Microorganism preparation method of low-molecular-weight water-soluble beta-glucan

Abstract

The invention discloses a microbial preparation method of low-molecular-weight water-soluble beta-glucan, and belongs to the technical field of water-soluble beta-glucan preparation, and the microbial preparation method specifically comprises the following steps: carrying out liquid fermentation culture on chaetomium JY25 with the preservation number of CGMCC No.6882 to obtain fermentation liquor; carrying out solid-liquid separation on the fermentation liquor to obtain crude enzyme containing a beta-glucan degrading enzyme system; carrying out enzymolysis reaction on the crude enzyme liquid and an insoluble yeast beta-glucan substrate under the conditions that the pH is 4.0-6.0 and the temperature is 50-65 DEG C to obtain an enzymolysis product; terminating the reaction, and separating and purifying an enzymolysis product to obtain the low-molecular-weight water-soluble beta-glucan with the weight-average molecular weight of 25kDa-30kDa; the invention provides an efficient, green and controllable new method for preparing the low-molecular-weight and water-soluble beta-glucan, and systematically solves multiple limitations of a traditional method in the aspects of efficiency, controllability, environmental protection property and product activity maintenance through combination of a specific functional strain, a synergistic compound enzyme system and an optimized reaction process.

Description

Technical Field

[0001] This invention belongs to the field of water-soluble β-glucan preparation technology, and relates to a microbial preparation method for low molecular weight, water-soluble β-glucan. Background Technology

[0002] β-glucan is a natural polysaccharide compound composed of glucose molecules linked by β-1,3-glycosidic bonds to form the backbone and β-1,6-glycosidic bonds to form the branches, meaning that glucose molecules branch out from the backbone via β-1,6-glycosidic bonds. β-glucan is widely found in plants, fungi, and bacteria, and possesses various physiological functions such as immune regulation, anti-tumor activity, lipid-lowering, and blood sugar-lowering effects. It has been widely applied in medicine, health products, and food industries.

[0003] Yeast β-glucan is a major component of the yeast cell wall, located in the inner layer of the cell wall and accounting for approximately 60% of its dry weight. It is an abundant source of natural glucans. However, due to its dense triple helix structure and high molecular weight, yeast β-glucan exhibits extremely poor solubility, limiting its applications. Currently, common methods to improve the water solubility of yeast β-glucan include physical and chemical methods. For example, acid degradation is used to prepare water-soluble yeast β-glucan, and it has been found that treatment with 45% sulfuric acid at 20°C degrades water-insoluble β-glucan into low-molecular-weight β-glucan. However, existing physical or chemical methods suffer from low conversion rates, product purity that fails to meet market demands, high energy consumption during production, and severe pollution.

[0004] In contrast, enzymatic hydrolysis is considered a more promising research direction due to its mild conditions, high specificity, and environmental friendliness. Existing technologies have publicly used β-1,3-glucanase for treatment, but this method currently typically relies on a single enzyme preparation, limiting its applicability to yeast β-glucan. This is because yeast β-glucan has a complex network structure composed of a β-1,3 backbone and β-1,6 side chains, resulting in very limited degradation efficiency and making efficient and controllable decomposition difficult. Furthermore, although some literature reports that *Chaetoceros* species can secrete β-1,3-glucanase, its research and application are concentrated in the field of biological control of agricultural plant diseases. There are no publicly reported studies on obtaining synergistic complex enzyme systems through microbial fermentation specifically for preparing yeast β-glucan with a specific low molecular weight range and high water solubility.

[0005] Therefore, developing a green and environmentally friendly microbial preparation method that is mild, efficient, controllable, and capable of targeted preparation of yeast β-glucan with specific low molecular weight and high solubility is of great value. Summary of the Invention

[0006] The purpose of this invention is to overcome the limitations of existing physical and chemical modification methods for yeast β-glucan, such as harsh conditions, process pollution, and uncontrollable product molecular weight, as well as the low degradation efficiency and high cost of existing enzymatic methods for its complex branched structure. This invention provides a mild, precise, controllable, efficient, and green microbial method for preparing low molecular weight water-soluble β-glucan.

[0007] The technical solution adopted in this invention specifically includes: A microbial method for preparing low molecular weight, water-soluble β-glucan, the key feature of which is that the preparation method specifically includes: S1. The *Chaetoceros* JY25 with preservation number CGMCC No. 6882 was subjected to liquid fermentation culture to obtain fermentation broth; S2. The fermentation broth obtained in step S1 is subjected to solid-liquid separation to obtain a crude enzyme solution containing β-glucan degrading enzyme system. S3. The above crude enzyme solution and insoluble yeast β-glucan substrate were subjected to enzymatic hydrolysis at pH 4.0-6.0 and temperature 50℃-65℃ to obtain the enzymatic hydrolysis product. S4. Terminate the reaction and separate and purify the enzymatic hydrolysis product to obtain low molecular weight, water-soluble β-glucan with a weight average molecular weight of 25kDa to 30kDa.

[0008] Furthermore, the culture medium used in the liquid fermentation culture of step S1 contains: 8 g / L to 12 g / L tryptone, 4 g / L to 6 g / L yeast extract, 8 g / L to 12 g / L sodium chloride, and the initial pH of the culture medium is 7.0 ± 0.2.

[0009] Specifically, the conditions for the liquid fermentation culture in step S1 are: temperature 26℃~30℃, shaking speed 160r / min~200r / min, and culture time 20h~28h.

[0010] Furthermore, in step S2, the solid-liquid separation is performed by centrifugation at 8000 r / min to 12000 r / min for 5 min to 15 min. After centrifugation, the supernatant is collected to obtain the crude enzyme solution containing the β-glucan-degrading enzyme system; simultaneously, the precipitate is collected, which mainly consists of insoluble yeast β-glucan substrate that has not been fully hydrolyzed, and can be directly returned to the reaction system in step S3 for recycling.

[0011] Specifically, the pH of the enzymatic hydrolysis reaction in step S3 is 4.5 to 5.5.

[0012] More specifically, in step S3, the temperature of the above enzymatic hydrolysis reaction is 57℃~63℃, and the time of the enzymatic hydrolysis reaction is 6h~10h.

[0013] It should be noted that in step S3, the above-mentioned insoluble yeast β-glucan substrate is added in the form of a suspension with a mass concentration of 1.7% to 2.3%.

[0014] It should be noted that the pH of the above enzymatic hydrolysis reaction is achieved by adjusting the initial pH of the insoluble yeast β-glucan substrate suspension, and the pH value of the reaction system is basically maintained at the initial pH of the suspension.

[0015] Preferably, the suspension contains 5% sorbitol by mass.

[0016] It should be noted that in step S3, the volume-to-mass ratio of the crude enzyme solution to the insoluble yeast β-glucan substrate is 100 mL: (15.3 g to 20.7 g).

[0017] Most importantly, the solubility of the aforementioned low molecular weight, water-soluble β-glucan in water at 25°C is not less than 100 g / L.

[0018] Compared with the prior art, the present invention has the following advantages: First, the specific Chaetomium species JY25 (CGMCC No. 6882) used in this invention has a clear plant endophytic origin and recognized biocompatibility, laying the foundation for the product's food and pharmaceutical applications. This strain originates from the endophytic fungus of the medicinal and edible plant Gynostemma pentaphyllum, and its source environment is natural and safe. More importantly, when applied, this strain can efficiently secrete a complex degradative enzyme system targeting the complex structure of yeast β-glucan even in ordinary fermentation media without the addition of specific inducers such as β-glucan. This phenomenon not only demonstrates its unique metabolic mechanism, distinguishing it from Chaetomium species, but also simplifies production steps, reduces production costs, and greatly facilitates its industrial application.

[0019] Secondly, this invention discloses a simplified process for enzymatic hydrolysis using the entire fermentation broth, eliminating the need for complex enzyme extraction and purification, significantly reducing production costs and operational complexity. Unlike traditional methods that require the separation and purification of enzyme preparations before the reaction, this invention directly utilizes the crude enzyme broth from the complete fermentation of Chaetomium JY25 for enzymatic hydrolysis. The crude enzyme broth of this invention, as a multifunctional whole, not only omits complex enzyme purification processes, making the production process more economical, efficient, and simplified, but also exhibits a significantly higher degradation efficiency than many commercially available β-glucanases.

[0020] Third, the enzymatic hydrolysis rate obtained by this invention has clear industrial application value and economic benefits. Under optimized process conditions, the enzymatic hydrolysis rate of insoluble yeast β-glucan by this invention remains at a high level, especially when using sorbitol as a promoter, the hydrolysis rate can reach 48.7%. This high conversion rate ensures the effective utilization of raw materials and meets the core requirement of yield for large-scale production.

[0021] Fourth, this invention successfully achieves precise control over the molecular weight of the product while simultaneously achieving efficient degradation, resulting in highly uniform weight-average molecular weight. The weight-average molecular weight of the products in all embodiments was precisely controlled within the target range of 25kDa to 30kDa. This high degree of controllability overcomes the technical challenge of product inhomogeneity in chemical methods and existing enzymatic methods, enabling the stable preparation of standardized low-molecular-weight β-glucan products that meet application requirements.

[0022] Fifth, the entire process of this invention is mild, environmentally friendly, and embodies the concept of a circular economy. The entire reaction is carried out in an aqueous phase, and can be performed efficiently over a wide temperature and pH range, without the need for strong acids, strong alkalis, or organic solvents. It is particularly noteworthy that the residual insoluble substrate after the enzymatic hydrolysis reaction (unfully hydrolyzed yeast β-glucan) can be easily recovered by centrifugation and recycled back into the enzymatic hydrolysis system. Therefore, this invention significantly improves the overall utilization rate of raw materials, reduces waste generation, and further enhances the greenness and economy of the process.

[0023] In summary, this invention not only provides an efficient, green, and controllable microbial method for preparing low molecular weight water-soluble β-glucan, but also comprehensively solves multiple technical problems of traditional methods in terms of safety, cost, efficiency, environmental protection, and product quality through the systematic combination of safe and specific strain resources, a simple process that does not require purification, high conversion rate and precise product control, and a recyclable green circular design. It has broad prospects for industrial application. Preservation information: China General Microbiological Culture Collection Center, CGMCC NO.6882, Chaetomium sp. 2012-11-26 Attached Figure Description

[0024] Figure 1 The total sugar content standard curve was plotted using the phenol-sulfuric acid method with glucose as the standard.

[0025] Figure 2 This is a photograph of sample 1 prepared in Example 1.

[0026] Figure 3 This is a photo of commercially available water-insoluble yeast beta-glucan.

[0027] Figure 4 This refers to the dissolution effect of sample 1.

[0028] Figure 5 This refers to the solubility of commercially available water-insoluble yeast β-glucan.

[0029] Figure 6This is the gel permeation chromatography (GPC / SEC) chromatogram of sample 1.

[0030] Figure 7 This is a gel permeation chromatography (GPC / SEC) chromatogram of commercially available water-insoluble yeast β-glucan.

[0031] Figure 8 The infrared spectra (IR spectra) are of water-soluble β-glucan, represented by sample 1, and commercially available insoluble yeast β-glucan.

[0032] In the attached figures, 1 represents the dissolution effect of sample 1 at a concentration of 5 g / L, 2 represents the dissolution effect of sample 1 at a concentration of 25 g / L, 3 represents the dissolution effect of sample 1 at a concentration of 50 g / L, 4 represents the dissolution effect of sample 1 at a concentration of 75 g / L, 5 represents the dissolution effect of sample 1 at a concentration of 100 g / L, 6 represents the dissolution effect of commercially available water-insoluble yeast β-glucan at a concentration of 5 g / L, 7 represents the dissolution effect of commercially available water-insoluble yeast β-glucan at a concentration of 25 g / L, 8 represents the dissolution effect of commercially available water-insoluble yeast β-glucan at a concentration of 50 g / L, 9 represents the dissolution effect of commercially available water-insoluble yeast β-glucan at a concentration of 75 g / L, and 10 represents the dissolution effect of commercially available water-insoluble yeast β-glucan at a concentration of 100 g / L. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0038] Unless otherwise specified in the examples, the procedures can be followed according to conventional conditions. If the manufacturers of the reagents or instruments used are not specified, such as yeast β-glucan (water insoluble), they are all conventional products that can be purchased commercially.

[0039] The *Chaetoceros* strain JY25 (hereinafter referred to as *Chaetoceros* JY25) with accession number CGMCC No. 6882 used in this invention was obtained from Henan University of Technology. After activation, the lyophilized powder of this strain was plate-cultured for 7 days. Mycelia from the colony edges were picked and inoculated into liquid seed culture medium (components included: 10 g / L tryptone; 5 g / L yeast extract; 10 g / L sodium chloride, pH=7.0, sterilized at 115℃ for 30 min, cooled for later use). The medium was cultured at 28℃ and 180 rpm for 24 h with shaking to obtain a seed culture with good mycelial growth and uniform appearance. The seed culture was aseptically aliquoted into 1 mL sterile centrifuge tubes and stored at -80℃ for long-term preservation.

[0040] To ensure the activity and genetic stability of the strains, the frozen strains are revived and retested every 6 months, and their morphological characteristics are verified by plate culture and microscopic observation to ensure that they can be used for subsequent fermentation.

[0041] In all embodiments of the present invention, the seed liquid prepared and activated by the above method is used directly for inoculation. Example 1

[0042] This embodiment provides a microbial method for preparing low molecular weight, water-soluble β-glucan. The specific process is as follows: S1. The *Chaetoceros* JY25 is subjected to liquid fermentation to obtain the fermentation broth, which specifically includes: Place the frozen Chaetomium JY25 in a clean bench for 1 hour and wait for it to return to room temperature. Then add one tube of Chaetomium JY25 seed culture to the liquid fermentation medium and incubate at 28°C with a shaking speed of 180 r / min for 24 hours. The liquid fermentation medium consisted of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride. The initial pH of the medium was 7.0. The medium was sterilized at 115°C for 30 min and then cooled before use.

[0043] S2. Centrifuge the fermentation broth obtained in step S1 at 10000 r / min for 10 min to obtain a crude enzyme solution containing β-glucan degrading enzyme system.

[0044] S3, enzymatic hydrolysis reaction, specifically including: The crude enzyme solution was added to a suspension containing 2.0% yeast β-glucan substrate at a volume ratio of 1:9 for enzymatic hydrolysis. The hydrolysis temperature was 60℃, the shaking speed was 180 r / min, and the hydrolysis time was 8 h to obtain the hydrolysate. The preparation process of a suspension containing 2.0% yeast β-glucan substrate is as follows: Take 1L of deionized water and adjust the pH of the solution to 5.0 with 1mol / L dilute hydrochloric acid; weigh 4g of yeast β-glucan and add it to a 500mL Erlenmeyer flask, and add 196mL of the above-prepared deionized water with pH=5.0 to the 500mL Erlenmeyer flask to prepare a suspension containing yeast β-glucan substrate for enzymatic hydrolysis.

[0045] S4. Terminate the reaction and separate and purify to obtain water-soluble β-glucan: Take 1g of enzyme hydrolysate into a 100mL centrifuge tube, add 59g of deionized water for dilution, centrifuge at 25℃ and 10000r / min for 10min, and collect the supernatant. The total sugar content in the supernatant was determined using the phenol-sulfuric acid method, and the enzymatic hydrolysis rate of β-glucan was calculated. Separate the enzymatic hydrolysate according to the above method to obtain the supernatant, add 6 times the volume of the collected supernatant of anhydrous ethanol, and let it stand at 4°C for 12 hours for alcohol precipitation. Centrifuge at 5000 r / min for 20 min, collect the precipitate, and wash the precipitate with anhydrous ethanol and acetone respectively; at the same time, collect the precipitate (mainly undigested insoluble yeast β-glucan substrate), which can be directly returned to the reaction system in step S3 for recycling; The precipitate was placed in a cold trap at a temperature of -40℃, a vacuum degree of 10Pa, and a drying time of 24h to obtain water-soluble β-glucan dry powder, which was designated as sample 1. Example 2

[0046] This embodiment provides a microbial method for preparing low molecular weight, water-soluble β-glucan. The specific process is as follows: S1. The *Chaetoceros* JY25 is subjected to liquid fermentation to obtain the fermentation broth, which specifically includes: Place the frozen Chaetomium JY25 in a clean bench for 1 hour and wait for it to return to room temperature. Then add one tube of Chaetomium JY25 seed culture to the liquid fermentation medium and incubate at 30°C with a shaking speed of 200 r / min for 20 hours. The liquid fermentation medium consisted of 12 g / L tryptone, 6 g / L yeast extract, and 12 g / L sodium chloride. The initial pH of the medium was 6.8. The medium was sterilized at 115°C for 30 min and then cooled before use.

[0047] S2. Centrifuge the fermentation broth obtained in step S1 at 12000 r / min for 5 min to obtain a crude enzyme solution containing β-glucan degrading enzyme system.

[0048] S3, enzymatic hydrolysis reaction, specifically including: The crude enzyme solution was added to a suspension containing 2.2% yeast β-glucan substrate at a volume ratio of 1:9 for enzymatic hydrolysis. The hydrolysis temperature was 63℃, the shaking speed was 200 r / min, and the hydrolysis time was 6 h to obtain the hydrolysate. The preparation process of a suspension containing 2.2% yeast β-glucan substrate is as follows: Take 1L of deionized water and adjust the pH of the solution to 5.5 with 1mol / L dilute hydrochloric acid; weigh 4g of yeast β-glucan and add it to a 500mL Erlenmeyer flask, and add 196mL of the above-prepared deionized water with pH=5.5 to the 500mL Erlenmeyer flask to obtain a suspension containing yeast β-glucan substrate for enzymatic hydrolysis.

[0049] S4. Terminate the reaction and separate and purify to obtain water-soluble β-glucan: Take 1g of enzyme hydrolysate into a 100mL centrifuge tube, add 59g of deionized water for dilution, centrifuge at 25℃ and 12000r / min for 5min, and collect the supernatant. The total sugar content in the supernatant was determined using the phenol-sulfuric acid method, and the enzymatic hydrolysis rate of β-glucan was calculated. Separate the enzymatic hydrolysate according to the above method to obtain the supernatant, add 8 times the volume of the collected supernatant of anhydrous ethanol, and let it stand at 4°C for 12 hours for alcohol precipitation. Centrifuge at 6000 r / min for 15 min, collect the precipitate, and wash the precipitate with anhydrous ethanol and acetone respectively; at the same time, collect the precipitate (mainly undigested insoluble yeast β-glucan substrate), which can be directly returned to the reaction system in step S3 for recycling. The precipitate was placed in a cold trap at a temperature of -35℃, a vacuum of 8Pa, and a drying time of 20h to obtain water-soluble β-glucan dry powder, which was designated as sample 2. Example 3

[0050] This embodiment provides a microbial method for preparing low molecular weight, water-soluble β-glucan. The specific process is as follows: S1. The *Chaetoceros* JY25 is subjected to liquid fermentation to obtain the fermentation broth, which specifically includes: Place the frozen Chaetomium JY25 in a clean bench for 1 hour and wait for it to return to room temperature. Then add one tube of Chaetomium JY25 seed culture to the liquid fermentation medium and incubate at 26°C with a shaking speed of 160 r / min for 28 hours. The liquid fermentation medium consisted of 8 g / L tryptone, 4 g / L yeast extract, and 8 g / L sodium chloride. The initial pH of the medium was 7.2. The medium was sterilized at 115°C for 30 min and then cooled before use.

[0051] S2. Centrifuge the fermentation broth obtained in step S1 at 8000 r / min for 15 min to obtain a crude enzyme solution containing β-glucan degrading enzyme system.

[0052] S3, enzymatic hydrolysis reaction, specifically including: The crude enzyme solution was added to a suspension containing 1.8% yeast β-glucan substrate at a volume ratio of 1:9 for enzymatic hydrolysis. The hydrolysis temperature was 57℃, the shaking speed was 160 r / min, and the hydrolysis time was 10 h to obtain the hydrolysate. The preparation process of a suspension containing 1.8% yeast β-glucan substrate is as follows: Take 1L of deionized water and adjust the pH of the solution to 4.5 with 1mol / L dilute hydrochloric acid; weigh 4g of yeast β-glucan and add it to a 500mL Erlenmeyer flask, and add 196mL of the above-prepared deionized water with pH=4.5 to the 500mL Erlenmeyer flask to obtain a suspension containing yeast β-glucan substrate for enzymatic hydrolysis.

[0053] S4. Terminate the reaction and separate and purify to obtain water-soluble β-glucan: Take 1g of enzyme hydrolysate into a 100mL centrifuge tube, add 59g of deionized water for dilution, centrifuge at 25℃ and 8000r / min for 15min, and collect the supernatant. The total sugar content in the supernatant was determined using the phenol-sulfuric acid method, and the enzymatic hydrolysis rate of β-glucan was calculated. Separate the enzymatic hydrolysate according to the above method to obtain the supernatant. Add anhydrous ethanol at 5 times the volume of the collected supernatant and let it stand at 4°C for 12 hours for alcohol precipitation. Centrifuge at 4000 r / min for 25 min, collect the precipitate, and wash the precipitate with anhydrous ethanol and acetone respectively; at the same time, collect the precipitate (mainly undigested insoluble yeast β-glucan substrate), which can be directly returned to the reaction system in step S3 for recycling; The precipitate was placed in a cold trap at a temperature of -45℃, a vacuum degree of 12Pa, and a drying time of 28h to obtain water-soluble β-glucan dry powder, which was designated as sample 3. Example 4

[0054] This embodiment provides a microbial preparation method for low molecular weight, water-soluble β-glucan. The specific process is the same as in Embodiment 1, except that: in step S3, the enzymatic hydrolysis temperature is 50℃, the enzymatic hydrolysis time is 10h, the pH of the yeast β-glucan substrate suspension is 4.0, the total sugar content in the supernatant is determined by the phenol-sulfuric acid method, the enzymatic hydrolysis rate of β-glucan is calculated, and water-soluble β-glucan dry powder is obtained, which is designated as Sample 4. Example 5

[0055] This embodiment provides a microbial preparation method for low molecular weight, water-soluble β-glucan. The specific process is the same as in Embodiment 1, except that: in step S3, the enzymatic hydrolysis temperature is 65℃, the enzymatic hydrolysis time is 6h, the pH of the yeast β-glucan substrate suspension is 6.0, the total sugar content in the supernatant is determined by the phenol-sulfuric acid method, the enzymatic hydrolysis rate of β-glucan is calculated, and water-soluble β-glucan dry powder is obtained, which is designated as Sample 5. Example 6

[0056] This embodiment provides a microbial preparation method for low molecular weight, water-soluble β-glucan. The specific process is the same as in Embodiment 1, except that: the yeast β-glucan substrate suspension prepared in step S3 contains 5% sorbitol by mass. The total sugar content in the supernatant is determined by the phenol-sulfuric acid method, and the enzymatic hydrolysis rate of β-glucan is calculated to obtain water-soluble β-glucan dry powder, which is designated as Sample 6. Comparative Example 1

[0057] This comparative example provides a method for preparing water-soluble β-glucan. The specific steps are the same as in Example 1, except that commercially available β-glucanases No. 1 to No. 3 are used instead of the crude enzyme solution used in Example 1. The specific steps include: S1. Preparation of commercially available enzyme solutions: Weigh 5g of β-glucanase No. 1 to No. 3 respectively, dissolve them in disodium hydrogen phosphate-citrate buffer at pH 5.0 and bring the volume up to 10mL to prepare commercially available enzyme solutions No. 1 to No. 3.

[0058] S2, Enzymatic hydrolysis reaction: Same as step S3 in Example 1, except that commercially available β-glucanase is used instead of the crude enzyme solution used in Example 1, and the amount of commercially available β-glucanase added (based on dry powder) is 0.3% of the dry weight of the yeast β-glucan substrate.

[0059] S3. Terminate the reaction and separate and purify to obtain water-soluble β-glucan: Same as step S4 in Example 1. Determine the total sugar content in the supernatant using the phenol-sulfuric acid method, calculate the enzymatic hydrolysis rate of β-glucan, and prepare β-glucan dry powder. Information on the commercially available β-glucanase used and the obtained reference standard number are as follows: β-glucanase No. 1: from Longkote Enzyme Preparation Co., Ltd., enzyme activity 50,000 U / g, the resulting β-glucan dry powder is designated as reference standard 1-1; β-glucanase No. 2: from Ningxia Xiasheng Industrial Group Co., Ltd., specification model BGS, enzyme activity 140,000 U / g, the obtained β-glucan dry powder is designated as reference standard 1-2; β-glucanase No. 3: from Ningxia Xiasheng Industrial Group Co., Ltd., specification SPE-005 for plant extraction, enzyme activity 120,000 U / g, the obtained β-glucan dry powder is designated as reference standard 1-3.

[0060] Note: The aforementioned commercially available β-glucanases are all commercially available enzyme preparations. Their product names, models, and nominal enzyme activities are all from publicly available information from the manufacturers. None of the manufacturers' instructions or publicly available information explicitly provide the specific enzyme type and proportions. Comparing commercially available enzymes from different sources, models, and activities is a routine and necessary practice in this field when evaluating the advancement of new technological solutions. The core purpose of this comparative example is to objectively compare the effect of the crude enzyme solution prepared by this invention with that of existing technology (i.e., commercially available enzymes) on the same substrate under the same conditions, in order to verify the relative advantages of the technical solution of this invention. The specific enzyme component proportions within the commercially available enzymes are technical details of the manufacturers and do not affect the rationality and legal validity of comparing them as a whole existing technology product in this patent, nor do they affect the ability of those skilled in the art to repeat this comparative experiment.

[0061] It should be noted that all enzymatic hydrolysis performance data (such as hydrolysis rate and product molecular weight) in this comparative example represent only the comparison results between the specific commercial enzyme and the crude enzyme solution produced by this invention under the specific reaction system and process parameters set in this invention. These results serve solely for the purpose of demonstrating patent inventiveness and do not constitute any evaluation of the overall quality of any product from any manufacturer or its performance in other application scenarios.

[0062] In addition, to illustrate the rationale for selecting these models, their typical industrial applications are briefly described: β-glucanase 2 (BGS type) is mainly used in the feed industry to reduce the viscosity of grains; β-glucanase No. 3 (plant extraction specific type) is mainly used in the extraction process of plant active ingredients.

[0063] By comparing them with general-purpose enzyme preparations (No. 1), the invention's degradation scheme for yeast β-glucan, a specific substrate, can be more comprehensively demonstrated to have unexpected technical advantages over various existing commercial enzymes designed for different purposes. Comparative Example 2

[0064] This comparative example provides a method for preparing water-soluble β-glucan. The specific steps are the same as in Example 1, except that the temperature parameters of the enzymatic hydrolysis reaction are adjusted. The specific steps include: S1. The *Trichoderma* JY25 is subjected to liquid fermentation culture to obtain fermentation broth: the specific steps are the same as step S1 in Example 1.

[0065] S2. Separate the fermentation broth obtained in step S1 into a solid-liquid mixture to obtain a crude enzyme solution: The specific steps are the same as step S2 in Example 1.

[0066] S3, Enzymatic hydrolysis reaction: The crude enzyme solution was added to a suspension containing 2.0% yeast β-glucan substrate at a volume ratio of 1:9 for enzymatic hydrolysis. The hydrolysis temperature was adjusted to 45°C, and the pH, shaking speed, and hydrolysis time were the same as in Example 1 to obtain the hydrolysate.

[0067] S4. Terminate the reaction and separate and purify to obtain water-soluble β-glucan: The steps are the same as S4 in Example 1. The total sugar content in the supernatant is determined by the phenol-sulfuric acid method, the enzymatic hydrolysis rate of β-glucan is calculated, and β-glucan dry powder is prepared and designated as control 2. Comparative Example 3

[0068] This comparative example provides a method for preparing water-soluble β-glucan. The specific steps are the same as in Example 1, except that the pH parameters of the enzymatic hydrolysis reaction are adjusted. The specific steps include: S1. The *Trichoderma* JY25 is subjected to liquid fermentation culture to obtain fermentation broth: the specific steps are the same as step S1 in Example 1.

[0069] S2. Separate the fermentation broth obtained in step S1 into a solid-liquid mixture to obtain a crude enzyme solution: The specific steps are the same as step S2 in Example 1.

[0070] S3, Enzymatic hydrolysis reaction: When preparing a 2% (w / w) yeast β-glucan substrate suspension for enzymatic hydrolysis, the pH was adjusted to 7.0. The crude enzyme solution was added to the suspension at a volume ratio of 1:9, and the hydrolysate was carried out for 8 hours at pH 7.0, temperature 60℃, and shaking speed 180 r / min to obtain the hydrolysate.

[0071] S4. Terminate the reaction and separate and purify to obtain water-soluble β-glucan: The steps are the same as S4 in Example 1. The total sugar content in the supernatant is determined by the phenol-sulfuric acid method, the enzymatic hydrolysis rate of β-glucan is calculated, and β-glucan dry powder is prepared and designated as reference standard 3. Analysis and Testing

[0072] I. Enzymatic hydrolysis rate analysis of β-glucan: This invention uses the phenol-sulfuric acid method to determine the total sugar content and calculate the enzymatic hydrolysis rate of β-glucan. The specific procedure is as follows: Weigh 5.064g of phenol into a beaker and add about 50mL of deionized water. Heat in a 70℃ water bath until the phenol is completely dissolved. After the phenol solution returns to room temperature, transfer it to a 100mL volumetric flask and dilute to 100mL with deionized water to obtain the phenol solution. Take 100 μL of phenol solution and 100 μL of the supernatant obtained after enzymatic hydrolysis and centrifugation into a glass test tube, add 500 μL of concentrated sulfuric acid, mix well, incubate at 30℃ for 30 min, and measure using an ELISA reader on A480. Calculate the total sugar concentration in the supernatant of the enzymatic hydrolysate according to the standard curve. Simultaneously, plot the total sugar content standard curve using glucose as the standard. See [link to standard curve]. Figure 1 .

[0073] According to the standard curve ( The total sugar content in the supernatant of the enzymatic hydrolysate was calculated, and the enzymatic hydrolysis rate of dextran was calculated according to Equation 1. The results are shown in Table 1.

[0074] Formula 1 in, C 0: Total sugar concentration in the reaction system before enzymatic hydrolysis, % V 0: Total volume of the reaction system before enzymatic hydrolysis, mL; C 1: The concentration of total sugar remaining in the reaction system after enzymatic hydrolysis, % V 1: Total volume of the reaction system after enzymatic hydrolysis, mL.

[0075] Table 1: Enzymatic hydrolysis rate of dextran in each example and comparative example

[0076] As can be seen from the results in Table 1, there are significant differences in the enzymatic hydrolysis efficiency between the various examples and the comparative examples, which fully verifies the necessity of the synergistic effect of the core strain, optimized process and specific promoter in the technical solution of the present invention.

[0077] In Examples 1 to 3, within the optimized parameter range, the enzymatic hydrolysis rate reached over 40%, demonstrating the effectiveness and stability of the core process of this invention. Example 6, by adding 5% sorbitol as an enzymatic hydrolysis promoter, further increased the hydrolysis rate to 48.7%. This is because sorbitol, through its osmotic pressure and hydration, effectively penetrates and relaxes the dense hydrogen bond network structure of the insoluble yeast β-glucan, increasing the contact area between the substrate and the complex enzyme system, thereby achieving a breakthrough improvement in enzymatic hydrolysis efficiency. In Examples 4 and 5, the hydrolysis rate was lower than in other examples, indicating that the hydrolysis pH and hydrolysis temperature are key process parameters of the preparation method of this invention, having a decisive influence on the hydrolysis effect.

[0078] Comparative Example 1, using commercially available β-glucanase, achieved a maximum hydrolysis rate of only 28.2%, significantly lower than all other examples. This demonstrates the technical limitations of commercially available β-glucanase in breaking down the complex network structure of yeast β-glucan. Notably, while enzyme No. 2 in Comparative Example 1 had the highest claimed enzyme activity (140,000 U / g) among the three, its hydrolysis rate was the lowest (21.6%). This indicates that for the specific substrate of yeast β-glucan, high enzyme activity does not equate to high efficiency; that is, degradation efficiency fundamentally depends on the specific matching between the enzyme system and the substrate structure, rather than simply the numerical value of activity units. The fact that all three commercially available enzymes with different designed applications in Comparative Example 1 showed low efficiency confirms that the degradation of yeast β-glucan requires a highly specific enzyme system. In contrast, the crude enzyme solution of Chaetomium JY25 used in this invention has a highly matched enzyme system with the substrate structure, thus enabling stable, efficient, and thorough degradation.

[0079] Reference standards two and three were subjected to unsuitable enzymatic hydrolysis temperatures and pH values, resulting in a sharp drop in the hydrolysis rate. This confirms that the specific temperature and acidic conditions used in this invention are indispensable key conditions for promoting the high efficiency of the crude enzyme solution of Chaetomium JY25.

[0080] II. Test of water solubility of β-glucan samples: Sample 1 (prepared in Example 1) of different masses was subjected to treatment at 25°C. Figure 2 ) and yeast β-glucan as a substrate (see Figure 3 Add the mixture to water, stir at 100 rpm for 2 minutes, and then let it stand at 25°C for 24 hours. Observe the dissolution effect and the results are shown below. Figure 4 and Figure 5 .

[0081] Depend on Figure 4 and Figure 5 The results show that the sample 1 prepared by the present invention can still completely dissolve at a concentration as high as 100 g / L, forming a homogeneous and stable solution, and no precipitation occurs after standing for 24 hours; while the insoluble yeast β-glucan, as a substrate, has already precipitated a large amount at a concentration as low as 5 g / L.

[0082] It is evident that the water solubility of β-glucan prepared by the method of this invention is fundamentally improved.

[0083] III. Molecular weight of β-glucan before and after enzymatic hydrolysis: The weight-average molecular weight of each sample and the reference standard was determined by gel permeation chromatography. Commercially available yeast β-glucan before enzymatic hydrolysis was used as the water-insoluble glucan sample. The results are shown in Table 2.

[0084] The GPC / SEC spectrum of sample 1 is shown below. Figure 6The GPC / SEC spectrum of commercially available water-insoluble yeast β-glucan is shown below. Figure 7 .

[0085] Table 2: Molecular weight of β-glucan before and after enzymatic hydrolysis

[0086] As can be seen from the results in Table 2, the weight-average molecular weight of samples 1 to 6 prepared by the present invention was precisely controlled within the range of 25kDa to 30kDa, which shows that the preparation method of the present invention has high specificity and repeatability.

[0087] It is particularly noteworthy that the commercially available yeast β-glucan used in this invention has an initial molecular weight of approximately 51 kDa, which, while not extremely high, makes it completely insoluble in water. This phenomenon stems from the unique high-order structure of yeast β-glucan. It is not a simple linear molecule, but rather a backbone composed of β-1,3 glycosidic bonds with numerous β-1,6 glycosidic bond branches, forming a highly complex and dense three-dimensional network structure. More importantly, these molecular chains are tightly packed together through strong hydrogen bonding, forming a stable triple helix conformation. This dense spatial network and strong intermolecular forces, rather than a single molecular weight, are the fundamental reasons for its extremely poor water solubility.

[0088] This invention is the first to discover the complex extracellular enzyme system produced by the specific species of Chaetomium JY25, which can effectively destroy its dense hydrogen bond network and branched structure, deconstructing the insoluble network into soluble fragments. Thus, while appropriately reducing the molecular weight to 25kDa to 30kDa, a highly soluble β-glucan product can be successfully prepared.

[0089] IV. Structural confirmation of β-glucan samples: Water-soluble β-glucan, represented by sample 1, and water-insoluble β-glucan, represented by commercially available yeast β-glucan as the substrate, were structurally confirmed using infrared spectroscopy. The results are shown in [Figure number missing]. Figure 8 .

[0090] Figure 8 In the middle, at 3375cm -1 2945cm -1 Both exhibit strong absorption peaks, primarily due to the -OH stretching vibration and the CH stretching vibration; these two absorption peaks are characteristic of polysaccharides. (1622 cm⁻¹) -1 The absorption peak is an aldehyde peak, caused by C=O stretching. 1416 cm⁻¹ -1 The absorption peak is due to the CH bending vibration; 1112 cm⁻¹ -1 The absorption peak is a characteristic absorption peak of the pyran ring, caused by the stretching vibration of the COH side group and the COC glycosidic bond, and is also at 1112 cm⁻¹. -1The absorption peak is also a characteristic absorption peak of glucose. 615 cm⁻¹ -1 The absorption peak indicates the presence of CH in the polysaccharide. (890-900 cm⁻¹) -1 The absorption peak is a characteristic absorption peak of β-glycosidic bonds.

[0091] Therefore, the microbial enzymatic hydrolysis preparation method provided by the present invention selectively destroys the higher physical structure that causes its insolubility in the process of successfully converting insoluble yeast β-glucan into a water-soluble product, while completely preserving its core chemical structure, that is, without changing the basic chemical skeleton of β-glucan, the glucose unit and the configuration of glycosidic bonds (β-type).

[0092] Therefore, this invention significantly improves the water solubility of the product while preserving the natural molecular structure and potential bioactivity of β-glucan to the greatest extent.

[0093] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A microbial method for preparing low molecular weight, water-soluble β-glucan, characterized in that, The preparation method specifically includes: S1. The *Chaetoceros* JY25 with preservation number CGMCC No. 6882 was subjected to liquid fermentation culture to obtain fermentation broth; S2. The fermentation broth obtained in step S1 is subjected to solid-liquid separation to obtain a crude enzyme solution containing β-glucan degrading enzyme system. S3. The crude enzyme solution and insoluble yeast β-glucan substrate are subjected to enzymatic hydrolysis at pH 4.0-6.0 and temperature 50℃-65℃ to obtain the enzymatic hydrolysis product. S4. Terminate the reaction and separate and purify the enzymatic hydrolysis product to obtain low molecular weight, water-soluble β-glucan with a weight average molecular weight of 25kDa to 30kDa.

2. The preparation method according to claim 1, characterized in that, The liquid fermentation culture described in step S1 uses a culture medium containing: 8 g / L to 12 g / L tryptone, 4 g / L to 6 g / L yeast extract, 8 g / L to 12 g / L sodium chloride, and an initial pH of 7.0 ± 0.

2.

3. The preparation method according to claim 1, characterized in that, The conditions for liquid fermentation culture in step S1 are: temperature 26℃~30℃, shaking speed 160r / min~200r / min, and culture time 20h~28h.

4. The preparation method according to claim 1, characterized in that, The solid-liquid separation described in step S2 is centrifugal separation, with centrifugation conditions of 8000 r / min to 12000 r / min for 5 min to 15 min.

5. The preparation method according to claim 1, characterized in that, The pH of the enzymatic hydrolysis reaction in step S3 is 4.5 to 5.

5.

6. The preparation method according to claim 1, characterized in that, The temperature of the enzymatic hydrolysis reaction in step S3 is 57℃~63℃, and the reaction time is 6h~10h.

7. The preparation method according to claim 1, characterized in that, The insoluble yeast β-glucan substrate mentioned in step S3 is added in the form of a suspension with a mass concentration of 1.7% to 2.3%.

8. The preparation method according to claim 7, characterized in that, The suspension contains 5% sorbitol by mass.

9. The preparation method according to claim 1, characterized in that, The volume-to-mass ratio of the crude enzyme solution to the insoluble yeast β-glucan substrate in step S3 is 100 mL: (15.3 g to 20.7 g).

10. The preparation method according to claim 1, characterized in that, The low molecular weight, water-soluble β-glucan obtained in step S4 has a solubility of not less than 100 g / L in water at 25°C.

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