A method for producing Tremella polysaccharide by fermentation

Abstract

The present invention relates to a method for preparing a high-yield tremella polysaccharide fermented. The present invention obtains a high-yield tremella polysaccharide strain by breeding tremella fruiting bodies, and the tremella polysaccharide yield is as high as 40-50 g / L by fermenting the strain; by using an ultrafiltration membrane to concentrate and remove impurities such as protein and pigment, and using spray drying to replace the traditional multiple alcohol flocculation product extraction process, finally, a white tremella polysaccharide with a total sugar content of more than 99.5%, a protein content of less than 0.3%, and a molecular weight of 100-3000 KDa can be prepared; the technology of this project has the advantages of high polysaccharide yield, high total sugar content of the product, low production cost, simple and safe operation, environmental friendliness, etc., and has great industrial significance.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for preparing high-yield tremella polysaccharide by fermentation. Background Art

[0002] Tremella polysaccharide is an important active substance present in Tremella fruiting bodies, mycelium and fermentation broth. Tremella polysaccharide is composed of xylose, mannose and glucuronic acid connected by α-1,3-glycosidic bonds, and the side chain is composed of galactose, arabinose and a small amount of fucose. In the past few decades, many studies have shown that Tremella polysaccharide has moisturizing, gelling, anti-inflammatory, wound healing, antioxidant, anti-aging and anti-radiation effects. As a safe and non-toxic natural active product, Tremella polysaccharide has been clinically used as an anti-tumor drug; in the cosmetics industry, Tremella polysaccharide can be used as a moisturizer.

[0003] Most of the methods for obtaining Tremella polysaccharides on the market are fruiting body extraction methods, which have obvious disadvantages, mainly including: limited raw materials, cumbersome extraction steps, high energy consumption, low yield, and high cost. Compared with the extraction method, the Tremella fuciformis spore fermentation method has the advantages of short production cycle, high polysaccharide purity, and easy industrialization. However, the current fermentation method for producing Tremella polysaccharides still has problems such as low yield, complex production process, and high cost, which cannot meet the growing market demand for Tremella polysaccharides. Summary of the invention

[0004] The purpose of the invention is to solve the problems of low yield, complicated production process and high cost of existing Tremella polysaccharide, and to provide a preparation method of high-yield Tremella polysaccharide by fermentation with high yield, simple process steps and low cost.

[0005] The overall technical concept of the present invention is:

[0006] The fermentation method for producing Tremella polysaccharide is composed of the following process steps:

[0007] (1) Place Tremella fuciformis ( Tremella fuciformis ) strain (strain number: CGMCC No.23287, strain deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, deposit date: October 12, 2021.) was inoculated into the slant medium in the test tube to prepare a slant strain;

[0008] (2) Expand the slant culture to use as seed solution;

[0009] (3) placing the seed liquid in a fermentation tank for fermentation to produce Tremella polysaccharide;

[0010] (4) Separation and purification of the product Tremella polysaccharide.

[0011] The specific process steps and process conditions of the present invention are:

[0012] The slant culture medium in step (1) is composed of the following components in parts by weight:

[0013] 10-20 parts of glucose, 1-5 parts of peptone, 1-2 parts of magnesium sulfate, 1-5 parts of potassium dihydrogen phosphate, 0.01-0.03 parts of vitamin B1, 15 parts of agar, add water to make up to 1000 parts;

[0014] The process conditions for preparing the slant strain are: culture temperature of 20-30°C and culture time of 12-48h.

[0015] The seed solution in step (2) and the fermentation solution in step (3) are composed of the following components in parts by weight:

[0016] 20-80 parts of sucrose, 1-5 parts of peptone, 1-2 parts of magnesium sulfate, 1-5 parts of potassium dihydrogen phosphate, 0.01-0.03 parts of vitamin B1 and 1000 parts of water;

[0017] The seed liquid culture conditions are: culture temperature 20-30℃, shaking table speed 100-200r / min, culture time 12-48h; then it is connected to the fermentation tank, and the fermentation process conditions of the fermentation tank are: inoculation amount 10%, fermentation temperature 20-30℃, ventilation volume 0.5-2vvm, stirring speed 100-900r / min, culture time 48-96h, and the yield of Tremella polysaccharide is 40-50g / L.

[0018] The separation and purification of the product Tremella polysaccharide in step (4) comprises the following three steps:

[0019] a. After the fermentation is completed, the fermentation liquid is centrifuged at 8000r / min for 20min to remove the bacteria;

[0020] b. The supernatant after step a is concentrated and impurities such as protein and pigment are removed by using an ultrafiltration membrane with a molecular weight cutoff of 60-80KDa;

[0021] c. Dry the concentrated solution obtained in step b in a spray dryer with a feed inlet temperature of 140-160° C. After drying, the total sugar content of the Tremella polysaccharide is more than 99.5%, the protein content is less than 0.3%, and the molecular weight is 100-3000 KDa.

[0022] Tremella fuciformis used in the present invention Tremella fuciformis ) The CGMCC NO.23287 strain has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number Tremella CGMCC NO.23287 and the deposit date of October 12, 2021.

[0023] The screening of Tremella CGMCC NO.23287 strain (strain number used: CGMCC No.23287, strain storage address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, storage date: October 12, 2021) consists of the following process steps:

[0024] (1) collecting mature and fresh wild Tremella fuciformis fruiting bodies that are large, white, thick, and free of bacteria and pests, and inoculating the slant to obtain Tremella fuciformis slant strains;

[0025] (2) Select Tremella fuciformis slant strains for plate coating to obtain Tremella fuciformis plate colonies;

[0026] (3) Inoculating Tremella plate colonies into shake flasks for fermentation and performing primary screening and rescreening;

[0027] (4) subjecting the rescreened Tremella fuciformis strains to compound mutagenesis to select high-yield Tremella fuciformis strains.

[0028] The slant culture medium in step (1) and the plate culture medium in step (2) are composed of the following components in parts by weight:

[0029] 20 parts of glucose, 2 parts of peptone, 1.5 parts of magnesium sulfate, 3 parts of potassium dihydrogen phosphate, 0.02 parts of vitamin B1, 15 parts of agar, add water to make up to 1000 parts;

[0030] The process conditions for preparing the slant strain were: culture temperature 25°C and culture time 24h.

[0031] The fermentation broth in step (3) is composed of the following components in parts by weight:

[0032] 60 parts of sucrose, 3 parts of peptone, 1.5 parts of magnesium sulfate, 3 parts of potassium dihydrogen phosphate, 0.02 parts of vitamin B1 and 1000 parts of water.

[0033] The fermentation process conditions are as follows: the fermentation liquid inoculation amount is 10%, the culture temperature is 25°C, the shaking speed is 150r / min, and the fermentation time is 72h.

[0034] The mutagenesis in step (4) includes the preparation of a seed suspension, the amount of mutagen added, the ultraviolet irradiation time,

[0035] Inoculate the slant strain into a test tube containing 5 mL of physiological saline, add 0.5%-2.0% of diethyl sulfate mutagen to the test tube, oscillate on a vortex oscillator for 1-2 minutes, take 0.5 mL of the bacterial suspension and spread it on a plate, place the plate under ultraviolet light for 20-60 seconds, and then culture the plate in the dark. The culture conditions are: culture temperature 25°C, culture time 24 hours.

[0036] Finally, get Tremella ( Tremella fuciformis) strain (the strain number used: CGMCC No.23287, strain storage address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, storage date: October 12, 2021).

[0037] The technical progress achieved by the present invention is:

[0038] (1) High yield: Using the technology of this project, each liter of fermentation liquid contains 40-50g of Tremella polysaccharide;

[0039] (2) High purity: This project uses ultrafiltration membrane to remove impurities such as protein and pigment. The total sugar content of the obtained Tremella polysaccharide is as high as 99.5% or more, laying a foundation for the application of Tremella polysaccharide in the fields of medicine and daily chemicals;

[0040] (3) Operational safety: This project adopts spray drying technology, which has the advantages of simple process, safe operation and low cost compared with the traditional ethanol multiple flocculation purification process.

[0041] In summary, the technology of this project has the advantages of high yield, high polysaccharide content of the product, low production cost, simple and safe operation, and environmental friendliness.

[0042] Embodiment: The present invention is further described below in conjunction with embodiment.

[0043] Example 1

[0044] (1) Slant culture of bacterial strains

[0045] The Tremella CGMCC NO.23287 strain (strain number used: CGMCC No.23287, strain storage address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, storage date: October 12, 2021.) was inoculated into the slant medium in the test tube to prepare a slant strain;

[0046] The bacterial slant culture medium is composed of the following components in parts by weight:

[0047] 10 parts of glucose, 5 parts of peptone, 1 part of magnesium sulfate, 5 parts of potassium dihydrogen phosphate, 0.03 parts of vitamin B1, 15 parts of agar, add water to make up to 1000 parts;

[0048] The process conditions for preparing the slant strain were: culture temperature 25°C and culture time 12h.

[0049] (2) Seed liquid fermentation

[0050] Expand the slant culture to serve as seed solution;

[0051] The seed liquid culture medium is composed of the following components in parts by weight:

[0052] 20 parts of sucrose, 5 parts of peptone, 1 part of magnesium sulfate, 5 parts of potassium dihydrogen phosphate, 0.03 parts of vitamin B1 and 1000 parts of water.

[0053] The culture conditions for preparing the seed solution were as follows: a culture temperature of 25°C, a shaking speed of 150 r / min, and a culture time of 12 h.

[0054] (3) Fermentation production of Tremella polysaccharide

[0055] The seed liquid is introduced into a fermentation tank for fermentation to produce Tremella polysaccharide;

[0056] The fermentation broth is composed of the following components in parts by weight:

[0057] 80 parts of sucrose, 3 parts of peptone, 1.5 parts of magnesium sulfate, 3 parts of potassium dihydrogen phosphate, 0.02 parts of vitamin B1 and 1000 parts of water.

[0058] The fermentation process conditions are as follows: inoculation amount of 10%, fermentation temperature of 25°C, ventilation volume of 2vvm, stirring speed of 100-900r / min, fermentation time of 72h, and Tremella polysaccharide yield of 48g / L.

[0059] (4) Separation and purification of the product Tremella polysaccharide, including the following three steps:

[0060] a. Dilute the fermentation liquid twice after fermentation and centrifuge at 8000r / min for 20min;

[0061] b. The supernatant obtained in step a is concentrated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 60-80KDa to remove impurities such as protein and pigment;

[0062] c. The concentrated solution obtained in step b is spray-dried to obtain white Tremella polysaccharide powder with a total sugar content of more than 99.5%, a protein content of less than 0.3%, and a molecular weight of 100-3000 KDa. The spray drying air inlet temperature is 150°C.

[0063] Example 2

[0064] (1) Slant culture of bacterial strains

[0065] The Tremella fuciformis strain (strain number used: CGMCC No. 23287, strain storage address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, storage date: October 12, 2021) was inoculated into the slant culture medium in the test tube to prepare a slant strain;

[0066] The bacterial slant culture medium is composed of the following components in parts by weight:

[0067] 15 parts of glucose, 3 parts of peptone, 2 parts of magnesium sulfate, 3 parts of potassium dihydrogen phosphate, 0.01 parts of vitamin B1, 15 parts of agar, add water to make up to 1000 parts.

[0068] The process conditions for preparing the slant strain were: culture temperature 20°C and culture time 48h.

[0069] (2) Seed liquid fermentation

[0070] The slant strain was expanded and cultured as seed liquid;

[0071] The seed liquid culture medium is composed of the following components in parts by weight:

[0072] 25 parts of sucrose, 3 parts of peptone, 2 parts of magnesium sulfate, 3 parts of potassium dihydrogen phosphate, 0.01 parts of vitamin B1 and 1000 parts of water.

[0073] The culture conditions for preparing the seed solution are as follows: a culture temperature of 20°C, a shaking speed of 100 r / min, and a culture time of 48 h.

[0074] (3) Fermentation to produce Tremella polysaccharide;

[0075] The seed liquid is introduced into a fermentation tank for fermentation to produce Tremella polysaccharide;

[0076] The fermentation broth is composed of the following components in parts by weight:

[0077] 70 parts of sucrose, 1 part of peptone, 2 parts of magnesium sulfate, 5 parts of potassium dihydrogen phosphate, 0.03 parts of vitamin B1 and 1000 parts of water.

[0078] The fermentation process conditions are as follows: inoculation amount of 10%, fermentation temperature of 30°C, ventilation volume of 0.5vvm, stirring speed of 100-900r / min, fermentation time of 96h, and Tremella polysaccharide yield of 45g / L.

[0079] (4) Separation and purification of the product Tremella polysaccharide, including the following three steps:

[0080] a. Dilute the fermentation liquid twice after fermentation and centrifuge at 10000r / min for 30min;

[0081] b. The supernatant obtained in step a is concentrated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 60-80KDa to remove impurities such as protein and pigment;

[0082] c. The concentrated solution obtained in step b is spray-dried to obtain white Tremella polysaccharide powder with a total sugar content of more than 99.5%, a protein content of less than 0.3%, and a molecular weight of 100-3000 KDa. The spray drying air inlet temperature is 160°C.

[0083] Example 3

[0084] (1) Slant culture of bacterial strains

[0085] The Tremella fuciformis strain (strain number used: CGMCC No. 23287, strain storage address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, storage date: October 12, 2021) was inoculated into the slant culture medium in the test tube to prepare a slant strain;

[0086] The bacterial slant culture medium is composed of the following components in parts by weight:

[0087] 20 parts of glucose, 1 part of peptone, 1.5 parts of magnesium sulfate, 1 part of potassium dihydrogen phosphate, 0.02 parts of vitamin B1, 15 parts of agar, add water to make up to 1000 parts.

[0088] The process conditions for preparing the slant strain were: culture temperature 25°C and culture time 24h.

[0089] (2) Seed liquid fermentation

[0090] Expand the slant culture to serve as seed solution;

[0091] The seed liquid culture medium is composed of the following components in parts by weight:

[0092] 20 parts of sucrose, 1 part of peptone, 1 part of magnesium sulfate, 5 parts of potassium dihydrogen phosphate, 0.03 parts of vitamin B1 and 1000 parts of water.

[0093] The culture conditions for preparing the seed solution were as follows: a culture temperature of 25°C, a shaking speed of 150 r / min, and a culture time of 24 h.

[0094] (3) Fermentation to produce Tremella polysaccharide;

[0095] The seed liquid is introduced into a fermentation tank for fermentation to produce Tremella polysaccharide;

[0096] The fermentation broth is composed of the following components in parts by weight:

[0097] 60 parts of sucrose, 3 parts of peptone, 1 part of magnesium sulfate, 1 part of potassium dihydrogen phosphate, 0.01 parts of vitamin B1 and 1000 parts of water.

[0098] The fermentation process conditions are as follows: inoculation amount is 10%, fermentation temperature is 20°C, ventilation volume is 1vvm, stirring speed is 100-900r / min, fermentation time is 48h, and Tremella polysaccharide yield is 40g / L.

[0099] (4) Separation and purification of the product Tremella polysaccharide, including the following three steps:

[0100] a. Dilute the fermentation liquid twice after fermentation and centrifuge at 8000r / min for 20min;

[0101] b. The supernatant obtained in step a is concentrated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 60-80KDa to remove impurities such as protein and pigment;

[0102] c. The concentrated solution obtained in step b is spray-dried to obtain white Tremella polysaccharide powder with a total sugar content of more than 99.5%, a protein content of less than 0.3%, and a molecular weight of 100-3000 KDa. The spray drying air inlet temperature is 140°C.

Claims

1. A method for producing Tremella polysaccharide by fermentation, characterized in that The production process of Tremella polysaccharide by fermentation with Tremella fuciformis consists of the following steps: (1) Tremella fuciformis CGMCC No. 23287 was inoculated into a slant culture medium in a test tube to prepare a slant culture; (2) Expanding the culture of the slant strain to serve as seed solution; (3) placing the seed liquid in a fermentation tank for fermentation to produce Tremella polysaccharide; (4) Separation and purification of the product Tremella polysaccharide.

2. A method for producing Tremella polysaccharide by fermentation according to claim 1, characterized in that: The slant culture medium of step (1) is composed of the following components in parts by weight: 10-20 parts of glucose, 1-5 parts of peptone, 1-2 parts of magnesium sulfate, 1-5 parts of potassium dihydrogen phosphate, 0.01-0.03 parts of vitamin B1, and 15 parts of agar, and water is added to make up to 1000 parts.

3. A method for producing Tremella polysaccharide by fermentation according to claim 1 or 2, characterized in that: The process conditions for preparing the slant strain are: culture temperature of 20-30°C and culture time of 12-48h.

4. A method for producing Tremella polysaccharide by fermentation according to claim 1, characterized in that: The seed solution of step (2) and the fermentation solution of step (3) are composed of the following components in parts by weight: 20-80 parts of sucrose, 1-5 parts of peptone, 1-2 parts of magnesium sulfate, 1-5 parts of potassium dihydrogen phosphate, 0.01-0.03 parts of vitamin B1 and 1000 parts of water.

5. The method for producing Tremella polysaccharide by fermentation according to claim 1, characterized in that: The seed liquid culture conditions of step (2) are: culture temperature 20-30°C, shaking speed 100-200r / min, and culture time 12-48h.

6. A method for producing Tremella polysaccharide by fermentation according to claim 1 or 4, characterized in that: The fermentation process conditions of the fermenter are: inoculation amount 10%, fermentation temperature 20-30℃, ventilation volume 0.5-2vvm, stirring speed 100-900r / min, fermentation time 48-96h, and the yield of Tremella polysaccharide is 40-50g / L.

7. The method for producing Tremella polysaccharide by fermentation according to claim 1, characterized in that: The separation and purification of the product Tremella polysaccharide in step (4) comprises the following three steps: a. Centrifuge the fermented liquid after fermentation to remove bacteria; b. Concentrate the supernatant after step a through ultrafiltration membrane and remove protein and pigment impurities; c. Spray-dry the concentrated solution obtained in step b.

8. The method for producing Tremella polysaccharide by fermentation according to claim 7, characterized in that: The centrifugal condition in step a is 8000r / min and the centrifugal time is 20min; the ultrafiltration membrane cut-off molecular weight in step b is 60-80KDa; the spray drying condition in step c is the feed inlet temperature of 140-160℃; after drying, white tremella polysaccharide with a total sugar content of more than 99.5%, a protein content of less than 0.3% and a molecular weight of 100-3000KDa can be obtained.

9. Tremella fuciformis CGMCC NO.23287.