Method for extracting p-coumaric acid from ginseng residues

Abstract

The invention provides a method for extracting p-coumaric acid from ginseng residues, and belongs to the technical field of comprehensive utilization of biological resources. The method comprises the following steps: (1) cooperatively pretreating the ginseng residues by adopting high-pressure microjet and acidic ionic liquid, the net structure of the lignin-p-coumaric acid-polysaccharide compound is destroyed through the combined action of a physical hole effect and chemical catalysis, ether bonds and ester bonds are promoted to be broken, and a porous matrix is formed; and (2) constructing a composite flora synergistic fermentation system of bacillus licheniformis, chromobacterium violaceum and rhodotorula glutinis, and converting macromolecular substances such as polysaccharide released after pretreatment into p-coumaric acid through multi-enzyme cascade catalysis. According to the invention, efficient release and biotransformation of coumaric acid in a composite binding state are realized through a physical-chemical biological synergistic strategy, the structural accessibility and resource transformation efficiency of ginseng residues are remarkably improved, and an innovative solution is provided for green high-value utilization of plant processing byproducts.

Description

Technical Field

[0001] The invention belongs to the technical field of comprehensive utilization of biological resources, and specifically relates to a method for extracting p-coumaric acid from ginseng residue. Background Art

[0002] Coumaric Acid is a derivative of cinnamic acid and a natural polyphenol active product, which is widely found in food and traditional Chinese medicine. There are three isomers of coumaric acid in nature, namely p-coumaric acid, o-coumaric acid and m-coumaric acid. Among them, p-coumaric acid (p-CA), also known as 4-hydroxycinnamic acid or 4-hydroxyphenylacrylic acid, is the most abundant coumaric acid isomer in nature and has good biological activity and pharmacological effects, such as antioxidant activity, anti-inflammatory, prevention and improvement of diabetes, liver and kidney protection, immune regulation and other effects.

[0003] Ginseng residue is a high-fiber byproduct produced during the extraction of ginseng active ingredients. Its components include cell wall materials rich in phenolic acid, dietary fiber, starch and other substances. However, most of the existing technologies use ginseng residue to make feed, which will cause a great waste of nutrients. Ginseng residue itself contains a large amount of dietary fiber and starch, which can be used as a good fermentation matrix. Therefore, using bacteria to ferment ginseng residue to extract bioactive ingredients can greatly improve the utilization value of ginseng residue.

[0004] p-Coumaric acid belongs to the phenolic acid class. Phenolic acid is mainly found in the plant cell walls of ginseng residue. It is connected to polysaccharides and lignin through ester bonds and ether bonds to form a lignin-phenolic acid-polysaccharide complex. The complex has a stable structure, which makes the extraction of phenolic acid more difficult. Existing methods of phenolic acid extraction mainly include acid hydrolysis, alkaline hydrolysis and enzymatic hydrolysis. Among them, heated acid hydrolysis easily leads to the loss of phenolic substances; it is difficult to screen microbial strains that can efficiently degrade ginseng residue to obtain phenolic acid, and the catalytic rate is relatively slow, so the practical application of enzymatic methods is very limited; the traditional alkaline hydrolysis method is inefficient. In addition, due to the diversity of natural substances, the different binding modes of phenolic substances in the cell wall and the easy oxidation of phenolic substances, many factors make the separation of phenolic acid complicated. Summary of the invention

[0005] Technical problems to be solved: In view of the above technical problems, the purpose of the present invention is to provide a method for extracting p-coumaric acid from ginseng residue, which belongs to the technical field of comprehensive utilization of biological resources. The method comprises: (1) using high-pressure micro-jet and acidic ionic liquid to synergistically pretreat ginseng residue, destroying the network structure of lignin-p-coumaric acid-polysaccharide complex through the combined action of physical cavitation effect and chemical catalysis, promoting the directional breakage of ether bonds and ester bonds and forming a porous matrix; (2) constructing a composite bacterial community synergistic fermentation system of Bacillus licheniformis, Chromobacterium violaceum and Rhodotorula glutinosus, and converting the polysaccharides and other macromolecular substances released after pretreatment into p-coumaric acid through multi-enzyme cascade catalysis. The present invention realizes the efficient release and bioconversion of composite bound p-coumaric acid through a physical-chemical-biological synergistic strategy, significantly improves the structural accessibility and resource conversion efficiency of ginseng residue, and provides an innovative solution for the green and high-value utilization of plant processing by-products.

[0006] Technical solution: A method for extracting p-coumaric acid from ginseng residue, comprising the following steps: S1. The ginseng residue was ultrafinely crushed, and the acidic ionic liquid was added and mixed to obtain a ginseng residue mixed solution; S2. The mixed solution of ginseng residue was subjected to high-pressure microfluidization treatment to obtain a pretreated mixed solution of ginseng residue; S3. The pretreated ginseng residue mixed solution was inoculated with a composite bacterial strain for fermentation to obtain a ginseng residue fermentation liquid; S4. Inactivate the bacteria by heating the fermented ginseng residue liquid, and separate and purify the obtained p-coumaric acid; The composite bacterial strain is Bacillus licheniformis+Chromobacterium violaceum+Rhodotorula glutinosus.

[0007] Furthermore, in step S1, the ratio of ginseng residue to acidic ionic liquid is 1 g: (5-10) mL; the acidic ionic liquid is prepared by stirring choline dihydrogen citrate, lactic acid and guaiacol at 70-80° C. for 30-45 min.

[0008] Furthermore, the molar ratio of choline dihydrogen citrate, lactic acid and guaiacol is 1:(1-3):(0.5-1.5).

[0009] Furthermore, the conditions of the high-pressure microfluidization treatment in step S2 are a treatment pressure of 80-150 MPa, a cycle number of 2-4 times, and a treatment temperature of 25-40°C.

[0010] Furthermore, the inoculation amount of the composite bacteria in step S3 is 4.5-7.5wt%; the ratio of Bacillus licheniformis: Chromobacterium violaceum: Rhodotorula glutinosus in the composite bacteria is (1.5-4): (1-3): (1-2.5).

[0011] Furthermore, the fermentation conditions in step S3 are fermentation temperature 25-35° C. and fermentation time 12-36 h.

[0012] Furthermore, the temperature for inactivating the bacteria in step S4 is 60-90°C.

[0013] Furthermore, the specific method for separation and purification in step S4 is as follows: ① centrifuge the inactivated ginseng residue fermentation liquid at 3500-5000rpm for 10-15min, and take the supernatant; ② adjust the pH of the supernatant to 3-5 and let it stand for 5-10min, then add ethyl acetate for extraction, and layer the organic phase; ③ rotary evaporate the organic phase to obtain p-coumaric acid.

[0014] Beneficial Effects

[0015] 1. The present invention establishes a dual-effect pretreatment mechanism of "dynamic solvent penetration-complex degradation" by the synergistic effect of high-pressure microjet and acidic ionic liquid (choline dihydrogen citrate / lactic acid / guaiacol composite system), wherein the acidic ionic liquid specifically cleaves the acid-sensitive ether bonds in the lignin-p-coumaric acid-polysaccharide composite through proton catalysis, and the instantaneous cavitation effect, turbulent shock wave and ultra-high shear force generated by the high-pressure microjet can effectively cut the polysaccharide chain and achieve physical relaxation of the composite network structure; the synergistic effect of the two not only significantly improves the penetration efficiency of the solvent, but also makes the ester bonds in the composite easily destroyed and broken by the multiple effects generated by the high-pressure microjet through the conformational exposure effect, promotes the dissolution of p-coumaric acid in ginseng residue, and makes the ginseng residue structure loose and porous, creating an ideal mass transfer interface for subsequent biotransformation.

[0016] 2. The present invention constructs a synergistic fermentation system of a composite bacterial species of Bacillus licheniformis + Chromobacterium violaceum + Rhodotorula glutinosus, forming a cascade metabolic pathway of "substrate conversion-intermediate synthesis-final product generation", which is specifically manifested as follows: ① Bacillus licheniformis realizes the saccharification conversion and ester bond cleavage of dietary fiber by secreting cellulase and lipase, and simultaneously generates the key precursor phenylalanine; ② Chromobacterium violaceum catalyzes the conversion of phenylalanine to tyrosine through phenylalanine hydroxylase; ③ Rhodotorula glutinosus uses tyrosine aminotransferase to complete the directional conversion of tyrosine to p-coumaric acid, thereby increasing the content of p-coumaric acid in ginseng residue.

[0017] 3. The present invention converts the insoluble complex bound p-coumaric acid in ginseng residue into a free product, while realizing the precise construction of the biotransformation pathway, which is beneficial to improving the utilization value and resource conversion of ginseng residue, providing a good theoretical reference for the high-value utilization of ginseng residue, and providing an industrializable solution for the high-value utilization of plant processing by-products. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The yield and purity of p-coumaric acid in Examples 1-14 and Comparative Examples 1-5; Figure 2 The DPPH free radical scavenging rate and ABTS+ free radical scavenging rate of Example 5 and Comparative Examples 1-5. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with embodiments, which are explanations of the present invention and are not limited to the following embodiments: The sources of the strains used in the present invention are as follows: Bacillus licheniformis CICC 22606 was purchased from the China Industrial Microbiological Culture Collection Center; Chromobacterium violaceum ATCC12540 was purchased from Sigma; and Rhodotorula glutinosus BJ-J12861 was purchased from Shanghai Bangjing Industrial Co., Ltd.

[0020] The composite strains described in the following examples and comparative examples are prepared by strain activation of Bacillus licheniformis CICC 22606+Chromobacterium violaceum ATCC12540+Rhodotorula glutinosus BJ-J12861. The strain activation process is as follows: Activation of Bacillus licheniformis CICC 22606 strain: inoculate Bacillus licheniformis CICC 22606 into LB liquid medium, culture at 30°C and 220rpm for 24h to obtain Bacillus licheniformis CICC 22606 seed solution, then inoculate 1% of the Bacillus licheniformis CICC 22606 seed solution into LB liquid medium, culture at 30°C and 220rpm for 24h, centrifuge and resuspend in sterile water until the strain concentration is greater than 10 8 CFU / mL to obtain the activated Bacillus licheniformis CICC 22606 bacterial solution; Activation of Chromobacterium violaceum ATCC12540 strain: inoculate Chromobacterium violaceum ATCC12540 into nutrient broth medium, culture at 28°C and 150rpm for 24h to obtain seed solution of Chromobacterium violaceum ATCC12540, then inoculate 1% seed solution of Chromobacterium violaceum ATCC12540 into nutrient broth medium, culture at 28°C and 150rpm for 24h, centrifuge and resuspend in sterile water until the strain concentration is greater than 10 8 CFU / mL to obtain the activated Chromobacterium violaceum ATCC12540 bacterial solution; Activation of red yeast BJ-J12861: Inoculate red yeast BJ-J12861 into YPD liquid medium, culture at 30°C and 200 rpm for 24 h to obtain red yeast BJ-J12861 seed solution, then inoculate 1% red yeast BJ-J12861 seed solution into YPD liquid medium, culture at 30°C and 200 rpm for 24 h, centrifuge and filter, and resuspend in sterile water until the strain concentration is greater than 10 8CFU / mL to obtain the activated Rhodotorula glutinosus BJ-J12861 bacterial liquid. Example 1

[0021] A method for extracting p-coumaric acid from ginseng residue comprises the following steps: S1. Stir 1 mol of choline dihydrogen citrate, 2 mol of lactic acid, and 0.5 mol of guaiacol at 80°C for 30 min until a clear and transparent solution is formed, which is an acidic ionic liquid; S2. 100g of ginseng residue was ultrafinely crushed, 800mL of acidic ionic liquid was added and mixed evenly to obtain a ginseng residue mixed solution; S3. The mixed solution of ginseng residue was circulated three times by a high-pressure microfluidizer at 30 ° C and 100 MPa to obtain a pretreated mixed solution of ginseng residue; S4. Inoculate 54 g of composite bacteria (27 g of Bacillus licheniformis CICC 22606 + 14 g of Chromobacterium violaceum ATCC12540 + 9 g of Rhodotorula glutinosus BJ-J12861) into the pretreated ginseng residue mixed solution and ferment at 30 ° C for 24 h to obtain a ginseng residue fermentation liquid; S5. The ginseng residue fermentation liquid is heated to 75°C to inactivate the bacteria, the inactivated ginseng residue fermentation liquid is centrifuged at 4000 rpm for 12 min, and the supernatant is taken; the pH of the supernatant is adjusted to 4 and allowed to stand for 5 min, and then ethyl acetate is added for extraction, and the organic phase is obtained by layering; the organic phase is subjected to rotary evaporation to obtain p-coumaric acid. Example 2

[0022] A method for extracting p-coumaric acid from ginseng residue comprises the following steps: S1. Stir 1 mol of choline dihydrogen citrate, 3 mol of lactic acid, and 0.5 mol of guaiacol at 75°C for 30 min until a clear and transparent solution is formed, which is an acidic ionic liquid; S2. 50 g of ginseng residue was ultrafinely crushed, 400 mL of acidic ionic liquid was added and mixed evenly to obtain a ginseng residue mixed solution; S3. The mixed solution of ginseng residue was circulated three times by a high-pressure microfluidizer at 30 ° C and 100 MPa to obtain a pretreated mixed solution of ginseng residue; S4. Inoculate 27 g of composite bacteria (13.5 g of Bacillus licheniformis CICC 22606 + 9.0 g of Chromobacterium violaceum ATCC12540 + 4.5 g of Rhodotorula glutinosus BJ-J12861) into the pretreated ginseng residue mixed solution and ferment at 30 ° C for 24 h to obtain ginseng residue fermentation liquid; S5. The ginseng residue fermentation liquid is heated to 80°C to inactivate the bacteria, the inactivated ginseng residue fermentation liquid is centrifuged at 5000 rpm for 10 min, and the supernatant is taken; the pH of the supernatant is adjusted to 4 and allowed to stand for 5 min, and then ethyl acetate is added for extraction, and the organic phase is obtained by layering; the organic phase is subjected to rotary evaporation to obtain p-coumaric acid. Example 3

[0023] A method for extracting p-coumaric acid from ginseng residue comprises the following steps: S1. Stir 1 mol of choline dihydrogen citrate, 2 mol of lactic acid, and 1.5 mol of guaiacol at 80°C for 30 min until a clear and transparent solution is formed, which is an acidic ionic liquid; S2. 100g of ginseng residue was ultrafinely crushed, 800mL of acidic ionic liquid was added and mixed evenly to obtain a ginseng residue mixed solution; S3. The mixed solution of ginseng residue was circulated three times by a high-pressure microfluidizer at 30 ° C and 100 MPa to obtain a pretreated mixed solution of ginseng residue; S4. Inoculate 54 g of composite bacteria (27 g of Bacillus licheniformis CICC 22606 + 14 g of Chromobacterium violaceum ATCC12540 + 9 g of Rhodotorula glutinosus BJ-J12861) into the pretreated ginseng residue mixed solution and ferment at 30 ° C for 24 h to obtain a ginseng residue fermentation liquid; S5. The ginseng residue fermentation liquid is heated to 80°C to inactivate the bacteria, the inactivated ginseng residue fermentation liquid is centrifuged at 4000rpm for 10min, and the supernatant is taken; the pH of the supernatant is adjusted to 4 and allowed to stand for 7min, and then ethyl acetate is added for extraction, and the organic phase is obtained by layering; the organic phase is subjected to rotary evaporation to obtain p-coumaric acid. Example 4

[0024] A method for extracting p-coumaric acid from ginseng residue comprises the following steps: S1. Stir 1 mol of choline dihydrogen citrate, 2 mol of lactic acid, and 0.5 mol of guaiacol at 80°C for 30 min until a clear and transparent solution is formed, which is an acidic ionic liquid; S2. 100g of ginseng residue was ultrafinely crushed, 500mL of acidic ionic liquid was added and mixed evenly to obtain a ginseng residue mixed solution; S3. The mixed solution of ginseng residue was circulated three times by a high-pressure microfluidizer at 25°C and 100MPa to obtain a pretreated mixed solution of ginseng residue; S4. Inoculate 36 g of composite bacteria (18 g of Bacillus licheniformis CICC 22606 + 12 g of Chromobacterium violaceum ATCC12540 + 6 g of Rhodotorula glutinosus BJ-J12861) into the pretreated ginseng residue mixed solution and ferment at 30 ° C for 24 h to obtain a ginseng residue fermentation liquid; S5. The ginseng residue fermentation liquid is heated to 80°C to inactivate the bacteria, the inactivated ginseng residue fermentation liquid is centrifuged at 3500rpm for 15min, and the supernatant is taken; the pH of the supernatant is adjusted to 4 and allowed to stand for 5min, and then ethyl acetate is added for extraction, and the organic phase is obtained by layering; the organic phase is subjected to rotary evaporation to obtain p-coumaric acid. Example 5

[0025] A method for extracting p-coumaric acid from ginseng residue comprises the following steps: S1. Stir 1 mol of choline dihydrogen citrate, 2 mol of lactic acid, and 0.5 mol of guaiacol at 80°C for 30 min until a clear and transparent solution is formed, which is an acidic ionic liquid; S2. 100g of ginseng residue was ultrafinely crushed, 1000mL of acidic ionic liquid was added and mixed evenly to obtain a ginseng residue mixed solution; S3. The mixed solution of ginseng residue was circulated three times by a high-pressure microfluidizer at 30 ° C and 100 MPa to obtain a pretreated mixed solution of ginseng residue; S4. The pretreated ginseng residue mixed solution was inoculated with 66 g of composite bacteria (33 g of Bacillus licheniformis CICC 22606 + 22 g of Chromobacterium violaceum ATCC12540 + 11 g of Rhodotorula glutinosus BJ-J12861), and fermented at 30 ° C for 24 h to obtain a ginseng residue fermentation liquid; S5. The ginseng residue fermentation liquid is heated to 75°C to inactivate the bacteria, the inactivated ginseng residue fermentation liquid is centrifuged at 4000 rpm for 12 min, and the supernatant is taken; the pH of the supernatant is adjusted to 4 and allowed to stand for 5 min, and then ethyl acetate is added for extraction, and the organic phase is obtained by layering; the organic phase is subjected to rotary evaporation to obtain p-coumaric acid. Example 6

[0026] A method for extracting p-coumaric acid from ginseng residue comprises the following steps: S1. Stir 1 mol of choline dihydrogen citrate, 2 mol of lactic acid, and 0.5 mol of guaiacol at 70°C for 45 min until a clear and transparent solution is formed, which is an acidic ionic liquid; S2. 100g of ginseng residue was ultrafinely crushed, 800mL of acidic ionic liquid was added and mixed evenly to obtain a ginseng residue mixed solution; S3. The mixed solution of ginseng residue was circulated three times by a high-pressure microfluidizer at 30 ° C and 80 MPa to obtain a pretreated mixed solution of ginseng residue; S4. Inoculate 54 g of composite bacteria (27 g of Bacillus licheniformis CICC 22606 + 14 g of Chromobacterium violaceum ATCC12540 + 9 g of Rhodotorula glutinosus BJ-J12861) into the pretreated ginseng residue mixed solution and ferment at 30 ° C for 24 h to obtain a ginseng residue fermentation liquid; S5. The ginseng residue fermentation liquid is heated to 80°C to inactivate the bacteria, the inactivated ginseng residue fermentation liquid is centrifuged at 3500rpm for 15min, and the supernatant is taken; the pH of the supernatant is adjusted to 4 and allowed to stand for 5min, and then ethyl acetate is added for extraction, and the organic phase is obtained by layering; the organic phase is subjected to rotary evaporation to obtain p-coumaric acid. Example 7

[0027] A method for extracting p-coumaric acid from ginseng residue comprises the following steps: S1. Stir 1 mol of choline dihydrogen citrate, 2 mol of lactic acid, and 0.5 mol of guaiacol at 80°C for 30 min until a clear and transparent solution is formed, which is an acidic ionic liquid; S2. 100g of ginseng residue was ultrafinely crushed, 800mL of acidic ionic liquid was added and mixed evenly to obtain a ginseng residue mixed solution; S3. The mixed solution of ginseng residue was circulated three times by a high-pressure microfluidizer at 35°C and 120MPa to obtain a pretreated mixed solution of ginseng residue; S4. Inoculate 54 g of composite bacteria (27 g of Bacillus licheniformis CICC 22606 + 14 g of Chromobacterium violaceum ATCC12540 + 9 g of Rhodotorula glutinosus BJ-J12861) into the pretreated ginseng residue mixed solution and ferment at 30 ° C for 24 h to obtain a ginseng residue fermentation liquid; S5. The ginseng residue fermentation liquid is heated to 75°C to inactivate the bacteria, the inactivated ginseng residue fermentation liquid is centrifuged at 4000 rpm for 12 min, and the supernatant is taken; the pH of the supernatant is adjusted to 4 and allowed to stand for 5 min, and then ethyl acetate is added for extraction, and the organic phase is obtained by layering; the organic phase is subjected to rotary evaporation to obtain p-coumaric acid. Example 8

[0028] A method for extracting p-coumaric acid from ginseng residue comprises the following steps: S1. Stir 1 mol of choline dihydrogen citrate, 2 mol of lactic acid, and 0.5 mol of guaiacol at 80°C for 30 min until a clear and transparent solution is formed, which is an acidic ionic liquid; S2. 100g of ginseng residue was ultrafinely crushed, 800mL of acidic ionic liquid was added and mixed evenly to obtain a ginseng residue mixed solution; S3. The mixed solution of ginseng residue was circulated three times by a high-pressure microfluidizer at 25°C and 100MPa to obtain a pretreated mixed solution of ginseng residue; S4. Inoculate 40.5 g of composite bacteria (20.25 g of Bacillus licheniformis CICC22606 + 13.5 g of Chromobacterium violaceum ATCC12540 + 6.75 g of Rhodotorula glutinosus BJ-J12861) into the pretreated ginseng residue mixed solution and ferment at 30 ° C for 24 h to obtain ginseng residue fermentation liquid; S5. The ginseng residue fermentation liquid is heated to 80°C to inactivate the bacteria, the inactivated ginseng residue fermentation liquid is centrifuged at 3500rpm for 15min, and the supernatant is taken; the pH of the supernatant is adjusted to 4 and allowed to stand for 10min, and then ethyl acetate is added for extraction, and the organic phase is obtained by layering; the organic phase is subjected to rotary evaporation to obtain p-coumaric acid. Example 9

[0029] A method for extracting p-coumaric acid from ginseng residue comprises the following steps: S1. Stir 1 mol of choline dihydrogen citrate, 2 mol of lactic acid, and 0.5 mol of guaiacol at 80°C for 30 min until a clear and transparent solution is formed, which is an acidic ionic liquid; S2. 100g of ginseng residue was ultrafinely crushed, 800mL of acidic ionic liquid was added and mixed evenly to obtain a ginseng residue mixed solution; S3. The mixed solution of ginseng residue was circulated three times by a high-pressure microfluidizer at 30 ° C and 100 MPa to obtain a pretreated mixed solution of ginseng residue; S4. Inoculate 67.5 g of composite bacteria (33.75 g of Bacillus licheniformis CICC22606 + 22.5 g of Chromobacterium violaceum ATCC12540 + 11.25 g of Rhodotorula glutinosus BJ-J12861) into the pretreated ginseng residue mixed solution and ferment at 30 ° C for 24 h to obtain ginseng residue fermentation liquid; S5. The ginseng residue fermentation liquid is heated to 75°C to inactivate the bacteria, the inactivated ginseng residue fermentation liquid is centrifuged at 4000 rpm for 12 min, and the supernatant is taken; the pH of the supernatant is adjusted to 4 and allowed to stand for 5 min, and then ethyl acetate is added for extraction, and the organic phase is obtained by layering; the organic phase is subjected to rotary evaporation to obtain p-coumaric acid. Example 10

[0030] A method for extracting p-coumaric acid from ginseng residue comprises the following steps: S1. Stir 1 mol of choline dihydrogen citrate, 2 mol of lactic acid, and 0.5 mol of guaiacol at 80°C for 30 min until a clear and transparent solution is formed, which is an acidic ionic liquid; S2. 100g of ginseng residue was ultrafinely crushed, 800mL of acidic ionic liquid was added and mixed evenly to obtain a ginseng residue mixed solution; S3. The mixed solution of ginseng residue was circulated three times by a high-pressure microfluidizer at 30 ° C and 100 MPa to obtain a pretreated mixed solution of ginseng residue; S4. Inoculate 54 g of composite bacteria (18 g of Bacillus licheniformis CICC 22606 + 18 g of Chromobacterium violaceum ATCC12540 + 18 g of Rhodotorula glutinosus BJ-J12861) into the pretreated ginseng residue mixed solution and ferment at 30 ° C for 24 h to obtain a ginseng residue fermentation liquid; S5. The ginseng residue fermentation liquid is heated to 75°C to inactivate the bacteria, the inactivated ginseng residue fermentation liquid is centrifuged at 4000 rpm for 12 min, and the supernatant is taken; the pH of the supernatant is adjusted to 4 and allowed to stand for 5 min, and then ethyl acetate is added for extraction, and the organic phase is obtained by layering; the organic phase is subjected to rotary evaporation to obtain p-coumaric acid. Embodiment 11

[0031] A method for extracting p-coumaric acid from ginseng residue comprises the following steps: S1. Stir 1 mol of choline dihydrogen citrate, 2 mol of lactic acid, and 0.5 mol of guaiacol at 80°C for 30 min until a clear and transparent solution is formed, which is an acidic ionic liquid; S2. 100g of ginseng residue was ultrafinely crushed, 800mL of acidic ionic liquid was added and mixed evenly to obtain a ginseng residue mixed solution; S3. The mixed solution of ginseng residue was circulated three times by a high-pressure microfluidizer at 30 ° C and 100 MPa to obtain a pretreated mixed solution of ginseng residue; S4. Inoculate 54 g of composite bacteria (21.6 g of Bacillus licheniformis CICC 22606 + 10.8 g of Chromobacterium violaceum ATCC12540 + 21.6 g of Rhodotorula glutinosus BJ-J12861) into the pretreated ginseng residue mixed solution and ferment at 30 ° C for 24 h to obtain ginseng residue fermentation liquid; S5. The ginseng residue fermentation liquid is heated to 75°C to inactivate the bacteria, the inactivated ginseng residue fermentation liquid is centrifuged at 4000 rpm for 12 min, and the supernatant is taken; the pH of the supernatant is adjusted to 4 and allowed to stand for 5 min, and then ethyl acetate is added for extraction, and the organic phase is obtained by layering; the organic phase is subjected to rotary evaporation to obtain p-coumaric acid. Example 12

[0032] A method for extracting p-coumaric acid from ginseng residue comprises the following steps: S1. Stir 1 mol of choline dihydrogen citrate, 2 mol of lactic acid, and 0.5 mol of guaiacol at 75°C for 30 min until a clear and transparent solution is formed, which is an acidic ionic liquid; S2. 100g of ginseng residue was ultrafinely crushed, 800mL of acidic ionic liquid was added and mixed evenly to obtain a ginseng residue mixed solution; S3. The mixed solution of ginseng residue was circulated three times by a high-pressure microfluidizer at 25°C and 100MPa to obtain a pretreated mixed solution of ginseng residue; S4. Inoculate 54 g of composite bacteria (27 g of Bacillus licheniformis CICC 22606 + 14 g of Chromobacterium violaceum ATCC12540 + 9 g of Rhodotorula glutinosus BJ-J12861) into the pretreated ginseng residue mixed solution and ferment at 35 ° C for 24 h to obtain a ginseng residue fermentation liquid; S5. The ginseng residue fermentation liquid is heated to 80°C to inactivate the bacteria, the inactivated ginseng residue fermentation liquid is centrifuged at 3500rpm for 15min, and the supernatant is taken; the pH of the supernatant is adjusted to 4 and allowed to stand for 10min, and then ethyl acetate is added for extraction, and the organic phase is obtained by layering; the organic phase is subjected to rotary evaporation to obtain p-coumaric acid. Embodiment 13

[0033] A method for extracting p-coumaric acid from ginseng residue comprises the following steps: S1. Stir 1 mol of choline dihydrogen citrate, 2 mol of lactic acid, and 0.5 mol of guaiacol at 80°C for 30 min until a clear and transparent solution is formed, which is an acidic ionic liquid; S2. 50 g of ginseng residue was ultrafinely crushed, 400 mL of acidic ionic liquid was added and mixed evenly to obtain a ginseng residue mixed solution; S3. The mixed solution of ginseng residue was circulated three times by a high-pressure microfluidizer at 30 ° C and 100 MPa to obtain a pretreated mixed solution of ginseng residue; S4. Inoculate 27 g of composite bacteria (13.5 g of Bacillus licheniformis CICC 22606 + 9 g of Chromobacterium violaceum ATCC12540 + 4.5 g of Rhodotorula glutinosus BJ-J12861) into the pretreated ginseng residue mixed solution and ferment at 30 ° C for 12 h to obtain ginseng residue fermentation liquid; S5. The ginseng residue fermentation liquid is heated to 75°C to inactivate the bacteria, the inactivated ginseng residue fermentation liquid is centrifuged at 4000 rpm for 12 min, and the supernatant is taken; the pH of the supernatant is adjusted to 4 and allowed to stand for 5 min, and then ethyl acetate is added for extraction, and the organic phase is obtained by layering; the organic phase is subjected to rotary evaporation to obtain p-coumaric acid. Embodiment 14

[0034] A method for extracting p-coumaric acid from ginseng residue comprises the following steps: S1. Stir 1 mol of choline dihydrogen citrate, 2 mol of lactic acid, and 0.5 mol of guaiacol at 75°C for 40 min until a clear and transparent solution is formed, which is an acidic ionic liquid; S2. 100g of ginseng residue was ultrafinely crushed, 800mL of acidic ionic liquid was added and mixed evenly to obtain a ginseng residue mixed solution; S3. The mixed solution of ginseng residue was circulated three times by a high-pressure microfluidizer at 25°C and 100MPa to obtain a pretreated mixed solution of ginseng residue; S4. Inoculate 54 g of composite bacteria (27 g of Bacillus licheniformis CICC 22606 + 14 g of Chromobacterium violaceum ATCC12540 + 9 g of Rhodotorula glutinosus BJ-J12861) into the pretreated ginseng residue mixed solution and ferment at 30 ° C for 36 h to obtain a ginseng residue fermentation liquid; S5. The ginseng residue fermentation liquid is heated to 80°C to inactivate the bacteria, the inactivated ginseng residue fermentation liquid is centrifuged at 4000rpm for 10min, and the supernatant is taken; the pH of the supernatant is adjusted to 4 and allowed to stand for 10min, and then ethyl acetate is added for extraction, and the organic phase is obtained by layering; the organic phase is subjected to rotary evaporation to obtain p-coumaric acid. Comparative Example 1

[0035] The difference between this comparative example and Example 5 is that no acidic ionic liquid is added.

[0036] A method for extracting p-coumaric acid from ginseng residue comprises the following steps: S1. 100g of ginseng residue was ultrafinely crushed, 1000mL of water was added and mixed evenly to obtain a ginseng residue mixed solution; S2. The mixed solution of ginseng residue was circulated three times by a high-pressure microfluidizer at 30 ° C and 100 MPa to obtain a pretreated mixed solution of ginseng residue; S3. Inoculate 66 g of composite bacteria (33 g of Bacillus licheniformis CICC 22606 + 22 g of Chromobacterium violaceum ATCC12540 + 11 g of Rhodotorula glutinosus BJ-J12861) into the pretreated ginseng residue mixed solution and ferment at 30 ° C for 24 h to obtain ginseng residue fermentation liquid; S4. The ginseng residue fermentation liquid is heated to 75°C to inactivate the bacteria, the inactivated ginseng residue fermentation liquid is centrifuged at 4000 rpm for 12 min, and the supernatant is taken; the pH of the supernatant is adjusted to 4 and allowed to stand for 5 min, and then ethyl acetate is added for extraction, and the organic phase is obtained by layering; the organic phase is subjected to rotary evaporation to obtain p-coumaric acid. Comparative Example 2

[0037] The difference between this comparative example and Example 5 is that high-pressure microfluidization treatment is not used.

[0038] A method for extracting p-coumaric acid from ginseng residue comprises the following steps: S1. Stir 1 mol of choline dihydrogen citrate, 2 mol of lactic acid, and 0.5 mol of guaiacol at 80°C for 30 min until a clear and transparent solution is formed, which is an acidic ionic liquid; S2. 100g of ginseng residue was ultrafinely crushed, 1000mL of acidic ionic liquid was added and mixed evenly to obtain a pretreated ginseng residue mixed solution; S3. Inoculate 66 g of composite bacteria (33 g of Bacillus licheniformis CICC 22606 + 22 g of Chromobacterium violaceum ATCC12540 + 11 g of Rhodotorula glutinosus BJ-J12861) into the pretreated ginseng residue mixed solution and ferment at 30 ° C for 24 h to obtain ginseng residue fermentation liquid; S4. The ginseng residue fermentation liquid is heated to 75°C to inactivate the bacteria, the inactivated ginseng residue fermentation liquid is centrifuged at 4000 rpm for 12 min, and the supernatant is taken; the pH of the supernatant is adjusted to 4 and allowed to stand for 5 min, and then ethyl acetate is added for extraction, and the organic phase is obtained by layering; the organic phase is subjected to rotary evaporation to obtain p-coumaric acid. Comparative Example 3

[0039] The difference between this comparative example and Example 5 is that only Bacillus licheniformis is used.

[0040] A method for extracting p-coumaric acid from ginseng residue comprises the following steps: S1. Stir 1 mol of choline dihydrogen citrate, 2 mol of lactic acid, and 0.5 mol of guaiacol at 80°C for 30 min until a clear and transparent solution is formed, which is an acidic ionic liquid; S2. 100g of ginseng residue was ultrafinely crushed, 1000mL of acidic ionic liquid was added and mixed evenly to obtain a ginseng residue mixed solution; S3. The mixed solution of ginseng residue was circulated three times by a high-pressure microfluidizer at 30 ° C and 100 MPa to obtain a pretreated mixed solution of ginseng residue; S4. The mixed solution of pretreated ginseng residue was inoculated with 66 g of Bacillus licheniformis and fermented at 30 ° C for 24 h to obtain a ginseng residue fermentation liquid; S5. The ginseng residue fermentation liquid is heated to 75°C to inactivate the bacteria, the inactivated ginseng residue fermentation liquid is centrifuged at 4000 rpm for 12 min, and the supernatant is taken; the pH of the supernatant is adjusted to 4 and allowed to stand for 5 min, and then ethyl acetate is added for extraction, and the organic phase is obtained by layering; the organic phase is subjected to rotary evaporation to obtain p-coumaric acid. Comparative Example 4

[0041] The difference between this comparative example and Example 5 is that only Chromobacterium violaceum is used.

[0042] A method for extracting p-coumaric acid from ginseng residue comprises the following steps: S1. Stir 1 mol of choline dihydrogen citrate, 2 mol of lactic acid, and 0.5 mol of guaiacol at 80°C for 30 min until a clear and transparent solution is formed, which is an acidic ionic liquid; S2. 100g of ginseng residue was ultrafinely crushed, 1000mL of acidic ionic liquid was added and mixed evenly to obtain a ginseng residue mixed solution; S3. The mixed solution of ginseng residue was circulated three times by a high-pressure microfluidizer at 30 ° C and 100 MPa to obtain a pretreated mixed solution of ginseng residue; S4. The pretreated ginseng residue mixed solution was inoculated with 66 g of Chromobacterium violaceum and fermented at 30 ° C for 24 h to obtain a ginseng residue fermentation liquid; S5. The ginseng residue fermentation liquid is heated to 75°C to inactivate the bacteria, the inactivated ginseng residue fermentation liquid is centrifuged at 4000 rpm for 12 min, and the supernatant is taken; the pH of the supernatant is adjusted to 4 and allowed to stand for 5 min, and then ethyl acetate is added for extraction, and the organic phase is obtained by layering; the organic phase is subjected to rotary evaporation to obtain p-coumaric acid. Comparative Example 5

[0043] The difference between this comparative example and Example 5 is that only Rhodotorula glutinosus is used.

[0044] A method for extracting p-coumaric acid from ginseng residue comprises the following steps: S1. Stir 1 mol of choline dihydrogen citrate, 2 mol of lactic acid, and 0.5 mol of guaiacol at 80°C for 30 min until a clear and transparent solution is formed, which is an acidic ionic liquid; S2. 100g of ginseng residue was ultrafinely crushed, 1000mL of acidic ionic liquid was added and mixed evenly to obtain a ginseng residue mixed solution; S3. The mixed solution of ginseng residue was circulated three times by a high-pressure microfluidizer at 30 ° C and 100 MPa to obtain a pretreated mixed solution of ginseng residue; S4. Inoculate 66 g of red yeast to the pretreated ginseng residue mixed solution and ferment at 30 ° C for 24 h to obtain ginseng residue fermentation liquid; S5. The ginseng residue fermentation liquid is heated to 75°C to inactivate the bacteria, the inactivated ginseng residue fermentation liquid is centrifuged at 4000 rpm for 12 min, and the supernatant is taken; the pH of the supernatant is adjusted to 4 and allowed to stand for 5 min, and then ethyl acetate is added for extraction, and the organic phase is obtained by layering; the organic phase is subjected to rotary evaporation to obtain p-coumaric acid. Performance Testing

[0045] (1) Extraction rate and purity of p-coumaric acid The p-coumaric acid yield of Examples 1-14 and Comparative Examples 1-5 was determined, wherein the p-coumaric acid yield (%) = M2 / M1×100%, wherein M2 represents the mass of p-coumaric acid, and M1 represents the mass of ginseng residue; the p-coumaric acid content of Examples 1-14 and Comparative Examples 1-5 was determined by high performance liquid chromatography, and the p-coumaric acid purity was calculated. The specific test method was as follows: ① Preparation of sample solution: Accurately weigh 0.5000 g of sample in a conical flask, add 20 mL of 70% ethanol solution, weigh the mass, heat and reflux for 30 min, take out and cool, weigh the mass again, make up the lost mass with 70% ethanol solution, shake well, and filter with a 0.22 μm syringe filter to obtain a sample solution; ② p-Coumaric acid reference solution: accurately weigh 6.91 mg of p-coumaric acid standard and place it in a 25 mL volumetric flask, dissolve it with 50% methanol solution and dilute to the mark, take 2.5 mL in a 25 mL volumetric flask, dilute to the mark with 50% methanol solution to prepare p-coumaric acid reference solution; ③ p-Coumaric acid series standard working solutions: accurately measure 0.1, 0.5, 1.0, 2.0, 3.0, and 5.0 mL of p-coumaric acid reference solution, respectively, and place them in 6 10 mL volumetric flasks, add 50% methanol solution to dilute to the mark, shake well, and prepare p-coumaric acid with mass concentrations of 0.28, 0.83, 1.38, 2.76, 5.51, and 13.78 mg / L, respectively. series of standard working solutions; ④ Sample determination: take p-coumaric acid series standard working solutions and sample solutions, and determine the content of p-coumaric acid in the samples according to the chromatographic conditions: Waters Atlantis T3 column (250 mm×4.6 mm, 5μm), column temperature 30℃, mobile phase acetonitrile-0.1% phosphoric acid aqueous solution (volume ratio 13:87), flow rate 1.0mL / min, injection volume 10μL, detection wavelength 310nm.

[0046] Depend on Figure 1It can be seen that the yield and purity of p-coumaric acid prepared in Examples 1-14 are higher than those in Comparative Examples 1-5, among which the yield and purity of p-coumaric acid in Example 5 are the highest, indicating that the use of high-pressure microfluidization in conjunction with acidic ionic liquid to treat ginseng residue can destroy the ether bonds and ester bonds in the cell wall lignin-p-coumaric acid-polysaccharide complex, making it easier for the composite strain of Bacillus licheniformis + Chromobacterium violaceum + Rhodotorula glutinosus to utilize the fermentation substrate; in addition, Bacillus licheniformis metabolizes to generate the precursor substance phenylalanine, which is converted into p-coumaric acid under the action of phenylalanine hydroxylase produced by Chromobacterium violaceum and tyrosine transaminase produced by Rhodotorula glutinosus, thereby improving the yield and purity of p-coumaric acid. However, in Example 1, water was used instead of the acidic ionic liquid, and in Example 2, high-pressure microfluidization treatment was not used. In Examples 1 and 2, the ginseng residue was not sufficiently pretreated, and the ether bonds and ester bonds in the lignin-p-coumaric acid-polysaccharide complex were not fully broken, thereby affecting the subsequent fermentation and utilization of the composite strains. In Examples 3, 4, and 5, only composite strains of Bacillus licheniformis, Chromobacterium violaceum, and Rhodotorula glutinosus were used, respectively, and the directional conversion of p-coumaric acid could not be completed, thereby affecting the yield and purity of p-coumaric acid.

[0047] (2) Antioxidant activity The antioxidant activity of p-coumaric acid prepared in Examples 1-14 and Comparative Examples 1-5 was analyzed as follows: ①DPPH free radical scavenging rate: 0.5 mL p-coumaric acid solution was added to 4 mL 2×10 -4 mol / L DPPH free radical solution, let it stand at room temperature for 30 min, and then measure the absorbance value A at 517 nm. 1 ; At the same time, 4 mL of 2×10 -4 mol / L DPPH free radical solution absorbance value A 0 ; The calculation formula for DPPH scavenging effect is DPPH free radical scavenging rate (%) = (A 0 -A 1 ) / A 0 ×100%; ②ABTS+ free radical scavenging rate: Dissolve ABTS in 7mM concentration water. Before use, react the ABTS stock solution with 2.45mM potassium persulfate to generate ABTS free radical cation (ABTS+), and place it in the dark at room temperature for 12-16 h; detect the ABTS+ solution at 734 nm by diluting it with ethanol to an absorbance of 0.7 ±0.02, and place it at 30°C; take p-coumaric acid and mix it with 2.9mL of the diluted ABTS+ solution, react at 30°C for 20 min, measure the absorbance at 734nm, and calculate the ABTS+ free radical scavenging rate of the sample.

[0048] Depend on Figure 2It can be seen that the DPPH radical scavenging rate and ABTS+ radical scavenging rate of Example 5 are higher than those of Comparative Examples 1-5. Since the yield and purity of p-coumaric acid prepared in Example 5 are higher than those of Comparative Examples 1-5, the p-coumaric acid prepared in Example 5 contains more active groups and thus has higher DPPH radical scavenging rate and ABTS+ radical scavenging rate.

[0049] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A method for extracting p-coumaric acid from ginseng residue, characterized in that: The following steps are involved: S1. The ginseng residue was ultrafinely crushed, and the acidic ionic liquid was added and mixed to obtain a ginseng residue mixed solution; S2. The mixed solution of ginseng residue was subjected to high-pressure microfluidization treatment to obtain a pretreated mixed solution of ginseng residue; S3. The pretreated ginseng residue mixed solution was inoculated with a composite bacterial strain for fermentation to obtain a ginseng residue fermentation liquid; S4. Inactivate the bacteria by heating the fermented ginseng residue liquid, and separate and purify the obtained p-coumaric acid; The composite bacterial strain is Bacillus licheniformis+Chromobacterium violaceum+Rhodotorula glutinosus.

2. The method for extracting p-coumaric acid from ginseng residue according to claim 1, characterized in that: In the step S1, the ratio of ginseng residue to acidic ionic liquid is 1 g: (5-10) mL; the acidic ionic liquid is prepared by stirring choline dihydrogen citrate, lactic acid and guaiacol at 70-80° C. for 30-45 minutes.

3. The method for extracting p-coumaric acid from ginseng residue according to claim 2, characterized in that: The molar ratio of the choline dihydrogen citrate, lactic acid and guaiacol is 1:(1-3):(0.5-1.5).

4. The method for extracting p-coumaric acid from ginseng residue according to claim 1, characterized in that: The conditions of the high-pressure microfluidization treatment in step S2 are a treatment pressure of 80-150 MPa, a cycle number of 2-4 times, and a treatment temperature of 25-40°C.

5. The method for extracting p-coumaric acid from ginseng residue according to claim 1, characterized in that: The inoculation amount of the composite bacteria in step S3 is 4.5-7.5wt%; the ratio of Bacillus licheniformis: Chromobacterium violaceum: Rhodotorula glutinosus in the composite bacteria is (1.5-4): (1-3): (1-2.5).

6. The method for extracting p-coumaric acid from ginseng residue according to claim 1, characterized in that: The fermentation conditions in step S3 are fermentation temperature 25-35° C. and fermentation time 12-36 h.

7. The method for extracting p-coumaric acid from ginseng residue according to claim 1, characterized in that: The temperature for inactivating the bacteria in step S4 is 60-90°C.

8. The method for extracting p-coumaric acid from ginseng residue according to claim 1, characterized in that: The specific method of separation and purification in step S4 is as follows: ① centrifuge the inactivated ginseng residue fermentation liquid at 3500-5000rpm for 10-15min, and take the supernatant; ② adjust the pH of the supernatant to 3-5 and let it stand for 5-10min, then add ethyl acetate for extraction, and separate the layers to obtain the organic phase; ③ rotary evaporate the organic phase to obtain p-coumaric acid.

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