Morchella esculenta preservation culture medium as well as preparation method and application thereof

Abstract

The invention relates to a morchella esculenta preservation culture medium which is prepared from the following components in parts by weight: 5-7 parts of soil, 1-2 parts of sawdust, 1 part of wheat, 1-2 parts of rice husk and 6-6.5 parts of water. The composition and proportion of the soil, the sawdust, the rice husk and the wheat are scientifically selected to prepare the morchella esculenta preservation culture medium, the morchella esculenta preservation culture medium is used for morchella esculenta preservation, does not need to be sealed, can be preserved for more than 7 years for a long time, improves the enzymatic activity in a morchella esculenta culture, can improve the morchella esculenta yield, the fruiting density and the sporocarp mature period when being used for field experiments, and is obviously superior to a traditional preservation method; the method is simple, high in operability and low in energy consumption and can be used for industrial production.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a Morchella preservation culture medium, a preparation method thereof and applications thereof. Background Art

[0002] Morchella is an edible mushroom belonging to the phylum Ascomycota, class Pechomycetes, orders Pechomycetes, family Morchaceae, genus Morchella. It is known as one of the four famous wild mushrooms, along with truffles, matsutake mushrooms, and chanterelles. Morchella is rich in crude protein, crude fat, carbohydrates, multiple amino acids and other nutrients. It has the effects of protecting the liver, lowering blood lipids, enhancing immunity, anti-tumor, anti-oxidation, digestion and stomach, anti-fatigue, and anti-bacteria. It can be used as a raw material to develop food flavor ingredients, nutritional supplements, and nutritional health products.

[0003] The cultivation of morels is a high-risk industry. The degeneration of morel strains is one of the main factors that lead to a sharp decline in fruiting body yields, so it is hoped that high-quality strains can be preserved for a long time. The existing preservation methods include the following: First, repeated subculture of multiple batches of strains. The longest life span of morel strains can grow continuously for about 2.5m, about 6000 hours, and the shortest life span is only 0.059m, which will die completely within a few days to more than ten days; second, new tissues or ascospores are isolated from fresh morels every year to produce morels, but due to spontaneous mutations of active cells, the genetic instability of morel varieties is easily increased; third, the freezing method requires strict freezing conditions and freezing facilities, which is costly; fourth, the morels are sealed and preserved with liquid protective agents such as liquid paraffin, which not only has high cost of liquid protective agents, but also has poor preservation effect. Therefore, it is necessary to study more efficient and stable morel preservation matrix and preservation method. Summary of the invention

[0004] The invention relates to a morel preservation culture medium, which consists of 5-7 parts of soil, 1-2 parts of sawdust, 1 part of wheat, 1-2 parts of husk and 6-6.5 parts of water in parts by weight.

[0005] In a preferred technical solution of the present invention, the preservation medium of the Morchella consists of 7 parts of soil, 1 part of sawdust, 1 part of wheat, 1 part of rice husk and 6 parts of water in parts by weight.

[0006] In a preferred technical solution of the present invention, the preservation medium of the Morchella consists of 6 parts of soil, 2 parts of sawdust, 1 part of wheat, 1 part of rice husk and 6 parts of water in parts by weight.

[0007] In a preferred technical solution of the present invention, the preservation medium of the Morchella consists of 5 parts of soil, 2 parts of sawdust, 1 part of wheat, 2 parts of rice husks and 6.5 parts of water in parts by weight.

[0008] In a preferred technical solution of the present invention, the soil is sandy loam.

[0009] In the preferred technical solution of the present invention, the soil particle size is 0.25-1 mm.

[0010] In a preferred technical solution of the present invention, the sawdust is from a broad-leaved tree of the genus Quercus, preferably any one of Quercus variabilis (Quercus variabilis), Quercus acutissima, Quercus sutchuenensis, or a combination thereof.

[0011] In the preferred technical solution of the present invention, the particle size of the wood chips is 30-50 meshes.

[0012] In the preferred technical solution of the present invention, the husks and wheat are fresh and free from mold.

[0013] In the preferred technical solution of the present invention, the method for preparing the Morchella preservation medium is to add water to the required amount of soil, sawdust, wheat, and husk, mix them evenly, and sterilize them.

[0014] The invention relates to a method for preparing a morel preservation medium. The morel preservation medium consists of 5-7 parts of soil, 1-2 parts of sawdust, 1 part of wheat, 1-2 parts of rice husks and 6-6.5 parts of water in parts by weight, and comprises the following steps: adding water to required amounts of soil, sawdust, wheat and rice husks, mixing them uniformly, and sterilizing them to obtain the preparation medium.

[0015] In a preferred technical solution of the present invention, the preservation medium of the Morchella consists of 7 parts of soil, 1 part of sawdust, 1 part of wheat, 1 part of rice husk and 6 parts of water in parts by weight.

[0016] In a preferred technical solution of the present invention, the preservation medium of the Morchella consists of 6 parts of soil, 2 parts of sawdust, 1 part of wheat, 1 part of rice husk and 6 parts of water in parts by weight.

[0017] In a preferred technical solution of the present invention, the preservation medium of the Morchella consists of 5 parts of soil, 2 parts of sawdust, 1 part of wheat, 2 parts of rice husks and 6.5 parts of water in parts by weight.

[0018] In a preferred technical solution of the present invention, the soil is sandy loam.

[0019] In the preferred technical solution of the present invention, the soil particle size is 0.25-1 mm.

[0020] In a preferred technical solution of the present invention, the sawdust is from a broad-leaved tree of the genus Quercus, preferably any one of Quercus variabilis (Quercus variabilis), Quercus acutissima, Quercus sutchuenensis, or a combination thereof.

[0021] In the preferred technical solution of the present invention, the particle size of the wood chips is 30-50 meshes.

[0022] In the preferred technical solution of the present invention, the husks and wheat are fresh and free from mold.

[0023] Another object of the present invention is to provide application of the Morchella preservation medium of the present invention in Morchella preservation.

[0024] In a preferred technical solution of the present invention, the application is to inoculate the mycelium culture onto a preservation medium.

[0025] In a preferred technical solution of the present invention, the application is to inoculate the mycelium culture onto the preservation medium without sealing.

[0026] In the preferred technical solution of the present invention, the application is to inoculate the mycelial culture onto the preservation medium without performing subculture during the preservation period.

[0027] In a preferred technical solution of the present invention, the application is to inoculate the mycelium culture onto a preservation medium, which can preserve the morels for not less than 5 years, preferably not less than 7 years, and more preferably not less than 10 years.

[0028] Another object of the present invention is to provide a method for preserving Morchella, comprising the following steps: inoculating the mycelium culture onto the Morchella preservation medium of the present invention.

[0029] In the preferred technical solution of the present invention, the preservation method does not require sealing.

[0030] In a preferred technical solution of the present invention, no subculture is performed during the preservation period.

[0031] In the preferred technical solution of the present invention, the method can preserve the morels for not less than 5 years, preferably not less than 7 years, and more preferably not less than 10 years.

[0032] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between liquids and solids, the percentage is volume / weight percentage; when the present invention relates to the percentage between solids and liquids, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0033] Compared with the prior art, the beneficial technical effects of the present invention include:

[0034] 1. The present invention scientifically selects the composition and ratio of soil, wood chips, husks and wheat to prepare a morel preservation medium for morel preservation, which can not only provide sufficient carbon source and matrix, but also improve the permeability of morel growth, does not need to be subcultured, does not need to be sealed, can be preserved for a long time for more than 7 years, and improves the activity of enzymes (such as cellulase, multifunctional peroxidase, manganese peroxidase) in morel cultures. It can be used for field experiments to increase the yield of morels, the density of mushroom production and the maturity period of fruiting bodies, which is significantly better than traditional preservation methods.

[0035] 2. The preservation method of the present invention is simple, highly operable, and low in energy consumption. It does not require specialized facilities and equipment for deep cryopreservation at -80°C or liquid nitrogen, and can be used for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Fig. 1 Field experiment results, a yield, b mushroom density, c maturity cycle. DETAILED DESCRIPTION

[0037] The present invention will be described below with reference to examples, but the present invention is not limited to the examples.

[0038] In the examples, Sichuan Morchella No. 1 (Sichuan Shenjun 2013007) from Sichuan Academy of Agricultural Sciences was used for the experiments.

[0039] Potato dextrose agar medium (PDA): contains 200g potato, 20g glucose, 20g agar and 1000mL distilled water. Take 20ml and put it into a test tube (test tube diameter 20mm×200mm), sterilize (121℃, 30min), and cool to obtain a test tube containing PDA solid medium. Take 20ml of the sterilized medium and put it into a culture dish (diameter 90mm), cool to obtain a culture dish containing PDA solid medium.

[0040] Preparation of Morchella mycelium culture: Place the mycelium of Morchella chuanxiensis No. 1 in a culture dish (90 mm in diameter) containing PDA solid culture medium and culture at 20° C. for 7 days.

[0041] A cultivation bag (17 cm × 35 cm × 0.005 cm) containing 500 g of cultivation medium: the composition of the cultivation medium by weight is 30 parts of wheat, 18.5 parts of soil, 20 parts of rice husk, 30 parts of sawdust, 1.5 parts of lime, and 150 parts of water. The cultivation bag is sterilized at 121°C for 2.5 hours and then cooled for use.

[0042] Example 1 Preparation of Morchella preservation medium of the present invention

[0043] (1) Select sandy loam with a particle size of 0.25 to 1 mm as soil;

[0044] (2) Select sawdust from the green loblolly tree and crush it, first pass it through a 10-mesh sieve, then pass it through a 30-50 mesh sieve, and take the sawdust on the 30-50 mesh sieve for later use;

[0045] (3) Choose fresh, mold-free husks and wheat of the current year;

[0046] (4) By weight, 7 parts of soil, 1 part of sawdust, 1 part of wheat, 1 part of rice husk, and 6 parts of water were mixed evenly, sterilized at 121°C for 2.5 hours, and cooled to obtain the product.

[0047] Example 2 Preparation of Morchella preservation medium of the present invention

[0048] (1) Select sandy loam with a particle size of 0.25 to 1 mm as soil;

[0049] (2) Select sawdust from the green loblolly tree and crush it, first pass it through a 10-mesh sieve, then pass it through a 30-50 mesh sieve, and take the sawdust on the 30-50 mesh sieve for later use;

[0050] (3) Choose fresh, mold-free husks and wheat of the current year;

[0051] (4) By weight, 6 parts of soil, 2 parts of sawdust, 1 part of wheat, 1 part of rice husk, and 6 parts of water are mixed evenly, sterilized at 121°C for 2.5 hours, and cooled to obtain the product.

[0052] Example 3 Preparation of Morchella preservation medium of the present invention

[0053] (1) Select sandy loam with a particle size of 0.25 to 1 mm as soil;

[0054] (2) Select sawdust from the green loblolly tree and crush it, first pass it through a 10-mesh sieve, then pass it through a 30-50 mesh sieve, and take the sawdust on the 30-50 mesh sieve for later use;

[0055] (3) Choose fresh, mold-free husks and wheat of the current year;

[0056] (4) By weight, 7 parts of soil, 2 parts of sawdust, 1 part of wheat, 2 parts of rice husk, and 6.5 parts of water were mixed evenly, sterilized at 121°C for 2.5 hours, and cooled to obtain the product.

[0057] Experimental Example 1 Study on the application of the Morchella preservation medium of the present invention in the preservation of Morchella

[0058] 1. Morel preservation experiment

[0059] Using a sterile cork puncher with an inner diameter of 5 mm, 18 Morchella mycelium cultures with a diameter of 5 mm were taken and divided into 3 groups, with 6 parallel replicates in each group, and inoculated onto different culture media. From February 2015 to February 2022, they were stored at 4°C for 7 years:

[0060] CK group: Morchella mycelium culture was inoculated into a test tube (test tube diameter 20mm×200mm) filled with PDA solid medium and sealed with a rubber stopper. The test tube strain was the starting test tube, and was subcultured to PDA solid medium twice a year, for a total of 14 times in the course of 7 years. Specific subculture method: Take an inoculation block with a diameter of 5mm from the Morchella mycelium culture in the starting test tube and transfer it to a new test tube filled with PDA solid medium.

[0061] Group T1: Morchella mycelium culture was inoculated into a test tube (test tube diameter 20 mm×200 mm) filled with the preservation medium of Example 1, sealed with a rubber stopper, and no subculture was performed during the 7-year test period.

[0062] T2 group: The Morchella mycelium culture was inoculated into a test tube (test tube diameter 20 mm × 200 mm) filled with the preservation medium of Example 1 and sealed with a rubber stopper. The test tube strain was the starting test tube and was subcultured to the preservation medium of Example 1 twice a year, for a total of 14 times in the course of 7 years. The specific subculture method was as follows: an inoculation block with a diameter of 5 mm was taken from the Morchella mycelium culture in the starting test tube and transferred to a new test tube filled with the preservation medium of Example 1.

[0063] Seven years later, the mycelium cultures of each group were inoculated into culture dishes (90 mm in diameter) filled with PDA solid culture medium, and activated and cultured at 20° C. in the dark for 7 days to obtain activated morel mycelium cultures.

[0064] 2. Field experiment

[0065] Cultivation bags containing GK group, TI group and T2 group morels were prepared respectively. The preparation method was as follows: a piece of activated morel mycelium culture was taken with a sterile puncher with an inner diameter of 5 mm, inoculated into the center of a culture dish (diameter 90 mm) filled with new PDA solid culture medium, and cultured at 20°C for 10 days, then 3 5 mm inoculation blocks were taken, inoculated into a cultivation bag filled with 500 g cultivation culture medium, and cultured at 20°C in the dark for 20 days.

[0066] The field experimental soil was located in the field cultivation test field of the Modern Agricultural Science and Technology Innovation Demonstration Park of Sichuan Academy of Agricultural Sciences in Taixing Town, Xindu District, Chengdu City (soil pH 6.5-7.5), and was divided into 3 groups, each with 6 plots of 5 m × 1 m, corresponding to 6 parallel repetitions during the preservation period.

[0067] Control group: The cultivation bags containing the GK group morels were sown into the soil at a sowing amount of 300 g / ㎡.

[0068] Experimental group 1: The cultivation bags containing T1 group morels were sown into the soil at a sowing amount of 300g / ㎡.

[0069] Experimental group 2: The cultivation bags containing T2 group morels were sown into the soil at a sowing amount of 300 g / ㎡.

[0070] Plant and harvest Morchella according to the conventional method and calculate its yield (g / m 2 , dry weight), mushroom density (per m 2 number of ascocarps), fruiting body maturity period (days from sowing to fruiting) (see Figure 1a , Figure 1b, Figure 1c ). Among them, * means there is a significant difference compared with the control group, P < 0.05, △ means there is a significant difference compared with the experimental group 2, P < 0.05.

[0071] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art may make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

Claims

1. A Morchella preservation medium, which is composed of 5-7 parts of soil, 1-2 parts of sawdust, 1 part of wheat, 1-2 parts of rice husks and 6-6.5 parts of water in parts by weight.

2. The culture medium according to claim 1, wherein the soil is sandy loam.

3. The culture medium according to any one of claims 1-2, wherein the soil particle size is 0.25 to 1 mm.

4. The culture medium according to any one of claims 1 to 3, wherein the sawdust is from a broad-leaved tree of the genus Quercus, preferably any one of Quercus variabilis, Quercus alpinus, Quercus acutissima, and Quercus sutchuenensis, or a combination thereof.

5. The culture medium according to any one of claims 1 to 4, wherein the sawdust particle size is 30-50 meshes.

6. The culture medium according to any one of claims 1 to 5, wherein the husks and wheat are fresh and free from mold.

7. The culture medium according to any one of claims 1 to 6, wherein the preparation method of the Morchella preservation culture medium is to uniformly mix required amounts of soil, sawdust, wheat, rice husks and water, and sterilize the mixture.

8. The method for preparing a Morchella preservation medium according to any one of claims 1 to 7, wherein the Morchella preservation medium is composed of 5 to 7 parts of soil, 1 to 2 parts of sawdust, 1 part of wheat, 1 to 2 parts of husks and 6 to 6.5 parts of water in parts by weight, comprising the following steps: Mix the required amount of soil, sawdust, wheat, rice husk and water evenly and sterilize them.

9. Use of the Morchella preservation medium according to any one of claims 1 to 7 in Morchella preservation, preferably, the mycelial culture is inoculated onto the Morchella preservation medium of the present invention.

10. A method for preserving Morchella, comprising the following steps: The mycelium culture is inoculated onto the Morchella preservation medium of the present invention. Preferably, the preservation method does not require sealing.

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