A preparation method of ganoderma lucidum and wormwood mixed fermented medicinal pieces

Abstract

The present invention belongs to the technical field of Chinese medicine fermentation, and relates to a method for preparing a mixed fermented slice of ganoderma lucidum and wormwood. First, Lactobacillus rhamnosus, Bacillus coagulans, and Saccharomyces cerevisiae are activated; the three activated strains are inoculated into the prepared mixed solution of ganoderma lucidum and wormwood, and cultured on a shaking table. After culture, fermentation is completed, centrifugal filtration is performed, and medicinal dextrin is added after concentration, vacuum drying, crushing, and sieving to obtain the mixed fermented slice of ganoderma lucidum and wormwood. The present invention utilizes excellent lactic acid bacteria, bacillus and yeast, and the three strains are combined to improve the activity of polysaccharides, thereby enhancing the antioxidant capacity and improving the taste of the mixed fermented slice of ganoderma lucidum and wormwood.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine fermentation, and in particular relates to a method for preparing a mixed fermented medicinal piece of ganoderma lucidum and wormwood. Background Art

[0002] Fermented Chinese medicine refers to the use of fermentation to change the medicinal properties, enhance the efficacy, and reduce toxicity of Chinese medicine slices through the biotransformation of microorganisms. For example, fermented slices such as light fermented black beans and Liushenqu, which are commonly used in clinical practice, are included in the "Chinese Pharmacopoeia" and the "Ministry-issued Standards". At present, with the continuous clarification and increasing application of the mechanism of action of biological fermentation technology in food, Chinese medicine, biopharmaceuticals, etc. at home and abroad, the conditions for developing a new type of Chinese medicine fermented slice industry have become mature.

[0003] Ganoderma lucidum (Leyss.ex Fr.) Karst. belongs to the class of Basidiomycetes, the family of Polyporaceae, and the genus of Ganoderma. The 2015 edition of the Pharmacopoeia states that Ganoderma has the effects of replenishing qi and calming the mind, relieving cough and relieving asthma. Pharmacological studies have shown that Ganoderma fruiting bodies are rich in a variety of essential vitamins and amino acids, polysaccharides, triterpenes and other bioactive substances. Among them, Ganoderma polysaccharides account for more than 25%, which have the effects of regulating blood lipids, lowering blood sugar, anti-oxidation and scavenging free radicals, and anti-aging. In cancer treatment, Ganoderma can be used as one of the auxiliary treatment methods.

[0004] Artemisia argyi Lévl.et Van. is a perennial herb or slightly semi-shrub in the Asteraceae family. The plant has a strong aroma. In China, except for extremely arid and high-altitude cold areas, it is almost everywhere. The whole plant is used as medicine, which has the effects of warming the meridians, removing dampness, dispelling cold, stopping bleeding, anti-inflammatory, relieving asthma, relieving cough, stabilizing pregnancy, and anti-allergic. The active ingredient in wormwood: wormwood polysaccharide is a compound with multiple biological activities, which has anti-tumor, immunity-enhancing, and antioxidant effects. It can inhibit the growth of tumor cells, enhance the synergistic anti-cancer effect of the immune system, and has a certain inhibitory effect on tumors such as liver cancer, gastric cancer, and breast cancer. In addition, wormwood polysaccharides can also promote cellular immune function and enhance the body's resistance.

[0005] In recent years, the relationship between intestinal flora and health has been continuously clarified, and new major achievements have been continuously reported. Among them, the interaction between drugs and intestinal flora, referred to as drug-microbiome interactions, has received more and more attention. Researchers have proved from multiple perspectives that the effect of drug-microbiome interactions on the metabolism and efficacy of drugs in vivo is inevitable. Traditional Chinese medicine is mainly administered through the digestive tract. Similarly, the drug-microbiome interaction effect in the body (in vivo fermentation process) is also critical to the effect of traditional Chinese medicine. In particular, the study of the metabolic effects of probiotics such as Bifidobacterium and Lactobacillus on traditional Chinese medicine has confirmed that the flora plays a vital role in the effect of traditional Chinese medicine, especially in enhancing the activity of traditional Chinese medicine. However, due to factors such as the number of bacterial species and the duration of action in the body, even the lack of probiotics that metabolize certain drugs in some patients or special populations will seriously affect the normal effect of drugs, not to mention the enhancement of activity. The method of moving drug-microbiome metabolism from the body to the outside (fermentation of traditional Chinese medicine) will be one of the best ways to improve the efficacy of traditional Chinese medicine. Summary of the invention

[0006] The invention uses ganoderma lucidum and wormwood with good medicinal value and broad application prospects as main raw materials, performs fermentation treatment through dominant strains, and provides a preparation method and application of ganoderma lucidum and wormwood mixed fermented medicinal pieces.

[0007] The present invention adopts three different types of probiotics as fermentation strains for mixed fermentation. The lactic acid bacteria used is Lactobacillus rhamnosus, which can ferment a variety of monosaccharides (glucose, arabinose). As a probiotic for the human intestinal tract, it is one of the most intensively studied probiotic species and is crowned with the reputation of "king of lactic acid bacteria"; the bacillus used is Bacillus coagulans, which can decompose sugars to generate L-lactic acid and has multiple resistances such as acid resistance, heat resistance, and salt resistance; the yeast used is Saccharomycopsis fibuligera. In addition to having strong saccharification ability and protease production ability, Saccharomycopsis fibuligera also has certain fragrance and ester production ability, which is a hot spot in the fermentation industry research in recent years. The present invention uses excellent fermentation strains to improve the activity of mixed polysaccharides of Ganoderma lucidum and wormwood, thereby enhancing antioxidant capacity and improving the taste of Ganoderma lucidum and wormwood slices. The technical solution of the present invention has important guiding significance for the promotion of fermented Chinese medicinal pieces of Ganoderma lucidum and Artemisia argyi.

[0008] The technical solution adopted by the present invention is:

[0009] The present invention provides a method for preparing a mixed fermented medicinal piece of ganoderma lucidum and wormwood, comprising the following steps:

[0010] (1) First, three strains of Lactobacillus rhamnosus, Bacillus coagulans, and Saccharomyces cerevisiae were selected, and a small amount of bacteria was lightly applied to each LP solid culture medium with an inoculation loop for purification and culture. Then, single colonies were transferred into each LP liquid culture medium for strain preparation to obtain activated Lactobacillus rhamnosus, Bacillus coagulans, and Saccharomyces cerevisiae strains.

[0011] (2) Ganoderma lucidum fruiting bodies and wormwood leaves are crushed and mixed, and 8 to 12 times the volume ratio of distilled water is added, and the mixture is heated to 80° C. and kept warm for 4 hours, then cooled to 35 to 40° C., 2% honey is added and mixed, and sterilized at 121° C. for 15 to 20 minutes and then allowed to stand for use.

[0012] (3) inoculating the activated strain obtained in step (1) into the mixed solution of ganoderma and wormwood prepared in step (2), culturing at a temperature of 35-37° C. and 90-120 rpm on a shaking table for 5-7 days, completing fermentation after culturing, centrifuging and concentrating into a paste using a vacuum concentrator at a temperature below 80° C., adding 30% medicinal dextrin and stirring evenly, vacuum drying at 80° C. to form a dry paste, crushing and passing through a 60-80 mesh sieve to obtain fermented ganoderma polysaccharide slices, and storing them at room temperature after packaging.

[0013] In a further embodiment, the preparation method of the LP liquid culture medium in step (1) is as follows: 10-15 g of glucose and 5-7 g of peptone are weighed, mixed evenly, and water is added to make up to 1 L, then sterilized at 121° C. for 15-20 min, and allowed to stand at room temperature to obtain the LP liquid culture medium.

[0014] The preparation method of the LP solid culture medium is consistent with the preparation conditions of the LP liquid culture medium, except that 18 to 20 g of agar powder is added to the original LP liquid culture medium, and then an appropriate amount of the prepared culture medium is poured into a culture dish, and the LP solid culture medium is obtained after being allowed to stand at room temperature for solidification, and then allowed to stand at room temperature for use.

[0015] In a further embodiment, when the single colonies are transferred to LP liquid culture medium for strain preparation in step (1), the ratio of the amount of the single colony to the amount of the culture medium is 0.01-0.1 g: 100 mL, and the culture time is 2-3 days.

[0016] In a further embodiment, the Ganoderma lucidum fruiting bodies and Artemisia argyi leaves in step (2) are crushed and mixed in a ratio of 3:1 (V:V).

[0017] In a further embodiment, in step (3), when the activated strain obtained in step (1) is inoculated into the mixed solution of Ganoderma lucidum and wormwood prepared in step (2), the ratio of the three strains is 1:1:1 (V:V:V), the inoculation amount is 5-7%, and the strain concentration before inoculation is 1×10 8 ~1×10 10 CFU / mL.

[0018] The positive beneficial effects of the present invention are:

[0019] 1. The present invention uses two food and medicine dual-purpose substances with extremely high application value: Ganoderma lucidum and Artemisia argyi as fermentation objects, and effectively utilizes the advantages and characteristics of the two to complement each other.

[0020] Among them, Ganoderma lucidum is rich in polysaccharides and has good anti-tumor effects, while Artemisia argyi polysaccharides have a similar effect to cellulose, which can promote gastrointestinal motility, lower blood lipids, and enhance immunity.

[0021] 2. The present invention utilizes excellent fermentation bacteria: Lactobacillus rhamnosus, Bacillus coagulans, and Saccharomyces cerevisiae for mixed fermentation.

[0022] Among them, Lactobacillus rhamnosus is a lactic acid bacteria, as the most studied probiotic in the world, it plays an important role in regulating the balance of intestinal flora and improving the body's immunity; Bacillus coagulans is a bacillus, but it can produce lactic acid, has many advantages such as high temperature resistance, acid resistance, oxygen resistance, and effective intestinal pathogens; Saccharomyces cerevisiae is a yeast, which not only has strong saccharification and protein production capabilities, but also can produce fragrance and fat. The three excellent strains complement each other, not only can balance the intestinal microenvironment and benefit health, but also significantly improve the antioxidant activity of the fermented object, and effectively improve the taste of the fermented material.

[0023] 3. The present invention improves the activity of the mixed polysaccharide of ganoderma lucidum and wormwood by three fermentation strains, thereby enhancing the antioxidant capacity and improving the taste of the fermented pieces of ganoderma lucidum and wormwood. The technical solution of the present invention has important guiding significance for the promotion of the fermented pieces of ganoderma lucidum and wormwood. DETAILED DESCRIPTION

[0024] The present invention is described in more detail below through specific implementation modes to facilitate understanding of the technical solution of the present invention, but is not intended to limit the protection scope of the present invention.

[0025] The following examples and comparative examples of the present invention were subjected to in vitro antioxidant tests, and the investigation group and the comparison group were subjected to odor and taste index evaluation investigations.

[0026] The preparation method of the LP liquid culture medium in the embodiment is as follows: 10 g of glucose and 5 g of peptone are weighed, mixed evenly, and water is added to make up to 1 L, then sterilized at 121° C. for 20 min and allowed to stand at room temperature to obtain the LP liquid culture medium.

[0027] Preparation method of LP solid culture medium: The preparation conditions are the same as those of LP liquid culture medium, except that 20 g of agar powder is added to the original LP liquid culture medium, and then an appropriate amount of the prepared culture medium is poured into a 90 mm culture dish. After standing at room temperature for solidification, the LP solid culture medium is obtained, and then standing at room temperature for use.

[0028] Embodiment 1:

[0029] A preparation method of a mixed fermented medicinal piece of Ganoderma lucidum and Artemisia argyi, the detailed steps are as follows (using a single strain for fermentation, specifically using Lactobacillus rhamnosus):

[0030] (1) First, select Lactobacillus rhamnosus as the fermentation strain, use an inoculation loop to lightly dip a small amount of the strain and spread it on LP solid culture medium for purification and culture, then take 0.05 g of a single colony and transfer it into 100 mL LP liquid culture medium and culture it for 2 days for strain preparation to obtain activated Lactobacillus rhamnosus strains.

[0031] (2) Ganoderma lucidum fruiting bodies and wormwood leaves were crushed and mixed in a ratio of 3:1 (V:V), and distilled water was added at a volume ratio of 10 times, so that the final volume exceeded 500 mL. The mixture was heated to 80°C for 4 hours, then cooled to 37°C, 2% honey was added and mixed, and sterilized at 121°C for 20 minutes and then allowed to stand for use.

[0032] (3) The activated strain Lactobacillus rhamnosus obtained in step (1) was inoculated into 500 mL of the mixed solution of Ganoderma lucidum and Artemisia argyi prepared in step (2), with a concentration of 1×10 8 CFU / mL, inoculation volume of 5%, temperature of 37°C, 110rpm shaking culture for 5 days, fermentation is completed after culture, centrifugal filtration, use a vacuum concentrator below 80°C to concentrate into a paste, add 30% medicinal dextrin and stir evenly, vacuum dry at 80°C to form a dry paste, crush and pass through a 70-mesh sieve to obtain Ganoderma lucidum polysaccharide fermented slices, and store at room temperature after packaging.

[0033] Embodiment 2:

[0034] This embodiment is basically the same as Embodiment 1, except that:

[0035] Step (1) selecting Bacillus coagulans as the fermentation strain;

[0036] In step (3), activated Bacillus coagulans is inoculated into the sterilized mixed solution obtained in step (2) for fermentation.

[0037] Embodiment 3:

[0038] This embodiment is basically the same as Embodiment 1, except that:

[0039] Step (1) selecting Saccharomyces cerevisiae as the fermentation strain;

[0040] In step (3), the activated Saccharomyces cerevisiae flavus is added to the sterilized mixed solution obtained in step (2) for fermentation.

[0041] Embodiment 4:

[0042] A preparation method of a mixed fermented medicinal piece of ganoderma lucidum and wormwood, the detailed steps are as follows (using two strains for fermentation, specifically using Lactobacillus rhamnosus and Bacillus coagulans):

[0043] (1) First, select Lactobacillus rhamnosus and Bacillus coagulans as fermentation strains, use an inoculation loop to lightly apply a small amount of strains on each LP solid culture medium for purification and culture, then take 0.05 g of a single colony and transfer it into 100 mL of LP liquid culture medium for 2 days to prepare strains, and obtain activated Lactobacillus rhamnosus and Bacillus coagulans strains.

[0044] (2) Ganoderma lucidum fruiting bodies and wormwood leaves were crushed and mixed in a ratio of 3:1 (V:V), and distilled water was added at a volume ratio of 10 times, so that the final volume exceeded 500 mL. The mixture was heated to 80°C for 4 hours, then cooled to 37°C, 2% honey was added and mixed, and sterilized at 121°C for 20 minutes and then allowed to stand for use.

[0045] (3) The activated strains of Lactobacillus rhamnosus and Bacillus coagulans obtained in step (1) were inoculated into 500 mL of the mixed solution of Ganoderma lucidum and Artemisia argyi prepared in step (2) at an inoculation ratio of 1:1 (V:V). The concentrations before inoculation were both 1×10 8 CFU / mL, the inoculation amount is 5%, and the temperature is 37°C, and the shaking table is 110rpm to culture for 5 days. After the culture, the fermentation is completed, centrifugal filtration is performed, and the paste is concentrated into a paste using a vacuum concentrator below 80°C. 30% medicinal dextrin is added and stirred evenly. The paste is vacuum dried at 80°C to form a dry paste, crushed and sieved through a 70-mesh sieve to obtain the fermented Ganoderma lucidum polysaccharide slices, which are packaged and stored at room temperature.

[0046] Embodiment 5:

[0047] This embodiment is basically the same as Embodiment 4, except that:

[0048] Step (1) selecting Lactobacillus rhamnosus and Saccharomyces cerevisiae as fermentation strains;

[0049] In step (3), activated Lactobacillus rhamnosus and Saccharomyces cerevisiae are added to the sterilized mixed solution obtained in step (2) for fermentation.

[0050] Embodiment 6:

[0051] This embodiment is basically the same as Embodiment 4, except that:

[0052] Step (1) selecting Bacillus coagulans and Saccharomyces cerevisiae as fermentation strains;

[0053] In step (3), activated Bacillus coagulans and Saccharomyces cerevisiae are added to the sterilized mixed solution obtained in step (2) for fermentation.

[0054] Embodiment 7:

[0055] A preparation method of a mixed fermented medicinal piece of ganoderma lucidum and wormwood, the detailed steps are as follows (three strains of bacteria are used for fermentation, namely, Lactobacillus rhamnosus, Bacillus coagulans, and Saccharomyces cerevisiae complex):

[0056] (1) First, select Lactobacillus rhamnosus, Bacillus coagulans, and Saccharomyces cerevisiae as fermentation strains, use an inoculation loop to lightly apply a small amount of strains on respective LP solid culture media for purification and culture, then take 0.05 g of a single colony and transfer it into respective 100 mL LP liquid culture media for culture for 2 days for strain preparation, and obtain activated Lactobacillus rhamnosus, Bacillus coagulans, and Saccharomyces cerevisiae strains.

[0057] (2) Ganoderma lucidum fruiting bodies and wormwood leaves were crushed and mixed in a ratio of 3:1 (V:V), and distilled water was added at a volume ratio of 10 times, so that the final volume exceeded 500 mL. The mixture was heated to 80°C for 4 hours, then cooled to 37°C, 2% honey was added and mixed, and sterilized at 121°C for 20 minutes and then allowed to stand for use.

[0058] (3) The activated strains of Lactobacillus rhamnosus, Bacillus coagulans, and Saccharomyces cerevisiae obtained in step (1) were inoculated into 500 mL of the mixed solution of Ganoderma lucidum and Artemisia argyi prepared in step (2), with an inoculation ratio of 1:1:1 (V:V:V), and the concentrations before inoculation were all 1×10 8 CFU / mL, the inoculation amount is 5%, and the temperature is 37°C, and the shaking table is 110rpm to culture for 5 days. After the culture, the fermentation is completed, centrifugal filtration is performed, and the paste is concentrated into a paste using a vacuum concentrator below 80°C. 30% medicinal dextrin is added and stirred evenly. The paste is vacuum dried at 80°C to form a dry paste, crushed and sieved through a 70-mesh sieve to obtain the fermented Ganoderma lucidum polysaccharide slices, which are packaged and stored at room temperature.

[0059] Comparative Example 1:

[0060] A preparation method of a mixed fermented medicinal piece of Ganoderma lucidum and Artemisia argyi, the detailed steps are as follows (using aseptic fermentation):

[0061] (1) Ganoderma lucidum fruiting bodies and wormwood leaves are crushed and mixed in a ratio of 3:1 (V:V), and distilled water is added at a volume ratio of 10 times, so that the final volume exceeds 500 mL. Heat to 80°C for 4 hours, then cool to 37°C, add 2% honey, mix, sterilize at 121°C for 20 minutes, and then stand for use.

[0062] (2) 500 mL of the mixed solution of Ganoderma lucidum and Artemisia argyi prepared in step (1) was cultured at 37° C. and 110 rpm on a shaker for 5 days to complete fermentation, centrifuged and concentrated into a paste using a vacuum concentrator at a temperature below 80° C., 30% of medicinal dextrin was added and stirred evenly, vacuum dried at 80° C. to form a dry paste, crushed and passed through a 70-mesh sieve to obtain fermented Ganoderma lucidum polysaccharide slices, and stored at room temperature after packaging.

[0063] Comparative Example 2:

[0064] This comparative example is basically the same as Example 1, except that:

[0065] Step (1) selecting Lactobacillus plantarum as the fermentation strain;

[0066] In step (3), the activated Lactobacillus plantarum is inoculated into the sterilized mixed solution obtained in step (2) for fermentation.

[0067] Comparative Example 3:

[0068] This comparative example is basically the same as Example 1, except that:

[0069] Step (1) selecting Saccharomyces cerevisiae as the fermentation strain;

[0070] In step (3): activated brewer's yeast is inoculated into the sterilized mixed solution obtained in step (2) for fermentation.

[0071] Comparative Example 4:

[0072] This comparative example is substantially the same as Example 4, except that:

[0073] Step (1) selecting Lactobacillus plantarum and Bacillus coagulans as fermentation strains;

[0074] In step (3), activated Lactobacillus plantarum and Bacillus coagulans are added to the sterilized mixed solution obtained in step (2) for fermentation.

[0075] Comparative Example 5:

[0076] This comparative example is substantially the same as Example 4, except that:

[0077] Step (1) selecting Lactobacillus plantarum and Saccharomyces cerevisiae as fermentation strains;

[0078] In step (3), activated Lactobacillus plantarum and Saccharomyces cerevisiae are added to the sterilized mixed solution obtained in step (2) for fermentation.

[0079] Comparative Example 6:

[0080] This comparative example is substantially the same as Example 4, except that:

[0081] Step (1) selecting Lactobacillus rhamnosus and Saccharomyces cerevisiae as fermentation strains;

[0082] In step (3), activated Lactobacillus rhamnosus and Saccharomyces cerevisiae are inoculated into the sterilized mixed solution obtained in step (2) for fermentation.

[0083] Comparative Example 7:

[0084] This comparative example is substantially the same as Example 4, except that:

[0085] Step (1) selecting Bacillus coagulans and Saccharomyces cerevisiae as fermentation strains;

[0086] In step (3), activated Bacillus coagulans and Saccharomyces cerevisiae are added to the sterilized mixed solution obtained in step (2) for fermentation.

[0087] Comparative Example 8:

[0088] This comparative example is substantially the same as Example 4, except that:

[0089] Step (1) selecting Lactobacillus plantarum and Saccharomyces cerevisiae as fermentation strains;

[0090] In step (3), activated Lactobacillus plantarum and Saccharomyces cerevisiae are inoculated into the sterilized mixed solution obtained in step (2) for fermentation.

[0091] Comparative Example 9:

[0092] This comparative example is substantially the same as Example 7, except that:

[0093] Step (1) selecting Lactobacillus plantarum, Bacillus coagulans, and Saccharomyces cerevisiae as fermentation strains;

[0094] In step (3), activated Lactobacillus plantarum, Bacillus coagulans and Saccharomyces cerevisiae are added to the sterilized mixed solution obtained in step (2) for fermentation.

[0095] Comparative Example 10:

[0096] This comparative example is substantially the same as Example 7, except that:

[0097] Step (1) selecting Lactobacillus rhamnosus, Bacillus coagulans and Saccharomyces cerevisiae as fermentation strains;

[0098] In step (3), activated Lactobacillus rhamnosus, Bacillus coagulans and Saccharomyces cerevisiae are added to the sterilized mixed solution obtained in step (2) for fermentation.

[0099] Comparative Example 11:

[0100] This comparative example is substantially the same as Example 7, except that:

[0101] Step (1) selecting Lactobacillus plantarum, Bacillus coagulans and Saccharomyces cerevisiae as fermentation strains;

[0102] In step (3), activated Lactobacillus plantarum, Bacillus coagulans and Saccharomyces cerevisiae are added to the sterilized mixed solution obtained in step (2) for fermentation.

[0103] In addition, the volume ratio of Ganoderma lucidum fruiting bodies and Artemisia argyi leaves added is adjusted based on Example 7:

[0104] Investigation Team 1:

[0105] A preparation method of a mixed fermented medicinal piece of ganoderma lucidum and wormwood, the detailed steps are as follows (three strains are used for fermentation, specifically Lactobacillus rhamnosus, Bacillus coagulans, and Saccharomyces cerevisiae complex):

[0106] (1) First, select Lactobacillus rhamnosus, Bacillus coagulans, and Saccharomyces cerevisiae as fermentation strains, use an inoculation loop to lightly apply a small amount of strains on respective LP solid culture media for purification and culture, then take 0.05 g of a single colony and transfer it into respective 100 mL LP liquid culture media for culture for 2 days for strain preparation, and obtain activated Lactobacillus rhamnosus, Bacillus coagulans, and Saccharomyces cerevisiae strains.

[0107] (2) Ganoderma lucidum fruiting bodies and wormwood leaves were crushed and mixed in a ratio of 1:1 (V:V), and distilled water was added at a volume ratio of 10 times, so that the final volume exceeded 500 mL. The mixture was heated to 80°C for 4 hours, then cooled to 37°C, 2% honey was added and mixed, and sterilized at 121°C for 20 minutes and then allowed to stand for use.

[0108] (3) The activated strains of Lactobacillus rhamnosus, Bacillus coagulans, and Saccharomyces cerevisiae obtained in step (1) were inoculated into 500 mL of the mixed solution of Ganoderma lucidum and Artemisia argyi prepared in step (2), with an inoculation ratio of 1:1:1 (V:V:V), and the concentrations before inoculation were all 1×10 8 CFU / mL, the inoculation amount is 5%, and the temperature is 37°C, and the shaking table is 110rpm to culture for 5 days. After the culture, the fermentation is completed, centrifugal filtration is performed, and the paste is concentrated into a paste using a vacuum concentrator below 80°C. 30% medicinal dextrin is added and stirred evenly. The paste is vacuum dried at 80°C to form a dry paste, crushed and sieved through a 70-mesh sieve to obtain the fermented Ganoderma lucidum polysaccharide slices, which are packaged and stored at room temperature.

[0109] Investigation Team 2:

[0110] This survey group is basically the same as survey group 1, except that:

[0111] Step (2) Ganoderma lucidum fruiting bodies and Artemisia argyi leaves are crushed and mixed in a ratio of 3:1 (V:V), and distilled water is added in a 10-fold volume ratio until the final volume exceeds 500 mL.

[0112] Investigation Team 3:

[0113] This survey group is basically the same as survey group 1, except that:

[0114] Step (2) Ganoderma lucidum fruiting bodies and Artemisia argyi leaves are crushed and mixed in a ratio of 1:3 (V:V), and distilled water is added in a 10-fold volume ratio until the final volume exceeds 500 mL.

[0115] Comparison Group 1:

[0116] This comparison group is basically the same as the investigation group 1, except that:

[0117] Step (2) After crushing the mugwort leaves, add 10 times the volume of distilled water until the final volume exceeds 500 mL.

[0118] Comparison Group 2:

[0119] This comparison group is basically the same as the investigation group 1, except that:

[0120] Step (2) After the Ganoderma lucidum fruiting body is crushed, distilled water is added at a volume ratio of 10 times, and the final volume exceeds 500 mL.

[0121] Comparison group 3:

[0122] This comparison group is basically the same as the investigation group 1, except that:

[0123] In step (2), no Ganoderma lucidum fruiting bodies and Artemisia argyi leaves were added, and 500 mL of distilled water was directly used.

[0124] In order to further illustrate that the mixed fermented medicinal pieces of Ganoderma lucidum and Artemisia argyi prepared by the technical solution of the present invention can achieve good technical effects, the following experiments and investigations were conducted:

[0125] The Ganoderma lucidum and wormwood mixed fermented pieces prepared in Examples 1 to 7 and Comparative Examples 1 to 11 were subjected to in vitro antioxidant index detection; and the odor and taste indexes of the Ganoderma lucidum and wormwood mixed fermented pieces obtained in the investigation groups 1 to 3 and the comparison groups 1 to 3 were evaluated and investigated; finally, the Ganoderma lucidum and wormwood mixed fermented pieces were subjected to an acute toxicity test, and the specific detection method was as follows:

[0126] (1) Antioxidant activity index of polysaccharides in mixed fermented slices of Ganoderma lucidum and Artemisia argyi: Nitrogen free radical (DPPH) scavenging ability analysis. The DPPH scavenging rate of polysaccharides in mixed fermented slices of Ganoderma lucidum and Artemisia argyi was determined by a nitrogen free radical (DPPH) scavenging ability test kit.

[0127] Operation steps: Grind the mixed fermented slices of Ganoderma lucidum and Artemisia argyi in a mortar, weigh 50 mg of the sample, add 1 ml of 75% ethanol and mix well, then place in a 40℃ water bath for 30 minutes; centrifuge at room temperature at 10000r / min for 10 minutes, take the supernatant, and place on ice for testing. Take 1 ml of DPPH solution, mix it with an equal amount of anhydrous ethanol and samples with different treatments, shake well, and place it at room temperature away from light for 30 minutes to allow it to react fully.

[0128] Then calculate using the following formula. (where A i is DPPH reagent + sample solution, A j DPPH reagent + anhydrous ethanol, A 0 Represents DPPH reagent + sample diluent. All three are absorbance values) measured at OD 517 All experiments were repeated three times.

[0129]

[0130] (2) Antioxidant activity index of polysaccharides in mixed fermented slices of Ganoderma lucidum and wormwood: Determination of iron ion reducing ability. The iron ion reducing ability test kit was used to determine the iron ion reducing ability of polysaccharides in mixed fermented slices of Ganoderma lucidum and wormwood.

[0131] Operation steps: After mixing phosphate buffer (0.2 mol / L) and potassium ferricyanide solution (1%) at a volume ratio (V:V) = 1:1, take 2 ml and add it to 1 ml, 0.5 mg / ml mixed fermented slices of Ganoderma lucidum and Artemisia argyi, incubate at 50°C for 20 min, cool in an ice bath, take 1 ml of trichloroacetic acid (10%) and mix thoroughly, centrifuge at 3000 rpm for 10 min, add 0.6 ml of ferric chloride (0.1%) to the supernatant and mix well, let stand for 10 min, and measure OD 700 The absorbance value was measured (the blank control had no sample solution added).

[0132] (3) Odor and taste indicators of mixed fermented slices of Ganoderma lucidum and Artemisia argyi: The relevant data were obtained in the form of a questionnaire.

[0133] Specific steps: 70 employees of the unit were selected as experience subjects; the fermented pieces prepared by each investigation group and the comparison group were tasted respectively; the tasting was divided into odor index and taste index, and the index levels were divided into three grades: good (good), medium (average), and poor (uncomfortable); the index evaluation formula was as follows:

[0134]

[0135]

[0136] For example, in the odor index of survey group 1, 49 people chose good, 10 people chose medium, and 11 people chose bad, so the odor index is marked as A(49)B(10)C(11); calculated by the index evaluation formula: the good odor evaluation is 70%, and the unpleasant odor evaluation is 16%.

[0137] (4) Acute toxicity test of mixed fermented pieces of Ganoderma lucidum and Artemisia argyi: LD50 test based on Koch's modified method.

[0138] Operation steps: 200 mice were weighed and randomly divided into 10 experimental groups according to their body weight according to the random number table method, with 20 mice in each group (see Table 3), half of them male and half female. Each medication group was given a single dose of the decoction piece (the lowest dose to the highest dose determined in the pre-experiment was set according to the common ratio method) (g / kg), and the decoction piece dose was 10ml / kg. After administration, whether poisoning or death occurred within 24 hours was observed and recorded, and the LD50 was calculated based on the death of the animals.

[0139] SPSS 25.0 software was used for data analysis, and the measured data were expressed as mean ± standard deviation. The t test was used for comparison among the groups, and P < 0.05 was considered statistically significant.

[0140] LD50 calculation: LD 50 =log -1 [Xm-i(Σp-0.5)]

[0141] Xm: logarithmic value of the dose in the maximum dose group;

[0142] i: the difference between the logarithmic values ​​of doses in two adjacent groups;

[0143] P: mortality rate of animals in each group.

[0144] The relevant test data obtained from the above example operations, questionnaires and toxicity tests are detailed in Tables 1, 2 and 3.

[0145] Table 1 Related test data obtained from the in vitro antioxidant experiment of the present invention

[0146] Test items DPPH clearance rate (%) <![CDATA[Ferric ion reducing power (OD 700 )]]> Example 1 70.75 1.28 Example 2 65.64 1.05 Example 3 68.72 1.12 Example 4 83.56 2.00 Example 5 88.37 2.28 Example 6 81.58 1.93 Example 7 94.33* 2.48 Comparative Example 1 43.25 0.49 Comparative Example 2 55.35 0.71 Comparative Example 3 62.46 0.99 Comparative Example 4 71.86 1.33 Comparative Example 5 75.41 1.77 Comparative Example 6 77.34 1.81 Comparative Example 7 73.63 1.47 Comparative Example 8 82.85 1.99 Comparative Example 9 89.74 2.31 Comparative Example 10 84.82 2.12 Comparative Example 11 85.71 2.22

[0147] (*: p<0.05) The result is extremely significant.

[0148] By comparing the experimental data of Examples 1 to 7 and Comparative Examples 1 to 11, it can be seen that the present invention uses Lactobacillus rhamnosus, Bacillus coagulans, and Saccharomyces cerevisiae to ferment the mixture of Ganoderma lucidum and Artemisia argyi, and the obtained mixed fermented pieces of Ganoderma lucidum and Artemisia argyi have the strongest antioxidant effect.

[0149] The specific test data are as follows:

[0150] DPPH removal rate: The dual-strain fermentation was better than the single-strain fermentation. The DPPH removal rate after mixed fermentation of three strains was the highest, while the DPPH removal rate after aseptic fermentation was the lowest. The DPPH removal rate of Example 7 was increased by 1 times compared with Comparative Example 1.

[0151] Iron ion reducing power: Similar to the DPPH clearance rate data, the mixed fermentation of three strains was higher than that of two strains and a single strain, among which Example 7 was the highest.

[0152] Table 2 Related index data obtained from the questionnaire of the present invention

[0153]

[0154] By comparing the index data between the above investigation groups 1 to 3 and the comparison groups 1 to 3, it can be seen that the present invention uses Ganoderma lucidum fruiting bodies and Artemisia argyi leaves to ferment in a volume ratio of 3:1 (V:V), and the prepared Ganoderma lucidum and Artemisia argyi mixed fermented medicinal pieces have the highest good evaluation degree and the lowest discomfort evaluation degree in terms of smell and taste.

[0155] The specific survey data are as follows:

[0156] Odor index: When the volume ratio of Ganoderma lucidum fruiting body to wormwood leaf was 1:1, the improvement of favorable evaluation was not significant. When the volume ratio of wormwood leaf increased, the favorable evaluation decreased significantly and the unpleasant evaluation increased. Among them, the favorable evaluation of survey group 2 was significantly better than that of other groups.

[0157] Taste index: In the control group, when there was a lack of Ganoderma lucidum fruiting bodies or mugwort leaves, the good evaluation was not high. After adding Ganoderma lucidum fruiting bodies and mugwort leaves, the good evaluation increased significantly and the unpleasant evaluation decreased. Among them, the good evaluation of investigation group 2 was significantly better than that of other groups.

[0158] Table 3 Acute toxicity test of the mixed fermented slices of Ganoderma lucidum and Artemisia argyi of the present invention

[0159]

[0160] By comparing the above acute toxicity test data, it can be seen that the 24-hour LD50 of the mixed fermented medicinal slices of Ganoderma lucidum and Artemisia argyi prepared by the present invention is 134.15 g / kg body weight, and the 7-day LD50 is 118.39 g / kg body weight, which is much higher than the actual non-toxic 5g / kg body weight of the highest level in the "National Food Safety Standard Acute Oral Toxicity Test" GB15193.3-2014, and meets the safety standards.

[0161] Therefore, it can be seen from Table 1, Table 2 and Table 3 that, in terms of comprehensive antioxidant activity index, odor index, taste index and acute toxicity test, the data of Example 7 of the present invention is the best, that is, the technical solution of the present invention achieves the most outstanding technical effect.

[0162] The embodiments described above are only preferred embodiments of the present invention and are not intended to limit the scope of implementation of the present invention. Therefore, any equivalent changes or modifications made according to the structures, features and principles described in the patent scope of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A method for preparing a mixed fermented medicinal piece of Ganoderma lucidum and Artemisia argyi, characterized in that: The following steps are involved: (1) Lactobacillus rhamnosus, Bacillus coagulans, and Saccharomyces cerevisiae are activated; (2) Ganoderma lucidum fruiting bodies and wormwood leaves are crushed and mixed, 8 to 12 times the volume ratio of distilled water are added, heated to 80°C and kept warm for 4 hours, then cooled to 35 to 40°C, 2% honey is added and mixed, and after sterilization, it is allowed to stand for use; the Ganoderma lucidum fruiting bodies and wormwood leaves are crushed and mixed in a volume ratio of 3:1; (3) inoculating the activated strain obtained in step (1) into the mixed solution of ganoderma lucidum and wormwood prepared in step (2), culturing on a shaking table at a temperature of 35 to 37°C for 5 to 7 days to complete fermentation, centrifuging and filtering, concentrating into a paste, adding 30% medicinal dextrin and stirring evenly, vacuum drying into a dry paste, crushing and sieving, and obtaining a mixed fermented slice of ganoderma lucidum and wormwood; when inoculating the activated strain obtained in step (1) into the mixed solution of ganoderma lucidum and wormwood prepared in step (2), the volume ratio of the three strains is 1:1:1, the inoculation amount is 5 to 7%, and the strain concentration before inoculation is 1×10 8 ~1×10 10 CFU / mL.

2. The preparation method according to claim 1, characterized in that: The activation in step (1) specifically comprises: taking three strains of Lactobacillus rhamnosus, Bacillus coagulans, and Saccharomyces cerevisiae, respectively applying them on LP solid culture medium for purification and culture, and then taking single colonies and transferring them into LP liquid culture medium for strain preparation, thereby obtaining activated Lactobacillus rhamnosus, Bacillus coagulans, and Saccharomyces cerevisiae strains; The preparation method of the LP liquid culture medium comprises: weighing 10-15 g of glucose and 5-7 g of peptone, mixing them evenly, adding water to make up to 1 L, sterilizing them at 121° C. for 15-20 min, and standing them at room temperature to obtain the LP liquid culture medium; The LP solid culture medium is prepared by adding 18-20 g of agar powder to the LP liquid culture medium.

3. The preparation method according to claim 2, characterized in that: When single colonies are transferred to LP liquid culture medium for strain preparation, the ratio of single colony usage to culture medium usage is 0.01-0.1 g:100 mL, and the culture time is 2-3 days.

4. The preparation method according to claim 1, characterized in that: The sterilization in step (2) is performed at 120°C for 20 minutes.

5. The preparation method according to claim 1, characterized in that: In step (3), the concentration into paste is carried out at a temperature below 80°C; and the vacuum drying is carried out at 80°C.

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