Preparation method and application of high-activity antioxidant product based on hericium erinaceus-hawthorn solid state fermentation

Abstract

The invention belongs to the technical field of biological fermentation, and particularly relates to a preparation method and application of a high-activity antioxidant product based on hericium erinaceus-hawthorn solid state fermentation. Activating the strain to prepare a seed solution; adding hawthorn into the rice solid culture medium, inoculating hericium erinaceus, performing constant-temperature culture, drying a fermentation product to constant weight at normal temperature and normal pressure after the culture is finished, crushing, and sieving for later use. The antioxidant effect of the fermentation product is comprehensively evaluated and verified through a systematic in-vitro chemical antioxidant experiment (ABTS < + >, DPPH free radical and hydroxyl free radical scavenging) and an in-vivo zebra fish oxidative stress model, and solid data support is provided for application of the fermentation product in healthy products. The resource high-value utilization way of the hawthorn and the hericium erinaceus is widened, and a technical scheme and a product prototype are provided for developing novel food raw materials, health-care products or medicine auxiliary materials with definite antioxidant functions.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, specifically relating to a method for preparing a highly active antioxidant product based on Hericium erinaceus-hawthorn solid-state fermentation and its application. More specifically, this invention relates to a method for significantly increasing the content of active ingredients such as total flavonoids, total phenols, and quercetin in fermentation products by optimizing fermentation process parameters, thereby endowing them with excellent in vitro and in vivo antioxidant functions, and the application of the thus obtained highly active fermentation product in functional foods, health products, or pharmaceutical excipients. Background Technology

[0002] hawthorn( Crataegus pinnatifida Hawthorn (Hericium erinaceus), a traditional food and medicinal resource, is rich in flavonoids, phenolic acids, and organic acids, and its antioxidant and lipid-lowering physiological functions have been widely confirmed. Currently, the development of hawthorn mainly focuses on physical processing (such as making hawthorn slices and candied fruit) and chemical extraction (such as solvent extraction to obtain total hawthorn flavonoids). In addition, Hericium erinaceus (… Hericium erinaceus As a precious edible and medicinal fungus, Hericium erinaceus (monkey head mushroom) is rich in polysaccharides, polypeptides, terpenes, and other active ingredients. It exhibits excellent physiological activities in enhancing immune function, protecting the digestive system mucosa, and providing antioxidant effects. Combining edible value with medicinal development potential, it has become one of the research hotspots in the field of edible and medicinal fungi. Currently, Hericium erinaceus is mainly produced through liquid fermentation or single-substrate solid-state fermentation.

[0003] Currently, the fermentation process of Hericium erinaceus (monkey head mushroom) faces challenges such as low fermentation efficiency and insufficient stability of active ingredient yield. Furthermore, traditional extraction processes, such as ethanol reflux extraction, suffer from drawbacks including the risk of residual organic solvents, high energy consumption, and potential damage to heat-sensitive active ingredients. Moreover, the potential for increasing extraction rates is limited, failing to achieve targeted transformation and enhanced efficacy of the components. Pure hawthorn physical products or chemical extracts primarily contain active ingredients in glycoside form, resulting in relatively low bioavailability and difficulty in achieving significant physiological effects at conventional dosages. Existing processing methods do not adequately explore the comprehensive utilization value of hawthorn, failing to effectively enhance its added value through biotransformation technology, thus limiting the application of this medicinal and edible resource in the high-end functional food sector. Currently, there are no records of using hawthorn to prepare a fermentation system for Hericium erinaceus fermentation. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of the prior art and provide a green, efficient method for preparing highly active antioxidant products based on Hericium erinaceus-hawthorn solid-state fermentation, which can significantly enhance the content and functionality of active ingredients in the products.

[0005] The present invention also provides applications of the highly active antioxidant products prepared using the above-described preparation method.

[0006] The technical scheme adopted by the present application to achieve the above-mentioned purpose is: The present application provides a preparation method of high-activity antioxidant product based on Hericium erinaceus-Hawthorn solid-state fermentation, comprising the following steps: (1) Strain activation process: inoculate Hericium erinaceus strain on PDA slant medium, and culture until the mycelium covers the slant; (2) Seed liquid preparation standard: inoculate the activated strain on PDA liquid medium, and shake culture to obtain seed liquid; (3) Mix rice and nutrient solution, sterilize and cool to obtain rice solid culture medium; sterilize the rice solid culture medium, add hawthorn, inoculate Hericium erinaceus strain, and perform constant temperature culture; after the culture is completed, dry the fermentation product at room temperature and normal pressure to constant weight, crush and sieve for use.

[0007] Preferably, in step (1), the composition of the PDA slant medium is: potato powder 5 g / L, glucose 20 g / L, and agar 15 g / L; the culture is performed in a 28±1℃ constant temperature incubator for 5-7 days.

[0008] Preferably, in step (2), the composition of the PDA liquid medium is: glucose 20 g / L, peptone 5 g / L, yeast extract powder 5 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, and vitamin B1 0.05 g / L; the shake culture is performed at 28±1℃ and 150±10 rpm for 120±12 h.

[0009] Preferably, in step (2), the mycelial pellet density of the seed liquid is ≥1×10 5 CFU / mL.

[0010] Preferably, in step (3), the composition of the nutrient solution is: glucose 20 g, peptone 5 g, yeast extract powder 5 g, potassium dihydrogen phosphate 1 g, vitamin B1 0.05 g, magnesium sulfate 0.5 g, L-asparagine 0.5 g, sodium chloride 0.5 g, manganese sulfate 0.01 g, zinc sulfate 0.01 g, and dissolved in 1000 mL of distilled water.

[0011] Preferably, in step (3), the ratio of the rice and the nutrient solution is 1 g:2 mL; and the mass ratio of the rice and the hawthorn is 50-90:10-50.

[0012] Preferably, in step (3), the culture is performed at 28℃ for 10-60 d.

[0013] The present invention also provides an application of the highly active antioxidant product based on Hericium erinaceus-hawthorn solid-state fermentation prepared by the above preparation method in the preparation of antioxidant products.

[0014] This invention provides a method for the targeted transformation and enrichment of hawthorn's active ingredients using bio-fermentation technology without the use of organic chemical solvents, thereby improving their content and bioavailability. This invention establishes a scientific and quantifiable system of fermentation process parameters, and through the specific culture medium of this invention, prepares a novel solid-state fermentation product that combines the beneficial components of both Hericium erinaceus and hawthorn, with synergistically enhanced antioxidant activity.

[0015] This invention comprehensively evaluates and verifies the antioxidant efficacy of the fermentation product through systematic in vitro chemical antioxidant experiments (ABTS+, DPPH free radical scavenging, and hydroxyl free radical scavenging) and an in vivo zebrafish oxidative stress model, providing solid data support for its application in health products. It also broadens the pathways for the high-value utilization of hawthorn and Hericium erinaceus resources, providing technical solutions and product prototypes for developing novel food ingredients, health products, or pharmaceutical excipients with clear antioxidant functions.

[0016] The Hericium erinaceus-hawthorn solid-state fermentation product prepared by this invention, based on its rich active ingredients and proven antioxidant function, can be widely used in the following fields: (1) As a natural food antioxidant: It can be directly added to oils, meat products, baked goods and beverages to delay food oxidation and deterioration, extend shelf life and replace some synthetic antioxidants (such as BHT and BHA).

[0017] (2) As a core ingredient in functional foods or health products: It can be further processed into dosage forms such as capsules, tablets, and powders, and used to develop products with functions such as anti-oxidation, anti-aging, enhancing immunity, and assisting in regulating blood lipids. It can also be added as a nutritional fortification ingredient to special dietary foods and sports nutrition products.

[0018] (3) As a functional ingredient in cosmetics: its antioxidant and anti-inflammatory properties can be used to develop skin care products with anti-skin aging and barrier repair functions.

[0019] (4) As a drug excipient or Chinese medicine raw material: It can be used as a drug excipient with antioxidant effect, or as a raw material in Chinese medicine compound prescriptions, to prepare drugs for treating diseases related to oxidative stress.

[0020] The beneficial effects of this invention are as follows: (1) Significantly increased content of active ingredients, achieving targeted transformation: This invention utilizes the fermentation of Hericium erinaceus to efficiently biotransform precursor substances in hawthorn. Under optimal conditions (20% hawthorn, fermentation for 40 days), compared to unfermented hawthorn, the total flavonoid content increased by up to 18.23%, the total phenol content increased by up to 163.20%, and the quercetin content achieved an astonishing maximum increase of 1093.09%. This indicates that this invention not only enriches the original components but also generates new active substances or transforms them into more active forms.

[0021] (2) It has comprehensive and excellent antioxidant properties, which have been fully verified in vivo and in vitro: In vitro antioxidant activity: The fermentation product exhibited strong scavenging ability against three major free radicals (ABTS, DPPH, and hydroxyl radicals), with scavenging rates ranging from 21.25% to 41.46%, 68.94% to 97.57%, and 22.09% to 47.17%, respectively, all significantly higher than that of unfermented hawthorn raw material. P <0.05).

[0022] In vivo antioxidant activity: Experiments using a zebrafish model confirmed that the fermentation products significantly enhanced the activity of the body's core antioxidant enzymes—superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST). P <0.05), and can effectively reduce the content of oxidized glutathione (GSSG) and the level of reactive oxygen species (ROS) in the body, proving that it has a clear physiological-grade antioxidant effect.

[0023] (3) The process is green and safe and suitable for industrialization: no organic chemical solvents are used in the whole process, avoiding the problem of solvent residue; solid-state fermentation is adopted, which has low energy consumption and conforms to the concept of green manufacturing; the selected strains and raw materials are all food and medicine homologous substances that are permitted to be used in the country, with high safety and easy scale-up and standardization of the production process.

[0024] (4) High application value and clear product positioning: The fermented product obtained integrates the dual beneficial components of hawthorn and Hericium erinaceus, and the activity is synergistically enhanced. It can be directly used as a high-value natural antioxidant, functional food ingredient or health product raw material, and applied to various product forms such as beverages, tablets, and capsules to meet the market demand for "natural, nutritious and healthy" products. Attached Figure Description

[0025] Figure 1 The changes in total flavonoid content of hawthorn before and after fermentation for 10, 20, 30, 40, 50, and 60 days were as follows: (A) Hawthorn addition 10% (B) Hawthorn addition 20% (C) Hawthorn addition 30% (D) Hawthorn addition 40% (E) Hawthorn addition 50% Figure 2 The changes in phenolic content of hawthorn before and after fermentation for 10, 20, 30, 40, 50, and 60 days are as follows: (A) 10% hawthorn added; (B) 20% hawthorn added; (C) 30% hawthorn added; (D) 40% hawthorn added; (E) 50% hawthorn added. Figure 3 The changes in quercetin content of hawthorn before and after fermentation for 10, 20, 30, 40, 50, and 60 days were as follows: (A) 10% hawthorn added; (B) 20% hawthorn added; (C) 30% hawthorn added; (D) 40% hawthorn added; (E) 50% hawthorn added. Figure 4 The change in ABTS free radical scavenging rate (%) before and after fermentation (10, 20, 30, 40, 50, and 60 days) of hawthorn fermentation with 20% hawthorn addition; Note: Different letters indicate significant differences ( P <0.05); Figure 5 The change in DPPH free radical scavenging rate (%) of hawthorn before and after fermentation for 10, 20, 30, 40, 50, and 60 days after adding 20% ​​hawthorn; Note: Different letters indicate significant differences ( P <0.05); Figure 6 The change in hydroxyl radical scavenging rate (%) of hawthorn before and after fermentation (10, 20, 30, 40, 50, and 60 days) after adding 20% ​​hawthorn; Note: Different letters indicate significant differences ( P <0.05); Figure 7 Changes in zebrafish mortality rate (%) after gradient administration of Hericium erinaceus-hawthorn solid-state fermentation product; Figure 8 The fluorescence area of ​​reactive oxygen species (ROS) in zebrafish was measured for gradient administration of Hericium erinaceus-hawthorn solid-state fermentation product; Note: Different letters indicate significant differences. P <0.05) Figure 9 The area of ​​reactive oxygen species (ROS) fluorescence signal in zebrafish after 10, 20, 30, 40, 50, and 60 days of solid-state fermentation of Hericium erinaceus and hawthorn; Note: Different letters indicate significant differences (…). P <0.05); Figure 10The figures show the fluorescence area of ​​reactive oxygen species in zebrafish under a fluorescence microscope: (A) blank group, (B) model group, (C) group treated with products after 10 days of fermentation, (D) group treated with products after 20 days of fermentation, (E) group treated with products after 30 days of fermentation, (F) group treated with products after 40 days of fermentation, (G) group treated with products after 50 days of fermentation, (H) group treated with products after 60 days of fermentation, (I) group treated with hawthorn, and (J) group treated with Hericium erinaceus. In the figures, reactive oxygen species in zebrafish oxidize Dichlorofluorescin (DCFH) to Dichlorofluorescein (DCF), which appears as a strong green fluorescent substance under a fluorescence microscope. Figure 11 Solid-state fermentation products of Hericium erinaceus and hawthorn after 10, 20, 30, 40, 50, and 60 days of drug administration, and the SOD activity (U / g) in zebrafish; Note: Different letters indicate significant differences ( P <0.05); Figure 12 Hericium erinaceus-hawthorn solid-state fermentation products after 10, 20, 30, 40, 50, and 60 days of drug administration; CAT activity (U / g) in zebrafish; Note: Different letters indicate significant differences. P <0.05); Figure 13 The content of GSSG in zebrafish (μg / g) of Hericium erinaceus-hawthorn solid-state fermentation products after 10, 20, 30, 40, 50, and 60 days of drug administration; Note: Different letters indicate significant differences ( P <0.05); Figure 14 Solid-state fermentation products of Hericium erinaceus and hawthorn after 10, 20, 30, 40, 50, and 60 days of drug administration; GST activity (U / g) in zebrafish; Note: Different letters indicate significant differences ( P <0.05). Detailed Implementation

[0026] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0027] This invention ensures the scientific validity and reliability of its effect assessment through the following multi-dimensional indicators, standardized testing methods, and rigorous data analysis: Judgment indicators: (1) Content of core active ingredients: total flavonoids, total phenols, quercetin.

[0028] (2) In vitro antioxidant capacity: scavenging rate of ABTS, DPPH free radicals and hydroxyl free radicals.

[0029] (3) Antioxidant effects in vivo: ROS level, SOD activity, CAT activity, GST activity and GSSG content in zebrafish.

[0030] (4) Safety indicators: maximum tolerated dose of zebrafish.

[0031] Detection method: (1) All chemical components were determined and in vitro antioxidant experiments were performed in strict accordance with national standards or industry-recognized reagent kit instructions, and a standard curve was plotted for quantification (R²>0.999).

[0032] (2) The in vivo experiments used the internationally recognized model organism, zebrafish, and the experimental procedures were standardized and the modeling conditions were optimized.

[0033] Results and Data Analysis: (1) All quantitative data are expressed as mean ± standard deviation, and each experiment is repeated at least 3 times.

[0034] (2) Statistical analysis was performed using IBM SPSS Statistics 20.0 software. One-way ANOVA was used for comparisons among multiple groups, and Tukey's test was used for pairwise comparisons between groups.

[0035] (3) A p-value < 0.05 is considered statistically significant. In graphs and tables, different letters are usually used to indicate significant differences.

[0036] (4) Use GraphPad Prism 10 software to draw charts to ensure that the data visualization is clear and accurate.

[0037] The hawthorn added in proportion during the solid-state fermentation process of this invention is hawthorn powder, which is prepared by grinding dried hawthorn into powder and then passing it through a 60-mesh sieve.

[0038] Example 1 This invention provides a method for preparing Hericium erinaceus-hawthorn solid-state fermentation products and their applications. The core of this method lies in achieving efficient conversion and enrichment of active ingredients through specific fermentation strains, optimized substrate formulation, and precise fermentation control. The specific technical solution is as follows: 1. Strain selection and seed culture preparation (1) Source and identification of the strain: The fermentation strain used in this invention is Hericium erinaceus, purchased from the China General Microbiological Culture Collection Center, with the number CGMCC 5.823. This strain was identified and confirmed by Beijing Qingke Biotechnology Co., Ltd. through ITS sequence analysis.

[0039] (2) Activation process of strain: Inoculate the Hericium erinaceus strain (inoculation amount: 0.5 cm x 0.5 cm) on PDA slant medium (formulation: potato extract powder 5 g / L, glucose 20 g / L, agar 15 g / L, pH natural) and incubate in a constant temperature incubator at 28±1℃ for 5-7 days until the mycelium covers the slant.

[0040] (3) Seed culture preparation standard: Activated bacterial strains were inoculated into PDA liquid medium (20 g / L glucose, 5 g / L peptone, 5 g / L yeast extract, 1 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.05 g / L vitamin B1), and cultured with shaking at 28±1℃ and 150±10 rpm for 120±12 h to obtain mycelial ball density ≥1×10⁻⁶. 5 Seed culture of CFU / mL.

[0041] 2. Preparation and optimization of solid-state fermentation substrate Take 5 g glucose, 5 g peptone, 5 g yeast extract, 1 g potassium dihydrogen phosphate, 1 g vitamin B1, 0.5 g magnesium sulfate, 0.5 g L-asparagine, 0.5 g sodium chloride, 0.01 g manganese sulfate, and 0.01 g zinc sulfate, and dissolve them in 1000 mL distilled water to prepare a nutrient solution. Take 30 g rice and 60 ml of nutrient solution at a ratio of 1:2 and place them in a fermentation glass jar. Cover with a sterile film and autoclave at 121 ℃ for 20 minutes. After cooling, set aside (this is the rice solid culture medium). Place the rice solid culture medium in a clean bench for ultraviolet sterilization. After sterilization, inoculate with Hericium erinaceus (spore density ≥ 1×10⁻⁶). 5 (CFU / mL), after inoculation, incubate at 28 ℃.

[0042] The solid-state fermentation process of Hericium erinaceus and hawthorn involved sterilizing rice solid culture medium and then adding hawthorn. Six experimental groups (AF groups) were established based on different amounts of hawthorn added. The ratio of rice to hawthorn is shown in Table 1. Samples were taken on days 10, 20, 30, 40, 50, and 60 of fermentation. The fermentation product was dried to constant weight at room temperature and pressure, pulverized, and passed through a 60-mesh sieve (250 μm pore size). It was then frozen and stored at -20℃ for later use.

[0043] Table 1. Ratio of rice to hawthorn added to the culture medium Based on the dry weight of the basal culture medium, the optimal addition amount is 20%. The total fermentation cycle is 10-60 days. Based on the dynamic changes of active ingredients and the evaluation of antioxidant activity, 40 days was determined to be the optimal fermentation endpoint.

[0044] Example 2: Determination and Quality Control of Active Ingredient Content in Hericium erinaceus-Hawthorn Solid-State Fermentation Products (I) Determination of total flavonoids and total phenols in solid-state fermentation products of Hericium erinaceus-hawthorn (1) Determination of total flavonoid content: The sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method was used. Rutin was used as the standard, and the absorbance was measured at a wavelength of 510 nm. The total flavonoid content in the fermentation product should not be less than 10.0 mg / g (based on dry weight).

[0045] (2) Construction of standard curves for rutin and gallic acid A standard curve for rutin and gallic acid was plotted with the concentration (X) of the standard solution as the abscissa and the absorbance (Y) as the ordinate, and the regression equation for the standard curve was obtained. The standard curves for rutin and gallic acid standards are shown in Table 3.

[0046] Table 2 Standard curves for rutin and gallic acid (3) Determination of total flavonoid content in Hericium erinaceus-hawthorn solid-state fermentation products Changes in total flavonoid content before and after hawthorn fermentation are as follows: Figure 1 As shown, the flavonoid content in hawthorn before fermentation was 9.93±0.0082 mg / g. After fermentation, the total flavonoid content in the fermentation product with 10% hawthorn addition was 13.59±1.68 mg / g, with a growth rate between 41.80% and 49.77%; the total flavonoid content in the fermentation product with 20% hawthorn addition was 10.34±0.93 mg / g, with a growth rate between -0.45% and 18.23%; the total flavonoid content in the fermentation product with 30% hawthorn addition was 10.19±0.78 mg / g, with a growth rate between -7.73% and 16.02%; the total flavonoid content in the fermentation product with 40% hawthorn addition was 9.77±1.10 mg / g, with a growth rate between -14.35% and 18.97%; and the total flavonoid content in the fermentation product with 50% hawthorn addition was 8.32±1.03 mg / g, without achieving positive growth in total flavonoid content, with a range between -23.36% and -1.89%.

[0047] (II) Determination of phenolic content in Hericium erinaceus-hawthorn solid-state fermentation products Total phenol content determination: The Folin-Ciocalteu method was used. Gallic acid was used as a standard, and the absorbance was measured at a wavelength of 765 nm. The total phenol content in the fermentation product should not be less than 2.0 mg / g (on a dry weight basis).

[0048] Changes in total phenol content before and after hawthorn fermentation are as follows: Figure 2As shown, the total phenol content in hawthorn before fermentation was 1.04 ± 0.0022 mg / g. After fermentation, the total phenolic content of the fermentation products was 2.91±0.19 mg / g for 10% hawthorn addition, with a growth rate between 153.15% and 204.10%; 2.37±0.25 mg / g for 20% hawthorn addition, with a growth rate between 104.51% and 163.20%; 2.23±0.19 mg / g for 30% hawthorn addition, with a growth rate between 85.14% and 135.34%; 2.04±0.076 mg / g for 40% hawthorn addition, with a growth rate between 88.21% and 109.73%; and 1.68±0.086 mg / g for 50% hawthorn addition, with a growth rate between 48.41% and 68.94%.

[0049] (III) Determination of Quercetin Content in Hericium erinaceus-Hawthorn Solid-State Fermentation Products (1) Determination of quercetin content: High performance liquid chromatography (HPLC) was used. Chromatographic conditions: InertSustain-C18 column (4.6 mm × 250 mm, 5 μm), column temperature 25 ℃, mobile phase was 0.1% formic acid aqueous solution (A) and acetonitrile (B), gradient elution: 0-10 min (10%B), 10-11 min (10-60%B), 11-12 min (60-50%B), 12-13 min (50-45%B), 13-14 min (45-30%B), 14-15 min (30-20%B), 15-16 min (20-10%B), 16-20 min (10%B), flow rate 1.0 mL / min, detection wavelength 370 nm, injection volume 20 μL. The quercetin content in the fermentation product was required to be not less than 0.06 mg / g (dry weight).

[0050] (2) Plotting the quercetin standard curve A standard curve for quercetin was plotted with the concentration (X) of the quercetin standard solution as the abscissa and the peak area (Y) as the ordinate, and the regression equation for the standard curve was obtained. The standard curves for the quercetin standard are shown in Table 4.

[0051] Table 3. Quercetin Standard Curve (3) Quercetin content in Hericium erinaceus-hawthorn solid-state fermentation products Changes in quercetin in hawthorn before and after fermentation are as follows: Figure 3As shown, the quercetin content in hawthorn before fermentation was 0.010±0.00031 mg / g. After fermentation, the quercetin content in the Hericium erinaceus-hawthorn solid-state fermentation product with 10% hawthorn addition was 0.040±0.0093 mg / g, with a growth rate between 186.84% and 447.66%; the quercetin content with 20% hawthorn addition was 0.070±0.030 mg / g, with a growth rate between 176.51% and 1093.09%; the quercetin content with 30% hawthorn addition was 0.079±0.018 mg / g, with a growth rate between 468.01% and 998.38%; and the quercetin content with 40% hawthorn addition was 0.081±0.014 mg / g. The growth rate was between 513.13% and 992.18%; the quercetin content of Hericium erinaceus-hawthorn solid-state fermentation product with 50% hawthorn addition was 0.071±0.019 mg / g, with a growth rate between 268.39% and 786.60%.

[0052] Example 3 (I) Determination of the proportion of hawthorn added to the solid-state fermentation product of Hericium erinaceus-hawthorn Based on the contents of quercetin (score 1), total flavonoids (score 2), and total phenols (score 3) in the fermentation products, the optimal amount of hawthorn was selected by weighting the comprehensive scores using the entropy method. The scores for different hawthorn addition amounts are shown in Table 5. Calculations showed that the fermentation product with 20% hawthorn addition had the highest comprehensive score of 0.659. Therefore, the optimal addition amount of hawthorn in the Hericium erinaceus-hawthorn solid-state fermentation process was determined to be 20%. Based on this, the in vitro and in vivo antioxidant activity of Hericium erinaceus-hawthorn solid-state fermentation products with different fermentation days was investigated.

[0053] (1) Standardize the data and use range standardization for calculation: Formula (1) (2) Calculate the specific gravity: Formula (2) (3) Calculate the entropy value: Formulas (3) and (4) are used to calculate the difference coefficient and weights: , Formulas (4) and (5) are used to calculate the overall score: Formula (5) Table 4. Comprehensive scores of Hericium erinaceus-hawthorn solid-state fermentation products under different hawthorn addition ratios. (II) Determination of in vitro antioxidant activity of Hericium erinaceus-hawthorn solid-state fermentation products In vitro antioxidant indicators: ABTS free radical scavenging rate: ≥40%, DPPH free radical scavenging rate: ≥90%, hydroxyl free radical scavenging rate: ≥45%. In vivo antioxidant indicators (zebrafish model): ROS scavenging rate: ≥75%, SOD activity increase: ≥80%, CAT activity increase: ≥150%, GST activity increase: ≥80%, GSSG content decrease: ≥40%.

[0054] (1) ABTS free radical scavenging ability of Hericium erinaceus-hawthorn solid-state fermentation products Take 0.01 g of sample (heric mushroom-hawthorn solid-state fermentation product), add 1 mL of extraction buffer, sonicate for 30 min, centrifuge at 10000 g and 4 ℃ for 10 min, and collect the supernatant for testing. Follow the instructions of the Total Antioxidant Capacity (ABTS method) kit for the remaining steps.

[0055] Total antioxidant capacity is expressed as free radical scavenging rate, ABTS free radical scavenging rate (%) = In the formula: A 空白 =Absorbance value of blank tube; A 测定 =Absorbance value of the sample measurement tube; Hericium erinaceus-hawthorn solid-state fermentation product ABTS free radical scavenging rate (%) Figure 4 As shown, the ABTS free radical scavenging rate (%) of Hericium erinaceus-hawthorn solid fermentation products fermented for 10-60 days with 20% hawthorn addition was stronger than that of unfermented hawthorn and Hericium erinaceus solid fermentation products of the same amount, with the ABTS free radical scavenging rate ranging from 21.25% to 45.46%.

[0056] The unfermented hawthorn group is hawthorn powder that has passed through a 60-mesh sieve.

[0057] The solid-state fermentation product of Hericium erinaceus is the fermentation product of Hericium erinaceus without inoculation with hawthorn, and the fermentation time is 40 days.

[0058] (2) DPPH free radical scavenging ability of Hericium erinaceus-hawthorn solid-state fermentation products Take 0.05 g of sample, add 0.8 mL of nitrogen free radical extraction solution, extract in a 40 ℃ water bath for 30-60 min, centrifuge at 10000 rpm for 10 min, and collect the supernatant for testing. Follow the instructions for the remaining steps according to the DPPH free radical scavenging assay kit manual.

[0059] DPPH free radical scavenging rate (%) = In the formula: A 0 = Absorbance value of blank tube; A1 = Absorbance value of the sample measurement tube; A 2 = Absorbance value of the sample control tube DPPH free radical scavenging rate (%) of Hericium erinaceus-hawthorn solid-state fermentation products Figure 5 As shown, the DPPH free radical scavenging rate (%) of the Hericium erinaceus-hawthorn solid fermentation product with 20% hawthorn addition after 10-60 days of fermentation was stronger than that of the same amount of unfermented hawthorn and Hericium erinaceus solid fermentation product. Its DPPH free radical scavenging rate (%) ranged from 68.94% to 97.57%.

[0060] (3) Hydroxyl radical scavenging ability of Hericium erinaceus-hawthorn solid-state fermentation products Take 0.05 g of sample (heric mushroom-hawthorn fermentation product), add 1 mL of 80% ethanol, and sonicate at 50 ℃ and 200-300 W for 30 min (shaking and mixing once every 5 min). If there is any loss, make up to 1 mL with 80% ethanol. Centrifuge at 12000 rpm for 10 min, and collect the supernatant for testing. Follow the instructions of the hydroxyl radical scavenging ability test kit for the remaining steps.

[0061] Hydroxyl radical scavenging rate (%) = In the formula: A 空白 =Absorbance value of blank tube; A 测定 =Absorbance value of the sample measurement tube; A 对照 =Absorbance value of sample control tube Hydroxyl radical scavenging rate (%) of Hericium erinaceus-hawthorn solid-state fermentation products Figure 6 As shown, the hydroxyl radical scavenging rate (%) of Hericium erinaceus-hawthorn solid-state fermentation products fermented for 10-60 days with 20% hawthorn addition was stronger than that of equal amounts of unfermented hawthorn and Hericium erinaceus solid-state fermentation products. The hydroxyl radical scavenging rate (%) ranged from 22.09% to 47.17%.

[0062] (III) Determination of the in vivo antioxidant activity of Hericium erinaceus-hawthorn solid-state fermentation products (1) 2,2'-Azobis(2-methylpropanediamine) dihydrochloride (AAPH-induced zebrafish oxidative stress model) Different concentration gradients (2, 4, 8, 10, 15, 20, 25, 30 mM) of AAPH solution and different modeling times (1 / 6 h, 1 / 2 h, 1 h, 2 h, 4 h, 24 h, 48 h) were set up to induce oxidative stress and explore the optimal modeling concentration and time. The survival and development of zebrafish larvae were observed under a microscope. Dead larvae were recorded and removed in a timely manner (dead larvae had no heartbeat, some had bent tails, and floated on the surface without swimming). The survival rate of larvae in each well was calculated 24 h after modeling (survival rate = number of surviving larvae at 6 dpf / total number of larvae).

[0063] The survival and development of zebrafish larvae were observed under a microscope, and the normal survival rate of zebrafish larvae in each well was counted (survival rate = number of surviving zebrafish / total number of zebrafish). The survival rates of zebrafish larvae induced by different concentrations of AAPH at different times are shown in Table 5. The zebrafish larvae induced by a concentration of 2 mM and an induction time of 24 h and 5 dpf were in good physical condition and had a high survival rate. Therefore, this condition was determined as the modeling condition and the next experiment was carried out.

[0064] Table 5. Survival rate of zebrafish juveniles induced by different concentrations of AAPH at different times. (2) Tolerance test After 72 hours of administration of Hericium erinaceus-hawthorn solid-state fermentation product, the mortality rate of zebrafish juveniles was as follows: Figure 7 As shown, the mortality rate of zebrafish was 0 when the drug concentration was less than 200 μg / mL (based on crude drug content). Therefore, the maximum tolerated dose of zebrafish to Hericium erinaceus-hawthorn solid-state fermentation product was determined to be 200 μg / mL.

[0065] (3) Determination of the optimal dosage of Hericium erinaceus-hawthorn solid-state fermentation product Gradual drug administration, the area of ​​reactive oxygen species (ROS) fluorescence signal in zebrafish is as follows Figure 8 As shown, the optimal dosage of Hericium erinaceus-hawthorn solid-state fermentation product was 160 μg / mL, at which point the reactive oxygen species fluorescence signal area was 2692.00±442.86. The optimal dosage was subsequently used as the experimental dosage for each fermentation group.

[0066] (4) Effect of Hericium erinaceus-hawthorn solid-state fermentation products on the area of ​​reactive oxygen species (ROS) positive fluorescence in zebrafish Hericium erinaceus-hawthorn solid-state fermentation product showed a strong reducing effect on reactive oxygen species (ROS) in zebrafish. The fluorescence signal area of ​​ROS in zebrafish before and after administration was as follows: Figure 9 As shown, the reactive oxygen species fluorescence signal area range decreased from a minimum of 7423.50±813.04 in the model group to 1725.20±583.40, which is slightly higher than that in the blank group (1496.90±350.75).

[0067] The area of ​​reactive oxygen species fluorescence signal in zebrafish observed under a fluorescence microscope is as follows: Figure 10 As shown, zebrafish in the model group (AAPH-induced zebrafish oxidative stress model) generated a large amount of reactive oxygen species (ROS), mainly concentrated in the abdomen and back, and showed large areas of green fluorescence after staining; zebrafish in the blank group only showed a small area of ​​fluorescence in the abdomen; after administration of fermentation products, the area and intensity of reactive oxygen fluorescence signals in zebrafish were significantly reduced; administration of hawthorn or Hericium erinaceus alone had a weak effect on reducing the content of reactive oxygen in zebrafish.

[0068] (5) Effect of Hericium erinaceus-hawthorn solid-state fermentation products on superoxide dismutase (SOD) activity in zebrafish Hericium erinaceus-hawthorn solid-state fermentation product has a restorative effect on SOD activity in zebrafish. Before and after administration, the SOD activity in zebrafish was as follows: Figure 11 As shown, SOD activity increased from 40.28±2.83 U / g in the model group to a maximum of 76.50±7.48 U / g, slightly lower than the control group (79.06±5.19 U / g). The product in the middle stage of fermentation (30-40 days) showed the most significant increase in SOD activity. In addition, the increase in SOD activity in the Hericium erinaceus group (44.75±6.07 U / g) was weak, and the recovery of SOD activity in the equal amount of hawthorn group (54.29±1.53 U / g) was less than that in the fermentation product group.

[0069] (6) Effect of Hericium erinaceus-hawthorn solid-state fermentation products on catalase (CAT) activity in zebrafish Before and after administration of Hericium erinaceus-hawthorn solid-state fermentation product, the CAT activity in zebrafish was as follows: Figure 12 As shown, CAT activity in zebrafish recovered to varying degrees after administration of the fermentation product. CAT activity increased from a maximum of 182.25±54.10 U / g in the model group to 500.75±36.46 U / g, which was lower than that in the blank group (628.16±26.02 U / g). The product group showed higher CAT activity between 10 and 40 days, indicating that the fermentation product in the early and middle stages had the most significant effect on increasing CAT activity, and its recovery ability was stronger than that of the equal-volume hawthorn group (344.02±26.76 U / g) and the Hericium erinaceus group (226.80±67.99 U / g).

[0070] (7) Effect of Hericium erinaceus-hawthorn solid-state fermentation products on the content of oxidized glutathione (GSSG) in zebrafish Hericium erinaceus-hawthorn solid-state fermentation product can reduce the GSSG content in zebrafish. The GSSG content in zebrafish before and after administration was as follows: Figure 13As shown, the GSSG content in zebrafish in the control group was 56.41±4.27 μg / g. After administration of the fermentation product, the GSSG content in all groups (the lowest being 70.66±6.53 μg / g) was lower than that in the model group (120.51±8.55 μg / g), indicating that the fermentation product can alleviate oxidative stress. The GSSG content in the 20-40 day product group was the lowest, indicating that the product in the middle stage of fermentation (20-40 days) had the best anti-oxidative damage effect. The GSSG content in the Hericium erinaceus group was 113.39±4.93 μg / g, and the GSSG content in the hawthorn group was 96.30±8.90 μg / g, both of which had lower antioxidant capacity than the fermentation product group.

[0071] (8) Effects of Hericium erinaceus-hawthorn solid-state fermentation products on glutathione-S-transferase (GST) activity in zebrafish Effects of Hericium erinaceus-hawthorn solid-state fermentation products on GST activity in zebrafish, such as Figure 14 As shown, the GST activity in zebrafish in the blank group was 2.7±0.086 U / g, and the GST activity in zebrafish in the model group was 0.87±0.11 U / g. After administration, the GST activity in the hawthorn group increased to 1.32±0.088 U / g, while the GST activity in the Hericium erinaceus group (0.82±0.17 U / g) did not increase. The GST activity in the fermentation product administration groups was significantly higher than that in the model group, with the highest increase reaching 1.66±0.10 U / g. Among them, the GST activity in the 40-day product group was the highest, indicating that the product had the strongest detoxification ability after 40 days of fermentation.

Claims

1. A method for preparing high-activity antioxidant product based on Hericium- hawthorn solid-state fermentation, characterized in that, The method comprises the following steps: (1) a strain activation process: inoculating Hericium erinaceus strains on PDA slant medium, and culturing until the mycelium covers the slant; (2) a seed liquid preparation standard: inoculating the activated strains on PDA liquid medium, and oscillating and culturing to obtain a seed liquid; (3) mixing rice and nutrient solution, sterilizing and cooling to obtain rice solid culture medium; sterilizing the rice solid culture medium, adding hawthorn, inoculating Hericium erinaceus strains, and performing constant-temperature culture; after the culture is completed, the fermentation product is dried at room temperature and normal pressure until the weight is constant, and then the product is crushed and sieved for use.

2. The production method according to claim 1, characterized by, In step (1), the PDA slant medium comprises potato powder 5 g / L, glucose 20 g / L, and agar 15 g / L; and the culture is performed in a 28±1℃ constant-temperature incubator for 5-7 days.

3. The preparation method according to claim 1, characterized in that, In step (2), the PDA liquid medium comprises glucose 20 g / L, peptone 5 g / L, yeast extract powder 5 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, and vitamin B1 0.05 g / L; and the oscillating culture is performed at 28±1℃ and 150±10 rpm for 120±12 h.

4. The production method according to claim 1 or 3, characterized by, In step (2), the seed liquid has a spore density of > 1 x 10 5 CFU / mL.

5. The preparation method according to claim 1, characterized in that, In step (3), the nutrient solution comprises glucose 5 g, peptone 5 g, yeast extract powder 5 g, potassium dihydrogen phosphate 1 g, vitamin B1 0.05 g, magnesium sulfate 0.5 g, L-asparagine 0.5 g, sodium chloride 0.5 g, manganese sulfate 0.01 g, zinc sulfate 0.01 g, and 1000 mL of distilled water.

6. The production method according to claim 1 or 5, characterized by, In step (3), the ratio of the rice and the nutrient solution is 1 g:2 mL; and the mass ratio of the rice and the hawthorn is 50-90:10-50.

7. The production method according to claim 1 or 6, characterized by, In step (3), the culture is performed at 28℃ for 10-60 d.

8. Application of a high-activity antioxidant product based on Hericium erinaceus-hawthorn solid-state fermentation, which is prepared by the preparation method of any one of claims 1-7, in the preparation of an antioxidant product.

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