CN113604514B - Method for biosynthesizing phenolic acid compound by using lignin and application thereof - Google Patents
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Abstract
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, drying, and redissolving to obtain a phenolic acid compound; the volume ratio of the bacterial liquid to the laccase is 1.5-2 (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 bacterial 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 has excellent antioxidant activity and bacteriostatic activity.
Description
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 year 14 About 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, a chemical method is mostly adopted for preparing phenolic acid compounds by utilizing lignin, the lignin is depolymerized into micromolecular phenolic compounds by methods such as pyrolysis, gasification, hydrolysis, reduction and oxidation, a homogeneous metal salt catalyst and an organic solvent are adopted in the preparation process, so that a large amount of waste liquid, waste residues 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 realize 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, which 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 1.5-2 (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 preparation method, siamese bacillus bacterial liquid and laccase are compounded according to a specific volume ratio, lignin is converted into a phenolic acid compound better, 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 (10-30 mg/g), and the phenolic acid compound also has excellent antioxidant activity and antibacterial activity.
As a preferred embodiment of the method for biosynthesizing the phenolic acid compound by utilizing the lignin, the Siamese Bacillus adopts Siamese Bacillus (Bacillus size) L5 with the patent number of CN111040961A, and the preservation number is 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 a phenolic acid compound from lignin according to the present invention, the volume ratio of the bacterial suspension to the laccase is 1.
When the bacterial liquid and the laccase are compounded in a volume ratio of 1.5 (v/v) 50 The 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 1000mg/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.
More preferably, the concentration of the Tris-HCl solution is 50mM, pH 7.5.
As a preferred embodiment of the method for biosynthesis of a phenolic acid compound using lignin according to the present invention, the mixed solution is cultured under shaking at a rate of 100 to 200r/min at a temperature of 20 to 40 ℃ for 0.5 to 1 day.
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 biosynthesizing a phenolic acid compound by using lignin, the method for culturing a bacillus siamensis bacterial solution comprises the following steps: selecting Siamese Bacillus (Bacillus siamensis) L5 single bacteria, falling the Siamese Bacillus L5 single bacteria into LB liquid culture medium for pre-culturing for 12-14 h to obtain premixed liquid, centrifuging the premixed liquid for 2-5 min under the condition of 4000-5000 r/min, then discarding supernatant, leaving the bacteria, washing with Tris-HCl solution and re-suspending to obtain the Siamese Bacillus L5 single bacteria with the concentration of 10 5 ～10 6 CFU/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, adding distilled water to a constant volume of 1L, and keeping the pH value to 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 phenolic acid compound with high content 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 (10-30 mg/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 simensis) L5 with the patent number of CN111040961A, and the preservation number is 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 SAE0050; the LB liquid culture medium comprises the following components: 10g of peptone, 5g of yeast extract and 10g of sodium chloride, adding distilled water to a constant volume of 1L, and keeping the pH value to 7.0; when an LB solid culture medium is required to be prepared, 15g/L agar powder is also required 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 Siamese Bacillus (Bacillus siamensis) L5 single bacteria, allowing the Siamese Bacillus L5 single bacteria to fall into an LB liquid culture medium for pre-culture for 12 hours to obtain premixed liquid, centrifuging the premixed liquid for 2 minutes under the condition of 4000r/min, then discarding supernatant, leaving the bacteria, washing with Tris-HCl solution and re-suspending to obtain Siamese Bacillus L5 bacteria liquid, wherein the concentration of the Siamese Bacillus L5 in the bacteria liquid is 10 5 ～10 6 CFU/mL, more preferably Siamese bacillus L5 in the bacterial liquid concentration is 10 6 CFU/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 25.6mg/g.
Example 2
A method for biosynthesizing phenolic acid compounds by utilizing lignin comprises the following steps:
1) Selecting Bacillus siamensis (Bacillus siamenn)sis) L5 single bacteria fall into LB liquid culture medium for pre-culture for 12h to obtain premixed liquid, centrifuging the premixed liquid for 2min under the condition of 4000r/min, then removing supernatant, leaving bacteria, washing with Tris-HCl solution and re-suspending to obtain Siamese bacillus L5 bacteria liquid, wherein the concentration of Siamese bacillus L5 in the bacteria liquid is 10 5 ～10 6 CFU/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 18.4mg/g.
Example 3
A method for biosynthesizing phenolic acid compounds by utilizing lignin comprises the following steps:
1) Selecting Siamese 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 premixed solution, centrifuging the premixed solution for 2 minutes under the condition of 4000r/min, then discarding supernatant, leaving the bacteria, washing with Tris-HCl solution and re-suspending to obtain Siamese Bacillus L5 bacteria solution, wherein the concentration of Siamese Bacillus L5 in the bacteria solution is 10 5 ～10 6 CFU/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, wherein the total content is 16.2mg/g.
Example 4
A method for biosynthesizing phenolic acid compounds by utilizing lignin comprises the following steps:
1) Selecting Siamese Bacillus (Bacillus simensis) L5 single bacteria, allowing the single bacteria to fall into an LB liquid culture medium for pre-culturing for 14h to obtain premixed solution, centrifuging the premixed solution for 5min under the condition of 5000r/min, discarding supernatant, leaving the bacteria, washing with Tris-HCl solution and re-suspending to obtain Siamese Bacillus L5 bacteria solution, wherein the concentration of Siamese Bacillus L5 in the bacteria solution is 10 5 ～10 6 CFU/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 23.5mg/g.
Comparative example 1
The comparative example 1 is different from example 1 in that Bacillus siamensis L5 and laccase are not added, and the rest parameters and steps are the same as those of 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 solution was inoculated into a 250mL triangular flask containing 50mL Tris-HCl solution, and the remaining parameters and steps were the same as in 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 5.3mg/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 1.1mg/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 a 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 the 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 uniformly, 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 replaces DPPH solution to determine the background absorbance value A2; the sample solvent was substituted for the sample to determine the blank value A0.DPPH · clearance (%) = (A0- (A1-A2))/A0 × 100%. Fitting curve of DPPH clearance and concentration of sample to be measured, obtaining IC by the concentration of sample to be measured when DPPH clearance is 50% 50 The value is obtained.
3. Method for evaluating bacteriostatic efficacy
The two-fold dilution method was used to determine the maximum inhibitory effect of the phenolic acid compounds prepared in examples 1-4 and comparative examples 1-3 on the bacterial strainSmall inhibitory concentration (MIC) value, and the experimental strains are 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 10 6 -10 7 And (4) 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 25.6mg/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 is 50 The 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.
Compared with the method in the example 1, in the method in the comparative example 1, no Bacillus siamensis (Bacillus siemensis) 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 addition of onlyWhen one of Siamese Bacillus (Bacillus siemensis) L5 and laccase is added, the total content of the obtained phenolic acid compounds is far worse than that of the phenolic acid compounds in examples 1-4, and IC is 50 The 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.
Claims (7)
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, drying, and redissolving to obtain a phenolic acid compound;
the volume ratio of the bacterial liquid to the laccase is 1: 0.5-2;
the Siamese Bacillus adopts Siamese Bacillus (Bacillus simensis) L5 with the patent number of CN111040961A, and the preservation number is as follows: CCTCC M2019885;
the cultivation method of Siamese bacillus bacterial liquid comprises the following steps: picking bacillus siamensis (Bac)illus siamensis) L5 single bacteria fall into LB liquid culture medium for pre-culture for 12-14 h to obtain premixed liquid, the premixed liquid is centrifuged for 2-5 min under the condition of 4000-5000 r/min, then supernatant is discarded, remained bacteria are washed by Tris-HCl solution and re-suspended to obtain the bacteria with the concentration of 10 5 ～10 6 CFU/mL Siamese bacillus bacterial liquid.
2. The method according to claim 1, wherein the volume ratio of the bacteria solution to the laccase is 1.
3. The method of claim 1, wherein the final concentration of alkali lignin in the mixed liquor is 1000mg/L.
4. The method of claim 1, wherein the mixed solution is cultured under shaking at a temperature of 20 to 40 ℃ and a rate of 100 to 200r/min for 0.5 to 1 day.
5. The method of claim 1, wherein the LB liquid medium comprises: 10g of peptone, 5g of yeast extract and 10g of sodium chloride, adding distilled water to a constant volume of 1L, and keeping the pH value to 7.0.
6. Use of phenolic acid compounds prepared according to any one of claims 1 to 5 for the preparation of bacteriostatic products.
7. Use of phenolic acid compounds prepared by the process according to any one of claims 1 to 5 for the preparation of antioxidant products.
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Citations (5)
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| CN104894079A (en) * | 2015-05-27 | 2015-09-09 | 华中科技大学 | Lignin degradation solution and preparation method thereof as well as method for degrading lignin by using lignin degradation solution |
| CN105255842A (en) * | 2015-11-18 | 2016-01-20 | 西南林业大学 | Laccase for degrading lignin |
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