Oat and lucid ganoderma fermentation liquor as well as preparation method and application thereof

Abstract

The invention provides oat and ganoderma lucidum fermentation liquor as well as a preparation method and application thereof, and belongs to the technical field of fermentation. The preparation method of the oat and ganoderma lucidum fermentation liquor comprises the following steps: S1, mixing oat and water according to a weight-volume ratio of 1: (16-25) (g: mL), sterilizing, cooling to room temperature, adding bifidobacterium liquid under an aseptic condition, mixing, and carrying out anaerobic fermentation to obtain a primary fermentation product; s2, mixing the primary leavening obtained in the step S1 with ganoderma lucidum solid fermentation hyphae, and culturing to obtain a secondary leavening; and S3, centrifuging the secondary fermentation product obtained in the step S2, wherein the supernatant is the oat and ganoderma lucidum fermentation liquor. The oat and ganoderma lucidum fermentation liquor has a remarkable anti-inflammatory function.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation, and relates to an oat ganoderma lucidum fermentation liquid and a preparation method and application thereof. Background Art

[0002] Oats are an annual herbaceous plant of the genus Avena in the Poaceae family, mainly produced in the temperate regions of the Northern Hemisphere. Oats are widely used as a food raw material. They are rich in unsaturated fatty acids, amino acids, minerals and dietary fiber. They have the effects of increasing satiety and lowering blood lipids. They are also used in other industries such as cosmetics and pharmaceuticals. The glucan in oats has a good moisturizing effect and is often used as an ingredient in cosmetics. The article "Research Progress and Application of Avena Alkaloids" (Tang Yaru et al., "Journal of Northern Agriculture", 2023, 51 (6): 37-50) pointed out that oats contain active ingredients such as avena alkaloids. Avena alkaloids have rich physiological activities and show broad application prospects in the fields of anti-oxidation, anti-inflammatory, anti-cancer, anti-proliferation, cardiovascular health, etc.

[0003] Chinese invention patent CN109430402A provides a method for preparing fermented oatmeal dairy products. The technology adopts a two-fermentation process. In the first fermentation, sweet wine koji is inoculated into steamed oats, and sweet embryos are obtained after fermentation at 35-45℃ for 10-12 hours, and the sweet embryos are sterilized; the mass ratio of sweet wine koji to steamed oats is 1: 900-1100; secondary fermentation: the starter is inoculated into sterilized raw milk and mixed with sweet additives, and then added to the sweet embryos prepared in the previous step for secondary fermentation to obtain fermented glutinous rice oatmeal yogurt; and then filtered and canned. This method not only reduces the acidity of the fermented liquid, improves stability, refines the bran, and improves the utilization rate of raw materials, but also brings a unique flavor.

[0004] Chinese invention patent CN103271274A provides a nutritional health porridge, which has the following composition (based on a total weight of 1000 parts by weight): 20-40 parts of corn, 10-15 parts of oats, 10-20 parts of yam, 5-10 parts of red dates, 10-20 parts of coix seeds, 5-10 parts of glutinous rice, 2-5 parts of ganoderma lucidum, 5-15 parts of walnuts, 2-6 parts of peanuts, 8-18 parts of kidney beans, 2-5 parts of spirulina, 50-100 parts of white sugar, 20-35 parts of honey, and 6-18 parts of probiotics. The nutritional health porridge of this technology has the effects of supplementing nutrition, promoting wound recovery, and regulating intestinal function. However, the method does not provide an oat ganoderma fermentation liquid, nor does it provide an application of an oat ganoderma fermentation liquid in an anti-inflammatory liquid.

[0005] In summary, the prior art does not provide a preparation method of oatmeal and Ganoderma lucidum fermentation liquid and its application in anti-inflammatory treatment. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide an oat ganoderma fermentation liquid and a preparation method and application thereof. The alkaloids in oats are mainly distributed in the oat aleurone layer, but the content is relatively low. Probiotics and ganoderma mycelium will secrete some cellulases, amylases and hemicellulases during the fermentation and growth process, which may promote the rupture of the oat cell wall and increase its dissolution. The fermentation liquid is acidic, which is conducive to the dissolution of the dissolved alkaloids. Oats contain glucan, which can promote the growth of probiotics and ganoderma mycelium. Probiotics and ganoderma themselves have anti-inflammatory and anti-allergic effects, and may have a synergistic effect. Therefore, by selecting probiotics (bifidobacteria) and ganoderma mycelium, and fermenting oats separately, it is expected to obtain an oat ganoderma fermentation liquid with better anti-inflammatory effect.

[0007] To achieve the above-mentioned object of the invention, on the one hand, the present invention provides a method for preparing oat ganoderma lucidum fermentation liquid, comprising the following steps: S1. Mix oats and water in a weight-to-volume ratio of 1:16-25, sterilize, cool to room temperature, add bifidobacterium liquid under aseptic conditions, mix, and perform anaerobically fermentation to obtain a primary fermentation product; S2, mixing the primary fermentation product obtained in step S1 with the liquid fermentation mycelium of Ganoderma lucidum, and culturing them to obtain a secondary fermentation product; S3, centrifuging the secondary fermentation product obtained in step S2, and the supernatant is the oatmeal Ganoderma lucidum fermentation liquid; Wherein, in step S1, the unit of the weight-to-volume ratio is g:mL.

[0008] Preferably, in step S1, the amount of the bifidobacterium liquid added is OD 600 1-3 mL of Bifidobacterium liquid with a value >0.7.

[0009] More preferably, the sterilization is performed by high temperature and high pressure sterilization using an autoclave.

[0010] As an example of the present invention, the high temperature and high pressure sterilization condition is 121° C. sterilization for 20 min at 103.4 kPa.

[0011] More preferably, and as an example of the present invention, in step S1, oats and water are stirred and mixed in a weight-to-volume ratio of 1:20 (g:mL), sterilized for 20 min, cooled to room temperature, and bifidobacterium liquid is added under sterile conditions (OD 200 g / g oats per gram of oats). 600 β-value>0.7 (2 mL) of Bifidobacterium liquid) and mix well.

[0012] The inventors found that the ratio of oats to water has an impact on the production process of the subsequent fermentation liquid. When the weight-to-volume ratio of oats to water is 1:10 (g:mL), gelatinization and overflow are serious during sterilization in a high-temperature and high-pressure autoclave, and the product after anaerobic fermentation is too thick, which is not conducive to the next step of cultivation.

[0013] Preferably, in step S1, the time of the anaerobic fermentation is 36-60 h, the temperature of the anaerobic fermentation is 36-37° C., and the oxygen concentration of the anaerobic fermentation is less than 0.1%.

[0014] More preferably, and as an example of the present invention, in step S1, the time of the anaerobic fermentation is 48 h, the temperature of the anaerobic fermentation is 36-37° C., and the oxygen concentration of the anaerobic fermentation is less than 0.1%.

[0015] Preferably, in step S2, the ratio of the volume of the primary fermentation product to the volume of the Ganoderma lucidum liquid fermentation liquid is 10:1; and the content of Ganoderma lucidum mycelium in the Ganoderma lucidum liquid fermentation mycelium is >7 g / L.

[0016] Preferably, in step S2, the culturing is carried out in a shaker, the culturing temperature is 25-27°C, more preferably 25°C, the culturing humidity is 30%-40%, more preferably 30%, and the culturing time is 3-7 days.

[0017] Preferably, in step S1, the type of bifidobacterium is selected from at least one of Bifidobacterium longum and Bifidobacterium pseudocatenulatum.

[0018] Preferably, in step S2, the type of Ganoderma lucidum is selected from at least one of Ganoderma lucidum and Ganoderma rubrum.

[0019] More preferably, in step S1, the bifidobacterium is Bifidobacterium longum (ATCC 15707); in step S2, the Ganoderma lucidum is Yuezhi No. 5.

[0020] In another aspect, the present invention provides an oat and ganoderma lucidum fermentation liquid, which is prepared by the above-mentioned preparation method.

[0021] In another aspect, the present invention provides the use of the oat and ganoderma lucidum fermentation liquid prepared by the above preparation method in the preparation of anti-inflammatory products.

[0022] The anti-inflammatory products include but are not limited to medicines, cosmetics, and the like.

[0023] Preferably, the anti-inflammatory product is a medicine or a cosmetic.

[0024] More preferably, the anti-inflammatory product is a cosmetic.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The invention provides a novel preparation method of oat ganoderma lucidum fermentation liquid. The oat ganoderma lucidum fermentation liquid prepared by the preparation method has good anti-inflammatory effect and can be applied to the fields of medicine, daily chemicals, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the HPLC-UV detection chart of the oat extract obtained in Comparative Example 2, the fermentation liquid obtained in Comparative Example 3, the fermentation liquid obtained in Comparative Example 4, the oat ganoderma lucidum fermentation liquid obtained in Example 1, and the oat alkaloid standard.

[0027] Figure 2 The figure is a diagram showing the anti-inflammatory effect of the oatmeal and Ganoderma lucidum fermentation liquid obtained in Example 1 in zebrafish; wherein (a) represents the blank control group, (b) represents the model control group, (c) represents the positive control group, (d) represents the 200-fold dilution group, (e) represents the 400-fold dilution group, and (f) represents the 800-fold dilution group.

[0028] Figure 3 is a quantitative statistical bar chart of the anti-inflammatory effect of oatmeal and Ganoderma lucidum fermented liquid obtained in Example 1; wherein the superscript represent p <0.05 (compared with the model control group), superscript represent p <0.001 (compared with the model control group), superscript ### represent p <0.001 (compared with the blank control group). DETAILED DESCRIPTION

[0029] Terms and Claims of the Present Invention: 1. The articles “a”, “an” and “the” include plural referents unless expressly limited to one or more referents otherwise.

[0030] 2. Numerical range: Unless otherwise expressly stated, all ranges or ratios disclosed herein will be understood to include any and all sub-ranges or sub-ratios contained therein. For example, a range or ratio of 1 to 30 stated should be considered to be included between a minimum value of 1 and a maximum value of 30, and any sub-range or sub-ratio, integer, decimal, or sub-range or sub-ratio consisting of integers or decimals including the end points.

[0031] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0032] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are all weight contents. Unless otherwise specified, it is understood that the process is carried out at room temperature.

[0033] In the following examples and comparative examples, the strain of Bifidobacterium used was Bifidobacterium longum ATCC 15707.

[0034] In the following examples and comparative examples, the strain of Ganoderma lucidum used is Ganoderma lucidum, sourced from Yuezhi No. 5 (provided by the Edible Fungi Center of the Institute of Microbiology, Guangdong Academy of Sciences).

[0035] In the following examples and comparative examples, the preparation method of the comprehensive PDA liquid culture medium used is as follows: Wash and peel the potatoes and cut them into small pieces. Take 200 g of the cut potatoes, add 1000 mL of distilled water and boil for 20-30 min. Filter with eight layers of gauze, add 20 g of glucose, stir to dissolve, add water to 1000 mL, and sterilize with high temperature and wet heat.

[0036] Example 1 The preparation of oat ganoderma lucidum fermentation liquid, the specific steps are as follows: (1) Stir and mix 10 g of oats with 200 mL of ultrapure water, place in an autoclave, and sterilize at 121°C for 20 min at 103.4 kPa. Cool to room temperature. Aseptically add 20 mL of OD 600 The bifidobacterium liquid with a value > 0.7 was mixed and placed in an anaerobic incubator for anaerobic fermentation for 48 hours. The temperature of the anaerobic fermentation was 37°C and the oxygen concentration of the anaerobic fermentation was < 0.1%. After the anaerobic fermentation was completed, 220 mL of the primary fermentation product was obtained.

[0037] (2) The Ganoderma lucidum strain (test tube strain) was transferred to the comprehensive PDA liquid culture medium, and fermented for 7 days at 27°C and 120 r / min in a shaking incubator until the mycelium was fully grown, which was the Ganoderma lucidum liquid fermentation mycelium. The content of Ganoderma lucidum mycelium in the Ganoderma lucidum liquid fermentation mycelium was determined to be >7 g / L.

[0038] 220 mL of the primary fermentation product and 22 g of Ganoderma lucidum liquid fermentation mycelium were mixed evenly under sterile conditions and cultured on a shaker under normoxic conditions for 3 days. The culture temperature was 25°C, the culture humidity was 30%, and the shaking speed of the shaker was 120 r / min. After the culture was completed, centrifuged at 10,000 r / min for 20 min, and the supernatant was taken to obtain the oat Ganoderma lucidum fermentation liquid.

[0039] Example 2 The preparation of oat ganoderma lucidum fermentation liquid, the specific steps are as follows: (1) Stir and mix 10 g of oats with 200 mL of ultrapure water, place in an autoclave, and sterilize at 121°C for 20 min at 103.4 kPa. Cool to room temperature. Aseptically add 20 mL of OD 600 The bifidobacterium liquid with a value > 0.7 was mixed and placed in an anaerobic incubator for anaerobic fermentation for 60 h. The temperature of the anaerobic fermentation was 37°C and the oxygen concentration of the anaerobic fermentation was < 0.1%. After the anaerobic fermentation was completed, 220 mL of the primary fermentation product was obtained.

[0040] (2) The Ganoderma lucidum strain (test tube strain) was transferred to the comprehensive PDA liquid culture medium, and fermented at 27°C and 120 r / min for 7 days until the mycelium was fully grown, which was the Ganoderma lucidum liquid fermentation mycelium. The content of Ganoderma lucidum mycelium in the Ganoderma lucidum liquid fermentation mycelium was determined to be >7 g / L.

[0041] 220 mL of the primary fermentation product and 22 g of Ganoderma lucidum liquid fermentation mycelium were mixed evenly under sterile conditions and cultured on a shaker under normoxic conditions for 3 days. The culture temperature was 25°C, the culture humidity was 30%, and the shaking speed of the shaker was 120 r / min. After the culture was completed, centrifuged at 10,000 r / min for 20 min, and the supernatant was taken to obtain the oat Ganoderma lucidum fermentation liquid.

[0042] Example 3 The preparation of oat ganoderma lucidum fermentation liquid, the specific steps are as follows: (1) Stir and mix 10 g of oats with 200 mL of ultrapure water, place in an autoclave, and sterilize at 121°C for 20 min at 103.4 kPa. Cool to room temperature. Aseptically add 20 mL of OD 600 The bifidobacterium liquid with a value > 0.7 was mixed and placed in an anaerobic incubator for anaerobic fermentation for 36 hours. The temperature of the anaerobic fermentation was 37°C and the oxygen concentration of the anaerobic fermentation was < 0.1%. After the anaerobic fermentation was completed, 220 mL of the primary fermentation product was obtained.

[0043] (2) The Ganoderma lucidum strain (test tube strain) was transferred to the comprehensive PDA liquid culture medium, and fermented at 27°C and 120 r / min for 7 days until the mycelium was fully grown, which was the Ganoderma lucidum liquid fermentation mycelium. The content of Ganoderma lucidum mycelium in the Ganoderma lucidum liquid fermentation mycelium was determined to be >7 g / L.

[0044] 200 mL of the primary fermentation product and 20 g of Ganoderma lucidum liquid fermentation mycelium were mixed evenly under sterile conditions and cultured on a shaking table for 3 days under normoxic conditions. The culture temperature was 25°C, the culture humidity was 30%, and the shaking speed of the shaking table was 120 r / min. After the culture was completed, centrifuged at 10,000 r / min for 20 min, and the supernatant was taken to obtain oat Ganoderma lucidum fermentation liquid.

[0045] Example 4 The preparation of oat ganoderma lucidum fermentation liquid, the specific steps are as follows: (1) Stir and mix 10 g of oats with 200 mL of ultrapure water, place in an autoclave, and sterilize at 121°C for 20 min at 103.4 kPa. Cool to room temperature. Aseptically add 20 mL of OD 600 The bifidobacterium liquid with a value of >0.7 was mixed and placed in an anaerobic incubator for anaerobic fermentation for 48 hours. The temperature of the anaerobic fermentation was 37°C and the oxygen concentration of the anaerobic fermentation was <0.1%. After the anaerobic fermentation was completed, 220 mL of the primary fermentation product was obtained.

[0046] (2) The Ganoderma lucidum strain (test tube strain) was transferred to the comprehensive PDA liquid culture medium, and fermented at 27°C and 120 r / min for 7 days until the mycelium was fully grown, which was the Ganoderma lucidum liquid fermentation mycelium. The content of Ganoderma lucidum mycelium in the Ganoderma lucidum liquid fermentation mycelium was determined to be >7 g / L.

[0047] 200 mL of the primary fermentation product and 20 g of Ganoderma lucidum liquid fermentation mycelium were mixed evenly under sterile conditions and cultured on a shaking table for 5 days under normoxic conditions. The culture temperature was 25°C, the culture humidity was 30%, and the shaking speed of the shaking table was 120 r / min. After the culture was completed, centrifuged at 10,000 r / min for 20 min, and the supernatant was taken to obtain oat Ganoderma lucidum fermentation liquid.

[0048] Example 5 The preparation of oat ganoderma lucidum fermentation liquid, the specific steps are as follows: (1) Stir and mix 10 g of oats with 200 mL of ultrapure water, place in an autoclave, and sterilize at 121°C for 20 min at 103.4 kPa. Cool to room temperature. Aseptically add 20 mL of OD 600 The bifidobacterium liquid with a value of >0.7 was mixed and placed in an anaerobic incubator for anaerobic fermentation for 48 hours. The temperature of the anaerobic fermentation was 37°C and the oxygen concentration of the anaerobic fermentation was <0.1%. After the anaerobic fermentation was completed, 220 mL of the primary fermentation product was obtained.

[0049] (2) The Ganoderma lucidum strain (test tube strain) was transferred to the comprehensive PDA liquid culture medium, and fermented at 27°C and 120 r / min for 7 days until the mycelium was fully grown, which was the Ganoderma lucidum liquid fermentation mycelium. The content of Ganoderma lucidum mycelium in the Ganoderma lucidum liquid fermentation mycelium was determined to be >7 g / L.

[0050] 200 mL of the primary fermentation product and 20 g of Ganoderma lucidum liquid fermentation mycelium were mixed evenly under sterile conditions and cultured on a shaker under normoxic conditions for 7 days. The culture temperature was 25°C, the culture humidity was 30%, and the shaking speed of the shaker was 120 r / min. After the culture was completed, centrifuged at 10,000 r / min for 20 min, and the supernatant was taken to obtain the oat Ganoderma lucidum fermentation liquid.

[0051] Comparative Example 1 10 g of oats and 100 mL of ultrapure water were thoroughly mixed and placed in a high pressure sterilizer for sterilization under the same sterilization conditions as in Example 1.

[0052] The experiment found that when sterilized in a high-pressure autoclave, the thick mixture gelatinized and overflowed severely, making it impossible to proceed to the next experimental operation, and it was impossible to obtain the final oatmeal Ganoderma lucidum fermentation liquid.

[0053] Comparative Example 2 Prepare unfermented oat extract.

[0054] Thoroughly stir and mix 10 g of oats with 150 mL of ultrapure water. Perform ultrasonic extraction at 60°C for 30 min, place in an autoclave, and sterilize at 121°C at 103.4 kPa for 20 min. Centrifuge and take the supernatant to obtain the oat extract.

[0055] Comparative Example 3 A fermentation broth of oats fermented only with bifidobacteria was prepared.

[0056] Stir and mix 10 g of oats with 150 mL of ultrapure water, place in an autoclave, and sterilize at 121°C for 20 min at 103.4 kPa. Cool to room temperature. Aseptically add 10 mL of OD 600 The bifidobacterium liquid with a value>0.7. Mix well and place in an anaerobic incubator for anaerobic fermentation for 48 hours. The temperature of anaerobic fermentation is 37°C and the oxygen concentration of anaerobic fermentation is less than 0.1%. After the anaerobic fermentation is completed, the supernatant is centrifuged to obtain the fermentation liquid.

[0057] Comparative Example 4 A fermentation liquid of oats fermented only with Ganoderma lucidum was prepared.

[0058] Stir and mix 10 g of oats with 150 mL of ultrapure water, place in an autoclave, and sterilize at 121°C for 20 min at 103.4 kPa. Cool to room temperature. Add Ganoderma lucidum solid fermentation mycelium under sterile conditions (1.0 g of Ganoderma lucidum solid fermentation mycelium per 150 mL of mixture). Mix well under sterile conditions and culture on a shaker under normoxic conditions for 5 days. The culture temperature is 25°C, the culture humidity is 30%, and the shaking speed of the shaker is 120 r / min. After the culture is completed, centrifuge and take the supernatant to obtain the fermentation liquid.

[0059] Effect example 1. Detection of active ingredient content in fermentation broth The oat ganoderma lucidum fermentation liquid obtained in Example 1, the oat extract obtained in Comparative Example 2, and the fermentation liquids obtained in Comparative Examples 3 and 4 were pretreated respectively: the pH was adjusted to 8-9 with a small amount of sodium hydroxide aqueous solution, and ethyl acetate was used for extraction. The ethyl acetate phases were combined and concentrated to dryness to obtain an ethyl acetate extract; 1 mL of chromatographic grade methanol was added to the ethyl acetate extract, the supernatant was centrifuged and filtered through a 0.22 μm filter membrane to obtain a liquid to be tested.

[0060] The preparation method of the test solution of oat alkaloid standard (brand: United Life Science, ULS; catalog number: SYNA092331) is as follows: Dissolve 0.5 mg of avenanthramide standard in 1 mL of chromatographic grade methanol and filter through a 0.22 μm filter membrane to obtain the test solution.

[0061] HPLC-UV was used for result analysis. HPLC elution conditions were as follows: Liquid phase model: SHIMADZU 20A. Chromatographic column: YMC-Pack ODS-AM (5 μm, 250 mm × 4.6 mm). Mobile phase A is acetonitrile, mobile phase B is water containing 0.1% formic acid, gradient elution: 0-10 min, 35%-40% A, 10-25min, 40%-100% A, flow rate 1 mL / min, injection volume 5 μL, detection wavelength 280 nm.

[0062] Results Figure 1 The test showed that the chemical complexity of Ganoderma lucidum and oats was significantly improved after fermentation, suggesting that fermentation with Ganoderma lucidum has a good effect on promoting the dissolution of phenolic components or other phenolic alkaloids in oats.

[0063] 2. Quality control index detection of oatmeal and Ganoderma lucidum fermentation liquid The appearance, smell, pH value, polysaccharide content, microorganism (bacteria), mold and yeast content of the oat ganoderma lucidum fermentation liquid obtained in Example 1 were tested. The results are shown in Table 1 below.

[0064] Table 1. Quality control index detection of oatmeal and Ganoderma lucidum fermentation liquid

[0065] Wherein, the detection method of the polysaccharide content is as follows: Accurately weigh 0.0100 g of glucose and dissolve it in water, and make up to 100 mL as the stock solution. Pipette 0 μL, 40 μL, 80 μL, 120 μL, 160 μL, and 200 μL of glucose stock solution into small test tubes, add water to 200 μL, then add 200 μL of 6% phenol solution and shake well. Use a pipette to accurately and quickly add 1000 μL of sulfuric acid, shake quickly and evenly, place at room temperature for 30 min, and measure the OD value at 490 nm. Use the absorbance value as the ordinate and the glucose mass as the abscissa to draw a standard curve, y=0.0247x-0.0025 (r 2 =0.9991). The samples were reacted and tested in the same way, and the sugar content was calculated according to the standard curve.

[0066] It can be seen that the oatmeal and Ganoderma lucidum fermentation broths prepared in Examples 1 to 5 all meet the quality control requirements.

[0067] 3. Effect of oatmeal and Ganoderma lucidum fermentation liquid on the anti-inflammatory effect of zebrafish 3.1 Experimental principle: The neutrophils of zebrafish embryos are highly similar to human neutrophils in morphology, biochemistry and physiological functions. Neutrophils are the first white blood cells to appear at the site of injury or invasion of pathogens, and their function is to remove infection or harmful substances. A model of neutrophil aggregation caused by copper sulfate-induced damage to neuromast cells in the lateral line region of zebrafish embryos was tested. The changes in the number of neutrophils in the lateral line region of fish embryos between the test substance treatment group and the model control group were compared, and the neutrophil inhibition rate was calculated to evaluate the anti-inflammatory effect of the sample.

[0068] 3.2 Experimental materials and equipment: 6-well plates, plastic pipettes, culture dishes, pipettes, fish embryo culture medium, copper sulfate, dexamethasone (2 mg / mL), 1% tricaine, and 1% agarose plates.

[0069] 3.3. Experimental instruments: constant temperature incubator, zebrafish culture system, continuous zoom stereo microscope, upright fluorescence microscope.

[0070] 3.4. Solution preparation: 40 μL of the oatmeal Ganoderma lucidum fermentation liquid prepared in Example 1 was taken, and 1960 μL of E3 culture liquid was added to prepare a 50-fold diluted oatmeal Ganoderma lucidum fermentation liquid solution of Example 1; 40 μL of the oatmeal Ganoderma lucidum fermentation liquid prepared in Example 1 was taken, and 3960 μL of E3 culture liquid was added to prepare a 100-fold diluted oatmeal Ganoderma lucidum fermentation liquid solution of Example 1; 600 μL of the 100-fold diluted oatmeal Ganoderma lucidum fermentation liquid solution of Example 1 was taken, and 2400 μL of E3 culture liquid was added to prepare a 500-fold diluted oatmeal Ganoderma lucidum fermentation liquid solution of Example 1.

[0071] Weigh 0.0039 g of dexamethasone powder and add 1 mL of dimethyl sulfoxide to prepare a 10 mmol / L dexamethasone stock solution. Take 6 µL of the dexamethasone stock solution and add 2.994 mL of E3 culture medium to prepare a 20 µmol / L dexamethasone solution.

[0072] Weigh 0.0160 g of copper sulfate powder and add 10 mL of pure water to prepare a 10 mmol / L copper sulfate stock solution. Take 30 µL of the copper sulfate stock solution and add 14.97 mL of E3 culture medium to prepare a 20 µmol / L copper sulfate solution.

[0073] Mix 3 mL of E3 culture medium and 3 mL of 20 µmol / L copper sulfate solution to prepare fish embryo culture medium containing 10 µmol / L copper sulfate.

[0074] Mix 3 mL of 20 µmol / L dexamethasone solution and 3 mL of 20 µmol / L copper sulfate solution to prepare a fish embryo culture medium containing 10 µmol / L copper sulfate and 10 µmol / L dexamethasone.

[0075] 3 mL of the oatmeal and Ganoderma lucidum fermented broth solution of Example 1 diluted 500 times and 3 mL of 20 µmol / L copper sulfate solution were mixed to prepare a fish embryo culture solution containing 10 µmol / L copper sulfate and the oatmeal and Ganoderma lucidum fermented broth of Example 1 diluted 1000 times.

[0076] 3 mL of the oatmeal and Ganoderma lucidum fermented broth solution diluted 50 times of Example 1 and 3 mL of 20 µmol / L copper sulfate solution were mixed to prepare a fish embryo culture solution containing 10 µmol / L copper sulfate and the oatmeal and Ganoderma lucidum fermented broth diluted 100 times of Example 1.

[0077] By mixing fish embryo culture medium containing 10 µmol / L copper sulfate and the oatmeal ganoderma fermentation liquid of Example 1 diluted 1000 times and fish embryo culture medium containing 10 µmol / L copper sulfate and the oatmeal ganoderma fermentation liquid of Example 1 diluted 100 times in different proportions, fish embryo culture medium containing 10 µmol / L copper sulfate and oatmeal ganoderma fermentation liquid at different dilution ratios (100-1000 times) can be obtained.

[0078] 3.5. Test grouping: Blank control group: 24 fish embryos were randomly selected and placed in one well of a 6-well plate, and 6 mL of E3 culture medium was added.

[0079] Model control group: 24 fish embryos were randomly selected and placed in one well of a 6-well plate, and 6 mL of fish embryo culture medium containing 10 µmol / L copper sulfate was added.

[0080] Positive control group: 24 fish embryos were randomly selected and placed in one well of a 6-well plate, and 6 mL of fish embryo culture medium containing 10 µmol / L copper sulfate and 10 µmol / L dexamethasone was added.

[0081] Test substance treatment: 24 fish embryos were randomly selected and placed in one well of a 6-well plate, and 6 mL of fish embryo culture medium containing 10 µmol / L copper sulfate and the oat ganoderma lucidum fermentation liquid of Example 1 diluted 200 times, 400 times or 800 times was added, which were respectively recorded as Example 1 diluted 200 times group, Example 1 diluted 400 times group, Example 1 diluted 800 times group (referred to as 200 times dilution group, 400 times dilution group, 800 times dilution group for short).

[0082] Place the 6-well plate in a 28 ± 1°C incubator and incubate for 40-45 min.

[0083] 3.6. Fish embryo fixation, staining, microscopic analysis and data statistics: After fixing the fish embryos in each test group in paraformaldehyde for at least 1 h, treat the fish embryos with PBST 3 times, 5 min each time, and then treat with 50% ethanol for 3 min. After the fish embryos were stained with Sudan black staining solution at room temperature for 1 h, they were rinsed with 70% ethanol 4 times, 5 min each time, and then treated with PBST 2 times, 5 min each time. Treat the fish embryos with bleach solution for 10 min. If they are treated in a test tube, keep the lid open. Then treat with 70% ethanol solution for 5 min, PBST for 1 min, clear solution 1 for 15 min, clear solution 2 for 10 min, and PBST for 3 min. Lay the fish embryos on their side. Then put them under a stereo microscope to take pictures of the tail of the fish embryos. Count the number of neutrophils in the lateral line area of ​​the tail three-quarters of the way from the anus for each fish embryo.

[0084] Calculate the neutrophil aggregation inhibition rate: Inhibition rate = (CS) ÷ C × 100%.

[0085] In the above formula, S represents the average number of neutrophils in fish embryos in the test substance treatment group; C represents the average number of neutrophils in fish embryos in the model control group.

[0086] The mean and standard error of each test group were calculated, and the statistical results were expressed as mean ± standard error. The data were analyzed by variance analysis using statistical software, and the number of neutrophils in each group was tested by two-tailed T test. p value. p <0.05 indicated a significant difference.

[0087] 3.7. Result analysis: The results are as follows Figure 2 As shown, there was obvious accumulation of neutrophils in the lateral line region of zebrafish embryos in the model control group; the positive control group ( Figure 2 The number of neutrophils in (c)) was compared with that in the model control group ( Figure 2 The distribution is sparser than that of (b) in the figure; the 200-fold diluted group ( Figure 2 (d) in the figure), the 400-fold dilution group ( Figure 2 The number of neutrophils in (e)) was compared with that in the model control group ( Figure 2 Compared with (b) in the figure, the 800-fold dilution group ( Figure 2 (f)) and the model control group ( Figure 2 There is little change compared to (b).

[0088] The bar graph of the quantitative test results of the anti-inflammatory effect is as follows Figure 3 As shown, the number of neutrophils in the 200-fold dilution group and the 400-fold dilution group was significantly reduced compared with the model control group. Although there was a statistical difference in the number of neutrophils in the 800-fold dilution group, the numerical change was not large.

[0089] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing oat ganoderma lucidum fermentation liquid, characterized in that: The following steps are involved: S1. Mix oats and water in a weight-to-volume ratio of 1:16-25, sterilize, cool to room temperature, add bifidobacterium liquid under aseptic conditions, mix, and perform anaerobically fermentation to obtain a primary fermentation product; S2, mixing the primary fermentation product obtained in step S1 with the liquid fermentation mycelium of Ganoderma lucidum, and culturing them to obtain a secondary fermentation product; S3, centrifuging the secondary fermentation product obtained in step S2, and the supernatant is the oatmeal Ganoderma lucidum fermentation liquid; Wherein, in step S1, the unit of the weight-to-volume ratio is g:mL.

2. The preparation method according to claim 1, characterized in that: In step S1, the amount of the bifidobacterium liquid added is OD 600 1-3 mL of Bifidobacterium liquid with a value >0.

7.

3. The preparation method according to claim 1, characterized in that: In step S1, the anaerobic fermentation time is 36-60 hours, the anaerobic fermentation temperature is 36-37° C., and the oxygen concentration of the anaerobic fermentation is less than 0.1%.

4. The preparation method according to claim 1, characterized in that: In step S2, the ratio of the volume of the primary fermentation product to the volume of the Ganoderma lucidum liquid fermentation mycelium is 10:1; and the content of Ganoderma lucidum mycelium in the Ganoderma lucidum liquid fermentation mycelium is >7 g / L.

5. The preparation method according to claim 1, characterized in that: In step S2, the culturing is carried out in a shaking incubator, the culturing temperature is 25-27° C., the culturing humidity is 30%-40%, and the culturing time is 3-7 days.

6. The preparation method according to claim 1, characterized in that: In step S1, the strain type of the bifidobacterium is selected from at least one of Bifidobacterium longum and Bifidobacterium pseudocatenulatum.

7. The preparation method according to claim 1, characterized in that: In step S2, the strain type of the Ganoderma lucidum is selected from at least one of Ganoderma lucidum and Ganoderma rubrum.

8. The preparation method according to claim 6 or claim 7, characterized in that: In step S1, the bifidobacterium is Bifidobacterium longum ATCC 15707; in step S2, the Ganoderma lucidum is Yuezhi No.

5.

9. An oat and ganoderma lucidum fermentation liquid, characterized in that: The method is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the oatmeal and ganoderma lucidum fermentation liquid prepared by the preparation method according to any one of claims 1 to 8 in the preparation of anti-inflammatory products.

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