procedure: HEK-Dectin-1 cells were incubated with different concentrations of FITC-labeled β-glucan at 4°C for 2 hours. After incubation, the cells were washed, and the mean fluorescence intensity (MFI) of cell binding was detected by flow cytometry.

[0067] Data processing: Untransfected HEK293 cells were used as a negative control. Given that the β-glucan prepared in Example 2 has a near-optimal molecular weight and β-1,6 branching degree, it exhibited the highest binding signal in the Dectin-1 receptor binding assay. Therefore, the binding capacity of Example 2 was set as 100% baseline, and the relative activity of other samples was calculated as (MFI). sample / MFI 实施例2 )×100%.

[0068] Table 2 Comparison of weight-average molecular weight, polydispersity index, and branching degree of β-1,6 branches for each example and comparative example.

[0069] Table 3 Comparison of dispersion stability index, immunogenicity, and sensory evaluation of each embodiment and comparative example.

[0070] Analyze the results in Tables 2 and 3: (1) The weight-average molecular weight of the products obtained in Examples 1-3 is between 105-188 kDa, and the molecular weight distribution is relatively narrow, with a PDI ≤ 2.1 and a branching degree of β-1,6 branches between 10.3-22.4%, as shown in the attached figure. Figure 3As shown in the figure. In contrast, the strong alkaline method in Comparative Example 1 resulted in severe debranching, with the β-1,6 branching degree being only 3.2% and an extremely wide molecular weight distribution, directly leading to extremely poor dispersion stability and immunomodulatory activity. This demonstrates that the mild extraction and precise enzymatic hydrolysis strategy of this invention has a fundamental advantage in protecting the active structure.

[0071] (2) The dispersion stability and immunomodulatory activity of Examples 1-3 were significantly higher than those of Comparative Example 1 prepared by the traditional process, as shown in the appendix. Figure 4 This demonstrates the superiority of the preparation method of the present invention compared to traditional processes. Although Example 1 and Comparative Example 2 have almost the same molecular weight and branching degree, the dispersion stability of Comparative Example 2 without physical modification is significantly reduced. This proves that ultrasonic-microfluidic synergistic treatment is a key step in breaking intermolecular hydrogen bond association and achieving high dispersibility; simply reducing the molecular weight is insufficient to solve application problems.

[0072] (3) Comparative Example 3 used only a single endonuclease, resulting in excessive branching of the PDI and β-1,6 branches in the product. This not only affected its dispersibility and stability but also led to a decrease in immunomodulatory activity, as shown in the appendix. Figure 4 This comparative example highlights the important role of the multi-enzyme synergistic system in achieving molecular weight uniformity and precise control of branching in this invention.

[0073] In summary, this invention provides an advanced, environmentally friendly, and high-performance yeast β-glucan preparation technology. Segmented and gentle extraction preserves the inherent structure of the raw materials; multi-enzyme synergy enables precise molecular-scale tailoring; and ultrasonic-microfluidic technology achieves a qualitative leap in macroscopic physical properties. This technology resolves the contradiction between high activity and high dispersibility in existing technologies, possessing significant industrial application potential and market value.

Claims

1. A method for preparing branched yeast β-glucan, comprising crude extraction, complex enzymatic hydrolysis, physical modification, fractional purification and drying, characterized in that, Includes the following steps: S1: Crude extraction of food-grade brewing yeast powder is carried out in sequence, including activation autolysis treatment, segmented water extraction treatment, enzymatic hydrolysis and salting out impurities, and finally, crude β-glucan is obtained by solid-liquid separation and washing. The specific method for the activation autolysis treatment is as follows: dilute food-grade brewer's yeast dry powder with pure water to a solid content of 10-20% (w / w), adjust the pH of the system to 5.0-6.0, and stir autolysis at 45-50°C for 12-24 hours; Add 0.01-0.1 wt% of papain to assist autolysis. After autolysis, rapidly heat to 85-95°C and keep warm for 10-15 minutes to inactivate endogenous enzymes. Centrifuge to collect the yeast precipitate. The segmented water extraction process involves first performing a low-temperature primary water extraction, followed by a medium-temperature secondary water extraction. The specific method for the low-temperature primary water extraction is as follows: the yeast precipitate is prepared into a suspension with a solid content of 8.0-12.0% (w / w) using pure water; the pH of the system is adjusted to 6.0-7.0; and the mixture is subjected to ultrasound at 45-55°C, with an ultrasound frequency of 20-40 kHz and an ultrasound power density of 0.1-0.3 W / cm³. 2 After stirring and extracting for 30-60 minutes, centrifuge to separate and collect the precipitate; the specific method of the medium-temperature secondary water extraction is as follows: the precipitate after the primary water extraction is prepared again with pure water to form a suspension with a solid content of 10-20% (w / w), the pH is adjusted to 7.0-8.0, the temperature is raised to 70-80°C, and the mixture is stirred and extracted for 1-2 hours. The specific method for enzymatic hydrolysis and salting out impurities is as follows: the suspension after segmented water extraction is cooled to 45-55°C, diluted with pure water to a solid content of 6% (w / w), stirred thoroughly, and 0.05-0.5 wt% of food-grade neutral protease is added. After enzymatic hydrolysis for 2-6 hours, 3-5 wt% sodium chloride is added for salting out, so that the dissolved proteins and nucleic acids flocculate and precipitate. S2: The crude β-glucan obtained in step S1 is prepared into a suspension with a solid content of 2-8% (w / w) using pure water. The suspension is fully dispersed by high-speed shearing. The pH of the system is adjusted to 5.5-6.5, and the temperature is controlled at 40-50°C. The compound enzyme solution is added to carry out the enzymatic hydrolysis reaction. During the reaction, the viscosity of the reaction system is monitored in real time to adjust the enzymatic hydrolysis time and temperature. When the viscosity of the system drops to 5-20% of the initial viscosity value, the temperature is rapidly increased to 85-95°C and maintained for 10-20 minutes to inactivate the enzyme, and the enzymatic hydrolysis solution is obtained. The compound enzyme solution is prepared by mixing β-1,3-glucan endonuclease, β-1,6-glucan endonuclease and β-glucosidase in a ratio of (10-50):(1-5):(0.5-2) activity units. The amount of the compound enzyme solution added is 500-4000 U / g substrate dry weight. S3: The enzymatically hydrolyzed solution obtained in S2 is physically modified by first performing ultrasonic dispersion treatment, followed by high-pressure microfluidic homogenization treatment to break down aggregates and construct a stable dispersion structure, thereby obtaining a physically modified solution. The conditions for ultrasonic dispersion treatment are: ultrasonic frequency of 20-40kHz, and total applied energy density of 50-200J / mL. The conditions for high-pressure microfluidic homogenization treatment are: homogenization pressure set at 100-180MPa, 3-5 cycles, and the solution temperature kept below 60°C throughout the process. S4: The physically modified liquid obtained in S3 is subjected to programmed cooling and fractionation precipitation to separate and collect the upper liquid phase rich in the target component; the upper liquid phase is purified by membrane separation, concentrated and spray-dried to obtain yeast β-glucan product with a weight average molecular weight of 100-200 kDa and a branching degree of 10-23% for β-1,6 branches; the specific method of programmed cooling and fractionation precipitation is as follows: at an initial temperature of 50-70°C, the liquid is cooled to 10-30°C at a programmed rate of 0.1-1.0°C / min, and the upper liquid phase is obtained by separation; the specific method of membrane separation purification is as follows: the upper liquid phase is separated by a membrane module with a molecular weight cutoff of 10 kDa to remove inorganic salts, small molecule sugars and peptide impurities.

2. The method for preparing branched yeast β-glucan as described in claim 1, characterized in that, The method for adjusting the pH of the system described in steps S1 and S2 is achieved by adding food-grade citric acid or food-grade sodium bicarbonate.

3. The method for preparing branched yeast β-glucan as described in claim 1, characterized in that, The conditions for spray drying in step S4 are: inlet air temperature of 160-190°C and outlet air temperature of 80-95°C.

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