A low phenylalanine enriched meal

Abstract

The present application relates to the technical field of phenylalanine removal, and discloses a low-phenylalanine-rich diet, specifically: a carboxyl group of a template molecule L-phenylalanine and a pyridyl group of a functional monomer pyridyl imino methyl acryloyloxy monomer are ionically combined, and an amino group of the L-phenylalanine and a carbonyl group of the pyridyl imino methyl acryloyloxy monomer are combined by hydrogen bond to self-assemble into a polymer molecule, and a magnetic L-phenylalanine adsorbent is prepared by a free radical polymerization method; the magnetic L-phenylalanine adsorbent is added into an L-phenylalanine fully dissociated rice protein solution, oscillation adsorption is performed, the magnetic L-phenylalanine adsorbent is magnetically separated, and a low-phenylalanine-rich rice protein base material is obtained by concentration. The present application provides a method for removing L-phenylalanine in rice protein by adsorption, and the obtained low-phenylalanine-rich rice protein base material is a protein component, which is supplemented with other nutrients to prepare a low-phenylalanine-rich diet.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of phenylalanine removal, in particular to a low-phenylalanine-rich diet. BACKGROUND

[0002] Phenylketonuria is a congenital, autosomal, single-gene recessive genetic disease. Due to the deficiency or reduced activity of phenylalanine hydroxylase or its coenzyme tetrahydrobiopterin in the liver of patients, the phenylalanine in the body cannot be converted into tyrosine along the normal metabolic pathway, but is converted into phenylpyruvic acid and phenyllactic acid through transamination under the catalysis of phenylalanine transaminase, and the generated phenylpyruvic acid and phenyllactic acid are transferred to urine and then excreted with urine to form phenylketonuria, so the disease is called phenylketonuria.

[0003] From the pathogenesis of phenylketonuria, the disease can be treated by reducing the concentration of phenylalanine in the blood of patients and increasing the activity of phenylalanine hydroxylase or tetrahydrobiopterin, and the main treatment methods include gene therapy, enzyme therapy and dietary therapy; among them, the dietary therapy controls and relieves the disease by limiting the content of phenylalanine in the diet, and is considered to be the safest, most feasible and effective treatment method.

[0004] Among them, phenylalanine has two isomers: L-phenylalanine and D-phenylalanine, L-phenylalanine is the form naturally existing in proteins, and D-phenylalanine is a non-natural amino acid.

[0005] Therefore, the development of special medical use formula food containing trace amounts of or no phenylalanine is of great significance to patients with phenylketonuria. SUMMARY

[0006] The present application provides a method for adsorbing and removing L-phenylalanine in rice protein, and a low-phenylalanine-rich rice protein base material is prepared, which is used as a protein component, and supplemented with other nutrients to prepare a low-phenylalanine-rich diet.

[0007] A method for adsorbing and removing L-phenylalanine in rice protein, comprising the following steps:

[0008] Step 1: preparing a magnetic L-phenylalanine adsorbent: the carboxyl group of the template molecule L-phenylalanine and the pyridyl group of the functional monomer pyridyl imine methyl acryloyloxy monomer are ionically bonded, and the amino group of L-phenylalanine and the carbonyl group of the pyridyl imine methyl acryloyloxy monomer are hydrogen-bonded to form a polymeric molecule, and a magnetic L-phenylalanine adsorbent is prepared by a free radical polymerization method;

[0009] Step two, the magnetic L-phenylalanine adsorbent is added into the L-phenylalanine fully free rice protein solution, and is placed in a constant temperature oscillator to oscillate adsorption, the magnetic L-phenylalanine adsorbent is separated by magnetism, and concentration is carried out to obtain the low-phenylalanine-rich rice protein base material.

[0010] Preferably, the preparation method of the pyridyl imine group methacryloyloxy monomer is:

[0011] Step S1, Schiff base reaction is generated between the primary amino functional group of aminoethyl piperazine and the aldehyde functional group of 4-pyridine formaldehyde, to generate a pyridyl imine group piperazine base monomer;

[0012] Step S2, using the Michael addition reaction mechanism, using the pyridyl imine group piperazine base monomer as a nucleophile, the amino (NH) functional group of the pyridyl imine group piperazine base monomer and the alpha, beta-alkyl functional group of ethylene glycol dimethacrylate are subjected to amino-alkene addition reaction to generate a pyridyl imine group methacryloyloxy monomer.

[0013] Preferably, the preparation method of the L-phenylalanine fully free rice protein solution is as follows: rice protein is prepared into a rice protein solution with distilled water, after ultrasonic cell crushing treatment, neutral protease (Neutrase) is added for enzymolysis to obtain an enzymolysis solution I; then chymotrypsin is added to continue the enzymolysis to obtain an enzymolysis solution II; then pronase is added to continue the enzymolysis to obtain an enzymolysis solution III; the neutral protease, chymotrypsin and pronase are inactivated, and the supernatant obtained by centrifugation is the L-phenylalanine fully free rice protein solution.

[0014] The low-phenylalanine-rich rice protein base material obtained according to the method for removing L-phenylalanine in rice protein is used as a protein component, and is mixed with glucose syrup, triglyceride, soy lecithin, fish oil, vitamins and minerals to prepare a low-phenylalanine-rich diet.

[0015] Compared with the prior art, the present application has the following beneficial technical effects:

[0016] The present application: a new functional monomer, pyridyl imine group methacryloyloxy monomer, is synthesized; L-phenylalanine is used as a template molecule, the carboxyl group of L-phenylalanine and the pyridyl group of pyridyl imine group methacryloyloxy monomer are combined through ionic interaction, and the amino group of L-phenylalanine and the carbonyl group of pyridyl imine group methacryloyloxy monomer are combined through hydrogen bond interaction to self-assemble into a polymer molecule; through a free radical polymerization method, a magnetic L-phenylalanine adsorbent is prepared, and experiments show that the magnetic L-phenylalanine adsorbent can adsorb and remove L-phenylalanine in rice protein. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1Chemical structure of pyridyl imine piperazinyl monomer;

[0018] Figure 2 Chemical structure of pyridyl imine methacryloyloxy monomer;

[0019] Figure 3 Chemical structure of L-phenylalanine;

[0020] Figure 4 Chemical structure of L-phenylalanine and pyridyl imine methacryloyloxy monomer self-assembled into polymeric molecules. DETAILED DESCRIPTION

[0021] Experimental Example 1:

[0022] Synthesis of pyridyl imine piperazinyl monomer, the reaction mechanism is that the primary amino functional group of aminoethyl piperazine reacts with the aldehyde functional group of 4-pyridine formaldehyde to generate pyridyl imine piperazinyl monomer through Schiff base reaction, and the chemical structure is as shown in Figure 1 ;

[0023] The specific steps of pyridyl imine piperazinyl monomer are as follows: 7.5 mL of aminoethyl piperazine is dissolved in 100 mL of deionized water, and 40 mL of anhydrous ethanol solution dissolved with 5.5 mL of 4-pyridine formaldehyde is added dropwise under magnetic stirring, after the dropwise addition is completed, it is refluxed at 60°C in an oil bath for 3 h, thin layer chromatography method is used to track the reaction to completion, it is placed at room temperature for 2 h, the solvent is removed by reduced pressure evaporation, recrystallized with anhydrous ethanol, and dried with calcium chloride to obtain pyridyl imine piperazinyl monomer.

[0024] Experimental Example 2:

[0025] Synthesis of pyridyl imine methacryloyloxy monomer, the reaction mechanism is that Michael addition reaction mechanism is used, pyridyl imine piperazinyl monomer is used as a nucleophile, and the amino (NH) functional group of pyridyl imine piperazinyl monomer reacts with the α, β-alkyl functional group of ethylene glycol dimethacrylate to generate pyridyl imine methacryloyloxy monomer through amino-alkene addition reaction, and the chemical structure is as shown in Figure 2 ;

[0026] The specific steps of synthesizing pyridyl imine methacryloyloxy monomer are as follows: under the protection of nitrogen, 3.5 g of pyridyl imine piperazinyl monomer and 20 mL of chloroform are placed in a four-necked flask, uniform stirring is started, 3 mL of ethylene glycol dimethacrylate is added dropwise through a constant pressure dropping funnel, after the dropwise addition is completed, it is heated and reacted in a 40°C constant temperature water bath for 4 h, and the chloroform is removed by normal pressure distillation to obtain pyridyl imine methacryloyloxy monomer, and the chemical structure characterization is as follows: CDCl3 is used as a solvent, 1 H NMR characterization result is:1 H NMR (400MHz, δ / ppm): 1.09-1.14 (d, 3H), 1.92-1.93 (s, 3H), 2.62-2.78 (m, 7H), 2.86-2.91 (m, 4H), 3.20-3.23 (m, 2H), 3.56 -3.66(m, 2H), 4.33-4.36(t, 2H), 4.45-4.48(t, 2H), 5.71-5.82(d, 2H), 7.59-7.60(d, 2H), 8.37(s, 1H), 8.64-8.66(d, 2H).

[0027] Example 1:

[0028] Preparation of 3-(methacryloyloxy)propyltrimethoxysilane (γ-MPS) modified Fe3O4 nanoparticles: Under argon protection and at a water bath of 75°C, 80 mL of 0.5 mol / L NaOH solution was added to the reactor, and 120 mL of a mixed solution (consisting of 1 volume part of ultrapure water and 1 volume part of anhydrous ethanol) containing 1.12 g of ferrous sulfate heptahydrate and 2.16 g of ferric chloride hexahydrate was added dropwise to the reactor. After the addition was completed, the mixture was kept at a constant temperature of 80°C in a water bath for 0.5 h, 1 mL of 3-(methacryloyloxy)propyltrimethoxysilane was added, the temperature was lowered to 50°C and matured for 0.5 h, cooled, magnetically separated, washed to neutrality, washed with anhydrous ethanol, and vacuum dried at 40°C for 24 h to obtain γ-MPS modified Fe3O4 nanoparticles.

[0029] Example 2:

[0030] Preparation of magnetic L-phenylalanine adsorbent: 83 mg L-phenylalanine (its chemical structure is as follows Figure 3 As shown), 0.832g of pyridyl imino methacryloxy monomer was added to 25mL of acetonitrile and stirred at a constant speed for 1h. The carboxyl group of L-phenylalanine and the pyridyl group of the pyridyl imino methacryloxy monomer self-assembled into a polymer molecule through ion interaction, and the amino group of L-phenylalanine and the carbonyl group of the pyridyl imino methacryloxy monomer self-assembled through hydrogen bonding to form a polymer molecule (its chemical structure is shown in FIG). Figure 4 As shown), 1 g of γ-MPS modified Fe3O4 nanoparticles, 2 mL of ethylene glycol dimethacrylate and 100 mg of azobisisobutyronitrile were added, nitrogen was passed for 10 minutes and then sealed, and the reaction was carried out at a constant temperature of 60°C for 24 hours. The product was magnetically separated and washed with anhydrous ethanol. Then, the product was extracted with an extractant consisting of 9 parts by volume of methanol and 1 part by volume of acetic acid for 24 hours to elute the L-phenylalanine molecules. The product was rinsed with acetone and vacuum dried at 60°C for 12 hours to obtain a magnetic L-phenylalanine adsorbent.

[0031] Example 3:

[0032] Preparation of L-phenylalanine fully dissociated rice protein solution: The rice protein was prepared into a rice protein solution with a concentration of 1 mg / mL using distilled water, and after ultrasonic cell crushing treatment, neutral protease (Neutrase) (enzyme activity: pH 7, 50°C, 4000 U / g protein) was added for enzymolysis to obtain an enzymolysis solution I; chymotrypsin (enzyme activity: pH 8, 55°C, 4000 U / g protein) was further added for enzymolysis to obtain an enzymolysis solution II; pronase (enzyme activity: pH 7, 37°C, 4000 U / g protein) was further added for enzymolysis to obtain an enzymolysis solution III; the neutral protease, chymotrypsin and pronase were inactivated, and the supernatant obtained by centrifugation was the L-phenylalanine fully dissociated rice protein solution, and the free L-phenylalanine content thereof was detected, and the result was 72.31%.

[0033] The determination method of free phenylalanine is as follows: 100 μL of the L-phenylalanine fully dissociated rice protein solution diluted with water by 5 times was taken into a sample tube; 25 μL of an L-phenylalanine standard solution was taken into a sample tube, and 100 μL of 0.06 mol / L trichloroacetic acid solution was added; water was added to 200 μL for both the sample tube and the reagent blank analysis, and then 0.3 mL of dipeptide-ninhydrin was added to each tube, which was placed in a 75°C water bath for 80 min, and then taken out and cooled, and 2.5 mL of copper reagent was added and uniformly shaken, and the fluorescence value of the sample was determined within 90 min under the conditions of an excitation wavelength of 385 nm and an emission wavelength of 472 nm, and compared with the L-phenylalanine standard sample to obtain the free phenylalanine content of the sample.

[0034] Static adsorption to remove L-phenylalanine from rice protein: 1 g of magnetic L-phenylalanine adsorbent was added to 10 mL of the L-phenylalanine fully dissociated rice protein solution (free L-phenylalanine content: 72.31%) described above, and placed in a constant temperature oscillator at a temperature of 25°C and a rotation speed of 100 r / min for 3 h, and then the magnetic L-phenylalanine adsorbent was separated by magnetism, and concentrated to obtain a low-phenylalanine-rich rice protein base material, and the free L-phenylalanine content thereof was detected, and the result was 0.08%.

[0035] Example 4:

[0036] Low-phenylalanine-rich diet: A low-phenylalanine-rich diet was prepared by mixing a low-phenylalanine-rich rice protein base material as a protein component with different proportions of glucose syrup, triglyceride, soy lecithin, fish oil, vitamins, minerals and other nutrients.

Claims

1. A method for removing L-phenylalanine from rice protein by adsorption, characterized in that, The method comprises the following steps: Step 1, preparation of a magnetic L-phenylalanine adsorbent, the specific method being: the carboxyl group of the template molecule L-phenylalanine and the pyridyl group of the functional monomer pyridyl imine group methacryloyloxy monomer are ionically combined, and the amino group of the L-phenylalanine and the carbonyl group of the pyridyl imine group methacryloyloxy monomer are combined by hydrogen bonding to self-assemble into a polymer molecule, under the action of ethylene glycol dimethacrylate crosslinking agent and azobisisobutyronitrile initiator, the polymer molecule is subjected to a free radical polymerization reaction on the surface of 3-(methacryloyloxy)propyl trimethoxysilane modified Fe3O4 nanoparticles, the L-phenylalanine template molecule is eluted, and the magnetic L-phenylalanine adsorbent is prepared; wherein the chemical structural formula of the pyridyl imine group methacryloyloxy monomer is: ； Step 2, the magnetic L-phenylalanine adsorbent is added to an L-phenylalanine fully free rice protein solution, and is placed in a constant-temperature oscillator for oscillation adsorption, the magnetic L-phenylalanine adsorbent is separated by magnetism, and a low-phenylalanine-rich rice protein base material is obtained by concentration.

2. The method for removing L-phenylalanine from rice protein by adsorption according to claim 1, characterized in that, The preparation method of the pyridyl imine group methacryloyloxy monomer is: Step S1, Schiff base reaction is generated between the primary amino functional group of the aminoethyl piperazine and the aldehyde functional group of 4-pyridine formaldehyde to generate a pyridyl imine group piperazinyl monomer; Step S2, using the Michael addition reaction mechanism, the pyridyl imine group piperazinyl monomer is used as a nucleophile, and an amino-alkene addition reaction is generated between the amino NH functional group of the pyridyl imine group piperazinyl monomer and the alpha, beta-alkyl functional group of ethylene glycol dimethacrylate to generate the pyridyl imine group methacryloyloxy monomer.

3. The method for removing L-phenylalanine from rice protein by adsorption according to claim 1, characterized in that, The preparation method of the L-phenylalanine fully free rice protein solution is: rice protein is prepared into a rice protein solution with distilled water, after ultrasonic cell crushing treatment, neutral protease Neutrase is added for enzymolysis to obtain an enzymolysis liquid I; pepsin is further added for enzymolysis to obtain an enzymolysis liquid II; pronase is further added for enzymolysis to obtain an enzymolysis liquid III; the neutral protease, the pepsin and the pronase are inactivated, and the supernatant obtained by centrifugation is the L-phenylalanine fully free rice protein solution.

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