WO2018095113A1

WO2018095113A1 - Morel cultivation strain and induction and cultivation methods therefor

Info

Publication number: WO2018095113A1
Application number: PCT/CN2017/101395
Authority: WO
Inventors: 秦小波
Original assignee: 秦小波
Priority date: 2016-11-25
Filing date: 2017-09-12
Publication date: 2018-05-31
Also published as: CN106544279B; CN106544279A

Abstract

A morel cultivation strain and induction and cultivation methods therefor, for performing morel induction by using a culture medium for inducing a mother culture of a morel cultivation strain, and cultivating morel by using a culture medium for cultivating the morel cultivation strain. The induction method comprises: extracting a strain preserved in a test tube, inoculating the strain in a mother culture induction culture medium plate, placing the culture medium plate in an incubator and incubating at 18°C to 22°C, and after hypha has grown over the entire plate, using the hypha as hypha for inoculation, and placing the hypha in the cultivation culture medium for subsequent use. The cultivation method comprises: sterilization, inoculation, and management at two stages of a mycelium growth phase and a fruiting body growth phase.

Description

Morchella cultivation strain and induction and cultivation method thereof

Technical field

The invention belongs to the field of industrialization and commercial cultivation of morel, and particularly relates to a species of Morchella cultivation and a method for inducing and cultivating the same.

Background technique

Morchella esculenta (L.) Pers belongs to the Morchella family Morchella. It is an internationally renowned precious edible fungus. It is the best-selling animal in the world and is exported to France and other European countries. product. And as the demand for morel in Germany, France, Italy, and the United States increases, its price continues to rise. Morchella was first recorded in the "Compendium of Materia Medica" and has a long history of use in China. Modern science has found that Morchella is rich in protein, fatty acids and carbohydrates, especially a variety of rare amino acids (such as cis-3-amino-L-proline, 2-aminoisobutyric acid, 2,4-triaminoiso) Butyric acid, etc., mineral elements (potassium, phosphorus, magnesium, calcium, iron, zinc, copper, manganese, etc.) and multivitamins (thiamine, riboflavin, nicotine, pantothenic acid, folic acid, pyridoxine, Biotin, ascorbic acid, VB12) and other ingredients give Morchella unique flavor and nutritional value, and become a high-end food in international catering. Moreover, morel foods and drugs are homologous, modern medical research shows that morel can tonify kidney, make up the brain, impotence, refreshing, lowering blood fat, anti-tumor and so on. In addition, morel can be used for sparkling raw materials, and its liquid fermentation culture can be used for the production of condiments, so it has broad application prospects in many fields such as food, health care products, pharmaceutical and chemical industries. At present, most of the morel exported to China are naturally wild, which is far from meeting the needs of domestic and international markets, and the excessive collection of wild resources has led to a sharp decline in its production, which has greatly damaged the biodiversity of morel. Morel is a precious edible fungus born in late spring and early summer. Exploring the artificial cultivation of morel has been around for nearly a hundred years. However, it was not until 1986 that Ron Ower of the Department of Biology of the Department of Biology of the University of San Francisco published a report on the success of artificial cultivation for the first time in the Journal of Mycology. In 1982 and 1986, two US patents for the cultivation of morel were obtained. Prior to this, there have been reports of successful cultivation of morel plants at home and abroad, but only occasionally the Morchella fruiting body has grown. In recent years, Chinese scientists and technicians have carried out many explorations on the artificial cultivation of morel. The Morchella fruiting bodies have been cultivated and the physiological conditions of some fruiting bodies have been explored. However, at present, artificial cultivation is a kind of bionic cultivation, which needs to be completed outdoors, and the repeatability is poor, the mushrooming is unstable, and mature commercial cultivation cannot be performed so far. Therefore, the artificial or semi-artificial cultivation techniques of Morchella have been the hotspot of international edible fungi research.

In summary, the prior art artificial cultivation of morel can not be completed indoors, and the prepared Morchella is poorly reproducible, the mushroom is unstable, and mature commercial cultivation cannot be carried out.

Summary of the invention

The object of the present invention is to provide a species of Morchella cultivation strain and a method for inducing and cultivating the same, which aims to solve the problem that the Morchella which can not be completed and prepared indoors in the prior art has poor repeatability, unstable mushroom, and cannot be commercialized. The problem of cultivation.

The present invention is achieved in the form of a Mortalella cultivating strain parental inducing medium, wherein the component of the Morchella cultivar parent strain induction medium is 2 g to 4 g by mass and 3 g of yeast extract by mass. 7g, glucose 15g～25g, agar 18g～30g, magnesium sulfate 0.2g～0.5g, potassium dihydrogen phosphate 0.2g～0.5g, potassium nitrate 1.0g～1.5g, sodium butyrate 0.1g～0.3g, folic acid 0.1g ～0.2g, vitamin B10.1g～0.3g, vitamin B60.1g～0.3g, vitamin C0.1g～0.3g; distilled water is added to a total volume of 1000mL.

The component of the induction medium of the Morchella cultivar culture substrate is 200 g to 250 g of water extract of potato, 15 g to 25 g of glucose, 18 g to 30 g of agar, 0.2 g to 0.5 g of magnesium sulfate, and phosphoric acid. Potassium hydrogen 0.2g to 0.5g, potassium nitrate 1.0g to 1.5g, sodium butyrate 0.1g to 0.3g, folic acid 0.1g to 0.2g, vitamin B10.1g to 0.3g, vitamin B60.1g to 0.3g, vitamin C0 .1g ~ 0.3g; add distilled water to a total volume of 1000mL.

Another object of the present invention is to provide a culture medium for the cultivation of Morchella species, which is composed of a dry culture base and a water culture medium of the Morchella cultivar culture medium; the dry culture base of the Morchella cultivation culture medium is composed of wheat by mass percentage 10% to 70%, corn granules 10% to 70%, wood chips 10% to 25%, soil 5% to 15%, peptone 0.5% to 2.0%, sucrose 1.0% to 3.0%, potassium dihydrogen phosphate 0.1% 0.3%, magnesium sulfate 0.1% to 0.3%, urea 0.1% to 0.3%, and vitamin B10.1% to 0.3%.

Further, the total amount of the wheat + corn is 60% to 80%; the amount of water added is 50% to 70%; and the cultivation medium has a pH of 6.0 to 7.5.

Another object of the present invention is to provide a method for inducing a mother seed induction medium for a Morchella cultivar, the method for inducing the mother medium to induce:

The preserved test tube is taken out, inoculated on a mother seed induction medium plate, and cultured in an incubator at 18 ° C to 22 ° C. After the hyphae are full, the inoculated hyphae are prepared for access to the cultivation medium.

Another object of the present invention is to provide a cultivation method for a cultivation medium of a Morchella cultivation strain, which is planted The cultivation method of the culture medium includes:

Sterilization: The culture medium is bottled, placed in a sterilizing pot, sterilized at 100 ° C for 8 h to 10 h for atmospheric pressure sterilization, or maintained at a pressure of 1.4 kg / cm ² for 1.5 h to 2.0 h for high pressure extinction. Bacteria; after sterilization, the culture medium in the bottle is consistent, and there is a transparent gap between the grains;

Inoculation: When the temperature of the bottle drops to 30 ° C and below after sterilization, the bacteria prepared after induction are connected under aseptic conditions;

After inoculation, the cultivated glass bottles are placed in a temperature-controlled cultivation room for management of the mycelium growth phase and the fruiting body growth phase.

Further, the management method of the mycelium growth period includes:

The inoculated bottle is placed in a clean and dark environment to maintain the air humidity of 50% to 70%;

In the initial stage, the indoor temperature is maintained at 16 ° C ~ 20 ° C; after the surface of the tomb is covered with morel hyphae, the temperature is raised to 20 ° C ~ 23 ° C, after 15 to 20 days of cultivation, the mycelial cover, after 20 to 25 days to grow At the bottom of the bottle, the hyphae penetrates the culture material and completes the vegetative growth stage.

Further, when the mycelium growth period is managed, when the hyphae appear in the hyphae, the primordium formation period is managed; specifically:

(1) Temperature difference stimulation, room temperature control at 20 ° C ~ 23 ° C during the day; 8 ° C ~ 12 ° C at night, stimulation 6 h ~ 10 h;

(2) Light stimulation, the light intensity is 50lx~200lx, and the daily light is 10h~14h;

(3) The relative humidity of the air is maintained at 60% to 75%; if the humidity is too large, the aerial hyphae will be too strong and the formation of the buds of the fruiting body will be affected, and the aerial hyphae in the base will be easily induced, which is unfavorable for the growth of the fruiting body; The humidity is too small, which makes the medium lose water early and affects the yield;

(4) Ventilation, ventilate every morning, noon and evening for 15min~20min, or ventilate for 30min in the morning and evening; in addition, if necessary, give mechanical stimulation. If the hyphae grow vigorously, disinfection budding can be used. A wire or a steel needle is placed on the surface of the medium at a distance of about 1 to 2 cm and a depth of 2 to 4 mm. For the formed sclerotia, the sclerotium of the surface layer or the sclerotium of the bottle wall is removed, and the carbonaceous nutrient is appropriately supplemented. Liquid, the primordium can be formed in about 10 days.

Further, the method of sub-entity growth management includes:

After the fruiting body grows out, first seal several holes in the sealing film, and control the room temperature at 20 °C ~ 25 °C; the light intensity is less than 500lx, the light time is not less than 10h every day; the morel has strong phototaxis, so in the child After the solid is formed, the relative direction of the culture bottle and the light source should be adjusted as appropriate, or the indoor light source should be adjusted. Direction, to ensure the normal growth of fruiting bodies, thereby increasing yield; air relative humidity is maintained at 70% to 90%, the maximum can not exceed 95%; daily ventilation time is continuously increased according to the growth of fruiting bodies; the growth period of this fruiting body The management phase is 15 to 20 days.

Further, after the collection of Morchella, the nutrient is still present in the medium, and the management of the fruiting body growth period is maintained, and the management of the Morchella esculenta is carried out.

The morel mushroom provided by the invention is a precious edible fungus with great development prospects. The invention has been explored for a long time, and provides a breakthrough at home and abroad, which can make the morel indoors The technical method of factory cultivation in the season is to optimize the induction of morel species to meet the requirements of factory cultivation, and at the same time, compared with the existing outdoor bionic cultivation, only one season of mushrooming is lagging behind, so that it can continuously produce all seasons. The integrated technology enables commercial production. In the prior art, only one season is produced in the outdoor, and the present invention is continuously produced indoors, and the number of mushrooms is not limited.

DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a flow chart showing a cultivation method of a culture medium for morel cultivation strains according to an embodiment of the present invention.

detailed description

In order to make the objects, technical solutions and advantages of the present invention more comprehensible, the present invention will be further described in detail below with reference to the embodiments. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The application principle of the present invention will be described in detail below with reference to the accompanying drawings.

As shown in FIG. 1 , the cultivation method of the culture medium for the Morchella cultivation strain provided by the embodiment of the present invention comprises:

S101: Sterilization: the culture medium is bottled, placed in a sterilization pot, sterilized at 100 ° C for 8 h to 10 h for atmospheric pressure sterilization, or maintained at a pressure of 1.4 kg / cm ² for 1.5 h to 2.0 h. Autoclaving; after sterilization, the culture medium in the bottle is consistent, and there is a transparent gap between the grains;

S102: Inoculation: when the temperature of the bottle drops to 30 ° C and below after sterilization, the bacteria prepared after induction are connected under aseptic conditions;

S103: After inoculation, the cultivated glass bottle is placed in a temperature-controlled cultivation room for management of the mycelium growth phase and the fruit body growth phase.

According to the embodiment of the present invention, the inoculating medium of the Morchella cultivation strain is determined by mass: peptone 2 g～4g, yeast extract 3g～7g, glucose 15g～25g, agar 18g～30g, magnesium sulfate 0.2g～0.5g, potassium dihydrogen phosphate 0.2g～0.5g, potassium nitrate 1.0g～1.5g, butyric acid Sodium 0.1g to 0.3g, folic acid 0.1g to 0.2g, vitamin B10.1g to 0.3g, vitamin B60.1g to 0.3g, and vitamin C 0.1g to 0.3g; distilled water is added to a total volume of 1000mL.

According to the embodiment of the present invention, the dry culture base of the cultivation medium of the Morchella cultivation strain is composed of: 10% to 70% of wheat and 10% to 70% of corn (grain) (Note: the total amount of wheat + corn is controlled at 60%) ～80%), wood chips 10% to 25%, soil 5% to 15%, peptone 0.5% to 2.0%, sucrose 1.0% to 3.0%, potassium dihydrogen phosphate 0.1% to 0.3%, magnesium sulfate 0.1% to 0.3% Urea 0.1% to 0.3%, vitamin B10.1% to 0.3%, Morchella cultivation culture medium, dry base and water to form a culture medium for morel cultivation.

The water content is controlled to be 50% to 70%, and the pH is 6.0 to 7.5.

The application principle of the present invention will be further described below in conjunction with the construction of a temperature-controlled cultivation room.

The construction of the temperature-controlled cultivation room of the invention:

The area of the culture room is usually 15 to 50 m ² , and the number of culture rooms is determined according to the cultivation scale. The foam wall of 3 to 5 cm thick is attached to the walls, ceilings, doors and windows of each room, and the floor is covered with a 5 cm thick foam board. Air conditioning and exhaust fans are also required for annual cultivation. An iron 4-layer bed frame with a length of 2m, a width of 1m and a height of 2.3m is placed in the room. The first layer is 30cm away from the ground, and each layer is 60cm apart. Install one 25W fluorescent lamp on each floor. A 15m ² room can accommodate 5 bed frames with a cultivation area of 40m ² .

The application principle of the present invention will be further described below in conjunction with the embodiments.

Induction of the parental induction medium of the invention:

The preserved test tube is taken out, inoculated on the mother seed induction medium plate, and cultured in an incubator at 18-22 ° C. After the hyphae are full, the cells can be used for inoculation.

Sterilization of the cultivation medium of the present invention:

Immediately after bottling, it should be placed in a sterilization pot for sterilization. Normal pressure sterilization should be maintained at 100 ° C for 8 to 10 h; autoclaving should be maintained at 1.5 kg / cm ² for 1.5 to 2.0 h. After sterilization, the medium in the bottle should be consistent, there is a gap between the grains, and the grains can be adhered but transparent.

Inoculation of the cultivation medium of the invention:

When the temperature of the bottle drops to 30 ° C after sterilization, the previously prepared strain is inserted under aseptic conditions. Be careful to ensure aseptic operation during inoculation, otherwise mold or bacterial infection will occur. Mold infection can produce different pigments and further spread, compete with the morel to nourish and inhibit the growth of morel hyphae; bacterial infection causes the culture of the bottle to be stinky and the morel grows.

The mycelium growth and mushroom management of the cultivation medium of the present invention include:

After inoculation, the cultivated glass bottles are directly placed in a temperature-controlled cultivation room for management. The cultivation of morel of the invention divides the cultivation management technology into two stages: the mycelium growth period and the fruit body growth period.

1) Management of mycelial growth stage

This stage takes 20 to 25 days, and the key to management is protection from light and low temperature. The inoculated bottle should be placed in a clean and dark environment to maintain the air humidity of 50% to 70%. The key is to protect from light and low temperature. In the initial stage, in order to reduce the contamination of bacteria, the indoor temperature should be kept between 16 and 20 °C. After the surface is covered with morel hyphae, the temperature is raised to 20-23 °C, and the hypha cover is cultivated after 15-20 days. After 20 to 25 days, grow to the bottom of the bottle, and the hyphae can be used to penetrate the culture material to complete the vegetative growth stage.

2) Management of primordium formation period

This phase takes 10 to 15 days. When there are more aerial hyphae in the hyphae, the conditions required for the formation of the primordium should be given:

(1) Temperature difference stimulation. The room temperature is controlled at 20 to 23 ° C during the day and 8 to 12 ° C at night, and the temperature is stimulated for 6 to 10 hours per day.

(2) Light stimulation. The light intensity is 50lx~200lx, and the light is 10h~14h per day. The light is too strong, the ventilation is poor, the primordial differentiation is dense, and even the hyphae aging and dying.

(3) Humidity. The relative humidity of the air is maintained at around 60% to 75%. If the humidity is too large, it will cause the aerial hyphae to be too strong and affect the formation of the buds of the fruiting body. It is easy to induce the aerial hyphae in the base, which is unfavorable for the growth of the fruiting body; the humidity is too small, which makes the medium lose water early and affects the yield.

(4) Ventilation. Every morning, noon, and evening, ventilate for 15 to 20 minutes, or ventilate for 30 minutes in the morning and evening. In addition, if necessary, mechanical stimulation should be given. For example, if the aerial hyphae grows vigorously, the disinfected budding, wire or steel needle can be used to divide the surface of the medium by about 1 to 2 cm and draw a square of 2 to 4 mm deep; The formed sclerotia should be removed from the surface sclerotium or the sclerotium growing on the bottle wall, and the carbon-containing nutrient solution should be appropriately supplemented to form the primordium for about 10 days.

3) Management of the growth period of the fruiting body:

This stage takes 15 to 20 days. After the fruiting body grows out, first seal several holes in the sealing film. The room temperature is controlled at 20-25 °C, and the maximum temperature cannot exceed 28 °C. The light intensity is within 500lx, and the illumination time is not less than 10h per day. Morel has a strong phototaxis, so after the formation of fruiting bodies, the relative direction of the flask and the light source should be adjusted according to the situation, or the direction of the indoor light source should be adjusted to ensure the normal growth of the fruiting body, thereby increasing the yield. The relative humidity of the air is maintained at 70% to 90%, and the maximum can not exceed 95%. Ventilation time per day increases ventilation time based on the growth of fruiting bodies.

Harvesting and preservation of the morel of the present invention:

When the fruit body of Morchella grows to about 10cm, when the pigment on the surface of the cap is darker, it indicates that the fruiting body is mature and can be harvested. After harvesting, the fruiting bodies are dried or dried at 80 °C, sealed in plastic bags, kept in a cool and dry place for short-term storage, and stored in a low-temperature freezer for long-term storage.

Management of the Morchella esculenta in the cultivation medium of the present invention:

After harvesting a morel of Morchella, there is still a lot of nutrients in the culture medium, and the growth of the fruiting body during the growth period will also grow Morpho.

The above is only the preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. Within the scope.

Claims

A Mortality Inducing Medium for Morchella sinensis cultivation strain, characterized in that the component of the induction medium of the Morchella cultivation strain is 2 g to 4 g by mass, 3 g to 7 g of yeast extract, glucose 15g～25g, agar 18g～30g, magnesium sulfate 0.2g～0.5g, potassium dihydrogen phosphate 0.2g～0.5g, potassium nitrate 1.0g～1.5g, sodium butyrate 0.1g～0.3g, folic acid 0.1g～0.2g , vitamin B1 0.1g ~ 0.3g, vitamin B6 0.1g ~ 0.3g, vitamin C 0.1g ~ 0.3g; add distilled water to a total volume of 1000mL.
The apparatus for inducing a moreon culture of the morel culture according to claim 1, wherein the component of the induction medium of the Morchella cultivar is extracted from water of 200 g to 250 g of potato by mass. Liquid, glucose 15g～25g, agar 18g～30g, magnesium sulfate 0.2g～0.5g, potassium dihydrogen phosphate 0.2g～0.5g, potassium nitrate 1.0g～1.5g, sodium butyrate 0.1g～0.3g, folic acid 0.1 g ~ 0.2g, vitamin B1 0.1g ~ 0.3g, vitamin B6 0.1g ~ 0.3g, vitamin C 0.1g ~ 0.3g; add distilled water to a total volume of 1000mL.
The invention relates to a method for inducing a mother seed induction medium according to claim 1, wherein the method for inducing the mother seed induction medium of the Morchella cultivation strain comprises:

The preserved test tube is taken out, inoculated on a mother seed induction medium plate, and cultured in an incubator at 18 ° C to 22 ° C. After the hyphae are full, the inoculated hyphae are prepared for access to the cultivation medium.
A cultivation medium for the culture medium of Morchella sinensis, characterized in that the culture medium for the cultivation medium of the Morchella is composed of the dry culture base of the culture medium of the Morchella cultivation strain and the water; the cultivation of the Morchella cultivation strain The dry weight of the medium is 10% to 70% by weight of wheat, 10% to 70% of corn granules, 10% to 25% of wood chips, 5% to 15% of soil, 0.5% to 2.0% of peptone, and 1.0% to 3.0% of sucrose. It is composed of potassium dihydrogen phosphate 0.1% to 0.3%, magnesium sulfate 0.1% to 0.3%, urea 0.1% to 0.3%, and vitamin B1 0.1% to 0.3%.
The Morchella cultivar culture medium according to claim 4, wherein the total amount of the wheat + corn is 60% to 80%; the amount of water added is 50% to 70%; pH 6.0 ~ 7.5.
A method for cultivating a culture medium for a Morchella cultivar culture according to claim 4, wherein the cultivation method of the Morchella cultivation culture medium comprises:

Sterilization: The culture medium is bottled, placed in a sterilization pot, sterilized at 100 ° C for 8 h to 10 h for atmospheric pressure sterilization, or maintained at a pressure of 1.4 kg / cm 2 for 1.5 h to 2.0 h. After sterilization, the culture medium in the bottle is uniform, and there is a transparent gap between the grains;

Inoculation: When the temperature of the bottle drops below 30 °C after sterilization, the bacteria prepared after induction are connected under aseptic conditions;

After inoculation, the cultivated glass bottles are placed in a temperature-controlled cultivation room for management of the mycelium growth phase and the fruiting body growth phase.
The method for cultivating a culture medium for a Morchella cultivar culture according to claim 6, wherein the method for managing the mycelium growth period comprises:

The inoculated bottle is placed in a clean, dark environment to maintain the air humidity of 50% to 70%; in the initial stage, the indoor temperature is maintained at 16 ° C ~ 20 ° C; after the surface is covered with morel hyphae, The temperature is raised to 20 ° C ~ 23 ° C, after 15 to 20 days of cultivation, the cover of the mycelium grows to the bottom of the bottle after 20 to 25 days, and the hyphae penetrates the culture material to complete the vegetative growth stage.
The method for cultivating a culture medium for a Morchella cultivar culture according to claim 6, wherein when the mycelium growth period is managed, when the hyphae have a plurality of aerial hyphae, the primordium formation period is performed. Management; specifically includes:

(1) Temperature difference stimulation, room temperature control at 20 ° C ~ 23 ° C during the day; 8 ° C ~ 12 ° C at night, stimulation 6 h ~ 10 h;

(2) Light stimulation, the light intensity is 50lx~200lx, and the daily light is 10h~14h;

(3) Maintaining the relative humidity of the air at 60% to 75%;

(4) Ventilation, ventilate every morning, noon and evening for 15min~20min, or ventilate for 30min in the morning and evening. For the formed sclerotia, remove the sclerotium from the surface layer or the sclerotium growing on the bottle wall, and add it appropriately. The carbon nutrient solution can form a primordium for about 10 days.
The method for cultivating a culture medium for a Morchella cultivar culture according to claim 6, wherein the method for managing the growth period of the fruiting body comprises:

After the fruiting body grows out, first seal the sealing film with several holes, the room temperature is controlled at 20 ° C ~ 25 ° C; the light intensity is within 500 lx, the daily light time is not less than 10 h; the relative humidity of the air is maintained at 70% to 90%;

The ventilation time per day increases the ventilation time according to the growth of the fruiting body; the life cycle of this fruiting body is 15 to 20 days.
The method for cultivating a culture medium for a Morchella cultivar culture according to claim 6, wherein after the harvesting of the morel, the nutrient is still present in the medium, and the management of the fruiting body growth period is maintained.茬Morel management.