Preparation method of rosemary medicinal fungus leavening capable of efficiently regulating intestinal flora, obtained product and application of rosemary medicinal fungus leavening

Abstract

The invention belongs to the field of development and utilization of traditional Chinese medicine resources, and particularly relates to a preparation method of a rosemary medicinal fungus fermentation product capable of efficiently regulating intestinal flora, an obtained product and application of the rosemary medicinal fungus fermentation product. The preparation method comprises the following steps: inoculating activated lucid ganoderma into a rosemary culture medium for sealed fermentation, then extracting with 70% ethanol, filtering, centrifuging, taking supernate, carrying out rotary evaporation and concentration, and freeze-drying a solid, so as to obtain the lucid ganoderma fermented rosemary extract. According to the invention, the medicinal and edible fungus lucid ganoderma is used for fermenting rosemary for the first time, effective substances in the fermented product are extracted to obtain an extract, the extract with a proper concentration of 200 mg / ml is used for carrying out intragastric therapy on a colitis model mouse, and a 16S rRNA sequencing technology is used for sequencing intestinal flora, so that compared with a model group, the result shows that the content of the effective substances in the model group is obviously reduced; the lucid ganoderma solid-state fermentation rosemary extract has the effects of obviously increasing the diversity of intestinal flora and recovering the steady state of the intestinal flora. According to the invention, two kinds of lucid ganoderma strains with anti-inflammatory effects and rosemary which is a traditional Chinese medicinal material are adopted for solid-state fermentation for the first time, and good results are achieved in the aspects of maintaining the steady state of intestinal flora and increasing the diversity of the flora.

Description

Technical Field

[0001] The invention belongs to the field of development and utilization of traditional Chinese medicine resources, and specifically relates to a preparation method of a rosemary medicinal fungus fermentation product for efficiently regulating intestinal flora, and the obtained product and application. Background Art

[0002] Lingzhi Ganoderma lucidum (Curtis) P. Karst. ) is a Polyporaceae fungus, rich in polysaccharides, triterpenes, sterols, proteins and various amino acids. It has antioxidant, anti-aging, anti-depression, anti-tumor, hypoglycemic, hypolipidemic, cellular immune function enhancement, liver protection, prevention and treatment of central nervous system diseases, and relief of chronic inflammation. It has great potential and value in the development of health foods and drugs.

[0003] rosemary( Rosmarinus officinalis L. Rosemary is a perennial herbaceous plant of the Lamiaceae family, also known as ocean dew and tansy. Rosemary contains flavonoids, terpenes, tannins, phenolic acids and other chemical substances, and has a variety of biological activities such as antibacterial, antioxidant, anti-inflammatory, anti-tumor, and immune regulation. Due to its outstanding antioxidant activity, it is widely used in stabilizing food properties, preventing oil rancidity, and preserving feed.

[0004] Intestinal microbial flora is divided into beneficial bacteria and harmful bacteria, which are divided according to phylum, mainly including beneficial bacteria Firmicutes ( Firmicutes ) 、 Bacteroidetes ( Bacteroidota ), Verrucomicrobia ( Verrucomicrobia ); and harmful bacteria Proteobacteria ( Proteobacteria ), Actinobacteria ( Actinobacteria ) etc. Further, according to the genus classification, beneficial bacteria mainly include Enterobacterium spp. ( Muribaculaceae )、Bacteroides( Bacteroides )、Helicobacter pylori( Lachnospiraceae ), Akkermansia muciniphila ( Akkermansia ), etc. Harmful bacteria mainly include Shigella ( Escherichia-Shigella ),staphylococcus( Staphylococcus ), Zürichbacillus ( Turicibacter )wait.

[0005] Modern fermentation technology has the advantages of improving the efficacy of traditional Chinese medicine, increasing the content of effective ingredients, improving the utilization rate of traditional Chinese medicine, reducing the content of toxic ingredients in drugs, and producing new active ingredients. However, in the prior art, there is no record of using Ganoderma lucidum to ferment rosemary and extracting the fermentation product to promote the increase of intestinal flora diversity. Summary of the invention

[0006] In view of the problems existing in the prior art, the object of the present invention is to provide a method for preparing a rosemary extract by solid-state fermentation of Ganoderma lucidum.

[0007] The present invention also provides a ganoderma lucidum solid-state fermented rosemary extract prepared by the method.

[0008] Another object of the present invention is to provide the use of the above-mentioned Ganoderma lucidum solid-state fermentation rosemary extract in the preparation of drugs for improving colitis and regulating intestinal flora.

[0009] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: The present invention provides a method for preparing a rosemary extract by solid-state fermentation of ganoderma lucidum, comprising the following steps: (a) taking a strain of Ganoderma lucidum, picking out hyphae and inoculating them on a PDA solid plate, and culturing them at a constant temperature to obtain activated Ganoderma lucidum; (b) adding rosemary powder to the PDA liquid culture medium, stirring while adding, and stirring evenly until the powder is neither loose nor clumped, and is in a state of being in a clumping state when tightly grasped but dispersed when loosened, thereby obtaining a rosemary culture medium; (c) sealing the rosemary culture medium and sterilizing it; (d) Rosemary fermentation: inoculating the activated Ganoderma lucidum into the bottom of the rosemary culture medium, sealing it, fermenting it, and drying it to a constant weight to obtain a dry fermentation product; (e) extracting the dried fermented product with 70% ethanol, filtering and centrifuging, collecting the supernatant and concentrating it by rotary evaporation, and freeze-drying the solid to obtain the Ganoderma lucidum fermented rosemary extract.

[0010] Furthermore, in step (a), the inoculation amount of the mycelium in the PDA solid culture medium is 5%; and the constant temperature culture is cultured at 27°C for 3 days.

[0011] Furthermore, in step (b), the solid-liquid ratio of the rosemary powder to the PDA liquid culture medium is 1:10; Furthermore, in step (c), the sterilization is performed by high pressure steam sterilization at a temperature of 115° C. for 30 min.

[0012] Furthermore, in step (d), the accession amount of the Ganoderma lucidum is 10%; and the fermentation culture is cultured at 27°C for 30 days.

[0013] Furthermore, in step (e), the volume ratio of the fermentation product to 70% ethanol is 1:20; and the extraction is performed at room temperature for 24 hours.

[0014] The present invention also provides a ganoderma lucidum solid-state fermentation rosemary extract prepared by the above preparation method.

[0015] Another object of the present invention is to provide the use of the above-mentioned Ganoderma lucidum solid-state fermentation rosemary extract in the preparation of drugs for improving colitis and regulating intestinal flora.

[0016] The rosemary powder used in the present invention is firstly obtained by crushing rosemary with a grinder and filtering it through a 60-mesh screen. In the fermentation extract provided by the present invention, in the process of regulating intestinal flora, the bacterial phyla that show changes in abundance include: Bacteroidetes, Firmicutes, Proteobacteria, Verrucomicrobia, etc.

[0017] The Ganoderma lucidum strain used in the present invention was purchased from Beina Biotechnology, and its appearance characteristics are as follows: in the early stage, the mycelium of Ganoderma lucidum is light white, and the surface has no obvious luster and is foggy; in the middle stage, colonies are formed on the culture medium, the colonies are white, and grow in a diffuse manner, and some mycelium grows longitudinally to the cover of the culture dish; when it grows to about 7-10 days, the mycelium ages and forms a light brown shape on the surface of the culture medium.

[0018] The beneficial effects of the present invention are (1) The present invention is the first to use the medicinal edible fungus Ganoderma lucidum to ferment rosemary, and extract the effective substances in the fermentation product to obtain an extract. The extract at a suitable concentration of 200 mg / ml was administered to colitis model mice for gavage treatment. The intestinal flora was sequenced using 16S rRNA sequencing technology. Compared with the model group, the Ganoderma lucidum solid-state fermentation rosemary extract of the present invention has the effect of significantly increasing the diversity of intestinal flora and restoring the homeostasis of intestinal flora.

[0019] (2) The present invention is the first to use two Ganoderma lucidum strains with anti-inflammatory effects and the Chinese medicinal material rosemary for solid-state fermentation, which has good results in maintaining the homeostasis of intestinal flora and increasing the diversity of flora; (3) The preparation method provided by the present invention can increase the active ingredients in the fermentation extract, has a high extraction rate, and is simple to operate; (4) The Ganoderma lucidum fermented rosemary extract provided by the present invention has a significant promoting effect on the increase in the diversity of beneficial bacteria such as Firmicutes and Bacteroidetes in the intestinal flora, and has a significant inhibitory effect on the increase in the diversity of harmful bacteria such as Proteobacteria caused by DSS, which provides a valuable reference for the subsequent development of anti-inflammatory drugs and maintaining the stability of the flora and protecting the gastrointestinal mucosal barrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Comparison of the Venn diagram of intestinal flora in the three experimental groups; Figure 2 Comparison of the α diversity index of the three groups of intestinal flora, among which (a) is the effect of rosemary extract fermented with Ganoderma lucidum on the Shannon index of intestinal flora; (b) is the effect of rosemary extract fermented with Ganoderma lucidum on the Simpson index of intestinal flora; (c) is the effect of rosemary extract fermented with Ganoderma lucidum on the Sobs index of intestinal flora; (d) is the effect of rosemary extract fermented with Ganoderma lucidum on the ACE index of intestinal flora Figure 3 Comparison of intestinal flora richness at the phylum level among the three groups; Figure 4 Comparison of intestinal flora richness at the genus level among the three groups. DETAILED DESCRIPTION

[0021] In order to facilitate the understanding of the present invention, the present invention is described in more detail below in conjunction with specific embodiments. Preferred embodiments of the present invention are given. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should be noted that the content of the present invention is not limited to the scope described in the embodiments.

[0022] Example 1 1. Activation of Ganoderma lucidum (1) Preparation of PDA comprehensive culture medium: Peel the potatoes, weigh 200 g, cut into small dices, add ultrapure water and cook over low heat until it becomes viscous, filter with gauze, add other culture medium components in the PDA comprehensive culture medium listed in Table 1, and make up to 1 L with ultrapure water.

[0023] Table 1 Culture medium formula (2) After the prepared PDA comprehensive culture medium is sterilized by high-pressure steam (115°C, 30 min), it is placed on a clean bench, sterilized by ultraviolet light for 15 min and protected from light for 5 min, and then poured into a plate. After the culture medium solidifies, take the Ganoderma lucidum strain, pick the mycelium and plant it in the center of the plate, place it in a constant temperature incubator at 27°C and invert for 3 days. Repeat this step to obtain activated Ganoderma lucidum.

[0024] 2. Rosemary fermentation (1) Crush the rosemary leaves with a grinder and pass them through a 60-mesh sieve to obtain rosemary powder.

[0025] (2) Add rosemary powder to PDA liquid culture medium (the solid-liquid ratio of rosemary powder to PDA liquid culture medium is 1:10), stirring while adding, and stir evenly until the rosemary powder is neither loose nor clumping, and is in a state of being tightly clumped but dispersed when released, thereby obtaining a rosemary culture medium. (3) Seal the rosemary culture medium with sealing film and sterilize it with high pressure steam (115 ℃, 30 min).

[0026] (4) Fermentation of rosemary: Ganoderma lucidum was inoculated into the bottom of rosemary culture medium (inoculation amount 10%), sealed with parafilm, and placed in an incubator at 27°C for 30 days for fermentation. The culture medium was dried to constant weight to obtain dried fermented material.

[0027] 3. Extraction of fermentation products The dried fermented material was extracted with 70% ethanol (V / V) (solid-liquid ratio 1:20), the filtrate was retained by gauze filtering, centrifuged for 5 minutes, the supernatant was placed in a rotary evaporator for rotary evaporation, and then freeze-dried with a freeze dryer, and ground to obtain a powdered Ganoderma lucidum fermented rosemary extract.

[0028] 4. Drug preparation Weigh the Ganoderma lucidum fermented rosemary extract separately, add 0.9% saline and ultrasonically shake until fully dissolved, prepare a drug concentration of 200 mg / ml, and prepare pure saline as a control. Both reagents are refrigerated in a 4°C refrigerator for use.

[0029] Effect Example 1 1. Colitis mouse model (1) Nine 6-week-old male C57 mice grown in the same environment were randomly divided into three groups, namely, a blank control group, a DSS (dextran sulfate sodium) model group, and a drug-treated group. Both the DSS group and the drug-treated group were supplied with 2.5% dextran sulfate sodium drinking water, while the control group was supplied with ordinary drinking water and the same feed supply. The inflammation model was established for 14 days.

[0030] (2) Before modeling, nine experimental mice were weighed and their initial weights were recorded. During the modeling period, the weight and feces of the mice (whether diarrhea, bloody stools, etc.) and activity levels were recorded every 24 hours to facilitate subsequent disease activity index (DAI) scoring.

[0031] 2. Drug treatment and 16S rRNA sequencing (1) On the 8th day of modeling, mice were treated with drugs by gavage at 0.2 ml / d / mouse. The blank control group and DSS group were gavaged with 0.9% saline, while the drug-treated group was gavaged with 200 mg / ml of the extract. The body weight and feces of the mice were also recorded every day.

[0032] (2) At the end of the experiment, the final weight of the mice was recorded. The mouse feces were collected under a sterile environment and placed in sterile cryopreservation tubes. After quick freezing with liquid nitrogen, they were stored in a -80°C refrigerator for subsequent sequencing. All experimental mice were killed by cervical dislocation, and colon tissues were collected and stored at -80°C for subsequent experiments.

[0033] (3) The frozen mouse feces were labeled and placed in dry ice in a low-temperature environment for 16S rRNA testing. The total DNA of the intestinal flora in the feces was extracted, and PCR amplification was performed to detect consistency and establish a library. The sequencing results were returned by Shanghai Meiji Biotechnology Co., Ltd. and the final results were obtained after analysis on the Meiji platform.

[0034] Figure 1The data show the number of unique and common populations and dominant species in the intestinal flora of the three groups of mice. There are 122 common populations in the three groups, 155 unique populations in the CK group, 16 in the DSS group, and 44 in the fermentation extract group. It can be seen that fermentation extract treatment can cause an increase in the number of populations.

[0035] like Figure 2 As shown in (a) and (b), DSS treatment caused a decrease in intestinal flora diversity (the Shannon index decreased and the Simpson index increased), while the fermentation extract treatment reversed this phenomenon, with the Shannon index increasing by 1.1 times and the Simpson index decreasing to 1 / 7 of the original value; (c) and (d) represent the community richness index, and the larger the value, the higher the community richness. It can be seen that compared with the blank control group, the community richness index of the model group decreased, while after treatment with the fermentation extract, the sobs index and ace index increased by 45.39% and 37.79%, respectively. This shows that the fermentation extract can significantly improve the phenomenon of decreased intestinal flora diversity caused by DSS.

[0036] like Figure 3 As shown, Firmicutes, Bacteroidota, Verrucomicrobia The intestinal beneficial bacteria categories all showed a trend of decreasing abundance ratio in the model group and increasing abundance ratio in the extract group; Proteobacteria this The harmful bacteria in the intestine increased significantly in the model group, and it was speculated that the increase in this phylum may be the key to causing colitis, but after treatment with the fermentation extract, the bacteria in this phylum decreased significantly, indicating that the fermentation extract can significantly increase the proportion of beneficial bacteria and reduce the proportion of harmful bacteria at the phylum level.

[0037] like Figure 4 As shown, Escherichia-Shigella, Staphylococcus, Turicibacter The abundance of harmful intestinal bacteria genera such as β-lactamase and β-lactamase increased significantly in the model group, but decreased significantly in the fermented extract group; Muribaculaceae, Akkermansia, Bacteroides The abundance of beneficial intestinal bacteria genera such as β-lactamase and β-lactamase decreased in the model group, but increased in the fermentation extract group, indicating that the fermentation extract can significantly reduce the proportion of harmful bacteria and increase the proportion of beneficial bacteria at the genus level.

[0038] In summary, the present invention used Ganoderma lucidum fermented rosemary extract to treat DSS-inflamed mice. Compared with the model group and the control group, it was found that Ganoderma lucidum fermented rosemary extract had a significant promoting effect on the increase in the diversity of beneficial bacteria such as Firmicutes and Bacteroidetes in the intestinal flora, and had a significant inhibitory effect on the increase in the diversity of harmful bacteria, Proteobacteria, caused by DSS, which provides a valuable reference for the subsequent development of anti-inflammatory drugs and maintaining the stability of the flora and protecting the gastrointestinal mucosal barrier.

[0039] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions may occur depending on design requirements and other factors. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a rosemary extract by solid-state fermentation of Ganoderma lucidum, characterized in that: The following steps are involved: (a) taking a strain of Ganoderma lucidum, picking out hyphae and inoculating them on a PDA solid plate, and culturing them at a constant temperature to obtain activated Ganoderma lucidum; (b) adding rosemary powder to the PDA liquid culture medium, stirring while adding, and stirring evenly until the powder is neither loose nor clumped, and is in a state of being in a clumping state when tightly grasped but dispersed when loosened, thereby obtaining a rosemary culture medium; (c) sealing the rosemary culture medium and sterilizing it; (d) Rosemary fermentation: inoculating the activated Ganoderma lucidum into the bottom of the rosemary culture medium, sealing it, fermenting it, and drying it to a constant weight to obtain a dry fermentation product; (e) extracting the dried fermented product with 70% ethanol, filtering and centrifuging, collecting the supernatant and concentrating it by rotary evaporation, and freeze-drying the solid to obtain the Ganoderma lucidum fermented rosemary extract.

2. The preparation method according to claim 1, characterized in that: In step (a), the inoculation amount of the mycelium in the PDA solid culture medium is 5%; and the constant temperature culture is cultured at 27° C. for 3 days.

3. The preparation method according to claim 1 or 2, characterized in that: In step (b), the solid-liquid ratio of the rosemary powder to the PDA liquid culture medium is 1:

10.

4. The preparation method according to claim 1, characterized in that: In step (c), the sterilization is performed by high pressure steam sterilization at a temperature of 115° C. for 30 min.

5. The preparation method according to claim 1 or 4, characterized in that: In step (d), the accession amount of the Ganoderma lucidum is 10%; and the fermentation culture is cultured at 27°C for 30 days.

6. The preparation method according to claim 1, characterized in that: In step (e), the volume ratio of the fermentation product to 70% ethanol is 1:20; and the extraction is performed at room temperature for 24 hours.

7. A Ganoderma lucidum solid-state fermentation rosemary extract prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the Ganoderma lucidum solid-state fermented rosemary extract as claimed in claim 7 in the preparation of drugs for improving colitis and regulating intestinal flora.

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