CN103477994B - A strain used for the production of Ganoderma lucidum polysaccharides by liquid fermentation of rice bran and bran - Google Patents

Abstract

The invention belongs to the field of microbial application technology and food biotechnology, and discloses a ganoderma lucidum strain used for producing polysaccharides by complete feed liquid fermentation of rice bran and wheat bran. Ganoderma lucidum JSU6161314 is stored in China Center for Type Culture Collection (CCTCC) on 25th, June, 2013, preservation number is CCTCC M2013287, and the bacterial strain is named as Ganoderma lucidum CCTCC M2013287. According to a method disclosed in a drawing of the invention, the novel bacterial strain, which is capable of realizing high yield of ganoderma lucidum polysaccharides, is obtained by modifying ganoderma lucidum CFCC6043. The novel bacterial strain is used for rapid fermentation in a rice bran and wheat bran complete feed liquid culture medium and high yield of ganoderma lucidum polysaccharides. After fermentation, hypha dry weight and hypha polysaccharide yield increase 23.0% and 45% respectively compared with that of the original strain.

Description

A strain used for the production of Ganoderma lucidum polysaccharides by liquid fermentation of rice bran and bran

technical field

The invention relates to the technical field of food microorganism application, in particular to a new mutagenic strain of a Ganoderma lucidum strain suitable for growing in a composite liquid medium of rice bran and bran whole material and high-yielding Ganoderma lucidum polysaccharide. the

Background technique

Since Chihara et al. (References: Chihara G, MaedaY, Hamuro J, et al.Inhibition of mouse sarcoma180by polysaccharides from Lentinus edodes (Berk.) sing[J].Nature, 1969, 222(5194): 687-688.) In 1969, he published a paper on "Nature" and revealed that lentinan has anti-tumor activity. More than 200 medicinal fungi with anti-tumor activity have been found, some of which are edible fungi, such as Grifola frondosa and Hericium erinaceus , Ganoderma lucidum, etc. The mycelium, fruiting bodies, sclerotia or spores of these edible fungi can produce various active ingredients such as amino acids, proteins, vitamins, polysaccharides, glycosides, flavonoids and antibiotics, or enrich trace elements such as selenium and zinc, etc. Improve human immunity, anti-tumor, enhance liver function, anti-oxidation and other effects. In the domestic market, edible fungus health food such as Ganoderma lucidum capsules, Grifola frondosa capsules and Cordyceps sinensis capsules are selling well. Internationally, New Zealand, Japan, and the United States all produce similar products. New Zealand's "GanoPoly" series products are made of polysaccharide compound chitosan extracted from Ganoderma lucidum and Yunzhi, and are mainly used to improve immunity and assist anti-tumor treatment. As far as Ganoderma lucidum polysaccharide is concerned, the research on it shows that: Ganoderma lucidum polysaccharide has good curative effects such as improving immunity and adjuvant anti-tumor therapy. Immunomodulatory effect and anti-tumor effect (for reference, see ① Huang S.Q., Ning Z.X. Extraction of polysaccharide from Ganoderma lucidum and its immune enhancement activity[J]. Int J Biol Macromol, 2010, 47(3): 336～341; ② Seto S.W., Lam T.Y, Tam H.L., et al.Novel hypoglycemic effects of Ganoderma lueidum water-extract in obese/diabetic(db/+db)mice[J].Phytomedicine, 2009, (16): 426～436; ③ Hikino H, Konno C , Mirin Y, et al.Isolation and hypoglycemic activity of ganoderans A and B, glycans of Ganoderma lucidum fruit bodies[J]. Planta Med, 1985, (12): 339～340)

Ganoderma luccidum (Ganoderma luccidum), also known as Zhicao and Ruicao, is a fungus belonging to the phylum Basidiomycotina, Phytomycetes, Phyllomycetes, Ganodermaceae, and the genus Ganoderma. It is mainly distributed in tropical and tropical regions of Asia, Australia, Africa and America. In subtropical regions, a few are also distributed in temperate regions. In Europe, which is located in the temperate zone of the northern hemisphere, there are only 4 species of Ganoderma lucidum, while in North America there are about 5 species. China is one of the countries with the richest species diversity of Ganoderma lucidum in the world. Traditionally, Ganoderma lucidum is mainly obtained through artificial cultivation or wild collection, and its fruiting body or spore powder is used as medicine. However, artificial cultivation of Ganoderma lucidum has a long cycle, low production efficiency, high labor intensity, and is subject to seasonal and environmental restrictions. It is vulnerable to pests and diseases, and its quality and output are unstable. However, the number of Ganoderma lucidum collected in the wild is limited, which is difficult to meet the requirements of large-scale production. Edible fungus liquid submerged fermentation technology has obvious advantages over the traditional fruiting body cultivation method. In order to efficiently produce Ganoderma lucidum polysaccharides, researchers have attached great importance to the study of liquid culture methods to promote industrial production, and have obtained many research results. Jia Xiaoyan, Zeng Xiaoxi, etc. conducted research on the conditions of liquid culture of Ganoderma lucidum. The main content of this type of research involves the production of strains, medium components and fermentation process parameters, and the research focuses on reducing production costs and increasing polysaccharide production. Most of these processes use starch, potato, glucose, etc. as the medium components. Even if the by-products of agricultural product processing such as rice bran or bran are used, they are still used as secondary components, and grain raw materials, glucose, or other raw materials are still used as the main ingredients. carbon and nitrogen sources. the

China is a large agricultural country with rich sources of agricultural and sideline products. Rice bran and bran are by-products of rice and wheat processing, not only rich in nutrients, but also cheap. Rice bran is rich in starch, cellulose and other substances, while bran is rich in protein, cellulose and other substances. Theoretically speaking, the composite of rice bran and bran already has the carbon and nitrogen sources needed for the growth of Ganoderma lucidum. Under the action of Ganoderma lucidum's own cellulase and other enzymes, Ganoderma lucidum can transform rice bran and bran into its own nutrients for growth and produce Ganoderma lucidum polysaccharides. Therefore, using cheap agricultural and sideline products such as rice bran and bran to completely replace the nutrients required for the liquid fermentation of Ganoderma lucidum such as glucose to produce Ganoderma lucidum sugar that can assist tumor treatment is of great significance to the high-value application of low-end raw materials and has extremely high economic value. the

According to the many years of research experience of the research group of Liu Weimin, the inventor of this patent, some edible fungus strains do not grow well on rice bran or bran liquid medium without adding glucose or other grain materials, and special treatment such as strain induction is required. Variation for good growth. For example, under the guidance of Liu Weimin, a number of graduate students conducted in-depth research on the liquid fermented rice bran or bran of the edible fungus Grifola frondosa, and obtained some results, forming the following literature: (1) Yang Suohua. Preparation of polysaccharides from fermented rice bran by Grifola frondosa [D] .Master's degree thesis. Zhenjiang: Jiangsu University. 2006; (2) Gu Huimin. Research on the production of polysaccharides and enrichment of organic selenium in liquid culture of Grifola frondosa in rice bran medium [D]. Master's degree thesis. Zhenjiang: Jiangsu University. 2009 ; (3) Zhang Jian. Study on the production of polysaccharides from rice bran bran produced by physical mutagenesis of Grifola frondosa [D]. Master's degree thesis. Zhenjiang: Jiangsu University, 2010; (4) Guo Chunmei. Transformation and liquid fermentation of rice bran and bran to produce polysaccharides and selenium-enriched research [D]. Master's degree thesis. Zhenjiang: Jiangsu University, 2011; Fermentation test of existing bacteria [D]. Master's degree thesis. Zhenjiang: Jiangsu University, 2012; (6) Liu Weimin, Zhang Jian, Guo Chunmei, etc. Grifola frondosa strains used in the production of polysaccharides from rice bran and bran composite materials [P ], 10579078.5, 2010; (7) Liu Weimin, Zhang Jian, Guo Chunmei, etc. The method of producing polysaccharides using rice bran compound raw materials and grifola frondosa mutagenic strain [P], 1010579048.4, 2010; (7) Liu Weimin, Guo Chunmei, Zhang Jian, et al. Strains used to ferment rice bran and bran extracts to produce Grifola frondosa polysaccharides [P], 10150888.3, 2011 (application); (8) Li Yanan. Mutagenesis, fermentation and product properties of Grifola frondosa strains [D]. Master's degree thesis. Zhenjiang: Jiangsu University, 2013 (the publication date of this paper is June 2013, which is the same as the date of submission of this patent application); (9) Zhao Li. Grifola frondosa compounded in rice bran bran Research on manganese enrichment by liquid fermentation in medium [D]. Master's degree thesis. Zhenjiang: Jiangsu University, 2012; (10) Liu Lili. Liquid fermentation of Ganoderma lucidum and strain mutagenesis for high-value transformation of rice bran bran [D]. Master Thesis. Zhenjiang: Jiangsu University, 2012; (11) Guo Tianlong. Mutagenesis of Ganoderma lucidum strains and high-value transformation of whole rice bran by liquid fermentation [D]. Master's degree thesis. Zhenjiang: Jiangsu University, 2013 (the publication date of the paper is June 2013, the same as the filing date of this patent). the

It can be seen from the above references that Liu Weimin, the inventor of this patent, has been researching on the topic of high-efficiency and high-value conversion of rice bran bran into rare edible fungus polysaccharides, realized multiple inventions and ideas, and proposed the use of Ganoderma lucidum to process rice bran bran as a whole material Liquid fermentation, high-efficiency and high-value transformation of rice bran bran into Ganoderma lucidum polysaccharide research ideas, mutagenesis treatment of the original strain, failed to guide Liu Lili's mutagenesis test (negative mutagenesis, see Liu Lili's master's thesis p46 for the analysis of Table 5.2 , Liu Lili’s master’s thesis all used rice bran and bran to filter and extract juice as the medium, not the rice bran and bran whole material liquid medium of the present invention), summed up experience, and then guided Guo Tianlong to conduct systematic research, and obtained positive induction Mutated strains, and a new method of liquid fermenting the whole material of rice bran and bran by mutagenizing the strain, and converting rice bran and bran into ganoderma polysaccharides with high efficiency and high value, formed the content of the patent of this invention and another patent of invention declared at the same time. The publication date of Guo Tianlong's master's thesis is June 2013, which is the same as the filing date of this patent application, which does not affect the novelty of the present invention. the

One of the key problems of the present invention is to obtain a new ganoderma lucidum strain, which can effectively grow on the whole rice bran material liquid medium and efficiently transform the whole rice bran material into ganoderma polysaccharide. The establishment of new strains and the new utilization of rice bran by new strains require innovation. The ratio of liquid medium composed of rice bran and bran suitable for the new Ganoderma lucidum strain and the process conditions of liquid fermentation need to be newly studied. There is no report, so the present invention has uniqueness, innovation and practicability, and possesses the basic features of an invention patent. the

At present, the physical breeding methods of microorganisms mainly include ultraviolet mutagenesis, microwave mutagenesis, and ion beam mutagenesis. In order to screen out Ganoderma lucidum strains that are suitable for growing on the whole rice bran compound medium and producing polysaccharides by mutagenesis, the present invention adopts a simple physical mutagenesis method and simultaneously carries out ultraviolet mutagenesis and microwave mutagenesis tests to expand the starting strains of Ganoderma lucidum tested. The range of mutation sites increases the possibility of obtaining positive mutant strains. The bacterial strain suitable for growing on whole rice bran bran compound medium and producing ganoderma lucidum polysaccharide induced by the present invention is invented for the first time. the

Contents of the invention

In order to reduce the use of land, reduce the production cycle, and reduce production costs, the present invention transforms and utilizes low-end raw materials into high-value products, which can ultimately reduce the price of Ganoderma lucidum polysaccharides and benefit the public. Considering the use of rice bran and bran, which are the by-products of bulk agricultural products rice and wheat processing The skin is used as a medium without adding other carbon sources such as glucose and nitrogen sources, and the liquid fermentation of Ganoderma lucidum is carried out to produce polysaccharides of Ganoderma lucidum. To achieve this goal, it is necessary to screen out Ganoderma lucidum strains suitable for growth on rice bran whole material liquid medium by mutagenesis. Therefore, the present invention selects high-yielding, low-cost Ganoderma lucidum strains based on the directional transformation of rice bran bran by means of ultraviolet and microwave mutagenesis, with high-yield polysaccharides as an index. Through the establishment of a series of screening methods such as stability and heredity, the excellent properties of Ganoderma lucidum are guaranteed, and the foundation is laid for the further large-scale use of low-cost rice bran and bran to produce high-value polysaccharides of Ganoderma lucidum. the

The technical scheme that the present invention takes is as follows:

The present invention starts from Ganoderma lucidum CFCC6043 (Ganoderma lucidum CFCC6043) of China Forestry Microbial Strain Preservation Management Center (CFCC), and provides a new Ganoderma lucidum mutagenic strain Ganoderma lucidum JSU6161314 (Ganoderma lucidum JSU6161314) through mutagenesis screening of ultraviolet rays and microwaves. It has high growth rate and high polysaccharide yield on rice bran bran whole material liquid medium without adding other carbon and nitrogen sources; the Ganoderma lucidum JSU6161314 has been preserved in the Chinese Type Culture Collection of Wuhan University, Wuhan, China on June 25, 2013 Center (CCTCC), the preserved strain number is CCTCC M2013287, and the name is Ganoderma lucidum CCTCC M2013287 (Latin name Ganoderma lucidum CCTCC M2013287). the

In one aspect of the present invention, the application of the above Ganoderma lucidum CCTCC M2013287 is provided for producing Ganoderma lucidum polysaccharides in a liquid medium for fermenting rice bran whole material. the

Beneficial effects of the present invention

The present invention adopts simple physical mutagenesis technology ultraviolet and microwave mutagenesis technology to select high-yield strains of Ganoderma lucidum. Starting from the existing Ganoderma lucidum CFCC6043 in the laboratory, parallel ultraviolet and microwave mutagenesis are carried out. Polysaccharides were screened as indicators, and Ganoderma lucidum CCTCC M2013287 was finally obtained. The polysaccharides produced by Ganoderma lucidum were produced in the rice bran bran whole material liquid medium without other carbon and nitrogen sources, and the yield was higher than that of the original strain Ganoderma lucidum CFCC6043. The resulting strain Ganoderma lucidum CCTCC M2013287 was fermented in tanks with mycelium dry weight and mycelial polysaccharide yield. The output was 6.3g/100mL medium and 0.203mg/100mL medium. The starting strain Ganoderma lucidum CFCC6043 mycelium dry weight and mycelial polysaccharide yield 5.12g/100mL medium and 0.14mg/100mL medium, the mycelial dry weight and mycelial polysaccharide yield of the mutagenized strain Ganoderma lucidum CCTCC M2013287 were higher than those of the original strain Ganoderma lucidum CFCC6043. up 23.0% and 45%. The antagonism test between Ganoderma lucidum CCTCC M2013287 and the original strain Ganoderma lucidum CFCC6043 proved that the genetic characteristics of the mutagenized strain were different from the original strain. The mutagenized strain is the desired new strain. This new strain Ganoderma lucidum CCTCC M2013287 is the first to be obtained, and it belongs to a new microbial resource with uniqueness and innovation. Based on the new strain Ganoderma lucidum CCTCC M2013287, a new method of fermenting rice bran and bran whole material liquid medium has been formed, which has changed the previous method of extracting juice from rice bran bran or extracting juice after cellulase treatment to improve the utilization rate of rice bran bran. Low disadvantages, efficient use of cheap rice bran, can reduce production costs, reduce resource consumption, and increase production. The mutagenic strain Ganoderma lucidum CCTCC M2013287 is under the same rice bran usage condition, and the mycelial dry weight and mycelial polysaccharide yield of the mutagenic strain Ganoderma lucidum CCTCC M2013287 are higher than the mycelial dry weight and mycelial polysaccharide yield of the starting strain Ganoderma CFCC6043. The yield of polysaccharides increased by 23.0% and 45% respectively, and the effect of increasing yield was remarkable. The invention produces the beneficial effect of technical progress. the

The final product is Ganoderma lucidum polysaccharide, which has the functions of enhancing immunity, assisting tumor treatment, etc., and can become a beneficial product for public health care. The high-value conversion and utilization of low-end raw materials saves costs and resources. Therefore, the present invention has good social benefits. the

The terminal product Ganoderma lucidum polysaccharide is currently relatively expensive in the market and is a high-value product. The present invention has good economic value. the

In summary, the present invention already possesses the uniqueness, creativity and practicability of an invention patent, and has produced beneficial technical, social and economic effects. the

Description of drawings

Fig. 1 is the flow chart of bacterial strain ultraviolet microwave parallel mutagenesis breeding method of the present invention;

Figure 2 is the effect of ultraviolet irradiation, where note: the left is irradiation 0s, the right is irradiation 90s;

Figure 3 is the effect of microwave irradiation, where note: the left is irradiation 0s, the right is irradiation 10s;

Figure 4 shows the growth status of the strains, where notes: from left to right are UV No. 9, Ganoderma lucidum CCTCC M2013287, and the starting strain;

Figure 5 is a comparison of the liquid seed morphology of the strains, where notes: from left to right are the starting strains, UV No. 9, Ganoderma lucidum CCTCC M2013287;

Figure 6 is the antagonism diagram between the mutant Ganoderma lucidum CCTCC M2013287 and the starting strain Ganoderma CFCC6043, where Note: the original strain Ganoderma lucidum CFCC6043 is on the left, and the mutant Ganoderma lucidum CCTCC M2013287 is on the right. the

Detailed ways

According to the process shown in accompanying drawing 1 of the description, the present invention provides ultraviolet and microwave mutagenesis breeding of Ganoderma lucidum strains with high growth rate and high polysaccharide yield on the rice bran bran composite medium without adding other carbon sources and nitrogen sources method, said method comprising the following steps:

·Take the ganoderma lucidum existing in the laboratory as the starting strain;

Inoculate the Ganoderma lucidum strain on a solid slant medium for normal culture;

The spore suspension was obtained by elution with physiological saline after bacterial culture;

After the above-mentioned spore suspension was irradiated with ultraviolet light and microwave radiation for mutagenesis, it was cultured in the absence of light and dark, and a strain with a faster growth rate and a relatively stable growth rate was screened out at one time;

Carry out fermentation on the whole rice bran and bran liquid fermentation medium, and perform secondary screening for strains with high growth rate and high polysaccharide yield;

Carry out stability and genetic analysis and identification;

In one embodiment, the solid slant medium used is potato 200g/L, glucose 20g/L, peptone 5g/L, potassium dihydrogen phosphate 1.5g/L, magnesium sulfate 0.75g/L, vitamin _B1 10mg/L , agar 20g/L, pH natural.

In one embodiment, the constant temperature is 28°C. the

In one embodiment, the ultraviolet mutagenesis adopts red light and dark operation, and two ultraviolet lamps with a power of 30W are used as the light source of the ultraviolet mutagenesis, and the distance is 20cm, and the irradiation is 30s. the

In one embodiment, the culture in the dark without light is at 28°C for 2-3 days in the dark. the

In one embodiment, the medium used for the culture without light and dark is rice bran, bran solid plate medium: rice bran 20g/L, bran 30g/L, potassium dihydrogen phosphate 1.5g/L, magnesium sulfate 0.75g/L L, vitamin _B1 10mg/L, agar 20g/L, pH natural.

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

In one embodiment, the microwave mutagenesis adopts red light and dark operation, the microwave power is 700W, the pulse frequency is 2450Hz, and the mutagenesis time is 20s. the

In one embodiment, the primary screening step is: picking well-grown single colonies from ultraviolet mutagenesis and microwave mutagenesis without light and dark culture plates and inoculating them into new rice bran bran solid plate medium respectively, and selecting 10 fast-growing and dense mutant strains were subcultured, and 3 strains with fast growth, good shape and high stability were selected from them compared with the starting strain. the

In one embodiment, the secondary fermentation screening step is as follows: the high-growth mutant strain determined for the first growth is used as the object of the secondary screening, and the fermentation screening is performed together with the starting strain to determine the strain that stably expresses the excellent traits of the mutant strain. The first screened strain and the starting strain were subjected to a shake flask fermentation test for 5 generations of continuous fermentation, and the target mutagenic strain was determined according to the index. the

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

In one embodiment, the rice bran and bran liquid fermentation medium is: rice bran 20g/L, bran 30g/L (rice bran and bran are boiled in water for 4 hours and fully utilized without extracting juice), potassium dihydrogen phosphate 1.5g /L, magnesium sulfate 0.75g/L, vitamin _B1 10mg/L, pH natural.

In one embodiment, the indicators are exopolysaccharides, hyphae (mixed with a small amount of raw materials) dry weight and hyphae polysaccharides. the

The analysis and identification method of the Ganoderma lucidum mutant strain transformed into rice bran and bran composite raw material in the present invention, described method comprises the following steps:

Comparison of colony morphology;

· Stability analysis;

In one embodiment, the colony morphology comparison is described. The Ganoderma lucidum strain and the JSU61613 bacterial strain on the slant are inoculated on the solid slant medium, and each of 6 replicates is cultivated in a constant temperature incubator at 28°C for 7 days, and observed every day. Once, observations include colony growth rate, color, colony shape, smell, etc. the

In one embodiment, the stability analysis is to investigate the stability by using the growth rate of mycelia as an index. Finally, 10 strains with better mutant strains are screened out, and after 5 passages, the optimal strain JSU6161314 is selected, and the The mutated strain was subcultured for 3 more generations, and the change of mycelial growth rate was observed to determine the stability, and compared with the starting strain. the

In one embodiment, said genetic analysis is antagonism analysis. the

Embodiment 1 Ganoderma lucidum ultraviolet-microwave mutagenesis

① Preparation of spore suspension

Inoculate Ganoderma lucidum on a 9cm sterilized solid medium plate and culture it for 4 days, then wash it with 10mL sterile physiological saline, use a sterilized brush to brush the vegetative body back and forth, and collect the washing solution. the

②UV mutagenic effect curve and selection of UV mutagenic dose

Take 20 9cm plates, each containing 5 mL of newly prepared spore suspension. Every two as a group, divided into 10 groups. Irradiate for 0, 5, 10, 15, 20, 25, 30, 45, 60, and 150s respectively (see Figure 2 for the effect of 90s irradiation), and then take 1mL from each dish for appropriate dilution and spread on a plate. The plates were wrapped with black cloth and then cultured in a dark 28°C incubator with no light. Count the colonies after the plate grows out. For each group, the average number of colonies in two dishes was used as the number of colonies in the group. The mutagenic lethality was calculated, and the mutagenic dose of ultraviolet light was selected as an index of the mutagenic lethality. the

In the formula, A is the number of colonies reproduced after mutagenesis; B is the number of colonies before mutagenesis. the

③UV mutagenesis

Turn on the ultraviolet light to preheat for about 20 minutes, take 2 sets of sterile culture dishes with a diameter of 9 cm, add 5 mL of the above-mentioned adjusted spore suspension respectively, and put them on the vibrator, open the plate cover, and put them under the ultraviolet lamp with a distance of 20 cm and a power of 30 W. Next, select the optimal irradiation dose according to the above and irradiate separately. Close the lid and turn off the UV lamp. The irradiation timing starts from opening the cover and ends when the cover is added. First turn on the vibrator switch, and then open the cover to irradiate, so that the cells in the spore suspension are irradiated evenly. the

④Microwave mutagenesis

Absorb the obtained spore suspension and pour it into a plate with a flat bottom. The suspension volume of each plate is 10mL. Adjust the microwave power to 700W and the pulse frequency to 2450Hz. According to different processing times, irradiate the spore suspension. (See Figure 3 for the 10s irradiation effect). Aspirate 0.3mL of spore suspension, spread rice bran and bran solid plate culture medium, and then place it in a 28°C incubator for 3 days. Viable counts were used to calculate the lethality. 10 batches of mutagenesis were carried out continuously, and the robust and pure bacterial strains that could grow first on the rice bran medium were selected. the

④ Screening of mutagenic strains

Take 0.2mL of the bacterial suspension irradiated by ultraviolet radiation and microwave radiation, and use a sterile glass coating stick to evenly coat the surface of the rice bran and bran solid plate culture medium, 5 plates per batch, and 10 batches of mutagenesis, and the regenerated single colony Inoculate them into new plates respectively, select 10 mutant strains with fast growth and denseness, and subculture them, and select 3 strains with fast growth, good shape and high stability compared with the original strain. the

The above-mentioned ganoderma lucidum mutant strains were subjected to a shake flask fermentation test, and the target mutant strains were screened out using the mixed dry weight of exopolysaccharides and mycelia as indicators. the

Table 1 The mixed dry weight of fermented mycelium of selected strains of each generation

Table 2 Selected strains fermenting exopolysaccharides in each generation

As can be seen from Tables 1 and 2, the mycelium and polysaccharide yields of the first-generation fermentation of the mutant strain JSU-10 (i.e. Ganoderma lucidum JSU6161314) were higher than those of the starting strain, but in the second and third generations, its traits changed. However, Ganoderma lucidum JSU6161314 was relatively stable, and its mixed dry weight of mycelia and exopolysaccharides were higher than those of the original strain, and the mixed dry weight of mycelia and the yield of exopolysaccharides in the third generation of the 100mL shake flask culture were increased by 1.8 % and 5.9%, Ganoderma lucidum JSU6161314 was preserved in the China Center for Type Culture Collection (CCTCC) on June 25, 2013, the preserved strain number is CCTCC M2013287, and the name is Ganoderma lucidum CCTCC M2013287 (the Latin name is Ganoderma lucidum CCTCC M2013287). the

Test 1 Analysis of mutant strains

1. Biomorphological analysis

Inoculate the ganoderma lucidum strains on the slant to the solid slant medium, 6 repetitions, culture in a constant temperature incubator at 28°C for 7 days, observe once a day, and observe the growth rate, color, shape and smell of the colonies obtained as follows: : The growth rate of Ganoderma lucidum CCTCC M2013287 is faster, and the plate is covered within 5 days. The plate growth rate of the starting strain is 0.83mm/h, and the plate growth rate of Ganoderma CCTCC M2013287 is 0.94mm/h, which has increased by 13.25%. See Figure 4. The colonies of Ganoderma lucidum CCTCC M2013287 are white, fluffy at first, and then become cotton-wool-like, somewhat felt-like, and the colonies are thick. The reverse side is slightly yellowish, with a slight milky aroma. See accompanying drawing 5 for the morphology of liquid first and second generation seeds. the

2. Stability analysis

The stability of the mycelial growth rate was used as an index to investigate the stability. After 5 subcultures, 10 better mutant strains were finally screened out, and the optimal Ganoderma lucidum CCTCC M2013287 strain was selected, and the mutant strain was passed down for another 3 generations. The change of silk growth speed was compared with the starting strain to determine the stability, and the results of subculture for 3 generations are shown in Table 3. the

Table 3 selects the growth rate of each generation of the strain

As can be seen from Table 3, the growth rate of the mutant strain JSU-10 (i.e. Ganoderma lucidum JSU6161314) is higher than that of the starting strain, and its growth rate degradation is not obvious. After 8 generations of subculture, it still has a growth rate of 0.911mm/h , it can be seen that the trait variation produced by the ultraviolet and microwave mutagenesis of Ganoderma lucidum can be inherited. the

3. Genetic Analysis

Antagonism Analysis

The mutant strain and the original strain were inoculated on the PDA plate at the same time, and the two strains were separated by a certain distance. They were cultured in an incubator at 28°C, and the antagonism between the colonies was observed after 4 days. the

During the growth and development of the mycelia of different species of edible fungi, the mycelia will restrict each other's growth and spread, produce antagonism, and form an antagonistic line at the junction, which is the phenomenon of antagonism. Antagonism exists not only between different species of edible fungi, but also between strains of the same species but with different genetic characteristics. The strength of antagonism between strains reflects the size of genetic differences between strains. It can be used to judge the genetic relationship between strains, and can be used for breeding to identify whether the strains produce variation. In this experiment, the starting strain and the mutant strain were inoculated on the plate to produce certain antagonism. It can be seen from the accompanying drawing 6 of the manual that the growth form of the mutant strain Ganoderma lucidum CCTCC M2013287 on the right has changed to a certain extent, and its hyphae grow densely, and are similar to The starting strain on the left forms an antagonistic line, indicating the genetic difference between the starting strain and the mutant strain. the

Yield comparison of the mutagenic strain Ganoderma lucidum CCTCC M2013287 and the starting strain Ganoderma lucidum CFCC6043 in the second experiment

The screened mutagenic strains and starting strains were fermented in a fermenter to compare their fermentation performance. the

Ganoderma lucidum strains were the mutagenic strain Ganoderma CCTCC M2013287 and the starting strain Ganoderma CFCC6043. The sample volume of the fermenter is 80% of the volume of the fermenter, the culture temperature is 28°C, the ventilation rate is 1:0.8v/v/mim, the stirring speed is 60r/min, the gauge pressure of the tank is 0.05MPa, the inoculation amount is 8%, and the culture time is 5d , the fermentation medium is rice bran 50g/L, bran 30g/L, potassium dihydrogen phosphate 1.5g/L, magnesium sulfate 0.75g/L, and the pH is natural. The weight method was used to determine the dry weight of mycelium, and the phenol-sulfuric acid method was used to determine mycelial polysaccharides. Centrifuge the mycelium mixture obtained from liquid culture, wash it three times with distilled water to remove the culture medium adhered to the surface of the mycelium, put it in a blast drying oven, and dry it to constant weight at 60°C , and the dry weight of mycelium mixed was obtained by weighing. Add a certain volume of distilled water to the dried mycelia, after grinding, extract in a water bath at 100°C for 3 hours, quantify to a certain volume, and use the phenol-sulfuric acid method to determine the content of mixed polysaccharides in mycelium. The test results are as follows: (1) The mutagenized strain Ganoderma lucidum CCTCC M2013287 and the starting strain Ganoderma lucidum CFCC6043 were fermented in the same conditions and medium, and the dry weights of mycelia were 63.0g/L and 51.2g/L respectively. Mycelium dry weight increased by 23% compared with the starting strain. (2) Ganoderma lucidum CCTCC M2013287 and Fa strain were fermented in the same conditions and medium, the mycelial polysaccharides were 2.03g/L and 1.40g/L respectively, and the mycelial polysaccharides of Ganoderma lucidum CCTCC M2013287 increased by 45% compared with the original strain. Fermentation tests showed that compared with Ganoderma lucidum CCTCC M2013287 and the original strain Ganoderma CFCC6043, the fermentation performance and mycelia polysaccharide production performance had a good change. the

Claims (1)

1. The strain used for the production of Ganoderma lucidum polysaccharide by rice bran and bran whole material liquid fermentation is characterized in that Ganoderma lucidum CCTCC M2013287 (Ganoderma lucidum CCTCC M2013287) preserved in Wuhan China Type Culture Collection Center (CCTCC for short).