Low-phenylalanine special dietary food

Abstract

The invention relates to the technical field of phenylalanine removal, and discloses a low-phenylalanine special dietary food. Specifically, carboxyl of template molecule L-phenylalanine and imidazolyl of a functional monomer methacryloyloxy di (imidazolyl) monomer are subjected to ionic interaction, amino of L-phenylalanine and carbonyl of the methacryloyloxy di (imidazolyl) monomer are self-assembled into a polymeric molecule through hydrogen-bond interaction, and through a free radical polymerization method, the polymer is prepared. Preparing a silica gel based L-phenylalanine adsorbent; and adding the silica gel-based L-phenylalanine adsorbent into the L-phenylalanine fully free rice protein solution, oscillating for adsorption, and concentrating to obtain the low-phenylalanine rice protein base material. The invention provides a method for adsorbing and removing L-phenylalanine in rice protein, the prepared low-phenylalanine rice protein base material is a protein component, and other nutrient substances are supplemented to prepare the low-phenylalanine special dietary food.

Description

Technical Field

[0001] This invention relates to the field of phenylalanine removal technology, specifically to a low-phenylalanine special dietary food. Background Technology

[0002] Phenylketonuria, also known as phenylalanine hydroxylase deficiency, is an autosomal recessive genetic disorder. Due to the lack or reduced activity of phenylalanine hydroxylase in the body, phenylalanine cannot be converted into tyrosine and other metabolites through the normal metabolic pathway. As a result, the alternative metabolic pathway is enhanced, producing phenylpyruvic acid, phenylacetic acid, and phenyllactic acid, which are excreted in large quantities in the urine, resulting in phenylketonuria.

[0003] Currently, the main treatments for this disease include dietary therapy and gene therapy. Dietary therapy, which restricts the phenylalanine content in the diet, is recognized as the safest and most effective method. Phenylalanine has two isomers: L-phenylalanine and D-phenylalanine. L-phenylalanine is the form that is naturally found in proteins, while D-phenylalanine is a non-natural amino acid.

[0004] Therefore, the development of low-phenylalanine special dietary foods is an important way to solve the problems encountered by patients with phenylketonuria in the process of dietary therapy. Summary of the Invention

[0005] This invention provides a method for adsorbing and removing L-phenylalanine from rice protein to prepare a low-phenylalanine rice protein base. This base is then used as a protein component, supplemented with other nutrients, to prepare a low-phenylalanine special dietary food.

[0006] A method for adsorbing and removing L-phenylalanine from rice protein includes the following steps:

[0007] Step 1: Preparation of silica-based L-phenylalanine adsorbent: The carboxyl group of the template molecule L-phenylalanine and the imidazole group of the functional monomer methacryloyloxydi(imidazolyl) monomer self-assemble into a polymer molecule through ionic interaction, and the amino group of L-phenylalanine and the carbonyl group of the methacryloyloxydi(imidazolyl) monomer self-assemble into a polymer molecule through hydrogen bonding. The silica-based L-phenylalanine adsorbent is then prepared by free radical polymerization.

[0008] Step 2: Add silica-based L-phenylalanine adsorbent to the fully free L-phenylalanine rice protein solution, place it in a constant temperature shaker, shake to adsorb, and concentrate to obtain low-phenylalanine rice protein base.

[0009] Preferably, the method for preparing the methacryloyloxydi(imidazolyl) monomer is as follows:

[0010] In step S1, sodium bisulfate is used as a catalyst and cyclohexane as a dehydrating agent. The carboxyl functional group of mercaptosuccinic acid reacts with the hydroxyl functional group of hydroxyethyl methacrylate to generate a di(methacryloyloxy)thiol monomer.

[0011] Step S2: Using the Michael addition reaction mechanism, imidazole is used as a nucleophile. 1,8-diazabicycloundec-7-ene catalyzes the amino (NH) functional group of imidazole to undergo an amino-ene addition reaction with the α,β-ene functional group of the bis(methacryloyloxy)thiol monomer to generate the bis(imidazolyl)thiol monomer.

[0012] Step S3: Using di(imidazolyl)thiol monomer and ethylene glycol dimethacrylate as raw materials, a photo-initiated thiol-olefin click chemistry reaction is used to prepare methacryloyloxy di(imidazolyl) monomer.

[0013] Preferably, the method for preparing the fully free L-phenylalanine rice protein solution is as follows: rice protein is prepared into a rice protein solution with distilled water, and after ultrasonic cell pulverization, neutral protease is added for enzymatic hydrolysis to obtain hydrolysate I; then chymotrypsin is added and enzymatic hydrolysis is continued to obtain hydrolysate II; then streptomycin is added and enzymatic hydrolysis is continued to obtain hydrolysate III; the neutral protease, chymotrypsin and streptomycin are inactivated, and the supernatant obtained by centrifugation is the fully free L-phenylalanine rice protein solution.

[0014] The low-phenylalanine rice protein base obtained by the adsorption and removal method of L-phenylalanine from rice protein is used as the protein component, and is supplemented with fat, dietary fiber, fruit powder, vitamins and minerals to prepare a low-phenylalanine special dietary food.

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

[0016] This invention synthesizes a novel functional monomer: methacryloyloxydi(imidazolyl) monomer. Using L-phenylalanine as a template molecule, the carboxyl group of L-phenylalanine and the imidazolyl group of the methacryloyloxydi(imidazolyl) monomer self-assemble into a polymer molecule through ionic interactions, and the amino group of L-phenylalanine and the carbonyl group of the methacryloyloxydi(imidazolyl) monomer self-assemble into a polymer molecule through hydrogen bonding. A silica-based L-phenylalanine adsorbent is prepared by free radical polymerization. Experiments show that the silica-based L-phenylalanine adsorbent can adsorb and remove L-phenylalanine from rice protein. Attached Figure Description

[0017] Figure 1 The chemical structural formula of the di(methacryloyloxy)thiol monomer;

[0018] Figure 2The chemical structural formula of the di(imidazolyl)thiol monomer;

[0019] Figure 3 The chemical structural formula of the methacryloyloxydi(imidazolyl) monomer;

[0020] Figure 4 The chemical structural formula of L-phenylalanine;

[0021] Figure 5 The chemical structural formula of the self-assembly of L-phenylalanine and methacryloyloxydi(imidazolyl) monomer into a polymer molecule. Detailed Implementation

[0022] Experimental Example 1:

[0023] The synthesis of bis(methacryloyloxy)thiol monomers follows a reaction mechanism: using sodium bisulfate as a catalyst and cyclohexane as a dehydrating agent, the carboxyl functional group of mercaptosuccinic acid undergoes an esterification reaction with the hydroxyl functional group of hydroxyethyl methacrylate to generate bis(methacryloyloxy)thiol monomers, the chemical structural formula of which is shown below. Figure 1 As shown;

[0024] The specific steps for synthesizing the di(methacryloyloxy)thiol monomer are as follows: 1.5 g of mercaptosuccinic acid, 7.5 mL of hydroxyethyl methacrylate, 0.3 g of sodium bisulfate monohydrate, and 5 mL of cyclohexane were added to a four-necked flask equipped with a thermometer and a reflux condenser with a water separator. The mixture was heated to reflux, and the timing was started when water droplets appeared in the water separator. The mixture was refluxed and separated for 2 hours. After cooling to room temperature, the mixture was first washed with saturated sodium bicarbonate until neutral, then washed with saturated brine. After drying, hydroxyethyl methacrylate and cyclohexane were removed by atmospheric distillation to obtain the di(methacryloyloxy)thiol monomer.

[0025] Experimental Example 2:

[0026] The synthesis of the di(imidazolyl)thiol monomer follows a reaction mechanism utilizing the Michael addition mechanism. Imidazole is used as a nucleophile, and 1,8-diazabicycloundec-7-ene catalyzes an amino-ene addition reaction between the amino (NH) functional group of the imidazolium and the α,β-enyl functional group of the di(methacryloyloxy)thiol monomer, yielding the di(imidazolyl)thiol monomer. Its chemical structure is shown below. Figure 2 As shown;

[0027] The specific steps for synthesizing the di(imidazolyl)thiol monomer are as follows: Under nitrogen protection, 1.87 g of di(methacryloyloxy)thiol monomer and 0.68 g of imidazole were dissolved in 30 mL of dimethyl sulfoxide, and then 0.25 mL of 1,8-diazabicycloundec-7-ene was added. The mixture was stirred at room temperature for 2 h. The reaction solution was first precipitated in anhydrous diethyl ether and then dissolved in chloroform. The di(imidazolyl)thiol monomer was obtained by rotary evaporation.

[0028] Experimental Example 3:

[0029] The synthesis of methacryloyloxydi(imidazolyl) monomers follows a reaction mechanism: using di(imidazolyl)thiol monomers and ethylene glycol dimethacrylate as raw materials, a photo-initiated thiol-olefin click chemistry reaction is employed to prepare methacryloyloxydi(imidazolyl) monomers. The chemical structural formula is shown below. Figure 3 As shown;

[0030] The specific steps for synthesizing methacryloyloxydi(imidazolyl) monomer are as follows: 0.6 mL of ethylene glycol dimethacrylate, 1.53 g of di(imidazolyl)thiol monomer, 20 mg of benzoyl dimethyl ether, and 20 mL of tetrahydrofuran are dissolved and then placed under ultraviolet light (365 nm, 10 mW / cm²). 2 The reaction was carried out for 1 hour, and tetrahydrofuran was removed by rotary evaporation. The product was then precipitated with anhydrous diethyl ether, filtered, and the resulting methacryloyloxydi(imidazolyl) monomer was given. Its chemical structure was characterized as follows: using CDCl3 as solvent... 1 The H NMR characterization results are as follows: 1 H NMR (400MHz, δ / ppm): 1.14-1.16 (d, 6H), 1.20-1.22 (d, 3H), 1.92-1.93 (t, 3H), 2.67-2.74 (m, 1H), 2.84-3.09 (m, 6H), 4.01-4.06 (m, 2H) , 4.15-4.20(m, 2H), 4.22-4.26(t, 1H), 4.33-4.68(m, 12H), 5.65-5.96(d, 2H), 7.05-7.06(m, 2H), 7.15-7.16(m, 2H), 7.69-7.70(s, 2H).

[0031] Example 1:

[0032] The specific preparation steps for 3-(methacryloyloxy)propyltrimethoxysilane (γ-MPS) modified silica gel are as follows:

[0033] Step S1-1: Soak 30g of silica gel with a particle size of 200~300 mesh in 100mL of 50% nitric acid solution for 24h. First wash with deionized water until neutral, then wash with acetone 3 times. Dry under vacuum at 110℃ for 12h to obtain activated silica gel.

[0034] Step S1-2: Add 5g activated silica gel, 5mL 3-(methacryloyloxy)propyltrimethoxysilane, 2mL triethylamine and 60mL toluene to the reactor, under nitrogen protection, stir and reflux for 6h, filter, wash with toluene, acetone and diethyl ether in sequence, and vacuum dry at 50℃ for 12h to obtain γ-MPS modified silica gel.

[0035] Example 2:

[0036] Preparation of silica-based L-phenylalanine adsorbent: 83 mg of L-phenylalanine (its chemical structural formula is shown below) was added. Figure 4 As shown), 1.416 g of methacryloyloxydi(imidazolyl) monomer was added to 25 mL of acetonitrile and stirred at a constant speed for 1 h. The carboxyl group of L-phenylalanine and the imidazolyl group of the methacryloyloxydi(imidazolyl) monomer self-assembled into a polymer molecule (its chemical structure is shown in Figure 1). Figure 5 As shown in the figure, 1g of γ-MPS modified silica gel, 2mL of ethylene glycol dimethacrylate and 100mg of azobisisobutyronitrile were added. After purging with nitrogen for 10min, the mixture was sealed and reacted at 60℃ with shaking for 24h. The product was washed with tetrahydrofuran, filtered, and then extracted with an extraction solution composed of 9 parts by volume of methanol and 1 part by volume of acetic acid for 24h to elute L-phenylalanine molecules. The product was washed with acetone and dried under vacuum at 60℃ for 12h to obtain silica gel-based L-phenylalanine adsorbent.

[0037] Example 3:

[0038] Preparation of L-phenylalanine-fully free rice protein solution: Rice protein was dissolved in distilled water to a concentration of 1 mg / mL. After ultrasonic cell disruption, neutral protease (Neutrase) (enzyme activity: pH 7, 50℃, 4000 U / g protein) was added for enzymatic hydrolysis to obtain hydrolysate I; then chymotrypsin (enzyme activity: pH 8, 55℃, 4000 U / g protein) was added for further enzymatic hydrolysis to obtain hydrolysate II; then streptomycin (enzyme activity: pH 7, 37℃, 4000 U / g protein) was added for further enzymatic hydrolysis to obtain hydrolysate III; after inactivating neutral protease, chymotrypsin, and streptomycin, the supernatant obtained by centrifugation was the L-phenylalanine-fully free rice protein solution. The free L-phenylalanine content was measured to be 72.31%.

[0039] The method for determining free phenylalanine is as follows: A fully free L-phenylalanine rice protein solution is precisely diluted 5 times with water, and 100 μL of the diluted solution is placed in a sample tube. 25 μL of L-phenylalanine standard solution is placed in the sample tube, and 100 μL of 0.06 mol / L trichloroacetic acid solution is added. Water is added to both tubes to a final volume of 200 μL, and a reagent blank analysis is performed simultaneously. Then, 0.3 mL of dipeptide-trisine is added to each tube, and the tubes are incubated in a 75℃ water bath for 80 min. After cooling, 2.5 mL of copper reagent is added, and the mixture is shaken thoroughly. The fluorescence value of the sample is measured within 90 min under conditions of excitation wavelength of 385 nm and emission wavelength of 472 nm. The result is compared with an L-phenylalanine standard to determine the content of free phenylalanine in the sample.

[0040] Static adsorption removal of L-phenylalanine from rice protein: 1g of silica-based L-phenylalanine adsorbent was added to 15mL of the above-mentioned fully free L-phenylalanine rice protein solution (free L-phenylalanine content 72.31%), and placed in a constant temperature shaker. The solution was shaken for 3h at 25℃ and 100r / min. The mixture was then concentrated to obtain a low-phenylalanine rice protein base. The free L-phenylalanine content was measured and found to be 0.12%.

[0041] Example 4:

[0042] Low-phenylalanine special dietary foods: These foods are prepared by mixing low-phenylalanine rice protein as the protein base with different proportions of nutrients such as fat, dietary fiber, fruit powder, vitamins, and minerals.

Claims

1. A method for adsorbing and removing L-phenylalanine from rice protein, characterized in that, Includes the following steps: Step 1: Preparation of silica-based L-phenylalanine adsorbent: The carboxyl group of the template molecule L-phenylalanine and the imidazole group of the functional monomer methacryloyloxydi(imidazolyl) monomer self-assemble into a polymer molecule through ionic interaction, and the amino group of L-phenylalanine and the carbonyl group of the methacryloyloxydi(imidazolyl) monomer self-assemble into a polymer molecule through hydrogen bonding. The silica-based L-phenylalanine adsorbent is then prepared by free radical polymerization. Step 2: Add silica-based L-phenylalanine adsorbent to the fully free L-phenylalanine rice protein solution, place it in a constant temperature shaker, shake to adsorb, and concentrate to obtain low-phenylalanine rice protein base.

2. The method for adsorbing and removing L-phenylalanine from rice protein according to claim 1, characterized in that, The method for preparing the methacryloyloxydi(imidazolyl) monomer is as follows: In step S1, sodium bisulfate is used as a catalyst and cyclohexane as a dehydrating agent. The carboxyl functional group of mercaptosuccinic acid reacts with the hydroxyl functional group of hydroxyethyl methacrylate to generate a di(methacryloyloxy)thiol monomer. Step S2: Using the Michael addition reaction mechanism, imidazole is used as a nucleophile. 1,8-diazabicycloundec-7-ene catalyzes the amino (NH) functional group of imidazole to undergo an amino-ene addition reaction with the α,β-ene functional group of the bis(methacryloyloxy)thiol monomer to generate the bis(imidazolyl)thiol monomer. Step S3: Using di(imidazolyl)thiol monomer and ethylene glycol dimethacrylate as raw materials, a photo-initiated thiol-olefin click chemistry reaction is used to prepare methacryloyloxy di(imidazolyl) monomer.

3. The method for adsorbing and removing L-phenylalanine from rice protein according to claim 1, characterized in that, The method for preparing the fully free L-phenylalanine rice protein solution is as follows: rice protein is prepared into a rice protein solution with distilled water, and after ultrasonic cell pulverization, neutral protease is added for enzymatic hydrolysis to obtain hydrolysate I; then chymotrypsin is added and enzymatic hydrolysis is continued to obtain hydrolysate II; then streptomycin is added and enzymatic hydrolysis is continued to obtain hydrolysate III; the neutral protease, chymotrypsin and streptomycin are inactivated, and the supernatant obtained by centrifugation is the fully free L-phenylalanine rice protein solution.

4. The low-phenylalanine rice protein base obtained by the adsorption and removal of L-phenylalanine from rice protein according to any one of claims 1-3 is used as the protein component, and is supplemented with fat, dietary fiber, fruit powder, vitamins and minerals to prepare a low-phenylalanine special dietary food.

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