CN107227263B

CN107227263B - White flammulina velutipes JK01 strain and application thereof

Info

Publication number: CN107227263B
Application number: CN201710555226.1A
Authority: CN
Inventors: 张根伟; 刘振国; 李书生; 马宏; 刘昆昂; 尹淑丽
Original assignee: Institute of Biology of Hebei Academy of Sciences
Current assignee: Institute of Biology of Hebei Academy of Sciences
Priority date: 2017-07-10
Filing date: 2017-07-10
Publication date: 2019-12-31
Anticipated expiration: 2037-07-10
Also published as: CN107227263A

Abstract

The invention belongs to the technical field of edible fungi, and particularly relates to a white flammulina velutipes JK01 strain and application thereof. The needle mushroom provided by the invention is named as JK01, is obtained by hybridizing strain Taiwan white and strain miscellaneous 19 serving as parents and performing systematic breeding, and belongs to needle mushroom (II)Flammulina velutipes) Deposited at the institute of microbiology, academy of sciences of china, address: no. 3 of Xilu No.1 of Beijing, Chaoyang, and CGMCC No.13877, with a preservation date of 20170614. Compared with the prior art, the flammulina velutipes JK01 strain provided by the invention is short in culture and cultivation period, consistent in growth, strong in stress resistance and high in yield, and is suitable for industrial cultivation; the stipe is thick, the appearance characteristic is excellent, the taste is crisp and refreshing, the product is very favorable for splitting and cooking, the comprehensive performance of JK01 is good, and the market prospect is wide.

Description

White flammulina velutipes JK01 strain and application thereof

Technical Field

The invention belongs to the technical field of edible fungi, and particularly relates to a white flammulina velutipes JK01 strain and application thereof.

Background

The golden mushroom is known as flammulina velutipes (Fr.) Sing, and is a delicious food and a better health-care food. The needle mushroom is cold in nature, sweet in taste and salty in flavor, has the effects of tonifying liver, benefiting intestines and stomach and resisting cancer, and is mainly used for treating diseases such as liver diseases, gastrointestinal inflammation, ulcer, tumors and the like. The flammulina velutipes has higher zinc content, and is helpful for preventing male prostate diseases. The needle mushroom is also a high-potassium low-sodium food, can prevent and treat hypertension, and is beneficial to the old. The content of the amino acid in the needle mushroom is very rich and higher than that in the common mushrooms, particularly the content of lysine is very high, and the lysine has the function of promoting the intelligence development of children. Recent research shows that the flammulina velutipes also has a good anticancer effect.

The needle mushroom belongs to low-temperature fruiting fungi, hypha is resistant to low temperature and weak in resistance to high temperature, and sporocarp differentiation is within the range of 3-18 ℃. The needle mushroom varieties are divided into yellow needle mushroom varieties and white needle mushroom varieties according to the color of the fruiting bodies. Yellow needle mushroom is generally cultivated by farmers, and has the characteristics of strong resistance, fast growth, thick stipe, high temperature resistance and good taste compared with white needle mushroom, such as 19, 6 and the like, but the yellow needle mushroom has dark yellow appearance, brown base and poor commodity; the white needle mushroom is mainly cultivated in factories, has more strains, is mostly a hybrid or variant variety, has white color and good commodity property, but has weaker strain resistance and lower required cultivation temperature.

At present, the industrialized cultivation of flammulina velutipes becomes the mainstream, and large-scale flammulina velutipes production enterprises such as Shanghai Xuezhi and Guangming Jiu mushroom mainly use Japanese white flammulina velutipes varieties, and a large amount of capital is spent to buy the strains every year. The strains have the characteristics of multiple branches, fine stipes, high yield and the like, but have some defects, such as multiple branches of sporocarp and adhesion of base parts, which brings inconvenience to splitting and cooking; the taste is not as crisp as yellow needle mushroom; the required growth and fruiting temperature is low, and the energy consumption is high; the requirement on equipment is high, and most domestic equipment cannot meet the ventilation and moisture preservation conditions, so that the yield is low; poor disease resistance, easy occurrence of rotten mushroom disease and the like. On the other hand, after 2-3 years of passage and production, the strains can degenerate, which is shown in that the number of chalspores is increased, the fruiting period is obviously delayed, the fruiting rate is low, the fruiting is irregular, the yield is reduced, and the like, and because of no know-how, foreign strains can only be introduced again.

Disclosure of Invention

The invention provides a white flammulina velutipes JK01 strain and application thereof, which are used for solving the problems of high requirement on strain equipment, poor resistance, long cultivation period, more branches, difficult splitting and the like.

The white flammulina velutipes strain provided by the invention is named as JK01, is obtained by hybridizing white and yellow varieties 19 of Taiwan white varieties serving as parents and performing systematic breeding, and belongs to flammulina velutipes (A)Flammulina velutipes) The culture medium is preserved in China general microbiological culture Collection center, and the address is as follows: no. 3 Xilu No.1 Beijing, Chaoyang, Beijing, has a preservation number of CGMCC No.13877 and a preservation date of 20170614.

Further, spores, mycelia and fruit bodies obtained by culturing the flammulina velutipes JK01 strain also belong to the scope of the present invention.

Furthermore, the application of the golden mushroom JK01 as a parent in crossbreeding and the application of the golden mushroom JK01 fruiting body in food processing also belong to the protection scope of the invention.

The golden mushroom JK01 provided by the invention has the following morphological characteristics:

the fruit body is white. The number of branches is 456 on average, the pileus is hemispherical and thick, the pileus is not easy to open, and the average diameter of the pileus is 4.13 mm; the average length of the stipe is 16.6cm, the average diameter of the stipe is 2.96mm, the size is uniform, and the base part has no villus and is not adhered; printing the spores with white; the meat is crisp and tender. The mycelia are dense, no powdery spores exist, and the aerial mycelia are not developed.

The invention also provides a culture method of the flammulina velutipes JK01, wherein the stock, liquid and cultivation temperature of the flammulina velutipes JK01 are 14-25 ℃.

The invention also provides a cultivation method of the needle mushroom JK01 fruiting body, wherein a cultivation medium comprises 34-36% of cultivation culture materials and 64-66% of water in percentage by mass, and the cultivation culture materials comprise 20-35% of rice bran, 5-15% of wheat bran, 5-10% of bean cake powder, 30-50% of corn cob, 3-5% of corn flour, 3-8% of cottonseed hull, 3-8% of beet pulp, 1-3% of calcium carbonate and 0.3-0.8% of shell fossil.

The specific cultivation process can refer to the prior art, and a specific process is also given below and comprises the following steps:

1) preparing liquid strains: and (3) inoculating the needle mushroom JK01 PDA test tube seeds into a triangular flask containing a liquid seed culture medium under the aseptic condition, carrying out shake cultivation at the temperature of 25 ℃ and the rotating speed of 140r/min, finishing shake cultivation when the liquid culture medium is filled with mycelium pellets, and uniformly stirring the mycelium pellets by using a magnetic stirrer to obtain a needle mushroom mixed mycelium liquid.

2) Spawn running: inoculating the mixed mycelium liquid of the flammulina velutipes on the culture material of a culture bottle under the aseptic condition, sealing the culture material with a ventilating cover, and placing the culture material in a culture chamber for dark culture; the early culture temperature is 18-20 deg.C, the late culture temperature is 12-16 deg.C, and the humidity is 55-65%. After the hypha grows over the cultivation bottle, scraping the hypha on the surface layer of the bottle mouth to remove the hypha to stimulate fruiting;

3) and (3) fruiting body formation: and (3) transferring the cultivation bottle subjected to mycelium stimulation into a growing room, wherein the initial temperature is 18 ℃, the cultivation bottle is gradually decreased according to days, and finally the cultivation bottle is maintained at 6-8 ℃ and the humidity is 85-95%. Sleeving when the sporophore is 2-3cm higher than the bottle mouth, and irradiating for 2 days with 150lux light for 8h each day. CO2 concentration 2000-9000 ppm, gradually increasing with the growth of fruiting body, and collecting when the stipe reaches 16-18cm。

Wherein, the liquid seed culture medium has the following formula: 3.3g/L of soybean meal, 20g/L of cane sugar, 0.66g/L of magnesium sulfate heptahydrate and 0.66g/L of dipotassium hydrogen sulfate, and adding water to fix the volume to 1L. pH = 6.2-6.5.

Compared with the prior art, the golden mushroom JK01 provided by the invention has the advantages of short culture and cultivation period, strong disease resistance, neat fruiting, high temperature resistance and high yield, and greatly improves the production efficiency of factories. And the stipe is coarse, the meat quality is crisp and tender, and the method is very beneficial to the resolution and cooking of the sporocarp. In conclusion, the golden mushroom JK01 is good in comprehensive performance and wide in market prospect.

Drawings

FIG. 1 is an ISSR method authenticity analysis of the white flammulina velutipes JK01 strain;

FIG. 2 shows the confronting culture of the strain JK01 and Trichoderma viride;

FIG. 3 shows the confronting culture of the white flammulina velutipes strain JK01 and Pseudomonas aeruginosa;

FIG. 4 shows the morphological characteristics of the white needle mushroom JK01 strain, trash 19 strain and Taiwan white mushroom picked in the same culture period;

FIG. 5 shows the fruiting morphological characteristics of the white needle mushroom JK01 strain in industrial cultivation.

FIG. 6 shows the fruiting body of commercially available needle mushroom.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The formulations of the various media used in the following examples are as follows:

mother culture medium (PDA medium): every 1L of culture medium is prepared from potato 200g, glucose 20g, and agar 12 g;

liquid seed culture medium: 3.3g/L of soybean meal, 20g/L of cane sugar, 0.66g/L of magnesium sulfate heptahydrate and 0.66g/L of dipotassium hydrogen sulfate, and adding water to fix the volume to 1L. pH = 6.2-6.5;

cultivation medium: 20-35% of rice bran, 5-15% of bran, 5-10% of bean cake powder, 30-50% of corncobs, 3-5% of corn flour, 3-8% of cottonseed hulls, 3-8% of beet pulp, 1-3% of calcium carbonate and 0.3-0.8% of shell fossil.

Example 1 hybridization JK01

A cross breeding method is adopted to obtain a flammulina velutipes strain JK 01.

The method comprises the following specific steps:

1) selecting a parent strain: selecting white and miscellaneous 19 of a strain Taiwan as parents, wherein sporocarp of the two parents can be purchased in the market;

2) preparing a mother culture medium, a liquid culture medium and a cultivation culture medium: the formulation of each culture medium is given, and is not described in detail herein;

3) obtaining 200 heterozygotes A by adopting a conventional crossbreeding method;

4) cultivation and screening: the obtained heterozygotes are subjected to cultivation tests according to a conventional method, and a target strain Jinza 9 is obtained from 200 heterozygotes A through systematic screening, wherein the strain has the advantages of fast hypha growth, strong resistance, early fruiting, high yield, light yellow color, thick stipe and difficult umbrella opening.

5) Collecting spores of the Jinza 9 strain, and performing selfing by the same method to obtain 80 heterozygotes B;

6) cultivation and screening: the obtained heterozygote B is subjected to a cultivation test according to a conventional method, a target strain JK01 is obtained from 80 heterozygotes B through systematic screening, and the strain has the advantages of quick hypha growth, early fruiting, high yield, pure white color, thick stipe, difficult umbrella opening and consistent fruiting, and meets the requirement of factory cultivation.

Example 2 Strain antagonism experiment and ISSR Authenticity analysis

The golden mushroom JK01 and the two parent strains Taiwan Baiza 19 are used as test materials, and the used culture medium is a PDA culture medium.

JK01 and Taiwan white, JK01 and miscellaneous 19 were inoculated and cultured on a plate medium in a manner of facing each other, the distance between the inoculation points was 4cm, and the culture was carried out at a constant temperature of 25 ℃ for 15 days. The results show that the antagonism of JK01 and Taiwan white and the antagonism of JK01 and miscellaneous 19 are obvious at the boundary of the culture colonies cultured on the opposite sides, which indicates that JK01 is the flammulina velutipes strain different from Taiwan white and miscellaneous 19.

ISSR method authenticity analysis of JK01 strain

In order to prove the differences of the JK01 strain from the Homophilus Kogyo Baizai 19 and the existing white flammulina velutipes variety F21 and Nikken gold, strain authenticity analysis is carried out. According to the edible fungus strain authenticity identification ISSR method of the agricultural industry standard of the people's republic of China and the existing literature reports, 8 primers with obvious amplification difference, high polymorphism and good reproducibility are screened from 15 primers. The names and sequences of the primers are shown in Table 1, and the clustering analysis results are shown in FIG. 1.

TABLE 1 primers

。

As can be seen from the results of the cluster analysis, JK01 has obvious differences from Taiwan Bai, Taiwan Bai 19, Taiwan F21 and Taijin, and belongs to different strains. In addition, the differences between Taibai and F21 are not significant, and whether the Taibai and F21 are the same strain or not is awaited for further study.

Example 3 disease resistance test

Trichoderma viride (Trichoderma viride) and pathogenic pseudomonas aeruginosa (p.aeroginosa) are major diseases in the cultivation of flammulina velutipes. The method is characterized in that flammulina velutipes pathogenic bacteria trichoderma viride and pseudomonas aeruginosa which are separated and preserved in our unit are used as indicators for opposite culture. The disease resistance is judged by observing the growth of the colony and the width of an antagonistic zone, and the narrower the antagonistic zone is, the stronger the disease resistance is.

Activating strains: respectively carrying out activation culture on the flammulina velutipes strain and the trichoderma viride strain in a PDA culture medium at 25 ℃, and growing in a full dish for later use. Before use as a seed, a cake was prepared using a punch with a diameter of 0.5 cm. The pseudomonas aeruginosa is activated in the PDA culture medium for 2 days at 25 ℃ for standby.

The method for measuring trichoderma resistance comprises the following steps:

a quantitative culture dish with a diameter of 90mm was used, and 35mL of PDA medium was added to each dish in a quantitative amount by a quantitative peristaltic pump and coagulated. Inoculating needle mushroom cake with diameter of 0.5cm 2cm away from the center, culturing at 25 deg.C for 4 days, inoculating Trichoderma viride cake 2cm away from the center, and culturing at 25 deg.C. The width of the antagonistic line, the width of colony growth were measured, and the color, size change of the inhibition zone was continuously observed.

The method for testing the capability of resisting the pseudomonas aeruginosa comprises the following steps:

a quantitative culture dish with a diameter of 90mm was used, and 35mL of PDA medium was added to each dish in a quantitative amount by a quantitative peristaltic pump and coagulated. Inoculating needle mushroom cake with diameter of 0.5cm to a position 1.75cm away from the center, inoculating activated Pseudomonas aeruginosa to a position 1.75cm away from the center, and culturing at 25 deg.C for 4 days. The growth and antagonism of hypha are observed and recorded, and the width of the antagonism line and the growth width of colony are measured.

The results are shown in the table 2, the figure 2 and the figure 3, and the results show that the flammulina velutipes JK01 has stronger capability of resisting trichoderma viride and pseudomonas aeruginosa. Particularly, the capability of resisting pathogenic pseudomonas aeruginosa is strong, and the disease resistance is very intuitively shown under the condition that the bending degree of the growth of the culture hyphae of the confronting golden mushroom and pseudomonas aeruginosa is large, the resistance is close to the hybrid 19, and the hybrid 19 is a strain which is recognized to be strong in resistance in production. In the industrialized cultivation process of the flammulina velutipes, bacterial diseases are easy to outbreak, rotten mushroom diseases appear, and the JK01 strain has strong antibacterial capacity and can reduce the diseases, so that the flammulina velutipes strain has wide application prospect.

TABLE 2 disease resistance of the strains

。

Example 4 cultivation experiment

The golden mushroom JK01 is subjected to a culture period and culture period optimization experiment in golden mushroom factory cultivation enterprises, each cultivation is carried out for 2 ten thousand bottles, the culture period and the unit yield are compared, and the advantages and the characteristics of the golden mushroom JK01 strain are summarized.

The cultivation conditions adopted in this example were as follows:

the culture medium refers to cultivation medium, and is not described in detail.

Volume of the cultivation bottle: 1100ml, fill (dry) 280 g/bottle.

Culturing: the early culture temperature is 18-20 deg.C, the late culture temperature is 12-16 deg.C, and the humidity is 55-65%.

Fruiting: and (3) transferring the cultivation bottle subjected to mycelium stimulation into a growing room, wherein the initial temperature is 18 ℃, the cultivation bottle is gradually decreased according to days, and finally the cultivation bottle is maintained at 6-8 ℃ and the humidity is 85-95%. Sleeving when the sporophore is 2-3cm higher than the bottle mouth, and irradiating for 2 days with 150lux light for 8h each day. CO2₂The concentration is 2000-9000 ppm, the fungus stalks are harvested when the fungus stalks reach 16-18cm length along with the gradual rise of the growth of the fruit bodies。Table 3 is a list of results of JK01, Taiwan Bai and miscellaneous 19 cultivation tests.

TABLE 3 cultivation test of Flammulina velutipes JK01, Taichang Baihe Haza 19 (first crop)

。

As is clear from Table 3, the cultivation period of JK01 was 25 days and the cultivation period was 49 days. Compared with the parent Taiwan white and the hybrid 19, JK01 shortens the cultivation period and the cultivation period compared with the white needle mushroom Taiwan white. In addition, the average yield per bottle of 367.4g is also obviously increased compared with the yield per bottle of 334.5g in Taiji province, and the yield difference between bottles is not large, so that the method is suitable for factory cultivation. The biological conversion rate reaches 131.2%, and the yield is higher in the fruiting mode of 1100ml cultivation bottles.

Example 5 morphological features

The morphological characteristics of JK01 fruiting bodies were recorded as their apparent characteristics when harvested under the conditions of factory production, 25-day cultivation period, 49-day cultivation period, see FIG. 5.

The fruit body is white. The number of branches is 456 on average; the pileus is hemispherical, thick and not easy to open, and the diameter of the pileus is 3.98mm-4.21mm, and the average diameter is 4.13 mm; the length of the stipe is 12.2cm-18.0cm, the average length is 16.6cm, the diameter is 2.95-2.97mm, the average diameter is 2.96mm, the size is uniform, the base part has no fluff and is not adhered; printing the spores with white; the meat is crisp and tender. The mycelia are dense, no powdery spores exist, and the aerial mycelia are not developed.

The morphological characteristics of Taiwan Bai and miscellaneous 19 fruit bodies were recorded as their apparent characteristics when harvested under the conditions of 49 days cultivation period, see FIG. 4.

Taiwan white fungus has white fruiting body color and average branch number of 565. The JK01 fruiting body has similar morphological characteristics with Taiwan Baizi fruiting body, thicker stipe, higher yield per unit, less branch number and more crisp and refreshing taste, thereby having wide market prospect. The mixture 19 is yellow needle mushroom variety, and morphological characteristics are not described excessively here.

The stipe was slightly thicker than the commercial white needle mushroom (right side) product, as shown in FIG. 6.

Example 6 temperature gradient test for hyphal growth

The growth rate was measured by inoculating JK01, Taiwan white and miscellaneous 19 blocks having a diameter of 0.5cm on a PDA medium petri dish and culturing at 10 deg.C, 15 deg.C, 20 deg.C, 25 deg.C and 30 deg.C for 7 days, respectively, and the test results are shown in Table 4. The test result shows that: the growth speed of the hyphae of the JK01 strain is higher than that of the Taiwan white bacterial strain, the growth speed is higher at 20 ℃ and 25 ℃, and the tolerance of the high temperature of 30 ℃ is stronger.

TABLE 4 influence of temperature on growth rate of different Flammulina velutipes hyphae (mm/day)

。

The above description is only an embodiment of the present invention, and expect can understand that simple modifications and substitutions of the present invention are included in the technical idea of the present invention without departing from the concept of the present invention.

Claims

1. A white needle mushroom JK01 strain is characterized in that the preservation number is CGMCC No. 13877; the strain is resistant to trichoderma and pseudomonas aeruginosa.

2. The method for culturing the strain as claimed in claim 1, wherein the culture temperature of the stock strain of the needle mushroom JK01 is 14-25 ℃ as well as that of the cultivated strain.

3. Use of a strain according to claim 1 as a parent for cross breeding.

4. A white needle mushroom spore obtained by culturing the strain according to claim 1.

5. A white Pleurotus ostreatus mycelium, wherein the mycelium is obtained by culturing the strain of claim 1.

6. A white needle mushroom fruit body obtained by cultivating the strain of claim 1.

7. A cultivation method of fruiting bodies as claimed in claim 6, wherein the cultivation medium comprises 34-36% by mass of cultivation culture medium and 64-66% by mass of water, and the cultivation culture medium comprises 20-35% by mass of rice bran, 5-15% by mass of bran, 5-10% by mass of bean cake powder, 30-50% by mass of corn cob, 3-5% by mass of corn flour, 3-8% by mass of cotton seed hulls, 3-8% by mass of beet pulp, 1-3% by mass of calcium carbonate and 0.3-0.8% by mass of shell fossil.

8. Use of the white needle mushroom fruiting body according to claim 6 in food processing.