Preparation method of black fungus mycelium polypeptide

Abstract

The invention provides a black fungus liquid fermentation culture medium and a black fungus polypeptide preparation method. The liquid fermentation culture medium comprises glucose, cane sugar, peptone, KH2PO4, MgSO4. 7H2O, vitamins and the like. The preparation method comprises the following steps: inoculating a black fungus mother strain into a black fungus mycelium liquid fermentation culture medium, and carrying out liquid fermentation to obtain black fungus mycelia; mixing the auricularia auricula mycelia with water according to proper comparison, performing ultrasonic treatment, adding protease, and performing enzymolysis reaction at 45-55 DEG C to obtain enzymatic hydrolysate; and centrifuging the enzymatic hydrolysate, concentrating, adding 95% ethanol of which the volume is 3-5 times that of the enzymatic hydrolysate, staying overnight at 4 DEG C, centrifuging, taking supernate, concentrating, and freeze-drying to obtain the black fungus polypeptide. According to the method, the yield of black fungus mycelia and the content of black fungus polypeptides can be remarkably increased.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a black fungus mycelium liquid fermentation medium and a preparation method of a black fungus polypeptide. Background Art

[0002] Black fungus (CAuricularia Auricularia) is a famous mountain delicacy. It can be eaten, used as medicine, and can be used as a tonic. It has the reputation of "the meat in the vegetarian food" and is known as the "black treasure in Chinese food" in the world. Black fungus, also known as wood ear, ear, light wood ear, belongs to the fungus of the genus Auricularia in the class of Basidiomycetes of the phylum Fungi. Black fungus is rich in nutrition and delicious. It is not only an edible fungus with high nutritional value, but also a medicinal fungus with high medicinal value. It is a health product recognized by the world. "Compendium of Materia Medica" records: "Auricularia grows on rotten wood, is sweet and flat in nature, and is mainly used to replenish qi and not be hungry, lighten the body and strengthen the will, and has the effect of treating hemorrhoids, bloody diarrhea, and bleeding." Modern medical research has found that black fungus has a variety of pharmacological effects such as lowering blood lipids, delaying aging, enhancing immunity and anti-tumor. Black fungus is a typical representative of "medicine and food are of the same origin". It is not only rich in nutrients, but also has extremely high medicinal value. Black fungus is rich in a large amount of carbohydrates and proteins.

[0003] Changbai Mountain black fungus has the advantages of excellent quality, high nutritional value, and rich resources, but its in-depth research and development is still insufficient. Studies have shown that the content of crude protein, crude fiber, polysaccharides, and polypeptides in black fungus mycelium is higher than that in black fungus fruiting bodies. At present, the research on the functional components of black fungus mainly focuses on polysaccharides, while there is little research on other components, especially black fungus polypeptides. Summary of the invention

[0004] Based on the problems existing in the above-mentioned prior art, the present invention aims to deepen the exploration of the functional activity of black fungus polypeptides, provide technical support for further deep processing of black fungus, and better develop functional foods that improve immunity, reduce fat and protect the liver. Extracting polypeptides from black fungus mycelium has the advantages of fast mycelium growth rate, simple fermentation steps, and easy control. Extracting polypeptides from mycelium can shorten production time and improve production efficiency. After ingesting mycelial protein, the human body can exert its health care function in the form of small molecule polypeptides, and mycelial macromolecular protein can form a large number of small molecule polypeptides after enzymatic hydrolysis.

[0005] The invention provides a novel black fungus mycelium liquid fermentation medium and a method for preparing a black fungus mycelium polypeptide.

[0006] The technical solution of the present invention is as follows:

[0007] A technical solution of the present invention provides a black fungus mycelium liquid fermentation medium, which includes 1% glucose, 1.5% sucrose, 0.3% peptone, 0.05% KH2PO4, 0.05% MgSO4·7H2O, 0.0001% vitamin B1 according to mass percentage, and distilled water is added to 1000mL.

[0008] The present invention also provides a method for preparing a black fungus mycelium polypeptide, comprising the following steps:

[0009] 1) inoculating the black fungus mother seed into the black fungus mycelium liquid fermentation medium, and performing liquid fermentation to obtain the black fungus mycelium;

[0010] 2) mixing the black fungus mycelium with water at a ratio of 1:10 to 1:50, soaking for 20 to 60 minutes, ultrasonically treating, adjusting the pH value to 7.0, adding protease, and performing enzymolysis at 45° C. to 55° C. to obtain an enzymolysis solution;

[0011] 3) The enzymatic hydrolysate was centrifuged, and the enzymatic hydrolysate supernatant was concentrated. 3 to 5 times the volume of 95% ethanol was added to the concentrate, and the concentrate was kept at 4° C. overnight. After centrifugation, the supernatant was collected;

[0012] 4) After the supernatant is appropriately concentrated, freeze-dried to obtain black fungus polypeptide.

[0013] Furthermore, in the above technical solution, in step 1), the conditions of the liquid fermentation are: fermentation temperature 25-27° C., ventilation and stirring, and fermentation time 6-10 days.

[0014] Furthermore, in the above technical solution, in step 1), the fermentation liquid obtained by liquid fermentation is filtered or centrifuged at 2800-6000 rpm to collect mycelium, which is then washed and dried to obtain black fungus mycelium.

[0015] Furthermore, in the above technical solution, in step 2), the protease is a neutral protease, an alkaline protease or a composite protease.

[0016] Furthermore, in the above technical solution, in step 2), the ultrasonic treatment is carried out at 100% ultrasonic power for 15 min to 60 min.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] First, the present invention provides a black fungus mycelium liquid fermentation medium, and uses the liquid fermentation medium to carry out liquid fermentation of the black fungus mycelium. According to the present invention, not only can the mycelium growth cycle be shortened and the operation steps be reduced, but also the efficiency is high and the production cost is low, and it is suitable for industrial production, and a simple preparation method is provided for the black fungus liquid fermentation, and a theoretical basis is provided for the later fermentation expansion culture of the black fungus mycelium, the extraction of active ingredients, the secondary metabolites and the like.

[0019] The contents of crude protein, crude fiber, polysaccharides, polypeptides, etc. in black fungus mycelium are higher than those in fruiting bodies. For black fungus mycelium obtained by liquid fermentation, ultrasonic pretreatment is used, combined with enzymatic extraction technology to reduce the loss of biologically active components in the material, and the extraction rate is high. In the product drying process, vacuum freeze drying technology is used to convert water directly from solid to gas. The material is kept below 0°C during the entire drying process, further reducing the loss of biological activity. This product can be dissolved in water at room temperature, which is convenient for rapid absorption by the human body.

[0020] According to the method of the present invention, the black fungus mycelium yield and the black fungus polypeptide content can be significantly improved. DETAILED DESCRIPTION

[0021] The specific implementation of the present invention is described below in conjunction with the examples. However, the protection scope of the present invention cannot be considered to be limited to the following specific implementation and specific examples. For ordinary technicians in the relevant technical field, several simple deductions or equivalent substitutions can be made without departing from the basic premise of the concept of the present invention, and these equivalent substitution schemes will still be regarded as within the protection scope of the present invention.

[0022] Example 1

[0023] Black fungus liquid fermentation medium: glucose 1%, sucrose 1.5%, peptone 0.3%, KH2PO4 0.05%, MgSO4·7H2O 0.05%, vitamin B1 0.0001%, add distilled water to make up to the specified amount, mix well and sterilize at 121℃ for 20min and then cool.

[0024] Under sterile conditions, 10-12 0.5 cm pieces of bacteria were separated from the test tube bacteria, inoculated into a conical flask, and cultured in a constant temperature oscillator at 26°C and 130 r / min for 5 days. The liquid culture bacteria were shaken and set aside.

[0025] The black fungus mother culture is inoculated and then liquid fermentation is carried out at a temperature of 26° C. with ventilation and stirring for 6 days. The cultured fermentation liquid is centrifuged at 6000 rpm, mycelium is collected, and black fungus mycelium is obtained through washing and freeze-drying.

[0026] Black fungus mycelium powder and water are mixed in a certain proportion, soaked for 30 minutes, ultrasonic power 100%, 20 minutes, adjust the pH value to 7, add an appropriate amount of neutral protease, react at 45°C, and inactivate the enzyme at 90°C for 10 minutes. Filter, evaporate and concentrate to 20 ml, add 4 times the volume of 95% ethanol, place in a 4°C refrigerator for alcohol precipitation overnight, centrifuge at 4000r / min for 10 minutes, and take the supernatant; the mycelium residue after centrifugation is extracted twice according to the above extraction steps, and the supernatant is combined; the supernatant is post-processed and freeze-dried to obtain black fungus polypeptide.

[0027] Example 2

[0028] Black fungus liquid fermentation medium: glucose 2%, sucrose 0.5%, peptone 0.1%, KH2PO4 0.025%, MgSO4·7H2O 0.05%, vitamin B1 0.0001%, add distilled water to make up to the specified amount, mix well and sterilize at 121℃ for 15min and then cool.

[0029] Under sterile conditions, 10-12 0.5 cm pieces of bacteria were separated from the test tube bacteria, inoculated into a conical flask, and cultured in a constant temperature oscillator at 26°C and 130 r / min for 6 days. The liquid culture bacteria were shaken and set aside.

[0030] The black fungus mother culture is inoculated and then liquid fermentation is carried out at a temperature of 27° C. with ventilation and stirring for 6 days. The cultured fermentation liquid is centrifuged at 6000 rpm, mycelium is collected, and black fungus mycelium is obtained through washing and freeze-drying.

[0031] Black fungus mycelium powder and water are mixed in a certain proportion, soaked for 30 minutes, ultrasonic power is 100%, 20 minutes, pH value is adjusted to 7, appropriate amount of alkaline protease is added, reaction is carried out at 50℃, enzyme is inactivated at 90℃ for 10 minutes. Filter, evaporate and concentrate to 20ml, add 4 times volume of 95% ethanol, place in a refrigerator at 4℃ for alcohol precipitation overnight, centrifuge at 4000r / min for 10 minutes, and take supernatant; extract the mycelium residue after centrifugation twice according to the above extraction steps, and combine the supernatant; the supernatant is post-processed and freeze-dried to obtain black fungus polypeptide.

[0032] Example 3

[0033] Black fungus liquid fermentation medium: glucose 3%, sucrose 1%, peptone 0.2%, KH2PO4 0.075%, MgSO4·7H2O 0.05%, vitamin B1 0.0001%, add distilled water to make up to the specified amount, mix well and sterilize at 121℃ for 30min and then cool.

[0034] Under sterile conditions, separate 10-12 0.5 cm2 bacterial blocks from the test tube bacteria, inoculate them into a conical flask, and culture them in a constant temperature oscillator at 26°C and 130 r / min for 7 days. Shake the liquid culture bacteria well and set aside.

[0035] The black fungus mother culture is inoculated and then liquid fermentation is carried out at a temperature of 27° C. with ventilation and stirring for 7 days. The cultured fermentation liquid is centrifuged at 6000 rpm, mycelium is collected, and black fungus mycelium is obtained through washing and freeze-drying.

[0036] Black fungus mycelium powder and water are mixed in a certain proportion, soaked for 30 minutes, ultrasonic power is 100%, 20 minutes, pH value is adjusted to 7, appropriate amount of composite protease is added, reaction is carried out at 55℃, enzyme is inactivated at 90℃ for 10 minutes. Filter, evaporate and concentrate to 20ml, add 4 times volume of 95% ethanol, place in a refrigerator at 4℃ for alcohol precipitation overnight, centrifuge at 4000r / min for 10 minutes, and take supernatant; extract the mycelium residue after centrifugation twice according to the above extraction steps, and combine the supernatant; the supernatant is post-processed and freeze-dried to obtain black fungus polypeptide.

Claims

1. A black fungus mycelium liquid fermentation medium, characterized in that: The content of the solution in terms of mass percentage includes 1% glucose, 1.5% sucrose, 0.3% peptone, 0.05% KH2PO4, 0.05% MgSO4·7H2O, 0.0001% vitamin B1, and distilled water is added to make the solution 1000 mL.

2. A method for preparing a black fungus mycelium polypeptide, comprising the following steps: 1) inoculating black fungus mother seed into the black fungus mycelium liquid fermentation medium of claim 1, and performing liquid fermentation to obtain black fungus mycelium; 2) mixing the black fungus mycelium with water at a ratio of 1:10 to 1:50, soaking for 20 to 60 minutes, ultrasonically treating, adjusting the pH value to 7.0, adding protease, and performing enzymolysis at 45° C. to 55° C. to obtain an enzymolysis solution; 3) The enzymatic hydrolysate was centrifuged, and the enzymatic hydrolysate supernatant was concentrated. 3 to 5 times the volume of 95% ethanol was added to the concentrate, and the concentrate was kept at 4° C. overnight. The supernatant was collected after centrifugation; 4) After the supernatant is appropriately concentrated, freeze-dried to obtain black fungus polypeptide.

3. The preparation method according to claim 2, characterized in that: In step 1), the conditions of the liquid fermentation are: fermentation temperature 25-27° C., ventilation and stirring, and fermentation time 6-10 days.

4. The preparation method according to claim 2, characterized in that: In step 1), the fermentation liquid obtained by liquid fermentation is filtered or centrifuged at 2800-6000 rpm to collect mycelium, which is then washed and dried to obtain black fungus mycelium.

5. The preparation method according to claim 2, characterized in that: In step 2), the protease is a neutral protease, an alkaline protease or a composite protease.

6. The preparation method according to claim 2, characterized in that: In step 2), the ultrasonic treatment is carried out at 100% ultrasonic power for 15 to 60 minutes.