Method for generating high-quality amino acid by hydrolyzing animal protein blood powder

Abstract

The invention discloses a method for generating a refined amino acid product by hydrolyzing animal protein blood powder, which comprises the following steps: adding an animal protein raw material into acid liquor for acid hydrolysis, precipitating a histidine crude product from protein hydrolysate, and separating the histidine crude product to obtain residual mother liquor; refining the histidine crude product to obtain a histidine refined product, adding a leucine precipitator into the residual mother liquor, and treating to obtain a leucine crude product; refining and recrystallizing the crude product to obtain high-purity leucine, and finally drying tail liquid by using a spray drying technology to obtain amino acid mixed powder after drying. According to the method, histidine and leucine can be directly separated from the protein hydrolysate, good crystal purity is achieved, the equilibrium adsorption rate of impurity amino acid in weakly acidic ion resin to histidine is overcome, meanwhile, the crystal grain size of leucine is controlled within a proper range, and good bulk density and fluidity are achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of amino acid preparation method, and particularly relates to a method for preparing amino acid fine products by hydrolyzing animal protein blood meal. BACKGROUND

[0002] Histidine is an alpha-amino acid. Histidine was first isolated by German physicist Albert Kossel in 1896. In the field of nutrition, histidine is considered as an essential amino acid for human, especially for children. After adulthood, human can synthesize histidine by themselves. Adding a small amount of histidine in the diet of chronic uremic patients can increase the speed of amino acid combination into hemoglobin, and reduce renal anemia, so histidine is also an essential amino acid for uremic patients. Histidine is decarboxylated to form histamine under the action of histidine decarboxylase. The solubility of histidine in water at 25 DEG C is 41.9 g / L, and histidine is slightly soluble in alcohol (solubility is very small and can be ignored), and insoluble in ether and chloroform. Therefore, histidine has a certain solubility in water, and commonly used histidine is histidine hydrochloride, which has good solubility.

[0003] The isoelectric point of amino acid refers to the pH value when the net charge of amino acid in solution is zero, at this time, the amino acid exists in the form of a zwitterion, and the solubility of the amino acid at the isoelectric point is the smallest. The isoelectric point precipitation is often used to obtain specific amino acids. The isoelectric points of common amino acids are as follows: amino acid isoelectric point amino acid isoelectric point glycine 5.97 alanine 6.00 valine 5.96 leucine 5.98 isoleucine 6.02 phenylalanine 5.48 serine 5.68 threonine 6.16 tyrosine 5.66 cysteine 5.05 methionine 5.74 proline 6.30 tryptophan 5.89 lysine 9.74 arginine 10.76 histidine 7.59 aspartic acid 2.77 glutamic acid 3.22 asparagine 5.41 glutamine 5.65 As can be seen from the above table, the isoelectric points of most amino acids are in weak acid, and the isoelectric point of histidine is in weak base, and the isoelectric point of lysine is in base. Therefore, histidine and lysine can be obtained by isoelectric point precipitation; however, the purity of amino acid obtained by isoelectric point precipitation is generally low, and therefore, the amino acid crude product is generally refined.

[0004] Document 1: Extraction of acid, base amino acid and leucine from pig blood hydrolysate, author Li Pingze.

[0005] In document 1, pig blood is treated and then hydrolyzed, and after hydrolysis, the solution is decolorized, filtered, and tyrosine is removed by precipitation, and then the solution is eluted by using resin. In the elution process, different acid eluent and base eluent are used to obtain different amino acids. However, the elution and separation are mainly realized by adjusting the pH value, and the pH values of the eluents are very close, which greatly increases the difficulty of the operation, and the actual effect is not good. At the same time, ammonia water is used for elution in document 1, and the resin used should be a strong acid resin.

[0006] Document 2: D113 weak acid ion exchange resin adsorbs L-histidine, author Xu Qingqing et al.

[0007] Document 2 measured the kinetic curve and adsorption isotherm of D113 weak acid ion exchange resin adsorbing L-histidine at 25℃, and investigated the effects of pH value, ammonium sulfate concentration, L-lysine concentration and L-arginine concentration in the solution on adsorption. The results showed that: D113 ion exchange resin adsorbed L-histidine for about 30 min to reach equilibrium; the equilibrium adsorption rate of L-histidine decreased with the decrease of pH value in the experimental pH range; when the pH decreased to 3.4, the adsorption capacity reached 133 mg / g; the presence of ammonia ions significantly reduced the adsorption capacity of L-histidine. The presence of L-lysine or L-arginine slightly reduced the adsorption capacity.

[0008] Document 3: D152 weak acid ion exchange resin adsorbing L-histidine, author Yang Xin et al.

[0009] Document 3 measured the kinetic curve and adsorption isotherm of D152 weak acid ion exchange resin adsorbing L-histidine at 25℃, and investigated the effects of pH value, ammonium sulfate concentration, L-lysine concentration and L-arginine concentration in the solution on adsorption.

[0010] Document 4: Characteristic of 732 cation exchange resin adsorbing L-histidine, author Zou Jianhui.

[0011] Document 4 measured the kinetic curve and adsorption isotherm of 732 cation exchange resin adsorbing L-histidine at 25℃, and investigated the effects of pH value, ammonium sulfate concentration, L-lysine and L-arginine on adsorption. The results showed that: the presence of ammonia ions significantly reduced the adsorption capacity of L-histidine. The presence of L-lysine or L-arginine slightly reduced the adsorption capacity.

[0012] From Document 2 to Document 4, it can be seen that: resin can be used as the main way of separating and purifying amino acids, but most of the existing processes for separating L-histidine from fermentation broth or protein hydrolysate use strong acid ion exchange resin, and the elution of L-histidine from this type of resin mainly uses ammonia water. Because the solution is alkaline, the eluent will cause partial decomposition of L-histidine during ammonia removal. If weak acid ion exchange resin is used to adsorb L-histidine, dilute acid can be used for elution, and concentration can avoid the decomposition of L-histidine. Therefore, weak acid resin is used for adsorption in Document 2 to Document 4, and dilute acid is used for elution.

[0013] At the same time, from Document 2 to Document 4, it can be seen that during the adsorption and elution purification process using ion resin, the remaining amino acids as impurities will have adverse effects on adsorption and elution, and will reduce the adsorption capacity.

[0014] Document 5: CN103755640A Separation method of L-histidine Document 5 discloses a separation method of L-histidine, comprising the following steps: A. The mixed amino acid mother liquor is subjected to decolorization treatment with activated carbon, and the pH value is adjusted to 0-7.0; B. Dichlorobenzenesulfonic acid precipitant is added, and crystallization is carried out at-10℃-50℃, and histidine benzene sulfonate precipitate is obtained by filtration; C. The histidine benzene sulfonate precipitate is added into boiling water, and after dissolution, the resolving salt is added for resolution, and the dichlorobenzenesulfonic acid resolving salt is removed by filtration; D. Sulfuric acid is added to the filtrate to remove excess resolving salt ions, and L-histidine is obtained by concentration.

[0015] Document 5 discloses a method for precipitating the amino acid hydrolysate using dichlorobenzenesulfonic acid precipitant, and separating histidine from other amino acids after precipitation.

[0016] Document 6: CN107629007A A method for separating and extracting leucine and histidine from blood powder.

[0017] Document 6 discloses that experimental research finds that after 100g blood powder is acid hydrolyzed, the hydrolysate contains 8.0g of leucine, 5.8g of histidine, and 3.6g of arginine. Since leucine, histidine, and arginine can all form precipitates with dichlorobenzenesulfonic acid, the separation of these three amino acids becomes a problem that must be solved. Further research finds that the concentration of arginine in the blood powder hydrolysate is relatively low, and as long as the precipitation conditions are properly controlled, arginine can not be precipitated, but using dichlorobenzenesulfonic acid as a precipitant, no matter how the precipitation conditions are changed, leucine and histidine in the blood powder hydrolysate are always precipitated together, and cannot be precipitated separately.

[0018] Therefore, when using special precipitants such as dichlorobenzenesulfonic acid or o-xylene-4-sulfonic acid to precipitate leucine and histidine, it is impossible to ensure that other amino acids will not be precipitated in small amounts.

[0019] Leucine, with the chemical name 2-amino-4-methylvaleric acid, is an organic compound with the chemical formula C6H13NO2, and has three isomers: L-leucine, D-leucine, and racemic leucine. L-leucine is a white crystalline or crystalline powder, which is a non-polar amino acid with a slightly bitter taste, soluble in water, dilute hydrochloric acid, alkaline solution, and carbonate solution, slightly soluble in alcohol, and insoluble in ether. The solubility of leucine in water at 20℃ is 23.7g / L, and the solubility in water at 100℃ can reach more than 40g / L.

[0020] Crystal form refers to the solid form of the same compound molecules formed by a specific arrangement, different crystal forms have different physical and chemical properties, which directly affect the stability, solubility and bioavailability. Different solvents or different crystallization conditions will get different crystal forms, which is the theoretical basis and operation method of crystal form screening. Crystal type mainly includes seven crystal systems (isometric, hexagonal, tetragonal, trigonal, orthorhombic, monoclinic and triclinic) and polymorphism (such as conformational polymorphism, solvate in pharmaceutical field), which is based on the symmetry of crystal or the arrangement of molecules.

[0021] Crystal habit refers to the macroscopic external morphology of crystal. Different crystallization methods of the same crystal may produce different crystal habits. For example, urea recrystallized in different solvents has the same crystal form, but the morphology is quite different. Adjectives commonly used to describe crystal habit include flat plate, flake, needle, isometric, cubic, octahedral, prismatic, pyramidal, spherical, etc. Crystal habit can provide quick and useful information about the performance of the crystallization process: for example, needle-shaped or flaky crystals usually have higher filtration resistance and worse solid flow characteristics in the preparation than cubic crystals. Therefore, it is necessary to obtain larger particle size crystals for needle-shaped or flaky crystal habits.

[0022] The main strategies to improve crystal habit are to select suitable solvents, control the growth rate of crystals, control the supersaturation, control the impurities in the crystallization system, adjust the pH, etc. Different systems have significant selectivity for improving crystal habit, and the scale-up effect of crystal habit improvement conditions is particularly obvious, which can only be determined by experiments. Improving crystal habit is often combined with changing crystal size, particle size distribution, etc. For example, improving fine needle-shaped crystals to thick rod-shaped crystals greatly improves product quality. Crystal habit depends not only on the internal structure of the crystal, but also on the environment of crystal growth, such as solvent, supersaturation, impurities, additives, temperature, solution pH, physical field, etc.

[0023] Crystal habit modifier is an additive that can control the external structure of crystal by adsorption, which can change the crystal growth habit without affecting the crystal system. Under the same external conditions, the same crystal often shows the same external shape, that is, each crystal has its own habit under certain external conditions. This property of crystal is called crystallization habit or crystal habit. Generally, the crystal face of crystal habit conforms to the crystal face with high net density, that is, the crystallization habit of crystal is mainly subject to Bravais law. Crystals of a given substance can be produced in completely different shapes by different methods, but they still belong to the same crystal system. For example, a crystallization method is conducive to the generation of needle-shaped habit, and another method can produce flaky habit.

[0024] Document 4: L-amino acid crystal habit engineering research, author Shi Jia-kang.

[0025] Document 4 studies the effects of different additives on the growth and crystal habit of L-aminopropanoic acid crystals through polycrystalline and single crystal growth experiments. The results show that additive A makes the crystal face perpendicular to the c-axis direction of L-aminopropanoic acid stop growing, and the crystal habit changes from rod-like to block-like. This is because the carboxyl and amino groups in additive A interact with the carboxyl and amino groups exposed on the crystal face, causing additive molecules to adsorb on the crystal face and inhibit its growth. The addition of additives C and D can accelerate the growth of the crystal face perpendicular to the c-axis direction of the crystal, and the crystal habit changes from rod-like to needle-like. This is because the long carbon chains contained in additives C and D are hydrophobic, and their adsorption on the crystal face has a desolvation effect, which accelerates the diffusion of solute to the crystal face and promotes the growth of the crystal face. This paper optimizes the process parameters (evaporation rate, seed, stirring, additive) in the evaporation crystallization process of L-aminopropanoic acid, effectively controls the particle size of L-aminopropanoic acid products, increases the mass ratio of particles in the 20-60 mesh range from 20% to 80%, and adjusts the crystal habit of L-aminopropanoic acid from long rod-like to block-like. The bulk density and flowability of the product are significantly improved.

[0026] As can be seen from document 7, the addition of additive A can affect the crystallization process of L-aminopropanoic acid, and additive A can effectively control the particle size of L-aminopropanoic acid products, adjust the crystal habit of L-aminopropanoic acid from long rod-like to block-like, and thus improve the bulk density and flowability of the L-aminopropanoic acid.

[0027] Document 8: Research on the nucleation and crystal transformation mechanism of L-glutamic acid polymorphs, authors: MX Yin et al.

[0028] Document 8 studies the effects of various amino acid additives on the crystallization and crystal transformation process of glutamic acid, and analyzes the effects of amino acids on the crystal transformation rate based on the molecular structure of the additives. The effects of additive concentration on the crystal habit and crystal transformation of glutamic acid are investigated, and the changes in the nucleation induction period and crystal growth during the crystal transformation process of glutamic acid under different additive concentrations are discussed. The residual amount of additive in glutamic acid crystals is determined by liquid chromatography, and the influence mechanism of the additive on the crystal transformation process of glutamic acid is discussed. For example, the effect of Tailor-made type additives on the crystal habit of benzamide, which can obtain flaky benzamide crystals when crystallized in ethanol, the normal growth mode of the crystals is disturbed when benzamide additives are added, thereby changing the crystal habit.

[0029] Document 8 classifies additives into the following types based on the differences in their influence mechanisms on the crystallization process: Tailor-made type additives, template additives (nylon, polyethylene, polypropylene, polytetrafluoroethylene, glutaric acid single crystal), and composite additives (phosphate, polycarbonate, polysulfate).

[0030] Document 9: Liu X. Effect of Additives on the Crystallization of L-Isoleucine Polymorphs from Solution. Master Thesis, Zhejiang University of Technology, 2016.

[0031] Document 9 studied the crystallization thermodynamic properties and nucleation process of different L-isoleucine polymorphs in solution, focusing on the effect of additives on the crystallization process of L-isoleucine polymorphs in solution. The effects of different additives (L-phenylalanine, L-histidine, glycine, sodium chloride, acetic acid, propionic acid, n-valeric acid) on the nucleation of L-isoleucine polymorphs were explored. The results showed that when sodium chloride, acetic acid, propionic acid, and n-valeric acid were present, the A crystal form was obtained. Finally, the minimum additive dosage that effectively affects the nucleation of L-isoleucine polymorphs was determined as 0.025 mol / L of sodium chloride, 0.125 mol / L of acetic acid, 0.1 mol / L of propionic acid, and 0.025 mol / L of n-valeric acid.

[0032] Document 10: Zhou X. Effect of Additives on the Growth of Amino Acid Crystals. Master Thesis, Zhejiang University of Technology, 2017.

[0033] Document 10 disclosed that most amino acids grow in needle-like or flaky crystal habits, which have low bulk density and poor flowability, seriously affecting the post-processing of the products. The research on the effect of additives on the growth of amino acid crystals proposed a perspective of using molecular simulation to design additives to control the growth of amino acid crystals and thus regulate the crystal habit from the perspective of crystal engineering.

[0034] As Poornachary et al. added custom additives aspartic acid to the glycine crystallization solution, they found that aspartic acid inhibited the growth of α-glycine crystals along the b-axis and c-axis directions. The crystal habit of α-glycine in aqueous solution was columnar bipyramid; after adding a small amount of L-aspartic acid, the crystal habit of α-glycine became prismatic bipyramid; with the increase of aspartic acid concentration, the α-glycine crystal became prismatic.

[0035] Han et al. studied the effect of inorganic salts (NaCl, NaAc, KNO3, CaCl2, MgSO4, etc.) on the growth process of γ-glycine in supersaturated aqueous solution, and found that these inorganic salts could inhibit the growth of γ-glycine crystals along the c-axis direction. The research in Document 7 found that the addition of polymer additives could change the crystal habit of DL-methionine from slender needles to hexagonal plates or blocks with smaller aspect ratios. These three polymers had strong inhibitory effect on the growth of DL-methionine crystals along the b-axis (long) and c-axis (wide) directions.

[0036] The factors affecting the growth of amino acid crystals by adding additives are not single, but the result of the joint regulation of multiple factors, and the mechanism of action is still in the stage of continuous development. Although additives have been widely studied in the regulation of crystal growth, due to the complex and variable mechanism of action of additives on crystal growth, there is still a lack of a regular and efficient crystal growth regulation strategy, and the selection of additives still relies on experience and trial and error.

[0037] From documents 7 to 11, it can be known that crystal form and crystal habit have a very significant influence on the physicochemical properties of the crystal. Due to the difference in crystallization process, different crystal habits can still be obtained under the condition of the same crystal form. Secondly, crystal habit is related to the concentration of the solution, the type and concentration of the solvent, the heating temperature, the stirring rate, the crystallization method and the type of additive during crystallization. At the same time, the type of additive added during the crystallization of each amino acid or each compound does not have commonality, for example, in document 7, additive A is added, and in document 8, the additives are nylon, polyethylene, polypropylene, polytetrafluoroethylene, glutaric acid monocrystal, phosphate, polycarbonate, polysulfate, etc.

[0038] Document 12: CN114773216A - L-leucine spherical crystal and its preparation method and application Document 12 discloses a kind of L-leucine spherical crystal and its preparation method and application, the preparation method includes the following steps: (1) L-leucine raw material 10-25 ℃ is dissolved in acid solution to obtain a mixed solution;(2) add isobutyl acetate to the mixed solution obtained in step (1) and mix, phase separation occurs, stirring at a speed of 250-650 rpm, to obtain a mixed solution dispersed with spherical droplets;(3) keep 250-650 rpm speed, add lye to the mixed solution dispersed with spherical droplets in step (2) until the pH reaches the isoelectric point of L-leucine, and keep for 1-2.5 h, to obtain L-leucine spherical crystal;The L-leucine prepared by the method has regular shape, adjustable size and good flowability.

[0039] Document 12 obtains spherical crystals of L-leucine by adding isobutyl acetate to a mixed solution, dispersing into spherical droplets under stirring treatment, and then precipitating near the isoelectric point. The size of the crystals is 1000-2000 μm. The method disclosed in document 12 produces spherical crystals, but the particle size is large, and the particle size distribution range is also very large. Crystal particle size and particle size distribution are one of the important indicators of crystal products, which mainly affect: ① solid-liquid separation process in production; ② product drying; ③ product purity; ④ product flowability and appearance; ⑤ product bioavailability (API dissolution rate and activity). In the preparation process, the particle size and its distribution are mainly controlled by crystallization and crushing. The large particle size of L-leucine reduces the dissolution rate, and the large particle size of the crystal form is easy to envelop part of the impurities. It is found through research that the particle size of L-leucine is controlled in the range of 500-1000 μm, which is the most suitable interval. This interval has good bulk density, flowability and solubility, and can obtain high-purity products.

[0040] In summary, the prior art has the following problems: (1) The prior art uses ion resin to purify histidine, but the strong acid ion resin is eluted with ammonia water, and ammonia needs to be removed during the treatment of the eluent. Histidine is easy to decompose during the ammonia removal process. If a weak acid ion resin is used, other amino acids, especially those in cationic state, will affect the adsorption process and reduce the equilibrium adsorption rate. (2) The particle size range of the prepared L-leucine is too large, and most of the crystal habit structures are not spherical. SUMMARY

[0041] The purpose of the present application is to provide a method for producing amino acid products from animal protein blood meal hydrolysis. The present application needs to solve the following technical problems: (1) Histidine and leucine can be separated from the protein hydrolysate; (2) When using acid ion resin, the equilibrium adsorption rate is improved; (3) The particle size of leucine crystals is controlled in the range of 500-1000 μm, and the crystal habit structure is spherical.

[0042] To achieve the above purpose, one embodiment of the present application provides a method for producing amino acid products from animal protein blood meal hydrolysis, comprising the following steps: Step S1, adding animal protein raw materials to acid solution for acid hydrolysis, adding alkali to adjust the pH value to 4.5-5.5 after hydrolysis, adding activated carbon for decolorization and filtration, and obtaining a protein hydrolysate after filtration; Step S2, the protein hydrolysate is cooled to normal temperature, and then the histidine crude product is precipitated from the protein hydrolysate, and the remaining mother liquor is obtained after the histidine crude product is separated; Step S3, the histidine crude product is refined to obtain the histidine fine product, and the histidine refining process is as follows: Step S31, the histidine crude product is added to anhydrous ethanol to remove impurities, and then filtered, and the filtered histidine crude product is washed several times with cold water, and the washing water is recovered, mixed with the filtered histidine crude product after the anhydrous ethanol is removed by heating and evaporation, and the histidine pretreated product is obtained. Step S32, the histidine pretreated product is dissolved and adsorbed by using a weak acid ion exchange resin, and then eluted by using a dilute hydrochloric acid solution, and the eluate is treated by activated carbon decolorization, and then evaporated and concentrated to supersaturation, and the histidine fine product is obtained by cooling and crystallization. Step S4, leucine precipitant is added to the remaining mother liquor, and the precipitate is obtained after standing and filtering, and the tail liquid is obtained after washing; the precipitate is added to water and neutralized by using ammonia to adjust the pH value, and the excess ammonia is removed by stirring to obtain the leucine crude product. Step S5, the leucine crude product is dissolved in hot water, concentrated after decolorization and filtration, and the crystal is naturally separated after cooling, and the leucine fine product is obtained by filtering the crystal. Step S6, the leucine fine product is recrystallized to obtain high-purity leucine. Step S7, the tail liquid is dried by using a spray drying technology, and the amino acid mixed powder is obtained after drying.

[0043] In the optimization scheme of the application, the specific method of step S1 is as follows: The animal protein raw material includes animal blood powder, animal hair or animal feather; the animal protein raw material is added into a 4mol / L-5mol / L hydrochloric acid solution, the solid-liquid ratio of the raw material and the hydrochloric acid solution is 1:3-5, the acid hydrolysis temperature is 100-120℃, the acid hydrolysis time is 9-12h, and then the pH value is adjusted to 4.5-5.5 by adding sodium hydroxide solution after hydrolysis is completed, and then the temperature is reduced to 50-70℃, and then activated carbon is added for decolorization and filtration, and then the protein hydrolysate is obtained after filtration.

[0044] In the optimization scheme of the application, in step S2, the pH value of the protein hydrolysate is adjusted to the isoelectric point of histidine by adding lye after the protein hydrolysate is cooled, and then the histidine is precipitated from the protein hydrolysate, and then the histidine crude product and the remaining mother liquor are obtained after standing for 5-10h and filtering.

[0045] In the optimization scheme of the present application, in step S32, the mass ratio of histidine pretreatment product to weak acid ion exchange resin is 1:3-8; the concentration of histidine pretreatment product solution is 15 g / L-30 g / L, the eluent flow rate is 1 ml / min-3 ml / min; and the elution temperature is 25 DEG C-35 DEG C.

[0046] In the optimization scheme of the present application, the specific process of leucine recrystallization in step S6 is as follows: S61, dissolve the leucine product into glycerol-acid solution, and filter using a microporous filter; the filtrate is concentrated by evaporation at 65 DEG C-75 DEG C; S62, under the condition of heat preservation, add alkali to adjust the pH value to 5.9-6.1, continue stirring for 10 min-20 min after the completion of alkali addition; S63, after heating to 80 DEG C-85 DEG C, add sodium alginate and continue stirring for 30 min-60 min, the addition amount of sodium alginate is 0.1%-0.5% of the mass of leucine product, and ultrasonic treatment is used during stirring; S64, then gradient cooling is carried out, the gradient cooling mode is: the temperature is reduced to 40 DEG C at a cooling rate of 20 DEG C / h-25 DEG C / h, then the temperature is reduced to 20 DEG C at a cooling rate of 10 DEG C / h-15 DEG C / h, then the temperature is reduced to 4 DEG C at a cooling rate of 5 DEG C / h-8 DEG C / h, then 4 DEG C is maintained for 12 h-24 h, and the process is ended when the crystal grows to 500 mu m-1000 mu m.

[0047] In the optimization scheme of the present application, the leucine precipitant added in step S4 is o-xylene-4-sulfonic acid, and the addition amount of leucine precipitant is 25%-35% of the mass of the raw material dry weight, the temperature is reduced to 0 DEG C-5 DEG C, the stirring action is 2 h-4 h, then heat preservation is carried out for 18 h-30 h, after the completion of static precipitation, filtration is carried out, the filter cake is washed and then filtered dry to obtain a precipitate and a tail liquid; The precipitate is added into 3-4 times the volume of ammonia water to adjust the pH to 7-8, the temperature is maintained at 70 DEG C-80 DEG C, excess ammonia is removed by vacuum stirring for 50 min-80 min, then the temperature is cooled to room temperature, and after standing for 12 h-20 h, filtration is carried out, the filter cake is washed several times with deionized water and then filtered dry to obtain a leucine crude product.

[0048] In the optimization scheme of the present application, in step S5, leucine is added to 30-40 times the mass of deionized water, heated to 50 DEG C-70 DEG C for dissolution, activated carbon is added for decolorization, after decolorization, filtration is carried out, the filtrate is concentrated under vacuum negative pressure at 70 DEG C-80 DEG C, and after cooling, the crystal is naturally precipitated, washed several times with deionized water, and then filtered dry to obtain a leucine product.

[0049] In the optimized scheme of the present invention, the glycerol-acidic solution in step S6 is a hydrochloric acid solution of glycerol, the concentration of hydrochloric acid in the glycerol-acidic solution is 3mol / L~4mol / L, and the concentration of glycerol is 0.1mol / L~0.3mol / L; the dissolution temperature of the refined leucine is 15℃~25℃.

[0050] In the optimized embodiment of the present invention, the pore size of the microporous membrane is 0.15μm~0.30μm; the filtrate is evaporated and concentrated to a leucine concentration of 25g / L~30g / L.

[0051] In the optimized scheme of this invention, the alkaline solution in step S6 is a sodium hydroxide solution with a concentration of 5 mol / L to 8 mol / L and a dropping rate of 10 ml / min to 20 ml / min; the stirring speed in step S6 is 350 rpm to 500 rpm; the ultrasonic power is 150 W to 200 W and the ultrasonic frequency is 20 kHz to 40 kHz; after step S43, the mixture is filtered and then dried under negative pressure at 50℃ to 60℃ to obtain leucine crystals. In steps S1 and S32, the amount of activated carbon added is 2% to 3% of the solution mass, and the decolorization time is 20 min to 30 min.

[0052] In summary, the present invention has the following advantages: This invention can directly separate histidine and leucine from protein hydrolysate, with good crystal purity. It overcomes the equilibrium adsorption rate of histidine by impurity amino acids in weakly acidic ion exchange resins. At the same time, by adjusting the recrystallization process of leucine, the crystal particle size of the prepared leucine is controlled within a suitable range, resulting in good bulk density and flowability. Attached Figure Description

[0053] figure 1 These are the experimental results of histidine equilibrium adsorption rate in Examples A1, A2, and Comparative Example A1 of the present invention; figure 2 This is a scanning electron microscope (SEM) image of embodiment C1 of the present invention; figure 3 This is a scanning electron microscope (SEM) image of embodiment C2 of the present invention; figure 4 This is a particle size distribution diagram of embodiment C1 of the present invention; figure 5 This is a particle size distribution diagram of embodiment C2 of the present invention; figure 6 Here is a SEM image of comparative experimental example 1 of this invention; figure 7 Here is a SEM image of comparative experimental example 1 of this invention; figure 8 The electron microscope SEM graph of the comparative experimental example 1 of the present application; figure 9 The electron microscope SEM graph of the comparative experimental example 1 of the present application. DETAILED DESCRIPTION

[0054] The present application provides a method for generating amino acid fine products by hydrolyzing animal protein blood meal, comprising the following steps: Step S1, adding animal protein raw materials into acid liquid for acid hydrolysis, after hydrolysis, adding alkali to adjust the pH value to 4.5-5.5, adding activated carbon for decolorization and filtration, obtaining protein hydrolysate after filtration; Step S2, reducing the protein hydrolysate to normal temperature, then precipitating histidine crude product from the protein hydrolysate, and obtaining remaining mother liquor after separating the histidine crude product; Step S3, refining the histidine crude product to obtain histidine fine product, and the histidine refining process is as follows: Step S31, adding the histidine crude product into anhydrous ethanol to remove impurities, then filtering, washing several times with cold water after filtration, recovering the washing water, mixing the washing water with the filtered histidine crude product after removing ethanol by heating and evaporation, to obtain histidine pretreated product; Step S32, dissolving the histidine pretreated product, then adsorbing by using weak acid ion exchange resin, after adsorption, eluting by using dilute hydrochloric acid solution, combining the eluate, then decolorizing the eluate by activated carbon, evaporating and concentrating the eluate to supersaturation, cooling and crystallizing to obtain histidine fine product; Step S4, adding leucine precipitant into the remaining mother liquor, filtering after standing and precipitating, then washing to obtain precipitate and tail liquid; adding the precipitate into water, neutralizing and adjusting the pH value by using ammonia, stirring to remove excess ammonia, to obtain leucine crude product; Step S5, dissolving the leucine crude product in hot water, concentrating after decolorizing and filtering, then naturally separating crystals after cooling, filtering and drying the crystals to obtain leucine fine product; Step S6, recrystallizing the leucine fine product to obtain high-purity leucine; Step S7, drying the tail liquid by using spray drying technology, to obtain amino acid mixed powder after drying.

[0055] In the present application, ion exchange resin is used for adsorption and purification process, the ion resin is packed into column, the solution flows through the ion resin, then the content of histidine in the effluent sample is detected. In the sample loading process, when histidine appears in the effluent sample, it indicates that the histidine adsorption process has been completed, then elution is carried out by using eluent, the eluate is collected, the content of histidine in the effluent sample is detected during the elution process, when the concentration of histidine in the elution sample is 0, it indicates that the elution is completed, then the elution can be stopped.

[0056] Example A: Preparation method of histidine fine product Step S1, the animal protein raw material includes animal blood meal, animal hair or animal feather; the animal protein raw material is added into a 4 mol / L-5 mol / L hydrochloric acid solution, the solid-liquid ratio of the raw material and the hydrochloric acid solution is 1:3-5, the acid hydrolysis temperature is 100°C-120°C, the acid hydrolysis time is 9 h-12 h, after hydrolysis is completed, a sodium hydroxide solution is added to adjust the pH value to 4.5-5.5, the temperature is reduced to 50°C-70°C, and then activated carbon is added for decolorization and filtration, and the protein hydrolysate is obtained after filtration.

[0057] Step S2, the protein hydrolysate is cooled to room temperature, and then a lye is added to adjust the pH value to the isoelectric point of histidine, so that the histidine is precipitated from the protein hydrolysate, and the histidine crude product and the remaining mother liquor are obtained after filtration after standing for 5 h-10 h.

[0058] Step S3, the histidine crude product is refined to obtain a histidine fine product, and the histidine refining process is as follows: Step S31, the histidine crude product is added to anhydrous ethanol to remove impurities, and then filtered, and the filtered histidine crude product is washed with cold water for several times, the washing water is recovered, and after the ethanol is removed by heating and evaporation, the washing water is mixed with the filtered histidine crude product to obtain a histidine pretreated product; Step S32, the histidine pretreated product is dissolved and adsorbed by using a weak acid ion exchange resin, after adsorption is completed, a dilute hydrochloric acid solution is used for elution, the eluate is combined, and then subjected to activated carbon decolorization treatment, and then the eluate is evaporated and concentrated to supersaturation, and cooled to crystallize to obtain a histidine fine product. In step S32, the mass ratio of the histidine pretreated product to the weak acid ion exchange resin is 1:3-8; the concentration of the histidine pretreated product solution is 15 g / L-30 g / L, the elution flow rate is 1 ml / min-3 ml / min; and the elution temperature is 25°C-35°C.

[0059] Example A1: Preparation method of histidine fine product Step S1, the animal protein raw material includes animal blood meal, animal hair or animal feather; the animal protein raw material is added into a 4 mol / L-5 mol / L hydrochloric acid solution, the solid-liquid ratio of the raw material and the hydrochloric acid solution is 1:3-5, the acid hydrolysis temperature is 100°C-120°C, the acid hydrolysis time is 9 h-12 h, after hydrolysis is completed, a sodium hydroxide solution is added to adjust the pH value to 4.5-5.5, the temperature is reduced to 50°C-70°C, and then activated carbon is added for decolorization and filtration, and the protein hydrolysate is obtained after filtration.

[0060] Step S2, the protein hydrolysate is cooled to room temperature, and an alkali solution is added to adjust the pH value of the protein hydrolysate to the isoelectric point of histidine, so that the histidine is precipitated from the protein hydrolysate, and the histidine crude product and the remaining mother liquor are obtained after standing for 8 hours and filtering.

[0061] Step S3, the histidine crude product is refined to obtain a histidine fine product, and the histidine refining process is as follows: Step S31, the histidine crude product is added to anhydrous ethanol to remove impurities, and then filtered, and the filtered histidine crude product is washed with cold water for several times, and the washing water is recovered, evaporated to remove ethanol, and then mixed with the filtered histidine crude product to obtain a histidine pretreated product; Step S32, the histidine pretreated product is dissolved and adsorbed by using a weak acid ion exchange resin, and then eluted by using a dilute hydrochloric acid solution, and the eluate is decolorized by activated carbon, and then evaporated and concentrated to supersaturation, and cooled to crystallize to obtain the histidine fine product. In step S32, the mass ratio of the histidine pretreated product to the weak acid ion exchange resin is 1:5; the concentration of the histidine pretreated product solution is 20 g / L, the elution flow rate is 2 ml / min; and the elution temperature is 28°C.

[0062] The yield of the histidine of example A1 is 7.5%.

[0063] Example A2: Step S1, the animal protein raw material includes animal blood powder, animal hair or animal feather; the animal protein raw material is added to a 4 mol / L hydrochloric acid solution, the solid-liquid ratio of the raw material to the hydrochloric acid solution is 1:3, the acid hydrolysis temperature is 100°C, the acid hydrolysis time is 12 hours, and after the hydrolysis is completed, a sodium hydroxide solution is added to adjust the pH value to 4.5, and then activated carbon is added for decolorization and filtration after the temperature is reduced to 60°C, and the protein hydrolysate is obtained after filtration.

[0064] Step S2, the protein hydrolysate is cooled to room temperature, and an alkali solution is added to adjust the pH value of the protein hydrolysate to the isoelectric point of histidine, so that the histidine is precipitated from the protein hydrolysate, and the histidine crude product and the remaining mother liquor are obtained after standing for 8 hours and filtering.

[0065] Step S3, the histidine crude product is refined to obtain a histidine fine product, and the histidine refining process is as follows: Step S31, the histidine crude product is added to anhydrous ethanol to remove impurities, and then filtered, and the filtered histidine crude product is washed with cold water for several times, and the washing water is recovered, evaporated to remove ethanol, and then mixed with the filtered histidine crude product to obtain a histidine pretreated product; Step S32, after the histidine pretreatment product is dissolved, it is adsorbed by using a weak acid ion exchange resin, after the adsorption is completed, it is eluted by using a dilute hydrochloric acid solution, the eluate is combined, the eluate is subjected to activated carbon decolorization treatment, and then the eluate is evaporated and concentrated to supersaturation, and cooled crystallization is performed to obtain the histidine fine product. In step S32, the mass ratio of the histidine pretreatment product to the weak acid ion exchange resin is 1:3; the concentration of the histidine pretreatment product solution is 15 g / L, the eluate flow rate is 1 ml / min; and the elution temperature is 25°C.

[0066] The yield of the histidine of example A1 is 8.3%.

[0067] The histidine fine products obtained from example A1 and example A2 are detected, and the detection method of the histidine is performed according to the method specified in the “People's Republic of China Pharmacopoeia” (2000 edition, part II). The results of the detection of the extracted histidine sample are as follows. From the above detection results, it can be seen that the histidine prepared by the hydrolysis method of the present application meets the requirements of the national pharmacopoeia.

[0068] Comparative example A1 Preparation method of histidine fine product Step S1, the animal protein raw material includes animal blood powder, animal hair or animal feather; the animal protein raw material is added into a 4.5 mol / L hydrochloric acid solution, the solid-liquid ratio of the raw material to the hydrochloric acid solution is 1:4, the acid hydrolysis temperature is 110°C, the acid hydrolysis time is 10 h, after the hydrolysis is completed, a sodium hydroxide solution is added to adjust the pH value to 4.8, the temperature is reduced to 65°C, and then activated carbon is added for decolorization and filtration. After filtration, a protein hydrolysate is obtained.

[0069] Step S2, the protein hydrolysate is cooled to room temperature, and then a lye is added to adjust the pH value to the isoelectric point of histidine, so that the histidine is precipitated from the protein hydrolysate. After standing for 8 h, filtration is performed to obtain a histidine crude product and a remaining mother liquor.

[0070] Step S3, the histidine crude product is refined to obtain a histidine fine product. The histidine refining process is as follows: Step S31, the histidine crude product is washed with cold water for several times, the washing water is recovered, the washing water is heated and evaporated to remove ethanol, and then the washing water is mixed with the filtered histidine crude product to obtain a histidine pretreatment product. Step S32, after the histidine pretreatment product is dissolved, it is adsorbed using a weakly acidic ion exchange resin, after the adsorption is completed, it is eluted using a dilute hydrochloric acid solution, the eluate is combined, the eluate is subjected to activated carbon decolorization treatment, and then the eluate is evaporated and concentrated to supersaturation, and cooled crystallization is performed to obtain a histidine fine product. In step S32, the mass ratio of the histidine pretreatment product to the weakly acidic ion exchange resin is 1:5; the concentration of the histidine pretreatment product solution is 20 g / L, and the eluate flow rate is 2 ml / min; the elution temperature is 28°C.

[0071] Experimental Example A: Effect of different purification methods on the equilibrium adsorption rate of histidine Method for measuring the equilibrium adsorption rate: According to a volume-to-mass ratio of 5:1 of the solution to the weakly acidic ion exchange resin, 500 ml of a histidine solution and 100 g of 732 cation exchange resin were added to a jacketed triangular flask, which was placed on an electromagnetic stirrer for stirring and mixing. The temperature was controlled at 30°C by a constant-temperature water tank, and the pH value was adjusted to 5.0 by 0.1 mol / L sulfuric acid. Every 20 min, 1 ml of sample was taken, and the mass concentration of histidine therein was analyzed to obtain the equilibrium adsorption rate. Equilibrium adsorption rate = (concentration before adsorption - concentration after adsorption) / concentration before adsorption.

[0072] The equilibrium adsorption rates of histidine of the three examples were calculated using the method disclosed in the above prior art for Example A1, Example A2, and Comparative Example A1, and the specific results are shown in Table 1. figure 1

[0073] From the above experimental results, it can be seen that after the crude histidine product is purified using anhydrous ethanol, alcohol-soluble amino acids and other impurities can be eliminated, and the equilibrium adsorption rate of histidine can be improved to some extent. The solubility of histidine in ethanol is very low, but it has a certain solubility in water, so the solvent for purification should not be an ethanol solution but anhydrous ethanol. During isoelectric point precipitation, the purity of the precipitate is not enough, so using anhydrous ethanol to remove alcohol-soluble impurities and other amino acids can remove impurities while preserving histidine as much as possible, avoiding the loss of histidine in the solvent, and thus improving the yield.

[0074] Example B1: Preparation of leucine fine product Step S4, leucine precipitant was added to the remaining mother liquor, and after standing and precipitation, filtration was performed, and the precipitate was obtained after washing. The precipitate was added to water and neutralized using ammonia to adjust the pH value, and excess ammonia was removed by stirring to obtain a leucine crude product.

[0075] ​The leucine precipitant added in step S4 is o-xylene-4-sulfonic acid, and the amount of leucine precipitant added is 30% of the dry weight of the raw material. The temperature is reduced to 3°C, stirring is performed for 3h, and then the temperature is maintained for 22h of standing and precipitation. After standing and precipitation is completed, filtration is performed, the filter cake is washed with deionized water, and then filtered dry to obtain the precipitate and tail liquid. The tail liquid produced after filtration in step S2 is dried using a spray drying technique, and after drying, an amino acid mixed powder is obtained.

[0076] The precipitate is added to 4 times the volume of ammonia water, the pH is adjusted to 7.5, the temperature is maintained at 75°C, vacuum stirring is performed for 60min to remove excess ammonia, then cooled to room temperature, and after standing for 16h, filtration is performed. The filter cake is washed several times with deionized water and then filtered dry to obtain the leucine crude product.

[0077] In step S5, the leucine crude product is added to hot water to dissolve, decolorized, filtered, and then concentrated. The amount of activated carbon added for decolorization is 2.5% of the mass of the solution, and the decolorization time is 25min. After cooling, the crystals are naturally precipitated, and the crystals are filtered dry to obtain the leucine fine product. In step S5, the leucine is added to 32 times the mass of deionized water, heated to 58°C to dissolve, activated carbon is added for decolorization, filtration is performed after decolorization, the filtrate is heated to 76°C and concentrated under vacuum negative pressure, and after cooling, the crystals are naturally precipitated. The crystals are washed several times with deionized water and then filtered dry to obtain the leucine fine product.

[0078] Example B2: Preparation of leucine fine product In step S4, a leucine precipitant is added to the remaining mother liquor, and after standing and precipitation, filtration is performed. After filtration, the precipitate is washed to obtain the precipitate; the precipitate is added to water, and ammonia water is used to adjust the pH value. Excess ammonia is removed by stirring to obtain the leucine crude product.

[0079] The leucine precipitant added in step S4 is o-xylene-4-sulfonic acid, and the amount of leucine precipitant added is 25% of the dry weight of the raw material. The temperature is reduced to 5°C, stirring is performed for 4h, and then the temperature is maintained for 30h of standing and precipitation. After standing and precipitation is completed, filtration is performed, the filter cake is washed with deionized water, and then filtered dry to obtain the precipitate and tail liquid. The tail liquid produced after filtration in step S2 is dried using a spray drying technique, and after drying, an amino acid mixed powder is obtained.

[0080] The precipitate is added to 3~4 times the volume of ammonia water, the pH is adjusted to 7, the temperature is maintained at 70°C, vacuum stirring is performed for 50min to remove excess ammonia, then cooled to room temperature, and after standing for 12h, filtration is performed. The filter cake is washed several times with deionized water and then filtered dry to obtain the leucine crude product.

[0081] Step S5, the leucine crude product is added into hot water to dissolve, and then concentrated after decolorization and filtration. The amount of activated carbon added for decolorization is 2% of the mass of the solution, and the decolorization time is 20 min. After cooling, the crystals are naturally precipitated, and the crystals are filtered and dried to obtain the leucine fine product. In step S5, the leucine is added into 30 times the mass of deionized water, heated to 50℃ to dissolve, activated carbon is added for decolorization, and then filtered after decolorization. The filtrate is heated to 70℃ and concentrated under vacuum negative pressure, and then the crystals are naturally precipitated after cooling. The crystals are washed several times with deionized water and then filtered and dried to obtain the leucine fine product.

[0082] Example B3: Preparation of leucine fine product Step S4, a leucine precipitant is added to the remaining mother liquor, and then the precipitate is obtained after standing and precipitation and filtration. The precipitate is added into water, neutralized with ammonia water to adjust the pH value, and then the excess ammonia is removed by stirring treatment to obtain the leucine crude product.

[0083] The leucine precipitant added in step S4 is o-xylene-4-sulfonic acid, and the amount of the leucine precipitant added is 35% of the mass of the dry weight of the raw material. The temperature is reduced to 5℃, stirred for 2 h, and then kept standing and precipitating for 18 h. After the standing and precipitation is completed, the filter cake is washed with deionized water and then filtered and dried to obtain the precipitate and tail liquid. The tail liquid produced after filtration in step S2 is dried using a spray drying technology, and then an amino acid mixed powder is obtained after drying.

[0084] The precipitate is added into 3-4 times the volume of ammonia water to adjust the pH value to 8, the temperature is kept at 80℃, the excess ammonia is removed by stirring treatment for 80 min under vacuum, and then cooled to room temperature. After standing for 20 h, the filter cake is washed several times with deionized water and then filtered and dried to obtain the leucine crude product.

[0085] Step S5, the leucine crude product is added into hot water to dissolve, and then concentrated after decolorization and filtration. The amount of activated carbon added for decolorization is 3% of the mass of the solution, and the decolorization time is 30 min. After cooling, the crystals are naturally precipitated, and the crystals are filtered and dried to obtain the leucine fine product. In step S5, the leucine is added into 40 times the mass of deionized water, heated to 70℃ to dissolve, activated carbon is added for decolorization, and then filtered after decolorization. The filtrate is heated to 80℃ and concentrated under vacuum negative pressure, and then the crystals are naturally precipitated after cooling. The crystals are washed several times with deionized water and then filtered and dried to obtain the leucine fine product.

[0086] Physical and chemical detection of leucine fine product: The detection method of leucine is carried out according to the method specified in the “People's Republic of China Pharmacopoeia” (2000 edition, part 2). The results of the detection of the L-leucine sample are as follows. From the above detection results, it can be seen that the leucine prepared by hydrolysis from animal hair or feather in the method of the present application meets the requirements of the national pharmacopoeia.

[0087] Example C: Recrystallization of leucine In Example C of the present application, a plurality of sub-examples are included, and the leucine fines used in each sub-example of Example C are prepared by the method of Example B1.

[0088] Step S6: Recrystallization of leucine fines to obtain high-purity leucine. The recrystallization steps are as follows: S61: Dissolve the leucine fines into a glycerol-acid solution, which is a hydrochloric acid solution of glycerol, with a hydrochloric acid concentration of 3 mol / L to 4 mol / L and a glycerol concentration of 0.1 mol / L to 0.3 mol / L. The dissolution temperature of the leucine fines is 15°C to 25°C.

[0089] Then filter using a microporous filter membrane with a pore size of 0.15 μm to 0.30 μm. Evaporate and concentrate the filtrate at 65°C to 75°C. Evaporate and concentrate the filtrate to a leucine concentration of 25 g / L to 30 g / L.

[0090] S62: Under the condition of heat preservation, add a lye to adjust the pH value to 5.9 to 6.1. The lye is a sodium hydroxide solution with a concentration of 5 mol / L to 8 mol / L. The dropwise addition rate of the sodium hydroxide solution is 10 ml / min to 20 ml / min. After the addition of the lye is complete, continue stirring for 10 min to 20 min at a stirring speed of 350 rpm to 500 rpm.

[0091] S63: After heating to 80°C to 85°C, add sodium alginate and continue stirring for 30 min to 60 min. The addition amount of sodium alginate is 0.1% to 0.5% of the mass of the leucine fines. Ultrasonic treatment is used during stirring. The power of the ultrasonic wave is 150 W to 200 W, and the frequency of the ultrasonic wave is 20 kHz to 40 kHz.

[0092] S64: Then perform gradient cooling. The gradient cooling method is as follows: start with a cooling rate of 20°C / h to 25°C / h to reduce the temperature to 40°C, then with a cooling rate of 10°C / h to 15°C / h to reduce the temperature to 20°C, then with a cooling rate of 5°C / h to 8°C / h to reduce the temperature to 4°C, and then maintain at 4°C for 12 h to 24 h. When the crystal grows to 500 μm to 1000 μm, stop. After stopping, filter and then perform negative pressure drying at 50°C to 60°C to obtain leucine crystals.

[0093] Example C1: Recrystallization of leucine S61, dissolve the leucine concentrate into a glycerol-acid solution, the glycerol-acid solution is a hydrochloric acid solution of glycerol, the concentration of hydrochloric acid in the glycerol-acid solution is 3 mol / L, and the concentration of glycerol is 0.18 mol / L; the dissolution temperature of the leucine concentrate is 20°C.

[0094] Then filter using a microporous filter membrane with a pore size of 0.20 μm; the filtrate is concentrated by evaporation at 70°C; the filtrate is concentrated by evaporation to a leucine concentration of 28 g / L.

[0095] S62, under the condition of heat preservation, add alkali to adjust the pH value to 5.9-6.1, the alkali is a sodium hydroxide solution, the concentration of the sodium hydroxide solution is 6 mol / L, and the dropwise adding speed of the sodium hydroxide solution is 12 ml / min. After the alkali is added, continue to stir for 15 min, and the stirring speed is 400 rpm.

[0096] S63, after heating to 82°C, add sodium alginate and continue to stir for 40 min, the amount of sodium alginate added is 0.3% of the mass of the leucine concentrate, and ultrasonic treatment is used during stirring; the power of the ultrasonic wave is 160 W, and the frequency of the ultrasonic wave is 30 kHz.

[0097] S64, then perform gradient cooling, the gradient cooling mode is: start with a cooling rate of 22°C / h to reduce the temperature to 40°C, then with a cooling rate of 12°C / h to reduce the temperature to 20°C, then with a cooling rate of 6°C / h to reduce the temperature to 4°C, then maintain at 4°C for 20 h, and the crystal growth is stopped when the crystal reaches 700 μm. After the end, filter, then perform negative pressure drying at 55°C to obtain leucine crystals.

[0098] Example C2: Recrystallization of leucine S61, dissolve the leucine concentrate into a glycerol-acid solution, the glycerol-acid solution is a hydrochloric acid solution of glycerol, the concentration of hydrochloric acid in the glycerol-acid solution is 4 mol / L, and the concentration of glycerol is 0.1 mol / L; the dissolution temperature of the leucine concentrate is 25°C.

[0099] Then filter using a microporous filter membrane with a pore size of 0.30 μm; the filtrate is concentrated by evaporation at 75°C; the filtrate is concentrated by evaporation to a leucine concentration of 25 g / L.

[0100] S62, under the condition of heat preservation, add alkali to adjust the pH value to 5.9-6.1, the alkali is a sodium hydroxide solution, the concentration of the sodium hydroxide solution is 5 mol / L, and the dropwise adding speed of the sodium hydroxide solution is 20 ml / min. After the alkali is added, continue to stir for 20 min, and the stirring speed is 500 rpm.

[0101] S63, after heating to 80℃-85℃, sodium alginate is added and stirring is continued for 60 min, the addition amount of sodium alginate is 0.1% of the quality of leucine concentrate, and ultrasonic treatment is used in the stirring process; the use power of the ultrasonic wave is 150W, and the ultrasonic wave frequency is 20khz.

[0102] S64, then gradient cooling is carried out, the gradient cooling mode is: the temperature is reduced to 40℃ at a cooling rate of 20℃ / h, then the temperature is reduced to 20℃ at a cooling rate of 10℃ / h, then the temperature is reduced to 4℃ at a cooling rate of 8℃ / h, then 4℃ is maintained for 24h, and the crystal growth is ended when the crystal is 600μm. After ending, filtration is carried out, then negative pressure drying is carried out at 60℃, and leucine crystals are obtained.

[0103] Experimental example: the sample of example C1 and example C2 is subjected to characterization analysis I. Sample electron microscope scanning First, the scanning electron microscope observation sample is prepared, an appropriate amount of sample is picked and fixed on a sample disc, gold spraying is carried out on the sample under the protection of nitrogen at a voltage of 15kV. The surface morphology of the sample is analyzed by using a TM3000 scanning electron microscope (Hitachi).

[0104] II. Particle size distribution Analysis method: (1) Image analysis software uses ImageJ software to process scanning electron microscope SEM pictures into any one of processing TIFF, PNG, GIF, JPEG, BMP formats. Click File-Open in the menu bar of ImageJ, find the TIF or JPG file to be opened, and open it. (2) Set the ruler, find the line drawing tool in the toolbar, and then draw a straight line that coincides with the length of the ruler. (3) Use the line drawing tool to draw the diameter of a particle, and the particle size statistics should be at least 100 nanometer particles. The analyze particles option of the application software can obtain the roundness (sphericity), diameter, area and perimeter of the spherical crystal image. The particle size distribution of the sample is calculated.

[0105] (3) Bulk density detection Detection method: the sample is dried to constant weight, 100g of the sample is weighed, the sample is filled into a 20mL standard container measuring cylinder, and is slowly added to the container through a funnel free-fall method, the filling height needs to exceed the container mouth, and then a ruler is used to scrape flat to be flush with the container mouth. The total mass of the container and the sample is weighed, the sample net weight is obtained by deducting the mass of the container, and then the bulk density of the sample is calculated.

[0106] Characterization analysis results: The electron microscope scanning SEM diagram of example C1 and example C2 of the application is as follows: figure 2 and figure 3The particle size cumulative distribution diagram is shown in Figure 2. figure 4 and figure 5 As shown in the figure, the crystal habit of the leucine obtained in the embodiment C of the present application is spherical, and the particle size distribution is mainly between 500 μm and 1000 μm. After testing, the bulk density of the sample of the embodiment C1 is 1.12 g / cm 3 , and the bulk density of the sample of the embodiment C2 is 1.13 g / cm 3 , both of which have good fluidity.

[0107] Comparative experimental example one: In order to verify the influence of different processes on the crystal habit in the recrystallization process, different process parameters and additives are used in different comparative experimental examples. All the comparative experimental examples of the present application are based on the embodiment C1, that is, all the experimental processes are changed on the basis of the embodiment C1, and the embodiment C1 is used as a comparison object. In the comparative experimental examples, the dissolution temperature, the micro-porous filter process, the concentration process, the addition method of the alkali solution and the stirring speed do not change, that is, these process procedures in each comparative experimental example remain consistent with the embodiment C1.

[0108] Process basis of comparative experimental example: S61, dissolving the leucine fine product into a glycerol-acid solution, the glycerol-acid solution is a hydrochloric acid solution of glycerol, the concentration of hydrochloric acid in the glycerol-acid solution is 3 mol / L, and the concentration of glycerol is 0.18 mol / L; the dissolution temperature of the leucine fine product is 20℃.

[0109] Then filter using a micro-porous filter, and evaporate and concentrate the filtrate.

[0110] S62, adding an alkali solution to adjust the pH value to 5.9-6.1, and continuing to stir after the addition of the alkali solution is completed.

[0111] S63, heating to 82℃ and then adding sodium alginate to continue stirring for 40 min, the addition amount of sodium alginate is 0.3% of the mass of the leucine fine product, and ultrasonic treatment is used during the stirring process; the power of the ultrasonic wave is 160W, and the frequency of the ultrasonic wave is 30khz.

[0112] S64, then gradient cooling, the gradient cooling method is: starting with a cooling rate of 22℃ / h to reduce the temperature to 40℃, then with a cooling rate of 12℃ / h to reduce the temperature to 20℃, then with a cooling rate of 6℃ / h to reduce the temperature to 4℃, and then maintaining at 4℃ for 20h, and ending when the crystal growth reaches 700μm. After ending, filtering, and then negative pressure drying at 55℃, the leucine crystal is obtained.

[0113] Comparative experimental example one: S61, dissolve the leucine concentrate into an acid solution with a concentration of 3 mol / L of hydrochloric acid. Then filter using a microporous filter and evaporate and concentrate the filtrate.

[0114] S62, add a lye to adjust the pH value to 5.9-6.1, and continue stirring after the lye is added.

[0115] S63, after heating to 82℃, add sodium alginate and continue stirring for 40 min, the amount of sodium alginate added is 0.3% of the mass of the leucine concentrate, and ultrasonic treatment is used during stirring; the power of the ultrasonic treatment is 160W, and the frequency of the ultrasonic treatment is 30khz.

[0116] S64, then perform gradient cooling, the gradient cooling method is: start with a cooling rate of 22℃ / h to reduce the temperature to 40℃, then with a cooling rate of 12℃ / h to reduce the temperature to 20℃, then with a cooling rate of 6℃ / h to reduce the temperature to 4℃, then maintain at 4℃ for 20h, and end when the crystal grows to 700μm. After ending, filter, then perform negative pressure drying at 55℃ to obtain leucine crystals.

[0117] Comparative Experimental Example 2: S61, dissolve the leucine concentrate into a glycerol-acid solution, the glycerol-acid solution is a glycerol hydrochloric acid solution, the concentration of hydrochloric acid in the glycerol-acid solution is 3 mol / L, and the concentration of glycerol is 0.18 mol / L; the dissolution temperature of the leucine concentrate is 20℃.

[0118] Then filter using a microporous filter and evaporate and concentrate the filtrate.

[0119] S62, add a lye to adjust the pH value to 5.9-6.1, and continue stirring after the lye is added.

[0120] S63, after heating to 82℃, continue stirring for 40 min, and ultrasonic treatment is used during stirring; the power of the ultrasonic treatment is 160W, and the frequency of the ultrasonic treatment is 30khz.

[0121] S64, then perform gradient cooling, the gradient cooling method is: start with a cooling rate of 22℃ / h to reduce the temperature to 40℃, then with a cooling rate of 12℃ / h to reduce the temperature to 20℃, then with a cooling rate of 6℃ / h to reduce the temperature to 4℃, then maintain at 4℃ for 20h, and end when the crystal grows to 700μm. After ending, filter, then perform negative pressure drying at 55℃ to obtain leucine crystals.

[0122] Comparative Experimental Example 3: S61, dissolve the leucine concentrate into a glycerol-acid solution, the glycerol-acid solution is a hydrochloric acid solution of glycerol, the concentration of hydrochloric acid in the glycerol-acid solution is 3 mol / L, and the concentration of glycerol is 0.18 mol / L; the dissolution temperature of the leucine concentrate is 20 DEG C.

[0123] Then, filtration is performed using a microporous filter, and the filtrate is concentrated by evaporation.

[0124] S62, add lye to adjust the pH value to 5.9-6.1, continue stirring after the lye is added.

[0125] S63, after heating to 82 DEG C, add sodium alginate and continue stirring for 40 min, the addition amount of sodium alginate is 0.3% of the mass of the leucine concentrate.

[0126] S64, then gradient cooling is performed, the gradient cooling mode is: the temperature is reduced to 40 DEG C at a cooling rate of 22 DEG C / h, then the temperature is reduced to 20 DEG C at a cooling rate of 12 DEG C / h, then the temperature is reduced to 4 DEG C at a cooling rate of 6 DEG C / h, then 4 DEG C is maintained for 20 h, and the crystal growth is stopped when the crystal reaches 700 mu m. After the end, filtration is performed, and then negative pressure drying is performed at 55 DEG C, to obtain leucine crystals.

[0127] Comparative experimental example 4: S61, dissolve the leucine concentrate into a glycerol-acid solution, the glycerol-acid solution is a hydrochloric acid solution of glycerol, the concentration of hydrochloric acid in the glycerol-acid solution is 3 mol / L, and the concentration of glycerol is 0.18 mol / L; the dissolution temperature of the leucine concentrate is 20 DEG C.

[0128] Then, filtration is performed using a microporous filter, and the filtrate is concentrated by evaporation.

[0129] S62, add lye to adjust the pH value to 5.9-6.1, continue stirring after the lye is added.

[0130] S63, after heating to 82 DEG C, add sodium alginate and continue stirring for 40 min, the addition amount of sodium alginate is 0.3% of the mass of the leucine concentrate.

[0131] S64, then gradient cooling is performed, the gradient cooling mode is: the temperature is reduced to 40 DEG C at a cooling rate of 22 DEG C / h, then the temperature is reduced to 20 DEG C at a cooling rate of 12 DEG C / h, then the temperature is reduced to 4 DEG C at a cooling rate of 6 DEG C / h, then 4 DEG C is maintained for 20 h, and the crystal growth is stopped when the crystal reaches 700 mu m. After the end, filtration is performed, and then negative pressure drying is performed at 55 DEG C, to obtain leucine crystals.

[0132] The difference control of the comparative experimental example and example C1 of the present application is shown as follows: The samples obtained in Comparative Examples 1 to 4 were subjected to electron microscopy, and the particle size distribution of each sample was determined by electron microscopy. The particle size distributions of Comparative Examples 1 to 4 are shown below: group particle size distribution range μm average particle size μm comparative experimental example 1 860-1530 1250 comparative experimental example 2 750-1230 960 comparative experimental example 3 680-1650 1050 comparative experimental example 4 930-1650 1180 The detection and analysis results show that none of the comparative experiments 1, 2, 3, and 4 were able to simultaneously obtain samples with the target particle size and crystal habit characteristics. figures 6 to 9 As can be seen from the data, the leucine crystals in Comparative Experiments 1, 2, and 4 did not exhibit a spherical crystal habit; most were plate-like or rod-shaped. However, the grain size distribution of these three examples ranged from 600 μm to 1700 μm, overlapping with the grain size distribution of 500 μm to 1000 μm, indicating that the aforementioned comparative experiments could reduce the grain size to some extent. The crystals in Comparative Experiment 3 exhibited a spherical crystal habit, but their grain size distribution range was wider. Compared to Example C1, this demonstrates that ultrasound can reduce the grain size to some extent in this case, resulting in a smaller grain size range and better uniformity.

[0133] In summary, it can be seen that Embodiment C1 of the present invention, by optimizing the solution to be a glycerol-acidic solution, adding sodium alginate, and employing a gradient cooling method, can obtain spherical crystal habit and reduce the grain size compared to the prior art. Furthermore, the ultrasonic treatment method used in S63 further reduces the particle size through ultrasonic oscillation, thereby achieving a crystal grain size of 500μm~1000μm. The ultrasonic power of the present invention cannot exceed 300W; high-power ultrasonic treatment easily creates strong cavitation, making crystal growth difficult, prolonging the crystallization time, or preventing the formation of a spherical crystal habit structure.

[0134] Comparative Experiment Example 2: Meanwhile, the present invention also conducted comparative experiments on the substitution of related reagents, using other materials to replace some of the materials in Example C1 for verification. The comparison of the experiments is shown below. The leucine crystals were produced using the method described in Example C1 of the above comparative experimental examples. The resulting leucine crystals were examined, and it was found that the leucine crystals from all the comparative experimental examples in Comparative Experiment 2 were not spherical. Therefore, as can be seen from the description in the background section of this invention, the additives used in the crystal nucleation and growth process are selective and not universal. Thus, the additives and related processes used in this invention selectively act on leucine crystals for production.

Claims

1. A method for producing an amino acid concentrate by hydrolysis of animal protein meal, characterized by, The method comprises the following steps: Step S1, adding animal protein raw material into acid solution for acid hydrolysis, after hydrolysis, adding alkali solution to adjust pH value to 4.5-5.5, adding activated carbon for decolorization and filtration, obtaining protein hydrolysate after filtration; Step S2, cooling the protein hydrolysate to normal temperature, then precipitating histidine crude product from the protein hydrolysate, and obtaining remaining mother liquor after separating the histidine crude product; Step S3, refining the histidine crude product to obtain histidine fine product, and the refining process of the histidine crude product is as follows: Step S31, adding the histidine crude product into anhydrous ethanol to remove impurities, then filtering, washing the filtered histidine crude product with cold water for several times, recovering the washing water, mixing the washing water with the filtered histidine crude product after removing ethanol by heating and evaporation, and obtaining histidine pretreated product; Step S32, dissolving the histidine pretreated product, then adsorbing by using weak acid ion exchange resin, eluting by using dilute hydrochloric acid solution after adsorption, combining the eluate, decolorizing the eluate by activated carbon, then evaporating and concentrating the eluate to supersaturation, cooling and crystallizing to obtain histidine fine product; Step S4, adding leucine precipitant into the remaining mother liquor, filtering after standing and precipitating, then washing to obtain precipitate and tail liquid; adding the precipitate into water, neutralizing and adjusting pH value by using ammonia water, stirring to remove excess ammonia, and obtaining leucine crude product; Step S5, dissolving the leucine crude product into hot water, concentrating after decolorizing and filtering, naturally separating crystals after cooling, and obtaining leucine fine product by filtering the crystals; Step S6, recrystallizing the leucine fine product to obtain high-purity leucine; Step S7, drying the tail liquid by using spray drying technology, and obtaining amino acid mixed powder after drying.

2. The method for producing amino acid concentrates using animal protein meal hydrolysis according to claim 1, characterized in that, The specific method of step S1 is as follows: The animal protein raw material comprises animal blood powder, animal hair or animal feather; the animal protein raw material is added into 4-5 mol / L hydrochloric acid solution, the solid-liquid ratio of the raw material to the hydrochloric acid solution is 1:3-5, the acid hydrolysis temperature is 100-120℃, the acid hydrolysis time is 9-12 h, sodium hydroxide solution is added to adjust pH value to 4.5-5.5 after hydrolysis, activated carbon is added for decolorization and filtration after the temperature is reduced to 50-70℃, and protein hydrolysate is obtained after filtration.

3. The method for producing amino acid concentrates using animal protein meal hydrolysis according to claim 1, characterized in that: In step S2, the pH value of the protein hydrolysate is adjusted to the isoelectric point of histidine after cooling, and histidine is precipitated from the protein hydrolysate, and the histidine crude product and the remaining mother liquor are obtained after standing for 5-10 h.

4. The method for producing amino acid concentrates using animal protein meal hydrolysis according to claim 1, characterized in that: In step S32, the mass ratio of the histidine pretreated product to the weak acid ion exchange resin is 1:3-8, the concentration of the histidine pretreated product solution is 15-30 g / L, the eluent flow rate is 1-3 ml / min, and the elution temperature is 25-35℃.

5. The method for producing amino acid concentrates using animal protein meal hydrolysis according to claim 1, wherein: the animal protein meal is selected from the group consisting of fish meal, meat meal, and bone meal. The specific process of leucine recrystallization in step S6 is as follows: S61, dissolving the leucine fine product into glycerol-acid solution, filtering by using a microporous filter; the filtrate is evaporated and concentrated at 65-75℃. S62, in the heat preservation state, add alkali liquor to adjust pH to 5.9~6.1, continue stirring for 10min~20min after the alkali liquor is added; S63, after heating to 80℃~85℃, add sodium alginate and continue stirring for 30min~60min, the adding amount of sodium alginate is 0.1%~0.5% of the mass of leucine concentrate, and ultrasonic treatment is used in the stirring process; S64, then gradient cooling is carried out, the gradient cooling mode is: the temperature is reduced to 40℃ at a cooling rate of 20℃ / h~25℃ / h, then the temperature is reduced to 20℃ at a cooling rate of 10℃ / h~15℃ / h, then the temperature is reduced to 4℃ at a cooling rate of 5℃ / h~8℃ / h, then the temperature is maintained at 4℃ for 12h~24h, and the crystal growth is ended when the crystal is grown to 500μm~1000μm.

6. The method for producing amino acid concentrates from animal protein meal hydrolysate according to claim 1, wherein: the animal protein meal is selected from the group consisting of fish meal, meat meal, and bone meal. The leucine precipitant added in step S4 is o-xylene-4-sulfonic acid, and the adding amount of the leucine precipitant is 25%~35% of the dry mass of the raw material, the temperature is reduced to 0℃~5℃, the stirring action is 2h~4h, then the temperature is maintained for 18h~30h, after the completion of the standing precipitation, filtration is carried out, the filter cake is washed and then filtered dry to obtain the precipitate and the tail liquid; The precipitate is added into 3~4 times the volume of ammonia water to adjust the pH to 7~8, the temperature is maintained at 70℃~80℃, the excess ammonia is removed by vacuum stirring for 50min~80min, then the temperature is cooled to room temperature, and after standing for 12h~20h, filtration is carried out, the filter cake is washed several times with deionized water and then filtered dry to obtain the leucine crude product.

7. The method for producing amino acid concentrates using animal protein meal hydrolysis according to claim 1, wherein: the animal protein meal is selected from the group consisting of fish meal, meat meal, and bone meal. In step S5, the leucine is added into 30~40 times the mass of deionized water, heated to 50℃~70℃ for dissolution, activated carbon is added for decolorization, after decolorization, filtration is carried out, the filtrate is heated to 70℃~80℃ for vacuum negative pressure concentration, after cooling, the crystal is naturally precipitated, the crystal is washed several times with deionized water and then filtered dry to obtain the leucine concentrate.

8. The method for producing amino acid concentrates using animal protein meal hydrolysis according to claim 5, characterized in that: In step S6, the glycerol-acid solution is a hydrochloric acid solution of glycerol, the concentration of hydrochloric acid in the glycerol-acid solution is 3mol / L~4mol / L, and the concentration of glycerol is 0.1mol / L~0.3mol / L; the dissolution temperature of the leucine concentrate is 15℃~25℃.

9. The method for producing amino acid concentrates using animal protein meal hydrolysis according to claim 5, wherein: the animal protein meal is selected from the group consisting of fish meal, meat meal, and bone meal. The filtration pore size of the microporous filter membrane is 0.15μm~0.30μm; the filtrate is evaporated and concentrated to a leucine concentration of 25g / L~30g / L.

10. The method for generating refined amino acids by hydrolyzing animal protein blood meal as described in claim 5, characterized in that: In step S6, the alkali liquor is a sodium hydroxide solution, the concentration of the sodium hydroxide solution is 5mol / L~8mol / L, and the dropwise adding rate of the sodium hydroxide solution is 10ml / min~20ml / min; the stirring speed in step S6 is 350rpm~500rpm; the power of the ultrasonic wave used is 150W~200W, and the frequency of the ultrasonic wave is 20khz~40khz; after step S43 is ended, filtration is carried out, then negative pressure drying is carried out at 50℃~60℃ to obtain the leucine crystal; In steps S1 and S32, the adding amount of activated carbon is 2%~3% of the mass of the solution, and the decolorization time is 20min~30min.

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