Disclosure of Invention
Aiming at the technical defects, the invention provides a purification and crystallization method of L-histidine with high purity, light transmittance of more than or equal to 99.0% and stable crystal form.
The technical scheme includes that the method comprises the steps of S1, preprocessing histidine solution, adjusting pH to 6.5-7.5 by acid, performing inorganic membrane filtration to obtain micro biao liquid, filtering micro filtrate by using an organic membrane to obtain nano filtrate, and controlling the filtering temperature below 90 ℃, wherein the filtering aperture of the inorganic membrane is 10-200 nm, and the aperture of the organic membrane is 100-200 daltons;
s2, adding acid or salt solution into the filtrate obtained in the step S1, heating to 60-80 ℃, concentrating under vacuum until the content of L-histidine in the filtrate obtained in the step S1 is 20-35%, stopping concentrating, and cooling to separate out solid to obtain a salt crude product, wherein the addition amount of the acid is 1.0-1.2 of the equivalent weight of histidine, and the addition amount of the salt solution is 5-25% of the weight of L-histidine;
S3, adding pure water into the salt crude product obtained in the step S2, heating to 60-90 ℃ until the salt crude product is completely dissolved, adding activated carbon for decolorization, filtering, collecting decolorized solution, adding salt solution to adjust pH to 7.4-7.7, adding a crystallization antioxidant, stirring, cooling and crystallizing the decolorized solution, wherein the cooling rate is 10-20 ℃ per hour, cooling to 2-15 ℃, and separating to obtain an L-histidine secondary crude product, wherein the adding amount of the pure water is 8-12 times the weight of the L-histidine secondary crude product, and the adding amount of the activated carbon is 5-12% of the weight of the L-histidine secondary crude product;
And S4, taking the secondary L-histidine crude product obtained in the step S3, adding pure water, heating to 70-85 ℃ until the secondary L-histidine crude product is completely dissolved, adding medicinal carbon for decolorization, filtering while the secondary L-histidine crude product is hot, collecting decolorized solution, stirring, cooling, crystallizing, separating and drying to obtain an L-histidine finished product, wherein the adding amount of the pure water is 8-12 times the weight of the secondary L-histidine crude product, and the adding amount of the medicinal carbon for decolorization is 2-6% of the weight of the secondary L-histidine crude product.
Further, in the method for purifying and crystallizing L-histidine, the inorganic membrane in the step S1 is a ceramic membrane or a silicon carbide membrane, the specification of the filter pore diameter is 20nm, and the organic membrane in the step S1 is a polyamide nanofiltration membrane, and the specification is 150 or 200 daltons. Nanofiltration membranes are organic membranes such as polyamide, polyethersulfone (PES), polyvinylidene fluoride (PVDF), sulfonated polyethersulfone, etc., preferably 200 daltons in size. The nanofiltration temperature is normal temperature, generally 20-40 ℃, preferably 30 ℃. The micro-filtrate is subjected to nanofiltration to further remove particles, colloid, impurity proteins and high-valence metal ions (Ca2+, mg2+, mn2+, and the like).
The acid for regulating the pH in the step S1 is at least one of hydrochloric acid, sulfuric acid, phosphoric acid and acetic acid. The histidine solution in the step S1 is obtained from microbial fermentation and needs to be heated to 80 ℃ for sterilization.
The acid added in the step S2 is at least one of hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, oxalic acid, malic acid, citric acid and organic sulfonic acid, and the addition amount of the acid solution is 1.05 of histidine equivalent number. The salt added in the step S2 is at least one of ammonium chloride, sodium chloride, potassium chloride, ammonium sulfate and ammonium acetate, and the addition amount of the salt is 10% of the weight of the L-histidine. Depending on the solubility of the salt in the solvent, a mass concentration of 10% is preferred.
And in the step S3, the heating and dissolving temperature is 70-75 ℃. The alkaline solution in the step S3 is one of potassium hydroxide solution, sodium hydroxide solution and ammonia water, and the acidic solution is one of acetic acid solution, sulfuric acid solution and hydrochloric acid solution. The antioxidant in the S3 is one or more of ascorbic acid, uric acid, tea polyphenol, sulfite or dithionite, and the addition amount is 0.1-0.5% of histidine by weight.
In the step S4, stirring and cooling are carried out, the stirring speed is preferably slow, the cooling speed is 10-20 ℃ per hour, and the temperature is reduced to 2-15 ℃. And the drying condition in the step S4 is 70-75 ℃ vacuum drying. In the step 4, the crystal form of the product is A type, and the quality index accords with the medicinal standard. The yield of the finished product in the step 4 is 50-66%.
Compared with the prior art, the method solves the problems that the L-histidine extraction wastewater is large in quantity and not friendly to environment, the extraction yield is low, the purity is low and the light transmittance is difficult to meet the requirements of pharmaceutical grade, and has the beneficial effects that the method improves the purity of the crude L-histidine by adding a specific salt solution to prepare the crude salt, removes most of impurities for preparing pharmaceutical grade products, shortens the production period, is simple to operate, does not use organic solvents, greatly reduces the wastewater quantity of the production process, and is suitable for industrial production. Meanwhile, a small amount of auxiliary agent is added in the crystallization process, so that the problem that L-histidine is easy to oxidize can be solved, the purity of the crystal form can be improved, and a single stable crystal form can be obtained. Compared with the traditional extraction and crystallization technology, the invention can obtain the L-histidine with high purity, the light transmittance is more than or equal to 99.0 percent, and other indexes meet the pharmaceutical grade standard. Meanwhile, a more stable single crystal form with high purity, and a crystal form A can be obtained. The method has the advantages of environmental friendliness, easiness in operation and controllable quality.
Detailed Description
The technical solutions of the present invention will be clearly and fully described below with reference to specific embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, any obvious modifications thereof by those skilled in the art without departing from the principles and spirit of the invention should be considered to be within the scope of the appended claims.
The raw materials used in the invention come from a laboratory or a production line of a factory, are histidine solutions obtained by synthesis, enzyme engineering transformation or microbial fermentation, and are particularly suitable for extraction and purification of fermentation liquor generated by fermentation.
Example 1:
S1, 36L (the content is about 3.5 percent, about 1.3 kg) of fermentation liquor is heated to 80 ℃, the pH is regulated to 7.0 by concentrated hydrochloric acid, the fermentation liquor passes through a 20nm ceramic membrane, the pressure is controlled to be 0.25-0.3 mpa, 30L of pure water is added for batch washing when 30L is filtered out, and filtrate washing water 60L are collected to obtain micro-filtrate. When the nanofiltration of the micro-filtrate 200D nanofiltration membrane is left to be 5L, washing is carried out by 40L pure water each time, and the total concentration of the collected filtrate and the washing filtrate is 100L (the content is about 1.25 percent).
S2, 2000mL (about 25g containing L-histidine) of nanofiltration solution is taken, 5.0g (20% of L-histidine weight) of potassium chloride is added, the mixture is heated to 70 ℃ and concentrated to 80mL (about 30% of L-histidine content) under vacuum, the solution is clarified, the temperature is reduced and crystallization is carried out to 10 ℃, and 29g of crude salt product is obtained through separation.
S3, taking a salt crude product, adding 250mL of pure water, heating and dissolving, regulating the pH to 7.6 by using potassium hydroxide, adding 2.0g of active carbon, decoloring for 30 minutes, filtering while the solution is hot, adding 0.1g of ascorbic acid into a 500mL reaction bottle, stirring at 75rpm and cooling and crystallizing at 15 ℃ per hour, when a large amount of crystallization occurs, regulating the stirring speed to 110rpm, continuously cooling to 5 ℃, standing for 2 hours, crystallizing, separating to obtain 14g of an L-histidine secondary crude product, and obtaining the yield of about 55%.
S4, taking 14g of secondary crude L-histidine, adding 120mL of pure water for dissolution, adding 0.7g of medicinal carbon for decolorization for 20 minutes, adding 0.1g of ascorbic acid into the decolorized solution, cooling at 15 ℃ per hour, stirring at 75rpm for cooling and crystallizing, adjusting the stirring speed to 100rpm when a large amount of crystallization occurs, cooling the decolorized solution to 10 ℃ for stirring for 2 hours, separating, and vacuum drying at 70 ℃ to obtain 8.5g of finished L-histidine with the yield of 60%.
The product is a uniform and stable L-histidine A crystal form, the purity measured by an amino acid analyzer is 99.9%, the light transmittance measured by the method of the 2020 edition of Chinese pharmacopoeia is 99.5%, the chloride is less than 0.02%, and no other amino acid is detected. Meets the requirements of medicine. The product is L-histidine in a single crystal form A, and a typical crystal form diagram under an optical digital microscope is shown in figure 1. The Differential Scanning Calorimeter (DSC) spectrum is shown in FIG. 19, which shows that no impurity peak exists and the crystal form is single.
Examples 2 to 11:
S2, taking 2000mL of histidine nanofiltration liquid obtained in the preparation process of the example 1, respectively adding 5.0 g of ammonium chloride, sodium chloride, potassium chloride, ammonium sulfate, potassium sulfate, ammonium acetate, potassium acetate, sodium acetate, ammonium dihydrogen phosphate and potassium dihydrogen phosphate into 2000mL of histidine nanofiltration liquid, concentrating to 80mL (content is 30%) under the vacuum condition at 70 ℃, cooling, crystallizing, and separating to obtain a crude salt product.
S3, dissolving the crude salt product with 250mL of water, regulating the pH to 7.6 by using sodium hydroxide, adding 2.0 g of charcoal for decoloration for 30 minutes, filtering while the solution is hot to obtain decolored solution, adding 0.1g of ascorbic acid, stirring at a speed of 15 ℃ per hour and a speed of 75rpm for every minute for cooling crystallization, regulating the stirring speed to 110rpm when a large amount of crystallization occurs, continuously cooling to 5 ℃, standing for 2 hours, and separating to obtain the secondary L-histidine crude product.
S4, dissolving the crude L-histidine product with 120mL of water, crystallizing and refining according to the example to obtain a finished product, and detecting the finished product as shown in the table 1.
The results of the various salts are shown in Table 1 below:
The tested product is uniform and stable L-histidine crystal form A, the light transmission is more than 99.5%, and the impurities and various indexes meet the requirements of drug standards. Typical crystal forms under an optical digital microscope are shown in fig. 2-11. The Differential Scanning Calorimeter (DSC) profile is similar to that of FIG. 19, showing no impurity peaks and a single crystalline form.
Examples 12 to 16
And S2, taking 2000mL of histidine nanofiltration solution prepared by the preparation process of the example 1, adjusting the pH value of the solution to be 2.0 by using hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid and benzenesulfonic acid, concentrating the solution to 30% under the vacuum condition at 70 ℃ respectively, cooling and crystallizing the solution, and separating to obtain a crude salt product.
And S3, dissolving the salt crude product by using 250mL, adding ammonia water to adjust the pH value to 7.6, adding charcoal to decolor for 30 minutes, filtering while the solution is hot to obtain decolored solution, stirring at a speed of 15 ℃ per hour and a speed of 75rpm for cooling crystallization every minute, adjusting the stirring speed to 110rpm when a large amount of crystallization occurs, continuously cooling to 5 ℃, standing for 2 hours, and separating to obtain the L-histidine secondary crude product.
S4, taking the secondary L-histidine crude product obtained in the step S3, adding pure water, heating to 70-85 ℃ until the secondary L-histidine crude product is completely dissolved, adding medicinal carbon for decolorization, filtering while the secondary L-histidine crude product is hot, collecting decolorized solution, stirring, cooling, crystallizing, separating and drying to obtain an L-histidine finished product, wherein the detection result of the finished product is shown in a table 2, the adding amount of the pure water is 8-12 times of the weight of the secondary L-histidine crude product, and the decolorizing adding amount of the medicinal carbon is 2-6% of the weight of the secondary L-histidine crude product.
Different acids were added and the results are shown in Table 2:
The tested product is a uniform and stable L-histidine crystal form A, the light transmission is more than 99.5%, and the impurities and various indexes meet the requirements of drug standards. Typical crystal forms under an optical digital microscope are shown in fig. 12-16. The Differential Scanning Calorimeter (DSC) profile is similar to that of FIG. 19, showing no impurity peaks and a single crystalline form.
EXAMPLE 17 implementation on a Mass production Scale
S1, fermenting in a fermentation tank with the concentration of 60m < 3 >, preparing histidine, heating fermentation liquor to 80 ℃ after fermentation, regulating the pH value to 7.2 by using concentrated hydrochloric acid, passing fermentation liquor with the concentration of 50m < 3 > (the content is about 3.8 percent and about 1900 kg) through a 20nm silicon carbide film, combining filtrate and washing water to obtain micro-filtrate with the concentration of 80m < 3 >, and passing through a 200D nanofiltration film;
S2, mixing the nanofiltration solution with nanofiltration water for about 140m < 3 > and concentrating in vacuum at 70 ℃ to 13% content, adding 190kg of sodium chloride, continuously concentrating to 6.5m < 3 > (about 30%), cooling to 5 ℃, and centrifuging to obtain 2000kg of crude salt.
S3, adding 12M3 water into the salt crude product, heating to dissolve completely, regulating the pH to 7.59 with sodium hydroxide solution, adding active carbon for decoloring for 30 minutes, adding 259g of sodium bisulphite, cooling the crystallization liquid to 10 ℃ at the speed of 10 ℃ per hour, preserving heat and crystallizing for 2 hours, and centrifuging to obtain 1294kg of L-histidine secondary crude product with the yield of 58.5%.
S4, adding 10M3 pure water into the secondary L-histidine crude product, heating to dissolve completely, adding active carbon to decolorize for 30 minutes, adding 200g of sodium bisulphite into the decolorized filtrate, stirring at a speed of 10 ℃ per hour, cooling to 10 ℃, centrifuging and drying to obtain 780kg of finished product, and the yield is 60%.
The product is a uniform and stable L-histidine A crystal form, the purity measured by an amino acid analyzer is 99.8%, the light transmittance measured by the method of the 2020 edition of Chinese pharmacopoeia is 99.6%, the chloride is less than 0.02%, and no other amino acid is detected. Meets the standard requirements of the 2020 edition of Chinese pharmacopoeia. The product is L-histidine A crystal form, is a single crystal form, and a typical crystal form diagram under an optical digital microscope is shown in figure 17. The Differential Scanning Calorimeter (DSC) spectrum is shown in figure 20, which shows that the impurity peak is not generated, and the crystal form is single.
In the above embodiments 1 to 17, the antioxidant in the S3 is one or more of ascorbic acid, uric acid, tea polyphenol, sulfite or dithionite, and the addition amount is 0.1 to 0.5% by weight of histidine. And in the step S4, stirring and cooling, wherein the cooling rate is 10-20 ℃ per hour, and cooling to 2-15 ℃. And the drying temperature in the step S4 is 70-75 ℃ and the vacuum drying is carried out.
The product is uniform and stable crystal form A, the light transmission is more than 99.5%, and the impurities and various indexes meet the requirements of drug standards.
Comparative example
2000ML of histidine nanofiltration solution prepared in the first example is heated to 70 ℃ and concentrated under vacuum, the histidine nanofiltration solution is about turbid when the histidine nanofiltration solution is about 200mL, the histidine nanofiltration solution is continuously concentrated until the histidine nanofiltration solution is about 80mL (the content is about 30%), the histidine nanofiltration solution is stopped concentrating, the histidine nanofiltration solution is cooled and crystallized to 10 ℃, and 25g of crude product is obtained through separation.
Taking 25g of crude salt, adding 250mL of pure water, heating and dissolving, regulating the pH to 7.6 by using potassium hydroxide, adding 2.0g of active carbon, decoloring for 30 minutes, filtering while the solution is hot, adding 0.1g of ascorbic acid into a 500mL reaction bottle, stirring at 75rpm and cooling and crystallizing at 15 ℃ per hour, when a large amount of crystallization occurs, regulating the stirring speed to 110rpm, continuously cooling to 5 ℃, standing for 2 hours, crystallizing, and separating to obtain 13g of crude product with the yield of about 52%.
13G of crude product is taken, 120mL of pure water is added for dissolution, 0.7g of medicinal carbon is added for decolorization for 20 minutes, 0.1g of ascorbic acid is added into the decolorized solution, the temperature is reduced at 15 ℃ per hour, the stirring speed is reduced to 100rpm when a large amount of crystallization occurs, the decolorized solution is reduced to 10 ℃ for stirring for 2 hours, separation and vacuum drying at 70 ℃ are carried out, and the finished product L-histidine of 7.6g is obtained, and the yield is 58.5%.
The histidine prepared by the conventional method is detected to be amorphous powder, the purity is 97.5%, the light transmittance is 93.6%, and 2.5% of other amino acids are not in accordance with the requirements of medicine. A typical crystal form diagram under an optical digital microscope is shown in FIG. 18, and a Differential Scanning Calorimeter (DSC) diagram is shown in FIG. 21, which shows that two L-histidine crystal forms exist and impurities exist.
It is to be understood that the examples are provided by way of illustration only and are not intended to limit the scope of the invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of the present invention as set forth in the appended claims.