CN109964731B

CN109964731B - High-yield and high-efficiency culture method of Armillaria mellea

Info

Publication number: CN109964731B
Application number: CN201910411214.0A
Authority: CN
Inventors: 王忠巧; 刘朝斌; 孙友强; 徐万雷; 李才汪; 唐静
Original assignee: Yiliang Gastrodia Elata Industrial Development Center
Current assignee: Yiliang Gastrodia Elata Industrial Development Center
Priority date: 2019-05-17
Filing date: 2019-05-17
Publication date: 2021-10-15
Anticipated expiration: 2039-05-17
Also published as: CN113615482A; CN113615482B; CN109964731A

Abstract

The invention relates to the technical field of armillaria mellea cultivation, in particular to a high-yield and high-efficiency cultivation method of Armillaria mellea. The method comprises inoculating Armillaria mellea (Armillaria gallica) small grass dam No.1 (CGMCC NO.16781) in culture medium of cultivation bag, placing the culture medium and sweet potato in cultivation bed together after mycelia are overgrown, and culturing after covering soil. The invention realizes the stable fruiting of the sporocarp of the Armillaria mellea cultivated by the artificial cultivation for the first time, can increase the fruiting amount, and can be popularized and applied in industrialization.

Description

High-yield and high-efficiency culture method of Armillaria mellea

Technical Field

The invention relates to the technical field of halimasch cultivation, in particular to a cultivation method of French halimasch small grass dam No. 1.

Background

Armillaria mellea (Armillaria gallica) is a fungus of Armillaria, belongs to the genera of Basidiomycotina, Agaricales and Armillaria, and fruiting bodies, hypha and rhizomorph can be used as medicines, and the fruiting bodies have delicious taste and rich nutrition. Armillaria mellea is a wild fungus, grows at the root of coniferous trees and broad-leaved trees, has a growth period of 8-9 months per year, and is distributed in Jilin, Liaoning, Hebei, Shanxi, Gansu, Qinghai and Sichuan. At present, the natural production and the disordered picking of forest lands in the field are carried out, the natural yield is lower, and the wild resources are gradually reduced. The Armillaria mellea is a fungus of Armillaria mellea which can be mixed and planted with gastrodia elata and grifola at present, the fruiting body of the Armillaria mellea is mainly obtained by wild cultivation at present, and no report of the fruiting body of Armillaria mellea artificially cultured is provided at present.

Disclosure of Invention

The inventor of the invention separates wild gastrodia elata in small grass dam town of Yi-E county of Yunnan province to obtain a new strain of Armillaria mellea, is suitable for high-quality and high-yield cultivation of Gastrodia elata Blume, and can improve the yield and quality of Gastrodia elata Blume. The new strain of Armillaria mellea (Armillaria gallica) is named as Xiaocaba No.1, and has been preserved in the China general microbiological culture Collection center in 2018, 10 and 31 months, with the addresses: the preservation number of the institute of microbiology of the Chinese academy of sciences, West Lu No.1, Beijing, Chaoyang, Beijing, and Beicheng district, is: CGMCC NO. 16781.

Experiments prove that the gastrodia elata is cultivated by adopting a new Armillaria gallica strain small grass dam No.1 (CGMCC NO.16781), so that the yield of the gastrodia elata can be improved, and the quality of the gastrodia elata can also be improved.

In the previous research, the new strain Armillaria gallica No.1 (CGMCC NO.16781) of the Armillaria mellea (Armillaria gallica) is used for culturing, and experiments prove that the strain has the advantages that the activity of extracellular laccase, the activity of extracellular cellulase, the activity of extracellular xylan, the activity of extracellular pectinase and the activity of extracellular amylase are obviously higher than those of other strains, the growth speed is higher, and the production period is shortened when the strain is used for strain production. In addition, the content of the polysaccharide of the strain is higher, and higher medicinal value is shown; can be used for preparing food, health food or medicine.

Because the artificially cultured fruiting bodies of the Armillaria mellea are difficult to obtain stably with high yield, in order to better research and apply the new strain Armillaria gallica No.1 (CGMCC NO.16781) of the Armillaria mellea and avoid strain degeneration caused by multiple asexual passages, the invention also deeply researches the method for artificially culturing the fruiting bodies of the Armillaria mellea, and unexpectedly obtains successful cultivation. The details are as follows.

The invention provides a high-yield and high-efficiency culture method of Armillaria mellea, which is characterized in that Armillaria mellea (Armillaria gallica) small grass dam No.1 (CGMCC NO.16781) is inoculated in culture medium of a culture bag, the culture medium (namely a culture seed) and sweet potatoes are placed in a culture ridge together after hypha rhizomes grow, and the culture is carried out after soil covering.

Wherein the culture medium (i.e., cultivar) overgrown with hypha fungi is preferably 10-15 cm deep. The weight ratio of the culture substrate to the sweet potatoes is 1 (0.2-1), preferably 1 (0.25-0.7). For example, it may specifically be 1:0.2, 1:0.25, 1:0.3, 1:0.4, 1:0.45, 1:0.52, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1: 1. Under the condition, the growth of Armillaria mellea is promoted, and the yield is stabilized.

The invention preferably selects fresh and mildew-free sweet potatoes.

Researches find that the sweet potato can be invaded, decomposed and absorbed by hypha (rhizomorph) of Armillaria mellea, so that various nutrients including carbon sources and nitrogen sources are supplemented for growth, the conversion from vegetative growth to reproductive growth is ensured, fruiting is promoted, and the yield of fruiting bodies is increased. In addition, the sweet potato has strong environmental adaptability, is easy to plant, is suitable for being planted in different areas in a matching way, is convenient to obtain materials and has low cost.

Further, the culture temperature after earthing is 15-25 ℃; the seeds can be matured and harvested after 35 to 60 days generally.

The culture method of Armillaria french of the invention can be managed according to conventional methods in the art, such as appropriate watering to keep the soil moist; appropriately shaded to control the culture temperature, etc.

According to the local climatic features of Yi-E county, the cultivation season is usually arranged from 2 late to 5 early months per year.

The invention also optimizes the culture medium of the cultivation bag, and the formula of the culture medium is as follows: 55-65 parts of sawdust, 20-30 parts of wheat grains, 10-15 parts of corncobs, 1-2 parts of white sugar, 1-1.5 parts of light calcium carbonate, 0.15-0.3 part of magnesium sulfate and 0.2-0.4 part of monopotassium phosphate; the pH is 5-6, and the final water content is 55-65%.

The preparation method can be carried out according to the conventional method in the field, for example, after the components are mixed, the pH and the water content are adjusted, and the wet heat sterilization is carried out at the temperature of 121-126 ℃. Culturing mycelium (mycelium) by conventional method in the art, such as inoculating Armillaria mellea according to aseptic manipulation, and culturing at 20-28 deg.C under dark condition for 30-50 days until the mycelium grows over the bag. And removing the plastic bag to cultivate the seeds in the soil.

The invention also comprises the france armillaria mellea sporocarp cultivated by the method, compared with the wild armillaria mellea, the france armillaria mellea sporocarp has the advantages of regular fruiting, large size, thick and strong handle, fresh and crisp taste, high content of polysaccharide in the armillaria mellea, high nutritional and medicinal values and good quality.

The invention realizes the stable fruiting of the sporocarp of the Armillaria mellea cultivated by the artificial cultivation for the first time, can increase the fruiting amount, and can be popularized and applied in industrialization.

Drawings

FIG. 1 is a phylogenetic N-J tree of four Armillaria mellea of example 1.

Fig. 2, 3 and 4 are graphs of gastrodia elata yield, gastrodia elata effective component content, gastrodine and p-hydroxybenzyl alcohol UPLC of gastrodia elata in example 2 cultivation test respectively.

Fig. 5, 6, 7, 8, 9 and 10 are graphs showing the change of extracellular laccase activity, extracellular cellulase activity, extracellular xylan activity, extracellular pectinase activity, extracellular amylase activity and polysaccharide content in different periods of AM1 and AM2 in example 3, respectively.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

Cultivation experiment

The Armillaria mellea strain used in the experiment is Armillaria mellea (Armillaria gallica) Xiaocaba No.1 (CGMCC NO. 16781).

The culture medium of the culture fungus bag comprises the following components: 60 parts of wood chips, 25 parts of wheat grains, 11 parts of corncobs, 2 parts of white sugar, 1.5 parts of light calcium carbonate, 0.15 part of magnesium sulfate and 0.35 part of monopotassium phosphate, and the pH value of the mixture is adjusted to 5.5. After being stirred evenly, water is added to the mixture, and the final water content is 65 percent. Subpackaging with 17cm by 33cm bags, performing damp heat sterilization at 121-126 deg.C, cooling, and inoculating Armillaria gallica No.1 (CGMCC NO.16781) by aseptic technique. Culturing at 25 deg.C in dark for 40 days until the bag is full of mycelia.

And (3) beginning to cultivate at the beginning of 3 months, selecting a field in a greenhouse, and digging a ridge bed with the ridge length of 10m, the width of 1m and the depth of about 15 cm. Experimental groups: putting the fungus bag full of hypha fungi cables (removing the plastic bag) and fresh sweet potatoes into the cultivation bed together, and covering soil about 3 cm. After covering soil for one week, if the weather is dry, watering is started properly, keeping the soil moist, and shading properly. Meanwhile, a control group is set: that is, only the bags filled with hypha fungi are planted in the soil without using fresh sweet potatoes. The experimental group and the control group were managed in the same manner.

As a result, 20 days later, primordia begin to form and expose out of the ground, and the Armillaria mellea sporophore is matured successively after about 15 days, and then the Armillaria mellea sporophore can be harvested. In contrast, the control group showed only a few primordia and no fruiting body was formed. The specific results are shown in the following table:

	the weight ratio of the culture medium to the sweet potato	Biological conversion rate
			Experimental group 1	1:0.25	44.2％
Experimental group 2	1:0.45	55.1％
			Experimental group 3	1:0.70	58.2％
Control group	Without sweet potato	Is substantially 0

Note: the biotransformation efficiency is the ratio of the fresh weight of Armillaria french sporocarp to the dry weight of the culture medium of the cultivation medium bag used, expressed as a percentage.

The Armillaria mellea (Armillaria gallica) Xiaocaba No.1 (CGMCC NO.16781) is further described below.

EXAMPLE 1 Strain acquisition, Strain identification

1.1 Strain isolation

Two Armillaria mellea strains are obtained by separating wild rhizoma Gastrodiae (rhizoma Gastrodiae tuber with cordia) from small Cabazhen town of Yi-E county of Yunnan province, and are numbered as AM1 and AM 4. The specific separation method is as follows:

cleaning soil on the gastrodia elata by using clear water, flowing tap water for 15 minutes, then washing with sterile water for 3 times, soaking with 0.1% mercuric chloride solution for 1-2 minutes, washing with sterile water for 2-3 times, removing residual disinfectant, then cutting a required part of tissue by using a sterile knife, placing the cut tissue in a sterile culture dish, absorbing surface water by using sterile filter paper, placing the cut tissue on a culture dish of a PDA culture medium added with chloramphenicol, and culturing for 5 days at the constant temperature of 25 ℃ to start to grow new funiculus.

The morphological characteristics of the strains AM1 and AM4 are as follows:

mycelium characteristics: the hyphae were milky white and fluorescent in the dark. The young hyphae end is brownish red, the strong hyphae surface is smooth, thick and dark red, and the old hyphae surface is dark brown.

And (3) fruiting body characteristics: pileus conical, convex or flat-topped. The pileus was brown-yellow to brown with a darker center color. The pileus surface is covered with fine cilia, either upright or obliquely upward. The lower surface of pileus of the primary fruiting body has a layer of flocculent tissue extending from the pileus border to the stipe. The mycorrhiza is initially white, gradually turning to cream or light orange, and covering the rusty spots. The shiffles co-grow or extend slightly down the stipe. The length of the stipe is 4-10 cm. Above the fungus ring, the stipe is light orange to brown; the stipe is white or light pink below the fungus ring; the base was gray-brown. The spore print appears white. The spores are ellipsoidal. The pileus cuticle consists of irregularly interlaced hyphae, protruding upward to form a squamous mass. Locked associations exist in hyphae.

Biological characteristics: the source of nutrient components can be preserved by using an improved PDA culture medium (200 g of potatoes, 33g of wheat bran, 20g of glucose, 3g of monopotassium phosphate, 1.5g of magnesium sulfate, 0.1g of thiamine hydrochloride, 10g of agar powder and 1000mL of distilled water, wherein the pH value is natural), and the modified PDA culture medium is suitable for low-temperature refrigeration preservation in a refrigerator at 4 ℃ and is switched for 1 time every 3 months. And (3) strain propagation production: the mother strain is suitable for being cultured by an improved PDA culture medium; the stock and cultivated species are suitable for producing culture medium which is composed of main materials of small branches and water and auxiliary materials of corn flour.

1.2 identification of the Strain

Two other Armillaria spp A9 and Jing 234 strains for cultivating rhizoma Gastrodiae are obtained from the institute of medicinal plants of Chinese academy of medical sciences, and are numbered AM2 (A9) and AM3 (Jing 234). Armillaria mellea A9 and Armillaria mellea 234 are the main current cultivars for cultivating rhizoma Gastrodiae at present.

The four Armillaria mellea strains of AM1, AM2, AM3 and AM4 were inoculated into modified PDA medium, and cultured at 25 deg.C. Stripping cultured Armillaria mellea from the culture medium, placing into a mortar, adding appropriate amount of quartz sand and liquid nitrogen, and grinding thoroughly, wherein the genome DNA extraction method is operated according to the instruction of the plant genome DNA extraction kit. The amplification sequences were rDNA-ITS (SEQ ID NO:1), β -tubulin (SEQ ID NO:2) and tef1- α (SEQ ID NO:3), and the primers used are shown in Table 1. PCR reaction (25. mu.L): reaction primer (10. mu.M) 1. mu.L, DNA template 1. mu.L, 2 XTaq PCR Mix 12.5. mu.L, ddH₂Make up to 25. mu.L of O. And (3) PCR reaction conditions: pre-denaturation at 94 deg.C for 3 min; circulation conditions are as follows: denaturation: 30s at 94 ℃; annealing: rDNA-ITS and tef 1-alpha at 55 deg.C for 30s, beta-tubulin at 53 deg.C for 40 s; extension at 72 ℃: rDNA-ITS is 120s, beta-tubulin is 90s, tef 1-alpha is 30s, and the above is circulated for 30 times; extension was carried out at 72 ℃ for 5 min.

Wherein the tef 1-alpha sequence is amplified by a two-step PCR reaction, primers EF526F and EF1567R are used in the first step reaction, and the PCR product of the first step is used as a template in the second step reaction. The second reaction was amplified using primers EF595F and EF 1160R.

TABLE 1 primer sequences and names

mu.L of PCR product was detected by 1% agarose gel electrophoresis, and the clear and unique PCR product was subjected to bidirectional sequencing. The other products are cut off the target geneThe fragments were recovered using agarose gel purification recovery kit, and the recovered DNA fragments were cloned using zero background pTOPO-TA cloning kit and E.coli DH5 alpha competent cells. Colonies cultured overnight at 37 ℃ were picked up, transferred to liquid LB medium (3mL) under aseptic conditions, and 3. mu.L of ampicillin (50 mg. multidot.mL) was added^-1) And (4) culturing for more than 4 hours in a constant-temperature incubator with 37 ℃ and 180rpm, and performing bidirectional sequencing on turbid bacterial liquid. Universal primers M13F (5'-TGTAAAACGACGGCCAGT-3') and M13R (5'-CAGGAAACAGCTATGACC-3') are adopted for sequencing bacterial liquid. Data were processed and analyzed using Excel 2016 and SPSS 19.0, graphepad 7.0 was plotted, DNAMAN was analyzed for gene homology, and MEGA7.0 drawn a phylogenetic N-J tree. The results are shown in FIG. 1 (the sequences of all strains of Armillaria mellea are the combined sequences of rDNA-ITS, β -tubulin and tef1- α).

After combining three gene fragments of rDNA-ITS, beta-tubulin and tef 1-alpha of strains AM1, AM2, AM3 and AM4 respectively, DNAMAN is used for analyzing homology and genetic distance (table 2) to find that the homology of AM1 and AM3 reaches 99.2 percent, and the genetic distance is closer to 0.005; AM2 is 98.9% and 98.7% respectively with AM1 and AM3, and is a little distant from the genetics; the AM4 strain has homology of only about 75% with the three strains, and has a distance of 0.246 to AM1, 0.242 to M2AM2, 0.251 to AM3 and a longer genetic distance.

TABLE 2 similarity and genetic distance Table for four different Armillaria mellea strains

Note: the top half of Table 2 is the genetic distance and the bottom half is the similarity, which is the separation line

The determined sequence fragments were clustered in one alignment at NCBI for AM1, AM2 and AM3, with a support rate of 99%. HKAS85563, HKAS85572, HKAS85567, HKAS86560, HKAS86559 and HKAS86564 are Armillaria mellea of France, and AM1 is gathered on the same branch, with a supporting rate up to 99%. Namely, the molecular identification result shows that the strain AM1 is Armillaria mellea.

The strain AM1 is named as Armillaria mellea (Armillaria gallica) Xiaocaba No.1, and is preserved in 31/10/2018 in China general microbiological culture Collection center, with the address: the preservation number of the institute of microbiology of the Chinese academy of sciences, West Lu No.1, Beijing, Chaoyang, Beijing, and Beicheng district, is: CGMCC NO. 16781.

Example 2 Gastrodia elata cultivation test

In this example, the influence of Armillaria mellea (Armillaria gallica) Xiaocaba No.1 (CGMCC NO.16781) on the yield and quality of rhizoma Gastrodiae by the use of the strain is examined. For ease of description, this strain was still numbered AM 1.

The above four strains were subjected to the gastrodia elata concomitant culture experiment using armillaria mellea AM2, AM3, and AM4 described in example 1 as controls.

Planting Gastrodia elata seeds: planting first generation of Gastrodia elata in Yiliang county, small grass dam local land; a cultivation site: gastrodia elata Blume of Xiaocaoba demonstrates a garden with an altitude of 2010m, 104 degrees, 12 '2' E, and 27 degrees, 45 '21' N. Adopting a 'three-nest-putting' cultivation mode, namely adopting a method of simultaneously placing cultivation holes on a tree stick, different Armillaria mellea strain production seeds and Gastrodia elata seeds for cultivation, and cultivating in 2017 within 3 and 20 days; the gastrodia elata cultivation holes are 60cm in length, 60cm in width and 60cm in depth, 45cm and 20cm in depth, 5 sticks with the diameter of 5-15 cm and the length of 25cm are placed in each hole, one layer of gastrodia elata is cultivated, 1 bottle of armillaria mellea strains is cultivated, and 200g of gastrodia elata seeds are cultivated. Imitating wild cultivation, and harvesting Gastrodia elata Blume No. 11 and 20 in 2017.

The result of rhizoma Gastrodiae yield is shown in figure 2, the content of effective components of rhizoma Gastrodiae is shown in figure 3, and gastrodine and p-hydroxybenzyl alcohol UPLC of rhizoma Gastrodiae is shown in figure 4. In fig. 3, a: gastrodin content; b: the content of p-hydroxybenzyl alcohol; c: total content of gastrodine and p-hydroxybenzyl alcohol; d: polysaccharide content;^**indicates the existence of P in comparison with each component<0.01 significantly different. In fig. 4, a: mixing standard substances; b: sample solutions a to d: AM 1-AM 4; 1: gastrodine; 2: p-hydroxybenzyl alcohol.

The result shows that the total yield of the gastrodia elata is 2.967kg/m by using the AM1 strain for concomitant cultivation²And the difference is obvious compared with the other three strains. The total yield of the AM3 strain is the lowest, and is 1.991kg/m²And compared with M2 and AM4 strains, the strain has significant difference. The yield of the kenaf of the AM1 strain with the rhizoma gastrodiae planted is 1.912kg/m²The same strain as AM4 (1.915 kg/m)²⁾Very close to each other, and has significant difference with AM2 and AM3 strains. In a word, the AM1 strain has obvious advantages of being accompanied by gastrodia elata, the total yield is higher than that of other three strains, and the yield of the gastrodia elata is higher than that of other two strains.

The content of gastrodin in the AM1 strain-cultured Gastrodia elata Blume is 5.822mg g^-14.931mg g higher than that of AM2 strain^-1And 3.882mg g of AM3 strain^-1. The content of gastrodin in the gastrodia elata grown with the AM4 strain is about 1/2 of that in the AM1 strain, and the gastrodin content is the lowest. The content of gastrodin accompanied with AM1 strain and the existence of P in AM4 strain<A very significant difference of 0.01. The highest content of the p-hydroxybenzyl alcohol AM3 strain is 0.188mg g^-1The second strain of AM1 was 0.151mg g^-1The AM2 strain and AM4 strain were found to be similar in content.

The total content of ephedrine and p-hydroxybenzol of the rhizoma Gastrodiae cultivated by different strains is higher than 0.25% standard specified in 2015 edition of Chinese pharmacopoeia, and the total content of AM1 strain cultivated by strains is 0.597% which is 2.388 times of the level of Chinese pharmacopoeia. The total content of the accompanied culture of the AM2, AM3 and AM4 strains is 0.501%, 0.407% and 0.312% respectively, and is significantly different from that of the AM1 strain, and the total content of the accompanied culture of the AM1 strain is 2 times that of the AM4 strain.

Polysaccharide content measurement results show that the content of the polysaccharide cultivated with the AM1 and AM3 strains is higher than that of the AM2 strain and AM4 strain, and the content of the polysaccharide cultivated with the AM1 strain is 21.39mg g^-121.42 mg. g of AM3 strain^-1The content is similar, the content of AM4 is similar to that of AM2 strain, and the content of AM4 strain is 19.39mg g^-1The amount of the AM2 strain was 17.62mg g^-1The contents are all lower.

AM1 has the highest yield in the four strains, and gastrodine content is as high as 5.822 mg.g^-1The total content of gastrodin and p-hydroxybenzyl alcohol is 5.973mg g^-1The cultivated gastrodia elata has the best quality. The yield of the gastrodia elata cultivated with the AM4 strain is similar to that of AM1, but the content of effective components is low, and the content and the total content of gastrodin are about 1/2 of AM 1. In conclusion, the yield and quality of the gastrodia elata mixedly planted by using the AM1 strain are optimal.

Example 3 comparison of biological Properties

This example examines the biological properties of Armillaria mellea (Armillaria gallica) Micrograss dius No.1 (CGMCC NO. 16781). For ease of description, this strain was still numbered AM 1.

Armillaria mellea AM2 described in example 1 was used as a control.

The improved solid PDA culture medium is respectively quantitatively inoculated with tender strains of the strains AM1 and AM2, cultured under the dark condition of 25 ℃, and experimental data are regularly observed and recorded.

Measurement of growth amount

The amount of growth of the strain was measured by the cross method from the fourth day of inoculation, and was measured every day until the strain was overgrown.

Extracellular enzyme Activity assay

Preparation of crude enzyme solution

The culture medium around the hyphae was randomly filled into a 2.0mL centrifuge tube with an inoculating needle, centrifuged at 12000r/min for 15min, and 1mL of the supernatant was aspirated into a 10mL volumetric flask and added to a constant volume with distilled water to prepare a diluted enzyme solution, which was used in the following experiment.

Enzyme activity assay

Determining laccase activity by adopting an ABTS method: 0.5mL of 0.5 mu mol/L ABTS and 1.0mL of disodium hydrogen phosphate-citric acid buffer solution (pH is 4.00.2 mol/L) are added into a 2.0mL enzyme activity reaction system and uniformly mixed, 0.5mL of diluted enzyme solution is added to start reaction, the change of the absorbance value at 420nm is recorded every 30s, the slope is calculated at the position which accords with the linear relation, and the enzyme activity is calculated. One unit of enzyme activity is defined as the amount of enzyme required to catalyze the oxidation of 1. mu. mol of ABTS per minute in the reaction system.

The DNS method is used for respectively measuring the activity of cellulase, xylanase, pectinase and amylase.

And (3) cellulase activity determination: 1.5mL of 1% CMC-Na solution prepared by 0.1mol/L of acetic acid buffer solution (pH is 4.6) and 0.1mL of diluted enzyme solution are respectively added into a graduated test tube, mixed uniformly and kept warm for 30min in a water bath at 40 ℃, taken out, immediately added with 1.5mL of DNS reagent, placed in a boiling water bath for heating for 5min, cooled, diluted to 10mL by distilled water, and the absorbance value is measured at 500 nm. The determination method of the activity of other enzymes is the same as the determination method of the activity of the cellulase, except that 1 percent of CMC-Na solution is changed into 1 percent of xylan solution, 1 percent of pectin solution and 1 percent of starch solution, and the preparation method is also the same.

The above methods all use the crude enzyme solution which is heated and inactivated as a reference, and calculate the contents of glucose, xylose and galacturonic acid of the substrate decomposed by the enzyme according to the glucose, xylose and galacturonic acid standard curves, wherein 1 enzyme activity unit is defined as the enzyme amount required for generating 1 mu mol of glucose, xylose and galacturonic acid per minute.

Drawing of standard curve

Glucose standard curve: accurately transferring 0.1-0.9 mL of glucose standard solution of 0.1mg/mL into a 10mL volumetric flask, and supplementing to 2.0mL with distilled water, and using 2.0mL of distilled water as a control. 1mL of 5% phenol solution was added, 5mL of concentrated sulfuric acid was added thereto, the mixture was shaken, and the volume of the mixture was adjusted to 10mL with distilled water. Placing in boiling water bath for 15min, taking out, cooling to room temperature, and cooling to A₄₉₀And (3) measuring an absorbance value at nm, taking the glucose concentration as an abscissa and the absorbance value as an ordinate, and drawing a glucose standard curve: y is 60.5X +0.0004(r is 0.9995), and the linear relationship of the curve is good in the range of 0.001-0.009 mg/mL.

Xylose standard curve: respectively adding 0.1-0.6 mL of 1.5mg/mL xylose standard solution into a 10mL volumetric flask, supplementing the xylose standard solution to 1.0mL by using an acetic acid buffer solution, and using 1.0mL of the acetic acid buffer solution as a blank control. Adding DNS reagent 2.0mL, shaking, placing in boiling water bath for developing color for 5min, cooling rapidly in cold water bath, diluting to 10mL with acetic acid buffer solution, shaking, and determining A₅₀₀The absorbance value of (a) is plotted as a xylose standard curve (Y ═ 18.26X-0.0022(r ═ 0.9993) with the xylose concentration as abscissa and the absorbance value as ordinate, and the curve has a good linear relationship in the range of 0.015 to 0.09 mg/mL.

Galacturonic acid standard curve: accurately transferring 1mg/mL galacturonic acid standard solution 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8mL into 10mL volumetric flask, adding 2mL DNS reagent after adding acetic acid buffer solution to 2.0mL, shaking up, placing in boiling water bath for color development for 5min, rapidly cooling in cold water bath, diluting to 10mL with acetic acid buffer solution, measuring A₅₀₀Values of galacturonic acid standard curve Y-12.763X-0.0091 (r-X) were plotted using galacturonic acid concentration as abscissa and absorbance as ordinate0.9992), the linear relationship of the curve is good in the range of 0.020 to 0.120 mg/mL.

Armillaria mellea polysaccharide assay

Weighing Armillaria mellea mycelium powder 0.1g dried to constant weight, adding 10mL 75% ethanol, heating in 50 deg.C water bath for 30min, and filtering. Adding 60mL of distilled water into filter residue, heating for 5min by microwave, cooling, filtering, and diluting the filtrate to 100mL by using distilled water. Accurately transferring 200 μ L of the extractive solution into 10mL volumetric flask, adding 5% phenol 1mL and concentrated sulfuric acid 5mL, adding distilled water to constant volume to 10mL, mixing, placing into boiling water bath for 15min, cooling at room temperature, and adding into A₄₉₀And (5) measuring the absorbance value at nm, and calculating the polysaccharide content according to a glucose standard curve.

Biomass of Armillaria mellea

As shown in Table 3, the growth of the strain was measured every day from day 4 of the growth of Armillaria mellea, and statistical analysis was conducted with every 4 days as a growth stage, and it was found that the growth rates of the two strains were maximized at day 8 of the inoculation and reached 0.812cm/d and 0.807cm/d, respectively, and were not different from each other, whereas the growth amounts of the two strains showed a very significant difference (P <0.01) between day 4 and day 16 of the inoculation, and the results showed that the AM1 strain was superior to the AM2 strain in growth rate. Compared with the dry breaking rate, the two strains have little difference. According to the growth observation of the two strains, the two strains grow on a culture dish after being inoculated for 20 days, and the AM1 strain is stronger in rhizomorph, more in branches and good in growth compared with the AM2 strain.

TABLE 3 Biomass of two strains AM1 and AM2 at different periods

Note:^*two strains are shown in P<Significant difference at 0.05 level^**Two strains are shown in P<Significant difference at the 0.01 level

Change in extracellular enzyme Activity of Armillaria mellea

Laccase activity

The change in the activity of extracellular laccase at different stages is shown in FIG. 5. As can be seen in FIG. 5, the activity of the laccase of Armillaria mellea increased with the increase of the culture time and then decreased, and reached the highest value on the 16 th day of inoculation. The laccase activity of the AM1 strain was stronger than that of the AM2 strain at 8,12,16,20 days, and there were differences: with differences at day 8 (p < 0.05); a significant difference was found at 16 days (p < 0.01); there were very significant differences (p <0.001) between days 12 and 20.

Cellulase activity

The change of the extracellular cellulase activity at different periods is shown in FIG. 6. The cellulase activities of the two strains AM1 and AM2 were significantly different at 4 days, 8 days, 12 days and 20 days of inoculation (P < 0.05). The cellulase activity of the two strains shows a trend of increasing and then decreasing, and reaches a maximum value on the 8 th day of inoculation; the AM2 cellulase activity decreased to a minimum on day 12 of inoculation, and then increased back.

Xylanase activity

The change of the activity of the extracellular xylanase in different periods is shown in FIG. 7. Therefore, the xylanase activities of the AM1 and AM2 strains have consistent variation trend, and both the xylanase activities increase along with the increase of the inoculation time, and then the xylanase activities decrease after the enzyme activities increase. Xylanase activity was highest at day 12 of inoculation, with significant differences between AM1 and AM2 at days 12 and 16 of inoculation (P < 0.05).

Pectase activity

The change of the activity of extracellular pectinase at different periods is shown in figure 8. Therefore, the change trend of the pectinase activity is different from that of other enzyme activities, the change of the pectinase activity of the armillaria mellea of the two strains AM1 and AM2 shows a trend of descending first and then ascending, the AM1 is the lowest on day 12, and the AM2 is the lowest on day 8 and is the largest on day 20. The pectinase activities of the AM1 and AM2 strains are different at 8, 16 and 2 days (P is less than 0.05). The pectinase activity of AM1 strain is higher than that of AM1 strain at all times.

Amylase Activity

The changes of the activity of the extracellular amylases AM1 and AM2 at different periods are shown in FIG. 9. It is known that the amylase activity also shows a trend of increasing and then decreasing. However, the AM1 strain had a maximum amylase activity at day 8 of inoculation, while the AM2 strain had a maximum activity at day 12 of inoculation. On days 8 and 12, the amylase activities of the AM1 and AM2 strains are obviously different (P is less than 0.05).

Polysaccharide content of Armillaria mellea

The change of polysaccharide content at different periods is shown in FIG. 10. The polysaccharide content of the two strains of AM1 and AM2 shows a trend of increasing firstly and then decreasing with the increase of the culture time. On day 16 of culture, the polysaccharide content reached a maximum. The polysaccharide content of the two strains is obviously different at 12 days and 16 days (P < 0.05).

Correlation of enzyme activity and Armillaria mellea hypha polysaccharide

Pearson correlation analysis shows that the polysaccharide content of the mycelia of the Armillaria mellea has strong correlation with the activity of xylanase, the correlation coefficient is 0.750, the enzyme activity of the Armillaria mellea and the enzyme activity of laccase show strong correlation, the correlation coefficient is 0.881, and the other enzyme activities have no correlation with the accumulation of the mycelia polysaccharide (P is less than 0.05). The variation curves of laccase activity and xylanase activity of the two strains are basically consistent with the variation trend of polysaccharide content of mycelia of the two strains, which shows that the growth and development of the two armillaria mellea strains are mainly related to the secretion of extracellular laccase and xylanase. The results are shown in Table 4.

TABLE 4 correlation of enzyme activity with mycelium polysaccharide

The growth rate of Armillaria mellea is reflected by Armillaria mellea polysaccharide and biomass. In the strain production, the production time can be saved and the cost can be reduced due to the higher growth speed in the growth, and the embodiment shows the advantage of the strain AM1, namely the Xiaocaoba No. 1. The armillaria genus mainly relies on decomposing lignin, cellulose, hemicellulose and the like in wood to obtain nutrition, laccase is mainly responsible for lignin degradation, cellulase is mainly responsible for cellulose degradation, xylanase is used for degrading xylan (the xylan is a main component of plant hemicellulose), pectinase is an enzyme for decomposing pectin which is a main component of plants, and amylase hydrolyzes starch, so the enzymes play important physiological functions in the growth and development process of higher fungi. This example also shows that strain AM1, i.e., Xiaocaoba No.1, exhibited better enzyme activity than strain AM2 when cultured.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> development center of Gastrodia elata industry in Yi-Liang county

High-yield and high-efficiency cultivation method of <120> Armillaria mellea

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 832

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ggaaggatca ttattgaaac ttgaatcgta gcatcgagaa ctgttgctga cctgttaaag 60

ggtatgtgca cgttcgacgt gttgcgttct attcatccac ctgtgcacct ttgtagactt 120

gattaacttt cgctttcgag cggttagaag ggttgctttc gagctccctt tgtctatcaa 180

gtctatgtct atataatctc ttgtatgtct agaatgtctt gtttatggga tgcaagtcct 240

ttaaatctta tacaactttc aacaacggat ctcttggctc tcgcatcgat gaagaacgca 300

gcgaaatgcg ataactaatg tgaattgcag aattcagtga atcatcgagt ctttgaacgc 360

accttgcgcc ctttggtatt ccgaagggca tgcctgtttg agtgtcatta aattctcaac 420

ctccccttct ttcattagga gtgcggcgga ttggatatgg gggtttgctg gtttctaacg 480

agatcagctc ctctgaaatg cattagcaga aaccgtttga ctttggctgc taggctgtga 540

taatatctac gccttgtagt tgggtcggaa tacgagtcat acagtggtaa ctaatcaggc 600

tttcgggtct ggcttagaat cggtttggaa ggtgcttaac ggctccttct gctttctccc 660

tttgcggaga tacttgtcca attctaagag aggagttgct tagcgcgggc ttagctttcc 720

ttgatatttc cctttgactt tgtagaagga ttcagcttct aaccgtccat tgacttggac 780

aatttattga ctatttgacc tcaaatcagg taggactacc cgctgaactt ag 832

<210> 2

<211> 877

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ataacaagtg tgcattttat gcgtttttgt accaactcgc actataggcc aagtcaatga 60

aaactcgccc tacagccacc aaaattgttg caaaccgatc ttgaccaatg gtttacttag 120

caaacctttc gtttcagaac cagttgccgt tcaaattttg aacgtagccc tcgaaagcta 180

agcaaacggt tgaaacggct aaaaaggtaa ccgtagctta acttacttag cttctaaggc 240

cttttcaatc atggcaacct tagggcaggc cctcaggctc actaacgatc cagaaaatca 300

gtcgtcgact cgccctttca aggatcttgg ctataccata tacgggatat cggccataag 360

gtcgatatca agtacaaggt aaagaccgtt ccaagtcagc gcacggttag agcttgctaa 420

ctaagtgcct tataagcctt caaggctcat aataactcgt gattcaaaag agaaaggtaa 480

caaaacacta aggcgttgtt gttagcctac cctttcgaat ctaagtaaca tgctgtcgcg 540

agtgcacaaa ggatactatc ccggccctca aggtctcagc atgtaaaaag aaaagagaaa 600

gtccaacaaa gttgaacaaa tctttagaac aagggcttaa cctcagtgga tcgtagcaac 660

aaggctactc taccacttac agtaccccgt tcccattcaa gtcgtctgca aaggattcac 720

cgccgcccat attatgatta caattcgtag acaagtcgcc caagcgtttc cgcaaagaca 780

acccagccca ccatatttgg ttcacgagca aagctctatt taaacacaag tctaatgtgc 840

gacaccaaca tcatcgtttc tagcacggat tctgact 877

<210> 3

<211> 581

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cgtgacttca tcaagaacat gatcaccggt acctcccagg ctgattgtgc catcctcatc 60

atcgctggtg gaactggtga gttcgaggcc ggtatctcca aggacggtca gacccgagag 120

cacgccctcc ttgccttcac cctcggtgtc aggcagctca ttgtcgccgt caacaagatg 180

gacaccacca aggtacgaga tctgctgctt tgtcttttgt ttagccaaat ctgactgtta 240

tctcagtgga gcgaggaccg gttcaacgaa atcgtcaagg aaacctctac cttcatcaag 300

aaggtcggct acaaccccaa ggccgttgct ttcgtcccca tctctggatg gcacggtgat 360

aacatgttgg aggagtccgc caagtaagtc tttacccaac tatgatcagt gctgtttctt 420

aacgttctct gtagcatgcc atggtacaag ggctggacca aggagaccaa ggccggtgtc 480

gtcaagggca agactctcct cgatgccatt gacgccattg agccccctgt ccgtccctcc 540

gacaagcctc tccgtctccc tctccaggac gtctacaaga t 581

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tccgtaggtg aacctgcgc 19

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tcctccgctt attgatatgc 20

<210> 6

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggtgcgggta actgggc 17

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gaggcagcca tcatgttctt 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gtcgtygtya tygghcaygt 20

<210> 9

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

achgtrccra taccaccrat ctt 23

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cgtgacttca tcaagaacat g 21

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ccgatcttgt agacgtcctg 20

<210> 12

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tgtaaaacga cggccagt 18

<210> 13

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

caggaaacag ctatgacc 18

Claims

1. A high-yield and high-efficiency culture method of Armillaria mellea is characterized in that Armillaria mellea (Armillaria gallica) small grass dam No.1 (CGMCC NO.16781) is inoculated in culture medium of a culture bag, the culture medium and sweet potatoes are placed in a culture ridge together after hypha rhizomes grow, and the culture is carried out after soil is covered.

2. The cultivation method according to claim 1, wherein the culture medium full of hypha cords is embedded in soil to a depth of 15 to 20 cm.

3. The cultivation method according to claim 1 or 2, wherein the weight ratio of the culture substrate to the sweet potato is 1 (0.2-1).

4. The cultivation method according to claim 3, wherein the weight ratio of the culture substrate to the sweet potato is 1 (0.25-0.7).

5. The cultivation method as claimed in any one of claims 1, 2 and 4, wherein the cultivation medium formula of the cultivation bag comprises the following components in parts by weight: 55-65 parts of sawdust, 20-30 parts of wheat grains, 10-15 parts of corncobs, 1-2 parts of white sugar, 1-1.5 parts of light calcium carbonate, 0.15-0.3 part of magnesium sulfate and 0.2-0.4 part of monopotassium phosphate; the pH is 5-6, and the final water content is 55-65%.