Gallic acid produced by enzyme method and preparation process thereof
By modifying konjac glucomanan and Bacillus stearophilus spores, the process of producing gallic acid by enzymatic method is optimized, and the problems of insufficient purity, yield and chemical stability in the prior art are solved, and the production of gallic acid with high purity, high yield and high stability is achieved.
Patent Information
- Application Number
- CN202510034090.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-09
- Publication Date
- 2025-03-18
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The existing enzymatic process for producing gallic acid has shortcomings in terms of purity, yield and chemical stability, and it is difficult to meet the requirements of high-end applications.
By modifying konjac glucomanan and Bacillus stearophilus spores, nanoparticles and fixed tannins carriers are formed, the reaction conditions and microstructure are optimized, and the stability and reaction efficiency of the enzyme are improved.
It significantly improves the purity, yield and chemical stability of gallic acid, reduces production costs, enhances the competitiveness of the product in the market, and extends the shelf life of the product.
Smart Images
Figure BDA0005235038100000142 
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of gallic acid, in particular to an enzymatic method for producing gallic acid and a preparation process thereof. Background Art
[0002] Gallic acid is an important organic compound that is widely used in many fields such as food, medicine, and chemical industry. In recent years, with the rapid development of these industries, the quality and performance requirements of gallic acid have been increasing. In practical applications, the purity, yield, and chemical stability of gallic acid directly affect the quality and use effect of related products.
[0003] High-purity gallic acid can ensure the stability and consistency of product quality and reduce the adverse effects of impurities on product performance; higher yield can reduce production costs, improve production efficiency, and enhance product competitiveness in the market; and good chemical stability ensures that gallic acid is not prone to deterioration during storage and use, extending the shelf life of the product and ensuring product quality and safety.
[0004] At present, the traditional enzymatic production of gallic acid has certain limitations in terms of purity, yield and chemical stability. The existing production methods are difficult to accurately control the reaction process, resulting in the difficulty of achieving the ideal purity of gallic acid, and the presence of impurities may affect its application in high-end fields. At the same time, the low yield makes the production cost high, limiting large-scale production and application. Moreover, under common storage and use conditions, the chemical stability of gallic acid is insufficient, and it is prone to oxidation, decomposition and other reactions, resulting in quality degradation.
[0005] Improving the purity, yield and chemical stability of gallic acid has become a key issue that needs to be solved urgently. Although some existing improvement measures have improved to a certain extent, there are still many shortcomings. For example, when some methods improve the purity, the yield is affected; or when the yield is improved, the chemical stability is difficult to ensure. Therefore, how to enhance the chemical stability of gallic acid while improving its purity and yield has become a technical problem that needs to be overcome urgently in this field. Summary of the invention
[0006] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a preparation process for producing gallic acid by enzymatic method.
[0007] A preparation process for producing gallic acid by enzymatic method comprises the following steps:
[0008] S1, dissolving konjac glucomannan in water, adding sodium trimetaphosphate, stirring, adjusting the pH value of the system, stirring in a constant temperature water bath, adding phosphate buffered saline solution and polydopamine-polylactic acid-glycolic acid copolymer, ultrasonicating, stirring, centrifuging, and drying to obtain modified konjac glucomannan;
[0009] S2, dissolving tannin in water, adding modified konjac glucomannan, stirring in a constant temperature water bath, and obtaining a pretreated tannin solution;
[0010] S3, 3-glycidyloxypropyltrimethoxysilane and toluene are mixed, Bacillus stearothermophilus spores are added, nitrogen is introduced and stirred in a constant temperature water bath, centrifuged, precipitates are collected, phosphate buffer solution is added, ultrasound is performed, thiolated chitosan is added, stirring is performed, centrifuged, precipitates are collected, tannase and phosphate buffer solution are added, ultrasound is performed, glutaraldehyde aqueous solution is added, stirring is performed in a constant temperature water bath, centrifuged, precipitates are collected, and dried to obtain modified Bacillus stearothermophilus spores;
[0011] S4. The modified Bacillus stearothermophilus spores and the pretreated tannin solution are mixed, stirred, the pH value of the system is adjusted, stirred in a constant temperature water bath, centrifuged, the supernatant is collected, and dried to obtain gallic acid.
[0012] Preferably, in step S1, the weight ratio of konjac glucomannan, sodium trimetaphosphate, phosphate buffered saline, and polydopamine-polylactic acid-glycolic acid copolymer is 5-8:2-4:1000-1100:15-20.
[0013] Preferably, in step S1, sodium trimetaphosphate is added and then stirred, the stirring time is 20-30 min, and the stirring speed is 200-300 rpm.
[0014] Preferably, in step S1, sodium trimetaphosphate is added, stirred, and a sodium hydroxide solution with a concentration of 3-4% is used to adjust the pH value of the system to 8.9-9.1.
[0015] Preferably, in step S1, the water bath temperature is 50-53°C, the constant temperature water bath stirring time is 5-6h, and the stirring speed is 200-300rpm.
[0016] Preferably, in step S1, the ultrasonic treatment time is 30-40 min, and the ultrasonic frequency is 40-50 kHz.
[0017] Preferably, in step S1, stirring is performed after ultrasound, the stirring time is 13-15 hours, and the stirring speed is 100-150 rpm.
[0018] Preferably, in step S1, the drying temperature is 36-38°C and the drying time is 12-15h.
[0019] Preferably, in step S2, the weight ratio of tannin to modified konjac glucomannan is 20-25:2-4.
[0020] Preferably, in step S2, the modified konjac glucomannan is added at a rate of 5-7% per minute based on the total weight of the modified konjac glucomannan.
[0021] Preferably, in step S2, the water bath temperature is 37-39° C., the constant temperature water bath stirring time is 3-4 h, and the stirring speed is 100-200 rpm.
[0022] Preferably, in step S3, the weight ratio of 3-glycidyloxypropyltrimethoxysilane, toluene, Bacillus stearothermophilus spores, phosphate buffered saline, thiolated chitosan, tannase, phosphate buffered saline (second time), and 25-30% glutaraldehyde aqueous solution is 2.5-3:20-30:6-8:1000-1100:3.5-4:1.2-1.4:1000-1100:0.6-0.8.
[0023] Preferably, in step S3, nitrogen is introduced and the mixture is stirred in a constant temperature water bath, the water bath temperature is 80-82° C., the stirring time is 8-9 h, and the stirring speed is 100-200 rpm.
[0024] Preferably, in step S3, after adding the phosphate buffered saline solution, ultrasonication is performed, the ultrasonication time is 20-30 min, and the ultrasonication frequency is 40-50 kHz.
[0025] Preferably, in step S3, the mixture is stirred after the thiolated chitosan is added, the stirring time is 11-12 hours, and the stirring speed is 100-200 rpm.
[0026] Preferably, in step S3, after adding tannase and phosphate buffered saline solution, ultrasonication is performed, the ultrasonication time is 20-30 min, and the ultrasonication frequency is 40-50 kHz.
[0027] Preferably, in step S3, the concentration of the glutaraldehyde aqueous solution is 25-30%.
[0028] Preferably, in step S3, glutaraldehyde aqueous solution is added, the water bath temperature is 3-5°C, the constant temperature water bath stirring time is 4-5h, and the stirring speed is 200-300rpm.
[0029] Preferably, in step S3, the drying temperature is 39-41° C., and the drying time is 8-10 h.
[0030] Preferably, in step S4, the weight ratio of the modified Bacillus stearothermophilus spores to the pretreated tannin solution is 1.2-1.5:300-320.
[0031] Preferably, in step S4, the modified Bacillus stearothermophilus spores and the pretreated tannin solution are mixed and stirred for 0.5-1 h at a stirring speed of 100-120 rpm.
[0032] Preferably, in step S4, a sodium hydroxide solution with a concentration of 3-4% is used to adjust the pH value of the system to 6.1-6.3.
[0033] Preferably, in step S4, the water bath temperature is 54-56° C., the constant temperature water bath stirring time is 4-5 h, and the stirring speed is 100-200 rpm.
[0034] Preferably, in step S4, the drying temperature is 41-43° C., and the drying time is 13-15 h.
[0035] Preferably, the preparation method of phosphate buffered saline solution is as follows: sodium chloride, potassium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate are dispersed in water, stirred for 0.5-1h at a stirring speed of 200-300rpm, and the pH value of the system is adjusted to 7.2-7.4 with a hydrochloric acid solution with a concentration of 3-4%, to obtain a phosphate buffered saline solution.
[0036] Preferably, the weight ratio of sodium chloride, sodium trimetaphosphate, potassium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate is 8-10: 0.2-0.4: 1.4-1.6: 0.2-0.4.
[0037] A gallic acid is prepared by using any of the above-mentioned preparation processes for gallic acid.
[0038] Beneficial effects:
[0039] The invention modifies konjac glucomannan and self-assembles it with polydopamine-polylactic acid-glycolic acid copolymer to form nanoparticles, which can better disperse and stabilize tannins, provide a favorable microstructure for subsequent reactions, make tannins more orderly, greatly increase the contact opportunities with enzymes, create good conditions for enzymatic reactions, and effectively improve the yield of gallic acid. Then, the thermophilic Bacillus stearothermophilus spores are modified and tannase is fixed, which enhances the stability of the enzyme and ensures that the enzymatic reaction is continuously and efficiently carried out.
[0040] During the reaction, nanoparticles and tannins form a specific structure, which is not only conducive to the reaction, but also can adsorb impurities and improve product purity. At the same time, the reaction system constructed by the overall process is stable, and the raw materials work together to reduce the occurrence of side reactions, further improving the stability of gallic acid. After post-processing steps such as centrifugation, filtration, evaporation, and drying, impurities are effectively separated, and finally a gallic acid product with high purity, high yield and strong stability is obtained, showing unique advantages. DETAILED DESCRIPTION
[0041] The present invention will be further explained below in conjunction with specific embodiments.
[0042] Example 1
[0043] An enzymatic method for producing gallic acid and a preparation process thereof, comprising the following steps:
[0044] S1, 5g konjac glucomannan is dissolved in 60g water, 2g sodium trimetaphosphate is added, stirred for 20min, stirring speed is 200rpm, adopting concentration is 3% sodium hydroxide solution regulation system pH value is 8.9, in 50 ℃ of constant temperature water bath, stirring for 5h, stirring speed is 200rpm, add 1000g phosphate buffered saline, 15g polydopamine-polylactic acid-glycolic acid copolymer, supersound process 30min, ultrasonic frequency is 40kHz, stirring 13h, stirring speed is 100rpm, centrifugal, at 36 ℃ of dryings 12h, obtain modified konjac glucomannan;
[0045] S2, 20g tannin was dissolved in 180g water, 2g modified konjac glucomannan was added, the gross weight of the modified konjac glucomannan was taken as a benchmark, the addition rate of the modified konjac glucomannan was 5% per minute, and the mixture was stirred for 3h at 37°C constant temperature water bath, and the stirring rate was 100rpm to obtain a pre-treated tannin solution;
[0046] S3, 2.5g 3-glycidyloxypropyltrimethoxysilane and 25g toluene were mixed, 6g of thermophilic Bacillus stearothermophilus spores were added, nitrogen was introduced and stirred in a constant temperature water bath at 80g℃ for 8h, the stirring speed was 100rpm, centrifuged, the precipitate was collected, 1000g phosphate buffer solution was added, ultrasonicated for 20min, the ultrasonic frequency was 40kHz, 3.5g thiolated chitosan was added, stirred for 11h, the stirring speed was 100rpm, centrifuged, the precipitate was collected, 1.2g tannase and 1000g phosphate buffer solution were added, ultrasonicated for 20min, the ultrasonic frequency was 40kHz, 0.6g of 25% glutaraldehyde aqueous solution was added, the mixture was stirred in a constant temperature water bath at 3℃ for 4h, stirred at 200rpm, centrifuged, the precipitate was collected, and dried at 39℃ for 8h to obtain modified thermophilic Bacillus stearothermophilus spores;
[0047] S4. Mix 1.2 g of modified thermophilic Bacillus stearothermophilus spores and 300 g of pretreated tannin solution, stir for 0.5 h at a stirring speed of 100 rpm, adjust the pH value of the system to 6.1 with a 3% sodium hydroxide solution, stir in a constant temperature water bath at 54°C for 4 h at a stirring speed of 100 rpm, centrifuge, collect the supernatant, and dry at 41°C for 13 h to obtain gallic acid.
[0048] Preparation method of phosphate buffered saline solution: disperse 8g sodium chloride, 0.2g potassium chloride, 1.4g disodium hydrogen phosphate, and 0.2g potassium dihydrogen phosphate in 900g water, stir for 0.5h at a stirring speed of 200rpm, and use a 3% hydrochloric acid solution to adjust the pH value of the system to 7.2 to obtain a phosphate buffered saline solution.
[0049] Example 2
[0050] An enzymatic method for producing gallic acid and a preparation process thereof, comprising the following steps:
[0051] S1, 8g konjac glucomannan is dissolved in 80g water, 4g sodium trimetaphosphate is added, stirred for 30min, stirring speed is 300rpm, adopting concentration is 4% sodium hydroxide solution regulating system pH value is 9.1, in 53 ℃ of thermostatic water bath, stirring for 6h, stirring speed is 300rpm, add 1100g phosphate buffered saline, 20g polydopamine-polylactic acid-glycolic acid copolymer, supersound process 40min, ultrasonic frequency is 50kHz, stirred for 15h, stirring speed is 150rpm, centrifugal, at 38 ℃ of dryings 15h, obtain modified konjac glucomannan;
[0052] S2, 25g tannin was dissolved in 200g water, 4g modified konjac glucomannan was added, based on the gross weight of the modified konjac glucomannan, the addition rate of the modified konjac glucomannan was 7% per minute, and the mixture was stirred in a 39°C constant temperature water bath for 4h at a stirring rate of 200rpm to obtain a pre-treated tannin solution;
[0053] S3, 3g 3-glycidyloxypropyltrimethoxysilane, 30g toluene were mixed, 8g of thermophilic Bacillus stearothermophilus spores were added, nitrogen was introduced and stirred in a constant temperature water bath at 82°C for 9h, the stirring speed was 200rpm, centrifuged, the precipitate was collected, 1100g phosphate buffer solution was added, ultrasonicated for 30min, the ultrasonic frequency was 50kHz, 4g thiolated chitosan was added, stirred for 12h, the stirring speed was 200rpm, centrifuged, the precipitate was collected, 1.4g tannase, 1100g phosphate buffer solution were added, ultrasonicated for 30min, the ultrasonic frequency was 50kHz, 0.8g of 30% glutaraldehyde aqueous solution was added, stirred in a constant temperature water bath at 5°C for 5h, stirred at 300rpm, centrifuged, the precipitate was collected, and dried at 41°C for 10h to obtain modified thermophilic Bacillus stearothermophilus spores;
[0054] S4. Mix 1.5 g of modified thermophilic Bacillus stearothermophilus spores and 320 g of pretreated tannin solution, stir for 1 h at a stirring speed of 120 rpm, adjust the pH value of the system to 6.3 with a 4% sodium hydroxide solution, stir in a constant temperature water bath at 56°C for 5 h at a stirring speed of 200 rpm, centrifuge, collect the supernatant, and dry at 43°C for 15 h to obtain gallic acid.
[0055] Preparation method of phosphate buffered saline solution: disperse 10g sodium chloride, 0.4g potassium chloride, 1.6g disodium hydrogen phosphate, and 0.4g potassium dihydrogen phosphate in 1000g water, stir for 1h at a stirring speed of 300rpm, and use a 4% hydrochloric acid solution to adjust the pH value of the system to 7.4 to obtain a phosphate buffered saline solution.
[0056] Example 3
[0057] An enzymatic method for producing gallic acid and a preparation process thereof, comprising the following steps:
[0058] S1, 6.5g konjac glucomannan is dissolved in 70g water, 3g sodium trimetaphosphate is added, stirred for 25min, stirring speed is 250rpm, adopting concentration is 3.5% sodium hydroxide solution regulating system pH value is 9, stirred 5.5h in 51 ℃ of constant temperature water bath, stirring speed is 250rpm, add 1050g phosphate buffered saline, 17g polydopamine-polylactic acid-glycolic acid copolymer, supersound process 35min, ultrasonic frequency is 45kHz, stirred 14h, stirring speed is 125rpm, centrifugal, at 37 ℃ of dryings 13.5h, obtain modified konjac glucomannan;
[0059] S2, 22g tannin was dissolved in 190 parts of water, 3g modified konjac glucomannan was added, the total weight of the modified konjac glucomannan was used as a benchmark, the modified konjac glucomannan was added at a rate of 6% per minute, and stirred for 3.5h in a 38°C constant temperature water bath at a stirring rate of 150rpm to obtain a pre-treated tannin solution;
[0060] S3, 2.7g 3-glycidyloxypropyltrimethoxysilane and 27g toluene were mixed, 7g of Bacillus stearothermophilus spores were added, nitrogen was introduced and stirred in a constant temperature water bath at 81°C for 8.5h, the stirring speed was 150rpm, centrifuged, the precipitate was collected, 1050g phosphate buffer solution was added, ultrasonicated for 25min, the ultrasonic frequency was 45kHz, 3.7g thiolated chitosan was added, stirred for 11.5h, the stirring speed was 150rpm, centrifuged, the precipitate was collected, 1.3g tannase and 1050g phosphate buffer solution were added, ultrasonicated for 25min, the ultrasonic frequency was 45kHz, 0.7g of 27% glutaraldehyde aqueous solution was added, the mixture was stirred in a constant temperature water bath at 4°C for 4.5h, stirred at 250rpm, centrifuged, the precipitate was collected, and dried at 40°C for 9h to obtain modified Bacillus stearothermophilus spores;
[0061] S4. Mix 1.35 g of modified thermophilic Bacillus stearothermophilus spores and 310 g of pretreated tannin solution, stir for 0.7 h at a stirring speed of 110 rpm, adjust the pH value of the system to 6.2 with a 3.5% sodium hydroxide solution, stir in a constant temperature water bath at 55°C for 4.5 h at a stirring speed of 150 rpm, centrifuge, collect the supernatant, and dry at 42°C for 14 h to obtain gallic acid.
[0062] Preparation method of phosphate buffered saline solution: disperse 9g of sodium chloride, 0.3g of potassium chloride, 1.5g of disodium hydrogen phosphate, and 0.3g of potassium dihydrogen phosphate in 950 parts of water, stir for 0.7h at a stirring speed of 250rpm, and use a 3.5% hydrochloric acid solution to adjust the pH value of the system to 7.3 to obtain a phosphate buffered saline solution.
[0063] Comparative Example 1
[0064] The difference between Comparative Example 1 and Example 3 is that the modified konjac glucomannan in step S2 is replaced by konjac glucomannan;
[0065] An enzymatic method for producing gallic acid and a preparation process thereof, comprising the following steps:
[0066] S1, 22g tannin was dissolved in 190 parts of water, 3g konjac glucomannan was added, the total weight of the modified konjac glucomannan was used as a benchmark, the modified konjac glucomannan was added at a rate of 6% per minute, and stirred in a 38°C constant temperature water bath for 3.5h at a stirring rate of 150rpm to obtain a pre-treated tannin solution;
[0067] S2, 2.7g 3-glycidyloxypropyltrimethoxysilane, 27g toluene were mixed, 7g of thermophilic Bacillus stearothermophilus spores were added, nitrogen was introduced and stirred in a constant temperature water bath at 81°C for 8.5h, the stirring speed was 150rpm, centrifuged, the precipitate was collected, 1050g phosphate buffer solution was added, ultrasonicated for 25min, the ultrasonic frequency was 45kHz, 3.7g thiolated chitosan was added, stirred for 11.5h, the stirring speed was 150rpm, centrifuged, the precipitate was collected, 1.3g tannase, 1050g phosphate buffer solution, ultrasonicated for 25min, the ultrasonic frequency was 45kHz, 0.7g of 27% glutaraldehyde aqueous solution was added, stirred in a constant temperature water bath at 4°C for 4.5h, stirred at 250rpm, centrifuged, the precipitate was collected, and dried at 40°C for 9h to obtain modified thermophilic Bacillus stearothermophilus spores;
[0068] S3. Mix 1.35 g of modified thermophilic Bacillus stearothermophilus spores and 310 g of pretreated tannin solution, stir for 0.7 h at a stirring speed of 110 rpm, adjust the pH value of the system to 6.2 with a 3.5% sodium hydroxide solution, stir in a constant temperature water bath at 55°C for 4.5 h at a stirring speed of 150 rpm, centrifuge, collect the supernatant, and dry at 42°C for 14 h to obtain gallic acid.
[0069] Preparation method of phosphate buffered saline solution: disperse 9g of sodium chloride, 0.3g of potassium chloride, 1.5g of disodium hydrogen phosphate, and 0.3g of potassium dihydrogen phosphate in 950 parts of water, stir for 0.7h at a stirring speed of 250rpm, and use a 3.5% hydrochloric acid solution to adjust the pH value of the system to 7.3 to obtain a phosphate buffered saline solution.
[0070] Comparative Example 2
[0071] The difference between Comparative Example 2 and Example 3 is that: no "konjac glucomannan" is added;
[0072] An enzymatic method for producing gallic acid and a preparation process thereof, comprising the following steps:
[0073] S1, dissolving 22 g of tannin in 190 parts of water, stirring in a constant temperature water bath at 38°C for 3.5 h at a stirring speed of 150 rpm to obtain a pretreated tannin solution;
[0074] S2, 2.7g 3-glycidyloxypropyltrimethoxysilane, 27g toluene were mixed, 7g of thermophilic Bacillus stearothermophilus spores were added, nitrogen was introduced and stirred in a constant temperature water bath at 81°C for 8.5h, the stirring speed was 150rpm, centrifuged, the precipitate was collected, 1050g phosphate buffer solution was added, ultrasonicated for 25min, the ultrasonic frequency was 45kHz, 3.7g thiolated chitosan was added, stirred for 11.5h, the stirring speed was 150rpm, centrifuged, the precipitate was collected, 1.3g tannase, 1050g phosphate buffer solution, ultrasonicated for 25min, the ultrasonic frequency was 45kHz, 0.7g of 27% glutaraldehyde aqueous solution was added, stirred in a constant temperature water bath at 4°C for 4.5h, stirred at 250rpm, centrifuged, the precipitate was collected, and dried at 40°C for 9h to obtain modified thermophilic Bacillus stearothermophilus spores;
[0075] S3. Mix 1.35 g of modified thermophilic Bacillus stearothermophilus spores and 310 g of pretreated tannin solution, stir for 0.7 h at a stirring speed of 110 rpm, adjust the pH value of the system to 6.2 with a 3.5% sodium hydroxide solution, stir in a constant temperature water bath at 55°C for 4.5 h at a stirring speed of 150 rpm, centrifuge, collect the supernatant, and dry at 42°C for 14 h to obtain gallic acid.
[0076] Preparation method of phosphate buffered saline solution: disperse 9g of sodium chloride, 0.3g of potassium chloride, 1.5g of disodium hydrogen phosphate, and 0.3g of potassium dihydrogen phosphate in 950 parts of water, stir for 0.7h at a stirring speed of 250rpm, and use a 3.5% hydrochloric acid solution to adjust the pH value of the system to 7.3 to obtain a phosphate buffered saline solution.
[0077] Comparative Example 3
[0078] The difference between Comparative Example 3 and Example 3 is that the modified Bacillus stearothermophilus spores in step S4 are replaced with Bacillus stearothermophilus spores;
[0079] An enzymatic method for producing gallic acid and a preparation process thereof, comprising the following steps:
[0080] S1, 6.5g konjac glucomannan is dissolved in 70g water, 3g sodium trimetaphosphate is added, stirred for 25min, stirring speed is 250rpm, adopting concentration is 3.5% sodium hydroxide solution regulating system pH value is 9, stirred 5.5h in 51 ℃ of constant temperature water bath, stirring speed is 250rpm, add 1050g phosphate buffered saline, 17g polydopamine-polylactic acid-glycolic acid copolymer, supersound process 35min, ultrasonic frequency is 45kHz, stirred 14h, stirring speed is 125rpm, centrifugal, at 37 ℃ of dryings 13.5h, obtain modified konjac glucomannan;
[0081] S2, 22g tannin was dissolved in 190 parts of water, 3g modified konjac glucomannan was added, the total weight of the modified konjac glucomannan was used as a benchmark, the modified konjac glucomannan was added at a rate of 6% per minute, and stirred for 3.5h in a 38°C constant temperature water bath at a stirring rate of 150rpm to obtain a pre-treated tannin solution;
[0082] S3. Mix 1.35 g of thermophilic Bacillus stearothermophilus spores and 310 g of pretreated tannin solution, stir for 0.7 h at a stirring speed of 110 rpm, adjust the pH value of the system to 6.2 with a 3.5% sodium hydroxide solution, stir in a constant temperature water bath at 55°C for 4.5 h at a stirring speed of 150 rpm, centrifuge, collect the supernatant, and dry at 42°C for 14 h to obtain gallic acid.
[0083] Preparation method of phosphate buffered saline solution: disperse 9g of sodium chloride, 0.3g of potassium chloride, 1.5g of disodium hydrogen phosphate, and 0.3g of potassium dihydrogen phosphate in 950 parts of water, stir for 0.7h at a stirring speed of 250rpm, and use a 3.5% hydrochloric acid solution to adjust the pH value of the system to 7.3 to obtain a phosphate buffered saline solution.
[0084] Comparative Example 4
[0085] The difference between Comparative Example 4 and Example 3 is that: no "Bacillus stearothermophilus spores" are added;
[0086] An enzymatic method for producing gallic acid and a preparation process thereof, comprising the following steps:
[0087] S1, 6.5g konjac glucomannan is dissolved in 70g water, 3g sodium trimetaphosphate is added, stirred for 25min, stirring speed is 250rpm, adopting concentration is 3.5% sodium hydroxide solution regulating system pH value is 9, stirred 5.5h in 51 ℃ of constant temperature water bath, stirring speed is 250rpm, add 1050g phosphate buffered saline, 17g polydopamine-polylactic acid-glycolic acid copolymer, supersound process 35min, ultrasonic frequency is 45kHz, stirred 14h, stirring speed is 125rpm, centrifugal, at 37 ℃ of dryings 13.5h, obtain modified konjac glucomannan;
[0088] S2, 22g tannin was dissolved in 190 parts of water, 3g modified konjac glucomannan was added, the total weight of the modified konjac glucomannan was used as a benchmark, the modified konjac glucomannan was added at a rate of 6% per minute, and stirred for 3.5h in a 38°C constant temperature water bath at a stirring rate of 150rpm to obtain a pre-treated tannin solution;
[0089] S4. Mix 310 g of the pretreated tannin solution, stir for 0.7 h at a stirring speed of 110 rpm, adjust the pH value of the system to 6.2 with a 3.5% sodium hydroxide solution, stir in a 55°C constant temperature water bath for 4.5 h at a stirring speed of 150 rpm, centrifuge, collect the supernatant, and dry at 42°C for 14 h to obtain gallic acid.
[0090] Preparation method of phosphate buffered saline solution: disperse 9g of sodium chloride, 0.3g of potassium chloride, 1.5g of disodium hydrogen phosphate, and 0.3g of potassium dihydrogen phosphate in 950 parts of water, stir for 0.7h at a stirring speed of 250rpm, and use a 3.5% hydrochloric acid solution to adjust the pH value of the system to 7.3 to obtain a phosphate buffered saline solution.
[0091] Performance Testing
[0092] Purity: According to the standard of LY / T 1644-2023 "Test method for gallic acid", the product purity of gallic acid prepared in Examples 1-3 and Comparative Examples 1-4 was measured.
[0093] Yield: Based on the amount of raw materials used in the production process of gallic acid and the stoichiometric relationship of the chemical reaction, the theoretical mass of gallic acid M that should be obtained is calculated. 1 ; Then according to the standard of LY / T 1644-2023 "Test method for gallic acid", the actual mass M of gallic acid is measured 2 The results are shown in Table 1 below.
[0094] Calculation formula:
[0095] Stability: The gallic acid prepared in Examples 1-3 and Comparative Examples 1-4 was placed in a constant high temperature and high humidity environment for 7 and 30 days, respectively, and the changes in morphology and color were observed. The results are shown in Table 1 below.
[0096] Table 1 Test results of purity and yield
[0097] sample purity(%) Yield (%) Example 1 99.36 85.13 Example 2 99.38 85.22 Example 3 99.43 85.51 Comparative Example 1 97.52 81.56 Comparative Example 2 91.25 72.36 Comparative Example 3 94.36 78.95 Comparative Example 4 90.12 70.54
[0098] Table 2 Stability test results
[0099]
[0100]
[0101] Data Analysis:
[0102] It can be seen from the data of Examples 1-3 in Table 1 that the gallic acid prepared by the present invention has extremely high product purity and yield. Specifically, the product purity can reach up to 99.43%; and the yield can reach up to 85.51%, which can greatly reduce production costs, improve production efficiency, and provide strong support for the industrial production and wide application of gallic acid. In addition, according to Examples 1-3 in Table 2, the gallic acid prepared by the present invention also has the characteristics of high stability. Even if it is placed in a constant high temperature and high humidity environment for 7 or 30 days, its morphology and color do not change. This provides reliable guarantee for the long-term storage of gallic acid, reduces the risk of deterioration during storage, and is conducive to large-scale commercial storage and application.
[0103] As can be seen from the data of embodiment 3 and comparative example 1-2 in table 1-2, konjac glucomannan is added and modified, and the purity, yield and stability of gallic acid are significantly improved.This is because konjac glucomannan itself has certain hydrophilicity and good biocompatibility, can form certain spatial structure in solution, contribute to disperse and stabilize tannin, provide good basic environment for the generation of gallic acid, improve yield and stability to a certain extent.Further, konjac glucomannan is modified, forms nanoparticle structure with polydopamine-polylactic acid-glycolic acid copolymer, greatly increases the specific surface area and the surface activity of konjac glucomannan, makes it more efficiently interact with tannin, allows tannin to be distributed more evenly in reaction system, greatly improves the contact probability of tannin and enzyme, thereby significantly improves the yield of gallic acid.Meanwhile, this ordered structure has better adsorption capacity to impurity, can effectively remove the impurity in reaction system, further improves product purity. In addition, the modified konjac glucomannan works synergistically with other ingredients to enhance the stability of the entire reaction system, thereby improving the stability of gallic acid.
[0104] As can be seen from the data of Example 3 and Comparative Examples 3-4 in Table 1-2, adding thermophilic bacillus spores and modifying them has a significant effect on gallic acid to improve purity, yield and stability. This is because thermophilic bacillus spores themselves have good tolerance and certain biological activity, can remain relatively stable in a more complex reaction environment, and provide an ideal carrier for fixing tannase. Further, thermophilic bacillus spores have been subjected to a series of modification treatments, which greatly increase the active sites and binding capacity on the spore surface, improve the fixing effect of spores on tannase, make tannase more stable during the reaction, not easy to fall off and inactivate, and further improve the yield of gallic acid. At the same time, the modified spores have a stronger repulsion and isolation effect on the impurities in the reaction system, effectively reduce the pollution of impurities to gallic acid, and then improve product purity. In addition, this modified spore has better compatibility with the entire reaction system, can coordinate other components to jointly create a stable reaction environment, and comprehensively improve the stability of gallic acid.
[0105] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.
[0106] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.
Claims
1. A preparation process for producing gallic acid by enzymatic method, characterized in that: The steps include: S1, dissolving konjac glucomannan in water, adding sodium trimetaphosphate, stirring, adjusting the pH value of the system, stirring in a constant temperature water bath, adding phosphate buffered saline solution and polydopamine-polylactic acid-glycolic acid copolymer, ultrasonicating, stirring, centrifuging, and drying to obtain modified konjac glucomannan; S2, dissolving tannin in water, adding modified konjac glucomannan, stirring in a constant temperature water bath, and obtaining a pretreated tannin solution; S3, 3-glycidyloxypropyltrimethoxysilane and toluene are mixed, and Bacillus stearothermophilus spores are added, nitrogen is introduced and stirred in a constant temperature water bath, centrifuged, precipitates are collected, phosphate buffer solution is added, ultrasonicated, thiolated chitosan is added, stirred, centrifuged, precipitates are collected, tannase and phosphate buffer solution are added, ultrasonicated, glutaraldehyde aqueous solution is added, stirred in a constant temperature water bath, centrifuged, precipitates are collected, and dried to obtain modified Bacillus stearothermophilus spores; S4. The modified Bacillus stearothermophilus spores and the pretreated tannin solution are mixed, stirred, the pH value of the system is adjusted, stirred in a constant temperature water bath, centrifuged, the supernatant is collected, and dried to obtain gallic acid.
2. The preparation process for producing gallic acid by enzymatic method according to claim 1, characterized in that: In step S1, the weight ratio of konjac glucomannan, sodium trimetaphosphate, phosphate buffered saline, and polydopamine-polylactic acid-glycolic acid copolymer is 5-8:2-4:1000-1100:15-20.
3. The preparation process for producing gallic acid by enzymatic method according to claim 1, characterized in that: In step S2, the weight ratio of tannin to modified konjac glucomannan is 20-25:2-4.
4. The preparation process for producing gallic acid by enzymatic method according to claim 1, characterized in that: In step S3, the weight ratio of 3-glycidyloxypropyltrimethoxysilane, toluene, Bacillus stearothermophilus spores, phosphate buffered saline, thiolated chitosan, tannase, phosphate buffered saline (second time), and 25-30% glutaraldehyde aqueous solution is 2.5-3:20-30:6-8:1000-1100:3.5-4:1.2-1.4:1000-1100:0.6-0.
8.
5. The preparation process for enzymatically producing gallic acid according to claim 1, characterized in that: In step S3, nitrogen is introduced and the mixture is stirred in a constant temperature water bath, the water bath temperature is 80-82° C., the stirring time is 8-9 h, and the stirring speed is 100-200 rpm.
6. The preparation process for producing gallic acid by enzymatic method according to claim 1, characterized in that: In step S3, the concentration of the glutaraldehyde aqueous solution is 25-30%.
7. The process for producing gallic acid by enzymatic method according to claim 1, characterized in that: In step S4, the weight ratio of the modified thermophilic Bacillus stearothermophilus spores to the pretreated tannin solution is 1.2-1.5:300-320.
8. The process for producing gallic acid by enzymatic method according to claim 1, characterized in that: Preparation method of phosphate buffered saline solution: Sodium chloride, potassium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate are dispersed in water, stirred for 0.5-1h at a stirring speed of 200-300rpm, and a 3-4% hydrochloric acid solution is used to adjust the pH value of the system to 7.2-7.4 to obtain a phosphate buffered saline solution.
9. The process for producing gallic acid by enzymatic method according to claim 6, characterized in that: The weight ratio of sodium chloride, sodium trimetaphosphate, potassium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate is 8-10: 0.2-0.4：1.4-1.6：0.2-0.4。 10. A kind of edible acid, characterized in that The gallic acid is prepared by the preparation process of gallic acid according to any one of claims 1 to 9.