Separation and purification method of fermentation product

Abstract

The embodiment of the invention provides a separation and purification method of a fermentation product, which comprises the following steps: redissolving the fermentation product based on p-toluene sulfonic acid according to a molar ratio of the fermentation product to the p-toluene sulfonic acid of 1: 1-1: 2; based on 0.5-1.5 M of inorganic acid, performing hydrogen bond network reconstruction on the redissolved fermentation product; decolorizing the cooled and crystallized fermentation product for 30-50 minutes on the basis of 1-2% of malic acid, 0.1-0.2% of tartaric acid and 0.05-0.15% of activated carbon; performing ion exchange resin and evaporative crystallization on the decolored fermentation product to obtain a purified fermentation product; before redissolution, a fermentation broth comprising the fermentation product is complexed and precipitated based on a composition of a polypeptide and a metal ion to precipitate the fermentation product. The separation and purification method can be used for efficiently and continuously separating and purifying the fermented branched chain amino acid at low cost, the product purity is improved, the impurity content is reduced, and the cyclic utilization of key raw materials is realized.

Description

Technical Field

[0001] This specification relates to biochemistry separation technology, and particularly to a method for separating and purifying fermentation products. Background Art

[0002] Branched Chain Amino Acids (BCAAs) include leucine, isoleucine and valine, which have important physiological functions for the human body. The production of BCAAs by fermentation is an efficient and environmentally friendly industrial production method. However, the fermentation broth contains a large amount of impurities and needs to go through complex separation and purification processes to obtain high-purity products. However, the commonly used separation and purification methods have a series of problems. For example, it is difficult to separate impurities. There are impurity amino acids with similar isoelectric points (such as L-α-aminobutyric acid, norvaline) in the fermentation broth, and the traditional ion exchange method has poor selectivity, resulting in insufficient purity (<99%); cost and environmental protection pressure. Chromatography equipment is expensive and has a small processing capacity. The amount of activated carbon used for decolorization is large, and the cost of treating waste acid solution is high; the process continuity is poor. The usual fermentation methods are difficult to achieve continuous production, resulting in low efficiency.

[0003] Therefore, it is necessary to provide a method for separating and purifying fermentation products to efficiently, low-costly and continuously separate and purify fermented BCAAs, improve product purity, reduce impurity content, and realize the recycling of key raw materials. Summary of the Invention

[0004] One or more embodiments of this specification provide a method for separating and purifying a fermentation product. The separation and purification method includes: redissolving the fermentation product based on p-toluenesulfonic acid, wherein the molar ratio of the fermentation product to the p-toluenesulfonic acid is 1:1 - 1:2; reconstructing the hydrogen bond network of the redissolved fermentation product based on 0.5 - 1.5 M inorganic acid; decolorizing the fermentation product after cooling crystallization with 1 - 2% malic acid, 0.1 - 0.2% tartaric acid, and 0.5 - 1.5‰ activated carbon for 30 - 50 min; obtaining the purified fermentation product by ion exchange resin and evaporation crystallization of the decolorized fermentation product.

[0005] In some embodiments, after passing through the ion exchange resin and before evaporation crystallization, the method further includes: eluting and recovering p-toluenesulfonic acid based on 0.3 - 0.8 M hydrochloric acid, and regenerating the resin based on 2.5 - 3.5 M hydrochloric acid.

[0006] In some embodiments, after the hydrogen bond network reconstruction and before decolorization, the method further includes: cooling the fermentation product after hydrogen bond network reconstruction to 9 - 11°C at a cooling rate of 8 - 12°C / h and seeding for 0.5 - 1.5 h.

[0007] In some embodiments, before reconstitution, the method further includes: based on the composition of the polypeptide and the metal ion, performing complexation precipitation on the fermentation broth containing the fermentation product to precipitate the fermentation product.

[0008] In some embodiments, the fermentation product is a branched-chain amino acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] This specification will be further illustrated by way of exemplary embodiments, which will be described in detail through the accompanying drawings. These embodiments are not restrictive. In these embodiments, the same numbers represent the same structures, where:

[0010] Figure 1 is an exemplary flowchart of a method for separating and purifying a fermentation product shown in some embodiments of this specification. DETAILED DESCRIPTION

[0011] To more clearly illustrate the technical solutions of the embodiments of this specification, the accompanying drawings required for the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some examples or embodiments of this specification. For those of ordinary skill in the art, without creative efforts, this specification can also be applied to other similar scenarios based on these drawings. Unless obvious from the language context or otherwise stated, the same reference numerals in the figures represent the same structures or operations.

[0012] As shown in this specification and the claims, unless the context clearly indicates an exception, words such as "a", "an", "one", and / or "the" are not specifically singular and may also include plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of the clearly identified steps and elements, and these steps and elements do not constitute an exclusive list. The method or device may also include other steps or elements.

[0013] Branched-chain amino acids (BCAAs), as important functional substances, have wide application values in the fields of food, medicine, cosmetics, and feed. Branched-chain amino acids include leucine, isoleucine, and valine. The industrial production of BCAAs has undergone a technological iteration from chemical synthesis to microbial fermentation - the early chemical synthesis method has been gradually replaced by microbial fermentation technology due to low yield and serious pollution. The production of branched-chain amino acids by fermentation is an efficient and environmentally friendly industrial production method, but the fermentation broth contains a large amount of impurities and requires complex separation and purification processes to obtain high-purity products.

[0014] In view of this, one embodiment of the present specification provides a method for separating and purifying a fermentation product. The separation and purification method includes: redissolving the fermentation product based on p-toluenesulfonic acid, wherein the molar ratio of the fermentation product to p-toluenesulfonic acid is 1:1 - 1:2; reconstructing the hydrogen bond network of the redissolved fermentation product based on 0.5 - 1.5 M inorganic acid; decolorizing the fermentation product after cooling crystallization for 30 - 50 min based on 1 - 2% malic acid, 0.1 - 0.2% tartaric acid, and 0.5 - 1.5‰ activated carbon; and obtaining the purified fermentation product by ion exchange resin and evaporation crystallization of the decolorized fermentation product.

[0015] In some embodiments, after passing through the ion exchange resin and before evaporation crystallization, the method further includes: eluting and recovering p-toluenesulfonic acid based on 0.3 - 0.8 M hydrochloric acid, and regenerating the resin based on 2.5 - 3.5 M hydrochloric acid.

[0016] In some embodiments, after the hydrogen bond network reconstruction and before decolorization, the method further includes: cooling the fermentation product after hydrogen bond network reconstruction to 9 - 11°C at a cooling rate of 8 - 12°C / h and seeding for 0.5 - 1.5 h.

[0017] In some embodiments, before redissolving, the method further includes: complexing and precipitating the fermentation broth containing the fermentation product based on a composition of polypeptide and metal ions to precipitate the fermentation product.

[0018] In some embodiments, the fermentation product is a branched-chain amino acid.

[0019] Figure 1 is an exemplary flowchart of the method for separating and purifying a fermentation product shown in some embodiments of the present specification. As Figure 1 shown, the method for separating and purifying a fermentation product may include the following steps.

[0020] Step S1, redissolving the fermentation product based on p-toluenesulfonic acid, wherein the molar ratio of the fermentation product to p-toluenesulfonic acid is 1:1 - 1:2.

[0021] In some embodiments, before redissolving, metal-MPN complexing precipitation may be performed, including: complexing and precipitating the fermentation broth containing the fermentation product based on a composition of polypeptide and metal ions to precipitate the fermentation product. That is, before step S1, there is also included: step S0, complexing and precipitating the fermentation broth containing the fermentation product based on a composition of polypeptide and metal ions to precipitate the fermentation product.

[0022] In some embodiments, the fermentation product includes branched-chain amino acids. In some embodiments, the fermentation product can be obtained by fermentation based on a branched-chain amino acid-producing strain.

[0023] The following are the specific steps for metal-MPN complex precipitation.

[0024] (1) MPN reagent preparation: Polypeptide (such as polyglutamic acid) and Fe3+ / Zn2+ are self-assembled at a molar ratio of 1:2 - 3 at pH 4.0 - 5.0 to form a soluble metal-MPN network solution (concentration 5 - 10% w / v).

[0025] (2) Complex addition: Add the MPN reagent (addition amount 0.5 - 2.0% v / v) to the supernatant filtered by a ceramic filter membrane, stir for 30 - 60 min. The target amino acids (leucine, isoleucine, valine) form insoluble MPN-amino acid complexes through coordination of amino groups with metal ions, and impurities are retained in the liquid phase due to charge repulsion.

[0026] (3) Separation: Separate the precipitate complex by centrifugation (3000 - 5000 rpm, 10 - 20 min) or membrane filtration, and collect the precipitate for the subsequent redissolution step. In some embodiments of this specification, by adding a soluble MPN reagent and using dynamic coordination complexation to selectively precipitate the target amino acids, replacing the traditional fixed-bed adsorption, the equipment can be simplified and adapted to high-impurity systems.

[0027] Redissolution is the process of redissolving the precipitated complex with an acidic reagent (such as p-toluenesulfonic acid, hydrochloric acid, citric acid, etc.) to release the target product and form a stable salt. The steps of redissolution include: mixing the MPN-amino acid complex precipitate with p-toluenesulfonic acid in proportion, releasing the target amino acid through acid dissociation, and at the same time, the MPN skeleton decomposes into polypeptide and metal ions (recoverable) under strong acid conditions. Mix the composite precipitate with p-toluenesulfonic acid at a molar ratio of 1:0.8 - 2.0, adjust the solid content to 40% - 60%, and dissolve it fully at 50 - 60 °C. P-toluenesulfonic acid forms a stable salt with the amino group of the target amino acid, significantly increasing the solubility and inhibiting the formation of mixed crystals of miscellaneous acids.

[0028] Step S2, based on 0.5 - 1.5 M inorganic acid, perform hydrogen bond network reconstruction on the redissolved fermentation product.

[0029] Hydrogen bond network reconstruction refers to the process of adjusting the solution pH and ionic strength to break the original intermolecular hydrogen bonds and reorganize the dissolution state of the target product to create conditions for subsequent crystallization.

[0030] When performing hydrogen bond network reconstruction, add 0.5 - 2.0 M hydrochloric acid in stages, adjust the system pH to 1.0 - 1.5, so that the amino acid and impurity amino acid molecules are fully protonated, breaking the original hydrogen bond network. Chloride ions (Cl-) play an ion shielding role in the solution, weakening the intermolecular electrostatic attraction and facilitating subsequent separation.

[0031] In some embodiments, after the reconstruction of the hydrogen bond network and before decolorization, the separation and purification method further includes: cooling the fermentation product after the reconstruction of the hydrogen bond network to 9-11°C at a cooling rate of 8-12°C / h, and carrying out crystal cultivation for 0.5-1.5 h.

[0032] Step S3, decolorizing the fermentation product after cooling crystallization with 1-2% malic acid, 0.1-0.2% tartaric acid, and 0.5-1.5‰ activated carbon for 30-50 min.

[0033] Specifically, it includes gradient cooling crystallization and weak acid complexation combined with activated carbon adsorption operation.

[0034] Gradient cooling crystallization: cooling from 55°C to 10°C at a rate of 10-15°C / h, combined with crystal cultivation at 200 rpm for 0.5-1 h to inhibit the formation of mixed crystals. After filtering and washing the crystal cake, redissolve it at a solid content of 40-50%.

[0035] Weak acid complexation combined with activated carbon adsorption: add 1%-3% malic acid and 0.5%-1.5% tartaric acid to the redissolved solution to form complexes with metal ions, pigment molecules, and impurity amino acids through carboxyl groups. Add 0.5‰-1‰ activated carbon, decolorize at 50-55°C for 30-40 min, and filter to obtain the decolorized solution. In some embodiments of the present specification, the synergistic effect of composite acid precipitation (p-toluenesulfonic acid salting + hydrochloric acid destroying hydrogen bonds + organic acid complexing impurities) is used, which can not only achieve trace control of impurity amino acids, control the single-category impurity amino acids to below 0.10%, but also reduce the activated carbon usage by 50%, reduce the later three-waste treatment cost, and reduce the environmental protection treatment pressure. In addition, the method of using a composite acid system combined with gradient crystallization can further avoid the formation of mixed crystals, improve the purity of the target amino acid in the crystal cake, control the residue of impurity amino acids, and at the same time improve the yield.

[0036] Step S4, obtaining the purified fermentation product from the decolorized fermentation product through ion exchange resin and evaporation crystallization.

[0037] Ion exchange resin refers to adsorbing impurities or target products using the charge characteristics of ion exchange resin. Ion exchange resin includes ion exchange resolution and recycling of p-toluenesulfonic acid: passing the decolorized solution through anion exchange resins YN201, YN269, and YN296 at 1-2 BV / h to adsorb p-toluenesulfonate and other organic acid complexes. Monitor the content of p-toluenesulfonate, and stop feeding when the content of the effluent is greater than 100 ppm. Wash with water until the solid content is 0, and regenerate the resin column with a gradient elution of 1-3 M hydrochloric acid to realize the recycling of p-toluenesulfonic acid. In some embodiments of the present specification, the recycling of p-toluenesulfonic acid is realized through gradient elution with different concentrations of hydrochloric acid, further reducing the wastewater treatment cost and environmental protection pressure.

[0038] Evaporative crystallization: The effluent is subjected to evaporative crystallization, and a high-purity amino acid crystal cake is obtained after filtration.

[0039] In some embodiments, after passing through the ion exchange resin and before evaporative crystallization, the separation and purification method further includes: recovering p-toluenesulfonic acid by elution with 0.3 - 0.8 M hydrochloric acid, and regenerating the resin with 2.5 - 3.5 M hydrochloric acid, which can reduce costs and the environmental burden. The separation and purification method of the fermentation product described in the embodiments of this specification can be applied to the separation and purification of branched-chain amino acids without changing the equipment, resin, and processing method. It has a small equipment investment, strong process applicability, and high continuity, and is suitable for industrial scale-up production.

[0040] The above-mentioned separation and purification method of the fermentation product will be elaborated in detail through multiple examples and comparative examples below. It should be noted that the reaction conditions, reaction materials, and the amounts of reaction materials in the examples are only for illustration and do not limit the protection scope of this specification. The comparative examples are the control groups of the examples.

[0041] In this specification, unless otherwise clearly stated, percentages and percentage contents are by mass. Unless otherwise specified, the experimental methods used are conventional methods, and the materials, reagents, etc. used can be purchased from commercial channels.

[0042] Example 1 MPN reagent precipitation method combined with a composite acid system to inhibit mixed crystals (purification of L-isoleucine)

[0043] Steps of Example 1:

[0044] Preparation of MPN reagent: Polyglutamic acid (10 kDa) and Fe3+ are mixed at a ratio of 1:2.5 to form a 5% w / v solution at pH 4.5.

[0045] Complexation precipitation: Add 1.5% MPN reagent to the ceramic filter clear liquid (100 L), stir for 40 min, and collect the precipitate by centrifugation (4000 rpm, 15 min).

[0046] Salt formation and redissolution: Take 100 kg of the complex precipitate, mix it with p-toluenesulfonic acid (molar ratio 1:1.5), with a solid content of 50%, and dissolve it at 60 °C.

[0047] Hydrogen bond reconstruction: Add 1 M hydrochloric acid in stages to adjust the pH to 1.2, and stir for 30 min.

[0048] Gradient crystallization: Cool down to 10 °C at a rate of 10 °C / h, incubate for 1 h, and filter to obtain a crystal cake.

[0049] Decolorization: Add 1.5% malic acid, 0.15% tartaric acid, and 1‰ activated carbon for decolorization for 40 min.

[0050] Ion exchange: Adsorption using YN201 resin and regeneration with gradient hydrochloric acid (elution with 0.5 M to recover p-toluenesulfonic acid → thorough regeneration of the resin with 3 M).

[0051] Crystallization: The finished product is obtained after evaporation crystallization.

[0052] Main results of Example 1:

[0053] Purity: 99.6% (L-isoleucine), impurity L-α-aminobutyric acid: 0.03%; L-norvaline: 0.04%.

[0054] Recovery rate of p-toluenesulfonic acid: 85%.

[0055] Comparative Example 1: Single hydrochloric acid system

[0056] Steps of Comparative Example 1: Omit the precipitating agent and the composite acid, and only redissolve with 1 M hydrochloric acid, with other conditions being the same.

[0057] Main results of Comparative Example 1: Purity 92.5% (impurity L-α-aminobutyric acid 0.54%; L-norvaline: 0.63%).

[0058] Conclusion: The composite acid system significantly reduces the impurity content (L-α-aminobutyric acid reduced by 94%, L-norvaline reduced by 94%).

[0059] Example 2: Gradient cooling crystallization to improve purity and yield (purification of L-leucine)

[0060] Steps of Example 2:

[0061] Salt formation and redissolution: 100 kg of crude L-leucine (purity 82%), molar ratio of p-toluenesulfonic acid 1:2, solid content 45%, dissolved at 55°C.

[0062] Hydrogen bond reconstruction: Add 1.5 M hydrochloric acid to adjust the pH to 1.5.

[0063] Gradient crystallization: Cool down to 15°C at a rate of 12°C / h and crystallize for 45 min.

[0064] Decolorization: Decolorize with 1% malic acid, 1% tartaric acid, and 0.8‰ activated carbon.

[0065] Ion exchange: Regenerate with gradient hydrochloric acid (0.5 M → 3 M).

[0066] Results of Example 2:

[0067] Yield: 93%;

[0068] Purity: 99.5% (impurity L-norvaline 0.04%).

[0069] Comparative Example 2: Rapid cooling

[0070] Gradient crystallization: The cooling rate is 20 °C / h, and there is no seeding step.

[0071] Results of Comparative Example 2: Purity 97.5% (impurity L-norvaline 0.32%), yield 83%.

[0072] Conclusion: Combining gradient cooling with seeding, the yield is increased by 10% and the purity is increased by 87.5%.

[0073] Example 3 Recycling of p-toluenesulfonic acid (purification of L-valine)

[0074] Steps of Example 3:

[0075] Salt formation and redissolution: 100 kg of crude L-valine (purity 80%), molar ratio of p-toluenesulfonic acid 1:1.8, solid content 55%.

[0076] Ion exchange: Adsorbed by YN269 resin, eluted with 0.5 M hydrochloric acid to recover p-toluenesulfonic acid.

[0077] Recycling: The recovered p-toluenesulfonic acid is directly used for the next batch.

[0078] Results of Example 4:

[0079] Recycling rate: 85% (purity ≥95% after 5 consecutive batches).

[0080] Cost comparison: The raw material cost is reduced by 40% (the traditional process requires a new input of precipitant for each batch).

[0081] Comparative Example 3 without recycling

[0082] Steps of Comparative Example 3: Use new p-toluenesulfonic acid for each batch and do not recover.

[0083] Results of Comparative Example 3: The raw material cost increases by 45%, and the COD of the waste liquid ≥800 mg / L.

[0084] Conclusion: The closed-loop regeneration technology reduces the raw material cost, and the COD of the waste liquid is reduced to below 200 mg / L.

[0085] Example 4 Optimization of activated carbon dosage (decolorization of mixed amino acids)

[0086] Steps of Example 4:

[0087] Salt formation and redissolution: Crude mixed amino acids (leucine + isoleucine + valine), molar ratio of p-toluenesulfonic acid 1:1.2.

[0088] Decolorization: Add 2% malic acid, 1% tartaric acid, and decolorize with 0.5‰ activated carbon for 30 min.

[0089] Results of Example 4:

[0090] Decolorization efficiency: 98% (absorbance ≤ 0.05).

[0091] Dosage of activated carbon: 0.5 kg per ton of product.

[0092] There is no organic acid in Comparative Example 4.

[0093] Steps of Comparative Example 4: Malic acid and tartaric acid are omitted, and only activated carbon is used for decolorization (3‰ is required).

[0094] Results of Comparative Example 4: Decolorization efficiency is 85% (absorbance ≥ 0.2), and the dosage of activated carbon is 3 kg per ton of product.

[0095] Conclusion: Organic acids synergistically reduce the dosage of activated carbon by 83% and improve the decolorization efficiency by 13%.

[0096] Optimal Example 5 Preferred Example Comprehensive Process Parameters (L-Isoleucine)

[0097] Specific process parameters of Example 5:

[0098] MPN reagent preparation: Polyglutamic acid (10 kDa) and Fe3+ are mixed at a ratio of 1:2.5 to form a 5% w / v solution at pH 4.5.

[0099] Complexation precipitation: Add 1.5% MPN reagent to the ceramic filter supernatant (100 L), stir for 40 min, and centrifuge (4000 rpm, 15 min) to collect the precipitate.

[0100] Salt formation and redissolution: Molar ratio 1:1.5, solid content 50%, dissolve at 60 °C.

[0101] Hydrogen bond reconstruction: Adjust the pH to 1.2 with 1 M hydrochloric acid.

[0102] Gradient crystallization: Cool down to 10 °C at a rate of 10 °C / h and crystallize for 1 h.

[0103] Decolorization: 1.5% malic acid + 0.15% tartaric acid, decolorize with 1‰ activated carbon for 40 min.

[0104] Ion exchange: YN201 resin, elute with 0.5 M hydrochloric acid to recover p-toluenesulfonic acid → regenerate the resin with 3 M hydrochloric acid.

[0105] Evaporation crystallization: Vacuum concentration temperature is 55 °C, and the vacuum degree is 0.09 MPa.

[0106] Results of Example 5:

[0107] Purity: 99.7% (L-isoleucine).

[0108] Impurity content: 0.02% of L-α-aminobutyric acid and 0.03% of L-norvaline.

[0109] Total yield: 95%.

[0110] Through the comparison between the above-mentioned examples and comparative examples, the dynamic complex precipitation technology, composite acid synergistic system, gradient crystallization control, and closed-loop regeneration technology of the metal-polypeptide network (MPN) provided in this specification significantly improve the purity and yield of branched-chain amino acids, while reducing the dosage of activated carbon and the cost of three-waste treatment, verifying its high efficiency and environmental protection advantages in industrial production.

[0111] The separation and purification method of the fermentation product provided in the examples of this specification has at least the following characteristics:

[0112] 1. Soluble metal-MPN complex precipitation technology: In the prior art, no precipitant or a single precipitant is used. In the examples of this specification, by adding soluble MPN reagents, the target amino acid is selectively precipitated by dynamic coordination complexation, replacing the traditional fixed-bed adsorption, simplifying the equipment and adapting to high-impurity systems.

[0113] 2. Composite acid impurity removal system: In the examples of this specification, the synergistic effect of composite acid precipitation (p-toluenesulfonic acid salting + hydrochloric acid breaking hydrogen bonds + organic acid complexing impurities) can achieve two beneficial effects: 1) Achieve trace control of impurity amino acids, and the single-category impurity amino acids can be controlled below 0.10%; 2) Reduce the activated carbon usage by 50%, reduce the cost of three-waste treatment in the later stage, and reduce the environmental protection treatment pressure.

[0114] 3. Recycling of p-toluenesulfonic acid: The prior art does not involve the recycling of key materials and requires multiple water or solvent activations or resin regenerations. In the examples of this specification, the recycling of p-toluenesulfonic acid is achieved through gradient elution with hydrochloric acid of different concentrations, further reducing the wastewater treatment cost and environmental protection pressure.

[0115] 4. Gradient cooling crystallization: In the prior art, it is mostly evaporation concentration crystallization. In the examples of this specification, it is a composite acid system combined with gradient crystallization, which can further avoid the formation of mixed crystals, improve the purity of the target amino acid in the crystallization cake, control the residue of impurity amino acids, and at the same time increase the yield.

[0116] 5. Wide applicability of the process flow: The prior art process flow is mostly for single amino acid products. In the examples of this specification, without changing the equipment, resin, and processing method, it can be applied to the separation and purification of three branched-chain amino acids, with small equipment investment, strong process applicability, and high continuity, suitable for industrial scale-up production.

[0117] The separation and purification method of the fermentation product provided in the examples of this specification has at least the following advantages:

[0118] 1. Further reducing the residue of impurity amino acids and further improving the purity of the main amino acid content: (1) The metal-MPN network, as a new adsorbent and precipitant, efficiently adsorbs and settles the target amino acid; (2) p-Toluenesulfonic acid forms a stable salt with the amino group of the target amino acid through the sulfonic acid group, significantly increasing the solubility; (3) The addition of hydrochloric acid fully protonates the amino acid and impurity amino acid molecules, destroying the original hydrogen bond network, and the chloride ion (Cl-) plays an ion shielding role in the solution to inhibit the formation of mixed crystals of heteracids; (4) The carboxylic acid group of the organic weak acid forms complexes with metal ions, pigment molecules, and impurity amino acids to further remove the content of impurity amino acids.

[0119] 2. Reducing the amount of activated carbon used: The carboxylic acid group of the organic weak acid forms complexes with metal ions, pigment molecules, and impurity amino acids, improving the decolorization effect of activated carbon and reducing the amount of activated carbon used.

[0120] 3. Reducing the cost of treating three wastes: After adsorbing p-toluenesulfonic acid with an ion exchange resin and performing gradient elution with hydrochloric acid, the recycling of key materials is realized, thereby reducing the post-treatment cost of treating the precipitant.

[0121] 4. The equipment and process have strong applicability, flexible product conversion, and low investment cost: Without changing the equipment, resin, and processing method, it can be applied to the separation and purification of three branched-chain amino acids, with a small equipment investment.

[0122] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only an example and does not constitute a limitation to this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are proposed in this specification, so such modifications, improvements, and corrections still fall within the spirit and scope of the exemplary embodiments of this specification.

[0123] At the same time, this specification uses specific terms to describe the embodiments of this specification. Such as "one embodiment", "an embodiment", and / or "some embodiments" mean a certain feature, structure, or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that the "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more at different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0124] Similarly, it should be noted that, in order to simplify the description disclosed in this specification and thus help the understanding of one or more embodiments of the invention, in the foregoing description of the embodiments of this specification, multiple features are sometimes merged into one embodiment, drawing, or description thereof. However, this disclosure method does not mean that the features required by the subject matter of this specification are more than those mentioned in the claims. In fact, the features of the embodiments are fewer than all the features of the individual embodiments disclosed above.

[0125] In some embodiments, numbers are used to describe components and the quantity of attributes. It should be understood that such numbers used for the description of embodiments are modified by the modifiers "about", "approximately", or "substantially" in some examples. Unless otherwise specified, "about", "approximately", or "substantially" indicate that the said numbers allow a variation of ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, and such approximate values may vary according to the characteristics required by individual embodiments. In some embodiments, the numerical parameters should consider the specified significant digits and adopt the method of retaining the general number of digits. Although the numerical ranges and parameters used in some embodiments of this specification to confirm the breadth of their scope are approximate values, in specific embodiments, the setting of such numerical values is as precise as possible within the feasible range.

[0126] For each patent, patent application, patent application publication, and other materials cited in this specification, such as articles, books, specifications, publications, documents, etc., their entire contents are hereby incorporated into this specification by reference. Except for the application history documents that are inconsistent with or conflict with the content of this specification, and except for the documents that limit the broadest scope of the claims of this specification (currently or subsequently attached to this specification). It should be noted that if there are inconsistencies or conflicts between the descriptions, definitions, and / or uses of terms in the supplementary materials of this specification and the content described in this specification, the descriptions, definitions, and / or uses of terms in this specification shall prevail.

[0127] Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, by way of example and not limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments clearly introduced and described in this specification.

Claims

1. A method for separating and purifying a fermentation product, the method comprising: Based on p-toluenesulfonic acid, the fermentation product is redissolved, wherein the molar ratio of the fermentation product to the p-toluenesulfonic acid is 1:1-1:2; Reconstructing the hydrogen bond network of the fermented product after redissolution based on 0.5-1.5M inorganic acid; Decolorizing the fermented product after cooling and crystallization for 30-50 minutes based on 1-2% malic acid, 0.1-0.2% tartaric acid, and 0.5-1.5‰ activated carbon; The decolorized fermentation product is subjected to ion exchange resin and evaporative crystallization to obtain the purified fermentation product.

2. The method according to claim 1, after passing through the ion exchange resin and before evaporation and crystallization, the method further comprises: The p-toluenesulfonic acid is recovered by eluting with 0.3-0.8 M hydrochloric acid, and the resin is regenerated by 2.5-3.5 M hydrochloric acid.

3. The method according to claim 1, after the hydrogen bond network reconstruction and before decolorization, the method further comprises: The fermented product after hydrogen bond network reconstruction is cooled to 9-11° C. at a cooling rate of 8-12° C. / h, and the crystals are incubated for 0.5-1.5 hours.

4. The method according to claim 1, before re-dissolving, further comprising: Based on the composition of polypeptide and metal ion, the fermentation broth containing the fermentation product is subjected to complex precipitation to precipitate the fermentation product. The method according to claim 1 , wherein the fermentation product comprises branched chain amino acids.

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