Hericium erinaceus beta-glucan degradation method and application of hericium erinaceus beta-glucan degradation method in gastric mucosa protection

Abstract

The invention provides a hericium erinaceus beta-glucan degradation method and application of hericium erinaceus beta-glucan in gastric mucosa protection, and belongs to the technical field of edible and medicinal fungal polysaccharide degradation. The method comprises the following steps: carrying out microwave irradiation and citric acid degradation on hericium erinaceus beta-glucan, carrying out alcohol precipitation and centrifugation on the degraded hericium erinaceus beta-glucan, respectively collecting a supernatant or a precipitate, freeze-drying to obtain a series of glucan components with different molecular weight segments, and carrying out in vitro activity evaluation to obtain the hericium erinaceus beta-glucan. The active hericium erinaceus beta-glucan with low molecular weight is obtained. Compared with a single degradation method, the method has the advantages that the molecular weight of the hericium erinaceus beta-glucan can be remarkably reduced through microwave irradiation and citric acid synergistic degradation, the in-vitro immunocompetence and gastric mucosa protection activity of degraded components are improved, and the hericium erinaceus beta-glucan can be used for functional food, health care products or medicines related to the hericium erinaceus beta-glucan.

Description

Technical Field

[0001] The invention belongs to the technical field of degradation of edible and medicinal fungal polysaccharides, and particularly relates to a method for degrading Hericium erinaceus β-glucan and its application in protecting gastric mucosa. Background Art

[0002] Hericium erinaceus is a rare edible and medicinal fungus. β-glucan, as its key active ingredient, has shown promising effects in immune regulation and gastric ulcer inhibition. However, the molecular weight of natural β-glucan prepared from Hericium erinaceus is as high as millions. For example, the molecular weight of β-glucan prepared in the early stage according to CN201410103882.4 is as high as 1.82×10 6 g·moL -1 Due to its triple helix conformation and high viscosity, its solubility is extremely low (<5 mg / mL), resulting in insufficient bioavailability, which seriously restricts its repair activity on the gastric mucosa.

[0003] Studies have shown that reducing the molecular weight of polysaccharides through controlled degradation can significantly improve their solubility and enhance their activity, and the concentration dependence is more prominent than that of low-molecular-weight polysaccharides. Therefore, degradation of high-molecular-weight Hericium erinaceus β-glucan is an effective method to improve its bioavailability and activity. Currently, the commonly used degradation methods mainly include chemical methods (such as acid and alkali methods), physical methods (such as ultrasound and microwave methods) and bio-enzymatic methods. In recent years, physical degradation technology has attracted much attention in the industry due to its significant advantages of high efficiency and environmental protection. At present, physical degradation is mostly carried out in the form of a single technology, and there is a general problem of limited degradation rate, which makes it difficult to meet the diverse needs for different molecular weights, especially low-molecular-weight polysaccharides. Therefore, the development of an effective Hericium erinaceus β-glycan degradation method is crucial to Hericium erinaceus and its application in the health industry. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for degrading Hericium erinaceus β-glucan and its application in protecting gastric mucosa.

[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: dissolving Hericium erinaceus β-glucan in citric acid, subjecting the Hericium erinaceus β-glucan to microwave irradiation to obtain synergistically treated Hericium erinaceus β-glucan, subjecting the treated Hericium erinaceus β-glucan to alcohol precipitation, centrifuging and collecting the supernatant or precipitate, and freeze-drying to obtain Hericium erinaceus β-glucan;

[0007] When the Hericium erinaceus β-glucan is dissolved in citric acid, the Hericium erinaceus β-glucan is dissolved in citric acid at a mass concentration of 2 mg·mL -1 Dissolved in citric acid solution with a mass concentration of 2% to 8%;

[0008] The microwave treatment conditions include a treatment power of 1200W to 1600W, a treatment temperature of 140°C to 180°C, and a treatment retention time of 30 to 50 minutes;

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

[0010] Preferably, the microwave treatment conditions include a treatment power of 1600W, a treatment temperature of 140°C, and a treatment retention time of 30min.

[0011] Preferably, when the Hericium erinaceus β-glucan is dissolved in citric acid, the Hericium erinaceus β-glucan is dissolved in citric acid at a mass concentration of 2 mg·mL -1 Dissolved in 6% citric acid solution.

[0012] Preferably, the ethanol comprises anhydrous ethanol.

[0013] Preferably, the alcohol precipitation time is greater than 8 hours.

[0014] Preferably, the supernatant is placed in a water bath to volatilize the ethanol, the water bath time is more than 1 hour, and the water bath temperature is more than 90°C.

[0015] Preferably, the precipitate after alcohol precipitation is completely dissolved by adding water at a material-liquid ratio of 1g:(0.5-1)mL.

[0016] The present invention also provides Hericium erinaceus β-glucan prepared by the method.

[0017] The present invention also provides the use of the method or the Hericium erinaceus β-glucan in preparing a product that improves immune activity or improves gastric mucosal protection.

[0018] Preferably, the product comprises a functional food, a health product or a medicine.

[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 Hericium erinaceus β-glucan is degraded using microwave irradiation in conjunction with citric acid. The degraded Hericium erinaceus β-glucan is then subjected to alcohol precipitation and centrifugation. The supernatant and precipitate are collected and freeze-dried to obtain a series of glucan fractions with different molecular weights. Low-molecular-weight active Hericium erinaceus β-glucan is obtained through in vitro activity evaluation. Compared to single degradation methods, the present invention significantly reduces the molecular weight of Hericium erinaceus β-glucan through synergistic degradation using microwave irradiation and citric acid, enhancing the in vitro immune activity and gastric mucosal protective activity of the degraded fraction. The degraded fraction can be used in functional foods, health products, or pharmaceuticals containing Hericium erinaceus β-glucan. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 These are the protective activity results of the samples on GES-1 under different microwave degradation conditions in Example 1; wherein a is the protective activity result of the samples degraded by microwave at different times on GES-1, b is the protective activity result of the samples degraded by microwave at different powers on GES-1; c is the protective activity result of the samples degraded by microwave at different temperatures on GES-1; CG represents the control group, MG represents the ethanol injury model group, and PC represents Sanjiu Weitai Granules, which is the positive control group; ns indicates that P>0.05 is not statistically significant, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001 vs MG; ### indicates P<0.001 vs CG.

[0022] Figure 2 The total weight average molecular weight graph of microwave irradiation combined with citric acid degradation in Example 1; wherein a is the weight average molecular weight graph of components degraded by microwave irradiation combined with citric acid, b is the weight average molecular weight graph of supernatant of 50% alcohol precipitation of each component degraded by microwave irradiation combined with citric acid, and c is the weight average molecular weight graph of 50% alcohol precipitation of each component degraded by microwave irradiation combined with citric acid; HEP is the largest molecular weight of Hericium erinaceus (1.82×10 6 g·moL -1 )β-glucan, 2% CAM, 4% CAM, 6% CAM, and 8% CAM represent the β-glucan components after microwave degradation of samples dissolved with different citric acids.

[0023] Figure 3 The in vitro immune activity of the samples degraded by microwave irradiation combined with citric acid in Example 2; wherein, a represents the in vitro immune activity results of each component degraded by microwave irradiation combined with citric acid, b represents the in vitro immune activity results of the supernatant after 50% alcohol precipitation of each component degraded by microwave irradiation combined with citric acid, and c represents the in vitro immune activity results of the precipitate after 50% alcohol precipitation of each component degraded by microwave irradiation combined with citric acid; HEP is the large molecular weight of Hericium erinaceus (1.82×10 6 g·moL -1 )β-glucan, PBS as negative control, LPS as positive control, the final concentration was 1 μg·mL -1 ; * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001 vs PBS; ### indicates P < 0.001 vs PBS.

[0024] Figure 4These are the protective activity results of the microwave irradiation combined with citric acid degradation samples on human gastric mucosal epithelial cells GES-1 in Example 2; wherein, a represents the protective activity result of each glucan component degraded by microwave irradiation combined with citric acid on GES-1 cells, b represents the protective activity result of the supernatant after 50% alcohol precipitation of each glucan component degraded by microwave irradiation combined with citric acid on GES-1 cells, and c represents the protective activity result of the precipitate after 50% alcohol precipitation of each glucan component degraded by microwave irradiation combined with citric acid on GES-1 cells; CG represents the control group, MG represents the ethanol injury model group, and PC represents Sanjiu Weitai Granules, which is the positive control group; * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001 vs MG; ### represents P < 0.001 vs CG. DETAILED DESCRIPTION

[0025] The present invention provides a method for degrading Hericium erinaceus β-glucan, comprising dissolving Hericium erinaceus β-glucan in citric acid, performing microwave treatment to obtain Hericium erinaceus β-glucan after synergistic treatment, subjecting the treated Hericium erinaceus β-glucan to alcohol precipitation, collecting supernatant and precipitate by centrifugation, and freeze-drying to obtain a series of Hericium erinaceus β-glucans of different molecular weight segments; the citric acid dissolution comprises: dissolving Hericium erinaceus β-glucan in citric acid at a mass concentration of 2 mg·mL -1 The β-glucan is dissolved in a citric acid solution with a mass concentration of 2% to 8%. The microwave treatment conditions include a treatment power of 1200W to 1600W, a treatment temperature of 140°C to 180°C, and a treatment retention time of 30 to 50 minutes. The alcohol precipitation includes precipitating the treated Hericium erinaceus β-glucan with ethanol; the final volume concentration of the ethanol is 50%. In the present invention, the high-molecular-weight Hericium erinaceus β-glucan sample is dissolved in citric acid and then subjected to microwave irradiation for degradation, and the two act synergistically.

[0026] In the present invention, the microwave treatment conditions include a treatment power of preferably 1600W, a treatment temperature of preferably 140°C, and a treatment retention time of preferably 30min.

[0027] In the present invention, the condition for dissolving Hericium erinaceus β-glucan with citric acid includes a citric acid solution with a preferred concentration of 6%.

[0028] In the present invention, when the microwave-degraded Hericium erinaceus β-glucan is subjected to alcohol precipitation, the ethanol is preferably anhydrous ethanol; and the alcohol precipitation time is greater than 8 hours. As an optional embodiment, the microwave-degraded Hericium erinaceus β-glucan is precipitated with ethanol at a volume ratio of 50%, that is, the microwave-degraded Hericium erinaceus β-glucan sample is mixed with anhydrous ethanol in a ratio of 1:1. During the mixing, anhydrous ethanol is slowly added to the sample while stirring until the ethanol concentration in the entire system reaches 50%, and the precipitation is carried out overnight.

[0029] In the present invention, the supernatant and precipitate are collected separately by centrifugation after alcohol precipitation. The centrifugal speed is preferably 7500-8500 rpm, more preferably 8000 rpm; the temperature is preferably 23-25° C., more preferably 24° C.; the centrifugal time is preferably 10-20 min, more preferably 15 min.

[0030] In the present invention, Hericium erinaceus β-glucan can be obtained by freeze-drying the supernatant or precipitate after centrifugation. Prior to freeze-drying, the supernatant is placed in a water bath to evaporate the ethanol. Prior to freeze-drying, the precipitate obtained is completely dissolved in water at a material-liquid ratio of 1 g:(0.5-1) mL. Degraded Hericium erinaceus β-glucan can be prepared from the freeze-dried supernatant or precipitate.

[0031] The present invention provides a method for degrading Hericium erinaceus β-glucan. A high-molecular-weight Hericium erinaceus β-glucan sample is subjected to microwave irradiation and synergistic degradation with citric acid, which can significantly reduce the molecular weight of the Hericium erinaceus β-glucan and improve the solubility. The molecular weight distribution range is as low as the thousandth level. After alcohol precipitation, a low-molecular-weight Hericium erinaceus β-glucan component with more uniform distribution, higher purity and better activity is obtained.

[0032] The present invention also provides the use of the method or the Hericium erinaceus β-glucan in preparing a product that improves immune activity or improves gastric mucosal protection.

[0033] The products described in the present invention include functional foods, health products, or pharmaceuticals; the applications are preferably to enhance human immune activity and / or enhance protective activity against human gastric epithelial GES-1 cells. To enhance immune activity, Hericium erinaceus β-glucan (6% CAMP) is selected from the precipitate after microwave degradation with 6% citric acid and precipitation with 50% ethanol. To enhance protective activity against human gastric epithelial GES-1 cells, Hericium erinaceus β-glucan (4% CAMS and / or 6% CAMS) is selected from the supernatant after degradation with 4% and / or 6% citric acid solutions and precipitation with 50% ethanol. Alternatively, Hericium erinaceus β-glucan is selected from the precipitate (6% CAMP) after degradation with 6% citric acid solutions and microwave degradation followed by precipitation with 50% ethanol. The gastric mucosal protective activity of 6% CAMS was the best, which was equivalent to or slightly better than that of the PC (Sanjiu Weitai) control group. The 6% CAMP sample precipitated by 50% ethanol had a gastric mucosal protective effect of 500 μg·mL -1 GES-1 also showed good protective activity under high concentration conditions, which was significantly better than the PC (Sanjiu Weitai) control group.

[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] The following examples used a microwave digester, model CEM Mars6, with an output power of 1 to 1800 W ± 15%, IEC. To configure the microwave digester application, select "Traditional Method" on the main interface; "Ramp to Temperature" for "Control Type"; "Standard" for "Mode"; "Xpress" for "Digestion Vessel Type"; and "Organic" for "Sample Type."

[0036] In the following examples, the default setting conditions for freeze drying are -50±5°C and a vacuum degree of about 1 mbar.

[0037] In the following examples, the GES-1 cells were purchased from Ningbo Mingzhou Biotechnology Co., Ltd., with the catalog number MZ-0779. The RAW 264.7 macrophage cell line was purchased from the Cell Center of the Chinese Academy of Medical Sciences - ATCC Cell Resource Center, with the catalog number CBP60533.

[0038] In the experimental data results of the following examples, ns means P>0.05 means there is no statistically significant difference, *P<0.05 means there is statistically significant difference, and **P<0.01 means there is 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] 1. Single microwave irradiation degradation of Hericium erinaceus β-glucan

[0042] The prepared high molecular weight Hericium erinaceus β-glucan (1.82×10 6 g·moL -1 ) with a mass concentration of 2 mg·mL -1 Dissolve in ultrapure water and place in a microwave digester tank to balance.

[0043] (1) Screening of microwave degradation time

[0044] The target temperature was set to 180°C, the ramp time was 30 min, the microwave power was 1000 W, and the retention times were 20 min, 30 min, 40 min, 50 min, and 60 min, respectively. The changes in the molecular weight of the degradation components under different microwave times were compared.

[0045] (2) Screening of microwave degradation temperature

[0046] The target microwave power was set to 1000W, the ramp time was 30min, the microwave power was 1000W, and the microwave action temperatures were 100℃, 120℃, 140℃, 160℃, and 180℃, respectively. The changes in the molecular weight of the microwave degradation components at different temperatures were compared.

[0047] (3) Screening of microwave degradation power

[0048] The target temperature was set to 180°C, the ramp time was 30 min, the retention time was 30 min, and the microwave action powers were 1000 W, 1200 W, 1400 W, and 1600 W, respectively. The changes in the molecular weight of the microwave degradation components under different powers were compared.

[0049] (4) Molecular weight distribution determination

[0050] The prepared Hericium erinaceus β-glucan was prepared into 5 mg·mL -1 The molecular weight distribution of the solution was determined by high performance gel size exclusion chromatography (HPSEC)-multi-angle laser spectroscopy (MALLS)-differential refractometer (RI) (referring to the method in the literature: Physical and chemical properties and in vitro immune activity of polysaccharides from fermentation mycelium of ARTP-induced Hericium erinaceus strain [J]. Journal of Mycology, 2018, 37(06): 794-804.).

[0051] Results and analysis of molecular weight distribution of components irradiated by microwave:

[0052] The results in Table 1 show that the weight average molecular weight of Hericium erinaceus β-glucan before degradation is 1.82×10 6 g·moL -1 With the extension of microwave degradation time, the response value of Peak1 also decreased and the peak area decreased, indicating that the high molecular weight polysaccharide was degraded; at the same time, a new Peak2 Mw of 2.14×10 4 ~2.94×10 4 g·moL -1 The molecular weight of the product was significantly decreased (Table 1). Therefore, the microwave degradation time setting had a certain degree of degradation effect on the molecular weight. When the degradation time was 30 min, the response value of Peak 1 decreased significantly, and the peak area ratio also decreased. The peak area of Peak 2 increased the most. Therefore, a microwave degradation retention time of 30 min was the optimal degradation time.

[0053] Table 1 Weight average molecular weight of microwave degradation at different times

[0054]

[0055] Table 2 shows the effect of different microwave degradation powers on the weight average molecular weight. The weight average molecular weight of Hericium erinaceus β-glucan before degradation is 1.82×10 6 g·moL -1 With the increase of microwave degradation power, the response value of Peak1 decreased and the peak area decreased accordingly, indicating that the degradation degree of large molecular weight polysaccharides was positively correlated with the microwave power. At the same time, a new Peak2 Mw of 1.68×10 4 ~5.31×10 4 g·moL -1 The degradation power of 1600W was significantly higher than that of Peak 1, indicating that microwave power can further degrade large β-glucans. At a degradation power of 1600W, the response value of Peak 1 decreased significantly, as did the peak area. Peak 2, with a peak area of 10,000W, accounted for more than half of the total area, demonstrating a significant degradation effect. Therefore, a microwave degradation power of 1600W was selected as the optimal degradation power.

[0056] Table 2 Weight average molecular weight of microwave degradation at different powers

[0057]

[0058] Table 3 shows the effect of different microwave degradation temperatures on the weight average molecular weight. The weight average molecular weight of Hericium erinaceus β-glucan before degradation is 1.82×10 6 g·moL -1 As the microwave degradation temperature increases, the overall response value and peak area of Peak 1 decrease, indicating the gradual degradation of high-molecular-weight polysaccharides. Peak 2 also exhibits a peak of magnitude 10,000. At 140°C, the peak area of Peak 1 decreases significantly, while the peak area of Peak 2 increases significantly, indicating a significant degradation effect at 140°C. Therefore, 140°C is the optimal microwave degradation temperature.

[0059] Table 3 Weight average molecular weight of microwave degradation at different temperatures

[0060]

[0061] 2. Protective activity of single microwave-degraded components on GES-1 cells

[0062] 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 samples were cultured in a 37°C, 5% CO2 constant temperature incubator for 12 h. 10 μL of each of the microwave-degraded Hericium erinaceus β-glucan samples with different action times, different action temperatures and different action powers were taken. The action concentrations were 50, 200 and 500 μg·mL -1The 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.

[0063] All the above experiments were repeated at least 3 times. The experimental data were expressed as mean ± standard deviation (Mean ± SD) and 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. P < 0.05 indicated that the results were significantly different and the differences were statistically significant.

[0064] Conclusion: Pass Figure 1 a in the figure is the protective activity of GES-1 by microwave degradation samples at different times. Figure 1 b in the figure is the protective activity of GES-1 by microwave degradation samples at different powers; Figure 1 The c in the figure is the protective activity of GES-1 by microwave degradation of samples at different temperatures. Figure 1 The degradation effect of β-glucan and its in vitro gastric mucosal protective activity under different microwave treatment conditions were comprehensively evaluated. The optimal microwave treatment conditions were: 1600w, 140℃, 30min.

[0065] 3. Molecular weight distribution of Hericium erinaceus β-glucan under optimal microwave treatment conditions

[0066] The optimal microwave treatment conditions were as follows: 1600W, 140℃, 30min for treating Hericium erinaceus β-glucan. Under these conditions, the molecular weight distribution of Hericium erinaceus β-glucan prepared is shown in Table 4.

[0067] Table 4 Molecular weight distribution of Hericium erinaceus β-glucan under preferred microwave treatment conditions

[0068]

[0069] The data in Table 4 show that microwave irradiation alone causes a certain degree of degradation in the weight-average molecular weight of Hericium erinaceus β-glucan, but the proportion of the original million-level molecular weight components is still very high, indicating that microwave irradiation alone has limited effect on the degradation of Hericium erinaceus million-level β-glucan.

[0070] 4. Molecular weight distribution of components degraded by microwave irradiation combined with citric acid

[0071] 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 -1The samples were dissolved in 2%, 4%, 6% and 8% citric acid solutions respectively, and then the Hericium erinaceus β-glucan dissolved in citric acid solution was microwave treated using the optimal conditions screened for microwave degradation. The samples were named 2% CAM, 4% CAM, 6% CAM and 8% CAM respectively. Part of the sample was retained and the remaining sample was mixed with 50% ethanol, that is, the sample and anhydrous ethanol were mixed in a ratio of 1:1. Anhydrous ethanol was slowly added to the sample while stirring during mixing to make the ethanol concentration in the whole system reach 50%. The alcohol was precipitated overnight at 8000 rpm and 24°C for 15 minutes. The supernatant and precipitate were collected separately. The supernatant was placed in a water bath (above 90°C for more than 1 hour, depending on the specific ethanol volatilization situation) to evaporate the ethanol. The precipitate was completely dissolved in water according to the material-liquid ratio (1:0.5-1:1). The samples were freeze-dried and then sampled to obtain the prepared samples. The supernatants were named 2% CAMS, 4% CAMS, 6% CAMS, and 8% CAMS; the precipitates were named 2% CAMP, 4% CAMP, 6% CAMP, and 8% CAMP.

[0072] The molecular weight distribution is measured by step (4) in 1.

[0073] Result analysis: The weight average molecular weight of the components degraded by microwave irradiation combined with citric acid is shown in Table 5. As shown in Table 5, the molecular weight distribution range of the components after microwave irradiation combined with citric acid treatment is 1.03×10 5 ~2.72×10 5 g·moL -1 (molecular weight of the order of 100,000); 2.52×10 4 ~4.95×10 4 g·moL -1 (molecular weight of ten thousand); and 1.92×10 3 ~7.00×10 3 g·moL -1 (Molecular weight of thousands). Figure 2 a( Figure 2 (a in the figure is the weight-average molecular weight of microwave combined with citric acid degradation) and Table 5 show that compared with single microwave irradiation treatment, microwave irradiation synergistically with citric acid degradation can significantly reduce the molecular weight of Hericium erinaceus β-glucan, and degradation can obtain components with relatively lower molecular weight.

[0074] Table 5 Microwave + citric acid degradation weight average molecular weight

[0075]

[0076] From the weight-average molecular weight test results of the supernatant and precipitate after 50% ethanol precipitation by microwave irradiation combined with citric acid in Table 6, it can be seen that after 50% alcohol precipitation, the sample components with relatively uniform distribution and high purity were obtained, and their molecular weight ranges were 1.37×10 4~3.61×10 4 g·moL -1 ;6.15×10 5 ~9.05×10 5 g·moL -1 . Figure 2 Figures b and c are the weight average molecular weight graphs of the supernatant and the precipitate after 50% alcohol precipitation after microwave irradiation combined with citric acid degradation, respectively. It can be seen that a more uniform Hericium erinaceus glucan component can be obtained after microwave combined with citric acid degradation and 50% alcohol precipitation treatment.

[0077] Table 6 Weight average molecular weight of supernatant and precipitate after microwave irradiation combined with citric acid degradation of 50% ethanol precipitation

[0078]

[0079] Example 2

[0080] In this example, the in vitro activity assay of the glucan components of 2% CAMS, 4% CAMS, 6% CAMS, 8% CAMS and precipitated 2% CAMP, 4% CAMP, 6% CAMP, 8% CAMP prepared in Example 1 was performed.

[0081] 1. In vitro immunoassay

[0082] (1) Cytotoxicity assay

[0083] The effects of different treatments of Hericium erinaceus β-glucan fractions on the viability of RAW 264.7 macrophage cells (ATCC Cell Resource Center, Chinese Academy of Medical Sciences, Cat. No. CBP60533) were determined using the MTT assay. Three replicates were performed for each group. The absorbance of each well was measured at OD 490 nm using an enzyme-linked immunosorbent assay (ELISA).

[0084] (2) Effects on NO release from RAW 264.7 macrophages

[0085] RAW 264.7 was diluted with DMEM culture medium to a suspension containing 1×105 cells per ml, added to a 96-well plate, 90 μL per well, and cultured at 37°C until the cells were completely attached. 10 μL of the test sample was added to the sample group (the final mass concentration was 50, 200, and 500 μg mL -1 ), LPS group was added with 10 μL LPS (10 μg mL -1), 10 μL PBS was added to the control group, and the cells were cultured in a constant temperature incubator for 24 h. Then, 80 μL of cell culture medium was taken and 40 μL of Griess reagent was added. The cells were reacted at room temperature for 10 min, and the absorbance was measured at 543 nm by a microplate reader. The effects of Hericium erinaceus β-glucan prepared by different treatments on the NO release of RAW 264.7 macrophages were calculated.

[0086]

[0087] 2. Determination of the protective effect on human gastric epithelial cells GES-1

[0088] 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 samples were cultured in a 37°C, 5% CO2 constant temperature incubator for 12 h. 10 μL of each sample was taken, and the 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.

[0089] All the above experiments were repeated at least 3 times. The experimental data were expressed as mean ± standard deviation (Mean ± SD) and 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. P < 0.05 indicated that the results were significantly different and the differences were statistically significant.

[0090] Result analysis:

[0091] In vitro immunoreactivity of different glucan fractions

[0092] 1) In vitro immunoreactivity of glucan fractions degraded by microwave irradiation combined with citric acid

[0093] Depend on Figure 3 The results of in vitro immunoreactivity test of the components degraded by microwave irradiation and citric acid showed that the immunoreactivity of the components was significantly improved after microwave irradiation and citric acid treatment. The components treated with microwave irradiation and citric acid at different concentrations showed better immunoreactivity than HEP (HEP is a high molecular weight β-glucan of Hericium erinaceus). Among them, the immunoreactivity of 6% CAM sample was the best. The immunoreactivity of CAMS in the supernatant after 50% ethanol precipitation was higher than that of low concentration (50 μg·mL -1 ), medium concentration (200 μg·mL -1 ) is better than HEP (HEP is a high molecular weight β-glucan from Hericium erinaceus); the immune activity of CAMP precipitated after 50% ethanol precipitation is better than HEP.

[0094] 2) Protective activity of microwave irradiation combined with citric acid degradation of glucan components on GES-1 cells

[0095] like Figure 4 The protective activity of the components degraded by microwave irradiation combined with citric acid on human gastric epithelial cells GES-1 was shown, while the components treated with microwave irradiation combined with citric acid alone did not show good protective activity on GES-1 ( Figure 4 a); However, the protective activity of GES-1 in the supernatant samples after precipitation with 50% ethanol was significantly improved, with 4% CAMS and 6% CAMS (supernatant) having the best gastric mucosal protective activity ( Figure 4 b) was better than the PC (Sanjiu Weitai) control group; the 6% CAMP sample precipitated by 50% ethanol also showed good GES-1 protective activity under high concentration conditions ( Figure 4 c), significantly better than the PC (Sanjiu Weitai) control group.

[0096] 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 method for degrading Hericium erinaceus β-glucan, characterized in that: The following steps are involved: dissolving Hericium erinaceus β-glucan in citric acid and subjecting it to microwave treatment to obtain a co-treated Hericium erinaceus β-glucan, subjecting the treated Hericium erinaceus β-glucan to alcohol precipitation, collecting the supernatant or precipitate by centrifugation, and freeze-drying to obtain Hericium erinaceus β-glucan; When the Hericium erinaceus β-glucan is dissolved in citric acid, the Hericium erinaceus β-glucan is dissolved in citric acid at a mass concentration of 2 mg·mL -1 Dissolved in citric acid solution with a mass concentration of 2% to 8%; The microwave treatment conditions include a treatment power of 1200W to 1600W, a treatment temperature of 140°C to 180°C, and a treatment retention time of 30 to 50 minutes; The alcohol precipitation comprises using ethanol to precipitate the treated Hericium erinaceus β-glucan; the final volume concentration of the ethanol is 50%.

2. The method according to claim 1, characterized in that The microwave treatment conditions include a treatment power of 1600W, a treatment temperature of 140°C, and a treatment retention time of 30min.

3. The method according to claim 1, characterized in that When the Hericium erinaceus β-glucan is dissolved in citric acid, the Hericium erinaceus β-glucan is dissolved in citric acid at a mass concentration of 2 mg·mL -1 Dissolved in 6% citric acid solution.

4. The method according to claim 1, wherein The ethanol includes anhydrous ethanol.

5. The method according to claim 1, wherein The alcohol precipitation time is greater than 8 hours.

6. The method according to claim 1, characterized in that The supernatant is placed in a water bath to volatilize the ethanol, the water bath time is more than 1 hour, and the water bath temperature is above 90°C.

7. The method according to claim 1, characterized in that The precipitate was completely dissolved by adding water at a material-liquid ratio of 1 g: (0.5-1) mL.

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

9. Use of the method according to any one of claims 1 to 7 or the Hericium erinaceus β-glucan according to claim 8 in the preparation of a product that improves immune activity or gastric mucosal protection.

10. The use according to claim 9, characterized in that: The products include functional foods, health products or medicines.

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