阅读说明：本技术 基于茶多酚加合脱氧糖酮抑制Amadori重排产物降解的机制提高其产率的方法 (Method for improving yield of Amadori rearrangement product based on mechanism of inhibiting degradation of Amadori rearrangement product by tea polyphenol adducted deoxyfuranone ) 是由 张晓鸣 于莙禾 崔和平 詹欢 翟昀 于静洋 夏书芹 于 2019-10-18 设计创作，主要内容包括：本发明公开了基于茶多酚加合脱氧糖酮抑制Amadori重排产物(ARP)降解的机制提高其产率的方法,其具体方法为：取氨基酸、糖和茶多酚加水溶解,调节混合溶液pH；将所得混合液置于反应瓶中,并在恒定水浴温度下加热得到反应液；将所得反应液在恒定温度下进行真空减压脱水反应,反应结束后使用冰浴冷却终止反应,得到固体反应物,用水复溶得到ARP溶液；本发明基于茶多酚通过捕获ARP的初级降解产物即脱氧糖酮化合物与真空减压脱水的协同作用,促进了ARP的形成并抑制其降解,从而使得ARP大量累积富集。本发明中所述方法操作简单、绿色、反应时间短,条件温和,所得ARP产率可达80％以上,具有较强的创新性和显著的进步性。(The invention discloses a method for improving the yield of an Amadori Rearrangement Product (ARP) based on a mechanism that tea polyphenol adducted deoxyfuranone inhibits the degradation of the Amadori rearrangement product, which comprises the following steps: dissolving amino acids, sugar and tea polyphenols in water, and adjusting pH of the mixed solution; placing the obtained mixed solution into a reaction bottle, and heating at a constant water bath temperature to obtain a reaction solution; carrying out vacuum reduced pressure dehydration reaction on the obtained reaction solution at a constant temperature, cooling by using an ice bath after the reaction is finished to terminate the reaction to obtain a solid reactant, and re-dissolving the solid reactant by using water to obtain an ARP solution; the invention promotes the formation of ARP and inhibits the degradation of the ARP based on the synergistic effect of capturing the primary degradation product of ARP, namely the deoxysugar ketone compound and vacuum decompression dehydration, thereby accumulating and enriching the ARP in a large quantity. The method disclosed by the invention is simple and green to operate, short in reaction time, mild in condition, high in ARP yield of more than 80%, strong in innovation and remarkable in progress.)

1. The method for improving the yield of Amadori rearrangement products based on a mechanism that tea polyphenol adducted deoxysugar ketone inhibits the degradation of the Amadori rearrangement products is characterized by comprising the following steps:

(1) dissolving amino acids, sugar and tea polyphenols in water, and adjusting pH of the mixed solution;

(2) placing the mixed solution obtained in the step (1) into a reaction bottle, and heating at a constant water bath temperature to obtain a reaction solution;

(3) and (3) carrying out vacuum reduced pressure dehydration reaction on the reaction liquid obtained in the step (2) at a constant temperature, cooling by using an ice bath after the reaction is finished to terminate the reaction, obtaining a solid reactant, and re-dissolving by using water to obtain an ARP solution.

2. The method of claim 1, wherein the tea polyphenols in step (1) comprise at least one of epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, catechin, gallocatechin, catechin gallate, or gallocatechin gallate.

3. The method according to claim 1, wherein the amino acid in step (1) is one or more of alanine, phenylalanine, serine, and methionine.

4. The method according to claim 1, wherein the sugar in step (1) is one or more of ribose, xylose, arabinose, glucose.

5. The method according to claim 1, wherein the amino acids, the sugars and the tea polyphenols in step (1) are prepared by the following steps: 10 parts of amino acid, 5-50 parts of sugar, 0.1-5 parts of tea polyphenol and 1200 parts of water by weight.

6. The method according to claim 1, wherein the pH value of the mixed solution in the step (1) is 6 to 9.

7. The method according to claim 1, wherein the constant water bath temperature in the step (2) is 60-90 ℃ and the heating time is 30-100 min.

8. The method according to claim 1, wherein the constant reaction temperature in the step (3) is 60 to 90 ℃ and the reaction time is 5 to 60 min; the ice bath conditions were: the ice bath temperature was maintained at 0 ℃.

9. The method according to claim 1, wherein the ARP yield in step (3) is greater than 80%.

Technical Field

The invention belongs to the field of food chemistry and food additives, and particularly relates to a method for improving the yield of Amadori rearrangement products based on a mechanism that tea polyphenol adducted deoxyfuranone inhibits the degradation of the Amadori rearrangement products.

Background

Amadori rearrangement product, Amadori Rearrangement Product (ARP), an important intermediate product in early Maillard reaction process, is widely studied, and is formed by cyclizing Schiff base (Shiff base) formed by addition between aldose carbonyl and amino and loss of a molecule of water to form corresponding N-substituted aldosamine, and then converting into reactive 1-amino-1-deoxy-2-ketose by Amadori rearrangement. They play an important role in the development of flavour and colour during food processing or storage. At present, the common ingredients of the salty seasoning in China are complete Maillard Reaction Products (MRP), although the flavor is strong, most of the ingredients are strong in volatility, stable aroma enhancement effect is difficult to maintain particularly under high-temperature processing conditions such as cooking and baking, the problems of aroma loss and non-lasting aroma are obvious, and the application is greatly limited. The ARP has stable physicochemical property at normal temperature, retains higher reaction activity under heating condition, and is easy to complete the subsequent Maillard reaction to generate volatile flavor substances. Thus, ARP can serve as an "active flavor precursor" that can be controllably formed for desirable organoleptic attributes during food processing by preparing such suitable flavor precursors.

However, the yield of such active flavour precursors in the aqueous phase is typically not more than 5%, making such flavour precursors difficult to produce commercially. At present, certain researches on the improvement of the ARP yield, such as synthesis in organic solvents such as anhydrous methanol and the like, exist, and the chemical methods have high physiological toxicity, pollute the environment and have higher production cost, are only suitable for theoretical research and cannot meet the requirement of large-scale production. Furthermore, the use of sodium sulfite also has the effect of increasing the ARP yield, however such chemical additives would leave sulfite in the food product. Recent research shows that sulfite can damage chromosomes and DNA, can also cause the increase of sister chromatid interchange (SCE) and Micronucleus (MN) rate of human blood lymphocytes, can delay the mitotic cycle of the lymphocytes and reduce the cell division index, and has obvious dose-effect relationship. Therefore, the addition of sulfite not only risks excessive intake but also has a great problem of food safety. Along with the conflict and rejection of people to the addition of chemical substances in food, the natural health food is urgently needed. The research on how to adopt a new natural, green and synthetic-free technical method with potential toxic and side effects to improve the yield of the ARP, breaks through the technical barrier of low yield of the aqueous phase, and has important significance for realizing the large-scale industrial production of the ARP active flavor precursor.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for improving the yield of Amadori rearrangement products based on a mechanism that tea polyphenol adductdeoxyfuranone inhibits the degradation of the Amadori rearrangement products. The method has the advantages of simple and convenient operation, mild conditions and high yield of the prepared product.

The technical scheme of the invention is as follows:

a method for inhibiting degradation and increasing yield of Amadori rearrangement products based on tea polyphenol adductdeoxyfuranone, the method comprising the steps of:

(1) dissolving amino acids, sugar and tea polyphenols in water, and adjusting pH of the mixed solution;

(2) placing the mixed solution obtained in the step (1) into a reaction bottle, and heating at a constant water bath temperature to obtain a reaction solution;

(3) and (3) carrying out vacuum reduced pressure dehydration reaction on the reaction liquid obtained in the step (2) at a constant temperature, cooling by using an ice bath after the reaction is finished to terminate the reaction, obtaining a solid reactant, and re-dissolving by using water to obtain an ARP solution.

The tea polyphenol in the step (1) comprises at least one of Epicatechin (EC), Epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), catechin (C), Gallocatechin (GC), Catechin Gallate (CG) or gallocatechin gallate (GCG).

The amino acid in the step (1) is one or more of alanine, phenylalanine, serine and methionine.

In the step (1), the sugar is one or more of ribose, xylose, arabinose and glucose.

The dosage of the amino acid, the sugar and the tea polyphenol in the step (1) is respectively as follows: 10 parts of amino acid, 5-50 parts of sugar, 0.1-5 parts of tea polyphenol and 1200 parts of water by weight.

The pH value of the mixed solution in the step (1) is 6-9.

In the step (2), the constant water bath temperature is 60-90 ℃, and the heating time is 30-100 min.

In the step (3), the constant reaction temperature is 60-90 ℃, and the reaction time is 5-60 min; the ice bath conditions were: the ice bath temperature was maintained at 0 ℃.

And (4) the ARP yield in the ARP solution in the step (3) can reach more than 80%.

The beneficial technical effects of the invention are as follows:

(1) the mechanism reason for adding tea polyphenol to improve the ARP yield in the ammonia carbonyl reaction mainly comprises the following three aspects of synergistic action:

① mechanism for inhibiting ARP degradation by tea polyphenol

Active sites C6 and C8 on the A ring of catechin compounds as the main components in tea polyphenol can capture the primary degradation product of ARP, namely deoxysugar ketone compounds (deoxysugars), to form phenol-sugar adducts with low reaction activity, thereby blocking the downstream reaction path of ARP and inhibiting the degradation of ARP. FIG. 14 shows the total ion chromatogram of sugar/amino acid system after adding tea polyphenols EGCG for thermal reaction. As shown in FIG. 14, tea polyphenol and deoxyketose DP can form di-DP-EGCG (a) double adduct and momo-DP-EGCG (d/e) single adduct, thereby blocking Maillard downstream reaction path and inhibiting degradation of ARP, and the capture mechanism is shown in FIG. 15, which is an important basis for improving ARP yield.

② vacuum decompression dehydration action for promoting conversion of ARP precursor substance Schiff base to ARP

The vacuum dehydration process can increase the conversion rate of ARP by promoting the formation of the precursor schiff base (Shiff base) of ARP. As shown in fig. 11, in the system without the addition of tea polyphenol EGCG, the ARP yield obtained by vacuum dehydration was 42.1%, which was 2.2% higher than that obtained by normal pressure water phase, and the yield was increased by 19 times, confirming that the vacuum dehydration process can promote the formation of ARP.

③ synergistic effect of the two

After the tea polyphenol is added, the conversion of the Schiff base of the ARP precursor to the ARP is promoted in the vacuum decompression dehydration process, and meanwhile, the degradation of the ARP is inhibited by the addition of the tea polyphenol, so that the ARP is accumulated and enriched in a short time, and the conversion rate of the ARP is obviously improved by the synergistic effect. As shown in fig. 1, the ARP yield increased to 94.8%, which is 225.2% higher than the ARP yield obtained by vacuum reduction of 42.1% (fig. 11). The dynamic model of ARP formation in the process is shown in FIG. 16, wherein the path A is the dynamic parameter of ARP in the vacuum reduced pressure dehydration process when no tea polyphenol is added, and the path B is the dynamic parameter of ARP in the vacuum reduced pressure dehydration process after the tea polyphenol is added. Meanwhile, compared with a system without tea polyphenol addition, the system has the activation energy Ea formed by ARP₂＝70.88kJ/mol＜Ea₂' -60.35 kJ/mol, which is probably due to the reduction of the activation energy of sugar/amino acid switch to ARP by tea polyphenols forming ARP-catechin (FIG. 15) like the ARP adduct ARP-EGCG (b/c) detected in FIG. 14 with ARP as unstable intermediate. Therefore, according to the catalytic kinetics principle, the tea polyphenol catalyzes the formation of ARP to a certain extent. Through dynamic simulation, the ARP generation rate k can be calculated and known₂′＞k₂Degradation rate k₄′＜k₄It was demonstrated that the addition of tea polyphenols promoted the formation of ARP and at the same time inhibited its degradation.

(2) In the prior art, the yield of the ARP aqueous phase is low, and most of the ARP aqueous phase is prepared and synthesized in an organic phase, and the technical method has the disadvantages of physiological toxicity, environmental pollution and complex operation, so that not only food-grade products cannot be obtained, but also industrial production is difficult to implement. The method for improving the yield of the ARP by using the tea polyphenol has the advantages of food-grade synthesis, simple and convenient operation, greenness, no pollution, low cost and easy industrial production, and the ARP obtained by the technology can be completely and directly applied to food ingredients or seasoning products.

(3) The tea polyphenol utilized by the invention is a full natural food additive which is extracted from tea and has stronger antioxidation, and has the functions of bacteriostasis and strong physiological activity, including anticancer, anti-mutation, in-vivo free radical removal and the like. The tea polyphenol has the characteristics of strong oxidation resistance, no peculiar smell, no potential toxic or side effect of a composition and the like in the application process, and has important significance for developing functional semi-finished food seasoning (active flavor precursor ARP). Therefore, the invention utilizes the technology that the tea polyphenol promotes the formation of the ARP and inhibits the degradation of the ARP, thereby not only obviously improving the yield of the ARP, but also increasing the functional activity and the shelf life of the food applied by the ARP.

(4) The technology for improving the yield of the aqueous phase ARP by using the tea polyphenol not only overcomes the defect of low yield of the aqueous phase ARP, but also adopts the natural auxiliary agent tea polyphenol, the raw materials are natural and easy to obtain, and compared with the processing auxiliary agents used by an organic phase method and a group protection method, the method greatly reduces the accounting cost, the safety cost and the environmental cost of production.

(5) The preparation process adopted by the invention has the advantages of simple and convenient operation, simple equipment, reaction at mild temperature, short preparation time and high yield. The process technology meets the concept of green, scientific and sustainable industrial design, and has higher edible safety. Therefore, the technology can be directly applied to actual production and has stronger actual application value.

Drawings

FIG. 1 is a chromatogram of an ARP solution prepared by an alanine/xylose reaction in an aqueous system with EGCG added in example 1;

FIG. 2 is a total ion chromatogram of the ARP prepared by the alanine/xylose reaction after purification in example 1, and a total ion mass spectrum of the ARP is qualitatively obtained by LC/MS/MS;

FIG. 3 is a chart of the hydrogen nuclear magnetic resonance spectrum of ARP prepared by the alanine/xylose reaction prepared in example 1;

FIG. 4 is a chart of the ARP NMR carbon spectrum prepared by the alanine/xylose reaction prepared in example 1;

FIG. 5 is a chromatogram of an ARP solution prepared by phenylalanine/xylose reaction in an aqueous system with EGCG added in example 2;

FIG. 6 is a total ion chromatogram of ARP prepared by the phenylalanine/xylose reaction after purification in example 2, and the total ion mass spectrum of the ARP is characterized by LC/MS/MS;

FIG. 7 is a chart of the ARP NMR spectrum prepared by the phenylalanine/xylose reaction prepared in example 2;

FIG. 8 is a nuclear magnetic resonance carbon spectrum of ARP prepared from the phenylalanine/xylose reaction prepared in example 2;

FIG. 9 is a chromatogram of the ARP solution prepared by the alanine/xylose reaction in the aqueous phase system with EGCG added in example 3;

FIG. 10 is a chromatogram of an ARP solution prepared by the alanine/xylose reaction in a normal pressure aqueous system without EGCG addition in comparative example 1;

FIG. 11 is a chromatogram of an ARP solution prepared by the reaction of alanine/xylose in a vacuum reduced pressure aqueous phase system without EGCG in comparative example 2;

FIG. 12 is a chromatogram of an ARP solution prepared by the alanine/xylose reaction in an anhydrous methanol system in comparative example 3;

FIG. 13 is a chromatogram of an ARP solution prepared by adding phenylalanine/xylose reaction to the aqueous phase system without EGCG in comparative example 4;

FIG. 14 is an adduct of tea polyphenols EGCG (f) with ARP: ARP-EGCG (b/c) and its degradation products DP adducts: di-DP-EGCG (a) and momo-DP-EGCG (d/e) total ion chromatograms of the double adducts;

FIG. 15 mechanism of the capture effect of tea polyphenols on ARP degradation products;

FIG. 16 the kinetic mechanism by which tea polyphenols promote ARP formation and inhibit its degradation.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings and examples.