Preparation method of wild mellea armillaria sporophore strain

Abstract

The invention discloses a preparation method of a wild mellea armillaria sporophore strain, and belongs to the technical field of wild edible mushrooms. The preparation method specifically comprises the following steps: (1) digging and disinfecting; (2) mother strain isolation culture; (3) culturing a liquid strain; and (4) preparing the low-temperature preserved strain. According to the method, the vigorously-growing young and tender cable tip parts of wild mellea armillaria sporophore funguses are used as separation materials and inoculated into a culture medium with a specially-made formula for tissue separation of mellea armillaria sporophore strains, purified mellea armillaria sporophore mother strains of the wild mellea armillaria sporophore are obtained, then the prepared liquid strains are inoculated into annual hardwood sections such as oak trees, and the wild mellea armillaria sporophore mother strains A bacterial material with high DPPH antioxidant activity is selected as a preservation strain for ultralow-temperature preservation. The method is easy to operate and high in separation success rate, the preservation period can reach 36 months or above, and the problems that in the prior art, the wild mellea armillaria sporophore strain is low in separation success rate and short in preservation period are solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of wild edible fungi, more particularly to a preparation method of wild Armillaria mellea mushroom strain. BACKGROUND

[0002] Armillaria mellea, Chinese name Armillaria mellea, belongs to the order of Agaricales, Armillaria genus, also known as Armillaria mellea, Armillaria mellea, Armillaria mellea, etc. In biological classification, it belongs to the fungal kingdom, basidiomycota, agaricomycetes, agaricomycetes, agaricomycetes, agaricomycetes, agaricomycetes, agaricomycetes. The Armillaria mellea in Northeast China is umbrella-shaped, light yellow, and brownish after aging. The small scales on the cap are flat or upright, and the mature cap is nearly smooth. The edge of the cap has radial stripes. The flesh is white. The stem is long and cylindrical, with a gill ring. The base of the stem is slightly thick, the stem is bent, the height is 5-13 cm, there are longitudinal stripes, and the inside is soft to hollow. Armillaria mellea has a strong aroma, a delicious taste, a smooth and refreshing taste, and is a high-grade food with high protein, low fat, and rich in minerals, vitamins and polysaccharides.

[0003] At present, the artificial cultivation technology of Armillaria mellea has not yet matured, and it is still not possible to realize large-scale and commercial artificial cultivation, but it can realize wild cultivation, and the benefit is considerable. The Armillaria mellea strain of wild cultivation is mainly prepared by collecting wild Armillaria mellea and artificially isolating and culturing to form wild Armillaria mellea strain, and then selected by fruiting test. However, the dosage of wild cultivation Armillaria mellea strain is large, and the wild Armillaria mellea strain will appear obvious degradation phenomenon after 3 generations of expansion, so it is necessary to isolate and prepare the strain from wild Armillaria mellea to ensure the yield and quality of artificial cultivation Armillaria mellea. Therefore, improving the success rate of wild Armillaria mellea strain isolation is crucial to ensure the demand for artificial cultivation Armillaria mellea strain. At the same time, wild Armillaria mellea is only produced in a specific season, and the fruiting body will mature and age in a few days after formation, and it is easy to breed diseases and pests. The young fruiting body with no diseases and pests is less and has a very low germination rate when used for tissue isolation. Therefore, it is very important to improve the success rate of strain isolation and reduce the infection rate in the process of wild Armillaria mellea strain preparation.

[0004] However, the existing wild hazel mushroom strains are all isolated from the inner flesh of the junction between the cap and stem of the young fruiting body. The hyphae are difficult to germinate from the tissue, and the wild hazel mushroom fruiting body is loose, rich in flavor and nutrients, and easy to breed pests and bacteria, so the contamination rate is high. Even if the hyphae of a few tissues germinate, they will lose the growth advantage compared with the hyphae of the bacteria due to their weak growth, and it is difficult to effectively purify them. Therefore, the success rate of strain separation using the tissue at the junction between the cap and stem of the wild hazel mushroom is low, the contamination rate is high, and the hyphae on the individual medium are difficult to purify due to the weak growth of the hyphae after being contaminated by the bacteria. The tip tissue of the wild hazel mushroom cord is active in cell growth and differentiation, and is usually buried in the ground or substrate. It is dense, colorless and odorless, has few pests and diseases, and carries few bacteria. Therefore, the germination rate is high, the contamination rate is low, and the success rate of separation is high. In addition, the preservation period of the existing strain slant preservation method can only reach several months. Even if the glycerol liquid strain is frozen in a ultra-low temperature refrigerator (-80℃), the preservation period is not more than 18 months, and the strain is frequently transferred, which is not only time-consuming and laborious, but also has a high contamination rate and is easy to mutate.

[0005] Therefore, how to improve the separation success rate of the wild hazel mushroom strain is a problem that those skilled in the art need to solve. SUMMARY

[0006] Therefore, the purpose of the present application is to provide a preparation method of wild hazel mushroom strain, which can effectively improve the separation success rate of wild hazel mushroom strain and prolong the preservation period to solve the problems in the prior art.

[0007] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows: The preparation method of the wild hazel mushroom strain comprises the following steps: (1) Digging and disinfection treatment Select wild hazel mushrooms with complete shape, robust fruiting body and no pests and diseases, cut the white part of the cord tip, remove impurities, wash and disinfect the surface; (2) Mother strain separation culture Cut 1-4 mm small sections from the white part of the cord tip, inoculate into the separation culture medium and culture to obtain the hazel mushroom mother strain; (3) Liquid strain culture Inoculate the hazel mushroom mother strain into the liquid culture medium and culture to obtain the liquid strain; (4) Preparation of low-temperature preserved strain Inoculate the liquid strain into the hard miscellaneous wood section, vacuum package and refrigerate.

[0008] Further, in the step (2), the formula of the separation medium is as follows: potato decoction 370-400 mL, fresh Armillaria decoction 130-150 mL, ArmiUaria growing soil soaking solution 450-500 mL, glucose 15-25 g / L, peptone 2-4 g, magnesium sulfate 1-2 g / L, potassium dihydrogen phosphate 1-3 g / L, VB1 5-15 mg, agar powder 18-20 g / L, pH 5-7.5.

[0009] Further, in the step (3), the formula of the liquid medium is as follows: potato decoction 370-400 mL, fresh ArmiUaria decoction 130-150 mL, ArmiUaria growing soil soaking solution 450-500 mL, glucose 15-25 g / L, peptone 2-4 g, magnesium sulfate 1-2 g / L, potassium dihydrogen phosphate 1-3 g / L, VB1 5-15 mg, pH 5-7.5, sterilized at 121°C for 20-30 min.

[0010] Further, the potato decoction is a filtrate obtained by mixing potato and water at a mass ratio of 1:(2.0-2.5) and boiling for 20-30 min.

[0011] Further, the fresh ArmiUaria decoction is a filtrate obtained by mixing fresh ArmiUaria fruiting body and water at a mass ratio of 1:(1.5-2.0) and boiling for 20-30 min.

[0012] Further, the ArmiUaria growing soil soaking solution is a clear filtrate obtained by mixing wild ArmiUaria growing soil and water at a mass ratio of 1:(2.0-2.5) and stirring thoroughly.

[0013] Further, in the step (2), the temperature for culture is 23-25°C, and the time is 15-21 d.

[0014] Further, in the step (3), the rotation speed for culture is 130-160 r / min, the temperature is 24-26°C, and the time is 15-21 d.

[0015] Further, in the step (4), the hard wood pieces are one-year-old and have bark, and are obtained from oak, birch, hazel or elm, have a diameter of 2 cm and a length of 10-15 cm, and are sterilized at 121°C for 30-40 min.

[0016] Further, in the step (4), the temperature for culture is 24-26°C, the humidity is 60%-70%, and the time is 15 d; and the method for cold storage is as follows: first, the low-temperature preserved strain is stored at 4°C for 12 h, then stored at -20°C for 12 h, and finally stored at -80°C.

[0017] Compared with the prior art, the present application has the following advantages: The present application uses the young and tender cord tip part of wild hazel mushroom to isolate materials, and inoculates the materials into a specially formulated culture medium to isolate hazel mushroom spores, so as to obtain purified hazel mushroom mother spores. Then, the prepared liquid spores are inoculated into annual hard miscellaneous wood segments of oak trees, and the spores with high DPPH antioxidant activity are selected as preservation spores for ultra-low temperature preservation. The present application has the advantages of simple operation, high isolation success rate, and preservation period of more than 36 months, which solves the problems of low isolation success rate and short preservation period of wild hazel mushroom spores in the prior art. BRIEF DESCRIPTION OF DRAWINGS

[0018] Fig. 1 Wild hazel mushroom for spore isolation; Fig. 2 Hazel mushroom mother spores; Fig. 3 Hazel mushroom mother spores in a conical flask; Fig. 4 Hazel mushroom cord in a culture medium; Fig. 5 Liquid hazel mushroom spores; Fig. 6 Bacterial balls in the liquid hazel mushroom spores. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0020] In the following embodiments, wild hazel mushrooms are collected from the Nanshan Oak Silkworm Research Base of the Zhalantun City Hulunbuir City Agricultural and Pastoral Science Research Institute.

[0021] Embodiment 1 The preparation method of wild hazel mushroom spores specifically includes the following steps: (1) Excavation and disinfection treatment Wild hazel mushrooms with complete shape, robust fruiting bodies, and no diseases and insect pests are selected. The humus layer and soil layer are excavated, and the cord in the growth substrate is found. The young and tender cord tip white part of the cord is cut off, impurities are removed, and the part is cleaned with water under sterile conditions. Then, the part is surface-disinfected by washing with sterile water for 3 times, soaking in 75% alcohol for 1 min, and washing with sterile water for 3 times. (2) Mother spore isolation and culture Using a scalpel on a clean bench, a 3 mm section of tissue was cut from the white tip of the young stem of wild hazel mushroom mycelium. The tissue was then inoculated into a sterilized isolation medium with forceps and cultured at 25°C for 15 days to obtain the mother culture of hazel mushroom. The formulation of the isolation culture medium is as follows: 400 mL potato decoction, 150 mL fresh hazel mushroom decoction, 450 mL hazel mushroom growing soil soaking solution, 20 g / L glucose, 3 g peptone, 1.5 g / L magnesium sulfate, 2 g / L potassium dihydrogen phosphate, 10 mg vitamin B1, 18 g / L agar powder, and pH 6.5. Potato decoction is the filtrate obtained by boiling diced potatoes and water at a mass ratio of 1:2.0 for 25 minutes. Fresh hazel mushroom decoction is the filtrate obtained by boiling wild hazel mushrooms and water at a mass ratio of 1:2.0 for 25 minutes. The hazel mushroom growing soil soaking solution is a clear, filtered liquid obtained by thoroughly mixing and stirring soil and water from wild hazel mushroom growing areas at a mass ratio of 1:2.0. (3) Liquid culture The agar powder in the raw materials of the isolation medium was removed to prepare a liquid culture medium, which was sterilized at 121℃ for 30 min under high pressure. After cooling, the mother culture of hazel mushroom was inoculated under aseptic conditions and cultured at 140 r / min and 25℃ for 15 days to obtain the liquid culture. (4) Preparation of strains for cryopreservation One-year-old oak tree segments with bark, measuring 2 cm in diameter and 15 cm in length, were placed in test tubes measuring 3 cm in diameter and 20 cm in length. The tubes were sterilized in an autoclave at 121°C for 35 min. After cooling, the tubes were inoculated with liquid culture under aseptic conditions and cultured in a sterile environment at 24–26°C and 60%–65% humidity for 15 days. After vacuum packaging with the tube opening facing upwards, the tubes were first stored in a 4°C refrigerator for 12 hours, then in a -20°C refrigerator for 12 hours, and finally refrigerated at -80°C.

[0022] In this embodiment, wild hazel mushrooms and fungal strains are described below. Figs. 1-6 .

[0023] Depend on Figs. 1-6 It is evident that selecting wild hazel mushrooms with intact shapes, robust fruiting bodies, and freedom from pests and diseases, and using the white tips of their mycelial cords for tissue separation in the isolation medium of this invention, results in a high success rate, vigorous mycelial growth, and well-developed mycelial cords. In the liquid culture medium, the mycelial solution is clear, and the mycelial balls exhibit a vigorous "starfish" shape, indicating that using the tips of wild hazel mushroom mycelial cords and the isolation medium of this invention for strain separation is a good method for preparing wild hazel mushroom strains.

[0024] Performance testing 1. Tissue isolation of different parts of wild C. sinensis The different parts of wild C. sinensis were sterilized on a clean bench using sterile operation according to the method of Example 1. A 3 mm block of tissue was cut from the tip of the young cord of wild C. sinensis using a scalpel, and a 3 mm block of tissue was taken from the inside of the mature cord (referred to as "mature cord"), the inside of the base of the stem (referred to as "base of stem"), the inside of the middle of the stem (referred to as "middle of stem"), and the inside of the junction between the stem and cap (referred to as "inside of junction") using tweezers. The strains were then prepared according to the method of Example 1, and 100 pieces of each of the different parts of tissue were inoculated into the isolation medium.

[0025] The results are shown in Table 1.

[0026] Table 1. Comparison of strain isolation from different parts of wild C. sinensis

[0027] Note: +++ indicates the best mycelial growth, ++ indicates better mycelial growth, and + indicates poorer mycelial growth.

[0028] As shown in Table 1, there were obvious differences in the mycelial germination rate, mycelial growth, contamination rate, and success rate of strain isolation from different parts of wild C. sinensis on the medium of Example 1. Although the contamination rate of the mature cord tissue was low (32%), the mycelial germination rate was low (24%), the germinated mycelial growth was poor, and the success rate of isolation was not high (16%). The mycelial germination rate of the base of the stem tissue was low (16%), the germinated mycelial growth was poor, and the contamination rate was the highest (86%), with the lowest success rate of isolation (2%). The mycelial growth of the middle of the stem tissue was better, but the germination rate was not high (26%), the contamination rate was high (75%), and the success rate of isolation was very low (6%). The germination rate of the inside of the junction was slightly high, and the mycelial growth was good, but the contamination rate was high (80%), so the success rate of isolation was also very low (9%). The mycelial growth of the young cord tip tissue was good, the germination rate was the highest (79%), the contamination rate was the lowest (12%), and the success rate was the highest (70%). Therefore, the young cord tip tissue of wild C. sinensis is suitable for strain tissue isolation.

[0029] 2. Tissue isolation of young cord tip of wild C. sinensis on different media Using a scalpel on a clean bench, 3mm pieces of tissue were cut from the tender tips of wild hazel mushrooms used in Example 1. The tissue was then inoculated into sterilized test tube slant PDA medium (hereinafter referred to as "PDA medium"), corn flour and sucrose medium (hereinafter referred to as "corn flour medium"), mixed potato medium (hereinafter referred to as "mixed medium"), potato sawdust extract medium (hereinafter referred to as "sawdust medium"), and the wild hazel mushroom isolation medium (hereinafter referred to as "isolation medium") from Example 1 of this invention. The incubation temperature was 25℃, and the incubation time was 15 days. 100 test tubes were inoculated into each of the above media.

[0030] The formula for PDA culture medium is as follows: 200 g potato (peeled), 20 g glucose, 16 g agar powder, and 1 L water. The formula for corn flour culture medium is: 40 g corn flour, 10 g sucrose, 16 g agar powder, and 1 L water; The formula for the comprehensive culture medium is as follows: 200 g potato (peeled), 20 g glucose, 3 g potassium dihydrogen phosphate, 1.5 g magnesium sulfate, 10 mg vitamin B1, 18 g agar powder, and 1 L water. The formula for the sawdust culture medium is as follows: 200 g potato (peeled), 20 g oak sawdust, 20 g sucrose, 10 g maltose, 18 g agar powder, and 1 L water.

[0031] The results are shown in Table 2.

[0032] Table 2. Comparison of tissue isolation strains from young, tender shoots of wild hazel mushrooms on different culture media.

[0033] Note: +++ indicates the best mycelial growth, ++ indicates good mycelial growth, and + indicates poor mycelial growth.

[0034] As shown in Table 2, when using young shoots of wild hazel mushrooms for tissue isolation on different culture media, the mycelial germination rates on the four conventional culture media were all low (12%, 11%, 16%, and 35%, respectively), while the germination rate on the isolation medium of this invention was as high as 79%, significantly higher than that on conventional culture media. The growth of the young shoots of hazel mushrooms also showed significant differences between the four conventional culture media and the isolation medium. Growth was poor on PDA medium, corn flour medium, and mixed medium, better on sawdust medium, and best on the isolation medium. The contamination rate of young shoots of wild hazel mushrooms was low on all different culture media, all below 20%, indicating that wild hazel mushrooms carry fewer contaminants, and a low contamination rate can be achieved through conventional disinfection methods. The success rate of tissue isolation of young shoots of wild hazel mushrooms on the four conventional culture media was very low or low (10%, 9%, 13%, and 30%, respectively), while the success rate was highest (70%) on the isolation medium. Therefore, the isolation medium of the present invention can promote the germination of hazel mushroom tips, and the medium is suitable for the isolation and preparation of wild hazel mushroom strains.

[0035] 3. DPPH antioxidant activity assay Following the method steps (4) of Example 1, 35 test tubes were inoculated and randomly divided into 7 groups of 5 test tubes each. Each group was incubated in a sterile environment at 24-26℃ and 60%-65% humidity for 12-18 days. The fungal material in the test tubes of the same group was pulverized and mixed. 5 g of each of the 12-day, 13-day, 14-day, 15-day, 16-day, 17-day, and 18-day cultured hazel mushroom samples were accurately weighed using an electronic balance. The samples were extracted with a mixture of chloroform, methanol, and water (volume ratio 1:2.5:1) at a material-to-liquid ratio of 15 mg:1 mL. The extraction was performed using ultrasound at 100 Hz for 30 min, allowed to stand for 4 h, and centrifuged at 5000 r / min. The supernatant was collected as the initial sample and stored at 4℃. 100 μL of each initial sample solution and 100 μL of 0.2 mg / mL DPPH solution were added to a 96-well microplate, with 3 replicates. After adding the sample, shake for 30 seconds, react in the dark at room temperature for 20 minutes, and then measure its absorbance (Ds) at a wavelength of 517 nm. At the same time, measure the absorbance of the blank sample (Dc) without DPPH and the absorbance (Dmax) with DPPH but without sample (using an equal volume of 100 μL of extract instead of sample).

[0036] DPPH free radical scavenging rate (%) = [1-(Ds-Dc) / Dmax]×100%, and the results are shown in Table 3.

[0037] Table 3. DPPH free radical scavenging rate (%) of fungal materials cultured for different days

[0038] As shown in Table 3, the free radical scavenging rate of the fungal material cultured for 15 days was the highest, indicating that the mycelium had the strongest antioxidant capacity at this time. Frozen fungal material cultured for 15 days has a strong ability to resist low temperature damage and is suitable for cryopreservation of fungal strains.

[0039] 4. Comparison of storage periods The same wild hazel mushroom strain was preserved using the preservation method of Example 1 of this invention and the existing preservation methods (low temperature slant and glycerol freezing), and the wild hazel mushroom strains preserved at different times were cultured using conventional methods.

[0040] The results are shown in Table 4.

[0041] Table 4. Effects of different preservation methods on the mycelial growth of wild hazel mushrooms

[0042] Note: +++ indicates the best mycelial growth, ++ indicates good mycelial growth, + indicates poor mycelial growth, and 0 indicates no mycelial growth.

[0043] As shown in Table 4, under 4℃ conditions, the strains preserved by slant culture dried out due to dehydration after more than six months, and the preservation period was only about six months; the strains preserved by freezing (-80℃) with glycerol mycelium had a preservation period of less than 18 months; while the strains preserved by Example 1 of this invention had a preservation period of at least 36 months.

[0044] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a wild hazel mushroom strain, characterized in that, Specifically, the following steps are included: (1) Excavation and disinfection Select wild hazel mushrooms that are intact, healthy, and free from pests and diseases. Cut off the white part of the tip of the mycelial cord, remove impurities, wash, and disinfect the surface. (2) Separation and culture of mother culture A 1-4 mm segment was cut from the white part of the tip of the mycelium cord and inoculated into an isolation medium for culture to obtain the mother culture of hazel mushroom; (3) Liquid culture The mother culture of hazel mushrooms was inoculated into a liquid culture medium and cultured to obtain a liquid spawn; (4) Preparation of strains for cryopreservation The liquid culture was inoculated into hardwood segments for cultivation, vacuum-packed, and refrigerated.

2. The method for preparing a wild hazel mushroom strain according to claim 1, characterized in that, In step (2), the formula of the isolation culture medium is as follows: 370-400 mL of potato decoction, 130-150 mL of fresh hazel mushroom decoction, 450-500 mL of hazel mushroom growing soil soaking solution, 15-25 g / L of glucose, 2-4 g of peptone, 1-2 g / L of magnesium sulfate, 1-3 g / L of potassium dihydrogen phosphate, 5-15 mg of vitamin B1, 18-20 g / L of agar powder, and pH value of 5-7.

5.

3. The method for preparing a wild hazel mushroom strain according to claim 1, characterized in that, In step (3), the liquid culture medium is formulated as follows: 370-400 mL of potato decoction, 130-150 mL of fresh hazel mushroom decoction, 450-500 mL of hazel mushroom growing soil soaking solution, 15-25 g / L of glucose, 2-4 g of peptone, 1-2 g / L of magnesium sulfate, 1-3 g / L of potassium dihydrogen phosphate, 5-15 mg of vitamin B1, pH 5-7.5, and sterilized at 121℃ for 20-30 min.

4. The method for preparing a wild hazel mushroom strain according to claim 2 or 3, characterized in that, The potato decoction is the filtrate obtained by mixing potatoes and water at a mass ratio of 1:(2.0~2.5) and boiling for 20~30 minutes.

5. A method for preparing a wild hazel mushroom strain according to claim 2 or 3, characterized in that, The decoction of fresh hazel mushrooms is the filtrate obtained by boiling fresh hazel mushroom fruiting bodies with water at a mass ratio of 1:(1.5~2.0) for 20~30 minutes.

6. A method for preparing a wild hazel mushroom strain according to claim 2 or 3, characterized in that, The hazel mushroom growing soil soaking solution is a clear filtrate obtained by thoroughly mixing and stirring soil and water from wild hazel mushroom growing areas at a mass ratio of 1:(2.0~2.5).

7. The method for preparing a wild hazel mushroom strain according to claim 1, characterized in that, In step (2), the culture temperature is 23~25℃ and the time is 15~21 days.

8. The method for preparing a wild hazel mushroom strain according to claim 1, characterized in that, In step (3), the culture rotation speed is 130~160 r / min, the temperature is 24~26℃, and the time is 15~21d.

9. The method for preparing a wild hazel mushroom strain according to claim 1, characterized in that, In step (4), the hardwood segment is a one-year-old oak, birch, hazel or elm tree with bark, with a diameter of 2 cm and a length of 10-15 cm, and is sterilized at 121℃ for 30-40 min.

10. The method for preparing a wild hazel mushroom strain according to claim 1, characterized in that, In step (4), the culture temperature is 24~26℃, the humidity is 60%~70%, and the time is 15 days; the refrigeration method is: the low-temperature preservation strain is first stored at 4℃ for 12h, then stored at -20℃ for 12h, and finally refrigerated at -80℃.

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