CN110283861B - Method for producing ganoderan by using ganoderma lucidum strains generated by mutagenesis - Google Patents

Abstract

The invention discloses a method for producing ganoderan by utilizing ganoderma lucidum strains generated by mutagenesis, and relates to the technical field of food microorganism application. In the method, a new strain of ganoderma lucidum is fermented in a liquid culture medium of rice bran and wheat bran complete materials which are inspected to meet the limit standard of pollutants, and after fermentation products of ganoderma lucidum mycelium raw materials and fermentation liquor are produced, qualified ganoderma lucidum polysaccharide products are obtained through multifrequency ultrasonic auxiliary extraction, freeze concentration, citric acid leaching to replace heavy metal ions, alcohol precipitation and alcohol washing and freeze drying. The invention adopts a new method to deeply convert low-value rice bran into the ganoderma lucidum polysaccharide which can be eaten by the ganoderma lucidum liquid state fermentation, has beneficial social and economic effects on reducing the production cost, efficiently and reasonably utilizing low-end resources, protecting the health of the public and the like, and has uniqueness, innovation and practicability.

Description

Method for producing ganoderan by using ganoderma lucidum strains generated by mutagenesis

Technical Field

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

Background

The fungi used as both medicine and food have long use history and wide use population in China. Modern science reveals that the mycelium, fruiting body or spore of the fungus such as Ganoderma, hericium erinaceus, cordyceps, maitake Mushroom, etc. can produce various active ingredients such as amino acids, proteins, vitamins, polysaccharides, nucleosides, flavonoids, etc. or enrich various microelements such as selenium, zinc, etc. with various effects of enhancing immunity, resisting tumor, enhancing liver function, resisting oxidation, etc. As such, there are many researchers and developers in the fields of medicine, food, health care, agriculture and industrial production to obtain a lot of research results or products. As for the rare edible fungi used for food, edible fungi health food such as ganoderma lucidum capsules, grifola frondosa capsules, ganoderma lucidum capsules and the like are popular in domestic markets, and the economic value of the edible fungi health food is very high. Hericium erinaceus is added into the biscuits to prepare Hericium erinaceus biscuits which are already common food sold in domestic supermarkets. Similar edible fungus health food products are produced internationally in New Zealand, japan, USA, korea, etc.

In the case of ganoderma lucidum, studies have shown that: ganoderma lucidum has effects such as liver protection, diabetes treatment, cardiovascular treatment, anti-tumor, and immunity enhancement (ref (1) Li Yingbo. Study of efficient isolation of anti-tumor ganoderic acid monomers from Ganoderma lucidum mycelia [ D ] Shanghai: university of Huadong, 2013 (2)Yuen J W M,Mak D S Y,Chan E S,et al.Tumor inhibitory effects of intravesical Ganoderma lucidum instillation in the syngeneic orthotopic MB49/C57bladder cancer mice model [ J ]. Journal of Ethnopharmacology,2018: S037887411811991 ] (3)Yongjun Kan,Tiqiang Chen,Yanbin Wu,Jianguo wu,Jinzhong Wu,et al.Antioxidant activity of polysaccharide extracted from Ganoderma lucidum using response surface methodology[J ]. International Journal of Biological Macromolecules (2015) 151-157. (4) Teng Baosong. Screening of Ganoderma lucidum effective hypoglycemic components, hypoglycemic mechanism study [ D ] Shanghai: double denier university, 2011 (5)Tang W,Liu J W,Zhao W M,et al.Ganodermic acid T from Ganoderma lucidum mycelia induces mitochondria mediated apoptosis in lung cancer cells[J). Life Sciences,2006,80 (3): 0-211.).

Ganoderma lucidum (Ganoderma lucidum) belongs to basidiomycetes, agaricus, polyporales, ganoderma lucidum, and Ganoderma lucidum, is a large fungus for both medicine and food, and is mainly grown beside wood piles of broad-leaved trees in forests, or on wood, standing trees and inverted trees, and sometimes on conifer trees, and is also grown in the shape of a pileus wood bolt, kidney or semicircle, reddish brown, red purple or dark purple, with lacquer-like luster, annular ribs and radial wrinkles, and has large size and shape variation, wherein the pileus of large individuals is 20×10cm, the thickness is about 2cm, the general individuals are 4×3cm, the thickness is 0.5-1 cm, countless small holes are arranged below, the pipe orifice is white or light brown, 4-5 pipe orifices are arranged in each millimeter, the inner wall is a fruiting layer, and the pipe orifice is produced at the top end of the spore basidiophore. The stipe grows laterally, rarely deviates from growth, grows in diameter of the pileus, is purple brown to black, has lacquer-like luster and is hard. Spore is oval, 8-11 x 7cm, two layers of walls, brown inner wall, small wart on surface and transparent and colorless outer wall. The ganoderma lucidum is distributed in Europe, america, africa, asia eastern region and the like, and the ganoderma lucidum in China is mainly distributed in Zhejiang Longquan, heilongjiang, jilin, hebei, shandong, anhui mountain, jiangsu, jiangxi, hunan, guizhou, fujian, guangdong, guangxi and other regions and has partial yield. The growth rate of ganoderma lucidum in the wild state is very slow and the price is high. Therefore, the wild ganoderma lucidum is very rare in resources, and the ganoderma lucidum is obtained mainly by artificial cultivation at present, but the artificial cultivation period is long, the production efficiency is low, the labor intensity is high, the ganoderma lucidum is limited by seasons, environment and the like, and is easy to suffer from diseases and insect pests, and the quality and the yield are unstable. 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 ganoderma lucidum fermented product and adopts deep processing application, thereby conforming to the development thought of innovation driving and having good prospect. The progress of research and development of the lucid ganoderma fermentation product can further promote the application of lucid ganoderma, and brings practical social and economic benefits. Because of this, it is necessary to study the production methods of new ganoderma lucidum fermented products and deep processed products thereof, for example, considering that some low-end agricultural product processing byproduct raw materials such as bran and rice bran are efficiently converted into high-value ganoderma lucidum fermented product raw materials, the production cost of ganoderma lucidum fermented products is expected to be reduced, and the price of final products such as ganoderma lucidum health-care foods or medicines is reduced, thereby being beneficial to the ordinary people and promoting the public health, which is one of the concerns of the patent of the invention.

China is a large country for producing rice and wheat, and rice bran and wheat bran resources are rich. 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, the wheat bran (the wheat bran refers to the whole 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 ganoderma lucidum. Under the action of glossy ganoderma cellulase and other enzyme system, glossy ganoderma converts rice bran and wheat bran into required nutritive matters for growth metabolism to synthesize glossy ganoderma mycelium and adenosine functional matters. Therefore, the rice bran and the wheat bran which are agricultural and sideline products are used as all carbon sources and nitrogen sources, and the ganoderma lucidum liquid fermentation is carried out to grow ganoderma lucidum health-care food raw materials, so that the method has technical feasibility and can bring better social and economic benefits.

Most of the liquid fermentation production methods of edible fungus powder adopt glucose, grain raw materials such as starch, potato or soybean powder and the like as culture mediums, and even agricultural and sideline products such as rice bran, wheat bran and the like are used as auxiliary components. The growth condition of the original strains of edible fungi, such as cordyceps sinensis, ganoderma lucidum, grifola frondosa and the like on a rice bran and bran total liquid medium without adding glucose or other grain raw materials is not optimal, and in addition, harmful substances such as lead, arsenic, aflatoxin and the like possibly contained in the rice bran and bran raw materials can be transferred into mycelium, so that innovative research on a liquid total material method of the fermented rice bran and bran of the strain is needed for preventing the transfer.

The research group of the patent inventor Liu Weimin has carried out a great deal of research on the liquid fermentation of grifola frondosa, ganoderma lucidum and cordyceps sinensis 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 Ganoderma strain (Ganoderma lucidum CFCC 6043) liquid fermented whole testa oryzae testa Tritici and polysaccharide activity (2014), etc. Liu Weimin the invention patents of the first inventor 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) A strain 201310274915.7 for producing ganoderan by full-liquid fermentation of rice bran and wheat bran, (9) a production method of 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.

Inventor Liu Weimin combes the guided papers of the Shuoshi research and the patents related to the invention, combines the problems which can not be answered when the related achievements of the enterprise negotiations are transferred, such as providing key support materials of gene maps without strain qualification, and the like, and the enterprises do not wish to develop industrialization, so that if the industrial road is to be reached, many problems still exist in the related patent technologies and research papers, and the problems 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 ganoderma lucidum fermentation product, utilizes original ganoderma lucidum strains which are different from the original ganoderma lucidum strains in source and have different fermentation performances to obtain new strains by mutagenesis, and ferments rice bran and wheat bran to produce qualified ganoderma lucidum polysaccharide products.

In summary, the invention relates to the field of using new strains of ganoderma lucidum to ferment bran and rice bran to produce ganoderma lucidum ferment, 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 original strains is improved by a mutagenesis method, a new strain of ganoderma lucidum which is not reported is obtained, and the new strain can be subjected to gene sequencing to ensure that the used strains are correct; 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 to obtain a new strain of ganoderma lucidum which is not reported, and the new strain can be subjected to gene sequencing to ensure that the used strains are correct. The background of the problem is that the inventor group expects to obtain a ganoderma lucidum strain which is never used and has good performance, and expects that the ganoderma lucidum strain can perform well in the biological conversion of bran and rice bran, so that the research on the method for producing ganoderma lucidum fermentation products by fermenting the bran and rice bran from ganoderma lucidum is considered. 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 ganoderma lucidum strain in this production method and improve the fermentation performance by strain mutagenesis method. 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 to be important in the prior art, and problems have been encountered in the re-determination of gene sequences in conventional strains. Therefore, the patent stares at the measurement of the ganoderma lucidum 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, besides the pollutant complexing agent or the additive which is helpful for degrading the pollutants, the separation and purification methods such as citric acid complexation, alcohol precipitation and alcohol washing of fermentation products after fermentation can be considered in the culture medium, so that the qualified ganoderma lucidum 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 invention needs to obtain a new ganoderma lucidum strain by a mutagenesis method. 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 new ganoderma lucidum strain of the full-liquid 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 ganoderan by fermenting the new ganoderma lucidum strain in a liquid culture medium of rice bran and wheat bran complete materials which are inspected to meet the limit standard of pollutants, which comprises the specific composition of the culture medium, fermentation conditions, extraction and impurity removal methods, hypha and polysaccharide yield and measurement values of polysaccharide quality indexes. The limit of mycotoxin aflatoxin B1, B2 and vomitoxin of the obtained ganoderma lucidum 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 the measurement.

Disclosure of Invention

The invention relates to a method for producing ganoderan by using a new ganoderma lucidum strain obtained by mutagenesis, wherein the new ganoderma lucidum strain is fermented in a liquid culture medium of rice bran and wheat bran complete materials which are inspected to meet the limit standard of pollutants, and qualified ganoderan products are obtained after multi-frequency ultrasonic auxiliary extraction, freeze concentration, citric acid leaching to replace heavy metal ions, alcohol precipitation and alcohol washing and freeze drying are carried out after fermentation of ganoderma lucidum mycelium raw materials and fermentation broth are produced.

The technical scheme adopted by the invention is as follows: fermenting in liquid culture medium of rice bran and wheat bran complete material meeting pollutant limit standard to produce fermented product of glossy ganoderma mycelium material and fermented liquid, multifrequency ultrasonic auxiliary extraction, freeze concentration, citric acid leaching to replace heavy metal ion, alcohol precipitation and alcohol washing, and freeze drying to obtain glossy ganoderma polysaccharide product.

In one aspect of the invention, the method for producing ganoderan by using the novel strain of ganoderma lucidum 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 whole bran are used as carbon source and nitrogen source to be directly used for a fermentation culture medium of ganoderma lucidum strain Ganoderma lucidum with preservation number CCTCC NO: M2018908, the concentration of the rice bran is 0.1-10g/100mL, the concentration of the bran is 0.1-10g/100mL, the total concentration of the rice bran and the bran is 10.1g/100mL, potassium dihydrogen phosphate is added to 0.015-0.25g/100mL, magnesium sulfate heptahydrate is 0.015-0.25g/100mL, pH is natural, 250mL shaking bottle material is 100mL, the material loading rate of a fermentation tank is 75%, the inoculum size is 10%, the culture temperature is 24-28 ℃, 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 5-12d;

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

(4) 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;

(5) 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 ganoderan product;

(6) The obtained Ganoderma mycelia polysaccharide and extracellular polysaccharide are measured by phenol sulfuric acid method, and the obtained Ganoderma polysaccharide is the sum of mycelia polysaccharide and extracellular polysaccharide, and is total polysaccharide.

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

In one aspect of the present invention, the new strain of ganoderma lucidum used in step (3) of the present invention is obtained by the inventor through ultraviolet mutagenesis screening of laboratory preserved ganoderma lucidum strains, and is determined to be a new strain of ganoderma lucidum by DNA sequencing of the division of biological engineering (Shanghai) and has been preserved in China Center for Type Culture Collection (CCTCC) at university of martial arts, chinese, at 12 months 19 in 2018, with a strain preservation number of cctccc NO: M2018908, and the proposed classification is named ganoderma lucidum JSU LIUYU18: ganoderma lucidum JSU LIUYU18, the new strains of Ganoderma lucidum described later in this specification are all referred to as this strain.

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 5-12d.

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, culturing at 24-28deg.C, stirring at 50-150r/min, and fermenting in a tank for 5-12d.

The beneficial effects of the invention are that

Fermenting the new strain of Ganoderma lucidum in liquid culture medium of testa oryzae and testa Tritici complete material meeting pollutant limit standard, when total concentration of testa oryzae and testa Tritici in the testa oryzae and testa Tritici complete material culture medium reaches 10.1g/100mL, KH ₂ PO ₄ 0.25g/100mL MgSO ₄ ·7H ₂ When O is 0.25g/100mL, the concentration of mycelium dry weight, mycelium polysaccharide and extracellular polysaccharide respectively reach 5.722g/100mL, 669.861mg/100mL and 1402.065mg/100mL during shake flask fermentation, and the total polysaccharide is 2071.926mg/100mL; the upper tank can reach 5.384g/100mL, 602.3mg/100mL, 1327.8mg/100mL and 1930.1mg/100 mL. Through detection, mycotoxins aflatoxins B1 and B2 and deoxynivalenol of total polysaccharide obtained through shake flask fermentation and upper tank fermentation are limited, heavy metal lead, arsenic, mercury, cadmium and chromium are limited, and pesticide carbendazim is preparedThe residual limit of the avermectin and the butachlor is not higher than the limit value or is not detected in GB2761-2017, GB2762-2017 and GB 2763-2016.

The invention realizes the method for producing ganoderan by the new ganoderma lucidum strain for the first time, in the method, the new ganoderma lucidum strain is fermented in a liquid culture medium of rice bran and wheat bran complete materials which are 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 ganoderan product is obtained. The new strain of ganoderma lucidum is a new strain of ganoderma lucidum 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 ganoderma lucidum polysaccharide, and the strain has uniqueness, so the invention also has uniqueness.

The invention also achieves substantial characteristics and significant progress innovations. When the total concentration of rice bran and wheat bran in the whole-liquid culture medium of the rice bran and wheat bran of the new strain of ganoderma lucidum reaches 10.1g/100mL, the dry weight concentration of mycelium, the concentration of mycelium polysaccharide and the concentration of crude polysaccharide in fermentation broth reach 5.722g/100mL, 669.861mg/100mL and 1402.065mg/100mL respectively, and the total polysaccharide is 2071.926mg/100mL. The total polysaccharide is the final ganoderan product, reaches the production level of 2071.926mg/100mL, shows that the new ganoderan strain has good fermentation performance and high ganoderan yield in the optimized culture medium, and has substantial characteristics and significant progress innovativeness.

The invention converts low-value rice bran and wheat bran into high-value ganoderan 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 shake flask liquid state fermentation rice bran and wheat bran complete medium with a new strain of Ganoderma lucidum with a preservation number of CCTCC NO: M2018908.

FIG. 2 is a diagram of mycelium product obtained by fermenting rice bran and wheat bran complete medium in liquid state with the preservation number of new strain of Ganoderma lucidum CCTCC NO: M2018908.

FIG. 3 is a tree of the new strain of Ganoderma lucidum.

Detailed Description

The invention provides a method for producing ganoderan by using a new ganoderma lucidum strain obtained by mutagenesis, in the method, the new ganoderma lucidum strain is fermented in a liquid culture medium of rice bran and wheat bran complete materials which are inspected to meet the limit standard of pollutants, and after the fermented product of ganoderma lucidum mycelium raw materials and fermentation liquor is produced, qualified ganoderan products are obtained through multifrequency ultrasonic auxiliary extraction, freeze concentration, citric acid leaching to replace 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;

the invention provides a method for breeding a new ganoderma lucidum strain with higher growth speed and higher polysaccharide yield on a bran and rice bran full-liquid state culture medium through ultraviolet mutagenesis, which comprises the following steps:

taking the original strain Ganoderma lucidum JSU LIU17 preserved in the laboratory as a 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 spore suspension by a required multiple, irradiating under ultraviolet lamp for mutagenesis, and inoculating onto testa oryzae and testa Tritici screening culture medium

Culturing in dark, and primarily screening strains with a relatively high growth rate and relatively stable growth rate; genetic stability testing of the initially screened Strain

Checking, namely re-screening strains with a relatively high growth rate and relatively stable growth rate;

shake flask fermentation with rice bran and wheat bran complete liquid medium, and screening strains 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 28 ℃.

In one embodiment, the UV mutagenesis is performed using a red light dark operation, each irradiated for 30s at a distance of 20cm from a UV lamp with a power of 30W.

In one embodiment, the light protected cultivation is at 28℃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 whole materials of the wheat bran and the rice bran are used, and the rice bran of the wheat bran must meet the use 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 respectively inoculated into a new rice bran and wheat bran solid plate culture medium, and strain with fast growth, good shape and high stability relative to the original strain is selected from the single colony.

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 a new rice bran and wheat bran solid plate culture medium, and selecting variant 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 ganoderma lucidum new strain and the original strain, continuously fermenting for 5 generations, and determining a target mutant strain according to indexes.

In one embodiment, the new strain Ganoderma lucidum of Ganoderma lucidum is strain No. 2, and the growth rate on the fifth generation plate of the re-screening is 1.18cm/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: 2.0g/100mL of rice bran, 3.0g/100mL of wheat bran, 0.15g/100mL of monopotassium phosphate, 0.075g/100mL of magnesium sulfate and natural pH.

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

In one embodiment, the antagonistic test is used for characterizing the genetic trait, the result is shown in figure 2, the antagonistic line is obvious, the right ganoderma lucidum mutant new strain grows densely and compactly, the growth performance of the ganoderma lucidum mutant new strain is stronger than that of the original strain, and the genetic trait is changed beneficially.

In one embodiment, the new strain of ganoderma lucidum has a preservation number CCTCC NO: M2018908, and is determined as a new strain of ganoderma lucidum by DNA sequencing of a division of biological engineering (Shanghai) stock.

The new strain of ganoderma lucidum has been preserved in China Center for Type Culture Collection (CCTCC) at university of Wuhan in China, the preservation number is CCTCC NO: M2018908, the proposed classification is named ganoderma lucidum strain JSULIUYU 18Ganoderma lucidum JSU LIUYU, and the new strain of ganoderma lucidum refers to the strain (see figure 3).

In one embodiment, the new strain of ganoderma lucidum has a preservation number CCTCC NO of M2018908, and is determined to be a new strain of ganoderma lucidum by DNA sequencing of a division of biological engineering (Shanghai) and is 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 bran is 0.1-10g/100mL, the total concentration of the rice bran and the bran is 10.1g/100mL, the potassium dihydrogen phosphate is added in the mixture of 0.015-0.25g/100mL, the magnesium sulfate heptahydrate is 0.015-0.25g/100mL, the pH is natural, the shaking flask material is 250mL, the material loading rate of a fermentation tank is 75%, the inoculum size is 10%, the culture temperature is 24-28 ℃, 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 5-12d;

in one embodiment, the ganoderma lucidum mycelia obtained by liquid culture are subjected to centrifugal separation to obtain mycelia 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 ganoderma lucidum mycelia is subjected to polysaccharide extraction, circularly leached with hot water at 60 ℃ and ultrasonic-assisted liquid of 35kHz and 1.5kW for 1.5 hours, centrifugally separated to obtain clear liquid, 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 fermentation results obtained by the new strains of Ganoderma lucidum obtained by the screening using the fermentation medium are: the dry weight concentration of mycelium, the concentration of mycelium polysaccharide and the concentration of crude polysaccharide in fermentation broth reach 5.722g/100mL, 669.861mg/100mL and 1402.065mg/100mL respectively, and the ganoderan is 2071.926mg/100mL.

In one embodiment, the yield of the ganoderan reaches 2071.926mg/100mL, which indicates that the new ganoderan strain has good fermentation performance and high yield in the optimized culture medium, and the invention has the substantial characteristics and the innovation of 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 strain of ganoderma lucidum 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 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 O0.25g/100mL, natural pH, adding water to the required volume, sterilizing at 121deg.C for 30min, and taking as culture medium for fermentation, shaking bottle sample amount of 40%, inoculation amount of 10%, culture temperature of 28deg.C, rotation speed of 150r/min, and culture time of 5d. The following steps (3) to (7) are performed in the embodiments described in the foregoing summary. The results were: mycelium dry weight 5.722g/100mL culture medium, mycelium polysaccharide 669.861mg/100mL culture medium, extracellular polysaccharide 1402.065mg/100mL culture medium, and Ganoderma total polysaccharide 2071.926mg/100mL culture medium. Through detection, 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.

Example two

The new strain of ganoderma lucidum 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 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 0.015g/100mL，MgSO ₄ ·7H ₂ O0.015g/100mL, natural pH, adding water to the required volume, sterilizing at 121deg.C for 30min, and taking as culture medium for fermentation, shaking bottle sample amount of 40%, inoculation amount of 10%, culture temperature of 24deg.C, rotation speed of 150r/min, and culture time of 12d. The following steps (3) to (7) are performed in the embodiments described in the foregoing summary. The results were: mycelium dry weight 2.4g/100mL cultureCulture medium, mycelium polysaccharide is 158mg/100mL culture medium, extracellular polysaccharide is 1150.8mg/100mL culture medium, and ganoderma lucidum total polysaccharide is 1308.8mg/100mL culture medium; through detection, 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.

Example III

The new strain of ganoderma lucidum 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 rice bran is used in an amount of 2.1g/100mL of culture medium, the wheat bran is used in an amount of 8.0g/100mL of culture medium, and KH is added ₂ PO ₄ 0.15g/100mL，MgSO ₄ ·7H ₂ O0.05g/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 27 ℃, the rotating speed is 150r/min, and the culture time is 6d. The following steps (3) to (7) are performed in the embodiments described in the foregoing summary. The results were: 2.8g/100mL of mycelium dry weight culture medium, 206mg/100mL of mycelium polysaccharide, 1336.8mg/100mL of extracellular polysaccharide and 1542.8mg/100mL of ganoderma lucidum total polysaccharide; through detection, 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.

Example IV

The new strain of ganoderma lucidum 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 rice bran is used in an amount of 1.1g/100mL of culture medium, the wheat bran is used in an amount of 9.0g/100mL of culture medium, and KH is added ₂ PO ₄ 0.2g/100mL，MgSO ₄ ·7H ₂ O0.18g/100mL, natural pH, adding water to the required volume, sterilizing at 121deg.C for 30min, and taking as culture medium for fermentation, shaking bottle sample amount of 40%, inoculation amount of 10%, culture temperature of 28deg.C, rotation speed of 150r/min, and culture time of 7d. The following is a summary of the inventionSteps (3) to (7) in the described technical scheme are implemented. The results were: 3.3g/100mL of culture medium with mycelium dry weight, 239.4mg/100mL of mycelium polysaccharide, 1368.3mg/100mL of extracellular polysaccharide and 1607.7mg/100mL of ganoderma lucidum total polysaccharide; through detection, 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.

Example five

The new strain of ganoderma lucidum 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 rice bran is used in an amount of 10g/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 0.05g/100mL，MgSO ₄ ·7H ₂ O0.05g/100mL, natural pH, adding water to the required volume, sterilizing at 121deg.C for 30min, and taking as culture medium for fermentation, shaking bottle sample amount of 40%, inoculation amount of 10%, culture temperature of 28deg.C, rotation speed of 150r/min, and culture time of 6d. The following steps (3) to (7) are performed in the embodiments described in the foregoing summary. The results were: 3g/100mL of mycelium dry weight culture medium, 221.9mg/100mL of mycelium polysaccharide, 1305.8mg/100mL of extracellular polysaccharide and 1527.7mg/100mL of ganoderma lucidum total polysaccharide; through detection, 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.

Example six

The new strain of ganoderma lucidum 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 24 ℃, and the ventilation rate is 1:0.8 v/v/mm, stirring speed 50r/min, tank gauge pressure 0.05MPa, inoculum size 10%, culture time 5d, fermentation medium of 1g/L rice bran, 100g/L wheat bran, KH ₂ PO ₄ 0.15g/L，MgSO ₄ ·7H ₂ O2.5g/L, pH is natural.The following steps (3) to (7) are performed in the embodiments described in the foregoing summary. The results were: mycelium dry weight 5.384g/100mL culture medium, mycelium polysaccharide 602.3mg/100mL culture medium, extracellular polysaccharide 1327.8mg/100mL culture medium, ganoderma lucidum total polysaccharide 1930.1mg/100mL culture medium; through detection, 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.

Example seven

The new strain of ganoderma lucidum 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 24 ℃, and the ventilation rate is 1:0.8 v/v/mm, stirring speed 150r/min, tank gauge pressure 0.05MPa, inoculum size 10%, culture time 12d, fermentation medium of 100g/L rice bran, 1g/L wheat bran, KH ₂ PO ₄ 2.5g/L，MgSO ₄ ·7H ₂ O0.15g/L, pH is natural. The following steps (3) to (7) are performed in the embodiments described in the foregoing summary. The results were: 3.2g/100mL of mycelium dry weight culture medium, 249.3mg/100mL of mycelium polysaccharide, 1323.5mg/100mL of extracellular polysaccharide and 1571.8mg/100mL of ganoderma lucidum total polysaccharide; through detection, 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.

Example eight

The new strain of ganoderma lucidum 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 26 ℃, and the ventilation rate is 1:0.8 v/v/mm, stirring speed 100r/min, tank gauge pressure 0.05MPa, inoculum size 10%, culture time 7.5d, fermentation medium of rice bran 26g/L, bran 75g/L, KH ₂ PO ₄ 1.8g/L，MgSO ₄ ·7H ₂ O1.8 g/L, and the pH is natural. The following is followed by the foregoing hairSteps (3) to (7) in the technical scheme described in the description are implemented. The results were: mycelium dry weight 2.9g/100mL culture medium, mycelium polysaccharide 216.5mg/100mL culture medium, extracellular polysaccharide 1234.6mg/100mL culture medium, ganoderma lucidum total polysaccharide 1451.1mg/100mL culture medium; through detection, 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.

Claims (1)

1. The method for producing ganoderan by using the new strain of ganoderma lucidum produced by mutagenesis comprises 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) Directly using testa oryzae and testa Tritici as carbon source and nitrogen source in fermentation culture medium of Ganoderma strain Ganoderma lucidum with preservation number CCTCC NO: M2018908, wherein testa oryzae is used in an amount of 0.1g/100mL culture medium, testa Tritici is used in an amount of 10.0g/100mL culture medium, adding KH ₂ PO ₄ 0.25g/100mL，MgSO ₄ ·7H ₂ O0.25g/100mL, natural pH, adding water to the required volume, sterilizing at 121deg.C for 30min, taking as culture medium for fermentation, shaking the bottle sample amount to 40%, inoculating 10%, culturing at 28deg.C at 150r/min for 5d;

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

(4) 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;

(5) 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 ganoderan product.

Images