A method for extracting p-coumaric acid from ginseng residues

Abstract

The present invention provides a method for extracting p-coumaric acid from ginseng residues, belonging to the technical field of comprehensive utilization of biological resources. The method includes: (1) synergistically pretreating ginseng residues with high-pressure microfluidics and acidic ionic liquids to destroy the network structure of the lignin-p-coumaric acid-polysaccharide complex through the combined action of physical cavitation effect and chemical catalysis, promoting the cleavage of ether bonds and ester bonds and forming a porous matrix; (2) constructing a co-fermentation system of Bacillus licheniformis, Chromobacterium violaceum and Rhodotorula glutinis to convert macromolecular substances such as polysaccharides released after pretreatment into p-coumaric acid through multi-enzyme cascade catalysis. The present invention realizes the efficient release and biological conversion of complex-bound p-coumaric acid through a physical-chemical-biological synergistic strategy, significantly improves the structural accessibility and resource conversion efficiency of ginseng residues, and provides an innovative solution for the green and high-value utilization of plant processing by-products.

Description

Technical Field

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

[0002] Coumaric acid is a derivative of cinnamic acid and a natural polyphenolic active product, which is widely present in foods and traditional Chinese medicines. 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 activities and pharmacological effects, such as antioxidant activity, anti-inflammatory, prevention and improvement of diabetes, liver and kidney protection, immunomodulation and other effects.

[0003] Ginseng residues are high-fiber by-products generated during the extraction of active ingredients from ginseng. Its components include cell wall substances rich in phenolic acids, dietary fiber, starch and other substances. However, in the prior art, most of the utilization of ginseng residues is used to make feed, which will cause a great waste of nutritional components. Ginseng residues themselves contain a large amount of dietary fiber and starch and can be used as good fermentation substrates. Therefore, using microbial strains to ferment ginseng residues to extract bioactive components can greatly improve the utilization value of ginseng residues.

[0004] P-coumaric acid belongs to phenolic acids, which mainly exist in the plant cell walls of ginseng residues and form lignin-phenolic acid-polysaccharide complexes through ester bonds and ether bonds with polysaccharides and lignin. The structure of this complex is stable, resulting in greater difficulty in extracting phenolic acids. The existing extraction methods of phenolic acids mainly include acid hydrolysis, alkali hydrolysis and enzymatic hydrolysis. Among them, heating acid hydrolysis is likely to cause the loss of phenolic substances; it is difficult to screen microbial strains that can efficiently degrade ginseng residues to obtain phenolic acids, and at the same time, the catalytic rate is relatively slow, and the practical application of the enzymatic method is very limited; the traditional alkali hydrolysis method has low efficiency. In addition, due to the diversity of natural substances, the different binding modes of phenolic substances in cell walls and the easy oxidation of phenolic substances and many other factors make the separation of phenolic acids complicated. Summary of the Invention

[0005] Technical problem to be solved: Aiming at the above technical problems, the purpose of the present invention is to provide a method for extracting p -coumaric acid from ginseng residues, belonging to the technical field of comprehensive utilization of biological resources. The method includes: (1) synergistically pretreating ginseng residues with high - pressure micro - jet and acidic ionic liquid to destroy the network structure of the lignin - p -coumaric acid - polysaccharide complex through the combined action of physical cavitation effect and chemical catalysis, promoting the directional cleavage of ether bonds and ester bonds and forming a porous matrix; (2) constructing a composite microbial community co - fermentation system of Bacillus licheniformis, Chromobacterium violaceum and Rhodotorula glutinis, and converting macromolecular substances such as polysaccharides released after pretreatment into p -coumaric acid through multi - enzyme cascade catalysis. The present invention realizes the efficient release and biological conversion of composite - bound p -coumaric acid through a physical - chemical - biological synergistic strategy, significantly improving the structural accessibility and resource conversion efficiency of ginseng residues, and providing 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 residues includes the following steps:

[0007] S1. Ultrafinely pulverize ginseng residues, add acidic ionic liquid and mix evenly to obtain a ginseng residue mixed solution;

[0008] S2. Perform high - pressure micro - jet treatment on the ginseng residue mixed solution to obtain a pretreated ginseng residue mixed solution;

[0009] S3. Inoculate a composite strain into the pretreated ginseng residue mixed solution for fermentation to obtain a ginseng residue fermentation broth;

[0010] S4. Heat up the ginseng residue fermentation broth to inactivate the strain, and separate and purify to obtain p -coumaric acid;

[0011] The composite strain is Bacillus licheniformis + Chromobacterium violaceum + Rhodotorula glutinis.

[0012] Furthermore, in step S1, the ratio of ginseng residues 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.

[0013] Furthermore, the molar ratio of choline dihydrogen citrate, lactic acid and guaiacol is 1:(1 - 3):(0.5 - 1.5).

[0014] Furthermore, the conditions for high - pressure micro - jet treatment in step S2 are treatment pressure 80 - 150 MPa, cycle number 2 - 4 times, and treatment temperature 25 - 40 °C.

[0015] Further, the inoculation amount of the composite strain in step S3 is 4.5 - 7.5 wt%; the ratio of Bacillus licheniformis: Chromobacterium violaceum: Rhodotorula glutinis in the composite strain is (1.5 - 4):(1 - 3):(1 - 2.5).

[0016] Further, the fermentation conditions in step S3 are a fermentation temperature of 25 - 35 °C and a fermentation time of 12 - 36 h.

[0017] Further, the temperature for inactivating the bacteria in step S4 is 60 - 90 °C.

[0018] Further, the specific method for separation and purification in step S4 is as follows: ① Centrifuge the inactivated ginseng residue fermentation broth at 3500 - 5000 rpm for 10 - 15 min, and take the supernatant; ② Adjust the pH of the supernatant to 3 - 5, let it stand for 5 - 10 min, then add ethyl acetate for extraction, and separate to obtain the organic phase; ③ Subject the organic phase to rotary evaporation to obtain p - coumaric acid.

[0019] Beneficial effects

[0020] 1. The present invention adopts the synergistic effect of high - pressure microfluidics and acidic ionic liquid (choline dihydrogen citrate / lactic acid / guaiacol composite system) to establish a "dynamic solvent penetration - complex degradation" dual - effect pretreatment mechanism. Among them, the acidic ionic liquid specifically cleaves the acid - sensitive ether bonds in the lignin - p - coumaric acid - polysaccharide complex through proton - catalyzed action, while the instantaneous cavitation effect, turbulent shock wave and ultra - high shear force generated by high - pressure microfluidics can effectively cut the polysaccharide chain, realizing the physical relaxation of the complex 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 complex vulnerable to being destroyed and broken under the multiple actions generated by high - pressure microfluidics through the conformational exposure effect, promoting the dissolution of p - coumaric acid in ginseng residue, and making the ginseng residue structure become porous, creating an ideal mass transfer interface for subsequent biotransformation.

[0021] 2. The present invention constructs a synergistic fermentation system of Bacillus licheniformis + Chromobacterium violaceum + Rhodotorula glutinis composite strain, forming a "substrate conversion - intermediate synthesis - end - product generation" cascade metabolic pathway, which is specifically manifested as follows: ① Bacillus licheniformis realizes the saccharification conversion of dietary fiber and the cleavage of ester bonds by secreting cellulase and lipase, and synchronously generates the key precursor phenylalanine; ② Chromobacterium violaceum catalyzes the conversion of phenylalanine to tyrosine through phenylalanine hydroxylase; ③ Rhodotorula glutinis uses tyrosine transaminase to complete the directional conversion of tyrosine to p - coumaric acid, thereby increasing the content of p - coumaric acid in ginseng residue.

[0022] 3. While the present invention converts the insoluble conjugated p -coumaric acid in ginseng residues into free - state products, it precisely constructs the biotransformation pathway, which is beneficial to improving the utilization value of ginseng residues and resource conversion, provides a good theoretical reference for the high - value utilization of ginseng residues, and provides an industrializable solution for the high - value utilization of plant processing by - products. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The yields and purities of p -coumaric acid for Examples 1 - 14 and Comparative Examples 1 - 5;

[0024] Figure 2 The DPPH radical scavenging rates and ABTS+ radical scavenging rates for Example 5 and Comparative Examples 1 - 5. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] The present invention will be further described below in conjunction with embodiments. The following embodiments are explanations of the present invention, and the present invention is not limited to the following embodiments:

[0026] Sources of the strains used in the present invention: Bacillus licheniformis CICC 22606 was purchased from the China Center for Industrial Culture Collection; Chromobacterium violaceum ATCC12540 was purchased from Sigma; Rhodotorula glutinis BJ - J12861 was purchased from Shanghai Bangjing Industrial Co., Ltd.

[0027] The composite strains described in the following embodiments and comparative examples were prepared by activating the strains Bacillus licheniformis CICC 22606 + Chromobacterium violaceum ATCC12540 + Rhodotorula glutinis BJ - J12861. The specific process of strain activation is as follows:

[0028] Activation of Bacillus licheniformis CICC 22606: Bacillus licheniformis CICC 22606 was inoculated into LB liquid medium and cultured at 30 °C and 220 rpm for 24 h to obtain the Bacillus licheniformis CICC 22606 seed solution. Then, 1% of the Bacillus licheniformis CICC 22606 seed solution was inoculated into LB liquid medium and cultured at 30 °C and 220 rpm for 24 h. After centrifugation and filtration, it was resuspended with sterile water until the strain concentration was greater than 10 8 CFU / mL to obtain the activated Bacillus licheniformis CICC 22606 bacterial solution;

[0029] Activation of Chromobacterium violaceum ATCC12540: Inoculate Chromobacterium violaceum ATCC12540 into nutrient broth medium and culture it at 28 °C and 150 rpm for 24 h to obtain the Chromobacterium violaceum ATCC12540 seed solution. Then inoculate 1% of the Chromobacterium violaceum ATCC12540 seed solution into nutrient broth medium and culture it at 28 °C and 150 rpm for 24 h. Centrifuge and filter, and resuspend with sterile water until the bacterial concentration is greater than 10 8 CFU / mL to obtain the activated Chromobacterium violaceum ATCC12540 bacterial solution;

[0030] Activation of Rhodotorula glutinis BJ-J12861: Inoculate Rhodotorula glutinis BJ-J12861 into YPD liquid medium and culture it at 30 °C and 200 rpm for 24 h to obtain the Rhodotorula glutinis BJ-J12861 seed solution. Then inoculate 1% of the Rhodotorula glutinis BJ-J12861 seed solution into YPD liquid medium and culture it at 30 °C and 200 rpm for 24 h. Centrifuge and filter, and resuspend with sterile water until the bacterial concentration is greater than 10 8 CFU / mL to obtain the activated Rhodotorula glutinis BJ-J12861 bacterial solution. Example 1

[0031] A method for extracting p-coumaric acid from ginseng residues, comprising the following steps:

[0032] 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 the acidic ionic liquid;

[0033] S2. Ultrafinely crush 100 g of ginseng residues, add 800 mL of acidic ionic liquid, and mix evenly to obtain a ginseng residue mixed solution;

[0034] S3. The ginseng residue mixed solution is cyclically treated 3 times by a high-pressure microfluidic device at 30 °C and 100 MPa to obtain a pretreated ginseng residue mixed solution;

[0035] S4. Inoculate 54 g of a composite strain (27 g of Bacillus licheniformis CICC 22606 + 18 g of Chromobacterium violaceum ATCC12540 + 9 g of Rhodotorula glutinis BJ-J12861) into the pretreated ginseng residue mixed solution, and ferment at 30 °C for 24 h to obtain a ginseng residue fermentation broth;

[0036] S5. Heat the ginseng residue fermentation broth to 75 °C to inactivate the strains. Centrifuge the inactivated ginseng residue fermentation broth at 4000 rpm for 12 min, and take the supernatant; adjust the pH of the supernatant to 4, let it stand for 5 min, then add ethyl acetate for extraction, and separate to obtain the organic phase; the organic phase is treated by rotary evaporation to obtain p-coumaric acid. Example 2

[0037] A method for extracting p - coumaric acid from ginseng residues, comprising the following steps:

[0038] 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 the acidic ionic liquid;

[0039] S2. Ultra - micro - pulverize 50 g of ginseng residues, add 400 mL of the acidic ionic liquid and mix evenly to obtain a ginseng residue mixed solution;

[0040] S3. The ginseng residue mixed solution is cyclically processed 3 times through a high - pressure micro - jet device at 30 °C and 100 MPa to obtain a pretreated ginseng residue mixed solution;

[0041] S4. Inoculate 27 g of a composite strain (13.5 g of Bacillus licheniformis CICC 22606+9.0 g of Chromobacterium violaceum ATCC12540+4.5 g of Rhodotorula glutinis BJ - J12861) into the pretreated ginseng residue mixed solution, and ferment at 30 °C for 24 h to obtain a ginseng residue fermentation broth;

[0042] S5. Heat the ginseng residue fermentation broth to 80 °C to inactivate the strains, centrifuge the inactivated ginseng residue fermentation broth at 5000 rpm for 10 min, and take the supernatant; adjust the pH of the supernatant to 4, let it stand for 5 min, then add ethyl acetate for extraction, and separate to obtain the organic phase; the organic phase is processed by rotary evaporation to obtain p - coumaric acid. Example 3

[0043] A method for extracting p - coumaric acid from ginseng residues, comprising the following steps:

[0044] 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 the acidic ionic liquid;

[0045] S2. Ultra - micro - pulverize 100 g of ginseng residues, add 800 mL of the acidic ionic liquid and mix evenly to obtain a ginseng residue mixed solution;

[0046] S3. The ginseng residue mixed solution is cyclically processed 3 times through a high - pressure micro - jet device at 30 °C and 100 MPa to obtain a pretreated ginseng residue mixed solution;

[0047] S4. Inoculate 54 g of a composite strain (27 g of Bacillus licheniformis CICC 22606+18 g of Chromobacterium violaceum ATCC12540+9 g of Rhodotorula glutinis BJ - J12861) into the pretreated ginseng residue mixed solution, and ferment at 30 °C for 24 h to obtain a ginseng residue fermentation broth;

[0048] S5. Heat the ginseng residue fermentation broth to 80 °C to inactivate the strains, centrifuge the inactivated ginseng residue fermentation broth at 4000 rpm for 10 min, and take the supernatant; adjust the pH of the supernatant to 4, let it stand for 7 min, then add ethyl acetate for extraction, and separate to obtain the organic phase; perform rotary evaporation on the organic phase to obtain p -coumaric acid. Example 4

[0049] A method for extracting p -coumaric acid from ginseng residue, comprising the following steps:

[0050] 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 the acidic ionic liquid;

[0051] S2. Ultra - micro - pulverize 100 g of ginseng residue, add 500 mL of the acidic ionic liquid and mix evenly to obtain a ginseng residue mixed solution;

[0052] S3. The ginseng residue mixed solution is circulated through a high - pressure micro - jet device at 25 °C and 100 MPa for 3 times to obtain a pretreated ginseng residue mixed solution;

[0053] S4. Inoculate 36 g of a composite strain (18 g of Bacillus licheniformis CICC 22606+12 g of Chromobacterium violaceum ATCC12540+6 g of Rhodotorula glutinis BJ - J12861) into the pretreated ginseng residue mixed solution, and ferment at 30 °C for 24 h to obtain a ginseng residue fermentation broth;

[0054] S5. Heat the ginseng residue fermentation broth to 80 °C to inactivate the strains, centrifuge the inactivated ginseng residue fermentation broth at 3500 rpm for 15 min, and take the supernatant; adjust the pH of the supernatant to 4, let it stand for 5 min, then add ethyl acetate for extraction, and separate to obtain the organic phase; perform rotary evaporation on the organic phase to obtain p -coumaric acid. Example 5

[0055] A method for extracting p -coumaric acid from ginseng residue, comprising the following steps:

[0056] 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 the acidic ionic liquid;

[0057] S2. Ultra - micro - pulverize 100 g of ginseng residue, add 1000 mL of the acidic ionic liquid and mix evenly to obtain a ginseng residue mixed solution;

[0058] S3. The ginseng residue mixed solution is circulated through a high - pressure micro - jet device at 30 °C and 100 MPa for 3 times to obtain a pretreated ginseng residue mixed solution;

[0059] S4. Inoculate 66 g of the composite strain (33 g of Bacillus licheniformis CICC 22606 + 22 g of Chromobacterium violaceum ATCC12540 + 11 g of Rhodotorula glutinis BJ-J12861) into the pretreated ginseng residue mixed solution, and ferment at 30 °C for 24 h to obtain the ginseng residue fermentation broth;

[0060] S5. Heat the ginseng residue fermentation broth to 75 °C to inactivate the strain. Centrifuge the inactivated ginseng residue fermentation broth at 4000 rpm for 12 min, and take the supernatant. Adjust the pH of the supernatant to 4 and let it stand for 5 min, then add ethyl acetate for extraction, and separate to obtain the organic phase. The organic phase is treated by rotary evaporation to obtain p -coumaric acid. Example 6

[0061] A method for extracting p -coumaric acid from ginseng residues, comprising the following steps:

[0062] 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 the acidic ionic liquid;

[0063] S2. Ultrafinely crush 100 g of ginseng residues, add 800 mL of the acidic ionic liquid and mix evenly to obtain the ginseng residue mixed solution;

[0064] S3. The ginseng residue mixed solution is circulated through a high - pressure microfluidic device at 30 °C and 80 MPa for 3 times to obtain the pretreated ginseng residue mixed solution;

[0065] S4. Inoculate 54 g of the composite strain (27 g of Bacillus licheniformis CICC 22606 + 18 g of Chromobacterium violaceum ATCC12540 + 9 g of Rhodotorula glutinis BJ-J12861) into the pretreated ginseng residue mixed solution, and ferment at 30 °C for 24 h to obtain the ginseng residue fermentation broth;

[0066] S5. Heat the ginseng residue fermentation broth to 80 °C to inactivate the strain. Centrifuge the inactivated ginseng residue fermentation broth at 3500 rpm for 15 min, and take the supernatant. Adjust the pH of the supernatant to 4 and let it stand for 5 min, then add ethyl acetate for extraction, and separate to obtain the organic phase. The organic phase is treated by rotary evaporation to obtain p -coumaric acid. Example 7

[0067] A method for extracting p -coumaric acid from ginseng residues, comprising the following steps:

[0068] 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 the acidic ionic liquid;

[0069] S2. Ultra - micro - pulverize 100 g of ginseng residue, add 800 mL of acidic ionic liquid and mix evenly to obtain a ginseng residue mixed solution;

[0070] S3. The ginseng residue mixed solution is cyclically processed 3 times through a high - pressure micro - jet device at 35 °C and 120 MPa to obtain a pretreated ginseng residue mixed solution;

[0071] S4. Inoculate 54 g of a composite strain (27 g of Bacillus licheniformis CICC 22606 + 18 g of Chromobacterium violaceum ATCC12540 + 9 g of Rhodotorula glutinis BJ - J12861) into the pretreated ginseng residue mixed solution, and ferment at 30 °C for 24 h to obtain a ginseng residue fermentation broth;

[0072] S5. Heat the ginseng residue fermentation broth to 75 °C to inactivate the strain, centrifuge the inactivated ginseng residue fermentation broth at 4000 rpm for 12 min, and take the supernatant; Adjust the pH of the supernatant to 4, let it stand for 5 min, then add ethyl acetate for extraction, and separate to obtain an organic phase; The organic phase is treated by rotary evaporation to obtain p - coumaric acid. Example 8

[0073] A method for extracting p - coumaric acid from ginseng residue, comprising the following steps:

[0074] 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 the acidic ionic liquid;

[0075] S2. Ultra - micro - pulverize 100 g of ginseng residue, add 800 mL of acidic ionic liquid and mix evenly to obtain a ginseng residue mixed solution;

[0076] S3. The ginseng residue mixed solution is cyclically processed 3 times through a high - pressure micro - jet device at 25 °C and 100 MPa to obtain a pretreated ginseng residue mixed solution;

[0077] S4. Inoculate 40.5 g of a composite strain (20.25 g of Bacillus licheniformis CICC22606 + 13.5 g of Chromobacterium violaceum ATCC12540 + 6.75 g of Rhodotorula glutinis BJ - J12861) into the pretreated ginseng residue mixed solution, and ferment at 30 °C for 24 h to obtain a ginseng residue fermentation broth;

[0078] S5. Heat the ginseng residue fermentation broth to 80 °C to inactivate the strain, centrifuge the inactivated ginseng residue fermentation broth at 3500 rpm for 15 min, and take the supernatant; Adjust the pH of the supernatant to 4, let it stand for 10 min, then add ethyl acetate for extraction, and separate to obtain an organic phase; The organic phase is treated by rotary evaporation to obtain p - coumaric acid. Example 9

[0079] A method for extracting p - coumaric acid from ginseng residues, comprising the following steps:

[0080] 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 the acidic ionic liquid;

[0081] S2. Ultrafinely pulverize 100 g of ginseng residues, add 800 mL of the acidic ionic liquid and mix evenly to obtain a ginseng residue mixed solution;

[0082] S3. The ginseng residue mixed solution is circulated through a high - pressure microfluidization device at 30 °C and 100 MPa for 3 times to obtain a pretreated ginseng residue mixed solution;

[0083] S4. Inoculate 67.5 g of a composite strain (33.75 g of Bacillus licheniformis CICC22606 + 22.5 g of Chromobacterium violaceum ATCC12540 + 11.25 g of Rhodotorula glutinis BJ - J12861) into the pretreated ginseng residue mixed solution, and ferment at 30 °C for 24 h to obtain a ginseng residue fermentation broth;

[0084] S5. Heat the ginseng residue fermentation broth to 75 °C to inactivate the strain, centrifuge the inactivated ginseng residue fermentation broth at 4000 rpm for 12 min, and take the supernatant; adjust the pH of the supernatant to 4, let it stand for 5 min, then add ethyl acetate for extraction, and separate the organic phase; the organic phase is treated by rotary evaporation to obtain p - coumaric acid. Example 10

[0085] A method for extracting p - coumaric acid from ginseng residues, comprising the following steps:

[0086] 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 the acidic ionic liquid;

[0087] S2. Ultrafinely pulverize 100 g of ginseng residues, add 800 mL of the acidic ionic liquid and mix evenly to obtain a ginseng residue mixed solution;

[0088] S3. The ginseng residue mixed solution is circulated through a high - pressure microfluidization device at 30 °C and 100 MPa for 3 times to obtain a pretreated ginseng residue mixed solution;

[0089] S4. Inoculate 54 g of a composite strain (18 g of Bacillus licheniformis CICC 22606 + 18 g of Chromobacterium violaceum ATCC12540 + 18 g of Rhodotorula glutinis BJ - J12861) into the pretreated ginseng residue mixed solution, and ferment at 30 °C for 24 h to obtain a ginseng residue fermentation broth;

[0090] S5. Heat the fermented solution of ginseng residue to 75 °C to inactivate the strains, centrifuge the inactivated fermented solution of ginseng residue at 4000 rpm for 12 min, and take the supernatant; adjust the pH of the supernatant to 4 and let it stand for 5 min, then add ethyl acetate for extraction, and separate to obtain the organic phase; perform rotary evaporation on the organic phase to obtain p -coumaric acid. Example 11

[0091] A method for extracting p -coumaric acid from ginseng residue, comprising the following steps:

[0092] 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 the acidic ionic liquid;

[0093] S2. Ultrafinely pulverize 100 g of ginseng residue, add 800 mL of the acidic ionic liquid and mix evenly to obtain a ginseng residue mixed solution;

[0094] S3. The ginseng residue mixed solution is cyclically processed 3 times by a high - pressure micro - jet device at 30 °C and 100 MPa to obtain a pretreated ginseng residue mixed solution;

[0095] S4. Inoculate 54 g of a composite strain (21.6 g of Bacillus licheniformis CICC 22606 + 10.8 g of Chromobacterium violaceum ATCC12540 + 21.6 g of Rhodotorula glutinis BJ - J12861) into the pretreated ginseng residue mixed solution, and ferment at 30 °C for 24 h to obtain a fermented solution of ginseng residue;

[0096] S5. Heat the fermented solution of ginseng residue to 75 °C to inactivate the strains, centrifuge the inactivated fermented solution of ginseng residue at 4000 rpm for 12 min, and take the supernatant; adjust the pH of the supernatant to 4 and let it stand for 5 min, then add ethyl acetate for extraction, and separate to obtain the organic phase; perform rotary evaporation on the organic phase to obtain p -coumaric acid. Example 12

[0097] A method for extracting p -coumaric acid from ginseng residue, comprising the following steps:

[0098] 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 the acidic ionic liquid;

[0099] S2. Ultrafinely pulverize 100 g of ginseng residue, add 800 mL of the acidic ionic liquid and mix evenly to obtain a ginseng residue mixed solution;

[0100] S3. The ginseng residue mixed solution is cyclically processed 3 times through a high-pressure microfluidization device at 25°C and 100 MPa to obtain a pretreated ginseng residue mixed solution;

[0101] S4. Inoculate 54 g of a composite strain (27 g of Bacillus licheniformis CICC 22606 + 18 g of Chromobacterium violaceum ATCC12540 + 9 g of Rhodotorula glutinis BJ-J12861) into the pretreated ginseng residue mixed solution, and ferment at 35°C for 24 h to obtain a ginseng residue fermentation broth;

[0102] S5. Heat the ginseng residue fermentation broth to 80°C to inactivate the strain, centrifuge the inactivated ginseng residue fermentation broth at 3500 rpm for 15 min, and take the supernatant; adjust the pH of the supernatant to 4, let it stand for 10 min, then add ethyl acetate for extraction, and separate the organic phase; the organic phase is subjected to rotary evaporation to obtain p -coumaric acid. Example 13

[0103] A method for extracting p -coumaric acid from ginseng residues, comprising the following steps:

[0104] 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;

[0105] S2. Ultrafinely pulverize 50 g of ginseng residues, add 400 mL of the acidic ionic liquid and mix evenly to obtain a ginseng residue mixed solution;

[0106] S3. The ginseng residue mixed solution is cyclically processed 3 times through a high-pressure microfluidization device at 30°C and 100 MPa to obtain a pretreated ginseng residue mixed solution;

[0107] S4. Inoculate 27 g of a composite strain (13.5 g of Bacillus licheniformis CICC 22606 + 9 g of Chromobacterium violaceum ATCC12540 + 4.5 g of Rhodotorula glutinis BJ-J12861) into the pretreated ginseng residue mixed solution, and ferment at 30°C for 12 h to obtain a ginseng residue fermentation broth;

[0108] S5. Heat the ginseng residue fermentation broth to 75°C to inactivate the strain, centrifuge the inactivated ginseng residue fermentation broth at 4000 rpm for 12 min, and take the supernatant; adjust the pH of the supernatant to 4, let it stand for 5 min, then add ethyl acetate for extraction, and separate the organic phase; the organic phase is subjected to rotary evaporation to obtain p -coumaric acid. Example 14

[0109] A method for extracting p -coumaric acid from ginseng residues, comprising the following steps:

[0110] 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 the acidic ionic liquid;

[0111] S2. Ultrafinely crush 100 g of ginseng residue, add 800 mL of acidic ionic liquid and mix evenly to obtain a ginseng residue mixed solution;

[0112] S3. The ginseng residue mixed solution is cyclically processed 3 times at 25 °C and 100 MPa through a high-pressure microfluidic device to obtain a pretreated ginseng residue mixed solution;

[0113] S4. Inoculate 54 g of a composite strain (27 g of Bacillus licheniformis CICC 22606 + 18 g of Chromobacterium violaceum ATCC12540 + 9 g of Rhodotorula glutinis BJ-J12861) into the pretreated ginseng residue mixed solution, and ferment at 30 °C for 36 h to obtain a ginseng residue fermentation broth;

[0114] S5. Heat the ginseng residue fermentation broth to 80 °C to inactivate the strain, centrifuge the inactivated ginseng residue fermentation broth at 4000 rpm for 10 min, and take the supernatant; adjust the pH of the supernatant to 4 and let it stand for 10 min, then add ethyl acetate for extraction, and separate the organic phase; the organic phase is treated by rotary evaporation to obtain p-coumaric acid. Comparative Example 1

[0115] The difference between this comparative example and Example 5 is that no acidic ionic liquid is added.

[0116] A method for extracting p-coumaric acid from ginseng residue, comprising the following steps:

[0117] S1. Ultrafinely crush 100 g of ginseng residue, add 1000 mL of water and mix evenly to obtain a ginseng residue mixed solution;

[0118] S2. The ginseng residue mixed solution is cyclically processed 3 times at 30 °C and 100 MPa through a high-pressure microfluidic device to obtain a pretreated ginseng residue mixed solution;

[0119] S3. Inoculate 66 g of a composite strain (33 g of Bacillus licheniformis CICC 22606 + 22 g of Chromobacterium violaceum ATCC12540 + 11 g of Rhodotorula glutinis BJ-J12861) into the pretreated ginseng residue mixed solution, and ferment at 30 °C for 24 h to obtain a ginseng residue fermentation broth;

[0120] S4. Heat the ginseng residue fermentation broth to 75 °C to inactivate the strain, centrifuge the inactivated ginseng residue fermentation broth at 4000 rpm for 12 min, and take the supernatant; adjust the pH of the supernatant to 4 and let it stand for 5 min, then add ethyl acetate for extraction, and separate the organic phase; the organic phase is treated by rotary evaporation to obtain p-coumaric acid. Comparative Example 2

[0121] The difference between this comparative example and Example 5 is that high-pressure microfluidization treatment is not used.

[0122] A method for extracting p-coumaric acid from ginseng residues, comprising the following steps:

[0123] 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 the acidic ionic liquid;

[0124] S2. Ultrafinely pulverize 100 g of ginseng residues, add 1000 mL of acidic ionic liquid, and mix evenly to obtain a pretreated ginseng residue mixed solution;

[0125] S3. Inoculate 66 g of a composite strain (33 g of Bacillus licheniformis CICC 22606 + 22 g of Chromobacterium violaceum ATCC12540 + 11 g of Rhodotorula glutinis BJ-J12861) into the pretreated ginseng residue mixed solution, and ferment at 30 °C for 24 h to obtain a ginseng residue fermentation broth;

[0126] S4. Heat the ginseng residue fermentation broth to 75 °C to inactivate the strain, centrifuge the inactivated ginseng residue fermentation broth at 4000 rpm for 12 min, and take the supernatant; adjust the pH of the supernatant to 4, let it stand for 5 min, then add ethyl acetate for extraction, and separate the organic phase; the organic phase is treated by rotary evaporation to obtain p-coumaric acid. Comparative Example 3

[0127] The difference between this comparative example and Example 5 is that only Bacillus licheniformis is used.

[0128] A method for extracting p-coumaric acid from ginseng residues, comprising the following steps:

[0129] 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 the acidic ionic liquid;

[0130] S2. Ultrafinely pulverize 100 g of ginseng residues, add 1000 mL of acidic ionic liquid, and mix evenly to obtain a ginseng residue mixed solution;

[0131] S3. The ginseng residue mixed solution is circulated through a high-pressure microfluidization device at 30 °C and 100 MPa for 3 times to obtain a pretreated ginseng residue mixed solution;

[0132] S4. Inoculate 66 g of Bacillus licheniformis into the pretreated ginseng residue mixed solution, and ferment at 30 °C for 24 h to obtain a ginseng residue fermentation broth;

[0133] S5. Heat the ginseng residue fermentation broth to 75 °C to inactivate the strains. Centrifuge the inactivated ginseng residue fermentation broth at 4000 rpm for 12 min, and take the supernatant. Adjust the pH of the supernatant to 4 and let it stand for 5 min, then add ethyl acetate for extraction, and separate to obtain the organic phase. The organic phase is subjected to rotary evaporation to obtain p -coumaric acid. Comparative Example 4

[0134] The difference between this comparative example and Example 5 is that only Chromobacterium violaceum is used.

[0135] A method for extracting p -coumaric acid from ginseng residues, comprising the following steps:

[0136] 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 the acidic ionic liquid;

[0137] S2. Ultrafinely crush 100 g of ginseng residues, add 1000 mL of the acidic ionic liquid and mix evenly to obtain a ginseng residue mixed solution;

[0138] S3. The ginseng residue mixed solution is cyclically processed 3 times by a high - pressure microfluidic device at 30 °C and 100 MPa to obtain a pretreated ginseng residue mixed solution;

[0139] S4. Inoculate 66 g of Chromobacterium violaceum into the pretreated ginseng residue mixed solution and ferment at 30 °C for 24 h to obtain a ginseng residue fermentation broth;

[0140] S5. Heat the ginseng residue fermentation broth to 75 °C to inactivate the strains. Centrifuge the inactivated ginseng residue fermentation broth at 4000 rpm for 12 min, and take the supernatant. Adjust the pH of the supernatant to 4 and let it stand for 5 min, then add ethyl acetate for extraction, and separate to obtain the organic phase. The organic phase is subjected to rotary evaporation to obtain p -coumaric acid. Comparative Example 5

[0141] The difference between this comparative example and Example 5 is that only Rhodotorula glutinis is used.

[0142] A method for extracting p -coumaric acid from ginseng residues, comprising the following steps:

[0143] 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 the acidic ionic liquid;

[0144] S2. Ultrafinely crush 100 g of ginseng residues, add 1000 mL of the acidic ionic liquid and mix evenly to obtain a ginseng residue mixed solution;

[0145] S3. The ginseng residue mixed solution is cyclically processed 3 times by a high-pressure microfluidization device at 30 °C and 100 MPa to obtain a pretreated ginseng residue mixed solution;

[0146] S4. 66 g of Rhodotorula glutinis is inoculated into the pretreated ginseng residue mixed solution and fermented at 30 °C for 24 h to obtain a ginseng residue fermentation broth;

[0147] S5. The temperature of the ginseng residue fermentation broth is raised to 75 °C to inactivate the strains. The inactivated ginseng residue fermentation broth 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. After stratification, the organic phase is obtained; the organic phase is rotary evaporated to obtain p-coumaric acid.

[0148] Performance Test

[0149] (1) Extraction rate and purity of p-coumaric acid

[0150] The yields of p-coumaric acid in Examples 1-14 and Comparative Examples 1-5 are determined. The yield of p-coumaric acid (%) = M2 / M1 × 100%, where M2 represents the mass of p-coumaric acid and M1 represents the mass of ginseng residue; the content of p-coumaric acid in Examples 1-14 and Comparative Examples 1-5 is determined by high-performance liquid chromatography, and the purity of p-coumaric acid is calculated. The specific test method is as follows: ① Preparation of sample solution: Accurately weigh about 0.5000 g of the sample in a conical flask, add 20 mL of 70% ethanol solution, weigh, heat under reflux for 30 min, take out and cool, weigh again, and make up the lost mass with 70% ethanol solution. Shake well and filter through a 0.22 μm needle filter to obtain the sample solution; ② p-coumaric acid reference solution: Accurately weigh 6.91 mg of p-coumaric acid standard product and place it in a 25 mL volumetric flask. Dissolve it with 50% methanol solution and make up to the mark. Take 2.5 mL and place it in a 25 mL volumetric flask, and make up to the mark with 50% methanol solution to obtain the p-coumaric acid reference solution; ③ Series of p-coumaric acid standard working solutions: Accurately measure 0.1, 0.5, 1.0, 2.0, 3.0, 5.0 mL of the p-coumaric acid reference solution respectively, place them in 6 10 mL volumetric flasks, add 50% methanol solution to make up to the mark, and shake well to prepare a series of standard working solutions with the mass concentrations of p-coumaric acid being 0.28, 0.83, 1.38, 2.76, 5.51, 13.78 mg / L respectively; ④ Sample determination: Take the series of p-coumaric acid standard working solutions and the sample solution, and determine the content of p-coumaric acid in the sample according to the chromatographic conditions. The chromatographic conditions are Waters Atlantis T3 chromatographic column (250 mm × 4.6 mm, 5 μm), column temperature 30 °C, mobile phase is acetonitrile - 0.1% phosphoric acid aqueous solution (volume ratio 13:87), flow rate 1.0 mL / min, injection volume 10 μL, detection wavelength 310 nm.

[0151] It can be seen from Figure 1 that the yields and purities of p -coumaric acid prepared in Examples 1 - 14 are higher than those in Comparative Examples 1 - 5. Among them, the yield and purity of p -coumaric acid in Example 5 are the highest, indicating that the use of high - pressure microfluidization in combination with acidic ionic liquids to treat ginseng residues can break the ether bonds and ester bonds in the cell wall lignin - p -coumaric acid - polysaccharide complex, facilitating the utilization of the fermentation substrate by the composite strain of Bacillus licheniformis + Chromobacterium violaceum + Rhodotorula glutinis; 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 aminotransferase produced by Rhodotorula glutinis, thereby increasing the yield and purity of p -coumaric acid. In Comparative Example 1, water was used to replace the acidic ionic liquid, and in Comparative Example 2, high - pressure microfluidization treatment was not used. The pretreatment of ginseng residues in Comparative Examples 1 and 2 was insufficient, and the ether bonds and ester bonds in the lignin - p -coumaric acid - polysaccharide complex were not fully broken, thus affecting the subsequent fermentation utilization by the composite strain; in Comparative Examples 3, 4, and 5, only the composite strains of Bacillus licheniformis, Chromobacterium violaceum, and Rhodotorula glutinis were used respectively, and the directional conversion of p -coumaric acid could not be completed, so the yield and purity of p -coumaric acid were affected.

[0152] (2)Antioxidant activity

[0153] Analyze the antioxidant activities of the p -coumaric acid prepared in Examples 1 - 14 and Comparative Examples 1 - 5, which are specifically as follows:

[0154] ① DPPH free radical scavenging rate: Add 0.5 mL of p -coumaric acid solution to 4 mL of 2×10 -4 mol / L DPPH free radical solution, shake well, and let it stand at room temperature for 30 min, then measure the absorbance value A1 at 517 nm; at the same time, measure the absorbance value A0 of 4 mL of 2×10 -4 mol / L DPPH free radical solution dissolved in 0.5 mL of 60% ethanol solution; the calculation formula for the DPPH scavenging effect is DPPH free radical scavenging rate (%) = (A0 - A1) / A0×100%;

[0155] ② ABTS+ free radical scavenging rate: Dissolve ABTS in water with a concentration of 7 mM. Before use, react the ABTS stock solution with 2.45 mM potassium persulfate to generate ABTS radical cations (ABTS+), and place it in the dark at room temperature for 12 - 16 h; detect the ABTS+ solution at 734 nm and dilute it with ethanol to an absorbance of 0.7 ± 0.02, and place it at 30℃; take p -coumaric acid and mix it with 2.9 mL of diluted ABTS+ solution, react at 30℃ for 20 min, measure the absorbance at 734 nm, and calculate the ABTS+ free radical scavenging rate of the sample.

[0156] It can be seen from Figure 2It can be seen that the DPPH free radical scavenging rate and ABTS+ free 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, thus having higher DPPH free radical scavenging rate and ABTS+ free radical scavenging rate.

[0157] As described above, it is only a preferred embodiment of the present invention, and does not impose any formal limitation on the present invention. Any person skilled in the art can make many possible changes and modifications to the technical solution of the present invention by using the disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution of the present invention still fall 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. Ginseng residue was ultrafinely ground, and an acidic ionic liquid was added and mixed uniformly to obtain a ginseng residue mixed solution; the ratio of the ginseng residue to the acidic ionic liquid was 1 g: (5-10) mL; the acidic ionic liquid was prepared by stirring choline dihydrogen citrate, lactic acid, and guaiacol at 70-80°C for 30-45 minutes; S2. The mixed solution of ginseng residue was subjected to high-pressure microfluidization to obtain a pretreated mixed solution of ginseng residue; S3. Inoculating the pretreated ginseng residue mixed solution with a composite bacterial strain for fermentation to obtain a ginseng residue fermentation liquid; the inoculation amount of the composite bacterial strain is 4.5-7.5wt%; the ratio of Bacillus licheniformis: Chromobacterium violaceum: Rhodotorula glutinosus in the composite bacterial strain is (1.5-4): (1-3): (1-2.5); S4. Inactivate the bacteria by heating the fermentation liquid of ginseng residue, and separate and purify the obtained p-coumaric acid; The composite bacterial species are Bacillus licheniformis, Chromobacterium violaceum and Rhodotorula glutinosus.

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

3. The method for extracting p-coumaric acid from ginseng residue according to claim 1, wherein: The conditions for 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.

4. The method for extracting p-coumaric acid from ginseng residue according to claim 1, wherein: The fermentation conditions in step S3 are a fermentation temperature of 25-35° C. and a fermentation time of 12-36 hours.

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

6. The method for extracting p-coumaric acid from ginseng residue according to claim 1, wherein: The specific method of separation and purification in step S4 is as follows: ① centrifuging the inactivated ginseng residue fermentation liquid at 3500-5000 rpm for 10-15 minutes and taking the supernatant; ② adjusting the pH of the supernatant to 3-5 and standing for 5-10 minutes, then adding ethyl acetate for extraction, and separating the layers to obtain the organic phase; ③ rotary evaporation of the organic phase to obtain p-coumaric acid.

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