Method for biosynthesizing phenolic acid compound by using lignin and application thereof
阅读说明：本技术 一种利用木质素生物合成酚酸类化合物的方法及其应用 (Method for biosynthesizing phenolic acid compound by using lignin and application thereof ) 是由 肖盈 陈佳志 张靖雯 麦裕良 于 2021-07-09 设计创作，主要内容包括：本发明涉及生物合成技术领域,具体公开了一种利用木质素生物合成酚酸类化合物的方法及其应用。方法包括以下步骤：将暹罗芽孢杆菌菌液和漆酶接种到Tris-HCl溶液中,并添加碱木质素得混合液,将混合液震荡培养,离心取上清液加入乙酸乙酯和盐酸溶液,超声提取,旋干,复溶,得到酚酸类化合物；所述菌液和漆酶的体积比为1:0.5～2(v/v)。利用本发明的方法其制备得到的高活性酚酸类化合物含量较高,且制备方法具有高效率、低成本及环境友好的优点,并且将暹罗芽孢杆菌菌液和漆酶以特定体积比复配,使得制备得到的酚酸类化合物含量较高,其酚酸类化合物还表现出优异的抗氧化活性和抑菌活性。(The invention relates to the technical field of biosynthesis, and particularly discloses a method for biosynthesizing a phenolic acid compound by using lignin and application thereof. The method comprises the following steps: inoculating Siamese bacillus bacterial liquid and laccase into a Tris-HCl solution, adding alkali lignin to obtain a mixed solution, carrying out shake culture on the mixed solution, centrifuging, taking supernate, adding ethyl acetate and a hydrochloric acid solution, carrying out ultrasonic extraction, carrying out spin drying, and redissolving to obtain a phenolic acid compound; the volume ratio of the bacterial liquid to the laccase is 1: 0.5-2 (v/v). The content of the high-activity phenolic acid compound prepared by the method is higher, the preparation method has the advantages of high efficiency, low cost and environmental friendliness, and the Siamese bacillus liquid and the laccase are compounded according to a specific volume ratio, so that the content of the prepared phenolic acid compound is higher, and the phenolic acid compound also shows excellent antioxidant activity and antibacterial activity.)
1. A method for biosynthesizing phenolic acid compounds by utilizing lignin is characterized by comprising the following steps:
inoculating Siamese bacillus bacterial liquid and laccase into a Tris-HCl solution, adding alkali lignin to obtain a mixed solution, carrying out shake culture on the mixed solution, centrifuging, taking supernate, adding ethyl acetate and a hydrochloric acid solution, carrying out ultrasonic extraction, carrying out spin drying, and redissolving to obtain a phenolic acid compound;
the volume ratio of the bacterial liquid to the laccase is 1: 0.5-2 (v/v).
2. The method of claim 1, wherein said Siamese Bacillus is Siamese Bacillus (Bacillus simensis) L5, patent No. CN111040961A, deposited under accession numbers: CCTCC M2019885.
3. The method of claim 1, wherein the ratio of the bacteria fluid to the laccase is 1:0.5(v/v) by volume.
4. The method of claim 1, wherein the final concentration of the alkali lignin in the mixed liquor is 1000 mg/L.
5. The method according to claim 1, wherein the mixed solution is cultured under shaking at a temperature of 20 to 40 ℃ and a speed of 100 to 200r/min for 0.5 to 1 day.
6. The method of claim 2, wherein the cultivation method of the Siamese bacillus solution comprises the following steps: selecting a Siamese Bacillus (Bacillus siamensis) L5 single bacterium, allowing the single bacterium to fall into an LB liquid culture medium for pre-culture for 12-14 h to obtain a premixed solution, centrifuging the premixed solution for 2-5 min under the condition of 4000-5000 r/min, then discarding a supernatant, leaving the bacterium, washing with a Tris-HCl solution and carrying out heavy suspension to obtain a solution with the concentration of 105～106CFU/mL Siamese bacillus bacterial liquid.
7. The method of claim 6, wherein the LB liquid medium comprises: 10g of peptone, 5g of yeast extract and 10g of sodium chloride, and adding distilled water to a constant volume of 1L and pH 7.0.
8. The phenolic acid compound prepared by the method of any one of claims 1 to 7.
9. Use of phenolic acid compounds according to claim 8 in bacteriostatic products.
10. Use of phenolic acid compounds according to claim 8 in antioxidant products.
Technical Field
The invention relates to the technical field of biosynthesis, in particular to a method for biosynthesizing a phenolic acid compound by utilizing lignin and application thereof.
Background
Phenolic acid compounds, including p-coumaric acid, caffeic acid, ferulic acid, protocatechuic acid, gallic acid, ellagic acid, danshensu, salvianolic acid, rosmarinic acid and the like, have excellent biological activities of resisting oxidation, resisting inflammation, resisting bacteria, whitening and fading spots, reducing blood fat, resisting tumors, preventing and treating cardiovascular and cerebrovascular diseases and the like, and are widely applied to the fields of medicines, food health care, cosmetics, animal and plant nutrition and the like.
Lignin is the most abundant aromatic compound in nature, and can generate 6 × 10 of lignin every year14About t lignin is a renewable carbon resource with great potential. In industrial production in China, the annual yield of lignin exceeds 1100 million tons, and part of lignin is used for combustion energy supply, low-value chemical products and the like, a large amount of lignin is abandoned, and the effective utilization rate is less than 20%, so that huge waste of biomass resources and ecological environment pollution are caused. Because the lignin intrinsic structure has a structural unit similar to that of the phenolic acid compound, the preparation of the phenolic acid compound by taking lignin as a raw material is an effective way for realizing high-value utilization of lignin wastes, and has potential technical feasibility.
In the prior art, the chemical method is mostly adopted for preparing the phenolic acid compound by utilizing lignin, the lignin is depolymerized into the micromolecule phenolic compound by the methods of pyrolysis, gasification, hydrolysis, reduction, oxidation and the like, and the homogeneous metal salt catalyst and the organic solvent are adopted in the preparation process, so that a large amount of waste liquid, waste residue and the like are generated in the subsequent separation and extraction operation, the environmental pollution is caused, the sewage and wastewater treatment cost in the later production period is high, and the biosynthesis method is environment-friendly and economical. Therefore, it is urgently required to develop a method for biosynthesizing phenolic acid compounds from lignin.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for biologically synthesizing phenolic acid compounds by using lignin and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
in a first aspect, the present invention provides a method for biosynthesizing phenolic acid compounds from lignin, comprising the following steps:
inoculating Siamese bacillus bacterial liquid and laccase into a Tris-HCl solution, adding alkali lignin to obtain a mixed solution, carrying out shake culture on the mixed solution, centrifuging, taking supernate, adding ethyl acetate and a hydrochloric acid solution, carrying out ultrasonic extraction, carrying out spin drying, and redissolving to obtain a phenolic acid compound;
the volume ratio of the bacterial liquid to the laccase is 1: 0.5-2 (v/v).
In the technical scheme of the invention, the Siamese bacillus bacterial liquid and the laccase are inoculated into the Tris-HCl solution, and the alkali lignin is added to biologically synthesize the phenolic acid compound, so that a large amount of waste liquid, waste residue and the like cannot be generated in the preparation process, and the method is beneficial to environmental protection.
According to the invention, Siamese bacillus bacterial liquid and laccase are compounded in a specific volume ratio, lignin is converted into a phenolic acid compound, the content of the prepared phenolic acid compound (including high-activity phenolic acid compounds such as protocatechuic acid, gallic acid, vanillin, syringaldehyde, p-hydroxybenzoic acid, cinnamic acid, p-coumaric acid, caffeic acid and ferulic acid) is higher (10-30mg/g), and the phenolic acid compound also has excellent antioxidant activity and antibacterial activity.
As a preferred embodiment of the method for biosynthesizing a phenolic acid compound by using lignin, the siamese Bacillus adopts siamese Bacillus (Bacillus simensis) L5 with the patent number of CN111040961A, and the preservation numbers are as follows: CCTCC M2019885.
When the Siamese Bacillus adopts the Siamese Bacillus (Bacillus siamensis) L5, the Siamese Bacillus is compounded with laccase, so that the content of the phenolic acid compounds can be increased, and the phenolic acid compounds have better oxidation resistance and bacteriostatic performance.
In a preferred embodiment of the method for biosynthesizing phenolic acid compounds by using lignin according to the invention, the volume ratio of the bacterial liquid to the laccase is 1:0.5 (v/v).
When the bacterial liquid and the laccase are compounded in a volume ratio of 1:0.5(v/v), lignin is better converted into the phenolic acid compound, the content of the prepared phenolic acid compound can reach 25.6mg/g, the antioxidant and bacteriostatic performance of the phenolic acid compound is optimal, and IC (integrated circuit) is obtained50The value is 3.9mg/L, and the minimum inhibitory concentration of the phenolic acid compounds on pseudomonas aeruginosa, staphylococcus aureus and escherichia coli is 1.2500mg/mL, 2.5000mg/mL and 0.6250mg/mL respectively.
In a preferred embodiment of the method for biosynthesis of a phenolic acid compound using lignin according to the present invention, the final concentration of the alkali lignin in the mixed solution is 1000 mg/L.
As a preferred embodiment of the method for biologically synthesizing the phenolic acid compound by utilizing the lignin, the volume ratio of the Siamese bacillus bacterial solution to the Tris-HCl solution is 1: 1.
More preferably, the concentration of the Tris-HCl solution is 50mM, pH 7.5.
As a preferred embodiment of the method for biologically synthesizing the phenolic acid compound by using the lignin, the mixed solution is subjected to shaking culture for 0.5 to 1 day under the conditions that the temperature is 20 to 40 ℃ and the speed is 100 to 200 r/min.
More preferably, the mixture is cultured with shaking at a rate of 150r/min at a temperature of 30 ℃ for 1 day.
As a preferred embodiment of the method for biologically synthesizing a phenolic acid compound by using lignin according to the present invention, the method for culturing a bacillus siameses bacterial solution comprises the following steps: selecting Siam bacillus (Bacil)lus siemensis) L5 single bacteria fall into LB liquid culture medium for pre-culture for 12-14 h to obtain premixed solution, the premixed solution is centrifuged for 2-5 min under the condition of 4000-5000 r/min, then the supernatant is discarded, the remained bacteria are washed by Tris-HCl solution and re-suspended to obtain the concentration of 105～106CFU/mL Siamese bacillus bacterial liquid.
As a preferred embodiment of the method for biosynthesizing phenolic acid compounds by using lignin according to the present invention, the LB liquid medium comprises: 10g of peptone, 5g of yeast extract and 10g of sodium chloride, and adding distilled water to a constant volume of 1L and pH 7.0.
In a second aspect, the present invention provides a phenolic acid compound prepared by the above method.
From the effect evaluation experiment result, the method is adopted to synthesize the high-content phenolic acid compound by utilizing lignin biosynthesis, and the phenolic acid compound has better antioxidant activity and bacteriostatic activity.
In a third aspect, the invention provides an application of the phenolic acid compound in a bacteriostatic product.
In a fourth aspect, the invention provides an application of the phenolic acid compound in an antioxidant product.
Compared with the prior art, the invention has the following beneficial effects:
1) the invention provides a method for biologically synthesizing phenolic acid compounds by utilizing lignin, which has the advantages of simple preparation method, less raw materials, low cost, high efficiency and environmental protection;
2) according to the invention, Siamese Bacillus (Bacillus siamensis) L5 bacterial liquid and laccase are compounded according to a specific volume ratio, lignin is converted into phenolic acid compounds well, and the content of the prepared phenolic acid compounds (including high-activity phenolic acid compounds such as protocatechuic acid, gallic acid, vanillin, syringaldehyde, p-hydroxybenzoic acid, cinnamic acid, p-coumaric acid, caffeic acid and ferulic acid) is higher (10-30mg/g), and the phenolic acid compounds also show excellent antioxidant activity and antibacterial activity.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
In the following examples and comparative examples, the experimental methods used were conventional ones unless otherwise specified, and the materials, reagents and the like used were commercially available ones unless otherwise specified.
In the invention, the Siamese Bacillus adopts Siamese Bacillus (Bacillus siamensis) L5 with the patent number of CN111040961A, and the preservation numbers are as follows: CCTCC M2019885, the preservation address is Wuhan university in Wuhan city, Hubei province, and the preservation date is 11 months and 1 day in 2019.
In the following examples and comparative examples, laccase was purchased from Sigma-Aldrich, Inc. under the trade name SAE 0050; the LB liquid culture medium comprises the following components: 10g of peptone, 5g of yeast extract and 10g of sodium chloride, and adding distilled water to a constant volume of 1L and a pH value of 7.0; when LB solid culture medium is needed, 15g/L agar powder is needed to be added, and the agar is obtained after high-pressure steam sterilization. The concentration of the Tris-HCl solution was 50mM, pH 7.5.
Example 1
A method for biosynthesizing phenolic acid compounds by utilizing lignin comprises the following steps:
1) selecting Siam Bacillus (Bacillus simensis) L5 single bacteria, allowing the single bacteria to fall into an LB liquid culture medium for pre-culture for 12 hours to obtain a premixed solution, centrifuging the premixed solution for 2 minutes under the condition of 4000r/min, discarding the supernatant, leaving the bacteria, washing with a Tris-HCl solution and re-suspending to obtain Siam Bacillus L5 bacteria solution, wherein the concentration of the Siam Bacillus L5 in the bacteria solution is 105～106CFU/mL, more preferably, Siamese bacillus L5 in the bacterial liquid concentration of 106CFU/mL；
2) Inoculating 500uL of Siamese bacillus bacterial liquid and 250uL of laccase into a 250mL triangular flask of 50mL Tris-HCl solution, adding alkali lignin to obtain a mixed solution, enabling the final concentration of the alkali lignin in the mixed solution to be 1000mg/L, carrying out oscillation culture on the mixed solution for 1 day under the conditions that the temperature is 30 ℃ and the speed is 150r/min, centrifuging to obtain a supernatant, adding 50mL ethyl acetate and 2mL hydrochloric acid solution, carrying out ultrasonic extraction for 30min, carrying out rotary drying at 45 ℃, and carrying out redissolution by adopting methanol to obtain the phenolic acid compound.
Analyzing phenolic acid compounds by HPLC-UV to obtain protocatechuic acid, gallic acid, vanillin, syringaldehyde, p-hydroxybenzoic acid, cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid with total content of 25.6 mg/g.
Example 2
A method for biosynthesizing phenolic acid compounds by utilizing lignin comprises the following steps:
1) selecting Siam Bacillus (Bacillus simensis) L5 single bacteria, allowing the single bacteria to fall into an LB liquid culture medium for pre-culture for 12 hours to obtain a premixed solution, centrifuging the premixed solution for 2 minutes under the condition of 4000r/min, discarding the supernatant, leaving the bacteria, washing with a Tris-HCl solution and re-suspending to obtain Siam Bacillus L5 bacteria solution, wherein the concentration of the Siam Bacillus L5 in the bacteria solution is 105～106CFU/mL；
2) Inoculating 500uL of Siamese bacillus bacterial liquid and 500uL of laccase into a 250mL triangular flask of 50mL Tris-HCl solution, adding alkali lignin to obtain a mixed solution, enabling the final concentration of the alkali lignin in the mixed solution to be 1000mg/L, carrying out oscillation culture on the mixed solution for 1 day under the conditions that the temperature is 30 ℃ and the speed is 150r/min, centrifuging to obtain a supernatant, adding 50mL ethyl acetate and 2mL hydrochloric acid solution, carrying out ultrasonic extraction for 30min, carrying out rotary drying at 45 ℃, and carrying out redissolution by adopting methanol to obtain the phenolic acid compound.
Analyzing phenolic acid compounds by HPLC-UV to obtain protocatechuic acid, gallic acid, vanillin, syringaldehyde, p-hydroxybenzoic acid, cinnamic acid, p-coumaric acid, caffeic acid and ferulic acid, wherein the total content is 18.4 mg/g.
Example 3
A method for biosynthesizing phenolic acid compounds by utilizing lignin comprises the following steps:
1) selecting Siam Bacillus (Bacillus simensis) L5 single bacteria, allowing the single bacteria to fall into an LB liquid culture medium for pre-culture for 12 hours to obtain a premixed solution, centrifuging the premixed solution for 2 minutes under the condition of 4000r/min, discarding the supernatant, leaving the bacteria, washing with a Tris-HCl solution and re-suspending to obtain Siam Bacillus L5 bacteria solution, wherein the concentration of the Siam Bacillus L5 in the bacteria solution is 105～106CFU/mL；
2) Inoculating 500uL of Siamese bacillus bacterial liquid and 1mL of laccase into a 250mL triangular flask of 50mL Tris-HCl solution, adding alkali lignin to obtain a mixed solution, enabling the final concentration of the alkali lignin in the mixed solution to be 1000mg/L, carrying out oscillation culture on the mixed solution for 1 day under the conditions that the temperature is 30 ℃ and the speed is 150r/min, centrifuging to obtain a supernatant, adding 50mL ethyl acetate and 2mL hydrochloric acid solution, carrying out ultrasonic extraction for 30min, carrying out rotary drying at 45 ℃, and carrying out redissolving by adopting methanol to obtain the phenolic acid compound.
Analyzing the phenolic acid compounds by HPLC-UV to obtain protocatechuic acid, gallic acid, vanillin, syringaldehyde, p-hydroxybenzoic acid, cinnamic acid, p-coumaric acid, caffeic acid and ferulic acid with total content of 16.2 mg/g.
Example 4
A method for biosynthesizing phenolic acid compounds by utilizing lignin comprises the following steps:
1) selecting Siam Bacillus (Bacillus simensis) L5 single bacteria, allowing the single bacteria to fall into an LB liquid culture medium for pre-culture for 14h to obtain a premixed solution, centrifuging the premixed solution for 5min under the condition of 5000r/min, discarding supernatant, leaving the bacteria, washing with a Tris-HCl solution and re-suspending to obtain Siam Bacillus L5 bacterial solution, wherein the concentration of the Siam Bacillus L5 in the bacterial solution is 105～106CFU/mL；
2) Inoculating 500uL of Siamese bacillus bacterial liquid and 250uL of laccase into a 250mL triangular flask of 50mL Tris-HCl solution, adding alkali lignin to obtain a mixed solution, enabling the final concentration of the alkali lignin in the mixed solution to be 1000mg/L, carrying out oscillation culture on the mixed solution for 1 day under the conditions that the temperature is 40 ℃ and the speed is 200r/min, centrifuging to obtain a supernatant, adding 50mL ethyl acetate and 2mL hydrochloric acid solution, carrying out ultrasonic extraction for 30min, carrying out rotary drying at 45 ℃, and carrying out redissolution by adopting methanol to obtain the phenolic acid compound.
Analyzing phenolic acid compounds by HPLC-UV to obtain protocatechuic acid, gallic acid, vanillin, syringaldehyde, p-hydroxybenzoic acid, cinnamic acid, p-coumaric acid, caffeic acid and ferulic acid with total content of 23.5 mg/g.
Comparative example 1
Comparative example 1 compared to example 1 differs in that no Bacillus siamensis L5 and laccase were added, and the remaining parameters and procedure were the same as in example 1.
By HPLC-UV analysis, no substances such as protocatechuic acid, gallic acid, vanillin, syringaldehyde, p-hydroxybenzoic acid, cinnamic acid, p-coumaric acid, caffeic acid, ferulic acid, etc. are detected in the product.
Comparative example 2
Comparative example 2 differs from example 1 in that step 2): 500uL of Siamese bacillus bacterial liquid is inoculated into a 250mL triangular flask of 50mL Tris-HCl solution, and the rest parameters and steps are the same as those in the example 1.
The phenolic acid compounds are analyzed by HPLC-UV to contain p-hydroxybenzoic acid, cinnamic acid, p-coumaric acid, caffeic acid and ferulic acid, and the total content is 5.3 mg/g.
Comparative example 3
Comparative example 3 differs from example 1 in that step 2): 250uL of laccase was added to a 250mL Erlenmeyer flask containing 50mL of Tris-HCl solution, and the rest of the parameters and steps were the same as in example 1.
HPLC-UV analysis is adopted to analyze that the phenolic acid compounds contain vanillin and syringaldehyde, and the total content is 1.1 mg/g.
Test examples
1. HPLC-UV analysis method for composition of phenolic acid compounds
Accurately weighing 50mg of phenolic acid compound samples prepared in examples 1-4 and comparative examples 1-3, fixing the volume to 25mL by using chromatographic grade methanol, carrying out ultrasonic treatment for 10min to completely dissolve the phenolic acid compound, and quantitatively analyzing the content of the active ingredients of the phenolic acid compound by using HPLC-UV: the chromatographic column adopts C18 column, acetonitrile/0.1% phosphoric acid water solution is used as mobile phase, 272nm is used as detection wavelength, and the amount of each active component is calculated by standard curve method.
2. Antioxidant efficacy evaluation method
DPPH free radical scavenging: preparing 0.01,0.025,0.05 and 0.1mg/mL of methanol solution of a sample to be tested, sequentially transferring 3.0mL of the sample solution of the phenolic acid compound to be tested in the examples 1-4 and the comparative examples 1-3 into a 10mL colorimetric tube, respectively adding 3.0mL of 0.15mM DPPH-methanol solution, shaking up, carrying out a dark reaction at room temperature for 30min, and measuring the absorbance value A1 of the sample at 517 nm; under the same conditions, methanol was used instead of DPPH for the solution measurementBackground absorbance value a 2; the sample solvent was substituted for the sample to determine the blank value a 0. DPPH · clearance (%) (a0- (a1-a2))/a0 × 100%. Fitting a curve of DPPH & clearance rate and the concentration of the sample to be detected, and obtaining the IC according to the concentration of the sample to be detected when the DPPH & clearance rate is 50 percent50The value is obtained.
3. Antibacterial efficacy evaluation method
The Minimum Inhibitory Concentration (MIC) values of the phenolic acid compound inhibitory strains prepared in examples 1-4 and comparative examples 1-3 were determined by a two-fold dilution method, and the experimental strains were Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli. Adding the phenolic acid compound extract with different mass concentrations into LB culture medium, shaking to obtain culture medium with final concentrations of phenolic acid compound of 40.0000, 20.0000, 10.0000, 5.0000, 2.5000, 1.2500, 0.6250, 0.3125 mg/mL. Taking 0.20mL of 106-107And (3) culturing the CFU/mL bacterial solution in a culture medium at the constant temperature of 30 ℃ for 24 hours. LB culture medium without phenolic acid compound is used as blank control, and the minimum concentration of sample liquid without bacterial growth is used as MIC.
The results are shown in Table 1.
TABLE 1
From the data in table 1, it can be seen that, according to HPLC-UV analysis, the phenolic acid compounds prepared by the methods of examples 1 to 4 of the present invention contain protocatechuic acid, gallic acid, p-coumaric acid, vanillin, syringaldehyde, p-hydroxybenzoic acid, cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid, and the total content can be as high as 25.6 mg/g; the phenolic acid compound has better oxidation resistance and bacteriostasis performance, wherein the phenolic acid compound prepared by the method of the embodiment 1 is used as the best embodiment in the embodiment 1, and the IC of the phenolic acid compound is50The value is 3.9mg/L, and the phenolic acid compounds have minimum effect on pseudomonas aeruginosa, staphylococcus aureus and escherichia coliThe inhibitory concentrations were 1.2500mg/mL, 2.5000mg/mL, and 0.6250mg/mL, respectively.
Compared with the method in the example 1, in the method in the comparative example 1, no Siamese Bacillus (Bacillus siamensis) L5 and laccase are added, and no protocatechuic acid, gallic acid, vanillin, syringaldehyde, p-hydroxybenzoic acid, cinnamic acid, p-coumaric acid, caffeic acid and ferulic acid are detected in the product through HPLC-UV analysis, which indicates that no lignin is converted to generate phenolic acid compounds; comparative examples 2 or 3 in which only one of Bacillus siamensis L5 and laccase was added, the total content of phenolic acid compounds obtained was far inferior to that of examples 1 to 4, and IC was determined50The value and the Minimum Inhibitory Concentration (MIC) value of the strain are higher, which indicates that the antioxidant activity and the bacteriostatic activity of the phenolic acid compound are poorer, and indicates that the compounding of Bacillus siamensis L5 and laccase can better convert lignin into the phenolic acid compound, so that the content of the prepared phenolic acid compound (comprising high-activity phenolic acid compounds such as protocatechuic acid, gallic acid, vanillin, syringaldehyde, p-hydroxybenzoic acid, cinnamic acid, p-coumaric acid, caffeic acid and ferulic acid) is higher, and the antioxidant activity and the bacteriostatic activity of the phenolic acid compound are improved. When the volume ratio of the bacteria liquid to the laccase is out of the range of the invention, the total content of the phenolic acid compounds is reduced, and the oxidation resistance and the antibacterial performance are weakened.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.