CN112047828B - Method for preparing protocatechuic acid by biochemical method - Google Patents
Method for preparing protocatechuic acid by biochemical method Download PDFInfo
- Publication number
- CN112047828B CN112047828B CN202010921942.9A CN202010921942A CN112047828B CN 112047828 B CN112047828 B CN 112047828B CN 202010921942 A CN202010921942 A CN 202010921942A CN 112047828 B CN112047828 B CN 112047828B
- Authority
- CN
- China
- Prior art keywords
- treatment
- acid
- protocatechuic acid
- drying
- crystallization
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/347—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
- C07C51/43—Separation; Purification; Stabilisation; Use of additives by change of the physical state, e.g. crystallisation
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Crystallography & Structural Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention provides a method for preparing protocatechuic acid by a biochemical method, which adopts the biochemical method combining biological fermentation dehydroshikimic acid and alkali-heat conversion technology to prepare protocatechuic acid, can reduce the growth influence of the product on a host, and also avoids the defects of cost rise and difficult extraction caused by single enzyme catalysis; the condition is optimized, so that the conversion rate of protocatechuic acid prepared by the method is greatly improved, and the method is simple to operate, good in stability, low in cost and easy to realize industrial production.
Description
Technical Field
The invention belongs to the field of drug intermediates, and particularly relates to a method for preparing protocatechuic acid by a biochemical method.
Background
Protocatechuic acid (PCA), 3, 4-dihydroxybenzoic acid, molecular formula: (HO) 2C6H3COOH, dissolved in hot water, ethanol, diethyl ether, acetone, ethyl acetate, slightly soluble in cold water, insoluble in benzene, chloroform. Is present in leaves of Stenoloma chusanum (Thunb.) Ching of the family Lepidoptera, leaves of ilex purpurea of the family Aquifoliaceae, etc.
Protocatechuic acid has antibacterial and bacteriostatic effects, and has different degrees of bacteriostatic effects on pseudomonas aeruginosa, escherichia coli, typhoid bacillus, dysentery bacillus, alcaligenes bacillus, bacillus subtilis and staphylococcus aureus during in vitro tests [ huruizhi, congrujia, plum-bosch, etc. ] the physiological function of protocatechuic acid and its application in livestock and poultry production [ J ]. Animakogaku, 2019,031 (011): 4979-4986 ]. In addition, the compound also has the pharmacological effects of obviously resisting oxidation, reducing myocardial oxygen consumption, improving myocardial oxygen resistance, inhibiting the growth of tumor cells, inducing apoptosis of HL60 leukemia cells and HepG2 liver cancer cells, slowing down heart and the like. It can also dispel phlegm and relieve asthma, and is clinically used for treating chronic tracheitis.
Protocatechuic acid is mainly applied to the fields of medicine synthesis, organic intermediate synthesis, dye synthesis, chemical reagents and the like, and is a core raw material of various medical products (such as antineoplastic agents erlotinib, sodium channel loss valve inhibitor veratridine, respiratory system drug mepivaline hydrochloride, drug for treating hepatitis B, picroside II, novel gastrointestinal motility promoting drug namely itopride hydrochloride, anesthetic, and the like).
In the aspects of food and feed, protocatechuic acid serving as a feed additive is used for promoting the growth and development of animals, improving the feed conversion rate, improving the carcass quality and meat quality flavor, comprehensively enhancing the immune function and disease prevention capability of the animals, overcoming the toxic and side effects and food potential safety hazards of chemically synthesized organic acid, and avoiding the defects of complex components, inconvenient use, difficult quality standard control and the like of the Chinese herbal medicine additive in the livestock and poultry breeding.
At present, the sources of protocatechuic acid are mainly plant extraction and chemical synthesis.
Protocatechuic acid is present in leaves of Stenoloma chusana belonging to the family of Pteridaceae and ilex purpurea Hassk belonging to the family of Aquifoliaceae. In addition, fructus Schisandrae chinensis, eucommiae cortex, strobilus Pini and folium Syringae can also be used as the extraction source of protocatechuic acid. However, the plant extraction has the disadvantages of high cost and low efficiency, and the raw materials are limited by the production area, season and yield, so that the large-scale production application is difficult to form.
The chemical synthesis method of protocatechuic acid is characterized by that it uses vanillin as substrate, and makes it and sodium hydroxide and potassium hydroxide aqueous solution undergo the processes of high-temp. and pressure reaction to obtain the invented product, then on the basis of said product the related technological improvement reports are reported, such as reducing reaction temp. to 200 deg.C and adopting catalyst mode, etc. Although protocatechuic acid is produced by chemical synthesis through a mature process for many years and large-scale production, the purity of the protocatechuic acid is not high, trace impurities and isomers are uncontrollable, and the protocatechuic acid is applied to the aspects of medicines and food and feed and has safety risks. And the reaction conditions are severe, so that high potential safety hazards exist.
With the continuous popularization of the application of protocatechuic acid in the fields of medicine, feed and the like, the yield and the demand of protocatechuic acid are steadily increased, the chemical method and the plant extraction method are difficult to meet the requirements of the future market, and the process for producing protocatechuic acid by biological fermentation is promoted. The protocatechuic acid produced by the biological enzyme method has high purity, few other micro impurities, definite harmlessness, white chromaticity, good dispersibility, high safety when being used in the aspect of medicine.
Patent CN 107058409 reports a method of whole-cell catalysis of natural anthocyanins to convert protocatechuic acid with microorganisms, but the product concentration is only 33mg/L. In 2015, okai et al expressed glutamate-pyruvate lyase from E.coli using Corynebacterium to produce protocatechuic acid, and the strain was fermented for 96h, the protocatechuic acid was expressed in an amount of 1140.0. + -. 11.6mg/L, and the space-time yield was too low { Naoko, okai, takanori, et al.production of protocatholic acid by Corynebacterium glutamicum expressing chloride-pyruvate lyase from Escherichia coli [ J ]. Applied Microbiology & Biotechnology,2016.}. Patent CN109477066 provides a microorganism capable of efficiently producing protocatechuic acid or a salt thereof using a saccharide as a raw material, and a method for efficiently producing protocatechuic acid or a salt thereof using the microorganism. By strengthening the activity of 3-dehydroshikimate dehydratase, chorismate pyruvate lyase and 4-hydroxybenzoic acid hydroxylase, the protocatechuic acid is produced by using glucose, and the highest yield can reach 82.5g/L. However, this method has by-products such as 4-hydroxybenzoic acid and a branched acid, which causes great difficulty in separation and purification.
The Escherichia coli engineering bacteria for producing protocatechuic acid by using glucose are constructed by Tianjin engineering materials of Chinese academy of sciences, and are fermented for 37 hours, wherein the yield is 33.3g/L. However, the organization reports that Escherichia coli engineering bacteria for producing protocatechuic acid precursor 3-dehydroshikimic acid can be constructed, and then 3-dehydroshikimic acid (DHS) fermentation liquor is used for producing 79g/L protocatechuic acid under the action of a whole-cell biocatalyst. The yield of 3-dehydroshikimic acid in CN201711002831 is known to be about 94g/L, but no relevant transformation experimental data are disclosed, and a whole-cell biocatalyst is introduced, so that not only is additional fermentation required, but also difficulty is brought to extraction, and the cost and the extraction complexity are increased.
CN109266591 discloses a genetic engineering bacterium for producing protocatechuic acid (3, 4-dihydroxybenzoic acid) by using phenol as a raw material and a construction method thereof, wherein the genetic engineering bacterium contains p-hydroxybenzoic acid decarboxylase gene yclBCD and hydroxylase gene pobA, phenol is used as a raw material, phenol is added with carboxyl by a biotransformation method to obtain p-hydroxybenzoic acid, and then the 3-position of the p-hydroxybenzoic acid is oxidized to obtain protocatechuic acid. However, the yield was not reported, and the substrate was phenol, which had a large environmental impact.
Yanxiuqing and the like report methods for synthesizing protocatechuic acid by using p-hydroxybenzoic acid as a substrate and using hydroxylase, but the yield and the substrate concentration are low, and the industrial production { Yanxiuqing-protocatechuic acid biotransformation [ J ]. Microbiological report, 2019,46 (12): 3378-3387 } is difficult to satisfy.
In patent CN110184288, it is disclosed that 60 g/L3-dehydroshikimic acid is used as a substrate, 3-dehydroshikimic acid dehydratase catalyzes the preparation of protocatechuic acid, the substrate conversion rate is more than 98% after 5h of reaction, although the substrate concentration is higher, extra enzyme is required to catalyze, and the fermentation cost and the extraction difficulty are increased.
In summary, in the existing protocatechuic acid biosynthesis technology, the product has bacteriostatic activity, so that the fermentation biomass and yield are difficult to increase, the problems of low yield, more byproducts and the like exist, and exogenous genes or enzymes are required to be introduced for catalytic preparation, so that the production cost is increased, the difficulty is brought to subsequent extraction and separation, and the industrial development is restricted.
Disclosure of Invention
The present invention aims to solve at least to some extent at least one of the technical problems of the prior art.
Therefore, the invention provides a method for preparing protocatechuic acid by a biochemical method, which comprises the following steps:
s1: carrying out membrane filtration treatment on microbial fermentation liquor containing dehydroshikimic acid to obtain first treatment liquid;
s2: adjusting alkali, filtering and heating the first treatment liquid, and then adjusting acid to obtain a second treatment liquid;
s3: carrying out decoloring treatment and drying treatment on the second treatment liquid to obtain a third treatment substance;
s4: extracting the third treated substance by using an organic solvent to obtain a fourth treated liquid;
s5: concentrating and dissolving the fourth treatment liquid to obtain a fifth treatment liquid;
s6: and crystallizing and drying the fifth treatment liquid to obtain high-purity protocatechuic acid.
The inventor finds that the protocatechuic acid is prepared by adopting a biochemical method combining biological fermentation and dehydroshikimic acid with an alkali-thermal conversion process, so that the growth influence of the product on a host can be reduced, and the defects of high cost and difficult extraction caused by single enzyme catalysis are avoided; the condition is optimized, so that the conversion rate of protocatechuic acid prepared by the method is greatly improved, and the method is simple to operate, good in stability, low in cost and easy to realize industrial production.
According to an embodiment of the present invention, the method for the biochemical preparation of protocatechuic acid may further have the following additional features:
according to an embodiment of the present invention, in step S1, the membrane filtration treatment is performed using a ceramic membrane having a pore size of 0.1 μm and/or a nanofiltration membrane having a cut-off of 1000Da and/or 50-150 Da.
According to the embodiment of the invention, in step S2, adjusting the pH value of the first treatment solution to 6-12, filtering and collecting filtrate, treating the filtrate at 50-90 ℃ for 30-90 min, and then adjusting the pH value to 3-4 to obtain a second treatment solution;
optionally, the base used for adjusting the base is sodium hydroxide, and the acid used for adjusting the base is sulfuric acid.
According to the embodiment of the invention, in the step S3, the second treatment liquid and activated carbon are heated and stirred, filtered, and the filtrate is collected, and then is subjected to drying treatment, so as to obtain a third treated substance;
optionally, the addition amount of the activated carbon is 0.1 to 2 mass%;
optionally, the drying treatment is spray drying, the air inlet of the spray drying is set to be 150-200 ℃, preferably 170-180 ℃, and the air outlet is set to be 50-100 ℃, preferably 70-90 ℃.
According to an embodiment of the present invention, in step S4, the organic solvent is ethyl acetate or 95% ethanol;
optionally, the volume ratio of the organic solvent to the third treatment substance is (5-10): 1;
optionally, the extraction time is 1-2 h.
According to the embodiment of the present invention, in step S5, the fourth treatment solution is concentrated to no fraction, and water is added and heated to dissolve 100 to 150g/L, so as to obtain a fifth treatment solution.
According to an embodiment of the present invention, in step S6, the crystallization treatment includes room temperature crystallization, low temperature crystallization, and water washing treatment;
optionally, the low temperature crystallization temperature is carried out at 0 to 10 ℃;
optionally, the time for room-temperature crystallization is 12 hours, and the time for low-temperature crystallization is 24 hours;
optionally, the drying treatment is carried out at 40 to 80 ℃.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 shows the determination of the content of a dehydroshikimic acid fermentation broth;
FIG. 2 shows the conversion of DHS to PCA intermediate detection;
FIG. 3 shows the PCA product content measurements.
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature.
The present invention proposes a biochemical method for preparing protocatechuic acid, which will be described in detail below.
According to an embodiment of the invention, the method comprises:
s1: carrying out membrane filtration treatment on microbial fermentation liquor containing dehydroshikimic acid to obtain first treatment liquid;
s2: adjusting alkali, filtering and heating the first treatment liquid, and then adjusting acid to obtain a second treatment liquid;
s3: carrying out decoloring treatment and drying treatment on the second treatment liquid to obtain a third treatment substance;
s4: extracting the third treated substance by using an organic solvent to obtain a fourth treated liquid;
s5: concentrating and dissolving the fourth treatment liquid to obtain a fifth treatment liquid;
s6: and crystallizing and drying the fifth treatment liquid to obtain high-purity protocatechuic acid.
According to an embodiment of the present invention, in step S1 the microbial fermentation broth containing dehydroshikimic acid is passed through a ceramic membrane with a pore size of 0.1 μm, nanofiltration membranes with a cut-off of 1000Da and 50-150Da in order to remove microbial solids and large particle impurities in order to obtain a clarified first process liquor containing dehydroshikimic acid.
According to the embodiment of the invention, in the step S2, sodium hydroxide is added into the first treatment liquid to adjust the pH value to 6-12, the filtrate is filtered and collected, the filtrate is treated at 50-90 ℃ for 30-90 min, and then the pH value is adjusted to 3-4 by using sulfuric acid, so that most of dehydroshikimic acid is stably converted into protocatechuic acid, and the reverse reaction is prevented.
According to an embodiment of the present invention, in step S3, activated carbon is added to the second treatment solution, heated and stirred, and the filtrate is collected by filtration, followed by spray drying to remove the discoloration of the treatment solution, so as to obtain a dried third treatment product containing protocatechuic acid;
according to the embodiment of the present invention, the amount of the activated carbon added is 0.1 to 2% by mass.
According to the embodiment of the invention, the air inlet is set to be 150-200 ℃, preferably 170-180 ℃, and the air outlet is set to be 50-100 ℃, preferably 70-90 ℃.
According to an embodiment of the present invention, in step S4, the third treatment substance is extracted using an organic solvent under stirring and refluxing. Thereby, protocatechuic acid is extracted from the third treated material to obtain a fourth treated liquid;
according to the embodiment of the invention, the organic solvent is ethyl acetate or 95% ethanol, and because some impurities such as dehydroshikimic acid and protocatechuic acid have different solubility in ethyl acetate or 95% ethanol, protocatechuic acid is effectively separated;
according to the embodiment of the invention, the volume ratio of the organic solvent to the third treatment substance is (5-10): 1.
According to an embodiment of the present invention, in step S5, the fourth treated liquid is concentrated until no fraction is produced, and is dissolved in water under heating to a concentration of about 100 to 150g/L. Thereby, a fifth treatment liquid is obtained;
according to an embodiment of the present invention, in step S6, the fifth treatment liquid is subjected to crystallization and drying treatment to obtain high-purity protocatechuic acid;
according to an embodiment of the present invention, the crystallization treatment includes room temperature crystallization and low temperature crystallization, the low temperature crystallization is performed at 0 to 10 ℃; protocatechuic acid can be well crystallized and separated out in water, so that protocatechuic acid is effectively separated out, the ethyl acetate effect is not as good as that of water, other impurities can be separated out in ethyl acetate, and the product purity is low;
according to the embodiment of the invention, the room temperature crystallization time is 12h, and the low temperature crystallization time is 24h;
according to an embodiment of the invention, the drying treatment is carried out at 40 to 80 ℃.
EXAMPLE 1 fermentation broth pretreatment
Carrying out biological fermentation on Escherichia coli engineering bacteria for producing dehydroshikimic acid (DHS) by using glucose to obtain 32kg of dehydroshikimic acid fermentation liquor (shown in figure 1), and carrying out pretreatment on the fermentation liquor by using a 0.1 mu m ceramic membrane and 1000Da and 50-150Da roll-type membranes to obtain 30L of clarified dehydroshikimic acid filtrate (the concentration of the dehydroshikimic acid is 80.11 g/L);
example 2 Condition examination of the alkaline thermal conversion
In experiments, it is found that only alkali-regulated dehydroshikimic acid can be partially converted into protocatechuic acid (as shown in figure 2), but reverse reaction occurs during acid regulation and decoloration, so that heating treatment is needed to solve the problem and achieve the purpose of stabilizing the product. The optimum treatment was determined by examining the effect of different pH, temperature and heating time on the conversion using the dehydroshikimic acid filtrate of example 1.
The reaction system is 500mL, the stirring speed is 350rpm, the pH value is adjusted by sodium hydroxide, the water bath is used for heating, and the reaction results are shown in the following table 1. As can be seen from the results in the table, at 80 ℃, pH11 and heating for more than 60min, a conversion of more than 95% of 80g/L or even higher substrate concentration can be achieved. Preferred conditions are 80 ℃, pH11 and heating for 90min.
TABLE 1 examination of temperature, pH, heating time and concentration
EXAMPLE 3 preparation of protocatechuic acid by alkaline thermal conversion
Using 10L of dehydroshikimic acid filtrate from example 1, adjust the pH to 11 with sodium hydroxide, filter to remove the precipitate, stir and heat at 80 ℃ and 350rpm for 90min, and adjust the pH to 3.5 with sulfuric acid. The conversion of dehydroshikimic acid to protocatechuic acid was 97.48%.
EXAMPLE 4 preparation of spray-dried powder
In the feed liquid of example 3, 100g of acidic activated carbon powder was added, heated and stirred at 50 ℃ for 60min to perform decolorization treatment, and the filtrate was collected by filtration through a positive pressure filter. And (3) carrying out spray drying operation on the feed liquid on a kilogram-level spray dryer, controlling the air inlet temperature to be 170-180 ℃ and the air outlet temperature to be 70-90 ℃, collecting dry powder in a drying cavity and a receiver, wherein the content of protocatechuic acid is 35.19%, and the single-step yield is about 92%.
EXAMPLE 5 Ethyl acetate extraction of protocatechuic acid
Protocatechuic acid has high solubility in ethyl acetate, and the solubility can reach 100g/L under the room temperature condition and is higher under the heating condition through experimental determination, so that the adoption of ethyl acetate for extracting protocatechuic acid is considered.
Group A: extracting 200g of spray-dried powder in example 4 by stirring and refluxing 6 times (v/w) of ethyl acetate under heating at 50 ℃ for 1h;
group B: extracting 200g of spray-dried powder in example 4 with 6 times (v/w) of ethyl acetate under stirring and refluxing at 50 ℃ for 2h;
group C: extracting 200g of spray-dried powder in example 4 with 6 times (v/w) of ethyl acetate under stirring and refluxing at 50 deg.C for 2h, and adding 2 times (v/w) of ethyl acetate to repeat extraction for 1h;
group D: extracting 200g of spray-dried powder in example 4 with 8 times (v/w) of ethyl acetate under stirring and refluxing at 50 ℃ for 3h;
group E: extracting 200g of spray-dried powder in example 4 by stirring and refluxing with 10 times (v/w) of ethyl acetate under heating at 50 ℃ for 2h;
and F group: extracting 200g of spray-dried powder in example 4 with 8 times (v/w) of 95% ethanol under stirring and refluxing at 50 deg.C for 2h;
the experimental results are shown in Table 2, and it can be seen that the effect is best when 8 times (v/w) of ethyl acetate is used and the extraction is performed in two times. Therefore, the preferable condition is 8 times (v/w) of ethyl acetate, and the extraction is divided into 3 times of extraction, the heating temperature is 50 ℃, and the total extraction time is 3 hours.
TABLE 2 extraction yield
| Grouping | Yield of |
| A | 81.51％ |
| B | 85.44％ |
| C | 96.27％ |
| D | 88.78％ |
| E | 97.35％ |
| F | 92.31％ |
EXAMPLE 6 aqueous crystallization of protocatechuic acid
500g of the spray-dried powder obtained in example 4 was extracted with 6 times (v/w) of ethyl acetate under stirring and refluxing at 50 ℃ for 2 hours, then 2 times (v/w) of ethyl acetate was added thereto and the extraction was repeated for 1 hour, the extract was concentrated under reduced pressure until no fraction was obtained, 1.2L of purified water was added thereto and dissolved by heating at 80 ℃. Stirring at room temperature for crystallization for 12h, stirring at 5 deg.C for crystallization for 24h, filtering the crystals, adding a small amount of precooled purified water to wash the crystals, and vacuum drying at 50 deg.C to obtain the product with crystallization yield of 85.77% and product content of more than 98%.
EXAMPLE 7 crystallization of protocatechuic acid ethyl acetate phase
200g of the spray-dried powder of example 4 was extracted with 6 times (v/w) of ethyl acetate under stirring and refluxing at 50 ℃ for 2 hours, the extraction was repeated for 1 hour with 2 times (v/w) of ethyl acetate, and the extract was concentrated under reduced pressure to about 200g/L. Stirring for crystallization at room temperature for 12h, stirring for crystallization at 5 ℃ for 24h, filtering the crystals, adding a small amount of pre-cooled ethyl acetate to wash the crystals, and performing vacuum drying at 50 ℃ to obtain the product with the crystallization yield of 63.26% and the product content of 85.32%.
EXAMPLE 8 preparation of protocatechuic acid
Taking 10kg of dehydroshikimic acid fermentation liquor, carrying out 0.1 mu m ceramic membrane, 1000Da and 50-150Da roll type membrane pretreatment to obtain clear dehydroshikimic acid filtrate with the concentration of 80.31g/L, adjusting the pH value to 11 by sodium hydroxide, filtering to remove precipitates, stirring and heating at the temperature of 80 ℃ and 350rpm for 90min, adjusting the pH value to 3.5 by sulfuric acid, adding 100g of acidic activated carbon powder, heating and stirring at the temperature of 50 ℃ for 60min, carrying out decoloration treatment, filtering by a positive pressure filter, and collecting filtrate. And (3) carrying out spray drying operation on the feed liquid on a kilogram-level spray dryer, controlling the air inlet temperature to be 175 ℃, controlling the air outlet temperature to be 80 ℃, and collecting dry powder in a drying cavity and a receiver, wherein the protocatechuic acid content is 35.65%.
Extracting spray-dried powder with 6 times (v/w) of ethyl acetate under stirring and refluxing at 50 deg.C for 2 hr, adding 2 times (v/w) of ethyl acetate, extracting for 1 hr, concentrating the extractive solution under reduced pressure until no fraction is obtained, adding 4.5L of purified water, and dissolving at 80 deg.C. Stirring at room temperature for crystallization for 12h, stirring at 5 deg.C for crystallization for 24h, filtering the crystals, adding a small amount of pre-cooled purified water to wash the crystals, vacuum drying at 50 deg.C to obtain PCA solid 550.48g, with total yield of 67.37% and product content greater than 98%, as shown in figure 3.
TABLE 3 product testing conditions
| Content (wt.) | 98.75％ | Loss on drying | <1.0％ | Appearance of the product | White-like powder |
| Single hetero compound | <0.5％ | Residue on ignition | <0.5％ | 20g/L conductance | 1025μS/cm |
| General miscellaneous | <2.0％ | Solvent residue | <0.05％ | Melting Point | 198.5℃ |
Comparative example 1 ethanol extraction of protocatechuic acid spray-dried powder
Taking 1kg of dehydroshikimic acid membrane treatment solution, stirring and heating at 100 deg.C and 350rpm for 60min, adjusting pH to 3.5 with sulfuric acid, adding 100g of acidic activated carbon powder, heating and stirring at 50 deg.C for 60min, decolorizing, filtering, and collecting filtrate. And (3) carrying out spray drying operation on the feed liquid on a spray dryer, controlling the air inlet temperature to be 180 ℃ and the air outlet temperature to be 80 ℃, and collecting dry powder in the drying cavity and the receiver.
Extracting spray-dried powder with 6 times (v/w) of ethyl acetate under stirring and refluxing at 50 deg.C for 2 hr, adding 2 times (v/w) of ethyl acetate, extracting for 1 hr, concentrating the extractive solution under reduced pressure until no fraction is obtained, adding 0.5L of purified water, and dissolving at 80 deg.C. Stirring at room temperature for crystallization for 12h, stirring at 5 deg.C for crystallization for 24h, filtering the crystals, adding a small amount of pre-cooled purified water to wash the crystals, vacuum drying at 50 deg.C to obtain protocatechuic acid solid 43.26g, with total yield of 27.04%, product content less than 50%, and large amount of gallic acid.
Comparative example 2 717 preparation of protocatechuic acid by anion exchange resin
Taking 1kg of dehydroshikimic acid membrane treatment solution, adjusting the pH value to 11 with sodium hydroxide, filtering to remove precipitate, stirring and heating at 80 ℃ and 350rpm for 90min, adjusting the pH value to 3.5 with sulfuric acid, adding 100g of acidic activated carbon powder, heating and stirring at 50 ℃ for 60min, carrying out decolorization treatment, filtering, and collecting filtrate. Adjusting pH to 6.0, loading 717 type resin (OH type) at flow rate of 1.5 times column volume, maximum adsorption capacity of 60g/L, water washing, and eluting with 90% ethanol to obtain an elution rate of about 40%.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (3)
1. A method for preparing protocatechuic acid by a biochemical method is characterized by comprising the following steps:
s1: carrying out membrane filtration treatment on microbial fermentation liquor containing dehydroshikimic acid to obtain first treatment liquid;
s2: adjusting alkali, filtering and heating the first treatment liquid, and then adjusting acid to obtain a second treatment liquid;
s3: carrying out decoloring treatment and drying treatment on the second treatment liquid to obtain a third treatment substance;
s4: extracting the third treated substance by using an organic solvent to obtain a fourth treated liquid;
s5: concentrating and dissolving the fourth treatment liquid to obtain a fifth treatment liquid;
s6: crystallizing and drying the fifth treatment liquid to obtain high-purity protocatechuic acid;
wherein in the step S1, the membrane filtration treatment is carried out by adopting at least one of a ceramic membrane with the aperture of 0.1 μm and a nanofiltration membrane with the interception amount of 1000Da and 50-150 Da;
in the step S2, adjusting the pH value of the first treatment solution to be 11, filtering and collecting filtrate, treating the filtrate at 80 ℃ for 90min, and then adjusting the pH value to be 3 to 4 to obtain a second treatment solution;
in the step S4, the organic solvent is ethyl acetate; and the volume ratio of the organic solvent to the third treatment substance is (5-10) to 1;
in the step S6, the crystallization treatment comprises room-temperature aqueous phase crystallization, low-temperature crystallization and water washing treatment, wherein the low-temperature crystallization temperature is 0 to 10 ℃, and the drying treatment is carried out at 40 to 80 ℃.
2. The method according to claim 1, wherein in step S3, the second treatment solution and activated carbon are heated and stirred, and the filtrate is filtered and collected, and then is subjected to a drying treatment to obtain a third treatment substance;
the drying treatment is spray drying;
the amount of the activated carbon added is 0.1 to 2 mass%.
3. The method according to claim 1, wherein in step S5, the fourth treatment solution is concentrated to no distillate, and is dissolved in water to 100 to 150g/L to obtain a fifth treatment solution;
the dissolution is carried out at 50 to 90 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010921942.9A CN112047828B (en) | 2020-09-04 | 2020-09-04 | Method for preparing protocatechuic acid by biochemical method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010921942.9A CN112047828B (en) | 2020-09-04 | 2020-09-04 | Method for preparing protocatechuic acid by biochemical method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112047828A CN112047828A (en) | 2020-12-08 |
| CN112047828B true CN112047828B (en) | 2023-02-07 |
Family
ID=73607329
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010921942.9A Active CN112047828B (en) | 2020-09-04 | 2020-09-04 | Method for preparing protocatechuic acid by biochemical method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112047828B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113981014A (en) * | 2021-08-30 | 2022-01-28 | 黄山科宏生物香料股份有限公司 | Method for producing protocatechuic acid |
| CN116327832B (en) * | 2023-03-21 | 2024-03-01 | 陕西嘉禾药业有限公司 | Industrial Li Ziduo phenol preparation method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001026567A (en) * | 1999-05-07 | 2001-01-30 | Toray Ind Inc | Purification of shikimic acid |
| CN102010338A (en) * | 2010-11-03 | 2011-04-13 | 中科院广州化学有限公司 | Method for preparing epicatechol gallate and protocatechuic acid ester compounds from natural shikimic acid |
| CN107266308A (en) * | 2017-07-20 | 2017-10-20 | 广西南宁桂知科技有限公司 | The technique that a kind of utilization longan pericarp prepares protocatechuic acid |
| JP2018150288A (en) * | 2017-03-15 | 2018-09-27 | 協和発酵バイオ株式会社 | Crystal of protocatechuic acid cation salt and manufacturing method therefor |
| CN111087296A (en) * | 2019-12-20 | 2020-05-01 | 东莞市东阳光生物合成药有限公司 | Method for extracting shikimic acid and shikimic acid extract |
-
2020
- 2020-09-04 CN CN202010921942.9A patent/CN112047828B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001026567A (en) * | 1999-05-07 | 2001-01-30 | Toray Ind Inc | Purification of shikimic acid |
| CN102010338A (en) * | 2010-11-03 | 2011-04-13 | 中科院广州化学有限公司 | Method for preparing epicatechol gallate and protocatechuic acid ester compounds from natural shikimic acid |
| JP2018150288A (en) * | 2017-03-15 | 2018-09-27 | 協和発酵バイオ株式会社 | Crystal of protocatechuic acid cation salt and manufacturing method therefor |
| CN107266308A (en) * | 2017-07-20 | 2017-10-20 | 广西南宁桂知科技有限公司 | The technique that a kind of utilization longan pericarp prepares protocatechuic acid |
| CN111087296A (en) * | 2019-12-20 | 2020-05-01 | 东莞市东阳光生物合成药有限公司 | Method for extracting shikimic acid and shikimic acid extract |
Non-Patent Citations (2)
| Title |
|---|
| 3-脱氢莽草酸生物合成的代谢工程;毛赟赟等;《中国生物工程杂志》;20071215;第27卷(第12期);第109-113页 * |
| Benzene-Free Synthesis of Catechol: Interfacing Microbial;Wensheng Li et al.;《J. AM. CHEM. SOC》;20051231;第27卷(第9期);第2874-2882页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112047828A (en) | 2020-12-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112047828B (en) | Method for preparing protocatechuic acid by biochemical method | |
| CN109207531B (en) | Biological preparation method of thiamphenicol and florfenicol key intermediate | |
| CN107418995B (en) | A kind of ellagic acid and preparation method thereof of granatanine liquid state fermentation preparation | |
| CN107083406A (en) | (R) production method of 3 hydroxybutyric acids | |
| CN111662942A (en) | Method for producing rebaudioside-A through double-enzyme fermentation catalysis | |
| CN110791538A (en) | Production method suitable for synthesizing sitagliptin phosphate by enzyme method | |
| CN109180674A (en) | Pyrroloquinoline quinone disodium salt isolation and purification method based on fermentation liquid compound salt analysis | |
| CN111187328B (en) | Method for preparing mogrol | |
| CN113005161A (en) | Preparation method of polysialic acid and polysialic acid product | |
| CN109020854B (en) | Method for extracting L-methionine from fermentation liquor | |
| CN116283553A (en) | Method for extracting 3, 4-dihydroxybenzoic acid from fermentation broth | |
| CN108047121B (en) | Production process of adrenal color hydrazone | |
| CN113045610B (en) | Method for extracting glucosamine from N-acetylglucosamine fermentation liquor | |
| CN110563782B (en) | Gentamicin C 1a Purification method of (2) | |
| CN109796333B (en) | Method for extracting and purifying pravastatin sodium from fermentation liquor | |
| CN108558799B (en) | Method for extracting 2, 5-furandicarboxylic acid from fermentation liquor | |
| CN112430634A (en) | Process for preparing L-tryptophan by fermentation method | |
| CN113929615B (en) | Method for purifying nojirimycin | |
| WO2020056714A1 (en) | Method for preparing high-purity riboflavin sodium phosphate | |
| CN112359028B (en) | Biosynthesis method of tomoxetine intermediate and carbonyl reductase | |
| CN116496330B (en) | Sialic acid extraction method and sialic acid extracted by same | |
| CN110937993B (en) | Method for separating and refining gallic acid by using macroporous resin | |
| CN115448829A (en) | Preparation method and application of (R) -3-hydroxybutyric acid with high optical purity | |
| CN113444103A (en) | Method for purifying dehydrated morronigenin from dogwood | |
| CN114410504A (en) | Method for extracting theanine by fermentation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |