CN110423788B - Method for producing grifola frondosa polysaccharide by using grifola frondosa strains generated by mutagenesis - Google Patents

Abstract

The invention discloses a method for producing grifola frondosa polysaccharide by using grifola frondosa strains generated by mutagenesis, and relates to the technical field of food microorganism application. A new grifola frondosa strain is obtained through ultraviolet mutagenesis, the preservation number is CCTCC NO: M2018907, liquid fermentation is carried out on a rice bran and wheat bran complete medium which is tested to meet the pollutant limit standard, and the rice bran and wheat bran are efficiently converted into grifola frondosa mycelium raw material and more extracellular crude polysaccharide. The fermentation product is subjected to multi-frequency ultrasonic extraction, freeze concentration, citric acid leaching to replace heavy metal ions, alcohol precipitation and alcohol washing to remove impurities, so that a qualified grifola frondosa polysaccharide product is obtained. The method for producing the grifola frondosa polysaccharide by the new grifola frondosa strain is realized for the first time, and meanwhile, the method converts low-value rice bran and wheat bran into high-value grifola frondosa polysaccharide with various health care functions, and has beneficial social and economic effects on reducing production cost, realizing high-efficiency, reasonable and high-value utilization of low-end resources and protecting public health.

Description

Method for producing grifola frondosa polysaccharide by using grifola frondosa strains generated by mutagenesis

Technical Field

The invention relates to the technical field of food microorganism application, in particular to a method for producing polysaccharide by using a new strain obtained by mutagenesis, wherein the new strain is fermented in a liquid culture medium of rice bran and wheat bran complete material which is inspected to meet the limit standard of pollutants, and after mycelium raw materials and fermented products of fermentation broth are produced, qualified polysaccharide products are obtained through multifrequency ultrasonic auxiliary extraction, freeze concentration, substitution of heavy metal ions by citric acid leaching, alcohol precipitation and alcohol washing and freeze drying.

Background

Many fungi for both medicine and food or medicinal fungi have a long history of use and a wide population of use in China. Modern science reveals that now, rare fungi such as mycelium, fruiting body or spore of Maitake Mushroom, hericium Erinaceus, ganoderma, inonotus obliquus, cordyceps etc. can produce various active ingredients such as amino acids, proteins, vitamins, polysaccharides, nucleosides, terpenoid, flavonoids, etc. or enrich various microelements such as selenium, zinc, etc., and has various effects of enhancing immunity, resisting tumor, enhancing liver function, resisting oxidation, etc. Because of this, various people in the world have great interest in researching and developing the fungi, and many researchers and developers exist in the fields of medicine, food, chinese medicine health care, agriculture and industrial production, so that many research results or products, such as ganoderma lucidum capsules, grifola frondosa capsules, paecilomyces hepiali capsules and the like, are obtained, and the economic value of the fungi is high. Hericium erinaceus is added into the biscuits to prepare Hericium erinaceus biscuits which are already common food sold in domestic supermarkets. Similar rare fungus health foods or pharmaceutical products are produced internationally in New Zealand, japan, america, korea, etc.

In terms of grifola frondosa, studies on it have shown that: the grifola frondosa has certain biological activity in the aspects of resisting tumor, resisting virus, reducing blood sugar, delaying aging, enhancing immunity and the like, and has higher medicinal value (reference (1)He Y,Li X,Hao C,et al.Grifola frondosa polysaccharide:a review of antitumor and other biological activity studies in China[J, discovery Medicine,2018,25 (138): 159-176. (2)Mao G H,Ren Y,Feng W W,et al.Antitumor and immunomodulatory activity of a water-soluble polysaccharide from Grifola frondosa [ J ]. Carbohydrate Polym,2015,134:406-412. (3)Chan Y Y,Chan E,Chan S W,et al.Enhancement of in vitro and in vivo anticancer activities of polysaccharide peptide from Grifola frondosa by chemical modifications[J), pharmaceutical Biology,2011,49 (11): 1114-20. (4)He X,Wang X,Fang J,et al.Polysaccharides in Grifola frondosa mushroom and their health promoting properties:A review[J ]. International journal of biological macromolecules,2017,101:910. (5) Zhang Zongqi, liu Liping. Structural characteristics of grifola frondosa polysaccharide and research on biological activity thereof are advanced [ J ]. Chinese brewing, 2018 (5)).

Grifola frondosa (Grifola frondosa) is a precious fungus used as both medicine and food, belonging to the genera Basidiomycota, hypsizygus, aphyllophorales, polyporaceae, grifola, also known as Polyporus frondosus, qianfos, lotus, chestnut mushroom, etc., and has different aliases depending on the place where it grows, and is called as lotus fungus in southwest, chestnut mushroom in northeast, and as "Maitake" in Zhejiang mountain, japan, and as "forest chicken" (hen of the wood). Grifola frondosa grows mostly in high mountain broadleaves Lin Xiangshu, chestnut trees or evergreen needles, and broad mixed forests. Parasitic on the root of the tree is a typical forest white rot fungus. In China, the Chinese medicinal materials are mainly distributed in regions such as Yunnan, sichuan, hebei, heilongjiang, jilin, guangxi, tibet, fujian and the like. The wild grifola frondosa is mainly produced in broad-leaved forests with the altitude of 800-1400 m, favors warm climates and moist soil, and the form of the wild grifola frondosa can be divided into two parts of mycelium and fruiting bodies, wherein the mycelium is used as a product of deep fermentation of the grifola frondosa, and has milky color and uniform spherical form; the fruit body as the edible part is a nutritional food rich in protein, vitamin E and various mineral elements. The artificial domestication cultivation of the grifola frondosa enters industrialization, the cultivation in the southern area of China is basically material-replacing cultivation, and the material-replacing buried cultivation is mainly used in the north. The fruiting body of Maitake Mushroom is gray to gray brown to gray white, a circle of irregular sharp protrusions are arranged at the edge, fungus meat is white, meat quality is crisp and tender, stems are short and are in coral branches, and the stems are overlapped into clusters. The appearance of the compound is similar to that of chrysanthemum, the appearance of the compound is similar to that of cloud flakes, and the name of 'cloud mushroom' is obtained. The existing method for obtaining the grifola frondosa mainly adopts artificial cultivation, and adopts fruiting body or spore powder as a medicine, but the artificial cultivation of the grifola frondosa has the advantages of long period, low production efficiency, high labor intensity, easiness in suffering from diseases and insect pests due to limitations of seasons, environment and the like, and unstable quality and yield. The rare fungus fermentation technology can overcome the defects of the traditional fruiting body cultivation, adopts a liquid or solid fermentation method to produce the grifola frondosa fermentation product, and adopts deep processing application to meet the development thought of innovation driving, thereby having good prospect. Advances made in research and development of the fermentation product of grifola frondosa will further promote the application of grifola frondosa, bringing practical social and economic benefits. Because of this, it is necessary to study new methods for producing the fermentation product of grifola frondosa and its deep processed products, for example, considering that some low-end agricultural product processing byproduct materials such as bran and rice bran are efficiently converted into high-value raw materials of the fermentation product of grifola frondosa, it is expected to efficiently utilize low-grade agricultural product resources, reduce the production cost of the fermentation product of grifola frondosa, thereby reducing the price of the final product such as health food or medicine of grifola frondosa, benefiting the general people and promoting the health of the public, which is one of the concerns of the patent of the present invention.

China is a large country of rice and wheat production, rice bran and wheat bran resources are rich, the annual yield of rice bran is about 1000 ten thousand tons, and wheat bran (the wheat bran in the specification refers to wheat bran) is about 3000 ten thousand tons. The rice bran and the wheat bran are used as by-products of rice or wheat processing, and have rich nutrition and low price. The rice bran contains rich and high-quality protein, active polysaccharide, fat, tocotrienol, tocopherol and other active substances with obvious physiological functions, and the wheat bran is rich in fat, protein, mineral substances, vitamins, cellulose and other nutritional ingredients, wherein the content of the cellulose can reach more than 18 percent of the total amount of the wheat bran, so that the rice bran and the wheat bran can provide sufficient carbon sources, nitrogen sources, vitamins and mineral substances for the growth of the grifola frondosa. Under the action of the grifola frondosa cellulase and other enzyme systems, rice bran and wheat bran can be converted into needed nutrient substances for growth and metabolism, and grifola frondosa mycelium and various functional substances are synthesized. Therefore, by using rice bran and wheat bran which are agricultural and sideline products as all carbon sources and nitrogen sources, the grifola frondosa liquid fermentation is carried out to produce the grifola frondosa fermentation product, and the grifola frondosa fermentation product is further processed into health-care food or medicine, thereby having technical feasibility and bringing better social and economic benefits.

Most liquid fermentation production methods of edible fungi use glucose, grain raw materials such as starch, potato or soybean powder and the like as culture mediums, and even agricultural byproducts such as rice bran, wheat bran and the like are used as auxiliary components. The growth condition of the original grifola frondosa on a rice bran and wheat bran whole liquid medium without adding glucose or other grain raw materials is not optimal, and the quality of the final product is affected by the fact that the rice bran and wheat bran raw materials contain harmful substances such as lead, arsenic, aflatoxin, vomit toxin, pesticide residues and the like, so that innovative research on a method for producing grifola frondosa fermentation product by fermenting the rice bran and wheat bran liquid whole material of the grifola frondosa is needed.

The research group of the first inventor Liu Weimin of the patent has conducted a great deal of research on liquid fermentation of ganoderma lucidum, grifola frondosa, cordyceps sinensis and the like for more than ten years, and a plurality of achievements such as a Shuoshi paper and an invention patent are formed successively. Liu Weimin guided master papers are: (1) Yang Suohua A polysaccharide is prepared by fermenting rice bran with Maitake Mushroom (2006); (2) Gu Huimin, research on polysaccharide production and organic selenium enrichment by liquid culture of grifola frondosa in rice bran medium (2009); (3) Zhang Jian, research on polysaccharide production from rice bran by liquid fermentation of grifola frondosa by physical mutagenesis (2010); (4) Guo Chunmei research (2011) on polysaccharide and selenium production from liquid fermentation of rice bran and wheat bran by strain mutagenesis of grifola frondosa; (5) Li Yanan, grifola frondosa strain mutagenesis, fermentation and performance studies (2013); (6) Liu Lili, ganoderma lucidum liquid fermentation and strain mutagenesis of high-value transformed rice bran and wheat bran (2012); (7) Guo Tianlong, the research on mutagenesis and liquid fermentation high-valued conversion of ganoderma lucidum strains into whole rice bran and wheat bran (2013); (8) Chen Jing, research on polysaccharide production by rice bran and wheat bran through cordyceps sinensis liquid fermentation high-value utilization (2014); (9) Zhang Xiaofei, studies of the activity of Ganoderma lucidum (Ganoderma lucidum CFCC 6043) liquid fermented whole rice bran and polysaccharide (2014); (10) Cao Xun, maitake Mushroom mutagenesis and New and old Strain liquid fermentation testa oryzae bran study (2015); (11) Gold mine, ganoderma lucidum mutagenesis and new and old strain liquid fermentation rice bran and wheat bran research (2015); (12) Wang Xuemei comparative study of liquid fermentation of rice bran and wheat bran complete with Cordyceps sinensis strain (2016); (13) Li Tingting, strain mutagenesis of Cordyceps sinensis and research on new culture medium of liquid fermentation of testa oryzae and testa Tritici by new strain (2017). Liu Weimin as the invention patents of the inventors are: (1) A method for producing polysaccharide using rice bran and wheat bran composite material and grifola frondosa mutant strain, 20101010579048.4; (2) Grifola frondosa strain 201010579078.5 for producing polysaccharide from rice bran and wheat bran composite raw materials; (3) A strain 201110150888.3 for fermenting rice bran and bran extract to produce grifola frondosa polysaccharide; (4) Bacterial strain 201310274913.8 for producing grifola frondosa polysaccharide by full liquid fermentation of rice bran and wheat bran; (5) A method for producing polysaccharide by fermenting rice bran and wheat bran complete materials in liquid state by using a ganoderma lucidum mutant strain, 201310275061.4; (6) A method for producing cordyceps sinensis polysaccharide by liquid state fermentation of rice bran and wheat bran complete material, 201310274914.2; (7) A method for producing polysaccharide by fermenting rice bran and wheat bran complete materials in liquid state by using grifola frondosa mutant strain, 201310274911.9; (8) Bacterial strain 201310274915.7 for producing ganoderan by full-liquid fermentation of rice bran and wheat bran; (9) A method for producing selenium-enriched ganoderma lucidum mycelium raw material, 201510996120.6; (10) A grifola frondosa mutant strain 201510990344.6 producing grifola frondosa mycelium; (11) Cordyceps sinensis mutant strain for producing Cordyceps sinensis mycelium, 201510996118.9, etc.

From the above description, the present inventors Liu Weimin have studied about the topic of converting rice bran and wheat bran to produce a fermentation product of rare edible fungi with high efficiency and high value, and have achieved the inventive idea and continuously improved the production method of various fermentation products of rare fungi.

The inventor Liu Weimin takes part in the research of the special project of the national thirteen-five-key research and development plan, namely the research and development of the key technical equipment for moderately processing the bulk rice products in the project of the modern food processing, grain collecting, transporting and equipment, and the research of the new technology and the new mode for the food utilization of the rice processing byproducts in the demonstration (2017 YFD 0401105), and takes charge of the research tasks of the research and development of the key technology for semi-solid fermentation of rice bran and the new product. Liu Weimin in combination with the above-mentioned research tasks, the guided papers of the Shuoshi research students and the patents of the inventions related to the present invention before are combed, consider the problem that the achievements related to the negotiation with enterprises cannot answer when transferring, for example, the previous explanation of strain sources does not meet the requirements of enterprises, the enterprises wish to develop industrially is not strong, etc., and it is found that if going to the industrialization road, many problems still exist in the related patent technologies and research papers, which need to be researched and solved and innovated. The problems include acquisition and confirmation of new efficient strains of rare fungi with different properties, establishment of standards of rice bran and wheat bran raw materials, innovation of fermentation methods of the rare fungi fermentation products, use of new methods for separating and purifying the rare fungi fermentation products, combination modes of objects needing innovation and the like. The inventive patent is designed with full consideration given to the uniqueness, outstanding substantive features and significant advances of innovation and practicality necessary to grant the inventive patent. Thus, the combined innovation of these problems should ensure that the related patent of the invention achieves the uniqueness, novelty and practicality necessary to grant the patent of the invention.

The inventor provides the application of the patent based on the above consideration and research on the production method of the grifola frondosa fermentation product, separates grifola frondosa original strains with different fermentation performances from the original grifola frondosa strains by utilizing the fruiting bodies of the grifola frondosa collected from the production site, carries out mutagenesis to obtain new strains, and ferments rice bran and wheat bran to produce qualified grifola frondosa polysaccharide products.

In summary, the invention relates to the field of using new strains for fermenting bran and rice bran to produce fermented products, and performing advanced processing treatment to obtain qualified products. Different from the previous research and achieved results: one of the key problems to be solved by the invention is that the fermentation capacity of the original strain is improved by a mutagenesis method, a new strain which is not reported is obtained, and the new strain can be subjected to gene sequencing, so that the correctness of the used strain is ensured; the second key problem to be solved by the invention is to determine the standard of the bran and the rice bran raw materials, because the sources of the bran and the rice bran are complex, the types of pollutants and the levels thereof are different, the previous researches and patents of the inventor do not pay enough attention in this aspect, and the invention brings uncertain results to the development of subsequent products; the third key problem to be solved by the invention is a production method for obtaining qualified products after deep processing treatment of fermentation mycelium and fermentation broth. The three points ensure that the patent of the invention is different from other inventors and the research team of the inventor Liu Weimin in various results, and has uniqueness, innovation and practicability.

The reasons presented for the key problems of the three aspects are further explained. The invention provides one of the key problems to be solved, namely, the invention provides a method for improving the fermentation capacity of original strains by mutagenesis, obtaining a new strain which is not reported, and carrying out gene sequencing on the new strain to ensure that the used strains are correct. The background of this problem is that the group of inventors expected a strain which was never used and had good performance, and expected to perform well in terms of bioconversion of bran, so consider the method of fermenting bran to produce a ferment. According to the previous research experience of the inventor, the problem of adaptability of strains needs to be solved firstly when the rare fungi ferment bran rice bran. Therefore, we need to examine the fermentation ability of the strain in this production method and improve the fermentation performance by strain mutagenesis. In addition, in the research process, after a large number of strain treatment steps, the strain is ensured not to have errors, and gene sequencing should be performed. This has not been considered in the past. Thus, the patent stares at the measurement of the gene series from the beginning, and ensures that the strain is correct.

The second key problem to be solved by the present invention is that the background of defining the standard of the bran and rice bran raw material is that our previous research and patent do not pay enough attention in this respect, and unexpected results are sometimes brought to the development of subsequent products. The previous research results always hope to be capable of processing various bran and rice bran, so that the use limit of the bran and rice bran raw materials is deliberately widened, the raw material standard is not formulated, but the result is that in the final product quality characterization, some abnormal values which are not in the research range appear, for example, the problem that the measurement result of certain heavy metal which is not measured in the previous research exceeds the standard is solved, the source of the bran and rice bran raw materials is complex, the quality is difficult to keep at similar level, and no obvious pollutant finally enters the final product. Thus, the inventive production process originally designed for certain specific contaminants and their possible contents, may have other contaminants in the product, so that the production process of the fermentation is redesigned to cope with the new contaminants that may have occurred. Thus, the problems of control and standard formulation of the quality of the bran and rice bran raw materials become the problems which must be considered in the design of the new invention patent. The invention provides a use standard of raw materials of bran and rice bran.

The third key problem to be solved by the invention is to carry out deep processing treatment on the fermented product to make the fermented product become a production method of a qualified product, and the background is to consider the reasonable rationality of the whole fermented product production method on the basis of solving the first two key problems. Since the bran and rice bran raw materials possibly bring in unknown pollutants, separation and purification methods such as freeze concentration, citric acid leaching to replace heavy metal ions, alcohol precipitation and alcohol washing are designed for the fermentation product after fermentation, so that a qualified grifola frondosa crude polysaccharide final product is obtained. Based on this knowledge, the present patent devised a more rational process for the production and treatment of fermentates.

The present invention requires the acquisition of new strains by mutagenesis methods. The breeding method of microorganism mainly includes physical mutagenesis, chemical mutagenesis and gene recombination, and in order to avoid using toxic and harmful chemical mutagen and genetic engineering method which is not accepted by the public in food field, the invention uses relatively simple and easy ultraviolet mutagenesis technology to make the original strain in the extreme environment of mutagenesis, furthest expands the range of mutation site and increases the possibility of obtaining positive mutant strain. The invention adopts the common mutagenesis method of ultraviolet mutagenesis, but the method has no innovation, but the strain performance obtained by the method is changed innovatively, and the method has uniqueness, innovation and practicability when the bran and rice bran complete medium is fermented.

The inventor mainly solves the three key new problems and constructs the content of the invention.

The novel strain of the full-liquid-state culture medium of the fermented bran and rice bran is obtained for the first time.

The wheat bran using standard of the invention is the wheat bran standard NY/T3218-2018 for executing the edible feed of the original department of agriculture, the pollution level of main heavy metals such as lead, arsenic, mercury, cadmium and chromium is not more than 3mg/kg at the highest, the pesticide applied in the wheat planting process accords with the agricultural technical standard, the wheat is free from serious scab in the growth process, the wheat is deteriorated due to no continuous rainwater in the harvesting season, and the wheat is not mildewed due to the storage and transportation of the wheat after the wheat is processed. The rice bran using standard is the standard NY/T122-1989 of the Ministry of agriculture of executing plus major heavy metals of lead, arsenic, mercury, cadmium and chromium, the pollution level limit is not more than 3mg/kg at maximum, the pesticide applied in the rice planting process accords with the agricultural technical standard, no serious diseases exist in the rice growing process, no continuous rainwater causes the deterioration of rice in harvesting season, and the rice bran storage and transportation after the rice processing can not cause mildew.

The invention provides a method for preparing polysaccharide by fermenting the novel strain in a liquid culture medium of rice bran and wheat bran complete materials which are inspected to meet the limit standard of pollutants, wherein the method comprises the specific composition of the culture medium, fermentation conditions, extraction and impurity removal methods, mycelium and polysaccharide yield and measurement values of polysaccharide quality indexes. The limit of mycotoxin aflatoxin B1, B2 and vomitoxin of the obtained total polysaccharide, the limit of heavy metals lead, arsenic, mercury, cadmium and chromium and the residual limit of pesticide carbendazim, abamectin and butachlor are not higher than the limit values specified in GB2761-2017, GB2762-2017 and GB2763-2016 or are not detected after the measurement.

Disclosure of Invention

The invention relates to a method for producing grifola frondosa polysaccharide by using a new grifola frondosa strain obtained by mutagenesis, wherein the new grifola frondosa strain is fermented in a liquid culture medium of a rice bran and wheat bran complete material which is tested to meet the limit standard of pollutants, and qualified grifola frondosa polysaccharide products are obtained after the fermentation of grifola frondosa mycelium raw materials and fermentation broth are produced, and the fermentation broth is subjected to multifrequency ultrasonic auxiliary extraction, freeze concentration, citric acid leaching to replace heavy metal ions, alcohol precipitation and alcohol washing and freeze drying.

The technical scheme adopted by the invention is as follows: fermenting the new strain of Grifola frondosa obtained by ultraviolet mutagenesis in a liquid culture medium of rice bran and wheat bran complete material which is inspected to meet the pollutant limit standard, producing the fermentation product of Grifola frondosa mycelium raw material and fermentation liquor, performing multifrequency ultrasonic auxiliary extraction, freeze concentration, citric acid leaching to replace heavy metal ions, alcohol precipitation and alcohol washing, and freeze drying to obtain qualified Grifola frondosa polysaccharide product.

In one aspect of the present invention, a method for producing a grifola frondosa polysaccharide using a novel strain of grifola frondosa produced by mutagenesis is performed according to the following steps:

(1) Inspecting wheat bran and rice bran, wherein the pollution level of lead, arsenic, mercury, cadmium and chromium which are main heavy metals is not more than 3mg/kg at the highest, the balance meets the wheat bran standard NY/T3218-2018 and the rice bran standard NY/T122-1989 for edible feed of the original agriculture department respectively, pesticides applied in the wheat planting process meet the agricultural technical specification, serious gibberellic disease is avoided in the wheat growing process, wheat deterioration is caused by no continuous rainwater in the harvesting season, mildewing is not caused by wheat storage and transportation after the wheat processing, serious diseases are avoided in the rice planting process, paddy deterioration is caused by no continuous rainwater in the harvesting season, and mildewing is not caused by rice storage and transportation after the rice processing;

(2) Sterilizing testa oryzae and testa Tritici with water 121 deg.C for 30min;

(3) The rice bran and the wheat bran complete material are used as carbon sources and nitrogen sources to be directly used as fermentation culture media of a new Grifola frondosa strain with the preservation number CCTCC NO: M2018907, the concentration of the rice bran is 0.1-10g/100mL, the concentration of the wheat bran is 0.1-10g/100mL, the sum of the concentration of the rice bran and the concentration of the wheat bran is 10.1g/100mL, 0.015-0.25g/100mL of monopotassium phosphate, 0.015-0.25g/100mL of magnesium sulfate heptahydrate, the pH is natural, 100mL of shaking bottle material is 250mL, the material loading rate of a fermentation tank is 75%, the inoculum size is 10%, the culture temperature of a shaking table is 20-25 ℃, the rotation speed of a shaking table is 150r/min or the rotation speed of a fermentation stirring paddle is 50-150r/min, and the fermentation time is 7-10d;

(4) Centrifugally separating the liquid culture to obtain mycelium and fermentation liquor, and measuring the yield of extracellular polysaccharide in the fermentation liquor;

(5) Circularly leaching the mycelium with ultrasonic-assisted liquid of 35kHz and 1.5kW at 70deg.C for 1.5 hr, centrifuging to obtain clear liquid, and measuring mycelium polysaccharide yield;

(6) Combining the clear solutions obtained by the centrifugal separation twice, freezing, concentrating, leaching and replacing heavy metal ions by citric acid, precipitating with ethanol, washing with ethanol, and freeze-drying to obtain a qualified grifola frondosa polysaccharide product;

(7) The obtained Maitake Mushroom mycelium polysaccharide and extracellular polysaccharide are measured by phenol sulfuric acid method, and the obtained Maitake Mushroom polysaccharide is the sum of mycelium polysaccharide and extracellular polysaccharide, and is total polysaccharide.

(8) With reference to national standards, the contents of pollutants such as lead, arsenic, mercury, cadmium, chromium, aflatoxin B1, deoxynivalenol, carbendazim, abamectin and butachlor in the grifola frondosa polysaccharide are measured.

In one aspect of the present invention, the new strain of grifola frondosa used in step (3) of the present invention, latin is Grifolafrondosa, preservation number is cctccc NO: M2018907, and is obtained by ultraviolet mutagenesis screening of a grifola frondosa strain isolated from grifola frondosa fruiting bodies picked from a grifola frondosa production site by the inventor, and is determined as a new strain of grifola frondosa by DNA sequencing of a division of biological engineering (Shanghai) and has been preserved at 19 days of 2018 in China center of type culture collection (cctccc) of the university of martial arts, preservation number is cccc NO: M2018907, and the proposed classification name is grifola frondosa strain jsuliung 18Grifola frondosa JSULIUWANG, which is the new strain of grifola frondosa.

In one aspect of the invention, the filling amount in shaking the flask in the step (3) is 40% of the volume of the shaking flask, the inoculation amount is 10% of the volume of the filling, the rotating speed is 150r/min, and the culturing time is 7-10d.

In one aspect of the invention, the sample loading amount in the fermentation in the step (3) is 75% of the volume of the fermentation tank, the ventilation amount in the fermentation tank is the ratio of ventilation volume per minute to the volume of the canning liquid under the ventilation condition of 1:0.8, the culture temperature is 20-25 ℃, the stirring speed is 50-150r/min, and the fermentation is carried out in a tank for 7-10d.

The beneficial effects of the invention are that

The new strain of grifola frondosa is utilized to ferment and produce grifola frondosa polysaccharide in a liquid culture medium of a rice bran and wheat bran complete material which meets the pollutant limit standard through inspection, wherein the rice bran in the rice bran and wheat bran complete material culture medium is 7.0g/100mL, the wheat bran is 3.1g/100mL, the monopotassium phosphate is 0.25g/100mL, the magnesium sulfate is 0.15g/100mL, and the dry weight concentration, the concentration of the crude polysaccharide of the mycelium, the concentration of the extracellular crude polysaccharide and the concentration of the total crude polysaccharide reach 3.151g/100mL, 156.861mg/100mL, 331.876mg/100mL and 488.737mg/100mL respectively. The upper tank can reach the levels of 3.821g/100mL, 183.021mg/100mL, 247.665mg/100mL and 430.686mg/100 mL. Through detection, the limit of mycotoxin aflatoxins B1 and B2 and deoxynivalenol of the total polysaccharide obtained through shake flask fermentation and upper tank fermentation, the limit of heavy metal lead, arsenic, mercury, cadmium and chromium and the residual limit of pesticide carbendazim, abamectin and butachlor are not higher than the limit values or undetected values specified in GB2761-2017, GB2762-2017 and GB 2763-2016.

The method for producing the grifola frondosa polysaccharide by using the novel grifola frondosa strain is realized for the first time, in the method, the novel grifola frondosa strain is fermented in a liquid culture medium of a rice bran and wheat bran complete material which is inspected to meet the limit standard of pollutants, and the fermentation product is subjected to multi-frequency ultrasonic extraction, freeze concentration, citric acid leaching to replace heavy metal ions, alcohol precipitation and alcohol washing to remove impurities, so that the qualified grifola frondosa polysaccharide product is obtained. The new strain of grifola frondosa is a new strain of grifola frondosa obtained by self mutagenesis of the inventor, can effectively grow in a new culture medium of liquid rice bran and wheat bran, and can efficiently convert the rice bran and wheat bran into grifola frondosa polysaccharide, and has uniqueness, so the invention also has uniqueness.

The invention also achieves substantial characteristics and significant progress innovations. The dry weight concentration, the mycelium crude polysaccharide concentration, the extracellular crude polysaccharide concentration and the total crude polysaccharide concentration of the mycelium of the new grifola frondosa strain reach 3.151g/100mL, 156.861mg/100mL, 331.876mg/100mL and 488.737mg/100mL respectively. The total polysaccharide is the final grifola frondosa polysaccharide product, reaches the production level of 488.737mg/100mL, has low content of pollutants in the grifola frondosa polysaccharide, meets the requirements of food raw materials, and shows that the novel grifola frondosa strain has good fermentation performance and high yield of the grifola frondosa polysaccharide in an optimized culture medium.

The invention converts low-value rice bran into high-value grifola frondosa polysaccharide with various health care functions, has beneficial social and economic effects on reducing production cost, realizing high-efficiency, reasonable and high-value utilization of low-end resources and protecting public health, and has practicability.

In view of the foregoing, the present invention has been developed to provide the unique, practical, and novel features necessary to deliver the inventive patent and to provide the technical, economic and social benefits that the inventive patent should possess.

Drawings

FIG. 1 is an external view of a new culture medium of a strain Grifola frondosa CCTCC NO: M2018907 shake flask liquid fermented rice bran and wheat bran.

FIG. 2 shows the mycelium product of the strain Grifola frondosa CCTCC NO: M2018907 in a novel medium for liquid fermentation of rice bran and wheat bran whole material.

FIG. 3 is a representation of a new strain of Grifola frondosa and a evolved tree thereof.

Detailed Description

The invention provides a method for breeding new grifola frondosa strains with higher growth speed and higher polysaccharide yield on a rice bran and wheat bran full-liquid state culture medium by ultraviolet mutagenesis, which comprises the following steps:

taking the original strain Grifola frontosa deposited in the laboratory as the starting strain;

Inoculating the extracted strain on PDA culture medium for activating culture;

after culturing the thalli, eluting the thalli by using sterile physiological saline to prepare spore suspension;

diluting the spore suspension by a required multiple, irradiating under an ultraviolet lamp for mutagenesis, inoculating to rice bran and wheat bran screening culture medium for light-proof culture, and primarily screening strains with a relatively high growth rate and relatively stable growth rate; carrying out genetic stability test on the strain subjected to primary screening, and re-screening the strain with a relatively high growth rate and relatively stable growth rate;

shake flask fermentation with rice bran and wheat bran complete liquid medium, screening strain with high growth rate and high polysaccharide yield;

the new strains screened were subjected to antagonism test and genetic sequencing.

In one embodiment, PDA culture medium is used with 200g/L potato, 20g/L glucose, 5g/L peptone, 1.5g/L potassium dihydrogen phosphate, 0.75g/L magnesium sulfate heptahydrate, 20g/L agar, and natural pH.

In one embodiment, the constant temperature is 23 ℃.

In one embodiment, the UV mutagenesis is performed using a red light dark operation, each irradiated for 40s at a distance of 20cm from a UV lamp with a power of 20W.

In one embodiment, the light protected cultivation is at 23℃for 7 days.

In one embodiment, the medium used for the light-shielding culture is a rice bran, wheat bran solid plate medium: 20g/L of rice bran, 30g/L of wheat bran, 1.5g/L of monopotassium phosphate, 0.75g/L of magnesium sulfate heptahydrate, 20g/L of agar, natural pH, and the rice bran and the wheat bran are used as whole materials, and the rice bran and the wheat bran all have to meet the standard.

In one embodiment, the screening method is a plate diameter assay.

In one embodiment, the preliminary screening step is: single colony with good growth is selected from ultraviolet mutagenesis light-shielding culture plates and inoculated into new rice bran and bran solid plate culture medium respectively, and strain with fast growth, good shape and high stability relative to the original strain is selected.

In one embodiment, the re-screening step is: and (3) respectively carrying out 5-generation plate subculture on the coarsely screened preferred strains, picking single colonies with good growth, respectively inoculating the single colonies into new rice bran and bran solid plate culture media, and selecting mutant strains with high growth speed, robustness and purity.

In one embodiment, the three screening steps are: and (3) taking the high growth rate variant strain determined by re-screening as a target of the triple screening, and carrying out shake flask fermentation screening with the original strain to determine the strain with stable expression of the excellent characters of the variant strain. And (3) carrying out shake flask fermentation test on the newly screened Grifola frondosa strain and the original strain Grifola frondosa, continuously fermenting for 5 generations, and determining the target mutant strain according to indexes.

In one embodiment, the new strain of Grifola frondosa is strain No. 6, and the growth rate on the rescreened fifth-generation plate is 1.738cm/d, which is significantly faster than the growth rate of strain No. 0 of the starting strain.

In one embodiment, the shake flask is a 250mL conical flask.

In one embodiment, the rice bran, wheat bran liquid fermentation medium is: 7.0g/100mL of rice bran, 1.6g/100mL of bran, 0.25g/100mL of monopotassium phosphate, 0.25g/100mL of magnesium sulfate and natural pH.

In one embodiment, water is added to the desired volume and sterilized at 121℃for 30min as a medium for fermentation.

In one embodiment, the shake flask sample size is 40% and the inoculum size is 10%.

In one embodiment, the incubation temperature is 23℃and the rotation speed is 150r/min for a period of 8d.

In one embodiment, the indicators are pure mycelium dry weight, mycelium crude polysaccharide weight, and broth extracellular crude polysaccharide weight for a fermentation volume of 100 mL.

In one embodiment, the crude mycelial polysaccharide and the crude extracellular polysaccharide are products of the polysaccharide prior to separation, as determined by the phenol-sulfuric acid method.

In one embodiment, the antagonism test is used for characterizing the genetic character, the result is shown in figure 2, the antagonism line is obvious, the new strain of the grifola frondosa on the right grows densely and compactly, and the growth performance of the new strain is stronger than that of the original strain, so that the genetic character is changed beneficially.

In one embodiment, the new strain of Grifola frondosa, having a preservation number of CCTCC NO: M2018907, is determined as a new strain of Grifola frondosa by DNA sequencing of the biological engineering (Shanghai) stock Co., ltd. The new strain of grifola frondosa has been preserved in China Center for Type Culture Collection (CCTCC) of university of Wuhan in China at 12 months 19 in 2018, the preservation number is CCTCC NO: M2018907, the proposed classification is named as a strain of grifola frondosa JSULIUWANG18 Grifola frondosa JSULIUWANG, and the new strains of grifola frondosa refer to the strain (see figure 3).

The invention provides a method for producing grifola frondosa polysaccharide by using a new grifola frondosa strain obtained by mutagenesis, wherein the new grifola frondosa strain is fermented in a liquid culture medium of a rice bran and wheat bran complete material which is tested to meet the limit standard of pollutants, and after the fermentation product of grifola frondosa mycelium raw material and fermentation liquor is produced, the qualified grifola frondosa polysaccharide product is obtained through multifrequency ultrasonic auxiliary extraction, freeze concentration, citric acid leaching displacement of heavy metal ions, alcohol precipitation and alcohol washing and freeze drying, and the method comprises the following steps:

in one embodiment, the pollution level of the main heavy metals of lead, arsenic, mercury, cadmium and chromium is not more than 3mg/kg at the highest, the balance respectively accords with the bran standard NY/T3218-2018 and the bran standard NY/T122-1989 for the edible feed of the original agriculture department, the pesticide applied in the wheat planting process accords with the farm technical specification, the wheat is not seriously scab harmful in the wheat growing process, the wheat is not deteriorated due to discontinuous rainwater in harvesting seasons, the wheat is not damaged due to the wheat storage and transportation in the mildew rice planting process, the rice is not seriously damaged in the rice growing process, the rice is deteriorated due to discontinuous rainwater in harvesting seasons, and the rice is not mildewed due to the rice storage and transportation after the rice processing;

In one embodiment, the new strain of Grifola frondosa, CCTCC NO: M2018907, is determined by DNA sequencing of the division of biological engineering (Shanghai) as a new strain of Grifola frondosa, used as a fermentation strain;

in one embodiment, the rice bran, wheat bran liquid fermentation medium is: the concentration of the rice bran is 0.1-10g/100mL, the concentration of the wheat bran is 0.1-10g/100mL, the sum of the concentration of the rice bran and the concentration of the wheat bran is 10.1g/100mL, the potassium dihydrogen phosphate is added to 0.015-0.25g/100mL, the pH is natural, the shaking bottle material is 100mL, the material loading rate of a fermentation tank is 75%, the inoculation amount is 10%, the culture temperature is 20-25 ℃, the rotation speed of a shaking table is 150r/min or the rotation speed of a fermentation stirring slurry is 50-150r/min, and the fermentation time is 7-10d;

in one embodiment, the mycelium of the grifola frondosa obtained by liquid culture is subjected to centrifugal separation to obtain mycelium and separation liquid respectively, and the separation liquid is used for measuring extracellular crude polysaccharide by a phenol sulfuric acid method;

in one embodiment, the mycelium of Grifola frondosa is subjected to a polysaccharide extraction operation, circularly leached with hot water at 60 ℃ plus ultrasonic-assisted liquid of 35kHz and 1.5kW for 1.5h, centrifugally separated to obtain a supernatant, and the mycelium polysaccharide yield is determined with phenol sulfuric acid;

in one embodiment, the obtained fermentation liquor and mycelium extracting solution are combined, and qualified extracellular polysaccharide is obtained by the methods of freeze concentration, substitution of heavy metal ions by citric acid leaching, alcohol precipitation and alcohol washing and freeze drying;

In one embodiment, the new strain of Grifola frondosa obtained by the screening utilizes a fermentation medium to obtain fermentation results of: the dry weight concentration of mycelium, the concentration of mycelium crude polysaccharide, the concentration of extracellular crude polysaccharide and the concentration of total crude polysaccharide reach 3.151g/100mL, 156.861mg/100mL, 331.876mg/100mL and 488.737mg/100mL respectively.

In one embodiment, the yield of the grifola frondosa polysaccharide reaches 488.737mg/100mL, which indicates that the novel grifola frondosa strain has good fermentation performance and high yield of grifola frondosa polysaccharide in an optimized culture medium, and the invention has substantial characteristics and innovations with remarkable progress.

In one embodiment, the limit of mycotoxin aflatoxin B1, B2 and deoxynivalenol of the total polysaccharide, the limit of heavy metal lead, arsenic, mercury, cadmium and chromium and the residual limit of pesticide carbendazim, abamectin and butachlor are not higher than the limit values or are not detected in GB2761-2017, GB2762-2017 and GB2763-2016 after detection.

Example 1

The new grifola frondosa strain obtained by the invention is adopted by the grifola frondosa strain. Rice bran and wheat bran materials meeting the use standards are used. The rice bran is used as a culture medium with the dosage of 7.0g/100mL, the wheat bran is used as a culture medium with the dosage of 3.1g/100mL, the monopotassium phosphate is added with 0.25g/100mL, the magnesium sulfate heptahydrate is added with 0.15g/100mL, the pH is natural, water is added to the required volume, the culture medium is sterilized for 30min at the temperature of 121 ℃, the sample amount of a shaking bottle is 40%, the inoculation amount is 10%, the culture temperature is 23 ℃, the rotating speed is 150r/min, and the culture time is 7d. The steps (4) to (8) according to the above-described embodiments are performed as follows. As a result, the dry weight concentration of the mycelium, the concentration of the mycelium crude polysaccharide, the concentration of the extracellular crude polysaccharide and the concentration of the total crude polysaccharide respectively reach 3.151g/100mL, 156.861mg/100mL, 331.876mg/100mL and 488.737mg/100mL, the limit of mycotoxin aflatoxins B1 and B2 and deoxynivalenol of the total polysaccharide, the limit of heavy metal lead, arsenic, mercury, cadmium and chromium and the residual limit of pesticide carbendazim, abamectin and butachlor are not higher than the limit values specified in GB2761-2017, GB2762-2017 and GB2763-2016 or are not detected after measurement.

Example 2

The new grifola frondosa strain obtained by the invention is adopted by the grifola frondosa strain. Rice bran and wheat bran materials meeting the use standards are used. The rice bran is used in an amount of 5.0g/100mL of culture medium, the wheat bran is used in an amount of 5.1g/100mL of culture medium, and KH is added ₂ PO ₄ 0.25g/100mL，MgSO ₄ ·7H ₂ O0.15 g/100mL, pH is natural, water is added to the required volume, sterilization is carried out for 30min at 121 ℃, the culture medium is used for fermentation, the sample amount of shaking bottle is 40%, the inoculation amount is 10%, the culture temperature is 23 ℃, the rotating speed is 150r/min, and the culture time is 8d. The following steps (4) to (8) are performed in the embodiments described in the foregoing summary. As a result, the dry weight concentration of the mycelium, the concentration of the mycelium crude polysaccharide, the concentration of the extracellular crude polysaccharide and the concentration of the total crude polysaccharide respectively reach 2.789g/100mL, 160.045mg/100mL, 364.586mg/100mL and 524.631mg/100mL, the limit of mycotoxin aflatoxins B1 and B2 of the total polysaccharide and deoxynivalenol, the limit of heavy metal lead, arsenic, mercury, cadmium and chromium and the residual limit of pesticide carbendazim, abamectin and butachlor are not higher than the limit values or undetected specified in GB2761-2017, GB2762-2017 and GB 2763-2016.

Example 3

The new grifola frondosa strain obtained by the invention is adopted by the grifola frondosa strain. Rice bran and wheat bran materials meeting the use standards are used. The rice bran is used in an amount of 10.0g/100mL of culture medium, the wheat bran is used in an amount of 0.1g/100mL of culture medium, and KH is added ₂ PO ₄ 0.015g/100mL，MgSO ₄ ·7H ₂ O0.015 g/100mL, pH is natural, water is added to the required volume, sterilization is carried out for 30min at 121 ℃, the culture medium is used for fermentation, the sample amount of shaking bottle is 40%, the inoculation amount is 10%, the culture temperature is 25 ℃, the rotating speed is 150r/min, and the culture time is 10d. Described below in the foregoing summary of the inventionSteps (4) to (8) in the technical scheme are implemented. As a result, the dry weight concentration of the mycelium, the concentration of the mycelium crude polysaccharide, the concentration of the extracellular crude polysaccharide and the concentration of the total crude polysaccharide respectively reach 3.206g/100mL, 124.712mg/100mL, 163.722mg/100mL and 288.434mg/100mL, the limit of mycotoxin aflatoxins B1 and B2 and deoxynivalenol of the total polysaccharide, the limit of heavy metal lead, arsenic, mercury, cadmium and chromium and the residual limit of pesticide carbendazim, abamectin and butachlor are not higher than the limit values or undetected specified in GB2761-2017, GB2762-2017 and GB 2763-2016.

Example 4

The new grifola frondosa strain obtained by the invention is adopted by the grifola frondosa strain. Rice bran and wheat bran materials meeting the use standards are used. 3.1g/100mL culture medium is used for rice bran, 7.0g/100mL culture medium is used for wheat bran, KH is added ₂ PO ₄ 0.15g/100mL，MgSO ₄ ·7H ₂ O0.075 g/100mL, pH is natural, water is added to the required volume, sterilization is carried out for 30min at 121 ℃, the culture medium is used for fermentation, the sample amount of shaking bottle is 40%, the inoculation amount is 10%, the culture temperature is 20 ℃, the rotating speed is 150r/min, and the culture time is 7d. The following steps (4) to (8) are performed in the embodiments described in the foregoing summary. As a result, the dry weight concentration of the mycelium, the concentration of the mycelium crude polysaccharide, the concentration of the extracellular crude polysaccharide and the concentration of the total crude polysaccharide respectively reach 2.117g/100mL, 145.143mg/100mL, 397.297mg/100mL and 542.44mg/100mL, the limit of mycotoxin aflatoxins B1 and B2 of the total polysaccharide and deoxynivalenol, the limit of heavy metal lead, arsenic, mercury, cadmium and chromium and the residual limit of pesticide carbendazim, abamectin and butachlor are not higher than the limit values or undetected specified in GB2761-2017, GB2762-2017 and GB 2763-2016.

Example 5

The new grifola frondosa strain obtained by the invention is adopted by the grifola frondosa strain. Rice bran and wheat bran materials meeting the use standards are used. The rice bran is used in an amount of 0.1g/100mL of culture medium, the wheat bran is used in an amount of 10.0g/100mL of culture medium, and KH is added ₂ PO ₄ 0.25g/100mL，MgSO ₄ ·7H ₂ O0.25 g/100mL, natural pH, adding water to the required volume, sterilizing at 121deg.C for 30min, the sample amount in shake flask is 40%, the inoculation amount is 10%, the culture temperature is 23 ℃, the rotating speed is 150r/min, and the culture time is 9d. The following steps (4) to (8) are performed in the embodiments described in the foregoing summary. As a result, the dry weight concentration of the mycelium, the concentration of the mycelium crude polysaccharide, the concentration of the extracellular crude polysaccharide and the concentration of the total crude polysaccharide respectively reach 1.481g/100mL, 106.819mg/100mL, 344.594mg/100mL and 451.413mg/100mL, the limit of mycotoxin aflatoxins B1 and B2 and deoxynivalenol of the total polysaccharide, the limit of heavy metal lead, arsenic, mercury, cadmium and chromium and the residual limit of pesticide carbendazim, abamectin and butachlor are not higher than the limit values or undetected specified in GB2761-2017, GB2762-2017 and GB 2763-2016.

Example 6

The new grifola frondosa strain obtained by the invention is adopted by the grifola frondosa strain. Rice bran and wheat bran materials meeting the use standards are used. The rice bran is used in an amount of 10.0g/100mL of culture medium, the wheat bran is used in an amount of 0.1g/100mL of culture medium, and KH is added ₂ PO ₄ 0.075g/100mL，MgSO ₄ ·7H ₂ O0.075 g/100mL, pH is natural, water is added to the required volume, sterilization is carried out for 30min at 121 ℃, the culture medium is used for fermentation, the sample amount of shaking bottle is 40%, the inoculation amount is 10%, the culture temperature is 23 ℃, the rotating speed is 150r/min, and the culture time is 8d. The following steps (4) to (8) are performed in the embodiments described in the foregoing summary. As a result, the dry weight concentration of the mycelium, the concentration of the mycelium crude polysaccharide, the concentration of the extracellular crude polysaccharide and the concentration of the total crude polysaccharide respectively reach 3.386g/100mL, 181.027mg/100mL, 282.810mg/100mL and 463.837mg/100mL, the limit of mycotoxin aflatoxins B1 and B2 of the total polysaccharide and deoxynivalenol, the limit of heavy metal lead, arsenic, mercury, cadmium and chromium and the residual limit of pesticide carbendazim, abamectin and butachlor are not higher than the limit values or undetected specified in GB2761-2017, GB2762-2017 and GB 2763-2016.

Example 7

The new grifola frondosa strain obtained by the invention is adopted by the grifola frondosa strain. Rice bran and wheat bran materials meeting the use standards are used. The rice bran is used in an amount of 0.1g/100mL culture medium, and the use of the branAdding KH into culture medium with the volume of 10.0g/100mL ₂ PO ₄ 0.15g/100mL，MgSO ₄ ·7H ₂ O0.075 g/100mL, pH is natural, water is added to the required volume, sterilization is carried out for 30min at 121 ℃, the culture medium is used for fermentation, the sample amount of shaking bottle is 40%, the inoculation amount is 10%, the culture temperature is 23 ℃, the rotating speed is 150r/min, and the culture time is 9d. The following steps (4) to (8) are performed in the embodiments described in the foregoing summary. As a result, the dry weight concentration of the mycelium, the concentration of the mycelium crude polysaccharide, the concentration of the extracellular crude polysaccharide and the concentration of the total crude polysaccharide respectively reach to 0.912g/100mL, 84.413mg/100mL, 358.737mg/100mL and 443.151mg/100mL, the limit of mycotoxin aflatoxins B1 and B2 of the total polysaccharide and deoxynivalenol, the limit of heavy metal lead, arsenic, mercury, cadmium and chromium and the residual limit of pesticide carbendazim, abamectin and butachlor are not higher than the limit values or undetected specified in GB2761-2017, GB2762-2017 and GB 2763-2016.

Example 8

New strain of Grifola frondosa is adopted. The use standard of the invention is met by checking that the bran and the rice bran are both in line with. The sample amount in the fermentation tank is 75% of the volume of the fermentation tank, the fermentation temperature is 20 ℃, the ventilation rate is the ratio of ventilation volume per minute to the volume of liquid in the tank under the ventilation condition is 1:0.8, stirring speed 50r/min, tank gauge pressure 0.05MPa, inoculum size 10%, culture time 7d, fermentation medium of 10.0g/100mL of rice bran, 0.1g/100mL of wheat bran, KH ₂ PO ₄ 0.015g/100mL，MgSO ₄ ·7H ₂ O0.015 g/100mL, pH is natural. The following steps (4) to (8) are performed in the embodiments described in the foregoing summary. The results were: the dry weight of mycelium is 3.424g/100mL, the mycelium polysaccharide is 176.315mg/100mL, the extracellular polysaccharide is 330.521mg/100mL of culture medium, the grifola frondosa polysaccharide is 506.836mg/100mL, the limit of mycotoxin aflatoxins B1 and B2 and deoxynivalenol of the total polysaccharide, the limit of heavy metal lead, arsenic, mercury, cadmium and chromium, and the residual limit of pesticide carbendazim, abamectin and butachlor are not higher than the limit values or undetected by GB2761-2017, GB2762-2017 and GB 2763-2016.

Example 9

New strain of Grifola frondosa is adopted. Testing branAnd rice bran are in accordance with the use criteria of the present invention. The sample amount in the fermentation tank is 75% of the volume of the fermentation tank, the fermentation temperature is 25 ℃, the ventilation rate is the ratio of ventilation volume per minute to the volume of liquid in the tank under the ventilation condition is 1:0.8, stirring speed 150r/min, tank gauge pressure 0.05MPa, inoculum size 10%, culture time 10d, fermentation medium of 0.1g/100mL rice bran, 10.0g/100mL wheat bran, KH ₂ PO ₄ 0.25g/100mL，MgSO ₄ ·7H ₂ O0.25 g/100mL, pH was natural. The following steps (4) to (8) are performed in the embodiments described in the foregoing summary. The results were: the dry weight of mycelium is 1.819g/100mL, the mycelium polysaccharide is 112.396mg/100mL, the extracellular polysaccharide is 258.248mg/100mL, the grifola frondosa polysaccharide is 370.644mg/100mL, the limit of mycotoxin aflatoxins B1 and B2 and deoxynivalenol of the total polysaccharide, the limit of heavy metals lead, arsenic, mercury, cadmium and chromium and the residual limit of pesticide carbendazim, avermectin and butachlor are not higher than the limit values or are not detected in the specifications of GB2761-2017, GB2762-2017 and GB 2763-2016.

Claims (1)

1. The method for producing the grifola frondosa polysaccharide by using the new grifola frondosa strain generated by mutagenesis is characterized by comprising the following steps:

(1) The bran and the rice bran were examined and,

(2) Sterilizing testa oryzae and testa Tritici with water 121 deg.C for 30min;

(3) The rice bran and the wheat bran complete material are used as carbon source and nitrogen source to be directly used as fermentation medium of new strain of grifola frondosa with preservation number CCTCC NO: M2018907, the rice bran usage amount is 10.0g/100mL of medium, the wheat bran usage amount is 0.1g/100mL of medium, KH is added ₂ PO ₄ 0.075g/100mL，MgSO ₄ ·7H ₂ O0.075 g/100mL, pH is natural, water is added to the required volume, sterilization is carried out for 30min at 121 ℃, the culture medium is used for fermentation, the sample amount of a shaking bottle is 40%, the inoculation amount is 10%, the culture temperature is 23 ℃, the rotating speed is 150r/min, and the culture time is 8d;

(4) Centrifugally separating the liquid culture to obtain mycelium and fermentation liquor, and measuring the yield of extracellular polysaccharide in the fermentation liquor;

(5) Circularly leaching the mycelium with 60 ℃ hot water, 35kHz and 1.5kW ultrasonic auxiliary liquid for 1.5 hours, centrifugally separating to obtain clear liquid, and measuring the yield of mycelium polysaccharide;

(6) And combining the clear solutions obtained by the centrifugal separation twice, freezing and concentrating, leaching and replacing heavy metal ions by citric acid, precipitating with ethanol, washing with ethanol, and freeze-drying to obtain a qualified grifola frondosa polysaccharide product.

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