CN110656050B - Inonotus obliquus strain obtained through ultraviolet mutagenesis - Google Patents

Abstract

The invention discloses an Inonotus obliquus strain obtained through ultraviolet mutagenesis, and relates to the field of application of food microbiology technology, wherein the preservation number of the new Inonotus obliquus strain is CCTCC NO: M2018906, the recommended Latin name is Inonotus obliquus JSU LIUK18, and the Latin name is Inonotus obliquus JSU LIUK 18. The new strain of the inonotus obliquus is used for liquid state fermentation of a culture medium which takes bran and rice bran complete materials which reach the pollutant limit use standard as main components through inspection, and after a fermentation product of a mycelium raw material and a fermentation liquid of the inonotus obliquus is produced, a qualified inonotus obliquus polysaccharide product is obtained through multi-frequency ultrasonic assisted extraction, freeze concentration, citric acid leaching and displacement of heavy metal ions, alcohol precipitation and alcohol washing and freeze drying.

Description

Inonotus obliquus strain obtained through ultraviolet mutagenesis

Technical Field

The invention relates to the field of food microbial technology application, in particular to a new inonotus obliquus strain obtained by ultraviolet mutagenesis, which is used for liquid state fermentation of a culture medium with bran and rice bran complete materials which reach the pollutant limit use standard as main components, and after a fermentation product of inonotus obliquus mycelium raw materials and fermentation liquor is produced, a qualified inonotus obliquus polysaccharide product is obtained through multi-frequency ultrasonic auxiliary extraction, freeze concentration, citric acid leaching and displacement of heavy metal ions, alcohol precipitation and alcohol washing and freeze drying.

Background

Many fungi used as both medicine and food or medicinal fungi have a long history of use and a wide range of people in China. Modern science reveals that various active ingredients such as amino acids, proteins, vitamins, polysaccharides, nucleosides, terpenoids, flavonoids and the like can be generated or various trace elements such as selenium, zinc and the like can be enriched in mycelia, sporophores or spores of currently common rare fungi such as hericium erinaceus, lucid ganoderma, grifola frondosa, inonotus obliquus and the like, and the traditional Chinese medicine composition has various effects of improving human immunity, resisting tumors, enhancing liver functions, resisting oxidation and the like. Because of this, there is a great interest in various international and domestic people in the research and development of fungi such as drugs, and there are many researchers and developers in the fields of medicine, food, health care in traditional Chinese medicine, agriculture and industrial production, so that many research results or products such as ganoderma lucidum capsules, grifola frondosa capsules and paecilomyces hepiali capsules are obtained, and the economic value of the research results or products is high. The hericium erinaceus added into the biscuit is made into hericium erinaceus biscuit, and the hericium erinaceus biscuit becomes common food and is sold in domestic supermarkets. Internationally, new zealand, japan, usa, korea, etc. produce similar rare fungal health food or pharmaceutical products.

In the case of Inonotus obliquus, studies on it have shown that: the Inonotus obliquus has certain biological activity in the aspects of resisting tumors, resisting cancers, resisting oxidation, reducing blood sugar, enhancing immunity, treating cardiovascular diseases and the like, and has higher medicinal value (the references: Liuping Fan, Shaodong Ding, Lianzhong Ai, et al.Antitumour and immunomodulating activity of water-soluble polysaccharide from inorganic bacteria obquus [ J ]. Carbohydrate Polymers, 2012, 90 (2): 870 and 874; Lishuai Ma, Haixia Chem, Peng Dong, Xueming Lu. Anflamator and anticancerosis of extracts and compounds [ 12 ] Inonotus obliquus [ J ]: Fr et J.: Fr.) Ohwi et al, (7) the research of Inonotus, J.: Fr.) and (7, 12; the research on the primary and secondary effects of Inonotus obliquus Pistan.: Fr.) of Inonotus, the research of the Inonotus, the research of Inonotus, the research of Inonotus, the research of Inonotus, the research, 2013; kim Yong Ook, Han Sang Bae, Lee Hong Woen, et al, immunological effect of the endo-polysaccharide produced by synthesized culture of Inonotus obliquus [ J ]. Life Sciences, 2005, 77: 2438-2456).

Inonotus obliquus (Inonotus obliquus) also called Inonotus obliquus belongs to Basidiomycotina, Hymenomycetes, Aphyllophorales, Polyporaceae, Aphyllophorales, is a large medicinal fungus parasitizing on birch, elm and other trees, and its fruiting body presents a block shape similar to carbon. The fruiting body of Inonotus obliquus is hard, has a yellowish brown to black color, and has a lot of cracking small pieces with remarkable unevenness. The sporocarp grows under bark in the initial stage, is prominent after peeling, is spread flatly, has different sizes, is white at first, slowly becomes yellow and finally becomes brown, the length of a fungus tube is 2-8 mm, and the front end of the fungus tube is cracked usually. The mushroom meat part consists of two hyphae, namely bristle strong hyphae and reproductive hyphae, and has tiny and invisible bad stripes. Inonotus obliquus is distributed in 40-50 deg. northern latitude regions of northern hemisphere, such as Finland, Russia, Japan, Korea, etc. Inonotus obliquus is mainly distributed in Jilin, Heilongjiang, Tibet, inner Mongolia and other places in China. The growth speed of the inonotus obliquus in a wild state is very slow, and the inonotus obliquus grows on birch trees for more than ten years and has remarkable medicinal value, so that the wild inonotus obliquus is expensive and is about 7-8 times of the price of phellinus igniarius. The inonotus obliquus is currently in an endangered environment, has a high preferential protection coefficient and is classified as a first-level protection grade line. It can be seen that the resource of the wild inonotus obliquus is very rare, so the problem of resource shortage of the inonotus obliquus has to be solved. At present, inonotus obliquus is obtained mainly by artificial cultivation, sporocarp or spore powder is used as a medicine, but the artificial cultivation of inonotus obliquus has long period, low production efficiency, high labor intensity, limitation by seasons, environment and the like, is easy to suffer from diseases and insect pests, and has unstable quality and yield. The rare fungus fermentation technology can overcome the defects of the traditional sporocarp cultivation, and the inonotus obliquus fermentation product is produced by adopting a liquid or solid fermentation method and is deeply processed and applied, thereby conforming to the development idea driven by innovation and having good prospect. The research and development progress of the inonotus obliquus fermentation product can further promote the application of the inonotus obliquus, and bring practical social and economic benefits. Therefore, it is necessary to research a new inonotus obliquus fermented product and a production method of a deep-processed product 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 inonotus obliquus fermented product raw materials, and it is expected that the production cost of the inonotus obliquus fermented product can be reduced, so that the price of a final product such as inonotus obliquus health food or medicine can be reduced, and the product is beneficial to common people and can promote public health, which is one of the concerns of the patent of the present invention.

China is a large producing country of rice and wheat, rice bran and wheat bran are abundant, the annual output of the rice bran is about 1000 ten thousand tons, and the annual output of the wheat bran (the wheat bran in the specification is about 3000 ten thousand tons). The rice bran and the wheat bran are used as byproducts in rice or wheat processing, and have rich nutrient components and low price. The rice bran contains rich and high-quality active substances with remarkable physiological functions, such as protein, active polysaccharide, fat, tocotrienol, tocopherol and the like, and the bran is rich in nutritional ingredients, such as fat, protein, mineral substances, vitamins, cellulose and the like, wherein the content of the cellulose can reach more than 18 percent of the total amount of the bran, so that the rice bran and the bran can provide sufficient carbon sources, nitrogen sources, vitamins and mineral substances for the growth of the inonotus obliquus. Under the action of the inonotus obliquus cellulase and other enzyme systems, the inonotus obliquus can convert rice bran and wheat bran into required nutrient substances for growth and metabolism, and synthesize inonotus obliquus mycelium and various functional substances. Therefore, the method has technical feasibility and can bring better social and economic benefits by using the agricultural and sideline products of rice bran and wheat bran as all carbon sources and nitrogen sources to carry out the liquid state fermentation of the inonotus obliquus to produce the inonotus obliquus fermentation product and further processing the inonotus obliquus fermentation product into health-care food or medicine.

Most of the liquid fermentation production methods of edible fungi adopt glucose and grain raw materials such as starch, potato or soybean meal and the like as culture media, and even agricultural byproducts such as rice bran, wheat bran and the like are added as auxiliary components. The original inonotus obliquus does not grow in a rice bran and bran complete material liquid culture medium without adding glucose or other grain raw materials, and the quality of a final product is influenced by harmful substances such as lead, arsenic, aflatoxin, vomitoxin, pesticide residues and the like contained in the rice bran and bran raw materials, so that innovative research needs to be carried out on a method for producing the inonotus obliquus fermentation product by using the fermented rice bran and bran complete material of the inonotus obliquus.

The research group of Liuwei people of the first invention of the patent carries out a great deal of research on the liquid fermentation of grifola frondosa, lucid ganoderma, cordyceps sinensis and the like for more than ten years, and a plurality of achievements such as Master thesis, invention patent and the like are formed successively. Master articles directed by liu weimin have: (1) poplar's Lock flower, maitake's fermented rice bran to prepare polysaccharide (2006); (2) study on how to produce polysaccharides and enrich organic selenium by liquid culture of maitake mushrooms in rice bran medium (2009) in view of psychic; (3) the research of creating polysaccharide by inducing liquid fermentation of grifola frondosa to produce rice bran and wheat bran by a physical method (2010); (4) inducing strain of Guochun plum and Grifola frondosa, performing liquid fermentation on rice bran and wheat bran to produce polysaccharide, and performing selenium enrichment research (2011); (5) mutagenesis and fermentation of a photinia glabra strain and performance research (2013); (6) liulili, ganoderma lucidum liquid fermentation and strain mutagenesis for high value conversion of rice bran and wheat bran (2012); (7) gutianlong, research on ganoderma lucidum strain mutagenesis and liquid state fermentation high-value transformation of whole rice bran and wheat bran (2013); (8) the research of producing polysaccharide by rice bran and wheat bran in a high-value way through liquid fermentation of cordyceps sinensis (2014); (9) zhang laughing fly, research on the activity of the liquid fermentation of the Ganoderma lucidum strain (Ganoderma lucidum CFCC6043) and the whole rice bran and the polysaccharide (2014); (10) gao, grifola frondosa mutagenesis and new and old strain liquid fermentation rice bran and bran research (2015); (11) jinlei, ganoderma lucidum mutagenesis and rice bran and bran fermentation research of new and old strain liquid (2015); (12) comparison study of Wang Xuemei (2016) on whole material of rice bran and wheat bran fermented with Cordyceps strain liquid; (13) litting, mutation of cordyceps sinensis strains and research on a new liquid fermentation rice bran and wheat bran complete feed culture medium of a new strain (2017). The invention patents of the inventor of Liuwei people include: (1) a method for producing polysaccharides using rice bran and wheat bran composite raw materials and grifola frondosa mutagenized strains, 20101010579048.4; (2) a grifola frondosa strain 201010579078.5 for producing polysaccharide from rice bran and wheat bran composite raw materials; (3) a strain for producing grifolan by fermenting rice bran and bran extracts, 201110150888.3; (4) a strain for producing grifolan by liquid fermentation of complete rice bran and bran, 201310274913.8; (5) a method for producing polysaccharide by liquid fermentation of rice bran and wheat bran complete material by ganoderma lucidum mutant strain 201310275061.4; (6) a method for producing cordyceps sinensis polysaccharide by liquid fermentation of whole rice bran and wheat bran, 201310274914.2; (7) a method for producing polysaccharide by liquid fermentation of rice bran and wheat bran complete feed with Grifola frondosa mutant strain, 201310274911.9; (8) a strain used for producing ganoderma lucidum polysaccharide by liquid state fermentation of complete rice bran and wheat bran, 201310274915.7; (9) a production method of a selenium-rich ganoderma lucidum mycelium raw material, 201510996120.6; (10) a Grifola frondosa mutant strain producing Grifola frondosa mycelia, 201510990344.6; (11) producing Cordyceps sinensis mycelium mutation strain 201510996118.9. From the above description, the inventor of the present invention, liuweimin, has been studying around the subject of high efficiency and high value production of rare edible fungus fermented products by using rice bran and wheat bran, to realize the idea of the invention and continuously improve the production methods of various rare fungus fermented products.

The article of the Master research institute guided by the inventor Liuwei people research institute and the invention patents related to the invention patents are combined with the problems which cannot be answered when related results are negotiated with enterprises, and the related patent technology and research article still have many problems to be researched, solved and innovated if the related patent technology and research article are to be moved to an industrialization way. The problems comprise acquisition of high-efficiency new strains of different rare fungi, establishment of rice bran and wheat bran raw material standards, innovation of a rare fungus fermentation product fermentation method, use of a new method for separating and purifying the rare fungus fermentation product, a combination mode of objects needing innovation and the like. The patents are designed with due consideration to the uniqueness necessary to patent the invention, the novelty and practicality of achieving the salient and essential characteristics and the significant improvements. Therefore, the combined innovation of the above problems should ensure that the related inventions achieve the uniqueness, novelty and practicability necessary for the patenting of the inventions.

Based on the consideration and the earlier research foundation of the special production method of the inonotus obliquus fermented product, the inventor provides an application of the inonotus obliquus new strain for producing the inonotus obliquus fermented product by fermenting rice bran and bran by using ultraviolet mutagenesis, because the inonotus obliquus is a medicine and food dual-purpose fungus which is different from ganoderma lucidum, grifola frondosa and cordyceps sinensis which are researched by the inventor, and the method for producing the inonotus obliquus fermented product by obtaining the new strain of the inonotus obliquus high-efficiency liquid state fermented rice bran and full material by using the ultraviolet mutagenesis method has not been researched by people before the application.

In conclusion, the invention relates to the field of inonotus obliquus used for fermenting bran and rice bran complete materials to produce inonotus obliquus fermentation products, and the inonotus obliquus fermentation products are subjected to deep processing treatment to obtain qualified products. One of the key problems to be solved by the invention is that the fermentation capacity of the original strain needs to be improved by a mutagenesis method, a new strain of inonotus obliquus which is not reported is obtained, and the new strain can be subjected to gene sequencing to ensure that the used strain is correct. The second key problem to be solved by the invention is to define the standard of the bran and rice bran raw material, because the sources of the bran and rice bran are complex, the types and the levels of pollutants are different, and the previous research and patent of the inventor do not pay enough attention in the aspect, thereby bringing about indefinite consequences to the subsequent product development. The invention also provides a production method for obtaining qualified products after deep processing of the fermented products. The three points ensure that the patent of the invention is different from various achievements of other inventors and the inventor's Liuwei people research team, and has uniqueness, innovativeness and practicability.

The reason for the key problems of the above three aspects is further explained. The method aims to solve one of the key problems, namely, the method needs to improve the fermentation capacity of the original inonotus obliquus strain by a mutagenesis method, obtain an unreported new inonotus obliquus strain, and perform gene sequencing on the new strain to ensure the correct strain. The background of the problem is that most of previous research results of an inventor team relate to rare edible fungi such as lucid ganoderma, grifola frondosa, cordyceps sinensis and the like, the original strain of inonotus obliquus is primarily related to bran fermentation, but the efficiency of the original strain to bran fermentation is not high. In view of the pharmacological disclosures of inonotus obliquus, the abundance of functional components, the scarcity of resources, the high price and other aspects, the research on the method for producing the inonotus obliquus fermented product by fermenting bran and rice bran with the new inonotus obliquus strain is considered. According to the past research experience of the inventor, the problem of adaptability of strains needs to be solved firstly by fermenting bran and rice bran with the rare fungi. Therefore, it is desirable to improve the fermentation performance by mutagenesis. In addition, in the research process, gene sequencing should be carried out to ensure that the strains do not have errors after a large number of strain processing steps.

The background of the invention which proposes the two key problems to be solved by the present invention, which is to define the standards of bran and rice bran raw materials, is that our previous research and patents have not paid enough attention in this respect, and sometimes have unexpected consequences for the subsequent product development. In the past, the research result is expected to process various kinds of bran and rice bran, so that the use limit of the bran and rice bran raw material is intentionally relaxed, and the raw material standard is not established, but the result is that some abnormal values which are not in the research range appear when the quality of the final product is characterized, such as the problem that the determination result of some heavy metals exceeds the standard, the reason for this is that the sources of the bran and rice bran raw materials are complex, the quality is difficult to maintain at a similar level, and no clear pollutant finally enters the terminal product. Thus, the original inventive production method designed for specific contaminants and their possible contents may have other contaminants in the product, so that the production method of the fermented product should be redesigned to cope with the new contaminants that may have occurred. Thus, the control and standardization of the quality of the bran and rice bran become the necessary consideration for the design of the new invention. The invention provides the use standards of raw materials of bran and rice bran.

The invention provides a production method for qualified products after deep processing of leavening, and aims to solve the three key problems. Since the bran and rice bran raw material may bring unknown pollutants, the aim of removing all pollutants cannot be achieved by considering the addition of enrichment inhibitors in the culture medium, and thus the hypha fermentation product can still have unknown pollutants and exceed the standard content when being used as a final product, a reasonable treatment method aiming at the possible occurrence condition is to design an extraction and separation method for the fermentation product after fermentation to obtain a qualified final product. Based on the recognition, the invention designs a more reasonable method for producing and treating the fermentation product.

The invention needs to obtain the new strain of the inonotus obliquus by a mutagenesis method. The breeding method of microorganism mainly includes physical mutagenesis, chemical mutagenesis, gene recombination and the like, in order to avoid using toxic and harmful chemical mutagens and a genetic engineering method which is not accepted by the public in the field of food, the invention uses a relatively simple and easy ultraviolet mutagenesis technology to enable an initial strain to be in an extreme environment of mutagenesis, expand the site range of mutation to the maximum extent and improve the possibility of obtaining a positive mutant strain. The invention adopts the common mutagenesis method of ultraviolet mutagenesis, although the method has no innovation, the performance of the strain obtained by the method is innovatively changed, and the method has uniqueness, innovation and practicability when fermenting the bran and rice bran complete feed culture medium. The new strain of the inonotus obliquus of the fermented bran and rice bran complete feed liquid culture medium is obtained by the invention for the first time.

Disclosure of Invention

In order to convert a complete liquid rice bran and bran material into a raw material of inonotus obliquus mycelium by utilizing an inonotus obliquus strain with high efficiency and high value, the invention needs to mutagenize and screen out a new inonotus obliquus strain suitable for growing on a new liquid culture medium of the complete rice bran and bran material. In order to avoid using toxic and harmful chemical mutagens and a genetic engineering method which is not accepted by the public in the food field, and consider that the ultraviolet physical mutagenesis technology is simple and easy, the ultraviolet mutagenesis method is adopted for mutagenesis, and a series of screening methods such as stability, heredity and the like are adopted, so that the excellent properties of the new strain of the inonotus obliquus are ensured, and a strain foundation is laid for industrially efficiently converting bran and rice bran with high value and low cost into the inonotus obliquus fermentation product with qualified quality. The invention establishes raw material standards for the used bran and rice bran raw materials and lays a raw material foundation for obtaining good fermentation products through fermentation, and the bran and rice bran with the raw material use standards are referred to in the specification unless special instructions are provided. The fermentation product of the invention, inonotus obliquus hypha and extracellular useful fermentation product, needs to be further extracted, separated and purified, and finally obtains the fermentation product finished product with qualified quality for the production of later derivative products.

The technical scheme adopted by the invention is as follows:

the new strain of Inonotus obliquus JSULUIUK 18 provided by the invention is a new strain of Inonotus obliquus obtained by ultraviolet mutagenesis, can quickly grow and produce high-yield fermentation products in a bran and rice bran complete material liquid culture medium, is determined to be a new strain of Inonotus obliquus by DNA sequencing of a biological engineering (Shanghai) member Limited company, and is preserved in China Center for Type Culture Collection (CCTCC) of Wuhan university in Wuhan, 12 and 19 days in 2018, with the preservation number of CCTCC NO: M2018906, and the recommended Latin literature is Inonotus obliquus JSU LIUK18Inonotus JSUUU LIUK18, and the new strain of Inonotus obliquus later described in the patent specification refers to the new strain.

In one aspect of the invention, the application of the new inonotus obliquus strain is provided, which is used for fermenting a rice bran and wheat bran complete feed liquid medium to produce an inonotus obliquus fermented product;

in one aspect of the invention, the obtained new strain of inonotus obliquus is used for liquid state fermentation of a culture medium which takes bran and rice bran raw material complete materials which are checked to reach the use standard as main components to produce a fermentation product of inonotus obliquus mycelium raw materials and fermentation liquor;

in one aspect of the invention, the inonotus obliquus mycelium raw material and the fermentation product of the fermentation broth are subjected to multi-frequency ultrasonic assisted extraction, freeze concentration, citric acid leaching and replacement of heavy metal ions, alcohol precipitation and alcohol washing and freeze drying to obtain a qualified inonotus obliquus polysaccharide product;

in one aspect of the invention, the use standard of the bran is the standard NY/T119-1989 of the bran for the edible feed of the department of executing the original agriculture, the maximum pollution level of the added main heavy metals of lead, arsenic, mercury, cadmium and chromium is not more than 3mg/kg, the pesticide applied in the wheat planting process meets the agricultural technology standard, the wheat growth process has no serious scab damage, no continuous rainwater in the harvesting season causes the wheat to deteriorate, and the storage and transportation of the bran after the wheat is processed can not cause the mildew;

in one aspect of the invention, the rice bran use standard is that the pollution level limit of main heavy metals of lead, arsenic, mercury, cadmium and chromium is not more than 3mg/kg at most according to the standard NY/T122-1989 of the original Ministry of agriculture, the pesticide applied in the rice planting process meets the agricultural technical specification, no serious diseases are caused in the rice growth process, continuous rainwater in the harvesting season causes rice deterioration, and the rice bran storage and transportation after rice processing does not cause mildew.

The invention has the advantages of

In order to meet the requirement of subsequent fermentation products for producing health-care food, the invention adopts a physical mutagenesis technology to breed a new strain of the Inonotus obliquus, the original strain is the Inonotus obliquus which is purchased from China forestry microorganism strain preservation center (English abbreviation name is CFCC) with the preservation number of CFCC 83414, the latin name of the strain is Inonotus obliquus with the preservation number of CFCC 83414, and the original strain is also named as the Inonotus obliquus LIUK0 in the research process and the specification. Starting from the original strain, the original strain is also the starting strain, carrying out ultraviolet mutagenesis, screening by taking the growth rate, the dry weight of pure hyphae and hypha polysaccharide as indexes, finally obtaining a new Inonotus obliquus strain obtained by mutagenesis, the Latin name is Inonotus obliquus, the preservation number is CCTCC NO: M2018906, the new Inonotus obliquus strain is determined as a new Inonotus obliquus strain through DNA sequencing of a company Limited in Biotechnology engineering (Shanghai), and the original strain is also named as Inonotus obliquus LIUK18in the research process and the specification. The new strain produces the Inonotus obliquus mycelium raw material in a bran and rice bran complete material liquid medium which does not add other carbon sources and nitrogen sources and meets the raw material use standard, compared with the original strain with the Inonotus obliquus preservation number of CFCC 83414, the production capacity is higher, the yield of pure mycelium is improved, and the mycelium polysaccharide and the polysaccharide in fermentation liquor are correspondingly improved.

During shaking flask fermentation, the bran and rice bran complete feed liquid culture medium used by the obtained new inonotus obliquus strain consists of 3g/100mL (referring to the volume of the culture medium, all the following are understood) and 2g/100mL of bran and rice bran respectively, the dry weight concentration of pure hyphae, the concentration of hyphae crude polysaccharide and the concentration of crude polysaccharide in fermentation liquor respectively reach 2.005g/100mL, 114.160mg/100mL and 240.105mg/100mL, and the total polysaccharide is 354.265mg/100 mL. And the dry weight concentration of hyphae, the crude polysaccharide concentration of the fermentation liquor and the total polysaccharide concentration of the original strain Inonotus obliquus CFCC 83414 under the same conditions are respectively 1.543g/100mL, 110.467mg/100mL, 155.542mg/100mL and 266.009mg/100 mL. The hypha dry weight concentration of the new bacterial strain is increased by 29.9%, hypha polysaccharide, extracellular polysaccharide and total polysaccharide obtained by every hundred mL of culture medium are respectively increased by 30.5%, 54.4% and 33.18%, which shows that the inonotus obliquus new bacterial strain obtained by mutagenesis has stronger capacity of fermenting bran and rice bran, and the fermentation performance of the bacterial strain is obviously and positively changed.

Through repeated ultraviolet mutagenesis screening research, a new inonotus obliquus strain with a preservation number of CCTCC NO: M2018906 is finally obtained, the new strain has higher growth speed and stronger capability of fermenting bran and rice bran in a bran and rice bran complete feed liquid culture medium which does not add other carbon sources and nitrogen sources and meets the use standard, so that the mycelium quantity and the mycelium polysaccharide yield in a specified fermentation volume are higher, and the fermentation characteristic of the new strain is changed compared with that of the original strain, thereby reflecting clear uniqueness. Based on the new strain of the inonotus obliquus, a new method for fermenting the bran and rice bran complete material liquid culture medium is formed, the bran and rice bran complete material are subjected to liquid fermentation and applied, the production capacity and the yield of the method are comprehensively superior to those of the original strain, and the new strain of the inonotus obliquus achieves 'outstanding substantive characteristics and remarkable progress innovativeness'. The invention efficiently utilizes the cheap bran and rice bran, can reduce the production cost, reduce the resource consumption, obviously increase the yield and has practicability.

In conclusion, the invention utilizes the uniqueness, the outstanding substantive features and the innovativeness and practicability of remarkable progress of the invention patent, and produces beneficial effects.

Drawings

FIG. 1 is a flow chart of the ultraviolet mutation breeding method of the new strain of inonotus obliquus obtained by the invention.

FIG. 2 is the antagonistic diagram of the new strain and the original strain of Fuscoporia obliqua, wherein the left side of the diagram is the original strain of Fuscoporia obliqua and the right side is the new strain of Fuscoporia obliqua.

FIG. 3 is a clade of a new strain of Inonotus obliquus.

Detailed Description

The invention provides a method for breeding a new inonotus obliquus strain with higher growth speed and higher polysaccharide yield on a bran and rice bran complete feed liquid culture medium by ultraviolet mutagenesis according to a flow shown in an attached figure 1 of a specification, and the method comprises the following steps:

taking an original strain Inonotus obliquus preserved in a laboratory, wherein the preservation number is CFCC 83414 as an original strain;

inoculating the taken out strain on a PDA culture medium for activated culture;

after the thalli is cultured, the thalli is eluted by sterile normal saline to prepare spore suspension;

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

shake flask fermentation is carried out by using a rice bran and wheat bran complete feed liquid culture medium, and strains with high growth rate and high polysaccharide yield are screened out;

the new strains selected were subjected to antagonism tests and gene sequencing.

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

In one embodiment, the constant temperature is 28 ℃.

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

In one embodiment, the light-protected culture is a light-protected culture at 28 ℃ for 7 days.

In one embodiment, the light-shielding culture medium is a rice bran, wheat bran solid plate culture 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, complete use of the wheat bran and the rice bran, and the wheat bran and the rice bran all need to meet the standard).

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

In one embodiment, the preliminary screening step is: and selecting single colonies with good growth from a flat plate subjected to ultraviolet mutagenesis and light-proof culture, and respectively inoculating the single colonies into a new rice bran and wheat bran solid plate culture medium, and selecting strains with fast growth, good shape and high stability relative to the original strains.

In one embodiment, the rescreening step is: respectively carrying out 5-generation plate subculture on the roughly screened superior strains, selecting well-grown single colonies, respectively inoculating the single colonies into a new rice bran and wheat bran solid plate culture medium, and selecting strong and pure variant strains with high growth speed.

In one embodiment, the three-sieving step is: taking the high-growth-rate variant strain determined by secondary screening as an object of the third screening, and carrying out shake flask fermentation screening together with the original strain to determine the strain with the excellent character and stable expression of the variant strain. And (3) carrying out shake flask fermentation test on the newly screened Inonotus obliquus strain and the original starting strain Inonotus with the preservation number of CFCC 83414, continuously fermenting for 5 generations, and determining a target mutagenic strain according to indexes.

In one embodiment, the novel mutant strain of inonotus obliquus, CCTCC NO. M2018906, is strain No. 27, and the growth rate on the fifth generation of re-screening plate is obviously faster than that of the original strain No. 0.

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

In one embodiment, the rice bran and wheat bran liquid fermentation medium is: 2.0g/100mL of rice bran, 3.0g/100mL of bran, 0.15g/100mL of monopotassium phosphate, 0.075g/100mL of magnesium sulfate and natural pH.

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

In one embodiment, the genetic character is characterized by an antagonism test, the result is shown in figure 2, the antagonism line is obvious, the right inonotus obliquus mutagenic new strain CCTCC NO: M2018906 grows densely and thickly, the growth performance of the inonotus obliquus mutagenic new strain is stronger than that of the original strain, and the genetic character is beneficially changed.

In one embodiment, the new strain of inonotus obliquus with the preservation number of CCTCC NO: M2018906 is determined as a new strain of inonotus obliquus by DNA sequencing of the company Limited in the biological engineering (Shanghai).

The new strain is preserved in China Center for Type Culture Collection (CCTCC) at Wuhan university in Wuhan, China at 19.12.2018, the preservation number is CCTCC NO: M2018906, the recommended Latin article name is Fuscoporia obliqua JSU LIUK18Inonotus obliquus JSU LIUK18, and the Fuscoporia obliqua new strains in the following patent specifications refer to the strain (see figure 3).

In one embodiment, the screened new mutant strain of inonotus obliquus, CCTCC NO, M2018906, is completely fermented by using a bran and rice bran fermentation medium, and the fermentation result is as follows: the dry weight concentration of pure hypha, the concentration of hypha crude polysaccharide, the concentration of extracellular crude polysaccharide in fermentation liquor and the total polysaccharide respectively reach 1.543g/100mL, 110.467mg/100mL, 155.542mg/100mL and 266.009mg/100 mL.

In one embodiment, the mycelium dry weight concentration of the inonotus obliquus mutant new strain is increased by 29.9%, and the mycelium polysaccharide, extracellular polysaccharide and total polysaccharide obtained per hundred mL of culture medium are respectively increased by 30.5%, 54.4% and 33.18%, which shows that the inonotus obliquus mutant new strain obtained by mutation has stronger capacity of fermenting bran and rice bran, and the fermentation performance of the strain is obviously and positively changed.

In one embodiment, the mycelium of the new strain of inonotus obliquus obtained after fermentation is subjected to centrifugal separation, multi-frequency ultrasonic assisted extraction, freeze concentration, citric acid leaching and replacement of heavy metal ions, alcohol precipitation and alcohol washing, and freeze drying to obtain qualified inonotus obliquus mycelium polysaccharide.

In one embodiment, the qualified extracellular polysaccharide of the new inonotus obliquus strain is obtained by performing freeze concentration, citric acid leaching and heavy metal ion replacement, alcohol precipitation and alcohol washing and freeze drying on fermentation liquor obtained after fermentation.

In one embodiment, the sum of qualified inonotus obliquus hyphal polysaccharides and qualified inonotus obliquus exopolysaccharides is total polysaccharides.

In one embodiment, the limit values of the mycotoxins aflatoxin B1, B2 and deoxynivalenol of the total polysaccharide, the limit values of heavy metals of lead, arsenic, mercury, cadmium and chromium and the residual limit values of pesticides of carbendazim, abamectin and butachlor are determined to be not higher than the limit values specified in GB2761-2017, GB2762-2017 and GB2763-2016 or are not detected.

Example 1

The inonotus obliquus strain adopts the new inonotus obliquus strain obtained by the invention. The using amount of rice bran is 2.0g/100mL of culture medium, the using amount of bran is 3.0g/100mL of culture medium, 0.15g/100mL of monopotassium phosphate and 0.075g/100mL of magnesium sulfate heptahydrate are added, the pH is natural, water is added to the required volume, sterilization is carried out at 121 ℃ for 30min, the obtained product is used as the culture medium for fermentation, the sample loading amount of a shake flask is 40%, the inoculation amount is 10%, the culture temperature is 28 ℃, the rotating speed is 150r/min, and the culture time is 6 d. The following steps (1) to (4) are carried out: (1) separating mycelium containing a small amount of rice bran and wheat bran obtained by liquid culture with a 20-mesh metal screen, and washing with water twice; (2) performing multi-frequency ultrasonic-assisted warm water extraction on the obtained wet mycelia at the water temperature of no more than 60 ℃, filtering to obtain clear liquid, and performing freeze concentration, citric acid leaching to replace heavy metal ions, alcohol precipitation, centrifugal filtration, alcohol washing and freeze drying to obtain qualified mycelia polysaccharide products; (3) filtering the obtained fermentation liquor, freezing and concentrating, leaching and replacing heavy metal ions by citric acid, precipitating with ethanol, centrifuging and filtering, washing with alcohol, and freeze-drying to obtain qualified extracellular polysaccharide product; (4) the polysaccharide content of the crude polysaccharide obtained by fermentation is measured by adopting a phenol-sulfuric acid method, and the result is that the dry weight concentration of pure hyphae, the concentration of hyphae crude polysaccharide, the concentration of extracellular crude polysaccharide and the concentration of total crude polysaccharide respectively reach the following results: the dry weight concentration of pure mycelium, the concentration of mycelium crude polysaccharide and the concentration of crude polysaccharide in fermentation liquor respectively reach 2.005g/100mL, 114.160mg/100mL and 240.105mg/100mL, and the total polysaccharide is 354.265mg/100 mL. The hypha dry weight concentration of the new strain is increased by 29.9 percent, and hypha polysaccharide, extracellular polysaccharide and total polysaccharide obtained by per hundred mL of culture medium are respectively increased by 30.5 percent, 54.4 percent and 33.18 percent; (5) through detection, the limit values of the mycotoxins aflatoxin B1 and B2 and deoxynivalenol of the total polysaccharide, heavy metal lead, arsenic, mercury, cadmium and chromium, and the residual limit values of pesticides carbendazim, abamectin and butachlor are not higher than the limit values specified in GB2761-2017, GB2762-2017 and GB2763-2016 or are not detected through determination.

Claims (1)

1. A strain of Inonotus obliquus has a preservation number of CCTCC NO: M2018906, and a recommended Latin name of Inonotus obliquus JSU LIUK 18.

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