Method for producing amino acid concentrate by hydrolyzing animal protein blood meal

Abstract

The application discloses a method for generating amino acid fine products by hydrolyzing animal protein blood meal, which comprises the following steps: adding animal protein raw materials into acid liquor to perform acid hydrolysis, precipitating histidine crude products from the protein hydrolysate, and separating the histidine crude products to obtain residual mother liquor; refining the histidine crude products to obtain histidine fine products, adding leucine precipitator into the residual mother liquor, and obtaining leucine crude products after treatment; obtaining high-purity leucine after refining and recrystallizing the leucine crude products; and finally drying the tail liquid by using a spray drying technology to obtain an amino acid mixed powder after drying. The application can directly separate histidine and leucine from the protein hydrolysate, and the separated histidine and leucine have good crystal purity; the balanced adsorption rate of impurity amino acids in the weak acid ion resin on the histidine is overcome; the crystal particle size of the leucine is controlled within a proper range, and the leucine has good bulk density and fluidity.

Description

Technical Field

[0001] This invention relates to the field of amino acid preparation methods, specifically to a method for generating refined amino acids by hydrolyzing animal protein blood meal. Background Technology

[0002] Histidine is an α-amino acid, first isolated in 1896 by German physicist Ebrich Kossel. In nutrition, histidine is considered an essential amino acid for humans, primarily children. Adults can synthesize histidine themselves. Adding small amounts of histidine to the diet of patients with chronic uremia increases the rate at which amino acids bind to hemoglobin, reducing renal anemia; therefore, histidine is also an essential amino acid for uremia patients. Histidine decarboxylates to histamine under the action of histidine decarboxylase. Histidine has a solubility of 41.9 g / L in water at 25°C, is very slightly soluble in alcohol (solubility is extremely low, almost negligible), and insoluble in ether and chloroform. Therefore, histidine has a certain solubility in water, and the commonly used form is histidine hydrochloride, which has good solubility.

[0003] The isoelectric point of an amino acid is the pH value at which the net charge of the amino acid in solution is zero. At this point, the amino acid exists as a zwitterion. The solubility of an amino acid is minimal at its isoelectric point. Isoelectric point precipitation is often used to obtain specific amino acids. The isoelectric points of common amino acids are shown below:

[0004] amino acids isoelectric point amino acids 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

[0005] As can be seen from the table above, most amino acids have isoelectric points in the weakly acidic region, histidine has an isoelectric point in the weakly basic region, and lysine has an isoelectric point in the basic region. Therefore, histidine and lysine can be obtained by isoelectric point precipitation; however, since the purity of isoelectric point precipitation is often low, it generally yields crude amino acids, so purification of crude amino acids is usually required.

[0006] Reference 1: Extraction of acidic, basic amino acids and leucine from pig blood hydrolysate, by Li Pingze.

[0007] Reference 1 discloses a process where pig blood is processed and hydrolyzed, followed by decolorization, filtration, and precipitation to remove tyrosine. Then, resin is used for elution. Different amino acids are obtained by adjusting the eluents to be acidic or alkaline. However, since the different eluents are mostly adjusted by changing the pH, and the pH values ​​of the eluents are very similar, this greatly increases the difficulty of the elution process, resulting in poor practical results. Furthermore, Reference 1 uses ammonia for elution, suggesting that the resin used is a strongly acidic resin.

[0008] Reference 2: Adsorption of L-histidine by D113 weakly acidic ion exchange resin, by Xu Qingqing et al.

[0009] Reference 2 determined the kinetic curve and adsorption isotherm of L-histidine adsorption using D113 weakly acidic ion exchange resin at 25℃, and investigated the effects of pH, ammonium sulfate concentration, L-lysine concentration, and L-arginine concentration on adsorption. The results showed that the D113 ion exchange resin reached equilibrium for L-histidine adsorption in approximately 30 minutes; within the experimental pH range, the equilibrium adsorption rate of L-histidine decreased with decreasing pH; when the pH dropped to 3.4, the adsorption capacity still 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.

[0010] Reference 3: Adsorption of L-histidine by D152 weakly acidic ion exchange resin, by Yang Xin et al.

[0011] Reference 3 determined the kinetic curve and adsorption isotherm of L-histidine adsorption by D152 weakly acidic ion exchange resin at 25℃, and investigated the effects of pH, ammonium sulfate concentration, L-lysine concentration and L-arginine concentration in the solution on adsorption.

[0012] Reference 4: Characteristics of L-histidine adsorption by 732 cation exchange resin, by Zou Jianhui.

[0013] Reference 4 determined the kinetic curve and adsorption isotherm of L-histidine adsorption on 732 cation exchange resin at 25℃, and investigated the effects of pH, ammonium sulfate concentration, L-lysine, and L-arginine on adsorption. The results showed that the presence of ammonium ions significantly decreased the adsorption capacity of L-histidine. The presence of L-lysine or L-arginine slightly reduced the adsorption capacity.

[0014] References 2 to 4 show that resins can be a primary method for amino acid separation and purification. However, most existing processes for separating L-histidine from fermentation broth or protein hydrolysate use strongly acidic ion exchange resins, which primarily elute L-histidine with ammonia. Since the eluent is alkaline, concentration to remove ammonia can cause partial decomposition of L-histidine. If a weakly acidic ion exchange resin is used for adsorption and elution of L-histidine, a dilute acid eluent can be used, and concentration can prevent decomposition. Therefore, References 2 to 4 employ a scheme of adsorption with a weakly acidic resin and elution with dilute acid.

[0015] Meanwhile, it can also be seen from references 2 to 4 that during the purification process using ion exchange resin adsorption and elution, the remaining amino acids, as impurities, will have an adverse effect on the adsorption and elution effect and reduce the adsorption amount.

[0016] Reference 5: CN103755640A A method for separating L-histidine

[0017] Reference 5 discloses a method for isolating L-histidine, including the following steps:

[0018] A. Decolorize the mixed amino acid mother liquor with activated carbon and adjust the pH value to 0~7.0;

[0019] B. Add dichlorobenzenesulfonic acid precipitant, let it stand at -10℃~50℃ to crystallize, and filter to obtain histidine benzenesulfonate precipitate;

[0020] C. Add histidine benzenesulfonate precipitate to boiling water, dissolve it, add leaching salt to leach, and filter to remove dichlorobenzenesulfonic acid leaching salt.

[0021] D. Add sulfuric acid to the filtrate to remove excess desorption salt ions, and concentrate to obtain L-histidine.

[0022] Reference 5 discloses a method for precipitating amino acid hydrolysate using dichlorobenzenesulfonic acid as a precipitant, and then separating histidine from other amino acids after precipitation.

[0023] Reference 6: CN107629007A A method for separating and extracting leucine and histidine from blood meal.

[0024] Reference 6 discloses experimental findings that 100g of blood meal 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 crucial problem. Further research revealed that the concentration of arginine in the blood meal hydrolysate is low, and arginine can be prevented from precipitating by appropriately controlling the precipitation conditions. However, when using dichlorobenzenesulfonic acid as the precipitant, regardless of changes in the precipitation conditions, leucine and histidine in the blood meal hydrolysate always precipitate simultaneously, making separate precipitation impossible.

[0025] Therefore, when using specialized precipitants such as dichlorobenzenesulfonic acid or o-xylene-4-sulfonic acid to precipitate leucine, histidine, etc., it cannot be guaranteed that other amino acids will not be precipitated in small amounts.

[0026] Leucine, chemically known as 2-amino-4-methylpentanoic acid, is an organic compound with the chemical formula C6H13NO2. It has three isomers: levorotatory, dextrorotatory, and racemic. L-Leucine is a white crystalline powder or crystalline crystalline amino acid with a slightly bitter taste. It is soluble in water, dilute hydrochloric acid, alkaline solutions, and carbonate solutions; slightly soluble in alcohol; and insoluble in ether. The solubility of leucine in water is 23.7 g / L at 20°C, and can reach over 40 g / L at 100°C.

[0027] Crystal form refers to the solid form of the same compound molecules arranged in a specific manner. Different crystal forms have different physicochemical properties, directly affecting stability, solubility, and bioavailability. Different solvents or different crystallization conditions result in different crystal forms, which is the theoretical basis and operational method for crystal form screening. Crystal form types mainly include the seven major crystal systems (isometric, hexagonal, tetragonal, trigonal, orthorhombic, monoclinic, and triclinic) as well as polymorphs (such as conformations and solvates in the pharmaceutical field), which are classified based on crystal symmetry or molecular arrangement.

[0028] Crystal habit refers to the macroscopic external morphology of a crystal. Different crystallization methods may yield different crystal habits for the same crystal. For example, recrystallizing urea in different solvents yields the same crystal form, but with significant differences in morphology. Common adjectives used to describe crystal habit include plate-like, lamellar, needle-like, equiaxed, cubic, octahedral, prismatic, pyramidal, and spherical. Crystal habit provides rapid and useful information about crystallization process performance: for example, needle-like or lamellar crystals generally have higher filtration resistance and worse solids flow characteristics in formulations than cubic crystals. Therefore, obtaining larger crystal sizes is essential for needle-like or lamellar crystal habits.

[0029] The main strategies for improving crystal habit include selecting suitable solvents, controlling the crystal growth rate, controlling supersaturation, controlling impurities in the crystallization system, and adjusting pH. Different systems exhibit significant selectivity for methods to improve crystal habit, and the scale-up effect of crystal habit improvement conditions is particularly pronounced, requiring experimental determination. Improving crystal habit is often combined with altering crystal grain size and size distribution; for example, changing from fine needle-like crystals to coarse rod-like crystals significantly improves product quality. Besides depending on the internal structure of the crystal, crystal habit is also related to the crystal growth environment, such as solvent, supersaturation, impurities, additives, temperature, solution pH, and physical field.

[0030] Crystal habit modifiers are additives that regulate the morphology and structure of crystals through adsorption, altering their growth habits without affecting their crystal system affiliation. Under the same external conditions, the same type of crystal often exhibits the same shape; that is, each crystal has its own habitual form under certain external conditions. This property of crystals is called crystal habit or crystal morphology. Generally, the crystal faces of a crystal habit conform to the crystal facets with the highest plane density, meaning that the crystal habit of a crystal mainly obeys Bravais's rule. A given substance can produce crystals with completely different shapes using different methods, but they still belong to the same crystal system. For example, one crystallization method favors needle-like habits, while another method yields platy habits.

[0031] Reference 4: Research on the crystallization process of L-aminopropionic acid, by Shi Jiakang.

[0032] Reference 4 investigated the effects of different additives on the growth and crystal habit of L-aminopropionic acid crystals through polycrystalline and single-crystal growth experiments. The results showed that additive A halted the growth of L-aminopropionic acid on the crystal face perpendicular to the c-axis, changing the crystal habit from rod-shaped to blocky. This was because the carboxyl and amino groups in additive A interacted with the exposed carboxyl and amino groups on the crystal face, causing the additive molecules to adsorb onto the crystal face and inhibiting its growth. However, the addition of additives C and D accelerated the growth of the crystal face perpendicular to the c-axis, changing the crystal habit from rod-shaped to needle-shaped. This was because the long carbon chains in additive molecules C and D are hydrophobic, and their adsorption on the crystal face has a desolvation effect, accelerating the diffusion of the solute to the crystal face and promoting crystal growth. This paper optimizes the process parameters (evaporation rate, seed crystals, stirring, and additives) in the evaporation and crystallization process of L-aminopropionic acid, achieving effective control over the particle size of L-aminopropionic acid products. The mass ratio of particles in the 20-60 mesh range in the product is increased from 20% to 80%, and the crystal structure of L-aminopropionic acid is changed from long rods to blocky shapes, significantly improving the bulk density and flowability of the product.

[0033] Reference 7 shows that the addition of additive A can affect the crystallization process of L-aminopropionic acid. Additive A can effectively control the particle size of L-aminopropionic acid, and change the crystal habit of L-aminopropionic acid from long rod shape to block shape, thereby improving the bulk density and flowability of L-aminopropionic acid.

[0034] Reference 8: Study on the nucleation and transformation mechanism of polymorphic L-glutamic acid, by Mo Yuxin et al.

[0035] Reference 8 investigated the effects of various amino acid additives on the crystallization and crystal form conversion process of glutamic acid, and analyzed the influence of amino acids on the crystal form conversion rate based on the molecular structure of the additives. The effects of additive concentration on the crystal habit and crystal form conversion of glutamic acid were examined, and the changes in the nucleation induction period and crystal growth of the two crystal forms during the crystal form conversion process under different additive concentrations were explored in detail. The residual amount of additives in glutamic acid crystals was determined using liquid chromatography, and the mechanism of the additives' influence on the crystal form conversion process of glutamic acid was discussed. For example, the effect of Tailor-made additives on the crystal habit of benzamide: crystallization in ethanol yields plate-like benzamide crystals; when benzoic acid additives are added, the normal crystal growth mode is disturbed, thus changing the crystal habit.

[0036] Based on the differences in the mechanism by which additives affect the crystallization process, Reference 8 classifies additives into the following types: Tailor-made additives, template additives (nylon, polyethylene, polypropylene, polytetrafluoroethylene, glutaric acid single crystals), and composite additives (phosphates, polycarbonates, polysulfates).

[0037] Reference 9: Study on the effect of additives on the crystallization process of L-isoleucine polymorphic solution, by Liu Xing.

[0038] Reference 9 investigated the thermodynamic properties and nucleation process of different L-isoleucine polymorphs in solution, focusing on the influence of additives on the crystallization process of L-isoleucine polymorphs. The effects of different additives (L-phenylalanine, L-histidine, glycine, sodium chloride, acetic acid, propionic acid, and valeric acid) on the nucleation of L-isoleucine polymorphs were explored. The results showed that the A-type polymorph was obtained in the presence of sodium chloride, acetic acid, propionic acid, and valeric acid. Finally, the minimum additive concentrations effectively influencing the nucleation of L-isoleucine polymorphs were determined to be 0.025 mol / L sodium chloride, 0.125 mol / L acetic acid, 0.1 mol / L propionic acid, and 0.025 mol / L valeric acid.

[0039] Reference 10: The effect of additives on the growth of amino acid crystals, by Zhou Xuan.

[0040] Reference 10 discloses that amino acids mostly grow into needle-like or plate-like crystals, which suffer from low bulk density and poor flowability, seriously affecting the subsequent processing of the product. Regarding the research on the influence of additives on amino acid crystal growth, from the perspective of crystal engineering, a prospect is proposed to regulate the crystal habit by directionally controlling the growth of amino acid crystals through additives designed using molecular simulation.

[0041] For example, Poornachary et al. added a custom additive, aspartic acid, to the glycine crystallization solution and found that aspartic acid inhibited the growth of α-glycine crystals along the b-axis and c-axis. α-Glycine crystals in aqueous solution are columnar bipyramidal; after adding a small amount of L-aspartic acid, the α-glycine crystals exhibit a prismatic bipyramidal shape; with increasing aspartic acid concentration, the α-glycine crystals become pyramidal.

[0042] Han et al. studied the effects of inorganic salts (NaCl, NaAc, KNO3, CaCl2, MgSO4, etc.) on the growth process of γ-glycine in supersaturated aqueous solutions and found that these inorganic salts inhibited the growth of γ-glycine crystals along the c-axis. Reference 7 found that adding polymer additives could change the crystal habit of DL-methionine from slender needle-like structures to hexagonal plates or blocks with a smaller aspect ratio. These three polymers had a strong inhibitory effect on the growth of DL-methionine crystals along both the b-axis (length) and c-axis (width).

[0043] The influence of additives on amino acid crystal growth is not singular; it is likely the result of multiple factors working together, and their mechanisms of action are still under development. Although there has been extensive research on additives in regulating crystal growth, due to the complexity and variability of their mechanisms, a standardized and efficient crystal growth regulation strategy is still lacking, and additive selection still relies on empirical trial and error.

[0044] From references 7 to 11, it can be concluded that crystal form and crystal habit have a significant impact on the physicochemical properties of crystals. Due to different crystallization processes, the same crystal form can still have different crystal habits. 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 additives used during crystallization. Furthermore, the types of additives added during crystallization are not common to each amino acid or compound; for example, reference 7 uses additive A, while reference 8 uses additives such as nylon, polyethylene, polypropylene, polytetrafluoroethylene, glutaric acid single crystals, phosphates, polycarbonates, and polysulfates.

[0045] Reference 12: CN114773216A - A spherical L-leucine crystal, its preparation method and application

[0046] The literature discloses an L-leucine spherical crystal, its preparation method and application. The preparation method includes the following steps: (1) dissolving L-leucine raw material in an acidic solution at 10-25℃ to obtain a mixed solution; (2) adding isobutyl acetate to the mixed solution obtained in step (1) for mixing, causing phase separation, and stirring at 250-650 rpm to obtain a mixed solution with dispersed spherical droplets; (3) maintaining a speed of 250-650 rpm, adding alkali solution to the mixed solution with dispersed spherical droplets in step (2) until the pH reaches near the isoelectric point of L-leucine, and maintaining this for 1-2.5 h to obtain L-leucine spherical crystals. The L-leucine prepared by this method has a regular shape, adjustable size and good fluidity.

[0047] Reference 12 describes a method for obtaining spherical L-leucine crystals with a size of 1000 μm to 2000 μm by adding isobutyl acetate to a mixed solution, dispersing it into spherical droplets under stirring, and then precipitating it near its isoelectric point. While the method disclosed in Reference 12 yields spherical crystals, the particle size is relatively large, and the particle size distribution range is also very wide. Crystal particle size and particle size distribution are important indicators of crystalline products, primarily affecting: ① the solid-liquid separation process during production; ② product drying; ③ product purity; ④ product flowability and appearance; and ⑤ product bioavailability (API dissolution rate and activity). In formulation processes, crystallization and pulverization are mainly used to control particle size and its distribution. Larger L-leucine crystals reduce the dissolution rate, and larger crystals tend to encapsulate some impurities during crystal formation. Studies have shown that controlling the L-leucine particle size within the range of 500 μm to 1000 μm is the most suitable range, as this range exhibits good bulk density, flowability, and solubility, resulting in a high-purity product.

[0048] In summary, the existing technology has the following problems:

[0049] (1) In the prior art, ion exchange resin is used to purify histidine. However, strong acid ion exchange resin is used to elute with ammonia. During the eluent treatment, ammonia needs to be removed, and histidine is easily decomposed during the ammonia removal process. If weak acid ion exchange resin is used, the other amino acids, especially those in cationic state, will affect the adsorption process and reduce the equilibrium adsorption rate.

[0050] (2) The particle size range of the L-leucine prepared is too large, and the crystal structure is mostly not spherical. Summary of the Invention

[0051] The purpose of this invention is to provide a method for generating refined amino acids by hydrolyzing animal protein blood meal. This invention aims to solve the following technical problems:

[0052] (1) Histidine and leucine can be isolated separately from protein hydrolysate;

[0053] (2) When using acidic ion exchange resin, improve the equilibrium adsorption rate;

[0054] (3) The particle size of leucine crystals is controlled within the range of 500μm~1000μm, and the crystal structure is spherical.

[0055] To achieve the above objectives, one embodiment of the present invention provides a method for generating refined amino acids by hydrolyzing animal protein blood meal, comprising the following steps:

[0056] Step S1: Add animal protein raw materials to acid solution for acid hydrolysis. After hydrolysis, add alkaline solution to adjust the pH value to 4.5~5.5, add activated carbon for decolorization and filter. After filtration, protein hydrolysate is obtained.

[0057] Step S2: Cool the protein hydrolysate to room temperature, then precipitate crude histidine from the protein hydrolysate, and separate the crude histidine to obtain the remaining mother liquor.

[0058] Step S3: The crude histidine is purified to obtain refined histidine. The histidine purification process is shown below:

[0059] Step S31: Add crude histidine to anhydrous ethanol to wash and remove impurities, then filter. After filtration, wash several times with cold water, recover the washing water, heat and evaporate the washing water to remove ethanol, and then mix it with the washed and filtered crude histidine to obtain pretreated histidine.

[0060] Step S32: After dissolving the pretreated histidine, it is adsorbed using a weak acid ion exchange resin. After adsorption, it is eluted with dilute hydrochloric acid solution. The eluents are combined and decolorized with activated carbon. Then, the eluent is evaporated and concentrated to supersaturation, and cooled to crystallize to obtain the refined histidine.

[0061] Step S4: Add leucine precipitant to the remaining mother liquor, let it stand to precipitate, filter it, wash it after filtration to obtain precipitate and tail liquor; add the precipitate to water, neutralize it with ammonia water to adjust the pH value, stir to remove excess ammonia, and obtain crude leucine.

[0062] Step S5: Dissolve crude leucine in hot water, decolorize, filter and concentrate, cool and crystals will naturally precipitate, filter the crystals to obtain refined leucine.

[0063] Step S6: Recrystallize the refined leucine to obtain high-purity leucine;

[0064] Step S7: The tail liquid is dried using spray drying technology to obtain an amino acid mixed powder.

[0065] In the optimized scheme of the present invention, the specific method of step S1 is as follows:

[0066] Animal protein raw materials include animal blood meal, animal hair, or animal feathers. The animal protein raw materials are added to a 4 mol / L to 5 mol / L hydrochloric acid solution, with a solid-liquid ratio of 1:3 to 5. The acid hydrolysis temperature is 100℃ to 120℃, and the acid hydrolysis time is 9h to 12h. After hydrolysis, sodium hydroxide solution is added to adjust the pH value to 4.5 to 5.5. The temperature is then lowered to 50℃ to 70℃, and activated carbon is added for decolorization and filtration. The filtered protein hydrolysate is obtained.

[0067] In the optimized scheme of the present invention, in step S2, after the protein hydrolysate is cooled, an alkaline solution is added to adjust the pH value to the isoelectric point of histidine, so that histidine is precipitated from the protein hydrolysate. After standing for 5h~10h, the solution is filtered to obtain crude histidine and the remaining mother liquor.

[0068] In the optimized scheme of the present invention, in step S32, the mass ratio of histidine pretreatment product to weakly acidic ion exchange resin is 1:3~8; the concentration of histidine pretreatment product solution is 15g / L~30g / L; the eluent flow rate is 1ml / min~3ml / min; and the elution temperature is 25℃~35℃.

[0069] In the optimized scheme of the present invention, the specific process of leucine recrystallization in step S6 is as follows:

[0070] S61. Dissolve the refined leucine in a glycerol-acidic solution and filter using a microporous membrane; concentrate the filtrate by evaporation at 65℃~75℃.

[0071] S62. While keeping warm, add alkali solution to adjust the pH value to 5.9~6.1. After the alkali solution is added, continue stirring for 10min~20min.

[0072] S63. After heating to 80℃~85℃, add sodium alginate and continue stirring for 30min~60min. The amount of sodium alginate added is 0.1%~0.5% of the mass of leucine concentrate. Ultrasonic treatment is used during the stirring process.

[0073] S64. Then, a gradient cooling process is performed. The gradient cooling method is as follows: first, the temperature is reduced to 40℃ at a cooling rate of 20℃ / h to 25℃ / h, then the temperature is reduced to 20℃ at a cooling rate of 10℃ / h to 15℃ / h, then the temperature is reduced to 4℃ at a cooling rate of 5℃ / h to 8℃ / h, and then maintained at 4℃ for 12h to 24h. The process ends when the crystal grows to 500μm to 1000μm.

[0074] In the optimized scheme of the present invention, the leucine precipitant added in step S4 is o-xylene-4-sulfonic acid, and the amount of leucine precipitant added is 25%~35% of the dry weight of the raw material. The temperature is lowered to 0℃~5℃, and the mixture is stirred for 2h~4h. Then, the mixture is kept warm and allowed to stand for precipitation for 18h~30h. After the precipitation is completed, the mixture is filtered, and the filter cake is washed and dried to obtain the precipitate and tail liquid.

[0075] Add 3-4 times the volume of ammonia water to neutralize and adjust the pH to 7-8. Maintain the temperature at 70℃-80℃ and vacuum stir for 50-80 minutes to remove excess ammonia. Then cool to room temperature, let stand for 12-20 hours, and filter. Wash the filter cake several times with deionized water and then filter dry to obtain crude leucine.

[0076] In the optimized scheme of the present invention, in step S5, leucine is added to 30-40 times its mass of deionized water and heated to 50°C-70°C to dissolve. Activated carbon is added for decolorization. After decolorization, the solution is filtered. The filtrate is heated to 70°C-80°C and concentrated under vacuum negative pressure. After cooling, crystals naturally precipitate. The crystals are washed several times with deionized water and then filtered dry to obtain high-quality leucine.

[0077] 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℃.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] In summary, the present invention has the following advantages:

[0082] 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

[0083] Figure 1 These are the experimental results of histidine equilibrium adsorption rate in Examples A1, A2, and Comparative Example A1 of the present invention;

[0084] Figure 2 This is a scanning electron microscope (SEM) image of embodiment C1 of the present invention;

[0085] Figure 3This is a scanning electron microscope (SEM) image of embodiment C2 of the present invention;

[0086] Figure 4 This is a particle size distribution diagram of embodiment C1 of the present invention;

[0087] Figure 5 This is a particle size distribution diagram of embodiment C2 of the present invention;

[0088] Figure 6 Here is a SEM image of comparative experimental example 1 of this invention;

[0089] Figure 7 Here is a SEM image of comparative experimental example 1 of this invention;

[0090] Figure 8 Here is a SEM image of comparative experimental example 1 of this invention;

[0091] Figure 9 This is a SEM image of comparative experimental example 1 of the present invention. Detailed Implementation

[0092] This invention provides a method for generating refined amino acids by hydrolyzing animal protein blood meal, comprising the following steps:

[0093] Step S1: Add animal protein raw materials to acid solution for acid hydrolysis. After hydrolysis, add alkaline solution to adjust the pH value to 4.5~5.5, add activated carbon for decolorization and filter. After filtration, protein hydrolysate is obtained.

[0094] Step S2: Cool the protein hydrolysate to room temperature, then precipitate crude histidine from the protein hydrolysate, and separate the crude histidine to obtain the remaining mother liquor.

[0095] Step S3: The crude histidine is purified to obtain refined histidine. The histidine purification process is shown below:

[0096] Step S31: Add crude histidine to anhydrous ethanol to wash and remove impurities, then filter. After filtration, wash several times with cold water, recover the washing water, heat and evaporate the washing water to remove ethanol, and then mix it with the washed and filtered crude histidine to obtain pretreated histidine.

[0097] Step S32: After dissolving the pretreated histidine, it is adsorbed using a weak acid ion exchange resin. After adsorption, it is eluted with dilute hydrochloric acid solution. The eluents are combined and decolorized with activated carbon. Then, the eluent is evaporated and concentrated to supersaturation, and cooled to crystallize to obtain the refined histidine.

[0098] Step S4: Add leucine precipitant to the remaining mother liquor, let it stand to precipitate, filter it, wash it after filtration to obtain precipitate and tail liquor; add the precipitate to water, neutralize it with ammonia water to adjust the pH value, stir to remove excess ammonia, and obtain crude leucine.

[0099] Step S5: Dissolve crude leucine in hot water, decolorize, filter and concentrate, cool and crystals will naturally precipitate, filter the crystals to obtain refined leucine.

[0100] Step S6: Recrystallize the refined leucine to obtain high-purity leucine;

[0101] Step S7: The tail liquid is dried using spray drying technology to obtain an amino acid mixed powder.

[0102] In this invention, the adsorption and purification process using ion exchange resin involves packing the ion exchange resin into a column. After the solution flows through the ion exchange resin, the histidine content in the eluted sample is detected. During the sample loading process, the presence of histidine in the eluted sample indicates that the histidine adsorption process is complete. Elution is then performed using eluent, and the eluent is collected. The histidine content in the eluted sample is monitored during the elution process. Elution is complete when the concentration of histidine in the eluted sample reaches 0, at which point elution can be stopped.

[0103] Example A: Preparation method of refined histidine

[0104] Step S1: Animal protein raw materials include animal blood meal, animal hair, or animal feathers; the animal protein raw materials are added to a 4 mol / L to 5 mol / L hydrochloric acid solution, the solid-liquid ratio of the raw materials to the hydrochloric acid solution is 1:3 to 5, the acid hydrolysis temperature is 100℃ to 120℃, the acid hydrolysis time is 9h to 12h, after hydrolysis, sodium hydroxide solution is added to adjust the pH value to 4.5 to 5.5, the temperature is lowered to 50℃ to 70℃, activated carbon is added for decolorization and filtration, and the protein hydrolysate is obtained after filtration.

[0105] Step S2: Cool the protein hydrolysate to room temperature. After cooling, add alkaline solution to adjust the pH to the isoelectric point of histidine, precipitate histidine from the protein hydrolysate, let it stand for 5-10 hours, and then filter to obtain crude histidine and the remaining mother liquor.

[0106] Step S3: The crude histidine is purified to obtain refined histidine. The histidine purification process is shown below:

[0107] Step S31: Add crude histidine to anhydrous ethanol to wash and remove impurities, then filter. After filtration, wash several times with cold water, recover the washing water, heat and evaporate the washing water to remove ethanol, and then mix it with the washed and filtered crude histidine to obtain pretreated histidine.

[0108] Step S32: After dissolving the histidine pretreatment sample, adsorb it using a weakly acidic ion exchange resin. After adsorption, elute with dilute hydrochloric acid solution, combine the eluents, decolorize the eluent with activated carbon, then evaporate and concentrate the eluent to supersaturation, and cool to crystallize to obtain purified histidine. In step S32, the mass ratio of histidine pretreatment sample to weakly acidic ion exchange resin is 1:3~8; the concentration of the histidine pretreatment sample solution is 15g / L~30g / L; the eluent flow rate is 1ml / min~3ml / min; and the elution temperature is 25℃~35℃.

[0109] Example A1: Preparation method of refined histidine

[0110] Step S1: Animal protein raw materials include animal blood meal, animal hair, or animal feathers; the animal protein raw materials are added to a 4.5 mol / L hydrochloric acid solution, the solid-liquid ratio of the raw materials to the hydrochloric acid solution is 1:4, the acid hydrolysis temperature is 110℃, the acid hydrolysis time is 10h, after the hydrolysis is completed, sodium hydroxide solution is added to adjust the pH value to 4.8, the temperature is lowered to 65℃, activated carbon is added for decolorization and filtration, and the protein hydrolysate is obtained after filtration.

[0111] Step S2: Cool the protein hydrolysate to room temperature. After cooling, add alkaline solution to adjust the pH to the isoelectric point of histidine, precipitate histidine from the protein hydrolysate, let it stand for 8 hours, and then filter to obtain crude histidine and the remaining mother liquor.

[0112] Step S3: The crude histidine is purified to obtain refined histidine. The histidine purification process is shown below:

[0113] Step S31: Add crude histidine to anhydrous ethanol to wash and remove impurities, then filter. After filtration, wash several times with cold water, recover the washing water, heat and evaporate the washing water to remove ethanol, and then mix it with the washed and filtered crude histidine to obtain pretreated histidine.

[0114] Step S32: After dissolving the histidine pretreatment sample, adsorb it using a weakly acidic ion exchange resin. After adsorption, elute with dilute hydrochloric acid solution, combine the eluents, decolorize the eluent with activated carbon, then evaporate and concentrate the eluent to supersaturation, and cool to crystallize to obtain purified histidine. In step S32, the mass ratio of histidine pretreatment sample to weakly acidic ion exchange resin is 1:5; the concentration of the histidine pretreatment sample solution is 20 g / L; the eluent flow rate is 2 ml / min; and the elution temperature is 28℃.

[0115] The histidine yield in Example A1 was 7.5%.

[0116] Example A2:

[0117] Step S1: Animal protein raw materials include animal blood meal, animal hair, or animal feathers; the animal protein raw materials are added to a 4 mol / L hydrochloric acid solution, the solid-liquid ratio of the raw materials to the hydrochloric acid solution is 1:3, the acid hydrolysis temperature is 100℃, the acid hydrolysis time is 12h, after the hydrolysis is completed, sodium hydroxide solution is added to adjust the pH value to 4.5, the temperature is lowered to 60℃, activated carbon is added for decolorization and filtration, and the protein hydrolysate is obtained after filtration.

[0118] Step S2: Cool the protein hydrolysate to room temperature. After cooling, add alkaline solution to adjust the pH to the isoelectric point of histidine, precipitate histidine from the protein hydrolysate, let it stand for 5 hours, and then filter to obtain crude histidine and the remaining mother liquor.

[0119] Step S3: The crude histidine is purified to obtain refined histidine. The histidine purification process is shown below:

[0120] Step S31: Add crude histidine to anhydrous ethanol to wash and remove impurities, then filter. After filtration, wash several times with cold water, recover the washing water, heat and evaporate the washing water to remove ethanol, and then mix it with the washed and filtered crude histidine to obtain pretreated histidine.

[0121] Step S32: After dissolving the histidine pretreatment sample, adsorb it using a weakly acidic ion exchange resin. After adsorption, elute with dilute hydrochloric acid solution, combine the eluents, decolorize the eluent with activated carbon, then evaporate and concentrate the eluent to supersaturation, and cool to crystallize to obtain purified histidine. In step S32, the mass ratio of histidine pretreatment sample to weakly acidic ion exchange resin is 1:3; the concentration of the histidine pretreatment sample solution is 15 g / L; the eluent flow rate is 1 ml / min; and the elution temperature is 25℃.

[0122] The histidine yield in Example A1 was 8.3%.

[0123] The histidine concentrates obtained in Examples A1 and A2 were tested according to the method specified in the Pharmacopoeia of the People's Republic of China (2000 Edition, Part II). The results of the histidine samples tested are shown below.

[0124] The test results above show that the histidine prepared by the method of the present invention meets the requirements of the National Pharmacopoeia.

[0125] Comparative Example A1

[0126] Preparation method of histidine fine product

[0127] Step S1: Animal protein raw materials include animal blood meal, animal hair, or animal feathers; the animal protein raw materials are added to a 4.5 mol / L hydrochloric acid solution, the solid-liquid ratio of the raw materials to the hydrochloric acid solution is 1:4, the acid hydrolysis temperature is 110℃, the acid hydrolysis time is 10h, after the hydrolysis is completed, sodium hydroxide solution is added to adjust the pH value to 4.8, the temperature is lowered to 65℃, activated carbon is added for decolorization and filtration, and the protein hydrolysate is obtained after filtration.

[0128] Step S2: Cool the protein hydrolysate to room temperature. After cooling, add alkaline solution to adjust the pH to the isoelectric point of histidine, precipitate histidine from the protein hydrolysate, let it stand for 8 hours, and then filter to obtain crude histidine and the remaining mother liquor.

[0129] Step S3: The crude histidine is purified to obtain refined histidine. The histidine purification process is shown below:

[0130] Step S31: Wash the crude histidine product with cold water several times, recover the washing water, heat and evaporate the washing water to remove ethanol, and then mix it with the washed and filtered crude histidine product to obtain the pretreated histidine product.

[0131] Step S32: After dissolving the histidine pretreatment sample, adsorb it using a weakly acidic ion exchange resin. After adsorption, elute with dilute hydrochloric acid solution, combine the eluents, decolorize the eluent with activated carbon, then evaporate and concentrate the eluent to supersaturation, and cool to crystallize to obtain purified histidine. In step S32, the mass ratio of histidine pretreatment sample to weakly acidic ion exchange resin is 1:5; the concentration of the histidine pretreatment sample solution is 20 g / L; the eluent flow rate is 2 ml / min; and the elution temperature is 28℃.

[0132] Experimental Example A: Effect of Different Purification Methods on the Equilibrium Adsorption Rate of Histidine

[0133] Methods for determining equilibrium adsorption rate:

[0134] Add 500 ml of histidine solution and 100 g of 732 cation exchange resin to a jacketed Erlenmeyer flask at a volume ratio of 5:1 (solution to weakly acidic ion exchange resin). Mix the flasks using a magnetic stirrer. Maintain the temperature at 30°C using a constant temperature water bath and adjust the pH to 5.0 with 0.1 mol / L sulfuric acid. Take a 1 ml sample every 20 minutes to analyze the mass concentration of histidine and determine the equilibrium adsorption rate.

[0135] Equilibrium adsorption rate = (concentration before adsorption - concentration after adsorption) / concentration before adsorption.

[0136] The equilibrium adsorption rates of histidine in Examples A1, A2, and Comparative Example A1 were calculated using the methods disclosed in the prior art described above. Specific results are as follows: Figure 1 As shown.

[0137] The experimental results above show that purifying crude histidine with anhydrous ethanol can eliminate alcohol-soluble amino acids and other impurities, thus improving the equilibrium adsorption rate of histidine to some extent. Histidine has very low solubility in ethanol but some solubility in water; therefore, anhydrous ethanol should be used instead of an ethanol solution for purification. During isoelectric point precipitation, the purity of the precipitate is insufficient. Therefore, using anhydrous ethanol to remove alcohol-soluble impurities and other amino acids can remove impurities while retaining as much histidine as possible, preventing histidine from being carried away by the solvent, thereby improving the yield.

[0138] Example B1: Preparation of high-quality leucine

[0139] Step S4: Add leucine precipitant to the remaining mother liquor, let it stand to precipitate, filter it, wash it after filtration to obtain the precipitate; add the precipitate to water, neutralize it with ammonia to adjust the pH value, stir to remove excess ammonia, and obtain crude leucine.

[0140] In step S4, the leucine precipitant added 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 lowered to 3°C, and the mixture is stirred for 3 hours, then kept at this temperature and allowed to stand for precipitation for 22 hours. After the precipitation is complete, the mixture is filtered, and the filter cake is washed with deionized water and then filtered dry to obtain the precipitate and tailings. The tailings obtained after filtration in step S2 are dried using spray drying technology to obtain an amino acid mixed powder.

[0141] The precipitate was neutralized and the pH was adjusted to 7.5 by adding 4 times its volume of ammonia water. The temperature was maintained at 75°C, and the mixture was vacuum stirred for 60 minutes to remove excess ammonia. The mixture was then cooled to room temperature, allowed to stand for 16 hours, and then filtered. The filter cake was washed several times with deionized water and then dried to obtain crude leucine.

[0142] Step S5: Dissolve crude leucine in hot water, decolorize, filter, and concentrate. The amount of activated carbon added for decolorization is 2.5% of the solution mass, and the decolorization time is 25 minutes. After cooling, crystals precipitate naturally. Filter the crystals to obtain refined leucine. Alternatively, in Step S5, dissolve leucine in 32 times its mass of deionized water and heat to 58°C. Add activated carbon for decolorization, filter, and heat the filtrate to 76°C for vacuum concentration. After cooling, crystals precipitate naturally. Wash the crystals several times with deionized water and filter to obtain refined leucine.

[0143] Example B2: Preparation of high-quality leucine

[0144] Step S4: Add leucine precipitant to the remaining mother liquor, let it stand to precipitate, filter it, wash it after filtration to obtain the precipitate; add the precipitate to water, neutralize it with ammonia to adjust the pH value, stir to remove excess ammonia, and obtain crude leucine.

[0145] In step S4, the leucine precipitant added 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 lowered to 5°C, and the mixture is stirred for 4 hours, then kept at this temperature and allowed to stand for 30 hours to precipitate. After the sedimentation is complete, the mixture is filtered, and the filter cake is washed with deionized water and then filtered dry to obtain the precipitate and tailings. The tailings obtained after filtration in step S2 are dried using spray drying technology to obtain an amino acid mixed powder.

[0146] The precipitate was neutralized and the pH was adjusted to 7 by adding 3-4 times its volume of ammonia water. The temperature was maintained at 70°C, and the mixture was vacuum stirred for 50 minutes to remove excess ammonia. Then, it was cooled to room temperature, allowed to stand for 12 hours, and then filtered. The filter cake was washed several times with deionized water and then dried to obtain crude leucine.

[0147] Step S5: Dissolve crude leucine in hot water, decolorize, filter, and concentrate. The amount of activated carbon added for decolorization is 2% of the solution mass, and the decolorization time is 20 minutes. After cooling, crystals precipitate naturally. Filter the crystals to obtain refined leucine. Alternatively, in Step S5, dissolve leucine in 30 times its weight of deionized water and heat to 50°C. Add activated carbon for decolorization, filter, and heat the filtrate to 70°C for vacuum concentration. After cooling, crystals precipitate naturally. Wash the crystals several times with deionized water and filter to obtain refined leucine.

[0148] Example B3: Preparation of high-quality leucine

[0149] Step S4: Add leucine precipitant to the remaining mother liquor, let it stand to precipitate, filter it, wash it after filtration to obtain the precipitate; add the precipitate to water, neutralize it with ammonia to adjust the pH value, stir to remove excess ammonia, and obtain crude leucine.

[0150] In step S4, the leucine precipitant added is o-xylene-4-sulfonic acid, and the amount of leucine precipitant added is 35% of the dry weight of the raw material. The temperature is lowered to 5°C, and the mixture is stirred for 2 hours, then kept at this temperature and allowed to stand for precipitation for 18 hours. After the precipitation is complete, the mixture is filtered, and the filter cake is washed with deionized water and then filtered dry to obtain the precipitate and tailings. The tailings obtained after filtration in step S2 are dried using spray drying technology to obtain an amino acid mixed powder.

[0151] The precipitate was neutralized and the pH was adjusted to 8 by adding 3-4 times its volume of ammonia water. The temperature was maintained at 80°C, and the mixture was vacuum stirred for 80 minutes to remove excess ammonia. Then, it was cooled to room temperature, allowed to stand for 20 hours, and then filtered. The filter cake was washed several times with deionized water and then dried to obtain crude leucine.

[0152] Step S5: Dissolve crude leucine in hot water, decolorize, filter, and concentrate. The amount of activated carbon added for decolorization is 3% of the solution mass, and the decolorization time is 30 minutes. After cooling, crystals precipitate naturally. Filter the crystals to obtain refined leucine. Alternatively, in Step S5, dissolve leucine in 40 times its weight of deionized water and heat to 70°C. Add activated carbon for decolorization, filter, and heat the filtrate to 80°C for vacuum concentration. After cooling, crystals precipitate naturally. Wash the crystals several times with deionized water and filter to obtain refined leucine.

[0153] Physicochemical testing of high-quality leucine: The detection method for leucine was carried out according to the method specified in the Pharmacopoeia of the People's Republic of China (2000 edition, Part II). The results of the L-leucine sample test are shown below.

[0154] The test results above show that the leucine prepared by the method of the present invention from animal hair or feathers by hydrolysis meets the requirements of the National Pharmacopoeia.

[0155] Example C: Recrystallization of leucine

[0156] Embodiment C of the present invention includes multiple sub-embodiments. The refined leucine used in each sub-embodiment of Embodiment C is the refined leucine prepared by the method in Embodiment B1.

[0157] Step S6: Recrystallize the refined leucine to obtain high-purity leucine. The recrystallization steps are as follows:

[0158] S61. Dissolve the refined leucine in a glycerol-acidic 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 refined leucine is 15℃ to 25℃.

[0159] Then, the solution is filtered using a microporous membrane with a pore size of 0.15μm to 0.30μm; the filtrate is then evaporated and concentrated at 65℃ to 75℃ until the concentration of leucine is 25g / L to 30g / L.

[0160] S62. While maintaining the temperature, add an alkaline solution to adjust the pH to 5.9-6.1. The alkaline solution is a sodium hydroxide solution with a concentration of 5 mol / L-8 mol / L and a dropping rate of 10 ml / min-20 ml / min. After the alkaline solution is added, continue stirring for 10-20 minutes at a stirring speed of 350 rpm-500 rpm.

[0161] S63. After heating to 80℃~85℃, add sodium alginate and continue stirring for 30min~60min. The amount of sodium alginate added is 0.1%~0.5% of the mass of leucine concentrate. Ultrasonic treatment is used during the stirring process. The ultrasonic power is 150W~200W and the ultrasonic frequency is 20khz~40khz.

[0162] S64. Then, a gradient cooling process is performed as follows: initially, the temperature is reduced to 40℃ at a cooling rate of 20℃ / h to 25℃ / h, then reduced to 20℃ at a cooling rate of 10℃ / h to 15℃ / h, then reduced to 4℃ at a cooling rate of 5℃ / h to 8℃ / h, and then maintained at 4℃ for 12h to 24h, stopping when the crystals grow to 500μm to 1000μm. After completion, the crystals are filtered and then dried under negative pressure at 50℃ to 60℃ to obtain leucine crystals.

[0163] Example C1: Recrystallization of leucine

[0164] S61. Dissolve the refined leucine in a glycerol-acidic solution, which is a hydrochloric acid solution of glycerol, with a hydrochloric acid concentration of 3 mol / L and a glycerol concentration of 0.18 mol / L; the dissolution temperature of the refined leucine is 20℃.

[0165] The solution was then filtered using a microporous membrane with a pore size of 0.20 μm; the filtrate was then evaporated and concentrated at 70 °C until the concentration of leucine was 28 g / L.

[0166] S62. While maintaining the temperature, add an alkaline solution to adjust the pH to 5.9-6.1. The alkaline solution is a sodium hydroxide solution with a concentration of 6 mol / L, and the dropping rate of the sodium hydroxide solution is 12 ml / min. After the alkaline solution is completely added, continue stirring for 15 minutes at a stirring speed of 400 rpm.

[0167] S63. After heating to 82℃, add sodium alginate and continue stirring for 40 minutes. The amount of sodium alginate added is 0.3% of the mass of the leucine concentrate. Ultrasonic treatment is used during the stirring process. The ultrasonic power is 160W and the ultrasonic frequency is 30kHz.

[0168] S64. Then, a gradient cooling process was performed: initially, the temperature was reduced to 40℃ at a cooling rate of 22℃ / h, then to 20℃ at a cooling rate of 12℃ / h, then to 4℃ at a cooling rate of 6℃ / h, and then maintained at 4℃ for 20h, stopping when the crystals grew to 700μm. After completion, the crystals were filtered and then dried under negative pressure at 55℃ to obtain leucine crystals.

[0169] Example C2: Recrystallization of leucine

[0170] S61. Dissolve the refined leucine in a glycerol-acidic solution, which is a hydrochloric acid solution of glycerol, with a hydrochloric acid concentration of 4 mol / L and a glycerol concentration of 0.1 mol / L; the dissolution temperature of the refined leucine is 25℃.

[0171] The solution was then filtered using a microporous membrane with a pore size of 0.30 μm; the filtrate was then evaporated and concentrated at 75 °C until the concentration of leucine was 25 g / L.

[0172] S62. While maintaining the temperature, add an alkaline solution to adjust the pH to 5.9-6.1. The alkaline solution is a sodium hydroxide solution with a concentration of 5 mol / L, and the dropping rate of the sodium hydroxide solution is 20 ml / min. After the alkaline solution is completely added, continue stirring for 20 minutes at a stirring speed of 500 rpm.

[0173] S63. After heating to 80℃~85℃, add sodium alginate and continue stirring for 60 minutes. The amount of sodium alginate added is 0.1% of the mass of the leucine concentrate. Ultrasonic treatment is used during the stirring process. The ultrasonic power is 150W and the ultrasonic frequency is 20kHz.

[0174] S64. Then, a gradient cooling process was performed: initially, the temperature was reduced to 40℃ at a cooling rate of 20℃ / h, then to 20℃ at a cooling rate of 10℃ / h, then to 4℃ at a cooling rate of 8℃ / h, and then maintained at 4℃ for 24 hours, stopping when the crystals grew to 600μm. After completion, the crystals were filtered and then dried under negative pressure at 60℃ to obtain leucine crystals.

[0175] Experimental Example: Characterization Analysis of Samples from Example C1 and Example C2

[0176] I. Sample scanning electron microscopy

[0177] First, a scanning electron microscope (SEM) sample was prepared. A suitable amount of sample was picked up, fixed on a sample tray, and sputtered with gold under nitrogen protection at 15 kV. The surface morphology of the sample was then analyzed using a TM3000 scanning electron microscope (Hitachi).

[0178] II. Particle Size Distribution

[0179] Analysis methods: (1) Image analysis software: Use ImageJ software to process the SEM images into any of the following formats: TIFF, PNG, GIF, JPEG, BMP. Click File—Open in the ImageJ menu bar, find the desired TIF or JPG file, and open it. (2) Set the ruler: Find the line tool in the toolbar, and use it to draw a straight line that coincides with the ruler length. (3) Use the line tool to draw the diameter of a certain particle. The particle size distribution should be counted for at least 100 nanoparticles. Use the analyze particles option of the software to obtain the roundness (sphericity), diameter, area, and perimeter of the spherulite image. Statistically analyze the particle size distribution of the sample.

[0180] (3) Bulk density testing

[0181] Test method: Dry the sample to constant weight, weigh 100g of sample, and fill the sample into a 20mL standard graduated cylinder. Slowly add the sample into the container using a funnel-type free-fall method, ensuring the filling height exceeds the container opening. Then, use a ruler to level the sample with the container opening. Weigh the total mass of the container and the sample, subtract the mass of the container to obtain the net weight of the sample, and then calculate the bulk density of the sample.

[0182] Characterization analysis results:

[0183] The scanning electron microscope (SEM) images of embodiments C1 and C2 of the present invention are as follows: Figure 2 and Figure 3 As shown, the cumulative particle size distribution diagram is as follows: Figure 4 and Figure 5 As shown in the attached figures, the leucine crystals obtained in Example C of this invention are spherical, with a particle size distribution mainly between 500 μm and 1000 μm. The bulk density of the sample from Example C1 was measured to be 1.12 g / cm³. 3 The bulk density of the sample in Example C2 was 1.13 g / cm³. 3 Both have good liquidity.

[0184] Comparative Experiment Example 1:

[0185] To verify the influence of different processes on crystal habit during recrystallization, different process parameters and additives were used in different comparative experimental examples. All comparative experimental examples of this invention are based on Example C1, meaning all experimental procedures are variations of Example C1, with Example C1 serving as the comparison object. In the comparative experimental examples, the dissolution temperature, microporous membrane filtration process, concentration process, alkali addition method, and stirring speed remained unchanged; that is, these process procedures were consistent with Example C1 in each comparative experimental example.

[0186] Comparative experimental example process basis:

[0187] S61. Dissolve the refined leucine in a glycerol-acidic solution, which is a hydrochloric acid solution of glycerol, with a hydrochloric acid concentration of 3 mol / L and a glycerol concentration of 0.18 mol / L; the dissolution temperature of the refined leucine is 20℃.

[0188] Then, a microporous membrane is used for filtration, and the filtrate is evaporated and concentrated.

[0189] S62. Add alkali solution to adjust the pH value to 5.9~6.1. Continue stirring after the alkali solution has been added.

[0190] S63. After heating to 82℃, add sodium alginate and continue stirring for 40 minutes. The amount of sodium alginate added is 0.3% of the mass of the leucine concentrate. Ultrasonic treatment is used during the stirring process. The ultrasonic power is 160W and the ultrasonic frequency is 30kHz.

[0191] S64. Then, a gradient cooling process was performed: initially, the temperature was reduced to 40℃ at a cooling rate of 22℃ / h, then to 20℃ at a cooling rate of 12℃ / h, then to 4℃ at a cooling rate of 6℃ / h, and then maintained at 4℃ for 20h, stopping when the crystals grew to 700μm. After completion, the crystals were filtered and then dried under negative pressure at 55℃ to obtain leucine crystals.

[0192] Comparative Experiment Example 1:

[0193] S61. Dissolve the refined leucine in an acidic solution with a hydrochloric acid concentration of 3 mol / L. Then filter the solution using a microporous membrane, and concentrate the filtrate by evaporation.

[0194] S62. Add alkali solution to adjust the pH value to 5.9~6.1. Continue stirring after the alkali solution has been added.

[0195] S63. After heating to 82℃, add sodium alginate and continue stirring for 40 minutes. The amount of sodium alginate added is 0.3% of the mass of the leucine concentrate. Ultrasonic treatment is used during the stirring process. The ultrasonic power is 160W and the ultrasonic frequency is 30kHz.

[0196] S64. Then, a gradient cooling process was performed: initially, the temperature was reduced to 40℃ at a cooling rate of 22℃ / h, then to 20℃ at a cooling rate of 12℃ / h, then to 4℃ at a cooling rate of 6℃ / h, and then maintained at 4℃ for 20h, stopping when the crystals grew to 700μm. After completion, the crystals were filtered and then dried under negative pressure at 55℃ to obtain leucine crystals.

[0197] Comparative Experiment Example 2:

[0198] S61. Dissolve the refined leucine in a glycerol-acidic solution, which is a hydrochloric acid solution of glycerol, with a hydrochloric acid concentration of 3 mol / L and a glycerol concentration of 0.18 mol / L; the dissolution temperature of the refined leucine is 20℃.

[0199] Then, a microporous membrane is used for filtration, and the filtrate is evaporated and concentrated.

[0200] S62. Add alkali solution to adjust the pH value to 5.9~6.1. Continue stirring after the alkali solution has been added.

[0201] S63. After heating to 82℃, continue stirring for 40 minutes, using ultrasonic treatment during the stirring process; the ultrasonic power is 160W and the ultrasonic frequency is 30kHz.

[0202] S64. Then, a gradient cooling process was performed: initially, the temperature was reduced to 40℃ at a cooling rate of 22℃ / h, then to 20℃ at a cooling rate of 12℃ / h, then to 4℃ at a cooling rate of 6℃ / h, and then maintained at 4℃ for 20h, stopping when the crystals grew to 700μm. After completion, the crystals were filtered and then dried under negative pressure at 55℃ to obtain leucine crystals.

[0203] Comparative Experiment Example 3:

[0204] S61. Dissolve the refined leucine in a glycerol-acidic solution, which is a hydrochloric acid solution of glycerol, with a hydrochloric acid concentration of 3 mol / L and a glycerol concentration of 0.18 mol / L; the dissolution temperature of the refined leucine is 20℃.

[0205] Then, a microporous membrane is used for filtration, and the filtrate is evaporated and concentrated.

[0206] S62. Add alkali solution to adjust the pH value to 5.9~6.1. Continue stirring after the alkali solution has been added.

[0207] S63. After heating to 82℃, add sodium alginate and continue stirring for 40 minutes. The amount of sodium alginate added is 0.3% of the mass of the leucine concentrate.

[0208] S64. Then, a gradient cooling process was performed: initially, the temperature was reduced to 40℃ at a cooling rate of 22℃ / h, then to 20℃ at a cooling rate of 12℃ / h, then to 4℃ at a cooling rate of 6℃ / h, and then maintained at 4℃ for 20h, stopping when the crystals grew to 700μm. After completion, the crystals were filtered and then dried under negative pressure at 55℃ to obtain leucine crystals.

[0209] Comparative Experiment Example 4:

[0210] S61. Dissolve the refined leucine in a glycerol-acidic solution, which is a hydrochloric acid solution of glycerol, with a hydrochloric acid concentration of 3 mol / L and a glycerol concentration of 0.18 mol / L; the dissolution temperature of the refined leucine is 20℃.

[0211] Then, a microporous membrane is used for filtration, and the filtrate is evaporated and concentrated.

[0212] S62. Add alkali solution to adjust the pH value to 5.9~6.1. Continue stirring after the alkali solution has been added.

[0213] S63. After heating to 82℃, add sodium alginate and continue stirring for 40 minutes. The amount of sodium alginate added is 0.3% of the mass of the leucine concentrate. Ultrasonic treatment is used during the stirring process. The ultrasonic power is 160W and the ultrasonic frequency is 30kHz.

[0214] S64. Then, a gradient cooling process was performed: initially, the temperature was reduced to 20℃ at a cooling rate of 35℃ / h, then further reduced to 4℃ at a cooling rate of 15℃ / h, and then maintained at 4℃ for 20 hours, ending when the crystals grew to 700μm. After completion, the crystals were filtered and then dried under negative pressure at 55℃ to obtain leucine crystals.

[0215] The differences between the comparative experimental example and Example C1 of the present invention are shown below:

[0216] 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:

[0217] Group Particle size distribution range μm Average particle size (μm) Comparative Experiment Example 1 860-1530 1250 Comparative Experiment Example 2 750-1230 960 Comparative Experiment Example 3 680-1650 1050 Comparative Experiment Example 4 930-1650 1180

[0218] 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.

[0219] 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.

[0220] Comparative Experiment Example 2:

[0221] 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 comparative experiments is shown below.

[0222] 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 generating refined amino acid products by hydrolyzing animal protein blood meal, characterized in that, Includes the following steps: Step S1: Add animal protein raw materials to acid solution for acid hydrolysis. After hydrolysis, add alkaline solution to adjust the pH value to 4.5~5.5, add activated carbon for decolorization and filter. After filtration, protein hydrolysate is obtained. Step S2: Cool the protein hydrolysate to room temperature, then precipitate crude histidine from the protein hydrolysate, and separate the crude histidine to obtain the remaining mother liquor. Step S3: The crude histidine is purified to obtain refined histidine. The histidine purification process is shown below: Step S31: Add crude histidine to anhydrous ethanol to wash and remove impurities, then filter. After filtration, wash several times with cold water, recover the washing water, heat and evaporate the washing water to remove ethanol, and then mix it with the washed and filtered crude histidine to obtain pretreated histidine. Step S32: After dissolving the pretreated histidine, it is adsorbed using a weak acid ion exchange resin. After adsorption, it is eluted with dilute hydrochloric acid solution. The eluents are combined and decolorized with activated carbon. Then, the eluent is evaporated and concentrated to supersaturation, and cooled to crystallize to obtain the refined histidine. Step S4: Add leucine precipitant to the remaining mother liquor, let it stand to precipitate, filter it, wash it after filtration to obtain precipitate and tail liquor; add the precipitate to water, neutralize it with ammonia water to adjust the pH value, stir to remove excess ammonia, and obtain crude leucine. Step S5: Dissolve crude leucine in hot water, decolorize, filter and concentrate, cool and crystals will naturally precipitate, filter the crystals to obtain refined leucine. Step S6: Recrystallize the refined leucine to obtain high-purity leucine; Step S7: The tail liquid is dried using spray drying technology to obtain an amino acid mixed powder. The specific process of leucine recrystallization in step S6 is as follows: S61. Dissolve the refined leucine in a glycerol-acidic solution and filter using a microporous membrane; concentrate the filtrate by evaporation at 65℃~75℃. S62. While keeping warm, add alkali solution to adjust the pH value to 5.9~6.

1. After the alkali solution is added, continue stirring for 10min~20min. S63. After heating to 80℃~85℃, add sodium alginate and continue stirring for 30min~60min. The amount of sodium alginate added is 0.1%~0.5% of the mass of leucine concentrate. Ultrasonic treatment is used during the stirring process. S64. Then, a gradient cooling process is performed. The gradient cooling method is as follows: first, the temperature is reduced to 40℃ at a cooling rate of 20℃ / h to 25℃ / h, then the temperature is reduced to 20℃ at a cooling rate of 10℃ / h to 15℃ / h, then the temperature is reduced to 4℃ at a cooling rate of 5℃ / h to 8℃ / h, and then maintained at 4℃ for 12h to 24h. The process ends when the crystal grows to 500μm to 1000μm.

2. The method for generating refined amino acids by hydrolyzing animal protein blood meal as described in claim 1, characterized in that, The specific method for step S1 is as follows: Animal protein raw materials include animal blood meal, animal hair, or animal feathers. The animal protein raw materials are added to a 4 mol / L to 5 mol / L hydrochloric acid solution, with a solid-liquid ratio of 1:3 to 5. The acid hydrolysis temperature is 100℃ to 120℃, and the acid hydrolysis time is 9h to 12h. After hydrolysis, sodium hydroxide solution is added to adjust the pH value to 4.5 to 5.

5. The temperature is then lowered to 50℃ to 70℃, and activated carbon is added for decolorization and filtration. The filtered protein hydrolysate is obtained.

3. The method for generating refined amino acids by hydrolyzing animal protein blood meal as described in claim 1, characterized in that: In step S2, after the protein hydrolysate is cooled, an alkaline solution is added to adjust the pH value to the isoelectric point of histidine, precipitating histidine from the protein hydrolysate. After standing for 5-10 hours, the mixture is filtered to obtain crude histidine and the remaining mother liquor.

4. The method for generating refined amino acids by hydrolyzing animal protein blood meal as described in claim 1, characterized in that: In step S32, the mass ratio of histidine pretreatment product to weakly acidic ion exchange resin is 1:3~8; the concentration of histidine pretreatment product solution is 15g / L~30g / L; the eluent flow rate is 1ml / min~3ml / min; and the elution temperature is 25℃~35℃.

5. The method for generating refined amino acids by hydrolyzing animal protein blood meal as described in claim 1, characterized in that: The leucine precipitant added in step S4 is o-xylene-4-sulfonic acid. The amount of leucine precipitant added is 25%~35% of the dry weight of the raw material. The temperature is lowered to 0℃~5℃, and the mixture is stirred for 2h~4h. Then, the mixture is kept at the temperature and allowed to stand for 18h~30h to settle. After the settling is complete, the mixture is filtered, the filter cake is washed and then filtered dry to obtain the precipitate and tail liquid. Add 3-4 times the volume of ammonia water to neutralize and adjust the pH to 7-8. Maintain the temperature at 70℃-80℃ and vacuum stir for 50-80 minutes to remove excess ammonia. Then cool to room temperature, let stand for 12-20 hours, and filter. Wash the filter cake several times with deionized water and then filter dry to obtain crude leucine.

6. The method for generating refined amino acids by hydrolyzing animal protein blood meal as described in claim 1, characterized in that: In step S5, leucine is added to 30-40 times its weight of deionized water and heated to 50-70°C to dissolve. Activated carbon is added for decolorization. After decolorization, the solution is filtered. The filtrate is heated to 70-80°C and concentrated under vacuum. After cooling, crystals naturally precipitate. The crystals are washed several times with deionized water and then filtered dry to obtain high-quality leucine.

7. The method for generating refined amino acids by hydrolyzing animal protein blood meal as described in claim 1, characterized in that: In step S6, the glycerol-acidic solution 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 refined leucine is 15℃ to 25℃.

8. The method for generating refined amino acids by hydrolyzing animal protein blood meal as described in claim 1, characterized in that: The filtration 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.

9. The method for generating refined amino acids by hydrolyzing animal protein blood meal as described in claim 1, characterized in that: 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 is completed, 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.

Images