Method for extracting resveratrol from fermentation liquor

Abstract

The invention discloses a method for extracting resveratrol from fermentation liquor, which comprises the following steps: (1) centrifuging the fermentation liquor, and collecting mushroom dregs; (2) adding ethanol, heating and extracting; adding ethanol into mushroom dregs, heating and extracting; (3) performing vacuum filtration on the extracting solution; (4) concentrating the filtrate under reduced pressure until the concentration of ethanol is 30-35%, and keeping the temperature at 50-60 DEG C for 1-3 hours; (5) centrifuging the concentrated solution; (6) dissolving the crude product with ethanol, performing suction filtration, and removing impurities; (7) adding activated carbon into the filtrate, stirring at room temperature, and filtering with a ceramic membrane; (8) carrying out evaporative crystallization and vacuum filtration on the decarburization liquid; and (9) carrying out vacuum drying on the crystal to obtain a resveratrol refined product. The resveratrol is directly extracted and purified from the fermentation liquor, and the defects of raw material shortage, seasonality, low content and the like are overcome; according to the method, fat-soluble impurities which are relatively difficult to remove in the fermentation liquor can be ingeniously removed by adjusting the concentration of the ethanol, so that a product with relatively high resveratrol content (98-99%) is prepared.

Description

Technical Field

[0001] The invention relates to the technical field of biological fermentation extraction, and in particular to a method for extracting resveratrol from fermentation broth. Background Art

[0002] Resveratrol is a natural polyphenol compound, mainly found in grape skin, red wine, peanut skin, knotweed and other plants, and has potential health benefits. Resveratrol has anti-cancer, anti-oxidation, anti-free radical, anti-bacterial and anti-fungal effects, liver protection, cardiovascular prevention and treatment effects, and estrogen-like effects; in addition, resveratrol also has beauty and weight loss effects. It is a new type of beauty and health care product that can delay human aging, maintain skin moisture, remove sores, chloasma, and protect against ultraviolet radiation. The development of products related to resveratrol has good market prospects and good economic benefits.

[0003] The traditional preparation method of resveratrol is to extract and purify it from plant materials such as knotweed and grape skin, but most of them have low yields and low production, which cannot meet market demand. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the technical defects of the background technology and provide a method for extracting resveratrol from a fermentation broth. The present invention directly extracts and purifies resveratrol from the fermentation broth, avoiding the shortcomings of raw material shortage, seasonality and low content; the present invention can skillfully remove fat-soluble impurities that are difficult to remove in the fermentation broth by adjusting the concentration of ethanol, thereby preparing a product with a high resveratrol content (98% to 99%).

[0005] The technical solution adopted by the present invention to solve the above technical problems is as follows:

[0006] A method for extracting resveratrol from a fermentation broth comprises the following steps:

[0007] (1) Collecting fermentation broth and bacterial residue: centrifuging the fermentation broth and collecting bacterial residue;

[0008] (2) Extraction: Add 3 to 5 times the amount of 75% to 85% ethanol to the collected fungus residue, heat it to 45 to 55° C. for extraction, and collect the extract; add 2 to 4 times the amount of 75% to 85% ethanol to the fungus residue, heat it to 45 to 55° C. for extraction, and collect the extract; combine the two extracts;

[0009] (3) Filtration: The combined extracts from the two steps are subjected to vacuum filtration to obtain a filtrate;

[0010] (4) Concentration: The filtrate collected in step 3 is concentrated under reduced pressure to an ethanol concentration of 30% to 35%, and kept at 50 to 60° C. for 1 to 3 hours to obtain a concentrated solution;

[0011] (5) Centrifugation: Centrifuge the concentrate from step 4 to obtain a precipitate, i.e., a crude product;

[0012] (6) Dissolving again: Dissolve the crude product from step 5 in ethanol, filter and remove impurities to obtain a filtrate;

[0013] (7) Carbon stripping: Add activated carbon to the filtrate from step 6, stir at room temperature, and filter through a ceramic membrane to obtain carbon stripping liquid;

[0014] (8) Recrystallization: evaporate and crystallize the carbonized liquid in step 8, and vacuum filter to obtain crystals;

[0015] (9) Drying: The crystals obtained in step 9 are vacuum dried to obtain resveratrol.

[0016] In step (1), the raw material fermentation liquid is made of yeast as a strain, glucose as a carbon source, amino acids as a nitrogen source, and trace elements such as calcium chloride as raw materials. The seeds are obtained through primary culture, secondary culture, and fermentation culture, and the fermentation liquid concentration is 15-20g / L.

[0017] Preferably, in step (2), the extraction time is 1 to 3 hours.

[0018] Preferably, in step (4), the conditions for reduced pressure concentration are -0.080 to -0.09 MPa and 50°C.

[0019] Preferably, in step (6), the concentration of ethanol is 90%.

[0020] Preferably, in step (7), the filtrate is added with 2‰ to 3‰ activated carbon; 2‰ means 2g of activated carbon is added to 1000ml of filtrate.

[0021] Preferably, in step (7), the stirring time at room temperature is 1 h.

[0022] Preferably, in step (7), the pore size of the ceramic membrane is 50 nm.

[0023] Preferably, in step (8), the temperature of the evaporation crystallization is 50°C.

[0024] Preferably, in step (9), the vacuum drying conditions are 45-60° C. for 4-6 hours.

[0025] In the above technical scheme, the ethanol concentration in the concentrated solution after 75% to 85% ethanol extraction is 30% to 35%, and it is kept at 50 to 60°C for 1 to 3 hours, which can remove most of the lipid viscous substances, which is greatly helpful for the extraction of high-purity resveratrol.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention directly extracts and purifies resveratrol from the fermentation broth, has sufficient raw materials, high yield, is relatively environmentally friendly, and can greatly meet market demand;

[0028] (2) There are some difficulties in purifying resveratrol from the fermentation broth, such as the presence of lipid viscous substances, p-coumaric acid, protein and other impurities in the fermentation broth; the present invention can improve this situation by adjusting the concentration of ethanol;

[0029] (3) Higher yield: The present invention can optimize and increase the yield of resveratrol, while the content in plants is usually relatively low and is greatly affected by environmental factors;

[0030] (4) Not restricted by seasons: Plant extraction is limited by the growth cycle and seasonal changes of crops, while fermentation can be carried out at any time of the year and is not restricted by natural conditions;

[0031] (5) Cost-effectiveness: Once an effective fermentation process and purification process are established, large-scale production can be achieved, which will be more economical than plant extraction because a large amount of agricultural land is not required to grow plants. DETAILED DESCRIPTION

[0032] In order to better understand the content of the present invention, further description is given below in conjunction with specific embodiments. It should be understood that these embodiments are only used to further illustrate the present invention, and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content described in the present invention, those skilled in the art make some non-essential changes or adjustments to the present invention, which still belong to the protection scope of the present invention.

[0033] The raw material fermentation broth of Examples 1 to 3 and Comparative Examples 1 to 3 is obtained by using yeast as a strain, glucose as a carbon source, amino acids as a nitrogen source, and trace elements such as calcium chloride as raw materials. The seeds are obtained through primary culture, secondary culture, and fermentation culture, and the fermentation broth concentration is 15 to 20 g / L.

[0034] Example 1

[0035] A method for extracting resveratrol from a fermentation broth, comprising the following steps:

[0036] (1) Collecting fermentation broth and bacterial residue: During the microbial metabolism process, resveratrol will continuously accumulate on the surface of the microorganisms or in the cells; since resveratrol is insoluble in water, it will precipitate in the fermentation broth. A tripod centrifuge is used to centrifuge 100 kg of the fermentation broth to collect the bacterial residue;

[0037] (2) Extraction: Add 3 times the amount of 80% ethanol to the collected fungus residue, heat it to 50°C and extract for 2 h, and collect the extract; add 2 times the amount of 80% ethanol to the fungus residue again, heat it to 50°C and extract for 2 h, and collect the extract; combine the two extracts;

[0038] (3) Filtration: The combined extracts from the two steps are subjected to vacuum filtration to obtain a filtrate;

[0039] (4) Concentration: The filtrate collected in step 3 was concentrated under reduced pressure at -0.08 MPa and 50°C to an ethanol concentration of 30%, and kept at 50°C for 2 h to obtain a concentrated solution;

[0040] (5) Centrifugation: Centrifuge the concentrate from step 4 to obtain a precipitate, i.e., a crude product;

[0041] (6) Dissolving again: Dissolve the crude product from step 5 in 90% ethanol, filter and remove impurities to obtain a filtrate;

[0042] (7) Carbon stripping: Add 2‰ activated carbon to the filtrate from step 6, stir at room temperature for 1 h, and filter through a 50 nm ceramic membrane to obtain carbon stripping solution;

[0043] (8) Recrystallization: evaporate the carbon solution in step 8 at 50°C and crystallize it, and vacuum filter it to obtain crystals;

[0044] (9) Drying: The crystals obtained in step 9 are dried under vacuum at 60° C. for 4 h to obtain resveratrol concentrate;

[0045] (10) Detection: The product was collected and sent for testing. The resveratrol content in the product was 99%.

[0046] Example 2

[0047] A method for extracting resveratrol from a fermentation broth, comprising the following steps:

[0048] (1) Collecting fermentation broth and bacterial residue: During the microbial metabolism process, resveratrol will be continuously enriched on the surface of microorganisms or in cells; since resveratrol is insoluble in water, it will precipitate in the fermentation broth. A tripod centrifuge is used to centrifuge 200 kg of the fermentation broth to collect the bacterial residue;

[0049] (2) Extraction: Add 4 times the amount of 75% ethanol to the collected fungus residue, heat it to 45°C, extract it for 1 hour, and collect the extract; add 3 times the amount of 75% ethanol to the fungus residue again, heat it to 45°C, extract it for 1 hour, and collect the extract; combine the two extracts;

[0050] (3) Filtration: The combined extracts from the two steps are subjected to vacuum filtration to obtain a filtrate;

[0051] (4) Concentration: The filtrate collected in step 3 was concentrated under reduced pressure at -0.09 MPa and 50°C to an ethanol concentration of 35%, and kept at 55°C for 1 h to obtain a concentrated solution;

[0052] (5) Centrifugation: Centrifuge the concentrate from step 4 to obtain a precipitate, i.e., a crude product;

[0053] (6) Dissolving again: Dissolve the crude product from step 5 in 90% ethanol, filter and remove impurities to obtain a filtrate;

[0054] (7) Carbon stripping: Add 2‰ activated carbon to the filtrate from step 6, stir at room temperature for 1 h, and filter through a 50 nm ceramic membrane to obtain carbon stripping solution;

[0055] (8) Recrystallization: evaporate the carbon solution in step 8 at 50°C and crystallize it, and vacuum filter it to obtain crystals;

[0056] (9) Drying: The crystals obtained in step 9 are dried under vacuum at 45° C. for 5 h to obtain resveratrol concentrate;

[0057] (10) Detection: The product was collected and sent for testing. The resveratrol content in the product was 99%.

[0058] Example 3

[0059] A method for extracting resveratrol from a fermentation broth, comprising the following steps:

[0060] (1) Collecting fermentation broth and bacterial residue: During the microbial metabolism process, resveratrol will be continuously enriched on the surface of microorganisms or in cells; since resveratrol is insoluble in water, it will precipitate in the fermentation broth. 300 kg of the fermentation broth is centrifuged using a tripod centrifuge to collect the bacterial residue;

[0061] (2) Extraction: Add 5 times the amount of 85% ethanol to the collected fungus residue, heat it to 55°C, extract for 3 h, and collect the extract; add 4 times the amount of 85% ethanol to the fungus residue again, heat it to 55°C, extract for 3 h, and collect the extract; combine the two extracts;

[0062] (3) Filtration: The combined extracts from the two steps are subjected to vacuum filtration to obtain a filtrate;

[0063] (4) Concentration: The filtrate collected in step 3 was concentrated under reduced pressure at -0.08 MPa and 50°C to an ethanol concentration of 32%, and kept at 60°C for 3 h to obtain a concentrated solution;

[0064] (5) Centrifugation: Centrifuge the concentrate from step 4 to obtain a precipitate, i.e., a crude product;

[0065] (6) Dissolving again: Dissolve the crude product from step 5 in 90% ethanol, filter and remove impurities to obtain a filtrate;

[0066] (7) Carbon stripping: Add 3‰ activated carbon to the filtrate from step 6, stir at room temperature for 1 h, and filter through a 50 nm ceramic membrane to obtain carbon stripping solution;

[0067] (8) Recrystallization: evaporate the carbon solution in step 8 at 50°C and crystallize it, and vacuum filter it to obtain crystals;

[0068] (9) Drying: The crystals obtained in step 9 are dried under vacuum at 50° C. for 6 h to obtain resveratrol concentrate;

[0069] (10) Detection: The product was collected and sent for testing. The resveratrol content in the product was 98%.

[0070] Comparative Example 1

[0071] Compared with Example 1, the only difference is that in step (4), the filtrate collected in step 3 is concentrated under reduced pressure until there is no alcohol taste, and kept at 50° C. for 2 hours; the content of resveratrol in the prepared product is 85%.

[0072] Comparative Example 2

[0073] Compared with Example 1, the only difference is that in step (4), the filtrate collected in step 3 is concentrated under reduced pressure to an ethanol concentration of 25%, and is kept at 50° C. for 2 h; the resveratrol content in the prepared product is 89%.

[0074] Comparative Example 3

[0075] Compared with Example 1, the only difference is that in step (4), the filtrate collected in step 3 is concentrated under reduced pressure to an ethanol concentration of 30%, and kept at 45° C. for 2 h; the content of resveratrol in the prepared product is 92%.

[0076] The method for determining the content of resveratrol in the products prepared in Examples 1 to 3 and Comparative Examples 1 to 3 of the present invention is as follows:

[0077] 1. Chromatographic conditions:

[0078] Chromatographic column: AGILENT-C 18 , 250mm×4.6mm, 5μm;

[0079] Detection wavelength: 315nm;

[0080] Flow rate: 1.0ml / min;

[0081] Column temperature: 40°C;

[0082] Injection volume: 10 μl;

[0083] Mobile phase: acetonitrile-water (20:80).

[0084] 2. Preparation of reference solution:

[0085] Take about 10 mg of resveratrol reference substance, weigh it accurately, put it in a 50 ml volumetric flask, add about 30 ml of 70% ethanol solution, dissolve it by ultrasonic treatment, take it out, and cool it to room temperature. Dilute it with 70% ethanol solution and make it up to the mark, shake it well. Take a 2 ml pipette, accurately pipette 2 ml of the above solution into a 50 ml volumetric flask, dilute it with 70% ethanol solution and make it up to the mark, shake it well, and you have it.

[0086] 3. Preparation of test solution:

[0087] Take about 10 mg of the test sample, weigh it accurately, put it in a 50 ml volumetric flask, add about 30 ml of 70% ethanol solution, dissolve it by ultrasonic treatment, take it out, and cool it to room temperature. Dilute it with 70% ethanol solution and make it up to the mark, shake it well. Take a 2 ml pipette, accurately pipette 2 ml of the above solution into a 50 ml volumetric flask, dilute it with 70% ethanol solution and make it up to the mark, shake it well, filter it with a 0.45 μm microporous filter membrane, and take the filtrate to obtain it.

[0088] 4. Determination method:

[0089] Accurately pipette 20 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine them by the method.

[0090] Calculated by peak area using the external standard method.

[0091] 5. Calculation

[0092]

[0093] A 样 : peak area of ​​test sample;

[0094] W 标 : Weight of reference substance (g);

[0095] V 样 : Dilution multiple of the test sample (m1);

[0096] A 标 : Peak area of ​​reference substance;

[0097] W 样 : Test sample weight (g);

[0098] V 标 : Dilution multiple of reference substance (ml).

[0099] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A method for extracting resveratrol from a fermentation broth, characterized in that: The steps include: (1) Collecting fermentation broth and bacterial residue: centrifuging the fermentation broth and collecting bacterial residue; (2) Extraction: Add 3 to 5 times the amount of 75% to 85% ethanol to the collected fungus residue, heat it to 45 to 55° C. for extraction, and collect the extract; add 2 to 4 times the amount of 75% to 85% ethanol to the fungus residue, heat it to 45 to 55° C. for extraction, and collect the extract; combine the two extracts; (3) Filtration: The combined extracts from the two steps are subjected to vacuum filtration to obtain a filtrate; (4) Concentration: The filtrate collected in step 3 is concentrated under reduced pressure to an ethanol concentration of 30% to 35%, and kept at 50 to 60° C. for 1 to 3 hours to obtain a concentrated solution; (5) Centrifugation: Centrifuge the concentrate from step 4 to obtain a precipitate, i.e., a crude product; (6) Dissolving again: Dissolve the crude product from step 5 in ethanol, filter and remove impurities to obtain a filtrate; (7) Carbon stripping: Add activated carbon to the filtrate from step 6, stir at room temperature, and filter through a ceramic membrane to obtain carbon stripping liquid; (8) Recrystallization: evaporate and crystallize the carbonized liquid in step 8, and vacuum filter to obtain crystals; (9) Drying: The crystals obtained in step 9 are vacuum dried to obtain resveratrol.

2. A method for extracting resveratrol from a fermentation broth according to claim 1, characterized in that: In step (2), the extraction time is 1 to 3 hours.

3. A method for extracting resveratrol from a fermentation broth according to claim 1, characterized in that: In step (4), the conditions for reduced pressure concentration are -0.080 to -0.09 MPa and 50°C.

4. A method for extracting resveratrol from a fermentation broth according to claim 1, characterized in that: In step (6), the concentration of ethanol is 90%.

5. A method for extracting resveratrol from a fermentation broth according to claim 1, characterized in that: In step (7), the filtrate is added with activated carbon with a carbon content of 2‰ to 3‰.

6. A method for extracting resveratrol from a fermentation broth according to claim 1, characterized in that: In step (7), the stirring time at room temperature is 1 h.

7. A method for extracting resveratrol from a fermentation broth according to claim 1, characterized in that: In step (7), the pore size of the ceramic membrane is 50 nm.

8. A method for extracting resveratrol from a fermentation broth according to claim 1, characterized in that: In step (8), the temperature of the evaporation crystallization is 50°C.

9. A method for extracting resveratrol from a fermentation broth according to claim 1, characterized in that: In step (9), the vacuum drying conditions are 45-60° C. for 4-6 hours.

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