An oat ganoderma fermentation broth, its preparation method and application

Abstract

The present invention provides a fermented liquid of oat and ganoderma lucidum, a preparation method thereof and an application thereof, belonging to the technical field of fermentation. The preparation method of the fermented liquid of oat and ganoderma lucidum provided by the present invention comprises the following steps: S1. Mix oat and water in a weight-to-volume ratio of 1:16-25 (g:mL), sterilize, cool to room temperature, add bifidobacterium bacterium liquid under aseptic conditions, mix, and perform anaerobic fermentation to obtain a primary fermented product; S2. Mix the primary fermented product obtained in step S1 with solid fermented mycelia of ganoderma lucidum, and culture to obtain a secondary fermented product; S3. Centrifuge the secondary fermented product obtained in step S2, and the supernatant is the fermented liquid of oat and ganoderma lucidum. The fermented liquid of oat and ganoderma lucidum has a remarkable anti-inflammatory function.

Description

Technical Field

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

[0002] Oat is an annual herbaceous plant of the genus Avena in the family Poaceae, mainly produced in the temperate regions of the Northern Hemisphere. As a food raw material, oats are widely used. It contains rich unsaturated fatty acids, amino acids, minerals, and dietary fiber, and has the effects of enhancing satiety and reducing blood lipids. It is also applied to other industries such as cosmetics and pharmaceuticals. The glucan in oats has good moisturizing effects and is often used as a component of cosmetics. The article "Research Progress and Application of Avenanthramides" (Tang Yaru et al., Journal of Northern Agriculture, 2023, 51(6): 37-50) points out that oats contain the active ingredient avenanthramides. Avenanthramides have rich physiological activities and show broad application prospects in the fields of antioxidant, anti-inflammatory, anti-cancer, anti-proliferation, and cardiovascular health care.

[0003] Chinese Patent CN109430402A provides a preparation method of a fermented oat dairy product. This technology adopts a two-step fermentation process. In the first fermentation, sweet yeast is inoculated into steamed oats and fermented at 35-45 °C for 10-12 h to obtain sweet embryos, and then the sweet embryos are sterilized; the mass ratio of the sweet yeast to the steamed oats is 1:900-1100; in the second fermentation: a fermenting agent is inoculated into sterilized raw milk, mixed with a sweetening additive, and then added to the sweet embryos prepared in the previous step for secondary fermentation to obtain fermented oat yogurt; and then it is obtained by filtration and canning. This method not only reduces the acidity of the fermentation broth, improves stability, refines wheat bran, and improves the utilization rate of raw materials, but also brings a unique flavor.

[0004] Chinese Patent CN103271274A provides a nutritious health porridge with the following composition (calculated based on a total weight of 1000 parts by weight): 20-40 parts of corn, 10-15 parts of oats, 10-20 parts of Chinese yam, 5-10 parts of red dates, 10-20 parts of coix seeds, 5-10 parts of black sticky 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 granulated sugar, 20-35 parts of honey, and 6-18 parts of probiotics. The nutritious health porridge of this technology has the effects of supplementing nutrition, promoting wound healing, and regulating intestinal function. However, this method does not provide an oat ganoderma fermentation broth, nor does it provide an application of an oat ganoderma fermentation broth in an anti-inflammatory liquid.

[0005] In summary, the prior art does not provide a preparation of an oat ganoderma fermentation broth and its application in anti-inflammatory. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide an oat ganoderma fermented liquid, a preparation method thereof and an application. The alkaloids in oats are mainly distributed in the aleurone layer of oats, but the content is relatively low. During the fermentation and growth process, probiotics and ganoderma mycelium secrete some cellulases, amylases and hemicellulases, etc., which may promote the rupture of the oat cell wall and increase its dissolution. The fermented liquid is acidic, which is beneficial to the dissolution of alkaloids. Oats contain glucan, which can promote the growth of probiotics and ganoderma. Probiotics and ganoderma itself have anti-inflammatory and anti-allergic effects, and there may be a synergistic effect. Therefore, probiotics (Bifidobacterium) and ganoderma mycelium are selected to ferment oats respectively, and it is expected to obtain an oat ganoderma fermented liquid with better anti-inflammatory effect.

[0007] To achieve the above object of the invention, on the one hand, the present invention provides a preparation method of an oat ganoderma fermented liquid, comprising the following steps:

[0008] S1. Mix oats and water in a weight-to-volume ratio of 1:16 - 25, sterilize, cool to room temperature, add Bifidobacterium bacterium solution under sterile conditions, mix, and perform anaerobic fermentation to obtain a primary fermentation product;

[0009] S2. Mix the primary fermentation product obtained in step S1 with ganoderma liquid fermentation mycelium, and culture to obtain a secondary fermentation product;

[0010] S3. Centrifuge the secondary fermentation product obtained in step S2, and the supernatant is the oat ganoderma fermented liquid;

[0011] Among them, in step S1, the unit of the weight-to-volume ratio is g:mL.

[0012] Preferably, in step S1, the addition amount of the Bifidobacterium bacterium solution is 1 - 3 mL of Bifidobacterium bacterium solution with an OD 600 value > 0.7 added per gram of oats.

[0013] More preferably, the sterilization is high-temperature and high-pressure sterilization using an autoclave.

[0014] As an example of the present invention, the conditions for the high-temperature and high-pressure sterilization are sterilization at 121 °C for 20 min under 103.4 kPa.

[0015] 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 bacterium solution (2 mL of Bifidobacterium bacterium solution with an OD 600 value > 0.7 added per gram of oats) is added under sterile conditions and mixed evenly.

[0016] The inventors found that the ratio of oats to water affects the subsequent production process of the fermentation broth. When the weight-to-volume ratio of oats to water is 1:10 (g:mL), severe gelatinization and overflow occur during sterilization using a high-temperature and high-pressure autoclave, and the product consistency after anaerobic fermentation is too high, which is not conducive to the next step of cultivation.

[0017] 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 < 0.1%.

[0018] 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 < 0.1%.

[0019] Preferably, in step S2, the ratio of the volume of the primary fermentation product to the volume of the Ganoderma lucidum liquid fermentation broth is 10:1; the content of Ganoderma lucidum mycelium in the Ganoderma lucidum liquid fermentation mycelium is > 7 g / L.

[0020] Preferably, in step S2, the cultivation is carried out in a shaker, the temperature of the cultivation is 25 - 27 °C, more preferably 25 °C, the humidity of the cultivation is 30% - 40%, more preferably 30%, and the time of the cultivation is 3 - 7 days.

[0021] Preferably, in step S1, the types of Bifidobacterium are selected from at least one of Bifidobacterium longum and Bifidobacterium pseudocatenulatum.

[0022] Preferably, in step S2, the types of Ganoderma lucidum are selected from at least one of Ganoderma lucidum (Reishi) and Ganoderma sinense.

[0023] More preferably, in step S1, the Bifidobacterium is Bifidobacterium longum (ATCC 15707); in step S2, the Ganoderma lucidum is Yuezhi No. 5.

[0024] On the other hand, the present invention provides an oat-Ganoderma lucidum fermentation broth prepared by the above preparation method.

[0025] On yet another hand, the present invention provides the application of the oat-Ganoderma lucidum fermentation broth prepared by the above preparation method in the preparation of anti-inflammatory products.

[0026] The anti-inflammatory products include, but are not limited to, medicines, cosmetics, etc.

[0027] Preferably, the anti-inflammatory products are medicines and cosmetics.

[0028] More preferably, the anti-inflammatory products are cosmetics.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention provides a novel method for preparing an oat ganoderma fermentation broth. The oat ganoderma fermentation broth prepared by this preparation method has good anti-inflammatory effects and can be applied to fields such as pharmaceuticals and daily chemicals. Description of the Drawings

[0031] Figure 1 It is an HPLC-UV detection chart of the oat extract obtained in Comparative Example 2, the fermentation broth obtained in Comparative Example 3, the fermentation broth obtained in Comparative Example 4, the oat ganoderma fermentation broth obtained in Example 1, and an oat alkaloid standard product.

[0032] Figure 2 It is an anti-inflammatory effect diagram of the oat ganoderma fermentation broth obtained in Example 1 in zebrafish; among them, (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.

[0033] Figure 3 It is a quantitative statistical bar chart of the anti-inflammatory effect of the oat ganoderma fermentation broth obtained in Example 1; among them, the superscript represents p <0.05 (compared with the model control group), and the superscript represents p <0.001 (compared with the model control group), and the superscript ### represents p <0.001 (compared with the blank control group). Detailed Embodiments

[0034] Terms and Statements of the Present Invention:

[0035] 1. Articles "a", "an", and "the": Include plural objects unless otherwise specifically limited to one (kind) of object.

[0036] 2. Numerical ranges: Unless otherwise specifically indicated, all ranges or ratios disclosed herein will be understood to include any and all sub-ranges or sub-ratios contained therein. For example, the stated range or ratio of 1 to 30 should be considered to include between the minimum value of 1 and the maximum value of 30, and any sub-range or sub-ratio, integer, decimal, or sub-range or sub-ratio composed of integers or decimals including the endpoints.

[0037] 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.

[0038] 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.

[0039] In the following examples and comparative examples, the strain of Bifidobacterium used was Bifidobacterium longum ATCC 15707.

[0040] 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).

[0041] In the following examples and comparative examples, the preparation method of the comprehensive PDA liquid culture medium used is as follows:

[0042] 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.

[0043] Example 1

[0044] The preparation of oat ganoderma lucidum fermentation liquid, the specific steps are as follows:

[0045] (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.

[0046] (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.

[0047] Mix 220 mL of the primary fermentation product evenly with 22 g of Ganoderma lucidum liquid fermentation mycelium under sterile conditions, and culture it in a shaking incubator for 3 days under normal oxygen conditions. The culture temperature is 25 °C, the culture humidity is 30%, and the shaking speed of the shaking incubator is 120 r / min. After the culture is completed, centrifuge at 10,000 r / min for 20 min, take the supernatant to obtain the oat-Ganoderma lucidum fermentation broth.

[0048] Example 2

[0049] The preparation of the oat-Ganoderma lucidum fermentation broth is as follows:

[0050] (1) Stir and mix 10 g of oats evenly with 200 mL of ultrapure water, place them in an autoclave, and sterilize at 121 °C under 103.4 kPa for 20 min. Cool to room temperature. Add 20 mL of Bifidobacterium bacterium liquid with an OD 600 value > 0.7 under sterile conditions. Mix well and place it in an anaerobic incubator for anaerobic fermentation for 60 h. The temperature of anaerobic fermentation is 37 °C, and the oxygen concentration of anaerobic fermentation < 0.1%. After the anaerobic fermentation is completed, 220 mL of the primary fermentation product is obtained.

[0051] (2) Transfer the Ganoderma lucidum strain (test tube strain) to a comprehensive PDA liquid medium, culture at 27 °C and 120 r / min for 7 days until the mycelium grows fully to obtain Ganoderma lucidum liquid fermentation mycelium. Measure that the content of Ganoderma lucidum mycelium in the Ganoderma lucidum liquid fermentation mycelium > 7 g / L.

[0052] Mix 220 mL of the primary fermentation product evenly with 22 g of Ganoderma lucidum liquid fermentation mycelium under sterile conditions, and culture it in a shaking incubator for 3 days under normal oxygen conditions. The culture temperature is 25 °C, the culture humidity is 30%, and the shaking speed of the shaking incubator is 120 r / min. After the culture is completed, centrifuge at 10,000 r / min for 20 min, take the supernatant to obtain the oat-Ganoderma lucidum fermentation broth.

[0053] Example 3

[0054] The preparation of the oat-Ganoderma lucidum fermentation broth is as follows:

[0055] (1) Stir and mix 10 g of oats evenly with 200 mL of ultrapure water, place them in an autoclave, and sterilize at 121 °C under 103.4 kPa for 20 min. Cool to room temperature. Add 20 mL of Bifidobacterium bacterium liquid with an OD 600 value > 0.7 under sterile conditions. Mix well and place it in an anaerobic incubator for anaerobic fermentation for 36 h. The temperature of anaerobic fermentation is 37 °C, and the oxygen concentration of anaerobic fermentation < 0.1%. After the anaerobic fermentation is completed, 220 mL of the primary fermentation product is obtained.

[0056] (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.

[0057] 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.

[0058] Example 4

[0059] The preparation of oat ganoderma lucidum fermentation liquid, the specific steps are as follows:

[0060] (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.

[0061] (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.

[0062] 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.

[0063] Example 5

[0064] The preparation of oat ganoderma lucidum fermentation liquid, the specific steps are as follows:

[0065] (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 600Bifidobacterium bacterium solution with a value > 0.7. Mix well and place in an anaerobic incubator for anaerobic fermentation for 48 h. The temperature of anaerobic fermentation is 37 °C, and the oxygen concentration of anaerobic fermentation < 0.1%. After anaerobic fermentation, 220 mL of the primary fermentation product is obtained.

[0066] (2) Transfer the Ganoderma lucidum strain (test tube strain) to the comprehensive PDA liquid medium, ferment at 27 °C and 120 r / min for 7 days until the mycelium grows fully, obtaining the Ganoderma lucidum liquid-fermented mycelium. Measure the content of Ganoderma lucidum mycelium in the Ganoderma lucidum liquid-fermented mycelium > 7 g / L.

[0067] Mix 200 mL of the primary fermentation product evenly with 20 g of Ganoderma lucidum liquid-fermented mycelium under sterile conditions and culture on a shaker for 7 days under normal oxygen conditions. The culture temperature is 25 °C, the culture humidity is 30%, and the shaker speed is 120 r / min. After the culture is completed, centrifuge at 10000 r / min for 20 min, take the supernatant to obtain the oat Ganoderma lucidum fermentation broth.

[0068] Comparative Example 1

[0069] Mix 10 g of oats with 100 mL of ultrapure water thoroughly and place in an autoclave for sterilization under the same conditions as in Example 1.

[0070] It was found in the experiment that when sterilizing in the autoclave, the thick mixture was severely gelatinized and overflowed, making it impossible to carry out the next experimental operation and obtain the final oat Ganoderma lucidum fermentation broth.

[0071] Comparative Example 2

[0072] Prepare the unfermented oat extract.

[0073] Mix 10 g of oats with 150 mL of ultrapure water thoroughly, extract with ultrasound at 60 °C for 30 min, place in an autoclave, and sterilize at 121 °C under 103.4 kPa for 20 min. Centrifuge and take the supernatant to obtain the oat extract.

[0074] Comparative Example 3

[0075] Prepare the fermentation broth of oats fermented only by Bifidobacterium.

[0076] Mix 10 g of oats with 150 mL of ultrapure water thoroughly, place in an autoclave, and sterilize at 121 °C under 103.4 kPa for 20 min. Cool to room temperature. Add 10 mL of Bifidobacterium bacterium solution with an OD 600 value > 0.7 under sterile conditions. Mix well and place in an anaerobic incubator for anaerobic fermentation for 48 h. The temperature of anaerobic fermentation is 37 °C, and the oxygen concentration of anaerobic fermentation < 0.1%. After anaerobic fermentation is completed, centrifuge and take the supernatant to obtain the fermentation broth.

[0077] Comparative Example 4

[0078] Prepare the fermentation broth of oats fermented only by Ganoderma lucidum.

[0079] Mix 10 g of oats with 150 mL of ultrapure water by stirring thoroughly, place it in an autoclave, sterilize at 121 °C under 103.4 kPa for 20 min. Cool to room temperature. Add Ganoderma lucidum solid fermentation mycelium under aseptic conditions (the addition amount is 1.0 g of Ganoderma lucidum solid fermentation mycelium per 150 mL of the mixture). Mix evenly under aseptic conditions and culture on a shaker for 5 days under aerobic conditions. 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 to obtain the supernatant, and obtain the fermentation broth.

[0080] Effect Example

[0081] 1. Detection of the content of active ingredients in the fermentation broth

[0082] Perform pretreatment on the oat-Ganoderma lucidum fermentation broth obtained in Example 1, the oat extract obtained in Comparative Example 2, and the fermentation broths obtained in Comparative Example 3 and Comparative Example 4 respectively: adjust the pH to 8 - 9 with a small amount of sodium hydroxide aqueous solution, extract with ethyl acetate, combine the ethyl acetate phases, concentrate to dryness to obtain the ethyl acetate extract; add 1 mL of chromatographic methanol to the ethyl acetate extract, centrifuge to obtain the supernatant, and filter through a 0.22 μm filter membrane to obtain the test solution.

[0083] The preparation method of the test solution of oat alkaloid standard (brand: United Life Science, ULS; product number: SYNA092331) is as follows:

[0084] Dissolve 0.5 mg of oat alkaloid standard in 1 mL of chromatographic methanol, filter through a 0.22 μm filter membrane to obtain the test solution.

[0085] Perform result analysis by HPLC-UV. The HPLC elution conditions are as follows:

[0086] 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 - 25 min, 40% - 100% A, flow rate 1 mL / min, injection volume 5 μL, detection wavelength 280 nm.

[0087] The results are shown in Figure 1After detection, the chemical complexity of Ganoderma lucidum and oats after fermentation has been significantly improved, suggesting that the fermentation of Ganoderma lucidum has a good effect on promoting the dissolution of phenolic components or other phenolic alkaloids in oats.

[0088] 2. Detection of quality control indicators of oat Ganoderma lucidum fermentation broth

[0089] The appearance, odor, pH value, polysaccharide content, microorganisms (bacteria), and the content of molds and yeasts of the oat Ganoderma lucidum fermentation broth obtained in Example 1 were detected. The results are shown in Table 1 below.

[0090] Table 1. Detection of quality control indicators of oat Ganoderma lucidum fermentation broth

[0091]

[0092] Among them, the detection method of the polysaccharide content is as follows:

[0093] Accurately weigh 0.0100 g of glucose, dissolve it in water, and make up the volume to 100 mL to obtain a stock solution. Respectively pipette 0 μL, 40 μL, 80 μL, 120 μL, 160 μL, and 200 μL of the glucose stock solution into small test tubes, make up the water to 200 μL, then add 200 μL of 6% phenol solution and shake well. Accurately and quickly add 1000 μL of sulfuric acid with a pipette, shake well immediately, place it at room temperature for 30 min, and measure the OD value at 490 nm. Take 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, detected in the same way, and the sugar content was calculated according to the standard curve.

[0094] It can be seen that the oat Ganoderma lucidum fermentation broths prepared in Examples 1 - 5 all meet the quality control requirements.

[0095] 3. Effect of oat Ganoderma lucidum fermentation broth on the anti - inflammatory effect of zebrafish

[0096] 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 pathogen invasion, and their function is to clear infections or harmful substances. A model of inducing the injury of neuromast cells in the lateral line region of zebrafish embryos by copper sulfate to cause neutrophil aggregation was used for testing. The number changes of neutrophils in the lateral line region of fish embryos in 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.

[0097] 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.

[0098] 3.3. Experimental instruments: constant temperature incubator, zebrafish culture system, continuous zoom stereo microscope, upright fluorescence microscope.

[0099] 3.4. Solution preparation:

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] Mix 3 mL of the diluted 50-fold Ganoderma lucidum-fermented oat solution of Example 1 with 3 mL of 20 μmol / L copper sulfate solution to prepare a fish embryo culture solution containing 10 μmol / L copper sulfate and the 100-fold diluted Ganoderma lucidum-fermented oat solution of Example 1.

[0107] By mixing the fish embryo culture solution containing 10 μmol / L copper sulfate and the 1000-fold diluted Ganoderma lucidum-fermented oat solution of Example 1 with the fish embryo culture solution containing 10 μmol / L copper sulfate and the 100-fold diluted Ganoderma lucidum-fermented oat solution of Example 1 in different proportions, fish embryo culture solutions with different dilution ratios (100 - 1000 times) containing 10 μmol / L copper sulfate and Ganoderma lucidum-fermented oat solution can be obtained.

[0108] 3.5. Test grouping:

[0109] Blank control group: Randomly select 24 fish embryos into one well of a 6-well plate and add 6 mL of E3 culture solution.

[0110] Model control group: Randomly select 24 fish embryos into one well of a 6-well plate and add 6 mL of fish embryo culture solution containing 10 μmol / L copper sulfate.

[0111] Positive control group: Randomly select 24 fish embryos into one well of a 6-well plate and add 6 mL of fish embryo culture solution containing 10 μmol / L copper sulfate and 10 μmol / L dexamethasone.

[0112] Test substance treatment: Randomly select 24 fish embryos into one well of a 6-well plate and add 6 mL of fish embryo culture solution containing 10 μmol / L copper sulfate and the 200-fold, 400-fold, or 800-fold diluted Ganoderma lucidum-fermented oat solution of Example 1, which are respectively recorded as the 200-fold dilution group of Example 1, the 400-fold dilution group of Example 1, and the 800-fold dilution group of Example 1 (abbreviated as the 200-fold dilution group, the 400-fold dilution group, and the 800-fold dilution group).

[0113] Place the 6-well plate in an incubator at 28 ± 1°C and culture for 40 - 45 min.

[0114] 3.6. Fixing, staining, microscopic analysis, and data statistics of fish embryos:

[0115] After fixing the fish embryos in each test group in paraformaldehyde for at least 1 h, the fish embryos were treated with PBST 3 times, 5 min each time, and then treated with 50% ethanol for 3 min. After staining the fish embryos with Sudan black staining solution at room temperature for 1 h, they were washed with 70% ethanol 4 times, 5 min each time, and then treated with PBST 2 times, 5 min each time. The fish embryos were treated with bleaching solution for 10 min. If treated in a test tube, the lid needs to be kept open. Then they were treated with 70% ethanol solution for 5 min, PBST for 1 min, clearing solution 1 for 15 min, clearing solution 2 for 10 min, and then PBST for 3 min. Place the fish embryos on their sides. Then place them under a stereomicroscope and take pictures of the tails of the fish embryos. Count the number of neutrophils in the three-quarter tail lateral line area starting from the anus of each fish embryo.

[0116] Calculate the neutrophil aggregation inhibition rate:

[0117] Inhibition rate = (C - S) ÷ C × 100%.

[0118] In the above formula, S represents the average number of neutrophils in the fish embryos of the test substance treatment group; C represents the average number of neutrophils in the fish embryos of the model control group.

[0119] Calculate the mean and standard error of each test group, and the statistical treatment results are expressed as mean ± standard error. Use statistical software to perform analysis of variance on the data and perform a two-tailed T-test on the number of neutrophils in each group to obtain p value. p <0.05 indicates significant difference.

[0120] 3.7. Result analysis:

[0121] The results are as Figure 2 shown. In the model control group, there was obvious aggregation of neutrophils in the lateral line area of zebrafish embryos in the zebrafish body; in the positive control group ( Figure 2 in (c)), the number of neutrophils was sparser compared to the model control group ( Figure 2 in (b)); in the 200-fold dilution group ( Figure 2 in (d)) and the 400-fold dilution group ( Figure 2 in (e)), the number of neutrophils decreased to a certain extent compared to the model control group ( Figure 2 in (b)), and there was little change in the 800-fold dilution group ( Figure 2 in (f)) compared to the model control group ( Figure 2 in (b)).

[0122] The bar chart of the quantitative test results of the anti-inflammatory effect is as Figure 3As shown, the number of neutrophils in the 200-fold dilution group and the 400-fold dilution group was significantly lower than that in the model control group. Although there was a statistically significant change in the number of neutrophils in the 800-fold dilution group, the numerical change was not large.

[0123] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than a limitation on the protection scope of the present invention. Any simple modification or equivalent replacement made by those of ordinary skill in the art to the technical solution of the present invention does not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A preparation method of an oat Ganoderma lucidum fermentation broth, characterized in that, Comprising the following steps: S1. Mix oats and water at a weight - volume ratio of 1:16 - 25, sterilize, cool to room temperature, add Bifidobacterium bacterial liquid under aseptic conditions, mix, and perform anaerobic fermentation to obtain a primary fermentation product; S2. Mix the primary fermentation product obtained in step S1 with Ganoderma lucidum liquid - fermented mycelia, culture to obtain a secondary fermentation product; S3. Centrifuge the secondary fermentation product obtained in step S2, and the supernatant is the oats - Ganoderma lucidum fermentation liquid; Among them, in step S1, the unit of the weight-to-volume ratio is g:mL, the sterilization condition is sterilization at 121°C for 20 min under 103.4 kPa, the OD 600 value of the Bifidobacterium bacterium solution > 0.7, the anaerobic fermentation time is 36 - 60 h, the anaerobic fermentation temperature is 36 - 37°C, and the oxygen concentration in the anaerobic fermentation < 0.1%; In step S2, the culture time is 3 days, the culture is carried out in a shaker, the culture temperature is 25 - 27 °C, and the culture humidity is 30% - 40%.

2. The preparation method according to claim 1, characterized in that, In step S1, the addition amount of the Bifidobacterium bacterial liquid is 1 - 3 mL of Bifidobacterium bacterial liquid per gram of oats.

3. The preparation method according to claim 1, wherein, In step S2, the volume ratio of the primary fermentation product to the volume of Ganoderma lucidum liquid - fermented mycelia is 10:1; the content of Ganoderma lucidum mycelia in the Ganoderma lucidum liquid - fermented mycelia > 7 g / L.

4. The preparation method according to claim 1, wherein In step S1, the strain type of the Bifidobacterium is selected from at least one of Bifidobacterium longum and Bifidobacterium pseudocatenulatum.

5. The preparation method according to claim 1, wherein In step S2, the strain type of the Ganoderma lucidum is selected from at least one of Ganoderma lucidum (Reishi) and Ganoderma sinense.

6. An oat ganoderma fermentation broth, characterized in that, Prepared by the preparation method according to any one of claims 1 - 5.

7. Use of the oats - Ganoderma lucidum fermentation liquid prepared by the preparation method according to any one of claims 1 - 5 in the preparation of anti - inflammatory products.

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