CN115039633A - A kind of artificial cultivation method of the fruiting body of Corynebacterium japonica - Google Patents

Abstract

The invention provides an artificial cultivation method of a sporocarp of Isaria japonica, which is characterized in that coix seeds are added into a cultivation material of the sporocarp, the addition proportion of the coix seeds is further optimized, the yield of the sporocarp of Isaria japonica is greatly improved, the process is simple, the production cost of artificially cultivating Isaria japonica sporocarp is obviously reduced, and the requirement of large-scale periodic production can be met; the prepared Isaria japonica sporocarp has richer nutrition, has the unique efficacy characteristic of the Spodoptera fruticosa, has higher contents of mannitol, ergosterol and other polysaccharides, and has wide market prospect.

Description

A kind of artificial cultivation method of the fruiting body of Corynebacterium japonica

technical field

The invention belongs to the technical field of microbial fermentation engineering, and in particular relates to a method for culturing medicinal bacteria for insects, in particular to a method for artificially culturing fruit bodies of Corynebacteria japonicus.

Background technique

Entomogenous fungi have high medicinal value. The amino acid composition of fungus crops is comprehensive and the utilization rate is high, and it is recognized as a "very good protein source" internationally. Fungus is also an important source of natural food vitamins. Among them, the human body is more likely to lack vitamin B, which is higher than ordinary vegetables, and vitamin B2 is higher than meat and cheese. It can prevent pernicious anemia, improve neurological function and effectively reduce blood lipids. The mineral content of edible mushrooms is twice that of vegetables and higher than that of beef and mutton. Edible mushrooms contain no starch and low fat content, making them ideal food for diabetics and obese patients. Edible fungi are also rich in steroids, triterpenes, coumarin glycosides, volatile oils, alkaloids, organic germanium and polysaccharides and other medicinal components, which are beneficial for regulating human function, improving immunity, lowering blood pressure and cholesterol, and resisting diseases and tumors. As well as anti-aging and other significant health care effects.

With the improvement of people's living standards and the enhancement of health care awareness, health care products produced with medicinal fungi as raw materials are more and more favored by the public. At present, the health care products of medicinal insects and fungi on the market are still mainly consumed by Cordyceps militaris and Cordyceps sinensis, and the products are too single. For example, the production methods used in my country, such as Cordyceps sinensis and Cordyceps sinensis, all use dried and fresh fungal fruiting bodies as raw materials. After dipping or grinding, a juice containing some active ingredients is obtained, and then After dilution, adding some flavoring accessories, and finally made into capsules or granules. However, this traditional production process of medicinal fungi has problems such as complex process, high production cost, and shortage of raw material resources, which cannot be adapted to large-scale periodic production. And this insect-borne fungus is rarely heard in the market.

Japanese Corynebacterium is also known as Macrosporidia, Japanese Cordyceps sinensis. The research work so far shows that many parasitic insects Paecilomyces and Corynebacterium can produce a variety of important biologically active substances. my country has abundant resources of Paecilomyces sp. and Trichosporium coryneformis, and some of them have been researched and developed, such as the Paecilomyces stipitides strains disclosed in CN102676396B, which have also been involved in many aspects of the national economy and people's life. Corynebacteria japonicum and Paecilomyces thaliana belong to different strains. Studies have shown that the extract of Corynebacterium japonicum has better anti-tumor and immunomodulatory effects, enhances lung and kidney function, prevents arteriosclerosis, prevents anemia, increases The mouse has the effects of hypoxia resistance under normal pressure, sedative, analgesic, and antibacterial.

At present, aiming at the artificial cultivation process of Corynebacterium japonica, CN97113190.2 discloses the artificial cultivation method of Cordyceps militaris and other Cordyceps militaris, and proposes that silkworm chrysalis can be used for the cultivation of Corynebacterium japonica; Cultivation, and published the document "Biological Characteristics and Domestication of Macrosporidia Flowers" in 2014 (Bacteria Research, Vol. 12, No. 4, 2014), but the output of its fruiting bodies is still low, and the cultivated The content of nutrients in the fruiting bodies is not high, resulting in high production costs, and it is difficult to achieve large-scale production.

Therefore, it is urgent to find out how to improve the artificial culture method of Japanese sporophyte production and reduce the cost, so as to help realize the industrialized production of Japanese sporozoites.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned problems, the present invention provides a method for culturing the fruiting body of Conychocystis japonicus. By adding coix seed in the cultivation material of the fruiting body, and further optimizing the addition ratio of Coix seed, the fruiting body of Conychocystis japonicus is greatly improved. The yield, the process is simple, the production cost of artificially cultivated Japanese sporozoites can be significantly reduced, and the requirement of large-scale periodic production can be met; Efficacy characteristics, polysaccharides such as mannitol and ergosterol are higher in content, and have broad market prospects.

The Japanese coryneform spore of the present invention was collected by the inventor in the Xianrendong National Nature Reserve, Zhuanghe City, Liaoning Province, and was identified as Macrosporidia (Japanese Corynebacterial tract) by Jilin Agricultural University according to its morphological characteristics and microstructure. spore, Isaria japonica), the inventor has realized the artificial cultivation of the Japanese coryneform spore for the first time, and after long-term passage, screening and domestication, the Japanese stick with more robustness, higher fruiting body yield and quality, and higher content of camp ingredients is selected. Trichosporium sp. ZQY001, and deposited in the General Microbiology Center of China Microbial Culture Collection Management Committee, the preservation number is CGMCC NO: 11610, the preservation name is Isaria japonica, and the preservation date is November 27, 2015, The address of the preservation unit is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

In one aspect, the present invention provides a cultivating material for culturing the fruiting body of Conychosporium japonicus, the cultivating material contains Coix seed.

The research team has proved through extensive experiments that Coix seed can significantly increase the fruiting body yield of C. japonicus, and the prepared C. japonicus fruiting body is more nutritious and contains higher polysaccharides such as mannitol and ergosterol.

Coix seed is a traditional health food for both medicine and food in my country. Coix seed has the functions of diuretic and swelling, invigorating spleen and removing dampness, clearing heat and expelling pus, anti-inflammatory and analgesic, and enhancing immunity. Coix seed is rich in polysaccharides, has the effect of lowering blood sugar, and contains more than 85% unsaturated fatty acids. The research team was surprised to find that the nutrients of coix seed are more easily utilized by C. japonicus, which can significantly improve the sporophyte of C. japonicus. output and quality.

Further, calculated by mass fraction, the addition amount of Coix seed in the cultivation material is 10-20%. Studies have shown that the addition of 10-20% of barley can significantly increase the yield of Japanese sporozoites.

Further, calculated by mass fraction, the addition amount of Coix seed in the cultivation material is 15-16%.

Further, calculated in parts by mass, the cultivation material comprises: glucose 2%, peptone 0.5%, ammonium nitrate 0.3%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, calcium carbonate 0.05%, agar 2%, plus Coix seed 15-16%, the rest is distilled water.

On the other hand, the present invention provides a method for culturing the fruiting body of Fasciola japonicus, which uses the above-mentioned culturing material to cultivate the fruiting body of Fasciola japonicus.

Further, the cultivating material is divided into a culturing container, the filling amount is 1/4 of the capacity of the culturing container, 120 ℃ of autoclave sterilization for 90min, access to 1% seed liquid, the culturing temperature is 28 ℃--35 ℃, the culture humidity is was 70%-85%, and the culture time was 10-15 days.

Further, the culture container is a fresh-keeping box or a culture bottle, wherein the fresh-keeping box is tightly closed by a lid for cultivation, and the culture bottle is cultivated by sealing with a plastic cloth.

Further, the culturing method further includes bacterial activation and seed liquid culture, and the seed liquid culture includes a first-stage medicine bottle seed liquid culture, a second-stage medicine bottle seed liquid culture and a seed tank fermentation culture.

Further, the culturing method comprises the following steps:

(1) Strain activation

Inoculate the Corynebacterium japonicum strain into the test-tube parent medium, and cultivate it in a constant temperature incubator at 23 to 25°C for 5 to 7 days to obtain the activated Corynebacterium japonicum strain;

The preparation method of the mother culture medium is as follows: take 200 grams of potatoes, wash them, cut them into small pieces, add distilled water, boil in boiling water for 30 minutes, take the boiled juice and 20 grams of glucose, 3 grams of potassium dihydrogen phosphate, and 1.5 grams of magnesium sulfate. gram, 20 grams of agar, add distilled water to make up to 1000 ml, pH is natural, autoclave at 120 ℃ for 90 minutes, and use it after the temperature drops below 26-30 ℃;

(2) First-class shake flask seed culture

Pick 3 to 4 pieces of 2mm medium-free mycelium from the activated Corynebacterium japonicum culture medium, transfer them to a conical flask equipped with a first-class shake flask medium, and culture for 3 for 2 to 3 days, then placed on a shaking table, and shaken at 23 to 25°C at 90 to 120 r/min for 2 to 3 days to obtain a secondary shake flask strain;

The preparation method of the first-stage shake flask medium is: weigh 20 grams of sucrose, 15 grams of corn flour, 5 grams of starch, 4 grams of glutamic acid, 4 grams of ammonium nitrate, 1 gram of potassium dihydrogen phosphate, and 0.5 grams of magnesium sulfate. , 0.5 g of calcium carbonate, add distilled water to make up to 1000 ml, divide the prepared medium into conical flasks, the filling amount is 1/5 to 2/5 of the capacity of the conical flask, use cotton plugs and kraft paper Seal the bottle mouth, autoclave at 120°C for 90 minutes, and use it after the temperature drops below 26-30°C;

(3) Secondary shake flask seed culture

Inoculate the secondary shake flask strain into the conical flask containing the secondary shake flask medium, the inoculation amount is 10-20%, place it on a shaker after inoculation, and shake at 23-25°C and 120 rpm. Cultivate for 2 to 3 days to obtain the seed tank strain liquid;

The preparation method of the secondary shake flask medium is: weigh 20 grams of sucrose, 15 grams of corn flour, 5 grams of starch, 5 grams of glutamic acid, 3 grams of ammonium nitrate, 1 gram of potassium dihydrogen phosphate, and 0.5 grams of magnesium sulfate. , 0.5 g of calcium carbonate, add distilled water to make up to 1000 ml, divide the prepared medium into conical flasks, the filling amount is 1/5 to 2/5 of the capacity of the conical flask, use cotton plugs and kraft paper Seal the bottle mouth, autoclave at 120°C for 90 minutes, and use it after the temperature drops below 26-30°C;

(4) Fermentation culture in seed tank

The seed tank strain liquid is inoculated into the seed tank equipped with the seed tank fermentation medium, and the inoculation amount is 10-20%. Fermentation and culture under the condition of min ratio of 0.5:1 for 3 to 4 days to obtain fermentor strain liquid;

The preparation method of the seed tank fermentation medium is: weighing 25 grams of sucrose, 15 grams of corn flour, 5 grams of starch, 7 grams of glutamic acid, 4 grams of ammonium nitrate, 1 gram of potassium dihydrogen phosphate, 0.5 grams of magnesium sulfate, 0.5 g of calcium carbonate, add distilled water to make up to 1000 ml, divide the prepared medium into conical flasks, the filling amount is 1/5 to 2/5 of the capacity of the conical flask, and tie with cotton plugs and kraft paper Seal the bottle mouth, autoclave at 120°C for 90 minutes, and use it after the temperature drops below 26-30°C;

(5) Cultivation of fruiting bodies

Use a pipette to extract 10 ml of the fermenter strain liquid, and inoculate it into a fresh-keeping box or a culture bottle containing the cultivation material. The fresh-keeping box is cultivated with the lid tightly closed, and the culture bottle is sealed with plastic cloth for cultivation; put it into the incubator 28°C--35°C, relative air humidity 70%-85%, culture for 10-15 days, take out the bottle, separate the fruiting body and the culture medium;

The preparation method of the described cultivation material is to take by weighing 20 grams of glucose, 5 grams of peptone, 3 grams of ammonium nitrate, 1 gram of potassium dihydrogen phosphate, 0.5 grams of magnesium sulfate, 0.5 grams of calcium carbonate, and 20 grams of agar, and add 150-100 grams of coix seed. 160g, add distilled water to make the volume to 1000ml, divide the prepared medium into glass bottles, the filling amount is 1/4 of the glass bottle capacity, seal the bottle with kraft paper, and autoclave at 120 ℃ for 90 minutes , and use it after the temperature drops below 26-30°C.

Further, the medium charging coefficient of the seed tank is 60-70%, and the gas introduced is a clean and sterile dry gas that has been sterilized, dedusted and dewatered.

In yet another aspect, the present invention provides the use of Coix seed in the preparation of fruiting body culture material for Conchosporium japonicus.

The beneficial effects of the present invention are:

1. By adding coix seed to the cultivation material for the cultivation of the fruiting bodies of Conychocystis japonicus, the yield of the fruiting bodies of Conychocystis japonicus is greatly improved, the process is simple, the production cost of artificially cultivated Concylosporium japonicum is significantly reduced, and the scale can be satisfied. cyclical production requirements;

2. The prepared fruit body of C. japonicus is more nutritious, has the unique efficacy characteristics of Macrosporidia, and has higher content of polysaccharides such as mannitol and ergosterol.

Description of drawings

Fig. 1 is the relational schematic diagram of the output of Coix seed amount provided in Example 3 and the output of Japanese coryneform sporozoites

Detailed ways

The present invention will be described in further detail below with reference to the examples. It should be noted that the following examples are intended to facilitate the understanding of the present invention, but do not have any limiting effect on it. The reagents and materials used in this example are all known products, obtained by purchasing commercially available products.

The artificial culture of embodiment 1 Japanese cortex sporozoite

The present embodiment adopts Corynebacterium japonica strain ZQY001, and the artificial cultivation process of fruiting body comprises the following steps:

(1) Strain activation

Cut 1 to 2 pieces of 2mm Corynebacterium japonica strain ZQY001 and inoculate them into the test-tube parent medium for bacterial activation and culture, and culture them in a constant temperature incubator at 23 to 25 °C for 5 to 7 days. Sporocystis flower thalli, to obtain activated Corynesporum japonicus species.

The preparation method of the mother culture medium is as follows: take 200 grams of potatoes, wash them, cut them into small pieces, add distilled water, boil in boiling water for 30 minutes, take the boiled juice and 20 grams of glucose, 3 grams of potassium dihydrogen phosphate, and 1.5 grams of magnesium sulfate. g, 20 g of agar, add distilled water to make up to 1000 ml, pH is natural, autoclave at 120°C for 90 minutes, and use it after the temperature drops below 26-30°C.

(2) First-class shake flask seed culture

Pick 3 to 4 pieces of 2mm medium-free mycelium from the activated Corynebacterium japonicum culture medium, transfer them to a conical flask equipped with a first-class shake flask medium, and culture for 3 For 2 to 3 days, the mixture is placed on a shaking table and shaken for 2 to 3 days at 23-25° C. and 90-120 r/min to obtain a secondary shake-flask inoculum containing a large amount of mycelium.

The preparation method of the first-stage shake flask medium is: weigh 20 grams of sucrose, 15 grams of corn flour, 5 grams of starch, 4 grams of glutamic acid, 4 grams of ammonium nitrate, 1 gram of potassium dihydrogen phosphate, and 0.5 grams of magnesium sulfate. , 0.5 g of calcium carbonate, add distilled water to make up to 1000 ml, divide the prepared medium into conical flasks, the filling amount is 1/5 to 2/5 of the capacity of the conical flask, use cotton plugs and kraft paper Seal the bottle mouth, autoclave at 120°C for 90 minutes, and use it when the temperature drops below 26-30°C.

(3) Secondary shake flask seed culture

Inoculate the secondary shake flask strain into the conical flask containing the secondary shake flask medium, the inoculation amount is 20%, place it on the shaker after inoculation, and shake it at 23-25°C and 120 rpm for 2 ~3 days, a seed pot inoculum solution containing a large amount of mycelium was obtained.

The preparation method of the secondary shake flask medium is: weigh 20 grams of sucrose, 15 grams of corn flour, 5 grams of starch, 5 grams of glutamic acid, 3 grams of ammonium nitrate, 1 gram of potassium dihydrogen phosphate, and 0.5 grams of magnesium sulfate. , 0.5 g of calcium carbonate, add distilled water to make up to 1000 ml, divide the prepared medium into conical flasks, the filling amount is 1/5 to 2/5 of the capacity of the conical flask, use cotton plugs and kraft paper Seal the bottle mouth, autoclave at 120°C for 90 minutes, and use it when the temperature drops below 26-30°C.

(4) Fermentation culture in seed tank

The seed tank strain liquid is inoculated into the seed tank equipped with the seed tank fermentation medium (the medium loading coefficient is 65%), and the inoculation amount is 15%. Fermentation and culture for 3 to 4 days under the condition that the ratio of V/V·min is 0.5:1 to obtain a fermentor strain liquid containing a large amount of mycelium; the gas introduced is sterilized, dedusted and removed. Clean and sterile dry gas for water treatment.

The preparation method of the seed tank fermentation medium is: weighing 25 grams of sucrose, 15 grams of corn flour, 5 grams of starch, 7 grams of glutamic acid, 4 grams of ammonium nitrate, 1 gram of potassium dihydrogen phosphate, 0.5 grams of magnesium sulfate, 0.5 g of calcium carbonate, add distilled water to make up to 1000 ml, divide the prepared medium into conical flasks, the filling amount is 1/5 to 2/5 of the capacity of the conical flask, and tie with cotton plugs and kraft paper Seal the bottle, autoclave at 120°C for 90 minutes, and use it after the temperature drops below 26-30°C.

(5) Cultivation of fruiting bodies

Use a pipette to extract 10 ml of the fermenter strain liquid, and inoculate it into a fresh-keeping box or a culture bottle containing the cultivation material. The fresh-keeping box is cultivated with the lid tightly closed, and the culture bottle is sealed with plastic cloth for cultivation; put it into the incubator 28°C--35°C, relative air humidity 70%-85%, culture for 10-15 days, take out the bottle, separate the fruiting body and the culture medium;

The preparation method of the described cultivation material is to take by weighing 20 grams of glucose, 5 grams of peptone, 3 grams of ammonium nitrate, 1 gram of potassium dihydrogen phosphate, 0.5 grams of magnesium sulfate, 0.5 grams of calcium carbonate, 20 grams of agar, and 160 grams of barley seed. Add distilled water to make the volume to 1000 ml, divide the prepared culture medium into glass bottles, the filling amount is 1/4 of the glass bottle capacity, seal the bottle mouth with kraft paper, autoclave at 120 ℃ for 90 minutes, wait for Use after the temperature drops below 26-30°C.

The effect of embodiment 2 Coix seed on Japanese cortex sporophyte culture

The present embodiment adopts the method for culturing the sporophyte of Japanese coryneform spores provided in Example 1, and simultaneously carries out the cultivation of multiple groups of comparative examples. , rice, soybean, silkworm chrysalis, corn, yam, potato were replaced, and the output of Japanese stick bunches with different cultivation materials, as well as the content of polysaccharide, mannitol, ergosterol and aspartic acid in the fruiting bodies were investigated.

The detection method of the polysaccharide content is as follows: the spore suspension with a concentration of 107/ml is placed in a liquid medium with a concentration of 20% with an inoculum of 7%, at pH 7, 27°C and a rotating speed of 150r/min. Fermentation was carried out for 7 days, and then the polysaccharide content was determined by the sulfuric acid-anthrone method.

The detection method of mannitol content is as follows: take about 1 g of the test sample, accurately weigh it, add 25 mL of water accurately, weigh it, sonicate at 50°C for 60 min, let it cool, weigh it again, make up for the lost weight with water, and centrifuge. , take 10 mL of the subsequent filtrate into an evaporating dish, evaporate to dryness in a water bath, dissolve the residue in a mobile phase and dilute to a 10 mL volumetric flask, filter through a 0.45 μm microporous membrane, and take the subsequent filtrate for HPLC analysis.

The detection method of ergosterol content is as follows: accurately weigh 1g of the sample, place it in a 10mL volumetric flask, add 1mol/L NaOH, 50% methanol solution, ultrasonically mix, and place in a 60°C water bath for 1hr, then centrifuge at 14000r/min for 20min, and take the supernatant. Add 5 mL of HCl3 to extract 2 mL of the extract, dry it with nitrogen and dissolve it in 1 mL of methanol, filter it through a 0.45 μm microporous membrane, and use the filtrate for HPLC analysis. Use chloroform as a solvent to prepare a series of ergosterol standards. solution.

The detection method of aspartic acid content is as follows: accurately weigh a certain amount of samples with good uniformity, put them into the hydrolysis tube, add 10-15mL of 6mol/L hydrochloric acid into the hydrolysis tube, and add an equal volume to the samples with high water content. Add 3-4 drops of freshly distilled phenol, then put the hydrolysis tube into the refrigerant, freeze for 3-5min, connect it to the suction tube of the vacuum pump, evacuate (close to 0Pa), and then fill with high-purity nitrogen , and then vacuum and fill with nitrogen. After repeating three times, seal or tighten the screw cap in the state of nitrogen filling. Put the sealed hydrolysis tube in a constant temperature box of 110 °C, and after hydrolysis for 22 hours, take it out and cool it; open the hydrolysis tube and filter the hydrolyzate. Then, rinse the hydrolysis tube several times with deionized water, transfer all the hydrolyzed solution to a 50mL volumetric flask and dilute to volume with deionized water; draw 1mL of green leaves into a 5mL volumetric flask, and use a vacuum desiccator to dry at 40-50 ℃, and the residual The compound was dissolved in 1-2 mL of water, then dried, repeated twice, evaporated to dryness, and dissolved in 1 mL of pH 2.2 buffer; 0.2 mL of mixed amino acid solution was accurately taken and diluted to 5 mL with pH 2.2 buffer; The amino acid content of the sample was determined by the external standard method of the automatic analyzer.

The specific investigation results are shown in Table 1.

Table 1. Effects of different cultivating materials on the culture of Japanese sporozoites

As can be seen from Table 1, compared with the blank control of the second group, when Coix seed was added to the cultivating material, the output of the fruiting bodies of Trichosporium japonicus was increased from 8.1g/50g medium to 25.3g/50g, and at the same time. The polysaccharides, mannitol, ergosterol, and aspartic acid in the fruiting bodies were all significantly improved; compared with groups 3-9, when wheat, rice, soybean, silkworm chrysalis, corn, yam, and potato were added, the effect of the Although the cultivation of the fruiting bodies of spores is also slightly effective, the effect is not as good as that of barley. This may be because the oil content of barley is lower, and the dietary fiber and fatty acids in it are more easily utilized by Trichosporium japonicus; The content of polysaccharide and fiber is high, which is difficult to be decomposed and utilized by C. japonicus; the oil content of soybean and silkworm chrysalis is high, and the types of fatty acids are not conducive to the cultivation of C. japonicus.

Example 3 The effect of different dosages of Coix seed on the cultivation of fruiting bodies

In this example, the culture method of Japanese coryneform spores provided in Example 1 is used for culture, and the addition ratio of Coix seed is 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17% respectively. , 18%, 19%, 20%, that is, in every 50g of cultivation material, the addition of Coix seed is 5g, 5.5g, 6g, 6.5g, 7g, 7.5g, 8g, 8.5g, 9g, 9.5g, 10g, respectively. The yield of the fruiting bodies of Japanese barley bunches, and the content of polysaccharides, mannitol, ergosterol and aspartic acid in the fruiting bodies of different addition ratios of Coix seed were investigated. The detection methods of polysaccharide content, mannitol, ergosterol and aspartic acid content are as described in Example 2. The specific investigation results are shown in Table 2, and the schematic diagram of the relationship between the output of Japanese sporozoites and the amount of Coix seed is shown in Figure 1.

Table 2. The effect of different dosages of Coix seed in the cultivation material on the culture of Japanese cortex sporozoites

It can be seen from Table 1 that different dosages of Coix seed have a certain influence on the culture of Japanese sporozoites. When the amount of barley increased from 10% to 16%, the output of Japanese sporozoites increased from 11.2g. /50g medium, increased to 25.3g/50g medium, the polysaccharide content, mannitol, ergosterol, and aspartic acid content in the fruiting body also increased to a certain extent, but with the continued addition of Coix seed When it continued to increase from 16% to 20%, the output of Japanese sporophytes began to show a downward trend, and the content of polysaccharides, mannitol, ergosterol, and aspartic acid in the fruit bodies also appeared to a certain extent. Decline. It can be seen that the best addition ratio of coix seed should be 15-16%, and the most preferred is 16%, that is, 8g coix seed/50g cultivation material.

Embodiment 4 Comparison of the nutritional components of Japanese coryneform sporophyte and natural cicada flower Cordyceps

In this example, the Corynebacterium japonica obtained in Example 1 and the natural Cordyceps sinensis (purchased from) were used to compare the contents of polysaccharide, mannitol, ergosterol and aspartic acid. The detection method of amino acid content is as described in Example 2. The specific investigation results are shown in Table 3.

Table 3. Comparison of nutritional components between the sporophyte and natural Cordyceps sinensis

serial number Variety Polysaccharide (mg/g) Mannitol (mg/g) Ergosterol (mg/g) Aspartic acid (%) 1 Corynebacterium japonicus 34.56±0.75 81.9±1.12 0.6256±0.0309 0.04 2 Natural Cicada Cordyceps 28.5±0.95 53.6±1.16 0.5743±0.018 0.02

As can be seen from Table 3, the present invention is compared with the natural Cordyceps sinensis by artificial cultivation of Trichosporium japonica, wherein the polysaccharide, mannitol, ergosterol, aspartic acid content are all significantly higher, and the functional component content is more, It will also have more health-care functions, and the market prospect is broad.

Although the present invention is disclosed above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be based on the scope defined by the claims.

Claims (10)

1. the cultivation material of the fruiting body culture of Trichosporium japonicus, it is characterised in that in the described cultivation material, contains Coix seed. 2. cultivating material as claimed in claim 1, is characterized in that, in the cultivating material, the addition of Coix seed is 10-20%, calculated by mass fraction. 3. cultivating material as claimed in claim 1, is characterized in that, in the cultivating material, the addition of Coix seed is 15-16%. 4. cultivating material as claimed in claim 3, is characterized in that, according to mass fraction, described cultivating material comprises: glucose 2%, peptone 0.5%, ammonium nitrate 0.3%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.05%, calcium carbonate 0.05%, agar 2%, barley 15-16%, and the rest are distilled water. 5 . A method for culturing the fruiting body of Trichosporium japonicum, characterized in that the cultivation material of any one of claims 1-4 is used to cultivate the fruiting body of Trichosporium japonicum. 6 . 6. culturing method as claimed in claim 5, is characterized in that, the cultivating material is sub-packed in the cultivating container, the filling amount is 1/4 of the culturing container capacity, 120 ℃ of autoclave sterilization 90min, insert 1% seed liquid , the culture temperature is 28°C--35°C, the culture humidity is 70%-85%, and the culture time is 10-15 days. 7. The culture method of claim 6, wherein the culture container is a fresh-keeping box or a culture bottle, wherein the fresh-keeping box is cultivated by tightly closing the lid, and the culture bottle is cultivated by sealing with plastic cloth. 8. The culturing method according to any one of claims 5-7, wherein the culturing method also comprises strain activation and seed liquid culture, and the seed liquid culture comprises first-level medicine bottle seed liquid culture, two Grade medicine bottle seed liquid culture and seed tank fermentation culture. 9. culture method as claimed in claim 8, is characterized in that, described culture method comprises the following steps: (1) Strain activation Inoculate the Corynebacterium japonicum strain into the test-tube parent medium, and cultivate it in a constant temperature incubator at 23 to 25°C for 5 to 7 days to obtain the activated Corynebacterium japonicum strain; The preparation method of the mother culture medium is as follows: take 200 grams of potatoes, wash them, cut them into small pieces, add distilled water, boil in boiling water for 30 minutes, take the boiled juice and 20 grams of glucose, 3 grams of potassium dihydrogen phosphate, and 1.5 grams of magnesium sulfate. gram, 20 grams of agar, add distilled water to make up to 1000 ml, pH is natural, autoclave at 120 ℃ for 90 minutes, and use it after the temperature drops below 26-30 ℃; (2) First-class shake flask seed culture Pick 3 to 4 pieces of 2mm medium-free mycelium from the activated Corynebacterium japonicum culture medium, transfer them to a conical flask equipped with a first-class shake flask medium, and culture for 3 for 2 to 3 days, then placed on a shaking table, and shaken at 23 to 25°C at 90 to 120 r/min for 2 to 3 days to obtain a secondary shake flask strain; The preparation method of the first-stage shake flask medium is: weigh 20 grams of sucrose, 15 grams of corn flour, 5 grams of starch, 4 grams of glutamic acid, 4 grams of ammonium nitrate, 1 gram of potassium dihydrogen phosphate, and 0.5 grams of magnesium sulfate. , 0.5 g of calcium carbonate, add distilled water to make up to 1000 ml, divide the prepared medium into conical flasks, the filling amount is 1/5 to 2/5 of the capacity of the conical flask, use cotton plugs and kraft paper Seal the bottle mouth, autoclave at 120°C for 90 minutes, and use it after the temperature drops below 26-30°C; (3) Secondary shake flask seed culture Inoculate the secondary shake flask strain into the conical flask containing the secondary shake flask medium, the inoculation amount is 10-20%, place it on a shaker after inoculation, and shake at 23-25°C and 120 rpm. Cultivate for 2 to 3 days to obtain a seed tank strain solution; The preparation method of the secondary shake flask medium is: weigh 20 grams of sucrose, 15 grams of corn flour, 5 grams of starch, 5 grams of glutamic acid, 3 grams of ammonium nitrate, 1 gram of potassium dihydrogen phosphate, and 0.5 grams of magnesium sulfate. , 0.5 g of calcium carbonate, add distilled water to make up to 1000 ml, divide the prepared medium into conical flasks, the filling amount is 1/5 to 2/5 of the capacity of the conical flask, use cotton plugs and kraft paper Seal the bottle mouth, autoclave at 120°C for 90 minutes, and use it after the temperature drops below 26-30°C; (4) Fermentation culture in seed tank The seed tank strain liquid is inoculated into the seed tank equipped with the seed tank fermentation medium, and the inoculation amount is 10-20%. Fermentation and culture under the condition of min ratio of 0.5:1 for 3 to 4 days to obtain fermentor strain liquid; The preparation method of the seed tank fermentation medium is: weighing 25 grams of sucrose, 15 grams of corn flour, 5 grams of starch, 7 grams of glutamic acid, 4 grams of ammonium nitrate, 1 gram of potassium dihydrogen phosphate, 0.5 grams of magnesium sulfate, 0.5 g of calcium carbonate, add distilled water to make the volume to 1000 ml, divide the prepared medium into conical flasks, the filling amount is 1/5 to 2/5 of the capacity of the conical flask, tie with cotton plugs and kraft paper Seal the bottle mouth, autoclave at 120°C for 90 minutes, and use it after the temperature drops below 26-30°C; (5) Cultivation of fruiting bodies Use a pipette to extract 10 ml of the fermenter strain liquid, and inoculate it into a fresh-keeping box or a culture bottle containing the cultivation material. The fresh-keeping box is cultivated with the lid tightly closed, and the culture bottle is sealed with plastic cloth for cultivation; put it into the incubator 28°C--35°C, relative air humidity 70%-85%, culture for 10-15 days, take out the bottle, separate the fruiting body and the culture medium; The preparation method of the described cultivation material is to take by weighing 20 grams of glucose, 5 grams of peptone, 3 grams of ammonium nitrate, 1 gram of potassium dihydrogen phosphate, 0.5 grams of magnesium sulfate, 0.5 grams of calcium carbonate, and 20 grams of agar, and add 150-100 grams of coix seed. 160g, add distilled water to make the volume to 1000ml, divide the prepared medium into glass bottles, the filling amount is 1/4 of the glass bottle capacity, seal the bottle with kraft paper, and autoclave at 120 ℃ for 90 minutes , and use it after the temperature drops below 26-30°C. 10 . The cultivation method according to claim 9 , wherein the medium charging coefficient of the seed tank is 60-70%, and the gas introduced is a clean and sterile drying process that has been sterilized, dust-removed and water-removed. 11 . gas.

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