A grifola frondosa liquid culture medium, a culture method thereof and application thereof

Abstract

The present application provides a grifola frondosa liquid culture medium and its culture method and application, relates to the fermentation technical field.The present application provides a grifola frondosa liquid culture medium, including glucose, proteose peptone, KH2PO4, MgSO4, vitamin B1, gibberellin and tyrosol, the culture medium can significantly improve grifola frondosa growth and the secretion of extracellular polysaccharide, and can be used for the preparation of food, medicine and health care product.

Description

Technical Field

[0001] This invention belongs to the field of fermentation technology, and relates to liquid culture media, specifically to a Grifola frondosa liquid culture medium and its culture method and application. Background Technology

[0002] Polysaccharides (PS) are polar, complex macromolecules with a degree of polymerization exceeding 10, formed by multiple identical or different monosaccharide groups linked by glycosidic bonds. Their molecular weight is generally in the tens of thousands, hundreds of thousands to millions, and some reports even reach tens of millions. Their basic structure is a macromolecular compound formed by the dehydration condensation of one or more monosaccharides with the hydroxyl groups of adjacent monosaccharides. They have a complex structure and are one of the four basic substances constituting life activities. Polysaccharides are the most abundant biopolymers in nature and exist in almost all organisms, including plants, animals and microorganisms (i.e., bacteria and fungi). Natural polysaccharides with certain functions can be roughly divided into several categories according to their source: (1) Plant polysaccharides: Angelica polysaccharide, pumpkin polysaccharide, tea polysaccharide, wolfberry polysaccharide, ginseng polysaccharide, jujube polysaccharide, etc. (2) Algal polysaccharides: Porphyra polysaccharide, red algae polysaccharide, kelp polysaccharide, spirulina polysaccharide, etc. (3) Fungal polysaccharides: Fungi are an extremely important component of my country's natural medicinal resources and traditional Chinese medicine. Among the more studied are lentinan from shiitake mushrooms, tremella mushrooms, Schizophyllum commune, Grifola frondosa, Poria cocos, Polyporus umbellatus, Ganoderma lucidum, as well as Auricularia auricula-judae, Cordyceps militaris, yeast glucan, etc. (4) Animal polysaccharides: These are mainly derived from various mammals, such as chitosan, chondroitin sulfate B, and heparin polysaccharides. The main biological activity of these polysaccharides is anticoagulant activity. (5) Bacterial polysaccharides: Examples include pneumococcal membrane polysaccharides, meningococcal membrane polysaccharides, and Haemophilus influenzae membrane polysaccharides. In biological systems, sugars not only serve as structural materials or energy storage, but also, along with their complexes, are key markers for cell recognition. With in-depth research on polysaccharides, researchers have gradually discovered their complex and multifaceted biological functions: polysaccharides possess immunomodulatory functions, such as treating rheumatism, chronic viral hepatitis, and other immune system diseases, and even having anti-AIDS effects; they also have anti-infective, anti-radiation, anticoagulant, hypoglycemic, and lipid-lowering effects, as well as promoting the biosynthesis of nucleic acids and proteins; they can control cell division and differentiation, and regulate cell growth and aging. Numerous pharmacological and clinical studies have shown that polysaccharide compounds possess complex and multifaceted biological functions without toxic side effects on normal cells.

[0003] The main monosaccharide component of Grifola frondosa polysaccharide is D-glucose, and it may also contain small amounts of D-xylose, D-fructose, D-fucose, L-arabinose, gentiobiose, galactose, etc. The biologically active Grifola frondosa polysaccharide is a β-(1-3)-D-glucan with a basic structure containing β-(1-6) side chains, with a branching degree of approximately 33%. The pharmacologically active components of Grifola frondosa polysaccharide are related to the content of its basic structure. Domestic and international scholars have conducted extensive research on it, extracting dozens of active polysaccharides from the fruiting bodies and mycelia of Grifola frondosa, including physiologically active components such as D-components, MD-components, Grifola frondosa, X-components, MT-2, and LELFD. Extensive chemical and pharmacological studies have demonstrated that Grifola frondosa polysaccharide has significant anti-tumor, anti-HIV, and immune system-improving effects. Therefore, it has been developed into various health products, including Baolisheng capsules and Virgil capsules in my country, MAIEXT in Japan, and Grifron series products in the United States.

[0004] Chinese patent CN105441334B discloses a method for producing Grifola frondosa polysaccharide: a specific strain of bacteria is made into seeds and inoculated into a culture medium containing a carbon source, a nitrogen source, inorganic salts and water, wherein the pH of the culture medium is 4-8, and fermentation is carried out continuously at 23-28℃ for 6-14 days; in the Grifola frondosa polysaccharide production stage, the carbon source of the fermentation culture medium is glucose and potato juice or corn flour, the nitrogen source is wheat bran juice and yeast powder, the ratio of potassium dihydrogen phosphate and magnesium sulfate is 2:1, and the mass percentage concentration of growth factor VB1 in the fermentation culture medium is 8-10 mg / L.

[0005] Chinese patent CN109355209A discloses a method for culturing *Grifola frondosa* strains. In the test tube culture stage, the culture medium includes a nitrogen source, a carbon source, phosphate, sulfate, agar, and a chestnut water extract. It also provides a method for culturing *Grifola frondosa* strains and a liquid fermentation method for *Grifola frondosa*, wherein the shake-flask seed culture medium and / or fermentation culture medium includes a nitrogen source, a carbon source, phosphate, sulfate, and a chestnut water extract. In artificial cultivation, adding a certain concentration of chestnut water extract as a nutrient supplement to the culture medium significantly promotes the growth of *Grifola frondosa* and the secretion of its effective components, increasing the dry weight of the mycelium by at least 1.5 times; the amount of *Grifola frondosa* polysaccharides obtained in the fermentation broth increases by at least 1.6 times. However, the preparation process of the chestnut water extract in the fermentation medium in this method is complex, resulting in a relatively low increase in *Grifola frondosa* polysaccharides. Summary of the Invention

[0006] This invention addresses the problems of low mycelial yield and extracellular polysaccharide secretion in existing technologies by providing a liquid culture medium for Grifola frondosa containing glucose, peptone, KH2PO4, MgSO4, vitamin B1, gibberellin, and tyrosol. This culture medium can significantly improve the growth of Grifola frondosa mycelium and the secretion of extracellular polysaccharides, and can be used in the preparation of food, pharmaceuticals, and health products.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: On one hand, the present invention provides a Grifola frondosa liquid culture medium, the components of which include glucose, peptone, KH2PO4, MgSO4, vitamin B1, gibberellin and tyrosol.

[0008] Preferably, the Grifola frondosa liquid culture medium comprises 20-40 g / L glucose, 2-6 g / L peptone, 0.5-1.5 g / L KH₂PO₄, 0.8-1.6 g / L MgSO₄, 8-12 mg / L vitamin B1, 10-20 mg / L gibberellin, and 0.3-1.0 mM tyrosol.

[0009] In some specific embodiments, the Grifola frondosa liquid culture medium comprises 22 g / L glucose, 3 g / L peptone, 0.8 g / L KH2PO4, 1.2 g / L MgSO4, 10 mg / L vitamin B1, 15 mg / L gibberellin and 0.6 mM tyrosol.

[0010] On the other hand, the present invention provides the application of the above-mentioned Grifola frondosa liquid culture medium in the cultivation of Grifola frondosa.

[0011] Preferably, the application includes increasing the dry weight of Grifola frondosa mycelium and / or increasing the secretion of Grifola frondosa extracellular polysaccharides.

[0012] On the other hand, the present invention provides a method for cultivating Grifola frondosa using the above-mentioned Grifola frondosa liquid culture medium.

[0013] Preferably, the method includes the following steps: S1. Inoculate the Grifola frondosa onto a solid culture medium and culture until the mycelium grows. S2. The mycelium obtained in step S1 is inoculated into seed culture medium and cultured to obtain seed fermentation broth; S3. The seed fermentation broth obtained in step S2 is inoculated into the Grifola frondosa liquid culture medium for culture.

[0014] Preferably, the solid culture medium in step S1 includes PDA solid culture medium, the components of which include 200 g / L potato, 20 g / L glucose, 5 g / L peptone, 2 g / L KH2PO4, 1 g / L MgSO4 and 20 g / L agar.

[0015] Preferably, the seed culture medium in step S2 comprises 200 g / L potato, 20 g / L glucose, 2 g / L peptone, 2 g / L KH2PO4 and 1 g / L MgSO4.

[0016] Preferably, the culture conditions in step S3 are 25°C and 140 r / min in a shaker.

[0017] In some specific embodiments, the Grifola frondosa strain has the accession number CGMCC No.14358 and was deposited at the China General Microbiological Culture Collection Center on July 25, 2017.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a simple liquid culture medium for Grifola frondosa that can significantly improve the growth of Grifola frondosa cells and the secretion of extracellular polysaccharides, and can be used in the preparation of food, medicine and health products.

[0019] Preservation information: Biomaterial: ZW-1; Classification and naming: Grifola frondosa ( Leafy tap ); Accession number: CGMCC No. 14358; Date of deposit: July 25, 2017; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0020] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers and the experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and the experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0022] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0023] This invention provides a Grifola frondosa liquid culture medium containing glucose, peptone, KH2PO4, MgSO4, vitamin B1, gibberellin and tyrosol.

[0024] In some embodiments, the Grifola frondosa liquid culture medium provided by the present invention contains 20-40 g / L glucose, 2-6 g / L peptone, 0.5-1.5 g / L KH2PO4, 0.8-1.6 g / L MgSO4, 8-12 mg / L vitamin B1, 10-20 mg / L gibberellin, and 0.3-1.0 mM tyrosol.

[0025] Example 1: Liquid fermentation of Grifola frondosa (1) Culture medium: 1.1 PDA solid culture medium (per liter): 200g potatoes, 20g glucose, 5g peptone, 2g KH2PO4, 1g MgSO4, 20g agar.

[0026] 1.2 Seed culture medium (per liter): 200g potatoes, 20g glucose, 2g peptone, 2g KH2PO4, 1g MgSO4.

[0027] 1.3 Fermentation medium (per liter): Glucose 22g, peptone 3g, KH2PO4 0.8g, MgSO4 1.2g, vitamin B1 10mg, gibberellin 15mg, tyrosol 0.6mM.

[0028] All the above culture sterilization conditions: autoclaving, 115℃, 30min.

[0029] (2) Experimental method: 2.1 Activation of microbial strains In this embodiment, the *Grifola frondosa* strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 25, 2017, with the accession number CGMCC No. 14358. PDA medium was prepared into slant culture medium using standard methods. A piece of *Grifola frondosa* mycelium, about the size of a soybean, was scooped out with an inoculation spatula and transferred to PDA slant culture medium. It was incubated at 25°C for 20 days. After the mycelium had grown, it was stored at 4°C for later use.

[0030] 2.2. Microbial Culture Prepare PDA culture medium into plates using standard methods. Use an inoculation spatula to scoop out soybean-sized pieces of activated Grifola frondosa mycelium and transfer them to PDA culture plates. Incubate at 25°C for 15 days. Once the mycelium has grown, store at 4°C for later use.

[0031] 2.3 Preparation of Seed Liquid Ten pieces of cultured Grifola frondosa mycelium, about the size of a soybean, were scooped out with an inoculation spatula and inoculated into 200ml Erlenmeyer flasks containing 60ml of seed culture medium. The flasks were then cultured at 25℃ and 140r / min on a shaker for 7 days.

[0032] 2.4 Preparation of Fermentation Broth The seed fermentation broth was inoculated into a 500ml baffled Erlenmeyer flask containing 150ml of fermentation culture at a 10% v / v inoculation rate, and cultured on a shaker at 25℃ and 140r / min for 7 days to obtain the fermentation broth.

[0033] Example 2: Liquid fermentation of Grifola frondosa The method of Example 1 was followed, except that the fermentation medium composition was changed to: glucose 20 g / L, peptone 6 g / L, KH2PO4 0.5 g / L, MgSO4 1.6 g / L, vitamin B1 8 mg / L, gibberellin 20 mg / L, and tyrosol 0.3 mM.

[0034] Example 3: Liquid fermentation of Grifola frondosa The method of Example 1 was followed, except that the fermentation medium components were changed to: Glucose 40 g / L, peptone 2 g / L, KH2PO4 1.5 g / L, MgSO4 0.8 g / L, vitamin B1 12 mg / L, gibberellin 10 mg / L, tyrosol 1.0 mM.

[0035] Comparative Example 1: Liquid Fermentation of Grifola frondosa The procedure was carried out according to Example 1, except that gibberellin was not added to the fermentation medium components.

[0036] Comparative Example 2: Liquid Fermentation of Grifola frondosa The procedure was carried out according to Example 1, except that tyrosol was not added to the fermentation medium components.

[0037] Comparative Example 3: Liquid Fermentation of Grifola frondosa The procedure was carried out according to Example 1, except that neither gibberellin nor tyrosol was added to the fermentation medium components.

[0038] Comparative Example 4: Liquid Fermentation of Grifola frondosa The procedure was carried out according to Example 1, except that gibberellin was replaced with an equimolar amount of naphthaleneacetic acid in the fermentation medium components.

[0039] Comparative Example 5: Liquid Fermentation of Grifola frondosa The procedure was carried out according to Example 1, except that tyrosol was replaced with an equimolar amount of farnesol in the fermentation medium components.

[0040] Test Example: Determination of Dry Weight and Extracellular Polysaccharides of Grifola frondosa Grifola frondosa fermentation broth was prepared according to the methods of the examples and comparative examples. Three parallel experiments were set up for each group. Then, the dry weight and extracellular polysaccharide content were determined, and the methods are as follows: (1) Determination of dry weight of Grifola frondosa mycelium The fermentation broth was filtered using a Buchner funnel. The mycelium was washed with pure water and then dried in a 55°C drying oven until constant weight. After weighing, the weight of the filter paper was subtracted to obtain the dry weight of the Grifola frondosa mycelium.

[0041] (2) Determination of extracellular polysaccharides The fermentation supernatant obtained by filtration in (1) was subjected to deproteinization. The deproteinization method was adopted by trichloroacetic acid, that is, trichloroacetic acid was directly added to the fermentation supernatant to make the mass percentage concentration of trichloroacetic acid in the liquid reach 20%. After standing, it was centrifuged, and the obtained supernatant was the extracellular polysaccharide solution. The polysaccharide concentration was then determined by phenol-sulfuric acid method.

[0042] Table 1

[0043] Note: In the same column of data, the same letter indicates that there is no significant difference between the data (P>0.05), and different letters indicate that there is a significant difference between the data (P<0.05).

[0044] The results are shown in Table 1. It can be seen that: in Comparative Example 3, neither gibberellin nor tyrosol was added to the culture medium, which represents the effect of the basal culture medium; in Comparative Examples 1 and 2, only one of tyrosol and gibberellin was added, respectively, resulting in an increase in mycelial dry weight and extracellular polysaccharide concentration compared to Comparative Example 3; Example 1 further increased the dry weight and polysaccharide concentration significantly compared to Comparative Examples 1 and 2, demonstrating the synergistic effect of tyrosol and gibberellin. Furthermore, in Comparative Example 4, naphthaleneacetic acid was used to replace gibberellin, and in Comparative Example 5, farnesol was used to replace tyrosol, neither of which achieved a synergistic effect. From the above, it is clear that the gibberellin and tyrosol in the culture medium provided by this invention have a significant and irreplaceable effect on increasing mycelial dry weight and extracellular polysaccharide concentration.

[0045] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A liquid culture medium for Grifola frondosa, characterized in that, The components include glucose 20-40 g / L, peptone 2-6 g / L, KH2PO4 0.5-1.5 g / L, MgSO4 0.8-1.6 g / L, vitamin B1 8-12 mg / L, gibberellin 10-20 mg / L and tyrosol 0.3-1.0 mM.

2. The Grifola frondosa liquid culture medium according to claim 1, characterized in that, The composition includes glucose 22 g / L, peptone 3 g / L, KH2PO4 0.8 g / L, MgSO4 1.2 g / L, vitamin B1 10 mg / L, gibberellin 15 mg / L and tyrosol 0.6 mM.

3. The use of the Grifola frondosa liquid culture medium according to any one of claims 1-2 in the cultivation of Grifola frondosa.

4. The application according to claim 3, characterized in that, The applications include obtaining Grifola frondosa mycelium and / or extracting Grifola frondosa extracellular polysaccharides.

5. A method for cultivating Grifola frondosa, characterized in that, The Grifola frondosa was cultured using the liquid culture medium according to any one of claims 1-2.

6. The method according to claim 5, characterized in that, Includes the following steps: S1. Inoculate the Grifola frondosa onto a solid culture medium and culture until the mycelium grows. S2. The mycelium obtained in step S1 is inoculated into seed culture medium and cultured to obtain seed fermentation broth; S3. Inoculate the seed fermentation broth obtained in step S2 into the Grifola frondosa liquid culture medium for culture.

7. The method according to claim 6, characterized in that, The solid culture medium mentioned in step S1 includes PDA solid culture medium, the components of which include 200 g / L potato, 20 g / L glucose, 5 g / L peptone, 2 g / L KH2PO4, 1 g / L MgSO4 and 20 g / L agar.

8. The method according to claim 6, characterized in that, The seed culture medium in step S2 consists of 200 g / L potato, 20 g / L glucose, 2 g / L peptone, 2 g / L KH2PO4 and 1 g / L MgSO4.

9. The method according to claim 6, characterized in that, The cultivation conditions described in step S3 are 25°C and 140 r / min in a shaker.

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