Preparation method and application of protein chelated with divalent metal ions

Abstract

The invention relates to a preparation method and application of divalent metal ion chelated protein, which comprises the following steps: A, dissolving divalent metal ion salt and L-lysine in water, heating and stirring to obtain a lysine metal chelated salt solution for later use; b, heating and dispersing protein in water to obtain a protein dispersion liquid; c, adding the lysine metal chelate salt solution into the protein dispersion liquid, adding glutamine transaminase, and performing heat preservation and stirring to obtain a cross-linking reaction liquid; d, continuously heating the cross-linking reaction solution and keeping the temperature to obtain an enzyme inactivation reaction solution; and E, homogenizing the enzyme inactivation reaction liquid to obtain the protein chelated with the divalent metal ions. Amino acid is adopted to chelate divalent metal ions, and the problems that a traditional special medical emulsion product is poor in stability, prone to precipitation and the like due to the fact that insoluble mineral substances are used are solved.

Description

Technical Field

[0001] The invention relates to the technical field of special medical formula foods, and in particular to a method for preparing a protein capable of chelating divalent metal ions and an application thereof. Background Art

[0002] Among the special medical products that have been successfully approved for marketing in my country today, powders account for the majority, while the protein content in emulsion products is generally low. The main reason is that during the preparation, sterilization, transportation and storage of special medical emulsions, insoluble mineral salts will spontaneously settle and flocculate with proteins during the sedimentation process, causing the product to break emulsions and flocculate. If soluble mineral ions are used, the protein will aggregate into clumps, destroying the stability of the system, commonly known as the "brine effect."

[0003] A common method of stabilizing minerals is to add a suitable colloid to the system. The principle of this method is to increase the stability of the system and slow down the sedimentation rate of minerals by cross-linking the colloid with the protein and increasing the viscosity of the system. However, the disadvantage of using this method is that the viscosity of the system increases, and the protein content is usually difficult to exceed 60g / L. When used in some situations where high protein supplementation is required but fluid is limited, it will cause water retention in patients. At the same time, increasing the viscosity of the system will also increase production energy consumption and cause esophageal discomfort in users. Summary of the invention

[0004] The purpose of the present invention is to use amino acid chelated divalent metal ions to solve the problem that traditional special medical emulsion products use insoluble minerals with poor stability and easy precipitation of the products, and use amino acid chelated metal ions to improve the mineral absorption efficiency, improve the thermal stability of proteins and extend the shelf life of the products.

[0005] The present invention adopts L-lysine as a chelating agent for divalent metal ions, and uses TG enzyme to catalyze a cross-linking reaction between L-lysine and glutamine in the protein. In addition to reacting with amino acid-metal ions, the protein also undergoes a certain degree of cross-linking within itself, thereby improving the thermal stability of the protein.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A method for preparing a protein that chelates divalent metal ions comprises the following steps:

[0008] A. Dissolve a divalent metal ion salt and L-lysine in water, and heat and stir to obtain a lysine metal chelate salt solution for later use;

[0009] B. Heat and disperse the protein in water to obtain a protein dispersion;

[0010] C. adding the lysine metal chelate solution to the protein dispersion, and adding glutamine aminotransferase, and stirring at a temperature of 40 °C to obtain a cross-linking reaction solution;

[0011] D. continuing to heat and maintain the cross-linking reaction solution to obtain an enzyme inactivation reaction solution;

[0012] E. Homogenize the enzyme inactivation reaction solution to obtain a protein that chelates divalent metal ions.

[0013] After amino acids and divalent metal ions are mixed, the temperature and pH value of the reaction system are adjusted to produce amino acid-metal ion chelates. TG enzyme is used to cross-link the reaction products with proteins. The peptide bonds with higher stability produced between proteins and amino acid-metal ion chelates have better stability than the insoluble minerals used in traditional special medical processes. At the same time, TG enzyme also catalyzes the formation of a gelatinous structure inside the protein, thereby improving the thermal stability of the protein itself.

[0014] Among the many amino acids, L-lysine can be linked with glutamine residues in proteins under the catalysis of glutamine transaminase to eventually form a stable product. Changing the type of amino acid or enzyme will cause other types of reactions and will not improve the stability of the system.

[0015] Preferably, in step A, the molar ratio of the divalent metal ion in the divalent metal ion salt to the L-lysine includes 1:2, and the molar mass ratio of the divalent metal ion in the divalent metal ion salt to water includes 0.01 mol~0.03 mol:50 g; in step B, the mass ratio of the protein to water includes 80~100 g:800~1000 g; in step C, the amount of glutamine transaminase added includes 4~6 U / g protein.

[0016] Preferably, in step A, the divalent metal ion salt includes water-soluble salts of one or more metal ions selected from the group consisting of magnesium ion, calcium ion, copper ion, ferrous ion, zinc ion, and manganese ion; in step B, the protein includes one or more selected from the group consisting of milk protein, whey protein, casein, caseinate, soy protein, corn protein, and pea protein; and in step E, the homogenization includes high-pressure homogenization.

[0017] Divalent metal ions often play an important role in the human body, such as Fe 2+ Participates in the formation of hemoglobin and is responsible for transporting oxygen; Zn 2+ With Mg 2+ It is an important component and enzyme agonist of enzymes in the human body, while calcium is an important component of human bones.

[0018] Among special medical foods, products with different formulas can be provided based on the patient's needs for different types of elements, which can be more conducive to the patient's recovery.

[0019] In food science, proteins are classified into fast proteins and slow proteins. The former are metabolized relatively quickly in the body and can generally be digested in half an hour to increase the level of amino acids in the blood. The most common one is whey protein. The latter are metabolized relatively slowly in the body and the overall digestion is relatively stable. It can maintain the stability of the amino acid level in the blood for a longer period of time and can provide a certain degree of satiety to the user. The most common one is casein.

[0020] The present invention uses one or more proteins to compound, which can respond to the needs of different patients for protein speed and animal and plant protein ratio, provide possibilities for products, and provide patients with nutritional ingredients that are more in line with their own needs.

[0021] Preferably, in step A, the specific conditions for heating and stirring include: heating to 65-85°C and stirring at 300rpm for more than 60 minutes; in step B, the specific conditions for heating and dispersing include: heating to 45-55°C and shearing at 3600rpm for 20 minutes; in step C, the specific conditions for heat preservation and stirring include: heat preservation at 45-55°C and stirring at 300rpm for 90-120 minutes; in step D, the specific conditions for continuing heating and maintaining include: heating to 65-75°C and maintaining for 10 minutes; in step E, the specific conditions for homogenization include: homogenization at 200-400Bar for 1-3 times.

[0022] Preferably, step A further comprises adding a pH adjuster during the heating and stirring process to adjust the pH to a weakly acidic state.

[0023] Weak acidity is the optimal reaction pH for enzymes, and excessive acidity or alkalinity is not conducive to the enzyme reaction.

[0024] Preferably, in step A, the pH is adjusted to 6-7; the pH adjuster comprises a gluconic acid solution.

[0025] Any pH adjuster can be conventionally selected for pH adjustment, but considering that the present invention is applied to food manufacturing and the chemical equilibrium constant between the anions of other acids and the metal ions in the system needs to be considered, gluconic acid solution should be considered as a better choice.

[0026] On the one hand, the gluconate radical and each metal cation in the present invention have a large equilibrium constant and are relatively stable in the reaction system. On the other hand, gluconic acid is also a food raw material. Using gluconic acid as an acid-base additive for chelating lysine with divalent metals also has the following advantages: one, gluconic acid can be used as a food additive, and gluconic acid is a carrier of the third generation of mineral supplements, and its safety is recognized by the market. Second, in some industries that use marine biological shells to produce amino acid chelates, hydrochloric acid is used to dissolve the shell and act as an acid-base regulator, but in the field of special medical foods, hydrochloric acid belongs to a processing aid, and it is required to be removed after the process flow while being introduced. Due to the complex ingredient system of special medical food products, the intermolecular balance is highly dependent on the pH value, and the stability of the system may be destroyed while removing hydrochloric acid, resulting in irreversible damage to the product. Third, lysine is an alkaline amino acid, and the reaction system is alkaline. Adding it to a protein dispersion will destroy the balance and cause protein precipitation, so it is necessary to adjust the pH of the chelated salt solution to be between 6 and 7.

[0027] A protein chelating divalent metal ions obtained by the method for preparing a protein chelating divalent metal ions as described above.

[0028] An application of the protein chelating divalent metal ions as described above is used to prepare a nanoemulsion of a special medical food.

[0029] Preferably, the preparation of the special medical food nanoemulsion comprises the following steps: after obtaining the protein that chelates the divalent metal ions, polysaccharides, oils, vitamins, and minerals are added, and the special medical food nanoemulsion is obtained through dispersion, homogenization, and high-temperature sterilization.

[0030] Preferably, the polysaccharide includes one or more of maltodextrin, resistant dextrin, oligofructose, oligogalactose, tapioca starch, cellulose, carrageenan, pectin, trehalose, and microcrystalline cellulose; the oil includes one or more of olive oil, soybean oil, rapeseed oil, corn oil, medium-chain triglycerides, and fish oil; the vitamin includes one or more of vitamin A palmitate, cholecalciferol, vitamin E, vitamin K1, thiamine nitrate, riboflavin, niacinamide, D-calcium pantothenate, pyridoxine hydrochloride, cyanocobalamin, L-ascorbic acid, D-biotin, folic acid, choline bitartrate, and inositol; the mineral includes one or more of potassium chloride, sodium chloride, sodium selenite, and potassium iodide.

[0031] The protein content of currently available special medical emulsion products generally does not exceed 60g / L, and the shelf life is usually 12 to 18 months. The main reason is that after the product is sterilized by a thermal process, too high a protein content will lead to instability of the emulsion system, and the interaction between the protein and the insoluble salt will cause the product to fail to maintain good appearance properties. The process of the present invention treats the protein and divalent metal ions, improves the heat resistance of the protein, and at the same time improves the protein's tolerance to salt, so that the maximum protein content of the emulsion can be increased to 72-90g / L, and the shelf life can be extended to 24 months, which is a significant breakthrough.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] By using amino acid chelating divalent metal ion technology and innovating traditional acid-base regulators, we have developed a method for preparing amino acid-metal ion chelates suitable for the food industry to improve the stability of special medical food systems.

[0034] The use of TG enzyme to crosslink the glutamine residues in the protein with amino acid-metal ion chelators can generate extremely stable peptide bonds compared to traditional processes. The reaction conditions are mild and controllable, and the enzyme can be inactivated by heating for a short period of time after the reaction without affecting the subsequent reaction system.

[0035] By using the special medical food nanoemulsion prepared by this patent, the protein content can be increased to 72-90g / L and the shelf life can be extended to 24 months. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a schematic diagram of the process of the present invention.

[0037] Figure 2 It is a comparison diagram of the appearance properties of the embodiments of the present invention and the comparative examples. DETAILED DESCRIPTION

[0038] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0039] Example 1

[0040] Examples 1-4

[0041] like Figure 1 As shown, the special medical food nanoemulsion is prepared by the following steps

[0042] 1. Weigh 1.665g calcium chloride, 0.955g magnesium chloride, 0.410g zinc chloride, 0.127g ferrous chloride, and 8.50g L-lysine, dissolve in 90g purified water, and adjust the pH to 6.0-7.0 with gluconic acid solution.

[0043] 2. Heat the reaction solution to 75°C and stir at 300 rpm for 120 min.

[0044] 3. As in step 2, weigh the milk protein according to the following table:

[0045]

[0046] Add to 800 g of water and use a high-speed disperser to shear for 20 min at 3600 rpm until the suspension has no obvious particles, and set aside.

[0047] 4. After the chelation reaction in step 2 reaches the time, add it to the protein suspension in step 3, weigh 500U of TG enzyme, heat it to 55°C, and stir it at 300rpm for 120min.

[0048] 5. Raise the reaction solution temperature to 75°C and maintain for 10 minutes.

[0049] 6. Homogenize the reaction solution once at 400 Bar pressure.

[0050] 7. Use special medical food emulsion preparation technology to prepare nanoemulsions, including adding polysaccharides, oils, vitamins, minerals, etc., and performing dispersion, homogenization, high-temperature sterilization, etc.

[0051] Comparative Example 1

[0052] Prepare the FSMP nanoemulsion as follows:

[0053] 1.1. Weigh 1.665 g of calcium chloride, 0.955 g of magnesium chloride, 0.410 g of zinc chloride, and 0.127 g of ferrous chloride and dissolve them in 90 g of purified water.

[0054] 2. Weigh 75g potassium caseinate and add it to 800g water. Use a high-speed disperser to shear for 20min at 3600rpm until there is no obvious particle in the suspension.

[0055] 3. Add the mineral solution from step 1 to the protein dispersion from step 2.

[0056] 4. Homogenize once at 400 Bar pressure.

[0057] 5. Use special medical food emulsion preparation technology to prepare nanoemulsions, including adding polysaccharides, oils, vitamins, minerals, etc., and performing dispersion, homogenization, high-temperature sterilization, etc.

[0058] Comparative Example 2

[0059] Prepare the FSMP nanoemulsion as follows:

[0060] 1. Weigh 1.551 g of tricalcium phosphate, 0.405 g of magnesium oxide, 1.370 g of zinc gluconate, and 0.152 g of ferrous sulfate and dissolve them in 90 g of purified water.

[0061] 2. Weigh 75g potassium caseinate and add it to 800g water. Use a high-speed disperser to shear for 20min at 3600rpm until there is no obvious particle in the suspension.

[0062] 3. Add the mineral solution from step 1 to the protein dispersion from step 2.

[0063] 4. Homogenize once at 400 Bar pressure.

[0064] 5. Use special medical food emulsion preparation technology to prepare nanoemulsions, including adding polysaccharides, oils, vitamins, minerals, etc., and performing dispersion, homogenization, high-temperature sterilization, etc.

[0065] Comparative Example 3

[0066] 1. Weigh 1.665g calcium chloride, 0.955g magnesium chloride, 0.410g zinc chloride, 0.127g ferrous chloride, and 8.50g L-lysine, dissolve in 90g purified water, and adjust the pH to 6.0-7.0 with gluconic acid solution.

[0067] 2. Heat the reaction solution to 75°C and stir at 300 rpm for 120 min.

[0068] 3. As in step 2, weigh 75g of milk protein, add it to 800g of water, and use a high-speed disperser to shear for 20min at 3600rpm until the suspension has no obvious particles, and set aside.

[0069] 4. After the chelation reaction in step 2 reaches the time, add it to the protein suspension in step 3, heat it to 55°C, and stir it at 300 rpm for 120 minutes.

[0070] 5. Homogenize the reaction solution once at 400 Bar pressure.

[0071] 6. Use special medical food emulsion preparation technology to prepare nanoemulsions, including adding polysaccharides, oils, vitamins, minerals, etc., and performing dispersion, homogenization, high-temperature sterilization, etc.

[0072] Comparative Example 4

[0073] 1. Weigh 1.665g calcium chloride, 0.955g magnesium chloride, 0.410g zinc chloride, 0.127g ferrous chloride, and 8.50g L-lysine, dissolve in 90g purified water, and adjust the pH to 6.0-7.0 with gluconic acid solution.

[0074] 2. Heat the reaction solution to 75°C and stir at 300 rpm for 120 min.

[0075] 3. As in step 2, weigh 75g of milk protein, add it to 800g of water, and use a high-speed disperser to shear for 20min at 3600rpm until the suspension has no obvious particles, and set aside.

[0076] 4. After the chelation reaction in step 2 reaches the time, add it to the protein suspension in step 3, weigh 500U of TG enzyme, heat it to 55°C, and stir it at 300rpm for 120min.

[0077] 5. Homogenize the reaction solution once at 400 Bar pressure.

[0078] 6. Use special medical food emulsion preparation technology to prepare nanoemulsions, including adding polysaccharides, oils, vitamins, minerals, etc., and performing dispersion, homogenization, high-temperature sterilization, etc.

[0079] Effect Example 1

[0080] The products prepared in Examples 1-4 and Comparative Examples 1-4 (such as Figure 2 ) were subjected to physical and chemical tests. The test results are shown in Table 1 and Figure 2 As shown:

[0081] Table 1

[0082]

[0083]

[0084] Note: *Instability coefficient: obtained by testing with a multifunctional stability analyzer. The principle of the equipment is to monitor the light transmittance curve of the product during the centrifugation process in real time. The supporting software combines Stokes' law and Lambert-Bell's law to calculate the instability coefficient of the system.

[0085] Combining from Table 1 Figure 2 It can be seen from the test results that the embodiment uses the process conditions of the present invention to combine divalent metal ions with lysine in the front-end reaction and use TG enzyme to cross-link them with proteins to form a more stable amino acid-metal ion chelate-protein complex system, which effectively improves the capacity of the system for divalent metals and improves the thermal stability of proteins.

[0086] As can be seen from Examples 1-3, the protein content in the special medical emulsion system can be increased to 9.5 g / 100 mL using the process conditions of the present invention, and the centrifugal stability at this protein concentration is good. In Example 4, we can see that the sterilized special medical emulsion has slight protein flocculation, and the emulsion particle size has also increased sharply from less than 500 nm in Examples 1-3 to 900 nm, which has broken through the stability of the nanoemulsion. Combining the above two results, we can infer that the emulsion system has broken through the critical value of stability.

[0087] Compared with Comparative Example 1, Comparative Example 1 selected soluble calcium and magnesium salts as element sources, divalent ionization, and the thermal stability of the system was affected to a certain extent. The complexation of L-lysine was lacking, and it could not react into a stable compound. The ionic strength of the system was high, and the nanoemulsion was unstable after thermal sterilization. The protein structure spontaneously agglomerated under the action of metal ions and finally formed floccules. Compared with Comparative Example 2, tricalcium phosphate and magnesium oxide were used as element sources of calcium and magnesium, which effectively reduced the ionic strength of the system and improved the thermal stability of the system to a certain extent. However, insoluble substances would continue to settle during the shelf storage of the emulsion, and would continue to combine with the macromolecules in the system during the settling process, resulting in the sinking of product nutrients and the destruction of the system. Figure 2 .

[0088] The special medical nanoemulsion prepared in Comparative Example 2 began to precipitate at the beginning of the stability analysis, while the special medical nanoemulsion prepared in the example slowly precipitated during the centrifugation process and had not completely precipitated by the end of the experiment.

[0089] Comparative Example 3 adds the same concentration of protein as Example 1, but lacks the crosslinking effect of TG enzyme, and the product has protein particles stratified, and the system is unstable.

[0090] In Comparative Example 4, the TG enzyme was not inactivated, resulting in a large amount of gelation inside the protein, and the protein agglomerated into large lumps.

[0091] It can be seen that the special medical food nanoemulsion prepared by the process of the present invention has the characteristics of high heat resistance and high stability.

[0092] The above disclosure is only the preferred embodiment of the present invention, which cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for preparing a protein that chelates divalent metal ions, characterized in that: The steps include: A. Dissolve a divalent metal ion salt and L-lysine in water, and heat and stir to obtain a lysine metal chelate salt solution for later use; B. Heat and disperse the protein in water to obtain a protein dispersion; C. adding the lysine metal chelate solution to the protein dispersion, and adding glutamine aminotransferase, and stirring at a temperature of 40 °C to obtain a cross-linking reaction solution; D. continuing to heat and maintain the cross-linking reaction solution to obtain an enzyme inactivation reaction solution; E. Homogenize the enzyme inactivation reaction solution to obtain a protein that chelates divalent metal ions.

2. The method for preparing a protein chelating divalent metal ions as claimed in claim 1, characterized in that: In step A, the molar ratio of the divalent metal ion in the divalent metal ion salt to the L-lysine includes 1:2, and the molar mass ratio of the divalent metal ion in the divalent metal ion salt to water includes 0.01 mol~0.03 mol:50 g; in step B, the mass ratio of the protein to water includes 80~100 g:800~1000 g; in step C, the added amount of the glutamine transaminase includes 4~6 U / g protein.

3. The method for preparing a protein chelating divalent metal ions as claimed in claim 1, characterized in that: In step A, the divalent metal ion salt includes water-soluble salts of one or more metal ions selected from the group consisting of magnesium ion, calcium ion, copper ion, ferrous ion, zinc ion, and manganese ion; in step B, the protein includes one or more selected from the group consisting of milk protein, whey protein, casein, caseinate, soy protein, corn protein, and pea protein; in step E, the homogenization includes high-pressure homogenization.

4. The method for preparing a protein chelating divalent metal ions as claimed in claim 1, characterized in that: In step A, the specific conditions for heating and stirring include: heating to 65-85°C and stirring at 300rpm for more than 60 minutes; in step B, the specific conditions for heating and dispersing include: heating to 45-55°C and shearing at 3600rpm for 20 minutes; in step C, the specific conditions for heat preservation and stirring include: heat preservation at 45-55°C and stirring at 300rpm for 90-120 minutes; in step D, the specific conditions for continuing heating and maintaining include: heating to 65-75°C and maintaining for 10 minutes; in step E, the specific conditions for homogenization include: homogenization at 200-400Bar for 1-3 times.

5. The method for preparing a protein chelating divalent metal ions as claimed in claim 1, characterized in that: In step A, a pH regulator is added during the heating and stirring process to adjust the pH to a weakly acidic state.

6. The method for preparing a protein chelating divalent metal ions as claimed in claim 5, characterized in that: In step A, the pH is adjusted to 6-7; the pH adjuster comprises a gluconic acid solution.

7. A protein chelating divalent metal ions obtained by the method for preparing a protein chelating divalent metal ions as claimed in claim 1.

8. Use of the protein for chelating divalent metal ions as claimed in claim 7, characterized in that: Used to prepare special medical food nanoemulsions.

9. The use according to claim 8, characterized in that The preparation of the special medical food nanoemulsion includes the following steps: after obtaining the protein that chelates the divalent metal ions, adding polysaccharides, oils, vitamins, and minerals, and obtaining the special medical food nanoemulsion through dispersion, homogenization, and high-temperature sterilization.

10. The use according to claim 9, characterized in that: The polysaccharide includes one or more of maltodextrin, resistant dextrin, oligofructose, oligogalactose, cassava starch, cellulose, carrageenan, pectin, trehalose, and microcrystalline cellulose; the oil includes one or more of olive oil, soybean oil, rapeseed oil, corn oil, medium-chain triglycerides, and fish oil; the vitamin includes one or more of vitamin A palmitate, cholecalciferol, vitamin E, vitamin K1, thiamine nitrate, riboflavin, nicotinamide, D-calcium pantothenate, pyridoxine hydrochloride, cyanocobalamin, L-ascorbic acid, D-biotin, folic acid, choline bitartrate, and inositol; the mineral includes one or more of potassium chloride, sodium chloride, sodium selenite, and potassium iodide.

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