L-alanine composite powder capable of eliminating nitrite and preparation method of L-alanine composite powder

Abstract

The invention discloses L-alanine composite powder capable of eliminating nitrite and a preparation method of the L-alanine composite powder. The L-alanine composite powder is prepared from ferrous chloride, calcium chloride, sodium alginate, L-alanine, gamma-aminobutyric acid, L-glutamic acid, cysteine, nitrite reductase, trehalose, calcium citrate and ascorbic acid. The preparation method comprises the following steps: preparing an amino acid mixed solution, embedding sodium alginate gel, preparing microspheres by an ionic cross-linking method, freeze-drying and crushing. The Fe < 2 + > / Ca < 2 + > bi-crosslinking gel is utilized to realize the balance between slow release and mechanical strength; a cysteine-nitrite reductase-Fe < 2 + > ternary system is constructed, nitrite can be synergistically catalyzed to generate nitric oxide, and the cysteine-nitrite reductase-Fe < 2 + > ternary system has the functions of removing nitrite and inhibiting production of nitrosamine; the obtained L-alanine composite powder has the capabilities of rapidly eliminating nitrite, slowly releasing nitric oxide, inhibiting generation of new nitrosamine and capturing existing nitrosamine.

Description

Technical Field

[0001] The present invention relates to the technical field of nitrite reduction in foods, and particularly to an L-alanine composite powder for eliminating nitrite and a preparation method thereof. Background Art

[0002] Nitrite (NO2 - ) widely exists in pickled foods, processed meats, and water bodies. Under acidic conditions, nitrite reacts with amine compounds (such as protein degradation products and exogenous amino acids) to form the strong carcinogen N-nitrosamine. Research shows that nitrosamine is closely related to various diseases such as gastric cancer and esophageal cancer. In addition, excessive nitrite may also cause methemoglobinemia, seriously threatening human health.

[0003] Currently, the main methods for removing nitrite and nitrosamine include the following several:

[0004] Physical adsorption method: such as activated carbon and molecular sieves, etc. These methods can only adsorb nitrosamine, cannot achieve degradation, and are easily saturated.

[0005] Chemical reduction method: such as Fe 2+ and Vc, etc. These methods require high dosages (usually > 50 mg / kg), may introduce metal residues, and are ineffective against nitrosamine.

[0006] Enzymatic hydrolysis method: such as nitrite reductase, etc. These methods are highly sensitive to the environment, the enzyme is easily inactivated, and the action time is short.

[0007] Sustained-release carrier technology: such as liposomes and polymer microspheres, etc. These methods have high costs, poor biocompatibility, and low sustained-release efficiency.

[0008] In traditional food processing, ascorbic acid (Vc) or nicotinamide is often used to inhibit the formation of nitrosamine, but the effects of these methods are limited and cannot effectively remove the already formed nitrosamine. Therefore, there is an urgent need to develop a new technology that can simultaneously remove nitrite and block the formation of nitrosamine. Summary of the Invention

[0009] The purpose of the present invention is to solve the deficiencies existing in the prior art, and to propose an L-alanine composite powder for eliminating nitrite and a preparation method thereof.

[0010] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0011] The present invention first proposes an L-alanine composite powder for eliminating nitrite, including raw materials with the following weights:

[0012] 99 - 134 g of alginate-based gel;

[0013] 9-11 g of trehalose (Xi'an Jinxiang Pharmaceutical Excipients Co., Ltd., purity 99%);

[0014] 0.3-0.6 g of calcium citrate (Shanghai Haohong Biopharmaceutical Technology Co., Ltd., purity 99%);

[0015] The preparation process of the alginate-based gel comprises the following steps:

[0016] S1. Preparation of amino acid mixture:

[0017] Dissolve L-alanine (Hebei Lihua Biotechnology Co., Ltd., purity 99%), γ-aminobutyric acid (Sichuan Jisheng Biomedicine Co., Ltd., purity 98.5%), L-glutamic acid (Hebei Jijie Biotechnology Co., Ltd., purity 99%), cysteine ​​(Wuxi Bikang Bioengineering Co., Ltd., purity 99%+), ascorbic acid (Shandong Fengtai Biotechnology Co., Ltd., purity 99%), and nitrite reductase (microbial nitrite reductase (NIR) [Product No.]: HB1136-QT, activity 5000-6000U / g) in PBS solution to obtain an amino acid mixture;

[0018] S2. Sodium alginate gel embedding:

[0019] Dissolve sodium alginate in deionized water, stir at 60°C until transparent, and cool to room temperature to obtain a sodium alginate solution;

[0020] S3. Preparation of microspheres by ionic crosslinking:

[0021] The powders of FeCl2·4H2O and CaCl2 were dissolved in deionized water to obtain FeCl2 / CaCl2 solution;

[0022] The amino acid mixture and the sodium alginate solution were gently mixed to avoid air bubbles to obtain a pre-gel solution;

[0023] The pre-gel solution was added dropwise into the FeCl2 / CaCl2 solution using a syringe to form microspheres, thereby enhancing the mechanical strength;

[0024] The microspheres were placed in FeCl2 / CaCl2 solution and solidified for 20 min to form Fe 2+ / Ca 2+ Double cross-linked network of gel beads;

[0025] The beads were washed three times with PBS solution to remove free ions and unentrapped components, and then freeze-dried to obtain alginate-based gel.

[0026] Preferably, the alginate-based gel specifically comprises 60 - 80 g of sodium alginate, 12 - 15 g of L-alanine, 6 - 8 g of γ-aminobutyric acid, 9 - 11 g of L-glutamic acid, 8 - 12 g of cysteine, 200 - 250 U of nitrite reductase, 0.8 - 1.2 g of ascorbic acid, 3 - 6 g of FeCl₂·4H₂O, and 0.5 - 1 g of CaCl₂.

[0027] Preferably, in the step S1, the mass ratio of the PBS solution to L-alanine, γ-aminobutyric acid, L-glutamic acid, cysteine, ascorbic acid, and nitrite reductase is 2:1, the pH of the PBS solution is 7.4, and the molar concentration is 0.01 mol / L.

[0028] Preferably, in the step S2, the mass ratio of sodium alginate to deionized water is 1:10.

[0029] Preferably, in the step S3, the mass ratio of the powders of FeCl₂·4H₂O and CaCl₂ to deionized water is 1:200.

[0030] Preferably, in the step S3, the mass ratio of the amino acid mixture solution to the sodium alginate solution is 1:10.

[0031] Preferably, in the step S3, the mass ratio of the pre-gel solution to the FeCl₂ / CaCl₂ solution is 1:3.

[0032] Preferably, in the step S3, the diameter of the microspheres is controlled to be 1 - 2 mm, and the dropping height is selected to be 8 - 12 cm.

[0033] Preferably, in the step S3, the freeze-drying conditions are pre-freezing at -80 °C until the solution is completely frozen; performing vacuum drying at -20 °C to -50 °C with a vacuum degree of 10 - 50 mTorr for 20 h in the primary drying; and at -10 °C to +20 °C with a vacuum degree of 1 - 2 mTorr for 4 h in the secondary drying.

[0034] The preparation method of the L-alanine composite powder for eliminating nitrite includes the following steps:

[0035] Dissolve trehalose and calcium citrate in deionized water and dissolve them thoroughly to obtain a trehalose mixed solution;

[0036] Soak the alginate-based gel in the trehalose mixed solution for 30 min, freeze-dry it, and then grind it into a powder of 100 meshes to obtain the L-alanine composite powder product, which is stored in a light-proof and sealed manner.

[0037] Preferably, the freeze-drying conditions are pre-freezing at -80°C until the solution is completely frozen; performing vacuum drying at -20°C to -50°C with a vacuum degree of 10 - 50 mTorr for 20 h in primary drying; and at -10°C to +20°C with a vacuum degree of 1 - 2 mTorr for 4 h in secondary drying.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0039] 1. The present invention can efficiently eliminate NO2 - , and generate NO and H2S:

[0040] Through the synergistic effect of cysteine and nitrite reductase, nitrite is eliminated through a dual pathway and converted into harmless NO; nitrite reductase directly catalyzes the reduction of nitrite, while cysteine binds to NO2 - to promote the formation of S-nitrosothiols, which are catalyzed by the Fe 2+ / Vc system to form NO and a disulfide intermediate (-S-S-), and NO and the disulfide intermediate (-S-S-) react with Fe 2+ gradually released from the crosslinked network to generate complexes such as Fe4S3(NO)7 - etc. These complexes gradually release NO and H2S under strong acidic conditions, further prolonging the slow release time of NO.

[0041] NO is an important vasodilator that can relax smooth muscle cells, thereby dilating blood vessels and reducing blood pressure. NO can also reduce arterial stiffness, improve blood flow in the carotid artery, and improve myocardial ischemia and reduce myocardial injury by increasing coronary blood flow; H2S has a vasodilatory effect similar to that of NO, can reduce blood pressure by regulating the function of vascular endothelial cells, and can also protect myocardial cells from ischemia-reperfusion injury by activating the antioxidant response and reducing oxidative stress.

[0042] 2. The present invention constructs a unique slow release system:

[0043] Fe 2+ / Ca 2+ The alginate-based gel microspheres formed by double crosslinking with sodium alginate form a three-dimensional network structure with high mechanical strength, which can resist the erosion of gastrointestinal fluid to a certain extent and provide protection for the nitrite reductase encapsulated therein.

[0044] After administration, the Fe 2+ crosslinked layer preferentially forms a dense structure on the surface of the microspheres, delaying the diffusion of internal active ingredients (such as nitrite reductase and cysteine); small molecule amino acids such as L-alanine and γ-aminobutyric acid on the surface of the microspheres are preferentially dissolved, neutralizing the local acidic environment and providing an appropriate pH for subsequent enzymatic reactions. Subsequently, the internal nitrite reductase and cysteine slowly diffuse through the gel network, continuously catalyzing NO2- Reduced to NO and form Fe4S3(NO)7 inside the gel - Complex

[0045] Ca 2+ The crosslinked layer enhances the internal stability and realizes gradient release. That is, in the weakly alkaline environment of the intestine (pH≥7), the swelling degree of the sodium alginate gel increases, and the embedded components are gradually released, avoiding the burst release in the gastric acid (pH≈2) environment. The Ca 2+ in the crosslinked part is retained, delaying the Fe 2+ release; in the intestine (pH = 6-7), the Ca 2+ gradually dissociates, and the encapsulated Fe4S3(NO)7 -

[0046] complex is exposed, triggering the slow release of NO / H2S

[0047] 3. The present invention can effectively block carcinogenic nitrosamines and capture existing nitrosamines

[0048] Aiming at the fact that nitrite in the digestive system and amino acids in the body or food are prone to produce highly carcinogenic nitrosamines in an acidic environment, the present invention captures nitrosamines through the synergistic effects of physical adsorption, chemical interaction and gel network structure of the alginate-based gel:

[0049] The three-dimensional crosslinked gel (Fe 2+ / Ca 2+ double crosslinking) formed by sodium alginate has micron-sized pores (1-2 mm microsphere structure), which can intercept nitrosamine molecules (molecular weight usually <200 Da) through surface adsorption and internal pores, and reduce their free concentration by physical adsorption

[0050] At physiological pH (7.4), the carboxyl group (-COO - ) of sodium alginate is negatively charged, while the N + =O - group of some nitrosamines (such as N-nitrosodimethylamine, NDMA) is weakly positively charged and is adsorbed through electrostatic interaction

[0051] The cysteine (-SH) and L-alanine embedded in the gel can react with nitrosamines by nucleophilic substitution to destroy their N-N=O carcinogenic structure. For example:

[0052] R1N(NO)R2+HS-CH2-CH(NH2)COOH→R1-NH-R2+S-CH2-CH(NH2)COOH+NO

[0053] The Fe 2+ released by the gel can catalyze the reduction and cleavage of nitrosamines to generate non-carcinogenic amines and NO

[0054] Meanwhile, the weakly acidic microenvironment of the alginate gel (regulated by calcium citrate, pH 5-6) inhibits the nitrosation reaction of nitrite (NO2 - ) with amines (optimal pH < 3), blocking the formation of nitrosamines as much as possible from the source.

[0055] 4. The present invention adopts a stabilization embedding technology:

[0056] Adopt alginate-based gel embedding and Fe 2+ / Ca 2+ double cross-linking technology to protect active ingredients such as nitrite reductase from the influence of the human digestive environment (such as pH, temperature) and improve storage stability. At the same time, trehalose is added as a lyoprotectant to further maintain enzyme activity and component stability.

[0057] In summary, the present invention is a more efficient, safe and economical L-alanine composite powder for removing nitrite that integrates elements such as effect, cost, and biocompatibility. Brief Description of the Drawings

[0058] Figure 1 It is a possible reaction mechanism diagram for the present invention to eliminate nitrite using alginate-based gel. Detailed Embodiments

[0059] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0060] Example 1:

[0061] An L-alanine composite powder for removing nitrite and its preparation method include the following steps:

[0062] S1. Preparation of amino acid mixture:

[0063] Dissolve 12 g of L-alanine, 8 g of γ-aminobutyric acid, 9 g of L-glutamic acid, 12 g of cysteine, 1.2 g of ascorbic acid, and 200 U of nitrite reductase in 50 ml of PBS solution (the pH of the PBS solution used this time and hereafter is 7.4, and the molar concentration is 0.01 mol / L) to obtain an amino acid mixture;

[0064] S2. Embedding with sodium alginate gel:

[0065] Dissolve 60 g of sodium alginate in 600 mL of deionized water, stir at 60 °C until transparent, and cool to room temperature to obtain a sodium alginate solution;

[0066] S3. Preparation of microspheres by ion cross-linking method:

[0067] Dissolve 3 g of FeCl₂·4H₂O and 1 g of CaCl₂ powder in 1500 mL of deionized water to obtain an FeCl₂ / CaCl₂ solution;

[0068] Gently mix the amino acid mixture and the sodium alginate solution to avoid air bubbles to obtain a pre-gel solution;

[0069] Use a syringe to drop the pre-gel solution into the FeCl₂ / CaCl₂ solution drop by drop to form microspheres, thereby enhancing the mechanical strength; to control the microsphere diameter at 1 - 2 mm, select a dropping height of 8 cm.

[0070] The microspheres are left to stand and solidify in the FeCl₂ / CaCl₂ solution for 20 min to form Fe 2+ / Ca 2+ Coagulated beads with a double cross-linked network;

[0071] Wash the coagulated beads 3 times with PBS solution to remove free ions and unembedded components, and obtain alginate-based gel after freeze-drying.

[0072] Dissolve 11 g of trehalose and 0.3 of calcium citrate in 100 mL of deionized water and dissolve thoroughly to obtain a trehalose mixed solution;

[0073] Soak the alginate-based gel in the trehalose mixed solution for 30 min, freeze-dry (the freeze-drying conditions are pre-freezing at -80 °C until the solution is completely frozen; perform vacuum drying, -20 °C to -50 °C, vacuum degree 10 - 50 mTorr, primary drying for 20 h; -10 °C to +20 °C, vacuum degree 1 - 2 mTorr, secondary drying for 4 h), grind into 100-mesh powder to obtain the L-alanine composite powder product, and store it in the dark in a sealed manner.

[0074] Reaction flow chart and mechanism explanation:

[0075] In the product L-alanine composite powder, L-alanine, γ-aminobutyric acid, L-glutamic acid, cysteine, ascorbic acid, and nitrite reductase are all encapsulated by the alginate-based gel and will not be suddenly released into the body environment, but can only be slowly released as the gel dissolves.

[0076] In the L-alanine composite powder, L-alanine, γ-aminobutyric acid, L-glutamic acid, cysteine, ascorbic acid, and nitrite reductase can all react with NO₂ - However, the reaction of L-alanine, γ-aminobutyric acid, L-L-alanine, γ-aminobutyric acid, L-glutamic acid, cysteine, ascorbic acid, and nitrite reductase glutamic acid with NO₂ - The reaction may produce carcinogenic amino acid-nitrosamine; compared with cysteine storing NO₂ -, due to the strong reducibility of ascorbic acid and the enzymatic catalysis of nitrite reductase, there is only one amino group on L-alanine, γ-aminobutyric acid, and L-glutamic acid, and the priority of reaction with NO2 - is the lowest, and the reaction path is blocked.

[0077] Nitrite reductase directly catalyzes the reduction of NO2 - to NO in the suitable environment provided by the gel.

[0078] Cysteine combines with NO2 - to form S-nitrosothiol, which is catalyzed by the Fe 2+ / Vc system to form NO and a disulfide intermediate (-S-S-), and NO and the disulfide intermediate (-S-S-) react with Fe 2+ gradually released from the cross-linked network to generate complexes such as Fe4S3(NO)7 - etc. This complex gradually releases NO and H2S under strong acidic conditions, further prolonging the slow release time of NO.

[0079] Different from Example 1, according to the differences in the formula and experimental conditions, Examples 2-3 were designed and completed:

[0080] Based on this, the following were also designed:

[0081] Comparative Example 1: The same as the formula and experimental method of Example 2, but without adding ferrous chloride;

[0082] Comparative Example 2: The same as the formula and experimental method of Example 2, but without adding calcium chloride;

[0083] Comparative Example 3: The same as the formula and experimental method of Example 2, but without adding sodium alginate;

[0084] Comparative Example 4: The same as the formula and experimental method of Example 2, but adding an excessive amount of ferrous chloride;

[0085] Comparative Example 5: The same as the formula and experimental method of Example 2, but adding an excessive amount of calcium chloride;

[0086] Comparative Example 6: The same as the formula and experimental method of Example 2, but adding an excessive amount of sodium alginate;

[0087] Comparative Example 7: The same as the formula and experimental method of Example 2, but without adding cysteine;

[0088] Comparative Example 8: The same as the formula and experimental method of Example 2, but without adding nitrite reductase;

[0089] Comparative Example 9: The same as the formula and experimental method of Example 2, but without adding trehalose;

[0090] Comparative Example 10: The formulation and experimental method are the same as those of Example 2, but calcium citrate is not added;

[0091] Comparative Example 11: The formulation and experimental method are the same as those of Example 2, but ascorbic acid is not added;

[0092] Comparative Example 12: The formulation and experimental method are the same as those of Example 2, but the dropping height during the formation of microspheres is reduced;

[0093] Comparative Example 13: The formulation and experimental method are the same as those of Example 2, but an excessive amount of cysteine is added;

[0094] Comparative Example 14: The formulation and experimental method are the same as those of Example 2, but an excessive amount of nitrite reductase is added;

[0095] Comparative Example 15: The formulation and experimental method are the same as those of Example 2, but an excessive amount of trehalose is added;

[0096] Comparative Example 16: The formulation and experimental method are the same as those of Example 2, but an excessive amount of calcium citrate is added;

[0097] Comparative Example 17: The formulation and experimental method are the same as those of Example 2, but an excessive amount of ascorbic acid is added;

[0098] Comparative Example 18: The formulation and experimental method are the same as those of Example 2, but the dropping height during the formation of microspheres is increased;

[0099] That is, Table 1. Among them, Comparative Example 3 did not form coagulated beads and did not participate in subsequent detections

[0100] Table 1. Formulation and production conditions of L-alanine composite powder

[0101]

[0102] Through multiple detections of Comparative Examples 1-18 and Examples 1-3, the detection items and detection methods of various performance data of the L-alanine composite powder are as follows:

[0103] 1. Nitrite scavenging rate (%)

[0104] Detection method: Griess reagent method (GB 5009.33-2016, National Food Safety Standard of China, mainly stipulates the determination methods of nitrite and nitrate in foods)

[0105] React the composite powder with a nitrite standard solution (NaNO2, 100 mg / L), 5 mg / L pepsin at 37 °C, in PBS with a pH of 7.4 at 37 °C for 7.5 h. Take the supernatant, add sulfanilamide and N-1-naphthylethylenediamine dihydrochloride, and measure the absorbance at 540 nm after color development.

[0106] Calculation formula:

[0107]

[0108] (C blank is the nitrite concentration in the solution without adding the composite powder, and C sample is the nitrite concentration in the solution with the composite powder added, calculated through the standard curve)

[0109] 2. Inhibition rate of nitrosamine formation (%)

[0110] Detection method: Gas chromatography - mass spectrometry (GC - MS) (GB 5009.26 - 2016, Determination method for N - dimethylnitrosamine content in foods)

[0111] Simulated nitrosamine formation system (dimethylamine + sodium nitrite, pH 2.0, reaction at 37°C for 2 h). After adding the composite powder, extract nitrosamine with dichloromethane and quantify by GC - MS. Theoretically, in this environment, dimethylamine reacts with sodium nitrite to form N - nitrosodimethylamine (NDMA). After adding the composite powder, cysteine and nitrite reductase contained in the composite powder will compete with dimethylamine for nitrosyl groups, thus inhibiting the formation of NDMA. Even if there is a small amount of NDMA, it will be trapped by the gel network.

[0112] Calculation formula:

[0113]

[0114] (C blank is the peak area of nitrosamine in the solution without adding the composite powder, and C sample is the peak area of nitrosamine in the solution with the composite powder added)

[0115] 3. Sustained - release time (h)

[0116] Detection method: Dynamic dialysis method (refer to USP <724>, Drug release testing)

[0117] Place the composite powder microspheres in a dialysis bag (MWCO 8 - 14 kDa) and immerse it in PBS solution (pH 7.4, 37°C). Take samples at regular intervals to measure the released Fe 2+ , until the release amount reaches 90%. In this product, the composition of the iron - ion gel network, as a catalyst to catalyze the formation of thiols, forms complexes to slowly release NO and H2S, which is one of the core components of the reaction and is used in a relatively large amount, facilitating determination.

[0118] Through this method, the released amount of Fe 2+ (mg / g composite powder microspheres) can also be measured simultaneously. React the sample solution with 1,10 - phenanthroline to form an orange - red complex, and measure the absorbance at 510 nm. Calculate the concentration of Fe 2+ through the FeSO4 standard curve.

[0119] Standard requirements:

[0120] The sustained-release preparation needs to maintain for 4 - 8 h (according to the Sustained Release Guiding Principles in Part Four of Chinese Pharmacopoeia).

[0121] The daily iron intake ≤ 18 mg (Reference Intakes for Dietary Nutrients of Chinese Residents).

[0122] 4. Enzyme activity retention rate (%)

[0123] Take nitrite reductase with the same amount as that contained in the L-alanine complex powder in each example and comparative example;

[0124] Place the L-alanine complex powder of each example and comparative example into 500 ml of 0.01 mol / L HCl solution with a pH of 2.0 and a temperature of 37 °C for 1 h (simulating the time and conditions of food existing in the stomach), add 500 ml of 0.01 mol / L NaCl solution to terminate the reaction, and re-freeze-dry.

[0125] React the prepared unreacted nitrite reductase and the re-freeze-dried L-alanine complex powder separately in a PBS solution with a pH of 7.4, 37 °C and a molar concentration of 100 mg / L of NaNO2 for 10 min, and terminate the reaction by vigorous shaking. Take the supernatant, add sufficient sulfanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride, measure the absorbance at 540 nm after color development, determine the amount of nitrite scavenged, calculate the enzyme activity of nitrite reductase after treatment and the enzyme activity before treatment through the amount of nitrite scavenged, and the enzyme activity of nitrite reductase after treatment and the enzyme activity before treatment are the enzyme activity retention rate (%).

[0126] Calculation formula:

[0127]

[0128] 4. Mechanical strength (kPa)

[0129] Detection method: Determination by texture analyzer

[0130] Compress the microspheres at a speed of 1 mm / s until rupture, record the maximum stress, and take the average value of 10 microspheres.

[0131] Standard requirements:

[0132] The mechanical strength of the microspheres of oral preparations needs to be ≥ 10 kPa (refer to Pharmaceutical Excipients Handbook).

[0133] 7. pH stability (ΔpH)

[0134] Detection method: pH meter monitoring method (GB / T 604 - 2002, General Method for the Determination of pH Color Change Range of Acid-Base Indicators in Chemical Reagents)

[0135] Dissolve the composite powder in deionized water (1:10 w / v) and shake at 37 °C for 24 h. Record the difference between the initial and final pH values (ΔpH = |pH0 - pH t |).

[0136] Standard requirement:

[0137] ΔpH ≤ 0.5 is considered stable (refer to the ICH Q1A stability guideline).

[0138] According to the above detection method and detection standard, the detection results are made into the following table:

[0139] Table 2. Formulation of L-alanine composite powder and its detected performance data

[0140]

[0141] Conduct a more precise detection of the key index of the product, the nitrite scavenging rate. Take the nitrite scavenging rate at different time points as the test result and draw Table 3:

[0142] Table 3. Nitrite scavenging effect table

[0143]

[0144] As can be seen from Table 3, cysteamine competes with nitrous acid reductase for nitrosyl. Cysteamine can directly react with nitrosyl to form thiol to store nitrosyl, so the reaction is faster. While nitrous acid reductase is an enzymatic reaction and is slower. Before the reaction of cysteamine is complete, it cannot compete with cysteamine; before 0.5 h, the alginate-based gel slowly dissolves and the drug embedded in it begins to be released, and the reaction is relatively slow.

[0145] There is a three-stage mechanism for the synergistic scavenging of nitrite: initiation period, steady-state reaction period, and maintenance period;

[0146] Among them, the initiation period is 0 - 2 h. At this time, the gel is initially dissolved, and the released cysteine will quickly combine with NO2 - to form amino acid thiol. After binding with Fe 2+ it obtains Fe4S3(NO)7 - , and the color of the gel deepens, which is the color of Fe4S3(NO)7 - , and it deepens continuously with the consumption of cysteine, which is used for the subsequent slow-release process (in Comparative Example 8, nitrite reductase was not added, but when the reaction reached 2 h, the conversion rate of the total nitrite had reached 48.3%);

[0147] The steady-state reaction period is 2 - 5 h. The free cysteine is exhausted, and the color of the gel begins to fade. This is the color change of Fe4S3(NO)7 -Consumed; the nitrite reductase embedded in the center of the gel begins to contact NO2 - , directly reducing it to nitrogen and nitric oxide. As an enzymatic reaction, the reaction rate of this process is not very fast, but the sodium alginate gel can protect the active ingredients from inactivation and can continuously catalyze stably before large-scale damage to the gel;

[0148] The maintenance period is in the time period of ≥5h. At this time, the alginate-based gel is decomposed, the nitrite reductase is digested, and the reaction rate decreases until the gel completely disappears and the reaction ends.

[0149] Key performance optimization analysis

[0150] (1) Nitrite clearance rate kinetics

[0151] Optimal groups: Comparative example 13 (clearance rate 95.1%), Comparative example 14 (clearance rate 95.5%) and Example 2 (94.8%) perform the best.

[0152] Mechanism: Comparative example 13 rapidly eliminates nitrite at the initial stage of the reaction through excessive cysteine, but the gap with Example 2 is not significant in the later stage, indicating that simply adding excessive cysteine cannot effectively improve the nitrite clearance rate of the product, resulting in waste of the drug.

[0153] Comparative example 14 accelerates the initial reaction through excessive enzyme (300U) (there is a large improvement in the clearance rate compared to Example 2 in the range of 0-2h), but is on par with Example 2 in the later stage (≥6h), indicating that there is a saturation threshold for enzyme activity.

[0154] Example 2 achieves stable and continuous clearance (7.2h sustained release) through the Fe 2+ -cysteine-nitrite reductase ternary system.

[0155] (2) Nitrosamine formation inhibition rate

[0156] Core factors: cysteine content (-SH directly attacks nitrosamine) and pH stability.

[0157] Comparative example 13 (15g of cysteine) has an inhibition rate of 86.4% and Comparative example 7 (without cysteine) has 60.2%, confirming the key role of sulfhydryl groups.

[0158] Example 2 (ΔpH = 0.2) has an inhibition rate of 90.1% and Comparative example 16 (ΔpH = 1.7)

[0159] 75.2%, showing the importance of pH stability.

[0160] (3) Balance between sustained release time and mechanical strength

[0161] Sodium alginate and Ca 2+Ratio:

[0162] Example 2 (70 g of sodium alginate + 0.75 g of Ca 2+ ): Sustained release for 7.2 h, strength 14.7 kPa (optimal).

[0163] Comparative Example 6 (100 g of sodium alginate): Sustained release for only 5.2 h (excessive crosslinking leads to a decrease in porosity).

[0164] Comparative Example 5 (5 g of Ca 2+ ): Strength 20.5 kPa but sustained release decreased by 4.8 h (excessive crosslinking inhibits release).

[0165] Abnormal data and special phenomena

[0166] Comparative Example 13 (15 g of cysteine):

[0167] The clearance rate is the highest at 95.1%, but the inhibition rate (86.4%) is lower than that of Example 2 (90.1%). Excessive cysteine may consume part of the Fe 2+ to form an inactive complex, weakening the blocking of nitrosamine.

[0168] Comparative Example 17 (1.5 g of ascorbic acid):

[0169] Fe 2+ release amount is 6.2 mg / g (the highest), but the clearance rate is only 87.9%. Excessive Vc causes Fe 2+ to be released too quickly, disrupting the gel system, with a violent initial reaction but exhaustion in the later stage.

[0170] Based on the examples and comparative examples conducted, the functions of each component are as shown in Table 4 below:

[0171] Table 4. Summary of the action mechanism of the core components

[0172]

[0173] In addition to their own efficacy as amino acids, the roles and mechanisms of L-alanine, γ-aminobutyric acid (GABA), and L-glutamic acid in the composite powder are analyzed as follows:

[0174] 1. L-alanine:

[0175] L-alanine synergizes with cysteine: L-alanine provides an amino group, and cysteine provides a mercapto group to jointly attack the N-N=O bond of nitrosamine. The mechanism of action is:

[0176] R1-N(NO)-R2 + HS-CH2CH(NH2)COOH - R1-NH-R2 + S-CH2CH(NH2)COOH + NO

[0177] Secondly, there are nitrosamine competitive inhibitors. As neutral amino acids, they preferentially combine with nitrite (NO2 - ) to block its reaction with secondary amines (such as protein degradation products) to form nitrosamines.

[0178] 2. γ-Aminobutyric acid (GABA)

[0179] The γ-amino group of GABA (pKa≈10.4) is protonated in an acidic environment to neutralize H + , protecting the activity of nitrite reductase (optimum pH 7-8) - in Comparative Example 10 (without calcium citrate but containing GABA), ΔpH = 1.4, which is better than the extreme group without buffer (ΔpH≥2.0).

[0180] GABA does not contain a carboxyl group and will not compete for the Ca 2+ crosslinking sites, avoiding interference with gel formation (when GABA coexists with sodium alginate in Comparative Example 6, the mechanical strength is 16.8 kPa).

[0181] 3. L-Glutamic acid

[0182] The dicarboxyl structure of L-glutamic acid (α-COOH and γ-COOH) forms a highly stable chelate with Ca 2+ to fill the pores of the sodium alginate gel and enhance the mechanical strength.

[0183] Secondly, the carboxyl group of L-glutamic acid coordinates with Fe 2+ to prevent its oxidation to Fe 3+ (especially when ascorbic acid is insufficient).

[0184] In summary, although adding excessive cysteine and nitrite reductase will increase the nitrite clearance rate, compared with the formulations given in Examples 1-3, there is no significant effective gap, and there is serious drug waste. It is worth mentioning that the -S-S- produced by the reaction of cysteine will produce H2S in the later stage. A small amount of H2S has the effect of sterilization and anti-inflammation, but once the concentration rises, it will cause negative states such as nausea and retching in people. Therefore, we can add excessive cysteine to produce an emergency drug that can be taken when people accidentally ingest foods containing a large amount of nitrite, and induce vomiting to quickly eliminate the residual nitrite (H2S concentration < 10 ppm); the formulations used in the examples can be used to supplement the amino acids required by the human body daily, as well as eliminate the ingested nitrite and nitrosamines formed by nitrite and amines.

[0185] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent replacements or changes, and should be covered by the protection scope of the present invention.

Claims

1. A compound powder of L-alanine for eliminating nitrite, characterized in that, It includes the following raw materials: 99 - 134 g of alginate-based gel; 9 - 11 g of trehalose; 0.3 - 0.6 g of calcium citrate; The preparation process of the alginate-based gel includes the following steps: S1. Preparation of amino acid mixture: Dissolve L-alanine, γ-aminobutyric acid, L-glutamic acid, cysteine, ascorbic acid, and nitrite reductase in PBS solution to obtain an amino acid mixture; S2. Encapsulation of sodium alginate gel: Dissolve sodium alginate in deionized water, stir at 60 °C until transparent, and cool to room temperature to obtain a sodium alginate solution; S3. Preparation of microspheres by ion crosslinking method: Dissolve the powders of FeCl2·4H2O and CaCl2 in deionized water to obtain an FeCl2 / CaCl2 solution; Gently mix the amino acid mixture and the sodium alginate solution to avoid bubbles to obtain a pre-gel solution; Dropwise add the pre-gel solution into the FeCl2 / CaCl2 solution with a syringe to form microspheres, thereby enhancing the mechanical strength; The microspheres were left to solidify in the FeCl2 / CaCl2 solution for 20 min to form beads with an Fe 2+ / Ca 2+ double-crosslinked network; Wash the coagulated beads 3 times with PBS solution to remove free ions and unencapsulated components, and obtain the alginate-based gel after freeze-drying.

2. The L-alanine composite powder for eliminating nitrite according to claim 1, wherein: The alginate-based gel specifically includes 60 - 80 g of sodium alginate, 12 - 15 g of L-alanine, 6 - 8 g of γ-aminobutyric acid, 9 - 11 g of L-glutamic acid, 8 - 12 g of cysteine, 200 - 250 U of nitrite reductase, 0.8 - 1.2 g of ascorbic acid, 3 - 6 g of FeCl2·4H2O, and 0.5 - 1 g of CaCl2.

3. The L-alanine composite powder for eliminating nitrite according to claim 1, characterized in that, In S1, the mass ratio of the PBS solution to L-alanine, γ-aminobutyric acid, L-glutamic acid, cysteine, ascorbic acid, and nitrite reductase is 2:1, the pH of the PBS solution is 7.4, and the molar concentration is 0.01 mol / L.

4. The L-alanine composite powder for eliminating nitrite according to claim 1, wherein In S2, the mass ratio of sodium alginate to deionized water is 1:

10.

5. A compound powder of L-alanine for eliminating nitrite according to claim 1, characterized in that, In S3, the mass ratio of the powders of FeCl2·4H2O and CaCl2 to deionized water is 1:

200.

6. The L-alanine composite powder for eliminating nitrite according to claim 1, characterized in that, In S3, the mass ratio of the amino acid mixture to the sodium alginate solution is 1:

10.

7. A compound powder of L-alanine for eliminating nitrite according to claim 1, characterized in that, In S3, the mass ratio of the pre-gel solution to the FeCl2 / CaCl2 solution is 1:

3.

8. The L-alanine composite powder for eliminating nitrite according to claim 1, characterized in that, In S3, control the diameter of the microspheres to be 1 - 2 mm, and select the dropping height to be 8 - 12 cm.

9. The L-alanine composite powder for eliminating nitrite according to claim 1, characterized in that, In S3, the freeze-drying conditions are: pre-freeze at -80 °C until the solution is completely frozen; perform vacuum drying, at -20 °C to -50 °C, with a vacuum degree of 10 - 50 mTorr, primary drying for 20 h; at -10 °C to +20 °C, with a vacuum degree of 1 - 2 mTorr, secondary drying for 4 h.

10. The preparation method of the L-alanine composite powder for eliminating nitrite according to any one of claims 1-9, characterized in that, It includes the following steps: Dissolve trehalose and calcium citrate in deionized water and dissolve them fully to obtain a trehalose mixed solution; Soak the alginate-based gel in the trehalose mixed solution for 30 min, after freeze-drying, grind it into a 100-mesh powder to obtain an L-alanine composite powder product, and store it in a light-proof and sealed manner.

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