(1) material
⒈ Strain: Korean Ganoderma lucidum mother strain, provided by Hunan Edible Fungi Research Institute.
2. Seed culture medium: 2% corn flour and 2% rice flour.
Sucrose 2% potassium dihydrogen phosphate o. 1g
0.05% magnesium sulfate is a natural ph value.
3. Fermentation medium: wheat bran extract 10% glucose 2%.
Ammonium sulfate 0.2%
The ph value of 0.2% of potassium dihydrogen phosphate is natural.
(2) Method 1. Seed culture:
1. Fermentation and culture to produce mycelium.
3. Determination of dry mycelium and wet mycelium.
Centrifuge the fermentation broth, then take its sediment, add 60% sucrose, perform high-speed (3,000 rpm) density gradient centrifugation for 5 minutes, take the upper mycelium, wash the sucrose, then press dry to obtain wet bacteria, and then dry the wet bacteria at high temperature to obtain dry bacteria. Weigh them separately.
2. Polysaccharide extraction.
Pretreatment of mycelium. Take a proper amount of Ganoderma lucidum wet mycelium and treat it with organic solvents such as ethanol to remove lipid substances in the dehumidified mycelium, and at the same time inactivate glycosidase to prevent polysaccharide degradation.
⑵ Hot water extraction of polysaccharides. The pretreated wet mycelia of Ganoderma lucidum were put into hot water (95℃) of 1: 20, and extracted continuously for three times. Combine the three water extracts, concentrate under reduced pressure to a certain volume, then mix with 95% ethanol with three times the volume, stand for 10- 12 hours, and then centrifuge. This precipitate is crude polysaccharide, mixed with small molecular impurities such as protein, pigment and oligosaccharide, and generally needs to be purified.
⑶ Polysaccharide, protein and deae cellulose were purified by column chromatography. Sevag method is generally used to remove protein: mix chloroform with 0.2 times the volume of polysaccharide solution and n-butanol with 0.04 times the volume, and shake for half an hour to separate until there is no precipitation at the interface between chloroform and water. Repeated treatment for 2-3 times can effectively remove protein in polysaccharide. Generally, the purification of polysaccharide is carried out by deae cellulose column chromatography: the deproteinized polysaccharide is eluted with 0.025mol/l, 0. 1mol/l borax and 0. 1mol/l sodium hydroxide solution respectively, and then the absorbance is measured with 0.2% copper sulfate solution, and the eluent containing polysaccharide is collected, and the desired polysaccharide is obtained after concentration and desalination.
⑷ Identification of polysaccharide purity.
It can be identified by electrophoresis, and if there is a single band, it is pure polysaccharide.
⒌ determination of protein in polysaccharides and polysaccharides;
Take a certain amount of mycelium crude polysaccharide, add water to boil and dissolve, deproteinize by sevag method, and determine the contents of protein and polysaccharide in crude polysaccharide by Coomassie brilliant blue method. The results showed that the crude polysaccharide content in mycelium was 17.0 1%, the polysaccharide content was 5.43%, and the protein content was 2. 16%.
Extraction of polysaccharide from ganoderma lucidum fruiting body
(1) material 1. Strain: Korean Ganoderma lucidum cultivated species.
4. Raw materials for fruiting body cultivation: sawdust 78%, rice bran 20%, sucrose 1%,
Gypsum 1%, natural ph value.
(2) Method
1. Culture of fruiting body.
4. Pretreatment of Ganoderma lucidum fruiting body.
3. Extraction of polysaccharide from fruiting body.
Take a proper amount of Ganoderma lucidum fruiting body, pound it into powder, sieve it with 80 meshes, and then extract it with hot water extraction. The specific operation is the same as the extraction of mycelium polysaccharide. The extracted crude polysaccharide contains small molecular impurities such as pigment and protein, which should also be removed.
4. Remove protein and pigment.
Selenium-negative method is also used to remove protein from crude polysaccharide of fruiting body. At present, no ideal method for removing pigment has been found. Generally, it is washed repeatedly with ethanol, and the effect is not ideal.
⒌ Identification of polysaccharide purity.
Identification of the purity of polysaccharide from the same mycelium.
6. Determination of crude polysaccharide, polysaccharide and protein in fruiting body.
The specific method is the same as the determination of polysaccharide and protein in mycelium. The content of crude polysaccharide in fruiting body is 7.5%, polysaccharide 1.5% and protein 0.6%, which are all lower than mycelium. It can be seen that the effective components of Ganoderma lucidum polysaccharide are formed in the mycelium stage, which provides a scientific basis for the submerged fermentation of Ganoderma lucidum to produce mycelium.