Hericium erinaceus beta-glucan composite degradation method and application thereof

Abstract

The invention provides a hericium erinaceus beta-glucan composite degradation method and application thereof, and belongs to the technical field of edible and medicinal fungus polysaccharide degradation. According to the method, a hericium erinaceus beta-glucan sample is firstly subjected to ultrasonic pretreatment and then is subjected to enzymolysis by using a compound enzyme, finally, degraded hericium erinaceus beta-glucan is subjected to alcohol precipitation and centrifugation, supernatant or precipitate is collected respectively, and freeze drying is performed to obtain hericium erinaceus beta-glucan, so that the molecular weight of the hericium erinaceus beta-glucan can be remarkably reduced, and the content of the hericium erinaceus beta-glucan is reduced. The reaction time is shortened, the molecular weight distribution range is as low as thousand magnitude, the particle distribution is more uniform, the size is reduced to nanoscale, and the biological activity of the hericium erinaceus beta-glucan is greatly improved while the molecular weight is reduced. The low-molecular-weight hericium erinaceus beta-glucan prepared by the method can be used for functional foods, medicines or health care products for improving the protective activity on human gastric mucosa.

Description

Technical Field

[0001] The invention belongs to the technical field of degradation of edible and medicinal fungal polysaccharides, and particularly relates to a composite degradation method of Hericium erinaceus β-glucan and an application thereof. Background Art

[0002] β-Glucans are a class of naturally occurring active polysaccharides found widely in cereals, algae, and fungi, and their biological activity is closely related to their structural characteristics. β-Glucans from different sources exhibit significant functional differentiation due to differences in glycosidic bond linkage and branching structure. Cereal β-glucans (such as oats and barley) are primarily linked by alternating β-1,3 and β-1,4 glycosidic bonds, forming a linear structure with lipid-regulating properties. Yeast β-glucans have a highly branched network structure composed of a β-1,3 main chain and β-1,6 side chains, primarily acting as immunomodulators. β-glucans from higher fungi such as Hericium erinaceus have a main chain composed of β-1,3 glycosidic bonds, with one β-1,6 glucose side chain connected to every three residues, forming a highly branched triple helical conformation, which imparts significant immunomodulatory and gastric mucosal protective activities. However, there are strong hydrogen bonding forces within and between β-glucan molecules, which causes it to exist in a tight triple-helical conformation. This greatly limits the effective contact between β-glucan molecules and water molecules, resulting in extremely low solubility and seriously affecting its application range.

[0003] Currently, the main strategies for β-glucan degradation include chemical, physical, and enzymatic methods. Enzymatic degradation is highly specific and can selectively sever the main chain glycosidic bonds, but is limited by the resistance of the side chain structure (β-1,6 glycosidic bonds) to enzymatic degradation, resulting in generally low degradation efficiency. Therefore, the effectiveness of targeted β-glucan degradation remains unknown. Breaking through this bottleneck of insufficient β-glucan degradation efficiency and significantly improving its degradation efficiency and activity is a technical challenge that urgently needs to be addressed in this field. Summary of the Invention

[0004] The purpose of the present invention is to provide a Hericium erinaceus β-glucan composite degradation method and application thereof.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a method for degrading Hericium erinaceus β-glucan, comprising the following steps: performing ultrasonic pretreatment on a Hericium erinaceus β-glucan sample to be processed to obtain an ultrasonically degraded Hericium erinaceus β-glucan sample, performing enzymatic hydrolysis on the ultrasonically degraded Hericium erinaceus β-glucan sample using a composite enzyme to obtain enzymatically hydrolyzed Hericium erinaceus β-glucan, precipitating the degraded Hericium erinaceus β-glucan with alcohol, centrifuging to collect a supernatant or precipitate, and freeze-drying;

[0007] The ultrasonic pretreatment conditions include an amplitude of 45-55% and a total ultrasonic working time of 30 minutes;

[0008] The complex enzyme comprises cellulase, chitosanase and complex plant hydrolase; the enzymolysis time is 1 to 16 hours;

[0009] The alcohol precipitation comprises using ethanol to precipitate the degraded Hericium erinaceus β-glucan; the final volume concentration of the ethanol is 50%.

[0010] Preferably, before ultrasonic pretreatment, Hericium erinaceus β-glucan is added to a concentration of 2 mg mL -1 The Hericium erinaceus β-glucan sample to be tested is dissolved in ultrapure water to obtain the Hericium erinaceus β-glucan sample to be tested.

[0011] Preferably, the ultrasonic pretreatment condition further includes operating the ultrasound at a frequency of 5 seconds on and 5 seconds off.

[0012] Preferably, the ultrasonic power is 1500W, the ultrasonic amplitude is 50%, and the ultrasonic frequency is 20kHz.

[0013] Preferably, the mass ratio of the cellulase, chitosanase and composite plant hydrolase is (0.8-1.2):(0.8-1.2):(0.8-1.2);

[0014] Preferably, the enzymatic activities of the cellulase, chitosanase and composite plant hydrolase are 700EGU / g, 250U / g and 100FBG / g; the mass volume ratio of Hericium erinaceus β-glucan after ultrasonic degradation to the composite enzyme is 100mL:(0.2-0.4)g.

[0015] Preferably, the ethanol includes anhydrous ethanol; and the alcohol precipitation time is greater than 8 hours.

[0016] Preferably, the precipitate is completely dissolved in water at a material-liquid ratio of 1 g: (0.5-1) mL, centrifuged, and the supernatant is collected.

[0017] The present invention also provides Hericium erinaceus β-glucan prepared by the method.

[0018] The present invention also provides the use of the method or the Hericium erinaceus β-glucan in foods, health products or medicines that improve the protective function of human gastric mucosa.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides a method for degrading Hericium erinaceus β-glucan. The method comprises the following steps: subjecting a high-molecular-weight Hericium erinaceus β-glucan sample to ultrasonic and complex enzyme synergistic degradation and then alcohol precipitation to obtain a series of degraded Hericium erinaceus β-glucans. The method can significantly reduce the molecular weight of Hericium erinaceus β-glucan, shorten the reaction time, make the degradation component particles more evenly distributed and reduce the size to the nanoscale, and greatly enhance the biological activity of Hericium erinaceus β-glucan while reducing the molecular weight. The Hericium erinaceus β-glucan can be used for functional foods, medicines or health products with improved protective activity on human gastric mucosa. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure is a weight-average molecular weight distribution diagram of the β-glucan component degraded by the composite enzyme in the test example; among them, Figure (a) is the weight-average molecular weight of the composite enzyme degradation; Figure (b) is the weight-average molecular weight of the supernatant of the composite enzyme degradation; Figure (c) is the weight-average molecular weight of the precipitate of the composite enzyme degradation.

[0022] Figure 2 Figure 2 is a weight-average molecular weight distribution diagram of ultrasound-assisted composite enzyme degradation in the test example; wherein, Figure (a) is the weight-average molecular weight of ultrasound-assisted composite enzyme degradation, Figure (b) is the weight-average molecular weight of the supernatant of ultrasound-assisted composite enzyme degradation, and Figure (c) is the weight-average molecular weight of the precipitate of ultrasound-assisted composite enzyme degradation.

[0023] Figure 3 The SEM images of β-glucan components of Hericium erinaceus treated with different methods in the experimental example, where 500× represents the magnification of the scanning electron microscope image is 500, and 2000× represents the magnification of the scanning electron microscope image is 2000.

[0024] Figure 4 The AFM spectra of the different treated Hericium erinaceus β-glucan components in the experimental example, where HEP represents the original high molecular weight Hericium erinaceus β-glucan.

[0025] Figure 5 These are the protective activity results of ultrasound-assisted composite enzymatic hydrolysis on human gastric mucosal epithelial cells GES-1 in the experimental example; wherein, Figure (a) shows the protective activity of the glucan component of ultrasound-assisted composite enzymatic hydrolysis on GES-1 cells, Figure (b) shows the protective activity of the supernatant glucan component after 50% alcohol precipitation by ultrasound-assisted composite enzymatic hydrolysis on GES-1 cells, and Figure (c) shows the protective activity of the precipitated glucan component after 50% alcohol precipitation by ultrasound-assisted composite enzymatic hydrolysis on GES-1 cells; CG represents the control group, MG represents the ethanol injury model group, and PC represents Sanjiu Weitai Granules, which is a positive control group; * represents P<0.05, ** represents P<0.01, *** represents P<0.001vsMG; ### represents P<0.001vs CG. DETAILED DESCRIPTION

[0026] The invention provides a Hericium erinaceus β-glucan composite degradation method, comprising the following steps: first subjecting a Hericium erinaceus β-glucan sample to be processed to ultrasonic pretreatment to obtain an ultrasonically degraded Hericium erinaceus β-glucan sample, then enzymatically hydrolyzing the ultrasonically degraded Hericium erinaceus β-glucan sample using a composite enzyme to obtain enzymatically degraded Hericium erinaceus β-glucan, ethanol-precipitating the degraded Hericium erinaceus β-glucan, centrifuging to collect a supernatant or precipitate, and freeze-drying to obtain the Hericium erinaceus β-glucan; the ultrasonic pretreatment conditions include an amplitude of 45-55% and a total ultrasonic working time of 30 minutes; the composite enzyme is cellulase, chitosanase and a composite plant hydrolase; the enzymatic hydrolysis time is 1-16 hours; the alcohol precipitation includes ethanol to precipitate the degraded Hericium erinaceus β-glucan; and the final volume concentration of the ethanol is 50%.

[0027] In the present invention, before ultrasonic pretreatment, Hericium erinaceus β-glucan was added to the mixture at a mass concentration of 2 mg·mL -1 The Hericium erinaceus β-glucan sample to be tested is dissolved in ultrapure water. The ultrasonic pretreatment conditions also include preferably operating at a frequency of 5 seconds on and 5 seconds off; preferably using 1500W of ultrasonic power, 20kHz of ultrasonic frequency, and 50% of amplitude. Performing ultrasonic pretreatment before the complex enzyme treatment produces a synergistic effect, resulting in more thorough enzymatic depolymerization, further facilitating the degradation of Hericium erinaceus β-glucan, and shortening the reaction time.

[0028] In the present invention, during the composite enzyme degradation, the mass ratio of the cellulase, chitosanase and composite plant hydrolase is preferably (0.8-1.2):(0.8-1.2):(0.8-1.2), more preferably (0.9-1.1):(0.9-1.1):(0.9-1.1), and most preferably 1:1:1; the enzymatic activities of the cellulase, chitosanase and composite plant hydrolase are 700EGU / g, 250U / g and 100FBG / g; the mass volume ratio of Hericium erinaceus β-glucan to the composite enzyme after ultrasonic degradation is 100mL:(0.2-0.4)g, preferably 100mL:0.3g. In the present invention, the enzymatic hydrolysis time is preferably any one of 1h, 2h, 3h, 4h, 5h, 6h, 7h and 8h.

[0029] In the present invention, the ethanol used for alcohol precipitation includes anhydrous ethanol; the alcohol precipitation time is greater than 8 hours. As an optional embodiment, the Hericium erinaceus β-glucan sample after complex enzyme degradation is mixed with anhydrous ethanol in a volume ratio of 1:1. During the mixing, anhydrous ethanol is slowly added to the sample while stirring until the final ethanol concentration in the entire system reaches 50%, and the alcohol precipitation is carried out overnight.

[0030] The present invention also provides Hericium erinaceus β-glucan prepared by the method.

[0031] The present invention also provides the use of the method or the Hericium erinaceus β-glucan in foods, health products or medicines that improve the protective function of human gastric mucosa.

[0032] The degradation method provided by the present invention is to subject a high-molecular-weight Hericium erinaceus β-glucan sample to ultrasonic and complex enzyme synergistic degradation and then alcohol precipitation to obtain a series of Hericium erinaceus β-glucans. The obtained Hericium erinaceus β-glucans can significantly reduce the molecular weight of Hericium erinaceus β-glucan, with the molecular weight distribution range as low as thousands of orders of magnitude. The particles of the degradation components are more evenly distributed and the size is reduced to the nanoscale. While the molecular weight is reduced, the biological activity of the Hericium erinaceus β-glucan is greatly improved.

[0033] The Hericium erinaceus beta-glucan prepared by the method of the present invention can be used as a functional food, health product or medicine with enhanced protective activity on human gastric mucosa.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional experimental methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0035] In the following examples, the cellulase, chitosanase and composite plant hydrolase were all purchased from Novozymes Biotechnology Co., Ltd., with product numbers: cellulase (Cellulast 1.5L), chitosanase (COANAH25), and composite plant hydrolase (Viscozyme L), with enzyme activities of 700 EGU / g, 250 U / g, and 100 FBG / g, respectively.

[0036] In the following examples, the freeze-drying conditions are set by default at -50±5°C and a vacuum degree of about 1 mbar. Conventional rotary evaporation conditions are used for the rotary evaporation without special restrictions.

[0037] In the following examples, the GES-1 cells were purchased from Ningbo Mingzhou Biotechnology Co., Ltd. with the product number MZ-0779.

[0038] In the following examples, all experiments were repeated at least 3 times, and the experimental data were expressed as mean ± standard deviation (mean ± SD). Data were processed using GraphPad Prism 9.5 software. T-test was used for comparison between two groups, and one-way analysis of variance was used for comparison between multiple groups. ns indicates that P>0.05 indicates no statistically significant difference, *P<0.05 indicates statistically significant difference, and **P<0.01 indicates highly statistically significant difference.

[0039] The technical solutions of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention.

[0040] Example 1

[0041] This embodiment provides a method for the ultrasound-assisted complex enzyme degradation of Hericium erinaceus β-glucan, comprising the following steps:

[0042] The large molecular weight (1.82×10 6 g·moL -1 ) Hericium erinaceus β-glucan was dissolved in ultrapure water at a mass concentration of 2 mg·mL-1 to obtain a high molecular weight Hericium erinaceus β-glucan solution.

[0043] The high-molecular-weight Hericium erinaceus β-glucan solution sample was first ultrasonically pretreated to obtain a Hericium erinaceus β-glucan sample after ultrasonic degradation. The ultrasonic equipment was a Sonics Uibra Cell VCX 1500, with a power of 1500W and a frequency of 20kHz. The ultrasonic conditions were: 50% amplitude, 5 seconds on, 5 seconds off, for a total ultrasonic operating time of 30 minutes.

[0044] The ultrasonically degraded Hericium erinaceus β-glucan sample was enzymatically hydrolyzed using a complex enzyme to obtain a hydrolyzed sample. The complex enzyme comprised cellulase, chitosanase, and a complex plant hydrolase (mass ratio 1:1:1). The complex enzyme was added at a concentration of 0.3% by mass to the high-molecular-weight Hericium erinaceus β-glucan solution, and the hydrolysis was performed for 0 h, 1 h, and 4 h, respectively. The hydrolyzed samples were named UI, UCEH1h, and UCEH4h, respectively. Part of the enzymatically hydrolyzed sample was retained, and the remaining enzymatically hydrolyzed samples were precipitated with 50% ethanol (the degraded sample and anhydrous ethanol were mixed in a volume ratio of 1:1. During the mixing, anhydrous ethanol was slowly added to the sample while stirring to make the final ethanol concentration in the whole system reach 50%). The supernatant and precipitate were collected separately, and the supernatant was placed on a rotary evaporator to evaporate the ethanol. The precipitate was completely dissolved in water at a material-liquid ratio of 1 g: (0.5-1) mL. After high-speed centrifugation, the supernatant was collected and freeze-dried (pre-freezing stage: laboratory equipment is usually -50°C to -80°C; sublimation drying stage: the shelf temperature needs to be controlled at -35°C to 10°C, and the temperature will fluctuate to a certain extent at different stages; the vacuum degree is 0.1 mbar to 1 mbar). The supernatants were named UCEH1hS and UCEH4hS respectively; the precipitates were named UCEH1hP and UCEH4hP respectively.

[0045] Example 2

[0046] Degradation of β-glucan from Hericium erinaceus by complex enzymes

[0047] The large molecular weight (1.82×10 6 g·moL -1 ) Hericium erinaceus β-glucan was prepared at a mass concentration of 2 mg·mL -1Dissolve in ultrapure water to obtain a solution of high-molecular-weight Hericium erinaceus β-glucan, which is then enzymatically hydrolyzed using a complex enzyme comprising cellulase, chitosanase, and a complex plant hydrolase (in a 1:1:1 mass ratio). The complex enzyme was added at a rate of 0.3% by mass to the high-molecular-weight Hericium erinaceus β-glucan solution and hydrolyzed for 1, 4, 8, and 16 hours, respectively. The samples after enzymatic hydrolysis were designated CEH1h, CEH4h, CEH8h, and CEH16h, respectively. Part of the enzymatically hydrolyzed sample was retained, and the remaining enzymatically hydrolyzed sample was precipitated with 50% ethanol by volume (the degraded sample and anhydrous ethanol were mixed in a volume ratio of 1:1. During the mixing, anhydrous ethanol was slowly added to the sample while stirring to make the ethanol concentration in the whole system reach 50%). The supernatant and precipitate were collected separately, and the supernatant was placed on a rotary evaporator to evaporate the ethanol. The precipitate was completely dissolved by adding water at a material-liquid ratio of 1 g: (0.5-1) mL. After high-speed centrifugation, the supernatant was collected and freeze-dried (pre-freezing stage: laboratory equipment is usually -50°C to -80°C; sublimation drying stage: the shelf temperature needs to be controlled at -35°C to 10°C, and the temperature will fluctuate to a certain extent at different stages; the vacuum degree is 0.1 mbar to 1 mbar). The supernatants were named CEH1hS, CEH4hS, CEH8hS, and CEH16HS; the precipitates were named CEH1hP, CEH4hP, CEH8hP, and CEH16HP.

[0048] Test example

[0049] 1. Molecular weight distribution determination

[0050] The Hericium erinaceus β-glucan prepared in Example 1 or Example 2 was prepared into a 5 mg·mL-1 solution, and its molecular weight distribution was determined by high performance gel size exclusion chromatography (HPSEC)-multi-angle laser spectroscopy (MALLS)-differential refractometer (RI) method.

[0051] 2. Surface morphology scanning electron microscopy observation

[0052] The high-molecular-weight Hericium erinaceus β-glucan HEP ​​described in Example 1, the ultrasound-enzymatically hydrolyzed polysaccharide UCEH1hS described in Example 1, and the enzymatically hydrolyzed polysaccharide CEH8hS described in Example 2 were examined using a Hitachi TM4000 Plus desktop microscope. Each sample was mounted on a sample stage and observed and recorded under high vacuum conditions at an accelerating voltage of 5 kV. Scanning electron microscope images were obtained at magnifications of 500 and 2000, respectively.

[0053] 3. Observation of droplet micromorphology

[0054] Using droplet deposition, the high-molecular-weight Hericium erinaceus β-glucan HEP ​​described in Example 1, the ultrasound-enzymatic composite polysaccharide UCEH1hS, and the composite enzymatic composite polysaccharide CEH8hS described in Example 2 were each diluted to 1.0 μg / mL in ultrapure water. The mixture was then dropped onto a fresh mica sheet and dried at room temperature for 48 hours. The polysaccharide chain structure was then observed using atomic force microscope (AFM). AFM images were acquired using a DimensionICON scanning probe microscope (Bruker Co., Karlsruhe, Baden-Württemberg, Germany) in tapping mode, with a scan frequency of 0.98 Hz and a resonance frequency and constant of 127 kHz and 12 N / m, respectively.

[0055] 4. Determination of the protective effect on human gastric epithelial cells GES-1

[0056] GES-1 cells (Ningbo Mingzhou Biotechnology Co., Ltd., catalog number MZ-0779) were seeded in a 96-well plate (90 μL / well) at a cell density of 1 × 10 5 The cells were cultured in a 37°C, 5% CO2 constant temperature incubator for 12 h. 10 μL of each of the HEP, UI, UCEH1h, UCEH4h, UCEH1hS, UCEH4hS, UCEH1hP, and UCEH4hP samples of Example 1 or 2 were taken, and the final concentrations were 50, 200, and 500 μg mL -1 The cells were cultured in a constant temperature incubator for 24 h and damaged with 1000 μM ethanol for 6 h, and the cell survival rate was determined using the MTT method.

[0057] Results and Analysis:

[0058] 1. Molecular weight distribution of composite enzyme degradation components

[0059] As shown in Table 1, two peaks, Peak 1 and Peak 2, appeared after the composite enzymatic hydrolysis treatment ( Figure 1 ), with the extension of enzymatic hydrolysis time, the β-glucan of Hericium erinaceus was 1.82×10 6 g·moL -1 Degraded to 1.22×10 6 ~1.42×10 6 g·moL-1 and 5.48×10 4 ~8.35×10 4 g·moL -1 In the two ranges, the high molecular weight component peak still accounts for more than 85% ( Figure 1 a) After precipitation with ethanol at a final concentration of 50%, the molecular weight of the CEHSs obtained from the supernatant ranged from 3.38×10 3 ~1.41×10 4 g·moL -1(Table 2, Figure 1 b); The molecular weight of precipitated CEHPs ranges from 2.27×10 5 ~1.28×10 6 g·moL -1 (Table 2, Figure 1 c) and a small amount of molecular weight of 6.20×10 4 ~8.33×10 4 g·moL -1 The results showed that the combined enzymatic hydrolysis had a certain degradation effect on Hericium erinaceus β-glucan, but the proportion of million-level components was still above 85%. This indicates that the single combined enzymatic hydrolysis has limited effect on β-glucan degradation. More combined degradation strategies are needed to maximize degradation efficiency and improve the solubility of Hericium erinaceus β-glucan.

[0060] Table 1 Complex enzyme degradation weight average molecular weight

[0061]

[0062]

[0063] Table 2 Weight average molecular weight of supernatant and precipitate after composite enzymatic hydrolysis + 50% ethanol precipitation

[0064]

[0065] 2. Molecular weight distribution of components degraded by ultrasound-assisted complex enzyme

[0066] As shown in Table 3, the molecular weight of the sample UI treated with ultrasound for only half an hour is 5.06×10 5 g·moL -1 After ultrasound-assisted composite enzymatic hydrolysis, the molecular weight of the sample was further degraded to 2.58×10 5 ~2.91×10 5 g·moL -1 and 9.50×10 3 ~4.80×10 4 g·moL -1 After precipitation with 50% ethanol, the molecular weight distribution of the supernatant was 3.70×10 4 ~1.56×10 4 g·moL -1 , and a low molecular weight component with a molecular weight of ten thousand was obtained; after 50% ethanol precipitation treatment, a molecular weight distribution of 2.24×10 5 ~2.07×10 5 g·moL-1, the molecular weight is reduced by 50% compared with the ultrasonic degradation component ( Figure 2), indicating that ultrasound-assisted composite enzymatic hydrolysis can effectively degrade Hericium erinaceus β-glucan and shorten the reaction time.

[0067] Table 3 Ultrasound-assisted composite enzyme degradation weight average molecular weight

[0068]

[0069]

[0070] 3. SEM analysis of different treatment components

[0071] The morphologies of the original Hericium erinaceus β-glucan and the components after composite enzymatic hydrolysis and ultrasound-assisted composite enzymatic hydrolysis: HEP, CEH8hS and UCEH1hS were observed by scanning electron microscopy (SEM). Figure 3 As shown in Figure 2, the morphology of Hericium erinaceus β-glucan components undergoes significant changes after treatment. HEP exhibits a flaky structure with a dense and compact surface. After combined enzymatic hydrolysis, the lamellar structure is disrupted, with small voids of varying degrees appearing on the surface. The specific surface area increases, and the surface becomes fluffy. Furthermore, combined ultrasound-assisted combined enzymatic hydrolysis further disrupts the lamellar structure, resulting in a finer particle structure and a reduction in aggregates or network-like interconnections.

[0072] 4. AFM analysis of different treatment components

[0073] In order to observe the actual chain behavior of β-glucan with different treatment components in solution, the micromorphology of the original β-glucan HEP ​​and the components after different treatments of CEH8hS and UCEH1hS were observed by AFM. Figure 4 It shows that HEP presents a typical large-scale aggregate morphology similar to a fishing net with cross-linked branches. The high molecular weight causes it to form a highly cross-linked network or fibrous structure through intermolecular hydrogen bonds and hydrophobic interactions. The root mean square particle size Rq and surface average roughness Ra are 0.591nm and 0.396nm, respectively, indicating that its surface aggregation degree is high. Relatively uniform macro-aggregates are formed in local areas due to the stacking of molecular chains, which is consistent with the SEM spectrum structure.

[0074] After composite enzymatic hydrolysis treatment, CEH8hS showed that composite enzymatic hydrolysis partially broke the glycosidic bonds of β-glucan, causing the macromolecular aggregates to dissociate into dispersed fragments or short chain fragments. Obvious structural depolymerization phenomenon was visible in the AFM image. The surface of the enzymatic hydrolysis product was uneven and the surface roughness increased. The root mean square particle size Rq and the average surface roughness Ra were 1.57 nm and 1.06 nm, respectively. The residual macromolecular chains that were not completely degraded (such as microfibril structures) may form higher local protrusions (Rmax reached 30.23 nm). The selective depolymerization effect of the enzymatic hydrolysis was incomplete, resulting in enhanced surface morphology heterogeneity.

[0075] After ultrasonic pretreatment and combined enzyme digestion, the AFM images of UCEH1hS showed that cavitation disrupted molecular chain entanglement, disrupting hydrogen bonds and hydrophobic interactions between molecular chains, exposing more cleavage sites and making digestion more efficient and thorough. Cross-linking structures were reduced in the AFM images, resulting in rounded granular morphology. The root mean square particle size (Rq) and surface average roughness (Ra) were 0.778 nm and 0.276 nm, respectively. Rq was slightly higher than that of the original sample (0.591 nm) but lower than that of the sample with simple enzyme digestion (1.57 nm), indicating a more uniform particle distribution and a reduction in size to the nanometer scale. The significantly reduced Ra (0.276 nm) reflects a highly homogenized surface. This suggests that ultrasound-assisted enzymatic digestion is more thorough and more conducive to the degradation of Hericium erinaceus β-glucan.

[0076] 5. Protective activity of ultrasound-assisted composite enzymatic dextran components on GES-1 cells

[0077] like Figure 5 As shown in the figure, ultrasound synergistically combined with enzymatic hydrolysis has a protective activity on human gastric epithelial cells GES-1. UI, UCEH1h, and UCEH4h showed better GES-1 protective activity than HEP ​​at high concentrations ( Figure 5 a); In the protective activity of GES-1 in the supernatant and precipitate samples after 50% ethanol precipitation, UCEH1hS and UCEH1hP showed a slight advantage over other treatments ( Figure 5 bc).

[0078] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A composite degradation method for Hericium erinaceus β-glucan, characterized in that: The method comprises the following steps: performing ultrasonic pretreatment on a Hericium erinaceus β-glucan sample to be processed to obtain an ultrasonically degraded Hericium erinaceus β-glucan sample, performing enzymatic hydrolysis on the ultrasonically pretreated Hericium erinaceus β-glucan sample using a composite enzyme to obtain enzymatically degraded Hericium erinaceus β-glucan, performing alcohol precipitation on the degraded Hericium erinaceus β-glucan, collecting the supernatant or precipitate by centrifugation, and freeze-drying; The ultrasonic pretreatment conditions include an amplitude of 45-55% and a total ultrasonic working time of 30 minutes; The complex enzyme comprises cellulase, chitosanase and complex plant hydrolase; the enzymolysis time is 1 to 16 hours; The alcohol precipitation comprises using ethanol to precipitate the degraded Hericium erinaceus β-glucan; the final volume concentration of the ethanol is 50%.

2. The method according to claim 1, characterized in that Before ultrasonic pretreatment, Hericium erinaceus β-glucan was added at a mass concentration of 2 mg·mL -1 The Hericium erinaceus β-glucan sample to be tested is dissolved in ultrapure water to obtain the Hericium erinaceus β-glucan sample to be tested.

3. The method according to claim 1, characterized in that The ultrasonic pretreatment conditions also include operating the ultrasound at a frequency of 5 seconds on and 5 seconds off.

4. The method according to claim 1, wherein The ultrasonic power is 1500W, the ultrasonic amplitude is 50%, and the ultrasonic frequency is 20kHz.

5. The method according to claim 1, wherein The mass ratio of the cellulase, chitosanase and composite plant hydrolase is (0.8-1.2):(0.8-1.2):(0.8-1.2).

6. The method according to claim 1, characterized in that The enzymatic activities of the cellulase, chitosanase and composite plant hydrolase are 700 EGU / g, 250 U / g and 100 FBG / g; the mass volume ratio of Hericium erinaceus β-glucan to the composite enzyme after ultrasonic degradation is 100 mL: (0.2-0.4) g.

7. The method according to claim 1, characterized in that The ethanol includes anhydrous ethanol; and the alcohol precipitation time is greater than 8 hours.

8. The method according to claim 1, characterized in that The precipitate was completely dissolved in water at a material-liquid ratio of 1 g: (0.5-1) mL, centrifuged, and the supernatant was collected.

9. Hericium erinaceus β-glucan prepared by the method according to any one of claims 1 to 7.

10. Use of the method according to any one of claims 1 to 7 or the Hericium erinaceus β-glucan according to claim 8 in functional foods, medicines or health products that enhance protection of human gastric mucosa.

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