CN109943490A - A kind of Guangdong Cordyceps prairie biomass preparation and recovery method - Google Patents

Abstract

The invention discloses a kind of preparation of Guangdong Cordyceps militaris protoplast and method for resuscitation.The preparation method includes Mycelium culture, protoplast preparation, protoplast purifying, is digested to obtain the protoplast of Guangdong Cordyceps militaris to the mycelium of Guangdong Cordyceps militaris using the mixing enzyme system containing lyases and driselase, and utilize TB₃Recovery medium recovers to Guangdong Cordyceps militaris protoplast.The present invention establishes the preparation of Guangdong Cordyceps militaris protoplast and recovery system for the first time, be conducive to the implementation of the technologies such as later period Study on Genetic Transformation or the protoplast fusion of Guangdong Cordyceps militaris, accelerate the research speed of its heredity and breeding etc., help to promote its research application progress in health food and field of medicaments, benefits society.

Description

A kind of Guangdong Cordyceps prairie biomass preparation and recovery method

technical field

The invention belongs to the biological field of fungi, and in particular relates to a method for preparing and recovering the biomass of Cordyceps sinensis in Guangdong.

Background technique

Cordyceps guangdongensis, a new species published in 2008, has been successfully domesticated and a mature fruiting body cultivation system has been established. Its fruiting body is safe and non-toxic, and was officially approved by the Ministry of Health in 2013 as "New Resource Food" (now renamed "New Food Raw Material"). Experiments have found that the fruiting body of Cordyceps guangdong has significant pharmacological effects, including anti-avian influenza virus, anti-fatigue and life extension, treatment of chronic renal failure, treatment of chronic bronchitis, and immune regulation. Previous studies have also found that Cordyceps in Guangdong is rich in metabolites, including polysaccharides, fatty acids, cordycepin, amino acids, adenosine, and trace elements. At present, through genome sequencing and analysis, it is predicted that there are 30 gene clusters involved in the synthesis of secondary metabolites, and the research on the related gene functions of active substances in Cordyceps guangdong is still in progress. By establishing a preparation and recovery system of Cordyceps guangdong, the present invention lays a foundation for realizing gene function research and breeding of Cordyceps guangdong, and is conducive to in-depth understanding of the synthesis pathway of unique metabolites and the function of key genes. Genetic modification of Cordyceps guangdong to increase the production of active metabolites and obtain more drug lead compounds.

Protoplasts refer to the part of the cell that is only surrounded by the protoplasmic membrane. Since there is no cell wall, it responds more strongly to inducers and is an ideal receptor for genetic engineering. In addition, protoplast fusion can overcome the problem of incompatibility between distant cells and realize hybridization and gene recombination. The key to the preparation of protoplasts is the digestion of the mycelial cell wall. There are many factors that affect the yield and quality of protoplasts, such as: bacterial age, digestive enzymes and their systems, enzymatic hydrolysis time, enzymatic hydrolysis temperature, osmotic stabilizers, etc. (Shen Huimin, Li Chao, etc. , Gao Li, et al. Research progress of protoplast-mediated fungal genetic transformation [J]. Plant Protection, 2017, 43(2): 25-28). Different fungi have their own enzymatic hydrolysis system and enzymatic hydrolysis conditions. At present, there have been researches on the preparation and recovery of protoplasts in the Cordyceps fungus Cordyceps sinensis, Cordyceps militaris, Paecilomyces cicadae, and Kaohsiung Cordyceps. However, the protoplasts of Cordyceps guangdong The preparation method has not yet been established, and the method of preparing Cordyceps prairie plastids using the mixed enzyme system of lyase and collapse enzyme has not been reported yet.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide a method for preparing the biomass of Cordyceps sinensis in Guangdong.

Described Guangdong Cordyceps prairie biomass preparation method, comprises the following steps:

S1, mycelium culture: inoculate the mycelium of Cordyceps guangdong into the YMPT liquid medium for cultivation to obtain the young mycelium of Cordyceps guangdong;

S2. Protoplast preparation: after washing the mycelium with sterile water and KC buffer in turn, adding a mixed enzyme solution for enzymolysis to obtain a mixed solution containing protoplasts;

S3. Purification of protoplasts: The protoplast-containing mixed solution obtained in step S2 is purified to obtain the protoplasts of Cordyceps sinensis.

The total concentration of the enzyme in the mixed enzyme solution of the step S2 is 0.015 g/mL, and the mixed enzyme is composed of a lyase and a collapse enzyme in a mass ratio of 1:1.

The lyophilized powder was purchased from Sigma with the CAS number of 85186-71-6, and the lyophilized powder with the product number of L1412-5G was purchased from Sigma.

The enzymatic hydrolysis in the step S2 is carried out at a rotational speed of 100 rpm, the enzymatic hydrolysis time is 5.5 h, and the enzymatic hydrolysis temperature is 28°C.

The formula of the YMPT liquid medium is as follows: each liter of medium contains 6 g of malt extract, 10 g of tryptone, 20 g of glucose, 6 g of yeast extract, and the solvent is deionized water.

The formula of the KC buffer is as follows: every 300 mL of the culture medium contains 13.42 g of KCl, 1.665 g of CaCl ₂ , and the solvent is deionized water.

The mycelium culture in the step S1 was cultured at 120 rpm and 24° C. for 12 days.

The purification of the step S3 is specifically as follows: filtering the mixed solution containing the protoplasts obtained in the step S2 with sterilized double-layer lens paper, taking the filtrate, centrifuging the filtrate at 4000 rpm and 4°C for 15 min, and decanting the supernatant The protoplast precipitate was retained, washed with KC buffer, centrifuged at 4000 rpm for 15 min at 4°C, and the supernatant was decanted to retain the protoplast precipitate to obtain Cordyceps guangdong protoplast.

The second object of the present invention is to provide a method for recovering the biomass of Cordyceps sinensis in Guangdong.

The described method for resuscitating Cordyceps in Guangdong includes the following steps: adding KC buffer to the protoplast protoplast obtained by the above _- mentioned preparation method for Cordyceps in Guangdong, and mixing the suspension in TB for regeneration Resuscitate culture in the medium to obtain regenerated colonies.

The formula of the TB3 regeneration medium is as follows: each liter of medium contains ₃ g of yeast extract, 3 g of acid hydrolyzed casein, 200 g of sucrose, 15 g of low melting point agarose, and distilled water as the solvent.

The recovery culture was incubated at a constant temperature of 24°C for 10 days.

The beneficial effects of the invention are as follows: the preparation and recovery system of Guangdong Cordyceps sinensis is established for the first time, which is beneficial to the late stage genetic transformation research of Guangdong Cordyceps sinensis or the implementation of protoplast fusion and other technologies, accelerates the research speed of its genetics and breeding, etc. In order to promote its research and application progress in the field of health food and medicine, and benefit the society.

Description of drawings

Figure 1 shows the young mycelium of Cordyceps guangdong.

Figure 2 shows the Guangdong Cordyceps bioplasts.

Figure 3 shows the recovery of Cordyceps in Guangdong on TB ₃ plates.

Detailed ways

The following examples are further illustrations of the present invention, rather than limitations of the present invention.

Example 1 Preparation of Guangdong Cordyceps Bioplasts

(1) Preparation of culture medium and KC buffer:

YMPT liquid medium: Take 6 g of malt extract, 10 g of tryptone, 20 g of glucose, and 6 g of yeast extract, mix, add deionized water to dilute to 1 L, 121 °C, and sterilize for 20 min.

TB ₃ regeneration medium: take 3 g of yeast extract, 3 g of acid hydrolyzed casein, 200 g of sucrose, and 15 g of low-melting agarose, add distilled water to make up to 1 L, and sterilize at 121 °C for 20 min.

KC buffer: take 13.42 g of KCl and 1.665 g of CaCl ₂ , add deionized water to dilute to 300 mL, and sterilize at 121 °C for 20 min.

(2) Preparation and recovery of Guangdong Cordyceps bioplasts:

The mycelium of Cordyceps guangdong was inoculated into 200 mL of YMPT liquid medium, and cultured at 120 rpm and 24°C for 12 d to obtain young mycelium (spheroids) of Cordyceps guangdong (Figure 1). To prepare an enzyme solution, add 0.15 g of a mixed enzyme of lyase and collapse enzyme to 10 mL of KC buffer (the mass ratio of lyase and collapse enzyme is 1:1), and filter and sterilize with a 0.22 μm disposable sterile filter for use. Take 0.5 g (about 40) of Cordyceps guangdong young fungus balls with a pipette, wash once with sterile water, and once with KC buffer. Then, the prepared enzyme solution was poured into the juvenile fungus ball, 100 rpm, 28 ° C, and enzymatic hydrolysis for 5.5 h to obtain a mixed solution containing protoplasts. Filter the mixture with sterilized double-layered lens paper, take the filtrate, centrifuge the filtrate at 4000 rpm for 15 min at 4°C, pour off the supernatant, keep the protoplast pellet, and wash the protoplast pellet with KC buffer. ; Continue to centrifuge at 4000rpm and 4°C for 15min, pour off the supernatant to retain the protoplast precipitate, dilute and suspend with KC buffer, and microscopic examination shows the protoplast of Cordyceps guangdong (Figure 2).

Dilute the Cordyceps in Guangdong to 10 ⁷ /mL with KC buffer, mix evenly in 45 mL of low-melting TB ₃ recovery medium, pour the plate, do three repetitions, and place it in a 24°C constant temperature incubator for 10 days. The hyphae grown from protoplast recovery can be obtained (Figure 3).

The above are only the preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limitations of the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For those skilled in the art, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims (10)

1. a kind of Guangdong Cordyceps militaris method for preparing protoplast, which comprises the following steps:

S1, Mycelium culture: Guangdong Cordyceps militaris mycelium being inoculated into YMPT fluid nutrient medium and is cultivated, and obtains Guangdong Cordyceps militaris The tender mycelium of children；

S2, protoplast preparation: after mycelium is successively washed with sterile water, KC buffer, being added mixed enzyme solution and digested, Obtain the mixed liquor containing protoplast；

S3, protoplast purifying: the mixed liquor containing protoplast that step S2 is obtained purifies, and it is primary to obtain Guangdong Cordyceps militaris Plastid.

2. Guangdong Cordyceps militaris method for preparing protoplast shown according to claim 1, which is characterized in that the mixing of the step S2 The total concentration of enzyme is 0.015g/mL in enzyme solution, and the mixed enzyme is that lyases and driselase are formed with mass ratio 1:1.

3. according to claim 1 or Guangdong Cordyceps militaris method for preparing protoplast shown in 2, which is characterized in that the step S2's Enzymatic hydrolysis is digested under 100rpm revolving speed, enzymolysis time 5.5h, and hydrolysis temperature is 28 DEG C.

4. Guangdong Cordyceps militaris method for preparing protoplast shown according to claim 1, which is characterized in that the YMPT liquid training Support the formula of base are as follows: every liter of culture medium contains malt extract 6g, tryptone 10g, glucose 20g, yeast extract 6g, molten Agent is deionized water.

5. Guangdong Cordyceps militaris method for preparing protoplast shown according to claim 1, which is characterized in that the KC buffer Formula are as follows: every 300mL culture medium contains KCl13.42g, CaCl₂1.665g, solvent are deionized water.

6. Guangdong Cordyceps militaris method for preparing protoplast shown according to claim 1, which is characterized in that the mycelia of the step S1 Body culture is 24 DEG C of culture 12d in 120rpm.

7. Guangdong Cordyceps militaris method for preparing protoplast shown according to claim 1, which is characterized in that the purifying of the step S3 Specifically: the mixed liquor containing protoplast for obtaining step S2 is filtered with the sterilized double-deck lens wiping paper, filtrate is taken, by filtrate It is centrifuged 15min at 4000rpm, 4 DEG C, the light reservation of falling supernatant protoplast pellet washs protoplast pellet with KC buffer, It is centrifuged 15min at 4000rpm, 4 DEG C, the light reservation of falling supernatant protoplast pellet obtains Guangdong Cordyceps militaris protoplast.

8. a kind of Guangdong Cordyceps militaris protoplast method for resuscitation, which comprises the following steps: will be described in claim 1 Guangdong Cordyceps militaris protoplast made from Guangdong Cordyceps militaris method for preparing protoplast is added KC buffer and Protoplast suspension is made, Suspension is mixed in TB₃Recovery culture is carried out in regeneration culture medium, obtains regeneration bacterium colony.

9. Guangdong Cordyceps militaris protoplast method for resuscitation according to claim 8, which is characterized in that the TB₃Regeneration culture The formula of base are as follows: every liter of culture medium contains yeast extract 3g, acid hydrolyzed casein 3g, sucrose 200g, low melting-point agarose 15g, solvent are distilled water.

10. Guangdong Cordyceps militaris protoplast method for resuscitation according to claim 8, which is characterized in that the recovery culture It is in 24 DEG C of constant temperature incubation 10d.