A method for cultivating hydrangea sphaerocephala strains

Abstract

A method for cultivating a hydrangea spp. strain comprises the following steps: 1) preparing a composite solid culture medium for standby use; 2) preparing a composite liquid culture medium for standby use; 3) taking a hydrangea spp. mother strain, inoculating it on the composite solid culture medium prepared in step 1), and culturing it for 20-25 days at 23-24° C. in a closed environment; 4) cutting an inoculation block, inoculating it on another composite solid culture medium, and culturing it for 15-20 days at 23-24° C. in a closed environment; 5) cutting a plurality of inoculation blocks from the extension end of the hyphae toward the center at 0.5-1.0 cm; 6) inoculating the inoculation block into the composite liquid culture medium prepared in step 2), and oscillating and culturing it for 10-15 days at 25-26° C. under a natural light environment to obtain a hydrangea spp. liquid fermentation strain. The present invention can effectively shorten the cultivation time of the hydrangea spp. strain, and the cultivated strain meets the needs of industrial and large-scale cultivation.

Description

Technical Field

[0001] The invention relates to the field of edible fungi, and in particular to a method for cultivating a hydrangea scalyptus strain. Background Art

[0002] Sparassis Crispa is a rare large fungus that can be used as both food and medicine.

[0003] At present, the mother strain (primary strain) of Hydrangea globulus is usually cultured on potato agar medium, and the culture time is usually 25-30 days. The original strain (secondary strain) is cultured on solid medium mainly composed of corn flour and pine sawdust, and the culture time reaches 50-55 days. The cultured strain is used for production, resulting in a long culture cycle for the entire strain. In addition, since the cultured original strain (secondary strain) is a solid strain with poor uniformity, it is also difficult to apply it in industry and has high production costs.

[0004] Therefore, designing a cultivation method with a short cultivation cycle and guaranteed strain quality is an urgent problem to be solved by technical personnel in this field. Summary of the invention

[0005] The purpose of the present invention is to provide a method for cultivating hydrangea globulus strains in view of the deficiencies of the prior art, which can effectively shorten the cultivation time of hydrangea globulus and the cultivated strains can meet the needs of industrial and large-scale cultivation.

[0006] The technical solution of the present invention is: a method for cultivating Pleurotus eryngii strains, comprising the following steps:

[0007] 1) Preparation of composite solid culture medium

[0008] Take 35-40 parts by weight of agar medium and 5-10 parts by weight of agar powder, add them to the Masson pine extract, adjust the pH to 5.0±0.5, and sterilize to prepare a solid culture medium for later use;

[0009] 2) Preparation of composite liquid culture medium

[0010] Take 10-20 parts by weight of glucose, 2.5-3.5 parts by weight of corn flour, and 0.4-0.7 parts by weight of magnesium sulfate, add them to the Masson pine extract, mix, adjust the pH to 5.0±0.5, and sterilize to prepare a liquid culture medium for later use;

[0011] 3) taking the mother strain of Pleurotus eryngii, inoculating it onto the composite solid culture medium prepared in step 1), and culturing it in a closed environment at 23-24° C. for 20-25 days;

[0012] 4) cutting a plurality of inoculated blocks on the solid culture medium of step 3), inoculating them onto another composite solid culture medium, and culturing them at 23-24° C. in a closed environment for 15-20 days;

[0013] 5) cutting a plurality of inoculation blocks from the solid culture medium of step 4) at a position 0.5-1.0 cm from the extension end of the hyphae toward the center;

[0014] 6) Inoculating the inoculated block into the composite liquid culture medium prepared in step 2), culturing with shaking at 25-26° C. under natural light for 10-15 days, and obtaining a liquid fermentation strain of Hydrangea spp.

[0015] Preferably, in step 1), the weight-to-volume ratio of the agar culture medium and the masson pine extract is 35-40:1000, and the thickness of the prepared solid culture medium is 3-4 mm.

[0016] Preferably, in step 2), the weight-to-volume ratio of glucose to the Masson pine extract is 1-2:100.

[0017] Furthermore, the preparation method of the Masson pine extract in step 1) and / or step 2) is:

[0018] 1) Take fresh and uniform Masson pine sawdust and add it into water;

[0019] 2) After boiling, cool and filter, and take the filtrate to obtain a Masson pine extract.

[0020] Preferably, in step 1), the weight-to-volume ratio of Masson pine sawdust to water is 1:25.

[0021] Preferably, sodium citrate is used to adjust the pH in step 1) and / or step 2).

[0022] Preferably, in step 6), the inoculation block is obtained by punching, and the diameter of the inoculation block is 6 mm.

[0023] Preferably, the oscillation frequency in step 7) is 150 r / min.

[0024] The above technical solution has the following beneficial effects:

[0025] 1. The cultivation method of the present invention comprises the following steps: the mother strain of Pleurotus eryngii is first activated by a composite solid culture medium, then transferred to a composite solid culture medium for rejuvenation, and finally inoculated into a composite liquid culture medium for expansion and cultivation to obtain a strain that can be used for conventional cultivation, so that the entire breeding cycle is shortened to 45-60 days, which can effectively shorten the cultivation time of Pleurotus eryngii.

[0026] 2. The composite liquid culture medium uses the fermentation technology to achieve efficient fermentation of liquid strains and shorten the cultivation period of hydrangea strains. The entire strain stage is cultivated for 45-60 days, which greatly shortens the breeding period compared with the traditional solid culture period of 75-85 days. The fermented bacterial liquid has a mycelium content of 1.2-1.5g / mL, which can be directly inoculated into hydrangea cultivation materials for cultivation and fruiting.

[0027] 3. Hydrangea sphaerocephala likes coniferous wood chips and is suitable for growth under slightly acidic conditions, with an optimum pH value of 4.5-6.0. The culture medium formulas in the present invention all use Masson pine extract to meet the demand of Hydrangea sphaerocephala for coniferous trees, and use sodium citrate to adjust the pH value to a weak acidic state to meet the mycelium growth conditions and promote the liquid fermentation of Hydrangea sphaerocephala under the optimum conditions, thereby achieving a higher fermentation efficiency.

[0028] 4. Liquid strains have the characteristics of short cycle, consistent age and convenient inoculation. After the liquid strains are inoculated, the liquid has strong fluidity, easy dispersion, fast germination, fast colonization, and consistent age, so the uniformity of fruiting is better. Liquid strains have good fluidity and dispersibility, can be dispersed to the center hole and bottom of the cultivation material, the mycelium grows at multiple points, germinates quickly, the overall uniformity is better, and the fruiting cycle and uniformity are also better.

[0029] The following is a further description in conjunction with specific implementation methods. DETAILED DESCRIPTION

[0030] In the present invention, the in vitro mother strain of Pleurotus eryngii propagated by Chongqing Hualu Biological Co., Ltd. is used.

[0031] Example 1

[0032] Preparation of Masson Pine Extract

[0033] Select fresh, uniform, dry, impurity-free and odor-free Masson pine sawdust, with no requirement for particle size and a moisture content of ≤50%. Add to pure water and mix in a mass volume ratio of 1:25 between Masson pine sawdust and pure water. Boil for 20 minutes, cool and filter, and take the filtrate for later use.

[0034] 1) Preparation of composite solid culture medium

[0035] Take 38.5g of agar medium and 6.0g of agar powder, add them to 1L of Masson pine extract and mix, use sodium citrate to adjust the pH to 5.0±0.5, and divide them into 1000mL conical flasks, with 500mL in each conical flask. After wrapping with cotton plugs and aluminum foil, sterilize at 121°C for 20min, cool naturally to 50-60°C, and divide them into test tubes and plates. Before the culture medium solidifies, tilt the test tubes to form a slant culture medium. The thickness of the plate culture medium is 3-4mm, and cool and solidify for use.

[0036] 2) Preparation of composite liquid culture medium

[0037] Take 15g of glucose, 3.0g of corn flour, and 0.5g of magnesium sulfate, add to 1L of Masson pine extract and mix, adjust the pH to 5.0±0.5 with sodium citrate, and divide into 1000mL conical flasks, with 500mL in each conical flask. After wrapping with cotton plugs and aluminum foil, sterilize at 121℃ for 20min and cool naturally.

[0038] 3) Take the company's self-propagated Hydrangea globulus mother culture test tube, cut the Hydrangea globulus mother culture into 3mm*3mm*3mm inoculation blocks, pick up the inoculation blocks with an inoculation needle, and place them in the center of the inclined composite solid culture medium of the test tube. The test tube is placed vertically in a biochemical incubator at 23.5℃ and cultured until the mycelium germinates and grows to meet the transfer requirements, about 23 days, to obtain the first-level seed.

[0039] 4) Cut the primary seed into 3mm*3mm*3mm inoculation blocks, pick up the inoculation blocks with an inoculation needle, place them in the center of the flat composite solid culture medium, seal them with a sealing film, and place them in a biochemical incubator at 23.5°C to cultivate until the mycelium germinates, grows and meets the transfer requirements, about 18 days, to obtain the secondary seed.

[0040] 5) Use a 6 mm diameter puncher to punch holes on the secondary solid culture medium from the extension end of the hyphae toward the center 0.5-1.0 cm, with each hole tangent to each other to eventually form a ring. Use an inoculation needle to gently pick up the inoculation block, and insert it into the shake flask composite liquid culture medium under flame protection. Five inoculation blocks are inserted into each triangular flask, and the triangular flask is placed on a constant temperature oscillator at 25.5±0.5°C. It is cultivated for 15 days under natural light conditions with an oscillation frequency of 150 r / min to obtain the third-level species of Hydrangea (i.e., liquid fermentation strain).

[0041] 6) Shake the liquid fermented strain of Hydrangea in the shake flask, take 5 ml of the pellet suspension, weigh it and place it in a centrifuge, centrifuge it at 2500 r / min for 2 min, pour out the supernatant and weigh it, and the mycelium content is 1.37 g / mL.

[0042] Example 2

[0043] The Hydrangea sphaerocephala strain prepared in Example 1 was taken and cultivated according to the technical regulations for factory-scale production of bag-grown Hydrangea sphaerocephala.

[0044] 1) The formula of the cultivation material is: 85% pine sawdust, 5% bran, 5% corn flour, 5% wheat grains (1mm≤particle size≤2mm), sodium citrate to adjust the pH value to 5.0±0.5. The bag weight is 950g / bag. After filling, the material surface is flat, leaving a center hole, the filling height is about 15cm, and the bag mouth is about 10-15cm away from the material surface. Each bag is inoculated with 35±5g of liquid bacteria.

[0045] 2) Culture conditions at the mycelium stage: culture temperature 23±0.5°C, relative humidity 50-65%, avoid light, ventilation frequency 1 / 25.

[0046] 3) Cultivation conditions during fruiting stage: After the mycelium is fully grown, the cultivation temperature is adjusted to 21±0.5℃, the air humidity is 75-85%, and 400lx white light is provided throughout the day. After the fruiting begins, the bag collar is removed and the bag mouth is straightened, and then the contact position between the bag mouth and the culture medium is fixed with a rubber band. When the fruiting body grows to the bag mouth, the bag mouth is cut. The fruiting body is harvested when it grows to the harvesting requirements.

[0047] The culture period for the mycelium stage is 35 days, and the culture period for the mushroom stage is 25 days. The fruiting bodies can be harvested after a total of 60 days.

[0048] Comparative Example 1

[0049] 1) Preparation of mother culture medium: 38.5g agar medium, 6.0g agar powder, add 1L pure water, and adjust pH to 5.0±0.5 with sodium citrate. Prepare test tube slant culture medium after sterilization. Inoculate the company's self-propagated mother culture medium into the mother culture medium, culture at 23.5±0.5℃ for 20-30 days, and prepare the primary culture.

[0050] 2) Preparation of solid seed culture medium: 85% pine sawdust, 5% bran, 5% corn flour, 5% wheat grains (1mm≤particle size≤2mm), sodium citrate to adjust pH value to 5.0±0.5, fill 450g / bag, leave a center hole, sterilize and inoculate the first-level seed prepared in step 1) into the solid culture medium to prepare the second-level seed. Cultivate at 23±0.5°C for 50-55 days, relative humidity of air 50-65%, avoid light, ventilate at 1 / 25, and prepare solid seed.

[0051] Comparative Example 2

[0052] The solid original seed prepared in Comparative Example 1 was used for fruiting culture using the same culture material formula, mycelium culture, and fruiting culture conditions as in Example 2. 15±5 g of solid seed was inoculated into each bag.

[0053] In comparative example 1, the culture period of the mycelium stage is 45 days, and the culture period of the mushroom stage is 30 days, and the fruiting bodies can be harvested after a total of 75 days.

Claims

1. A method for cultivating Pleurotus eryngii strains, characterized in that: The following steps are involved: 1) Preparation of composite solid culture medium Take 35-40 parts by weight of agar medium and 5-10 parts by weight of agar powder, add them to the Masson pine extract, adjust the pH to 5.0±0.5, and sterilize to prepare a solid culture medium for later use; 2) Preparation of composite liquid culture medium Take 10-20 parts by weight of glucose, 2.5-3.5 parts by weight of corn flour, and 0.4-0.7 parts by weight of magnesium sulfate, add them to the Masson pine extract, mix, adjust the pH to 5.0±0.5, and sterilize to prepare a liquid culture medium for later use; 3) taking the mother strain of Pleurotus eryngii, inoculating it onto the composite solid culture medium prepared in step 1), and culturing it in a closed environment at 23-24° C. for 20-25 days; 4) cutting a plurality of inoculated blocks on the solid culture medium of step 3), inoculating them onto another composite solid culture medium, and culturing them at 23-24° C. in a closed environment for 15-20 days; 5) cutting a plurality of inoculation blocks from the solid culture medium of step 4) at a position 0.5-1.0 cm from the extension end of the hyphae toward the center; 6) Inoculating the inoculated block into the composite liquid culture medium prepared in step 2), culturing with shaking at 25-26° C. under natural light for 10-15 days, and obtaining a liquid fermentation strain of Hydrangea spp.

2. The cultivation method according to claim 1, characterized in that Step 1) The weight-to-volume ratio of the agar culture medium and the masson pine extract is 35-40:1000, and the thickness of the prepared solid culture medium is 3-4 mm.

3. The cultivation method according to claim 1, characterized in that Step 2) The weight-to-volume ratio of glucose to Masson pine extract is 1-2:

100.

4. The cultivation method according to claim 1, characterized in that The preparation method of the Masson pine extract in step 1) and / or step 2) is: 1) Take fresh and uniform Masson pine sawdust and add it into water; 2) After boiling, cool and filter, and take the filtrate to obtain a Masson pine extract.

5. The method for cultivating Pleurotus eryngii strains according to claim 4, characterized in that: Step 1) The weight volume ratio of Masson pine sawdust and water is 1:

25.

6. The method for cultivating Pleurotus eryngii strains according to claim 1, characterized in that: Sodium citrate is used to adjust the pH in step 1) and / or step 2).

7. The method for cultivating Pleurotus eryngii strains according to claim 1, characterized in that: Step 4) A seeding block is obtained by punching, and the diameter of the seeding block is 6 mm.

8. The method for cultivating Pleurotus eryngii strains according to claim 1, characterized in that: Step 5) The oscillation frequency is 150 r / min.