阅读说明：本技术 高纯度呈味核苷酸二钠的制备方法 (Preparation method of high-purity flavor nucleotide disodium ) 是由 吴涛 王成 常利斌 龚华 唐艳 王海雷 刘月明 于 2020-07-17 设计创作，主要内容包括：本发明公开了高纯度呈味核苷酸二钠的制备方法,其包括如下步骤：(1)酶催化反应制得酶解液；(2)酶解液过滤除不溶性杂质制得滤液；(3)滤液经纳滤、电渗析去除磷酸盐制得核苷酸二钠清液；(4)核苷酸二钠清液浓缩制得核苷酸二钠初次浓缩液；(5)核苷酸二钠初次浓缩液降温结晶制得呈味核苷酸二钠。本发明采用酶催化法生产核苷酸,用纳滤膜和电渗析膜相结合,去除酶解液中的蛋白、色素、磷酸盐和金属离子等杂质,达到产品提纯的目的,同时优化了具体的分离、结晶工艺,在保证产品收率不低于105％(g/g)的情况下,使得产品纯度能够达到99.5％；本发明工艺流程短,操作简便,原料成本低,且更加绿色环保。(The invention discloses a preparation method of high-purity flavor nucleotide disodium, which comprises the following steps: (1) enzyme-catalyzed reaction to prepare enzymatic hydrolysate; (2) filtering the enzymolysis solution to remove insoluble impurities to obtain a filtrate; (3) nano-filtering the filtrate, and removing phosphate by electrodialysis to obtain nucleotide disodium clear solution; (4) concentrating the clear solution of the nucleotide disodium to prepare a primary concentrated solution of the nucleotide disodium; (5) and cooling and crystallizing the primary nucleotide disodium concentrated solution to obtain the flavor nucleotide disodium. The method adopts an enzyme catalysis method to produce nucleotide, combines a nanofiltration membrane with an electrodialysis membrane to remove impurities such as protein, pigment, phosphate, metal ions and the like in enzymatic hydrolysate, achieves the aim of product purification, optimizes specific separation and crystallization processes, and ensures that the product purity can reach 99.5 percent under the condition that the product yield is not lower than 105 percent (g/g); the method has the advantages of short process flow, simple and convenient operation, low raw material cost and greenness and environmental protection.)

1. The preparation method of the high-purity flavor nucleotide disodium is characterized by comprising the following steps of:

(1) enzyme-catalyzed reaction to prepare enzymatic hydrolysate;

(2) filtering the enzymolysis solution to remove insoluble impurities to obtain a filtrate;

(3) removing phosphate from the filtrate by a nanofiltration membrane and an electrodialysis membrane in sequence to obtain nucleotide disodium clear solution;

(4) concentrating the clear solution of the nucleotide disodium to prepare a primary concentrated solution of the nucleotide disodium;

(5) and cooling and crystallizing the primary nucleotide disodium concentrated solution to obtain the flavor nucleotide disodium.

2. The method for preparing high-purity disodium ribonucleotide according to claim 1, wherein the enzymatic reaction in the step (1) is used for preparing an enzymatic hydrolysate, and the enzymatic hydrolysate is specifically: adding water into inosine or guanosine and a phosphate donor, uniformly mixing, adding phosphorylase to perform an enzyme catalytic reaction, and collecting an enzymatic hydrolysate; wherein, the pH value of the enzyme catalytic reaction is maintained at 4.1-4.9, the reaction temperature is controlled between 31 ℃ and 39 ℃, and the reaction time is 8-24 hours.

3. The method for producing highly pure disodium ribonucleotide according to claim 2, wherein the molar ratio of the phosphate donor to the inosine or guanosine is (0.5-2.5): 1.0; the mass ratio of the phosphorylase to the inosine is (0.5-2.0) to 1.0; the mass ratio of the phosphorylase to the guanosine is (1.0-4.0): 1.0.

4. The method for preparing disodium high-purity flavor nucleotide according to claim 3, wherein the phosphate donor is at least one of acid pyrophosphate, tripolyphosphate, tetrapolyphosphate, and hexametaphosphate; wherein the pyrophosphate, the tripolyphosphate, the tetrapolyphosphate and the hexametaphosphate are sodium salts or potassium salts.

5. The method for preparing high-purity disodium ribonucleotide according to claim 1, wherein the filtering of the enzymatic hydrolysate obtained in step (2) to remove insoluble impurities is specifically as follows: and filtering the enzymolysis liquid by using a plate and frame filter, circularly feeding, and starting to receive materials when the light transmittance of the filtrate reaches 80-90% to prepare the filtrate.

6. The method for preparing disodium ribonucleotide according to claim 1, wherein the operating conditions of the nanofiltration membrane and the electrodialysis membrane in step (3) are as follows: the mass percent concentration of the feed filtrate is 5-15%, wherein the membrane inlet pressure of the nanofiltration membrane is 0.2-0.3Mpa, the membrane outlet pressure of the nanofiltration membrane is 0.2-0.28Mpa, the operating temperatures of the nanofiltration membrane and the electrodialysis membrane are both 40-60 ℃, the feed filtrate further removes small molecular impurities through the nanofiltration membrane, enters the electrodialysis membrane and is added with water for dialysis, and the water addition is stopped when the purity of the nucleotide disodium in the trapped fluid is detected to be higher than 90%, so that the nucleotide disodium clear solution with the purity of more than 90% is prepared.

7. The method for preparing disodium ribonucleotide with high purity and flavor according to claim 1, wherein in the step (4), the concentration of the primary concentrated solution of disodium ribonucleotide is 20-35% by mass.

8. The preparation method of the high-purity disodium ribonucleotide according to claim 1, wherein the step (5) of cooling and crystallizing the primary concentrated solution of the disodium ribonucleotide to prepare the disodium ribonucleotide comprises the following steps: adjusting the pH value of the primary nucleotide disodium concentrate to 7.3-7.5, then continuously concentrating to obtain a secondary nucleotide disodium concentrate, wherein the mass percentage concentration of the secondary nucleotide disodium concentrate is 30-40%, transferring the secondary nucleotide disodium concentrate into a crystallizer, adding seed crystals, and performing gradient cooling crystallization, wherein the crystallization temperature is reduced to 5-10 ℃, and is reduced by 3-5 ℃ per hour.

9. The method for producing highly pure disodium ribonucleotide according to claim 8, wherein 1 to 1.5 parts by mass of seed crystals are added per 100 parts by mass of the disodium ribonucleotide secondary concentrate.

The technical field is as follows:

the invention relates to a preparation method of disodium ribonucleotide, in particular to a preparation method of high-purity disodium ribonucleotide.

Background art:

nucleotides are the basic building blocks of nucleic acids, consisting of a phosphate, a ribose, and a base. Ribose and base form nucleosides through glycosidic linkages, and nucleosides and phosphates form nucleotides through ester linkages. In life, many nucleotides are often found as food additives. The taste of the food is formed by water-soluble components in the food, wherein the water-soluble components comprise nucleotide, amino acid, organic base and inorganic ions; the above components have at least one of salty, sweet, sour, bitter and fresh tastes; wherein, the flavour development nucleotide comprises inosinic acid, guanylic acid, cytidylic acid, uridylic acid, xanthylic acid and the like. The flavor nucleotide has synergistic effect on sweet taste, meat taste and mellow taste, and has effect in eliminating or inhibiting bad flavors such as sour taste, bitter taste, fishy smell and burnt taste, so as to reduce addition amount and cost, and provide more mellow taste. Therefore, the flavor-developing nucleotide is widely added into various condiments such as chicken essence, chicken powder, fresh-increasing monosodium glutamate, soy sauce, seasoning bags, soup bases, tomato sauce, mayonnaise and the like to enhance the taste and improve the mouthfeel.

Disodium flavour development nucleotide, also known as disodium 5 ' -flavour development nucleotide, comprises disodium 5 ' -Inosinate (IMP) and disodium 5 ' -Guanylate (GMP), is a new generation of nucleotide food freshener, can be directly added into food to play a role in freshening, and is one of the main flavour development components of instant noodle seasoning packets and seasonings such as chicken essence, chicken powder, fresh sauce and the like. The flavour development disodium nucleotide also has the effects of assisting in resisting tumors, improving the immunity of the organism, regulating the nutrition metabolism of the organism and the like, and has wide application in the fields of food, medicine and agriculture industry.

At present, most of the foreign countries produce nucleotide by enzyme catalysis, and domestic production enterprises produce nucleotide by chemical synthesis. Compared with the enzyme catalysis method, the chemical synthesis method not only has complex process route and high cost of raw materials, but also needs to use highly toxic reactants and use organic solvent as a reaction carrier, thereby not only causing serious environmental pollution, but also influencing the body health of production personnel. In the enzyme catalysis method, according to the analysis of enzyme reaction products, the main products of enzymatic hydrolysate of the enzyme conversion method are IMP and phosphate, and part of impurities such as protein, pigment, metal ions and the like are also contained. The methods for separating and purifying nucleotide according to the different composition of the enzymatic hydrolysate are different, for example, a separation and purification method disclosed in chinese patent document CN 101265490B, in which sodium hydroxide is added to the enzymatic conversion solution, phosphate is removed by phosphate isoelectric method, and inosinic acid disodium is obtained by adjusting inosinic acid isoelectric point; the method has the defects of incomplete phosphate removal and low product purity.

The invention content is as follows:

the invention aims to provide a preparation method of flavour development disodium ribonucleotide, which has the advantages of low cost, environmental protection, simple and convenient operation, high purity and high yield.

The purpose of the invention is implemented by the following technical scheme: the preparation method of the high-purity flavor nucleotide disodium comprises the following steps:

(1) enzyme-catalyzed reaction to prepare enzymatic hydrolysate;

(2) filtering the enzymolysis solution to remove insoluble impurities to obtain a filtrate;

(3) removing phosphate from the filtrate by a nanofiltration membrane and an electrodialysis membrane in sequence to obtain nucleotide disodium clear solution;

(4) concentrating the clear solution of the nucleotide disodium to prepare a primary concentrated solution of the nucleotide disodium;

(5) and cooling and crystallizing the primary nucleotide disodium concentrated solution to obtain the flavor nucleotide disodium.

Further, the enzymatic reaction in the step (1) to prepare the enzymatic hydrolysate specifically comprises: adding water into inosine or guanosine and a phosphate donor, uniformly mixing, adding phosphorylase to perform an enzyme catalytic reaction, and collecting an enzymatic hydrolysate; wherein, the pH value of the enzyme catalytic reaction is maintained at 4.1-4.9, the reaction temperature is controlled between 31 ℃ and 39 ℃, and the reaction time is 8-24 hours.

Further, the molar ratio of the phosphate donor to the inosine or guanosine is (0.5-2.5): 1.0; the mass ratio of the phosphorylase to the inosine is (0.5-2.0) to 1.0; the mass ratio of the phosphorylase to the guanosine is (1.0-4.0): 1.0.

Further, the phosphoric acid donor is at least one of acid pyrophosphoric acid, pyrophosphate, tripolyphosphoric acid, tripolyphosphate, tetrapolyphosphoric acid and hexametaphosphate; wherein the pyrophosphate, the tripolyphosphate, the tetrapolyphosphate and the hexametaphosphate are sodium salts or potassium salts.

Further, the filtering of the enzymatic hydrolysate in the step (2) to remove insoluble impurities to obtain a filtrate specifically comprises: and filtering the enzymolysis liquid by using a plate and frame filter, circularly feeding, and starting to receive materials when the light transmittance of the filtrate reaches 80-90% to prepare the filtrate. By controlling the light transmittance of the filtrate, the plate-and-frame filter fully intercepts impurities such as thalli, protein, pigments and the like in the enzymatic hydrolysate, and the subsequent physicochemical indexes such as light transmission, content and the like of the finished product are ensured to be qualified.

Further, in the step (3), the operating conditions of the nanofiltration membrane and the electrodialysis membrane are as follows: the mass percent concentration of the feed filtrate is 5-15%, wherein the membrane inlet pressure of the nanofiltration membrane is 0.2-0.3Mpa, the membrane outlet pressure of the nanofiltration membrane is 0.2-0.28Mpa, the operating temperatures of the nanofiltration membrane and the electrodialysis membrane are both 40-60 ℃, the feed filtrate further removes small molecular impurities through the nanofiltration membrane, enters the electrodialysis membrane and is added with water for dialysis, and the water addition is stopped when the purity of the nucleotide disodium in the trapped fluid is detected to be higher than 90%, so that the nucleotide disodium clear solution with the purity of more than 90% is prepared. The nanofiltration membrane is matched with the electrodialysis membrane, and the operating conditions of the nanofiltration membrane and the electrodialysis membrane are reasonably set, so that the nanofiltration is stably operated, the optimal decolorization effect is achieved, the light transmittance of a filtrate is improved to more than 95%, meanwhile, the electrodialysis achieves the optimal effect, and the removal rate of anions and cations reaches more than 92%.

Further, in the step (4), the concentration of the primary nucleotide disodium concentrate is 20-35% by mass.

Further, the step (5) of cooling and crystallizing the primary nucleotide disodium concentrated solution to prepare the flavor nucleotide disodium, which comprises the following steps: adjusting the pH value of the primary nucleotide disodium concentrate to 7.3-7.5, then continuously concentrating to obtain a secondary nucleotide disodium concentrate, wherein the mass percentage concentration of the secondary nucleotide disodium concentrate is 30-40%, transferring the secondary nucleotide disodium concentrate into a crystallizer, adding seed crystals, and performing gradient cooling crystallization, wherein the crystallization temperature is reduced to 5-10 ℃, and is reduced by 3-5 ℃ per hour. The primary concentration and the secondary concentration are used for improving the concentration of the flavor nucleotide disodium to reach supersaturation and separate out in a crystal form; the pH value is adjusted by one step in the middle of two times of concentration to achieve the effect of adjusting isoelectric point crystallization, so that crystals are stably grown, and the purity and yield of subsequent products are improved.

Further, 1 to 1.5 parts by mass of seed crystal is added to 100 parts by mass of the secondary concentrated solution.

Interpretation of terms:

1. phosphorylase: phosphate groups may be added to the acceptor with the inorganic acid phosphoric acid as the phosphate group donor.

2. A plate and frame filter: also known as plate and frame filter presses, are commonly used in chemical dewatering machinery, and the main components include: a filter frame, a filter plate, and a filter cloth. It features clear filtrate and high recovery rate of solid.

3. Light transmittance: light transmittance is a physical term that reflects the ability of light to transmit through a medium and refers to the percentage of the amount of light transmitted through a transparent or translucent body as compared to the amount of light incident upon it. The degree of clarity used in the reaction filtrate in this patent.

4. Electrodialysis membrane: the process of separating different solute particles by the selective permeability of a semi-permeable membrane is called dialysis. When dialysis is carried out under the action of an electric field, under the drive of an external direct current electric field, anions and cations respectively move to an anode and a cathode by utilizing the selective permeability of an ion exchange membrane (namely, the cations can permeate a cation exchange membrane, and the anions can permeate an anion exchange membrane), thereby realizing the purpose of separating and concentrating substances.

5. And (3) crystallization: the cooling of the hot saturated solution results in supersaturation of the solution with the solute due to its reduced solubility, whereby the solute precipitates in the form of crystals, a process known as crystallization. During the preparation of inosinic acid, finished disodium inosinate often appears in crystal form.

The invention has the advantages that:

the method adopts an enzyme catalysis method to produce nucleotide, combines a nanofiltration membrane and an electrodialysis membrane to remove impurities such as protein, pigment, phosphate, metal ions and the like in enzymatic hydrolysate, achieves the aim of product purification, optimizes specific separation and crystallization processes, and ensures that the product purity can reach 99.5 percent under the condition that the product yield is not lower than 105 percent; the method has the advantages of short process flow, simple and convenient operation, low raw material cost and greenness and environmental protection.

The specific implementation mode is as follows:

a preparation method of high-purity flavor nucleotide disodium comprises the following steps:

(1) enzyme catalysis reaction to prepare enzymolysis liquid: adding water into inosine or guanosine and a phosphate donor, uniformly mixing, adding phosphorylase to perform an enzyme catalytic reaction, and collecting an enzymatic hydrolysate; wherein, the pH value of the enzyme catalytic reaction is maintained at 4.1-4.9, the reaction temperature is controlled between 31 ℃ and 39 ℃, and the reaction time is 8-24 hours; the molar ratio of the phosphate donor to the inosine or guanosine is (0.5-2.5): 1.0; the mass ratio of the phosphorylase to the inosine is (0.5-2.0) to 1.0; the mass ratio of the phosphorylase to the guanosine is (1.0-4.0): 1.0. Wherein the phosphoric acid donor is at least one of acid pyrophosphoric acid, pyrophosphate, tripolyphosphoric acid, tripolyphosphate, tetrapolyphosphoric acid salt and hexametaphosphate; wherein, pyrophosphate, tripolyphosphate, tetrapolyphosphate and hexametaphosphate are sodium salt or potassium salt.

(2) Filtering the enzymolysis solution to remove insoluble impurities to obtain a filtrate, specifically, filtering the enzymolysis solution by using a plate and frame filter, circularly feeding, and starting to receive the material when the light transmittance of the filtrate reaches 80-90% to obtain the filtrate; in some embodiments, the light transmittance reaches 80% to begin to receive material; in some embodiments, the light transmittance reaches 90% to begin to meet.

(3) Removing phosphate from the filtrate by a nanofiltration membrane and an electrodialysis membrane in sequence to obtain nucleotide disodium clear solution, which comprises the following steps: the mass percent concentration of the feed filtrate is 5-15%, wherein the membrane inlet pressure of the nanofiltration membrane is 0.2-0.3Mpa, the membrane outlet pressure of the nanofiltration membrane is 0.2-0.28Mpa, the operating temperatures of the nanofiltration membrane and the electrodialysis membrane are both 40-60 ℃, the feed filtrate further removes small molecular impurities through the nanofiltration membrane, enters the electrodialysis membrane and is added with water for dialysis, and the water addition is stopped when the purity of the nucleotide disodium in the trapped fluid is detected to be higher than 90%, so that the nucleotide disodium clear solution with the purity of more than 90% is prepared.

In some embodiments, nanofiltration membranes with a molecular weight cut-off of 500-.

In some embodiments, the mass percent concentration of the feed filtrate is 5%, the membrane inlet pressure of the nanofiltration membrane is 0.3Mpa, the membrane outlet pressure of the nanofiltration membrane is 0.28Mpa, the operating temperatures of the nanofiltration membrane and the electrodialysis membrane are both 60 ℃, and the water addition is stopped when the purity of the nucleotide disodium in the trapped fluid is detected to be 95%, so as to prepare the nucleotide disodium clear solution with the purity of 95%. In some embodiments, the mass percent concentration of the feed filtrate is 5%, the membrane inlet pressure of the nanofiltration membrane is 0.25Mpa, the membrane outlet pressure of the nanofiltration membrane is 0.23Mpa, the operating temperatures of the nanofiltration membrane and the electrodialysis membrane are both 40 ℃, and the water addition is stopped when the purity of the nucleotide disodium in the trapped fluid is detected to be 90%, so that the nucleotide disodium clear solution with the purity of 90% is prepared. In some embodiments, the mass percent concentration of the feed filtrate is 10%, the membrane inlet pressure of the nanofiltration membrane is 0.22Mpa, the membrane outlet pressure of the nanofiltration membrane is 0.20Mpa, the operating temperatures of the nanofiltration membrane and the electrodialysis membrane are both 50 ℃, and when the purity of the nucleotide disodium in the trapped fluid is detected to be 93%, water is stopped to be added, so that the nucleotide disodium clear solution with the purity of 93% is prepared. In some embodiments, the mass percent concentration of the feed filtrate is 10%, the membrane inlet pressure of the nanofiltration membrane is 0.22Mpa, the membrane outlet pressure of the nanofiltration membrane is 0.20Mpa, the operating temperatures of the nanofiltration membrane and the electrodialysis membrane are both 50 ℃, and the water addition is stopped when the purity of the nucleotide disodium in the trapped fluid is detected to be 90%, so as to prepare the nucleotide disodium clear solution with the purity of 90%. In some embodiments, the mass percent concentration of the feed filtrate is 15%, the membrane inlet pressure of the nanofiltration membrane is 0.22Mpa, the membrane outlet pressure of the nanofiltration membrane is 0.20Mpa, the operating temperatures of the nanofiltration membrane and the electrodialysis membrane are both 55 ℃, and when the purity of the nucleotide disodium in the trapped fluid is detected to be 93%, water is stopped to be added, so that the nucleotide disodium clear solution with the purity of 93% is prepared.

(4) Concentrating the clear nucleotide disodium liquid to prepare a primary nucleotide disodium concentrated solution, wherein the mass percentage concentration of the primary nucleotide disodium concentrated solution is 20-35%;

(5) the method comprises the following steps of (1) cooling and crystallizing the primary nucleotide disodium concentrated solution to obtain flavor nucleotide disodium, which specifically comprises the following steps: adjusting the pH value of the primary nucleotide disodium concentrated solution to 7.3-7.5, then continuously concentrating to obtain a secondary nucleotide disodium concentrated solution, wherein the mass percentage concentration of the secondary nucleotide disodium concentrated solution is 30-40%, transferring the secondary nucleotide disodium concentrated solution into a crystallizer, adding seed crystals, and adding 1-1.5 parts by mass of seed crystals into every 100 parts by mass of the secondary nucleotide disodium concentrated solution; gradient cooling crystallization is adopted, the crystallization temperature is reduced to 5-10 ℃, and the crystallization temperature is reduced by 3-5 ℃ per hour.

In some embodiments, the pH is adjusted to 7.3 ± 0.02 at a concentration of 30% by mass, and 1 part by mass of seed crystals is added per 100 parts by mass of the disodium nucleotide secondary concentrate. In some embodiments, the pH is adjusted to 7.5 ± 0.02 at a concentration of 30% by mass, and 1.5 parts by mass of seed crystals are added per 100 parts by mass of the disodium nucleotide secondary concentrate. In some embodiments, the pH is adjusted to 7.5 ± 0.02, the concentration is 40% by mass, and 1 part by mass of seed crystals is added per 100 parts by mass of the disodium nucleotide secondary concentrate. In some embodiments, the pH is adjusted to 7.7 ± 0.02 at a concentration of 40% by mass, and 1 part by mass of seed crystals is added per 100 parts by mass of the disodium nucleotide secondary concentrate. In some embodiments, the pH is adjusted to 7.7 ± 0.02, the concentration is 40% by mass, and 1.5 parts by mass of seed crystals are added per 100 parts by mass of the disodium nucleotide secondary concentrate.

For a further understanding of the invention, reference will now be made to the following examples which are included to illustrate the invention. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention. Appropriate modifications to the process parameters, which may be apparent to those skilled in the art in view of the disclosure herein, are also considered to be within the scope of this patent. Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.