Method for preparing 3-hydroxybutyric acid by directly crystallizing hydrolysate

Abstract

The invention provides a method for preparing 3-hydroxybutyric acid from hydrolysate through direct crystallization, and belongs to the technical field of biosynthesis. The method comprises the following steps: (1) primary crystallization: adding a crystallization solvent into poly-3-hydroxybutyrate hydrolysate, cooling, crystallizing, carrying out suction filtration, and carrying out top washing to obtain a 3-hydroxybutyric acid crude product; and (2) secondary crystallization: adding the 3-hydroxybutyric acid crude product into a crystallization solvent, heating for dissolution, cooling for crystallization, suction filtration, top washing and drying to obtain the 3-hydroxybutyric acid. The solid 3-hydroxybutyric acid product is obtained through direct crystallization of hydrolysate of poly-3-hydroxybutyrate, and the method has the advantages of being high in content (98% or above), high in purity (99% or above), basically free of impurity crotonic acid residues (smaller than 0.01%), short in production period, low in equipment requirement, low in raw material cost, simple in production operation and the like, is suitable for industrial production and has good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biosynthesis technology, specifically relating to a method for preparing 3-hydroxybutyric acid by direct crystallization from hydrolysate. Background Technology

[0002] 3-Hydroxybutyric acid (3HB), also known as β 3-Hydroxybutyric acid (HHBEA) is an organic compound with the molecular formula C4H8O3 and a molecular weight of 104.11. This substance generally exists in three forms: the L-form, D-form, and DL-form, each with different physical properties and physiological activities. For example, D-3-hydroxybutyric acid, as a core component of ketone body metabolism in mammals, is not only a potential drug precursor for treating neurological diseases such as epilepsy and Alzheimer's disease, but can also improve the pathological state of diabetes by regulating glucose and lipid metabolism. L-3-hydroxybutyric acid is a normal human metabolite, and its levels are elevated in elderly patients with depression. In the ketogenic diet field, both the D-form and L-form are widely studied as energy supplements. Furthermore, the L-form is deliquescent and forms monoclinic crystals, while the D-form tends to form syrupy substances, making the preparation of stable crystals relatively difficult. Currently, there is a lack of mature and economical reports on the large-scale preparation technology of high-quality, easily operable D-form or L-form single-chiral 3-hydroxybutyric acid solid crystals. Therefore, the current industrial-scale production process for high-purity, single-chiral 3-hydroxybutyric acid (especially the D-type) faces significant bottlenecks.

[0003] 3-Hydroxybutyric acid (3-HHB) is currently mainly obtained through chemical and fermentation methods. Chemical synthesis uses petroleum-based derivatives as raw materials, such as the alkaline hydrolysis of ethyl 3-hydroxybutyrate. While this method can achieve a yield of 95.1%, the purity or content is generally below 90%, failing to meet the stringent optical purity requirements for food-grade and especially pharmaceutical applications. Furthermore, the strong acids and alkalis used in the reaction process can easily lead to environmental pollution and equipment corrosion. Microbial fermentation relies on recombinant strains to convert sugars into (R)-3-hydroxybutyric acid. For example, patent application CN118813510A describes a method using *E. coli* BL21(DE3) strain, which yields 75.7 g / L in a 5L fermentation system after 60 hours. However, subsequent purification requires multiple steps including ultrafiltration, ion exchange, and vacuum distillation, resulting in separation costs accounting for over 60% of the overall production cost, making large-scale production economically unviable. Against this backdrop, PHB hydrolysis, as an intermediate route connecting biosynthesis and chemical transformation, has gradually attracted attention. Poly-3-hydroxybutyrate (PHB), a polyester synthesized intracellularly by microorganisms, can be produced on a large scale through the fermentation of agricultural waste. The 3-hydroxybutyric acid produced by its hydrolysis naturally has a single configuration depending on the raw materials used. However, the existing PHB hydrolysis process faces multiple technical barriers in industrial application, and the purification process of the hydrolysis products is a key bottleneck restricting industrialization. (1) The limitations of traditional hydrolysis process lead to high purification costs: After acid hydrolysis, traditional hydrolysis process neutralizes to form a high-salt system, which requires desalination by electrodialysis, ion exchange resin, etc., and adsorption products by adsorption resin, activated carbon, inorganic adsorption materials, etc., and then eluted with solvent multiple times (such as the patent application with publication number CN115448829A, invention name is a method for preparing and applying (R)-3-hydroxybutyric acid with high optical purity). The solvent consumption is large, the efficiency is low, the cost is high and the content is low (42.13%), making it difficult to achieve continuous production.

[0004] (2) Low product purity and difficulty in application: 3-hydroxybutyric acid is very easy to dehydrate and condense during the production process to produce oligomers such as dimers and polymers, and is accompanied by the by-product crotonic acid (impurity). The existing crystallization process is difficult to achieve efficient separation of oligomers, crotonic acid and product 3-hydroxybutyric acid, resulting in low optical purity. For example, in the patent application with publication number CN119822950A, after resin adsorption, filtration, drying, elution and concentration, there are still oligomer residues in the product. Therefore, the purity is low at around 95%, which is not conducive to its subsequent application.

[0005] (3) The product is unstable and deliquescent: 3-hydroxybutyric acid has a melting point of 48~50℃. The lower the purity and content, the lower the melting point. It is also easy to absorb moisture and deliquesce, making it difficult to produce and transport. Therefore, there are few solid 3-hydroxybutyric acid products at present. Most of them are prepared into salts. As a popular sports energy supplement in the European and American markets, 3-hydroxybutyric acid has a large recommended daily intake. If the salt intake is high, it will affect the electrolyte balance in the body, which is not good for human health and limits its industrial production and application.

[0006] To address the aforementioned pain points, there is an urgent need for a method to prepare high-purity solid 3-hydroxybutyric acid from PHB hydrolysis concentrate. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing 3-hydroxybutyric acid (3-HHB) directly from hydrolysate. Addressing the aforementioned challenges, this invention proposes an integrated process of direct concentration of PHB hydrolysate followed by solvent-induced crystallization (using cooling). By simplifying the process and reducing energy consumption, it achieves a balance between ease of operation, cost-effectiveness, and high product quality, providing a practical and feasible technical path for the large-scale industrial production of 3-HHB.

[0008] This invention provides a method for preparing 3-hydroxybutyric acid by direct crystallization from a hydrolysate, comprising the following steps: (1) Primary crystallization: Add crystallization solvent to the hydrolysate of poly(3-hydroxybutyrate), cool and crystallize, filter and wash to obtain crude 3-hydroxybutyric acid; (2) Secondary crystallization: Add crude 3-hydroxybutyric acid to the crystallization solvent, heat to dissolve, cool to crystallize, filter, wash, and dry to obtain 3-hydroxybutyric acid.

[0009] Further, in step (1), the poly-3-hydroxybutyrate hydrolysate is a hydrolysate containing 3-hydroxybutyric acid obtained by acidic or alkaline hydrolysis, neutralization and concentration of poly-3-hydroxybutyrate.

[0010] Preferably, the content of 3-hydroxybutyric acid in the poly-3-hydroxybutyrate hydrolysate is 40%~85%; More preferably, the content of 3-hydroxybutyric acid in the poly-3-hydroxybutyrate hydrolysate is 50% to 65%.

[0011] Further, in steps (1) and (2), the crystallization solvent is independently selected from C1-C5 alcohol solvents, C1-C5 ketone solvents, and C1-C5 ketone solvents, respectively. 10 Ether solvents, C1~C 10 One or a combination of two or more of the following: ester solvents and C1-C5 nitrile solvents.

[0012] Further, the C1-C5 alcohol solvents are selected from one or a combination of two or more of methanol, ethanol, and isopropanol; and / or, the C1-C5 ketone solvents are selected from one or a combination of two or more of acetone and methyl ethyl ketone; and / or, the C1-C5 ketone solvents are selected from one or a combination of two or more of acetone and methyl ethyl ketone; 10 The ether solvent is selected from one or a combination of two or more of diethyl ether, propyl ether, tetrahydrofuran, isopropyl ether, isobutyl ether, and methyl tert-butyl ether; and / or, the C1~C 10 The ester solvent is selected from one or a combination of two or more of ethyl formate, ethyl acetate, and butyl acetate; and / or, the C1-C5 nitrile solvent is selected from acetonitrile.

[0013] Preferably, in step (1), the crystallization solvent is selected from methanol, ethanol, isopropanol, acetone, methyl ethyl ketone, diethyl ether, propyl ether, tetrahydrofuran, isopropyl ether, isobutyl ether, methyl tert-butyl ether, ethyl formate, ethyl acetate, butyl acetate, or acetonitrile; and / or, in step (2), the crystallization solvent is selected from methanol, ethanol, isopropanol, acetone, methyl ethyl ketone, diethyl ether, propyl ether, tetrahydrofuran, isopropyl ether, isobutyl ether, methyl tert-butyl ether, ethyl formate, ethyl acetate, butyl acetate, or acetonitrile; More preferably, in step (1), the crystallization solvent is selected from ethanol; and / or, in step (2), the crystallization solvent is selected from ethanol.

[0014] Furthermore, in step (1), the cooling crystallization process is divided into two stages: the first stage is cooling down to 10℃~-15℃ for crystallization, and the second stage is cooling down to -5℃~-30℃ for crystallization. And / or, in step (1), the method of vacuum filtration and top washing is to use one or more of C5~C8 alkane solvents and aromatic hydrocarbon solvents for top washing during vacuum filtration, collect the filter cake, and obtain crude 3-hydroxybutyric acid. Preferably, in step (1), the cooling crystallization process is divided into two stages. The first stage involves cooling to 5℃ ~ -10℃ for crystallization, with a crystallization time of 1~5h. The second stage involves cooling to -10℃ ~ -20℃ for crystallization, with a crystallization time of 1~10h. And / or, in step (1), the solvent used for top washing is one or a combination of two or more of n-heptane, 2-methylhexane, n-pentane, and n-hexane.

[0015] More preferably, in step (1), the cooling crystallization process is divided into two stages. In the first stage, the temperature is lowered to -2℃ to -9℃ for crystallization, and the crystallization time is 1 to 5 hours. In the second stage, the temperature is lowered to -15℃ to -18℃ for crystallization, and the crystallization time is 1 to 10 hours.

[0016] More preferably, in step (1), the volume-to-mass ratio of the crystallization solvent to the PHB hydrolysis concentrate is 0.05 to 0.3:1.

[0017] Furthermore, in step (2), decolorization is also included between heating and dissolving and cooling and crystallizing; after dissolving the crude product, activated carbon is added for decolorization and filtration to obtain a decolorized solution, and then the solution is cooled and crystallized.

[0018] Further, in step (2), the cooling crystallization process is divided into one or two stages; when the cooling crystallization process is one stage, the temperature is lowered to 30℃ ~ -10℃ for crystallization; when the cooling crystallization process is two stages, the first stage is lowered to 30℃ ~ 10℃ for crystallization, and the second stage is lowered to 10℃ ~ -10℃ for crystallization. And / or, in step (2), the method of vacuum filtration and top washing is to use one or more of C5~C8 alkane solvents and aromatic hydrocarbon solvents for top washing during vacuum filtration, collect the filter cake, and dry it to obtain crude 3-hydroxybutyric acid. Preferably, In step (2), the cooling crystallization process is divided into one or two stages; when the cooling crystallization process is a single stage, the temperature is lowered to 25℃ ~ 0℃ for crystallization, and the crystallization time is 1~5h; when the cooling crystallization process is a two-stage process, the first stage is lowered to 25℃ ~ 10℃ for crystallization, and the crystallization time is 1~5h, and the second stage is lowered to 10℃ ~ -5℃ for crystallization, and the crystallization time is 1~5h. And / or, in step (2), the solvent used for top washing is one or a combination of two or more of n-heptane, 2-methylhexane, n-pentane, n-hexane, and toluene.

[0019] More preferably, in step (2), the cooling crystallization process is divided into one or two stages; when the cooling crystallization process is one stage, the temperature is lowered to 2℃ ~ -5℃ for crystallization, and the crystallization time is 1~5h; when the cooling crystallization process is two stages, the first stage is lowered to 22℃ ~ 19℃ for crystallization, and the crystallization time is 1~5h, and the second stage is lowered to 10℃ ~ -5℃ for crystallization, and the crystallization time is 1~5h.

[0020] Furthermore, in step (2), during the secondary crystallization process, the mother liquor obtained after filtration is concentrated, and then crystallized again according to the method provided in step (1), and filtered to obtain the crude product; And / or, in step (2), during the secondary crystallization process, the mother liquor obtained after filtration is added to the poly-3-hydroxybutyrate hydrolysate, and then crystallized once according to the method provided in step (1), and filtered to obtain the crude product.

[0021] Furthermore, the poly-3-hydroxybutyrate is selected from one or a combination of two or more of the following: poly-3-hydroxybutyrate racemic mixture, poly(R)-3-hydroxybutyrate, and poly(S)-3-hydroxybutyrate.

[0022] Furthermore, in the 3-hydroxybutyric acid obtained in step (2), the content of 3-hydroxybutyric acid is >98%, the purity is >99%, and there is no crotonic acid residue; Preferably, the 3-hydroxybutyric acid is selected from one or a combination of two or more of the racemic 3-hydroxybutyric acid, (R)-3-hydroxybutyric acid, and (S)-3-hydroxybutyric acid.

[0023] This invention provides a crystallization method for 3-hydroxybutyric acid by screening crystallization processes. The method of this invention uses a simple crystallization process to obtain high-purity 3-hydroxybutyric acid solid with low crotonic acid content by using 3-hydroxybutyric acid hydrolysate obtained from poly-3-hydroxybutyrate obtained by bio-fermentation as raw material and then crystallizing it twice, thus expanding the application scope and development prospects.

[0024] Compared with the prior art, the present invention has the following beneficial effects: Green and environmentally friendly: All raw materials are food-grade, avoiding the use of toxic and harmful reagents, which expands its potential application scenarios in food, health products, medicine, cosmetics and biomaterials; at the same time, no wastewater is generated during the production process, which meets the requirements of sustainable development.

[0025] The process is simple: the product is obtained by crystallization under low temperature conditions, without the need for complex equipment or additional purification steps, which greatly reduces production costs and improves production efficiency.

[0026] High purity: A simple impurity removal process is used to separate difficult-to-remove oligomers, crotonic acid and other impurities from the product. The solvent used for impurity removal can be recycled after a simple separation operation, achieving efficient and green impurity removal. The product contains 3-hydroxybutyric acid with a content of >98% and a purity of >99%, and crotonic acid content of <0.01%, which is significantly better than traditional methods.

[0027] It has great potential for industrialization: the reaction conditions are mild, the equipment and operation are simple, the cost is low, it is easy to scale up production, and it is suitable for large-scale applications.

[0028] Although the process and equipment of this invention are extremely simple, through innovative optimization of the solvent system and crystallization conditions, it achieves for the first time the preparation of high-purity 3-hydroxybutyric acid in solid form, significantly improving its content and purity, and providing technical support for large-scale production. This invention also provides a mother liquor recovery process, thereby improving the crystallization yield of 3-hydroxybutyric acid, realizing solvent recycling, and solving the problem of waste. Compared with existing methods, this invention has significant technological advancements and market leadership, providing a completely new solution for the industrial production of 3-hydroxybutyric acid, and also offering an efficient, green, and safe industrialization solution for the application of 3-hydroxybutyric acid in food, health products, medicine, cosmetics, and biomaterials, especially in the fields of health products and medicine.

[0029] The successful development of this invention not only fills the technological gap in the green and efficient production of 3-hydroxybutyric acid, but also provides an important reference for the sustainable development of bio-based chemicals, and has broad application prospects and market competitiveness.

[0030] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0031] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0032] Figure 1 This is a trend diagram of crystal growth rate during the crystallization process of the 3-hydroxybutyric acid system in Example 11.

[0033] Figure 2 This is a trend chart of the number of crystals during the crystallization process of the 3-hydroxybutyric acid system in Example 11.

[0034] Figure 3 This is a temperature trend graph of the 3-hydroxybutyric acid system during crystallization in Example 11.

[0035] Figure 4 This is a graph showing the relative transmittance trend of the 3-hydroxybutyric acid system during the crystallization process in Example 11.

[0036] Figure 5 This is a particle size distribution diagram of the crystals at the end of crystallization in the 3-hydroxybutyric acid system in Example 11.

[0037] Figure 6 This is a diagram showing the distribution of crystal diameter-to-length ratio at the end of crystallization in the 3-hydroxybutyric acid system in Example 11.

[0038] Figure 7 This is a crystal imaging image of the 3-hydroxybutyric acid system at the moment of completion of crystallization in Example 11.

[0039] Figure 8 The image shows the 3-hydroxybutyric acid product obtained in Example 9.

[0040] Figure 9 This is a liquid chromatogram of a mixed standard of 3-hydroxybutyric acid and crotonic acid.

[0041] Figure 10 The liquid chromatogram of the 3-hydroxybutyric acid product obtained in Example 9 is shown.

[0042] Figure 11 This is a microscopic crystal morphology diagram of the 3-hydroxybutyric acid product obtained in Example 9.

[0043] Figure 12 The image shows the XRD pattern of the 3-hydroxybutyric acid product obtained in Example 9. Detailed Implementation

[0044] Unless otherwise specified, the raw materials and equipment used in the specific embodiments of the present invention are all known products and were obtained by purchasing commercially available products.

[0045] In a specific embodiment of the present invention, the content and purity of the substances are detected by HPLC.

[0046] Note: The poly-3-hydroxybutyrate (PHB, Yili Weining Biotechnology Co., Ltd.) hydrolysate concentrate used in the following examples and test cases was prepared using a similar method to that in the patent application No. 202511323092.1. The specific method is as follows: Weigh poly-3-hydroxybutyrate, add 2-10 times (w / v) of 80%-85% aqueous acetic acid or formic acid solution and 0.5%-2% (v / v) concentrated sulfuric acid, heat to dissolve and react for 2-10 hours, distill under reduced pressure at 50-80°C to remove the solvent and obtain the first hydrolysate, add 0.5-3 BV of purified water, heat to 70-90°C and react for 2-10 hours, add calcium hydroxide and activated carbon and stir for 1 hour, filter and distill under reduced pressure at 50-70°C to remove water and solvent and obtain the second hydrolysate concentrate.

[0047] Example 1: Preparation of 3-hydroxybutyric acid from PHB hydrolysis concentrate Weigh 20.23g of concentrated hydrolysate of poly-3-hydroxybutyrate (3-hydroxybutyric acid content 80.15%, crotonic acid content 1.64%), add 10mL of isopropanol, stir well, and start cooling. Crystallization begins at -5℃. Crystallize for 1 hour, continue cooling until a large amount of crystals precipitate. At this point, the internal temperature is -14℃. Stop cooling and crystallize for 3 hours. Filter until no filtrate flows out, and wash the filter cake with 10mL of n-heptane. Collect the filter cake to obtain 7.74g of crude 3-hydroxybutyric acid, with a 3-hydroxybutyric acid content of 94.85% and a purity of 95.19%, and a crotonic acid content of 0%.

[0048] Example 2: Preparation of 3-hydroxybutyric acid from PHB hydrolysis concentrate Weigh 100.05g of concentrated hydrolysate of poly-3-hydroxybutyrate (3-hydroxybutyric acid content 64.01%, crotonic acid content 1.73%), add 50mL of ethyl acetate, start stirring and cool, crystallize at -6℃ and grow crystals for 2h, continue cooling to internal temperature -15℃, a large amount of crystals precipitate, stop cooling, grow crystals for 2h, filter until no filtrate flows out, wash the filter cake with 20mL of 2-methylhexane, collect the filter cake, and obtain 38.48g of crude 3-hydroxybutyric acid, with a 3-hydroxybutyric acid content of 84.87%, a 3-hydroxybutyric acid purity of 94.12%, and a crotonic acid content of 0%. Add 19 mL of ethyl acetate to the crude product, heat to 40 °C to dissolve, then cool to 2 °C to allow crystals to form. Allow the crystals to grow for 2 hours, filter, and wash the filter cake with 15 mL of 2-methylhexane. Dry the filter cake under vacuum at room temperature for 2 hours to obtain 3-hydroxybutyric acid. Liquid chromatography analysis showed that the 3-hydroxybutyric acid content was 98.05%, the purity was 99.17%, and the crotonic acid content was 0%.

[0049] Example 3: Preparation of 3-hydroxybutyric acid from PHB hydrolysis concentrate Weigh 50.01g of concentrated hydrolysate of poly-3-hydroxybutyrate (3-hydroxybutyric acid content 67.54%, crotonic acid content 1.65%), add 10mL of acetone, stir for 10min and then begin cooling. Crystals precipitate at an internal temperature of -5℃. Crystallize at this temperature for 1h, and the number of crystals gradually increases. Continue cooling to -18℃, and a large amount of crystals precipitate. Stop cooling and crystallize for 3h. Filter and wash the filter cake with 30mL of n-hexane to obtain 19.58g of crude wet crystals of 3-hydroxybutyric acid, with a 3-hydroxybutyric acid content of 93.98%, a 3-hydroxybutyric acid purity of 96.136%, and a crotonic acid content of 0%.

[0050] Example 4: Preparation of 3-hydroxybutyric acid from PHB hydrolysis concentrate Weigh 51.26g of concentrated hydrolysate of poly-3-hydroxybutyrate (3-hydroxybutyric acid content is 63.50%, crotonic acid content is 1.81%), add 15mL of ethyl acetate, stir for 20min, and then cool to crystallize. A small amount of crystals precipitated at -7℃. The crystals were allowed to grow for 1h, and then the temperature was lowered to -15℃. The cooling was stopped and the crystals were allowed to grow for another 2h. The crystals were homogeneous. The mixture was filtered, and the filter cake was washed with 30mL of n-pentane. After drying the filter cake, 18.30g of crude monocrystalline product was obtained. Add 15 mL of ethanol (industrial ethanol, ethanol content ≥95%) to the crude product, heat to 45℃ to dissolve, add 0.11 g of activated carbon, stir for 30 min, filter, cool the filtrate to crystallize, crystallize at 5℃ and grow crystals for 3 h, filter by suction and wash the filter cake with 30 mL of n-pentane, dry to obtain 7.64 g of 3-hydroxybutyric acid product. The 3-hydroxybutyric acid content was tested to be 98.75%, the purity of 3-hydroxybutyric acid was 99.29%, and the crotonic acid content was 0%.

[0051] Example 5: Preparation of 3-hydroxybutyric acid from PHB hydrolysis concentrate Weigh 100.00g of concentrated hydrolysate of poly-3-hydroxybutyrate (3-hydroxybutyric acid content: 55.19%, crotonic acid content: 1.15%), add 30mL of isopropyl ether, stir well, start cooling, crystallization begins at -8℃, crystallize for 4h, continue cooling to -15℃, stop cooling, crystallize for 2h, filter until no filtrate flows out, wash the filter cake with 50mL of n-heptane and collect the filter cake to obtain 15.49g of crude 3-hydroxybutyric acid, with a 3-hydroxybutyric acid content of 91.16%, a 3-hydroxybutyric acid purity of 93.72%, and a crotonic acid content of 0%.

[0052] Example 6: Preparation of 3-hydroxybutyric acid from PHB hydrolysis concentrate Weigh 7.50 kg of concentrated hydrolysate of poly-3-hydroxybutyrate (3-hydroxybutyric acid content 62.38%, crotonic acid content 1.71%), add 1.5 L of ethanol (industrial ethanol, ethanol content ≥95%), mix well, and start cooling until crystals precipitate. At this time, the internal temperature is -2℃. Stop cooling and continue stirring for 3 hours. The crystals gradually increase in size. Continue cooling to -18℃, and a large number of crystals precipitate. Allow crystals to grow for 2 hours, filter until no filtrate flows out, and wash the filter cake with 1 L of n-hexane to obtain 2.45 kg of crude product (3-hydroxybutyric acid content 88.99%, crotonic acid content 0%). Collect the filtrate and let it stand for phase separation, and recover n-hexane. Add 0.49L of ethanol (industrial ethanol, ethanol content ≥95%) to the crude product, dissolve it at 45℃, add 7.35g of activated carbon, stir to decolorize, filter, take the filtrate and cool to crystallize. Crystallization occurs at an internal temperature of 25℃, crystallize for 2 hours, continue to cool to -5℃, crystallize for another 2 hours, filter, and wash the filter cake with the recovered n-hexane from the previous step. After drying, 1.30kg of 3-hydroxybutyric acid product is obtained. The 3-hydroxybutyric acid content is 99.54%, the purity is 100.000%, and the crotonic acid content is 0%.

[0053] Example 7: Preparation of 3-hydroxybutyric acid from PHB hydrolysis concentrate Weigh 500.10g of concentrated hydrolysate of poly-3-hydroxybutyrate (3-hydroxybutyric acid content: 53.80%, crotonic acid content: 1.03%), add 150mL of isopropanol, mix well, and then cool to crystallize. Crystallization occurred at -9℃ and was maintained for 1.5h. The temperature was further lowered to -15℃ and maintained for 2h. The mixture was filtered and the filter cake was washed with 100mL of n-heptane to obtain 172.50g of crude product. Then, 86mL of isopropanol was added, and the mixture was heated to 45℃ to dissolve the crude product. After decolorization with 1.86g of activated carbon, the mixture was cooled to crystallize. Crystallization occurred at 2℃ and was maintained for 4h. The mixture was filtered and the filter cake was washed with 100mL of n-heptane. After drying the filter cake, 65.90g of 3-hydroxybutyric acid product was obtained. The 3-hydroxybutyric acid content was 96.79%, the purity was 99.856%, and the crotonic acid content was 0%. The filtrate was concentrated by vacuum distillation at 45℃ and then cooled again for crystallization. Crystals were generated at -10℃ and cultured for 1.5 hours. The temperature was then further reduced to -15℃, resulting in the precipitation of a large amount of crystals. The mixture was stirred at this temperature for another 2 hours. After filtration and washing the filter cake with 100 mL of n-heptane, 63.81 g of 3-hydroxybutyric acid was obtained. The content of 3-hydroxybutyric acid was 98.19%, the purity of 3-hydroxybutyric acid was 99.53%, and the content of crotonic acid was 0%.

[0054] Example 8: Preparation of 3-hydroxybutyric acid from PHB hydrolysis concentrate Primary crystallization: 320.19 kg of concentrated hydrolysate of poly-3-hydroxybutyrate (3-hydroxybutyric acid content of 65.12%, 3-hydroxybutyric acid purity of 86.33%, and crotonic acid content of 1.45%) and 80 L of ethanol (industrial ethanol, ethanol content ≥95%) were added to the crystallization tank. Stirring was started and the temperature was lowered. Crystals precipitated when the internal temperature was -3℃. The cooling was stopped and the crystals were allowed to grow for 3 hours. A large number of crystals precipitated. The temperature was lowered to -17℃ and the crystals were allowed to grow for 2 hours. The mixture was filtered until no filtrate flowed out. The filter cake was washed with 100 L of n-hexane. The filter cake was weighed to obtain 140.84 kg of wet crude crystals. The filtrate was collected and allowed to stand for phase separation. The n-hexane was recovered.

[0055] Secondary crystallization: Add 42L of ethanol (industrial ethanol, ethanol content ≥95%) to the crude product, heat to 45℃, stir until dissolved, stop heating, add 1000g of activated carbon, stir for 1h, filter, remove activated carbon, put the filtrate into a crystallization tank, turn on stirring and cool down, crystals appear at an internal temperature of 22℃, stop cooling and grow crystals for 3h, the crystal particles gradually grow, continue cooling to 10℃, a large number of crystals precipitate, grow crystals for 3h, stop stirring, filter to dryness, wash the filter cake with n-hexane, dry the filter cake under vacuum at 25℃, and obtain 58.50kg of 3-hydroxybutyric acid product. Liquid chromatography analysis shows that the 3-hydroxybutyric acid content is 99.85%, the 3-hydroxybutyric acid purity is 99.62%, and the crotonic acid content is 0%.

[0056] Example 9: Preparation of 3-hydroxybutyric acid from PHB hydrolysis concentrate Primary crystallization: 325.60 kg of concentrated hydrolysate of poly-3-hydroxybutyrate (3-hydroxybutyric acid content 63.53%, 3-hydroxybutyric acid purity 85.90%, crotonic acid content 1.40%) and 65 L of ethanol (industrial ethanol, ethanol content ≥95%) were added to the crystallization tank. Stirring was started and the temperature was lowered. Crystals precipitated when the internal temperature was -2℃. The cooling was stopped and the crystals were allowed to grow for 3 hours. A large number of crystals precipitated. The temperature was lowered to -15℃ and the crystals were allowed to grow for 2 hours. The mixture was filtered until no filtrate flowed out. The filter cake was washed with 100 L of n-hexane. The filter cake was weighed to obtain 141.65 g of wet crude crystals. The filtrate was collected and allowed to stand for phase separation. The n-hexane was recovered.

[0057] Secondary crystallization: Add 35L of ethanol (industrial ethanol, ethanol content ≥95%) to the crude product, heat to 45℃, stir until dissolved, stop heating, add 838g of activated carbon, stir for 1h, filter, remove activated carbon, put the filtrate into a crystallization tank, turn on stirring and cool down, crystals emerge at an internal temperature of 19℃, stop cooling and grow crystals for 3h, crystal particles gradually grow, continue cooling to 5℃, a large number of crystals precipitate, grow crystals for 3h, stop stirring, filter to dryness, wash the filter cake with n-hexane, let the filtrate stand and separate phases, recover n-hexane, and collect 108.32kg of crystallization mother liquor, vacuum dry the filter cake at 25℃ to obtain 67.40kg of 3-hydroxybutyric acid product, the 3-hydroxybutyric acid content is 98.28% and the purity is 99.97%, and the crotonic acid content is 0% by liquid chromatography.

[0058] Example 10: Preparation of 3-hydroxybutyric acid from PHB hydrolysis concentrate The mother liquor obtained in Example 9 was put into a crystallization tank, stirred and cooled until crystals precipitated. At this time, the internal temperature was -5°C. After crystallization for 3 hours, the temperature was further lowered to -18°C and a large amount of crystals precipitated. After crystallization for 2 hours, the mixture was filtered and the filter cake was washed with the recovered n-hexane from Example 9 to obtain 22.10 kg of crude 3-hydroxybutyric acid. Liquid chromatography analysis showed that the 3-hydroxybutyric acid content was 93.85%, the 3-hydroxybutyric acid purity was 96.24%, and the crotonic acid content was 0%.

[0059] Example 11: Imaging and monitoring the crystallization process using a PCM crystallization monitoring system. I. Primary crystallization Weigh 1005.10g of concentrated hydrolysate of poly-3-hydroxybutyrate (3-hydroxybutyric acid content 65.40%, crotonic acid content 1.05%), add 200mL of ethanol (industrial ethanol, ethanol content ≥95%), stir well, start cooling, crystallization begins at -2℃, crystallize for 1h, continue cooling until a large amount of crystals precipitate, at which point the internal temperature is -16℃, stop cooling, crystallize for 2h, filter until no filtrate flows out, wash the filter cake with 500mL of n-hexane and collect the filter cake to obtain 420.06g of crude 3-hydroxybutyric acid, 3-hydroxybutyric acid content 92.32%, 3-hydroxybutyric acid purity 95.34%, crotonic acid content 0%.

[0060] II. Secondary crystallization Weigh 400.05g of crude monocrystalline product, add 160mL of ethanol (industrial ethanol, ethanol content ≥95%), heat to 43℃ to dissolve, cool to crystallize, and use a PCM crystallization monitoring system (online video microscope and particle size analysis system) to image and monitor the crystallization process. The test parameters are as follows: detection size 1~1000μm, data acquisition interval 60s, image saving interval 2s.

[0061] III. Experimental Results The crystallization process and its final stage in this system, as well as the imaging, are as follows: Figures 1-7 As shown in Table 1, the key parameters are: crystallization temperature 14.29℃, average growth rate during crystallization 15 µm / h, cooling dominates crystal growth, the rate is stable, the final crystallization temperature is 7.69℃, at which point the average particle size is medium to large, and the standard deviation of particle size distribution is narrow (coefficient of variation ≈30%), the nucleation density is medium, the plate-like / plate-like habit is obvious, the morphology consistency is acceptable, and it is suitable for downstream filtration and drying.

[0062] Table 1. Key parameters during crystallization process Example 12: Identification of 3-hydroxybutyric acid I. Experimental Methods The product prepared in Example 9 was subjected to detection of relevant indicators such as liquid chromatography, quantitative nuclear magnetic resonance, elemental analysis, ash content, and specific rotation.

[0063] II. Experimental Results The results are shown in Table 2. The product appears as a white crystalline solid. Figure 8 The content of the substance was 98.28% and the purity was 99.97% as determined by liquid chromatography. The specific rotation was -24.73° and the moisture content was 0.57%. Heavy metals such as lead, mercury, and cadmium were not detected.

[0064] Among them, the liquid chromatography detection results are as follows Figure 9 , Figure 10 As shown, Figure 9 The image shows the liquid chromatography chromatogram of a mixture of R-3-hydroxybutyric acid standard and crotonic acid standard. Figure 10 The image shows the liquid chromatogram of 3-hydroxybutyric acid prepared using the method in Example 9 of this invention. The response time of the 3-hydroxybutyric acid product in the chromatogram is consistent with that of the 3-hydroxybutyric acid standard. The results indicate that this invention successfully prepared 3-hydroxybutyric acid.

[0065] Table 2. Identification results of 3-hydroxybutyric acid prepared in Example 9 The product obtained in Example 9 of this invention was subjected to quantitative nuclear magnetic resonance (QNMR) analysis. The results are shown in Table 3. The results showed good consistency, with an average content of 98.70%, which was basically consistent with the liquid phase detection results.

[0066] Table 3. Quantitative nuclear magnetic resonance (QNMR) analysis results of the product obtained in Example 9 Example 13: Characterization of 3-hydroxybutyric acid I. Experimental Methods The product prepared in Example 9 was subjected to microscopic magnification imaging and X-ray diffraction (XRD).

[0067] II. Experimental Results Upon testing, the product obtained in Example 9 appeared as a thick, transparent sheet with good flowability. Figure 11 X-ray diffraction results are as follows: Figure 12 As shown, the 3-hydroxybutyric acid prepared in Example 9 has characteristic peaks at 2theta values ​​of 12.3°±0.2° and 21.7°±0.2°.

[0068] The following specific experimental examples demonstrate the beneficial effects of the present invention.

[0069] Experimental Example 1: Preparation of 3-hydroxybutyric acid from PHB hydrolysis concentrate Using 500g of PHB hydrolysis concentrate (containing 63.83% 3-hydroxybutyric acid and 3.50% crotonic acid) as raw material, crystallization was carried out under different solvents and process conditions to prepare crude crystalline product. The specific crystallization method is as follows: Weigh 500g of PHB hydrolysis concentrate, add crystallization solvent, stir well, and begin cooling. Crystallization begins at x1 ℃, and crystals are allowed to grow for y1 h. Continue cooling until a large number of crystals precipitate, at which point the internal temperature is x2 ℃. Stop cooling and allow crystals to grow for y2 h. Filter until no filtrate flows out, and wash the filter cake with 100mL of n-heptane. Collect the filter cake to obtain the crude product. The crystallization parameters and crystallization results are shown in Table 4.

[0070] Table 4. Selection of crystallization parameters and crystallization results for the preparation of 3-hydroxybutyric acid from PHB hydrolysis concentrate. During the research, it was found that solid 3-hydroxybutyric acid could not be successfully obtained from the PHB hydrolysis concentrate using dichloromethane, n-hexane, toluene, petroleum ether, chloroform, and n-heptane as crystallization solvents. That is, the PHB hydrolysis concentrate cannot crystallize under all conditions; its crystallization process is highly condition-dependent. Only under specific process conditions can high-purity, high-yield solid 3-hydroxybutyric acid be successfully obtained.

[0071] Based on a comprehensive assessment of key industrialization factors such as production feasibility, process safety, production cost, and product quality, this invention prioritizes ethanol as the crystallization solvent. Compared to other effective solvents, ethanol has the following outstanding advantages: (1) High safety: low toxicity and volatility (compared to acetone), high flash point (compared to ethyl acetate), which is more conducive to safe production and operation; (2) High product purity: as shown in Table 4, the purity of 3HB obtained by crystallization using ethanol can reach over 97%, and the removal effect on the impurity crotonic acid is significant; (3) Good feasibility: ethanol is inexpensive, widely available, easy to recycle and reuse, and solvent residue is easy to control, making it very suitable for large-scale industrial production.

[0072] Therefore, through extensive experimentation and screening, the present invention has determined the optimal crystallization process, which can stably produce high-quality solid 3-hydroxybutyric acid products while ensuring safety and economy.

[0073] In summary, this invention has obtained the optimal crystallization process for 3-hydroxybutyric acid through experimental screening, enabling the direct crystallization of solid 3-hydroxybutyric acid products from the hydrolysate of poly-3-hydroxybutyrate. This process offers advantages such as high content (over 98%), high purity (over 99%), virtually no residual crotonic acid (<0.01%), short production cycle, low equipment requirements, low raw material costs, and simple production operation. It is suitable for industrial production and has promising application prospects.

Claims

1. A method for preparing 3-hydroxybutyric acid by direct crystallization from hydrolysate, characterized in that: Includes the following steps: (1) Primary crystallization: Add crystallization solvent to the hydrolysate of poly(3-hydroxybutyrate), cool and crystallize, filter and wash to obtain crude 3-hydroxybutyric acid; (2) Secondary crystallization: Add crude 3-hydroxybutyric acid to the crystallization solvent, heat to dissolve, cool to crystallize, filter, wash, and dry to obtain 3-hydroxybutyric acid.

2. The method according to claim 1, characterized in that: In step (1), the poly(3-hydroxybutyrate) hydrolysate is a hydrolysate containing 3-hydroxybutyric acid obtained by acidic or alkaline hydrolysis, neutralization and concentration of poly(3-hydroxybutyrate).

3. The method according to claim 1, characterized in that: In steps (1) and (2), the crystallization solvent is independently selected from C1-C5 alcohol solvents, C1-C5 ketone solvents, and C1-C5 ketone solvents, respectively. 10 Ether solvents, C1~C 10 One or a combination of two or more of the following: ester solvents and C1-C5 nitrile solvents.

4. The method according to claim 3, characterized in that: The C1-C5 alcohol solvents are selected from one or more combinations of methanol, ethanol, and isopropanol; and / or, the C1-C5 ketone solvents are selected from one or more combinations of acetone and methyl ethyl ketone; and / or, the C1-C5 ketone solvents are selected from one or more combinations of acetone and methyl ethyl ketone. 10 The ether solvent is selected from one or a combination of two or more of diethyl ether, propyl ether, tetrahydrofuran, isopropyl ether, isobutyl ether, and methyl tert-butyl ether; and / or, the C1~C 10 The ester solvent is selected from one or a combination of two or more of ethyl formate, ethyl acetate, and butyl acetate; and / or, the C1-C5 nitrile solvent is selected from acetonitrile. Preferably, in step (1), the crystallization solvent is selected from methanol, ethanol, isopropanol, acetone, methyl ethyl ketone, diethyl ether, propyl ether, tetrahydrofuran, isopropyl ether, isobutyl ether, methyl tert-butyl ether, ethyl formate, ethyl acetate, butyl acetate, or acetonitrile; and / or, in step (2), the crystallization solvent is selected from methanol, ethanol, isopropanol, acetone, methyl ethyl ketone, diethyl ether, propyl ether, tetrahydrofuran, isopropyl ether, isobutyl ether, methyl tert-butyl ether, ethyl formate, ethyl acetate, butyl acetate, or acetonitrile; More preferably, in step (1), the crystallization solvent is selected from ethanol; and / or, in step (2), the crystallization solvent is selected from ethanol.

5. The method according to claim 1, characterized in that: In step (1), the cooling crystallization process is divided into two stages: the first stage is cooling down to 10℃ ~ -15℃ for crystallization, and the second stage is cooling down to -5℃ ~ -30℃ for crystallization. And / or, in step (1), the method of vacuum filtration and top washing is to use one or more of C5~C8 alkane solvents and aromatic hydrocarbon solvents for top washing during vacuum filtration, collect the filter cake, and obtain crude 3-hydroxybutyric acid. Preferably, in step (1), the cooling crystallization process is divided into two stages. The first stage involves cooling to 5℃ ~ -10℃ for crystallization, with a crystallization time of 1~5h. The second stage involves cooling to -10℃ ~ -20℃ for crystallization, with a crystallization time of 1~10h. And / or, in step (1), the solvent used for top washing is one or a combination of two or more of n-heptane, 2-methylhexane, n-pentane, and n-hexane.

6. The method according to claim 1, characterized in that: In step (2), decolorization is also included between heating and dissolving and cooling and crystallizing; after dissolving the crude product, activated carbon is added for decolorization and filtration to obtain a decolorized solution, and then the solution is cooled and crystallized.

7. The method according to claim 6, characterized in that: In step (2), the cooling crystallization process is divided into one or two stages; when the cooling crystallization process is one stage, the temperature is lowered to 30℃ ~ -10℃ for crystallization; when the cooling crystallization process is two stages, the first stage is cooled to 30℃ ~ 10℃ for crystallization, and the second stage is cooled to 10℃ ~ -10℃ for crystallization. And / or, in step (2), the method of vacuum filtration and top washing is to use one or more of C5~C8 alkane solvents and aromatic hydrocarbon solvents for top washing during vacuum filtration, collect the filter cake, and dry it to obtain crude 3-hydroxybutyric acid. Preferably, In step (2), the cooling crystallization process is divided into one or two stages; when the cooling crystallization process is a single stage, the temperature is lowered to 25℃ ~ 0℃ for crystallization, and the crystallization time is 1~5h; when the cooling crystallization process is a two-stage process, the first stage is lowered to 25℃ ~ 10℃ for crystallization, and the crystallization time is 1~5h, and the second stage is lowered to 10℃ ~ -5℃ for crystallization, and the crystallization time is 1~5h. And / or, in step (2), the solvent used for top washing is one or a combination of two or more of n-heptane, 2-methylhexane, n-pentane, n-hexane, and toluene.

8. The method according to claim 1, characterized in that: In step (2), during the secondary crystallization process, the mother liquor obtained after filtration is concentrated, and then crystallized again according to the method provided in step (1), and filtered to obtain the crude product. And / or, in step (2), during the secondary crystallization process, the mother liquor obtained after filtration is added to the poly-3-hydroxybutyrate hydrolysate, and then crystallized once according to the method provided in step (1), and filtered to obtain the crude product.

9. The method according to claim 1, characterized in that: The poly-3-hydroxybutyrate is selected from one or a combination of two or more of the following: poly-3-hydroxybutyrate racemic, poly(R)-3-hydroxybutyrate, and poly(S)-3-hydroxybutyrate.

10. The method according to any one of claims 1 to 9, characterized in that: The 3-hydroxybutyric acid obtained in step (2) has a content of >98%, a purity of >99%, and no crotonic acid residue; Preferably, the 3-hydroxybutyric acid is selected from one or a combination of two or more of the racemic 3-hydroxybutyric acid, (R)-3-hydroxybutyric acid, and (S)-3-hydroxybutyric acid.

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