WO2021017781A1 - 一种通过还原型谷胱甘肽指示氨基酸美拉德反应制备中间体的方法 - Google Patents

一种通过还原型谷胱甘肽指示氨基酸美拉德反应制备中间体的方法 Download PDF

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WO2021017781A1
WO2021017781A1 PCT/CN2020/100797 CN2020100797W WO2021017781A1 WO 2021017781 A1 WO2021017781 A1 WO 2021017781A1 CN 2020100797 W CN2020100797 W CN 2020100797W WO 2021017781 A1 WO2021017781 A1 WO 2021017781A1
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reaction
temperature
maillard reaction
stage
reduced glutathione
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PCT/CN2020/100797
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French (fr)
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张晓鸣
卢思芸
崔和平
于莙禾
詹欢
翟昀
唐微
于静洋
夏书芹
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江南大学
安徽强旺调味食品有限公司
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Priority to US17/251,207 priority Critical patent/US11802137B2/en
Publication of WO2021017781A1 publication Critical patent/WO2021017781A1/zh

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    • C07H1/00—Processes for the preparation of sugar derivatives
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • G01N30/02—Column chromatography
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    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N2021/3125—Measuring the absorption by excited molecules
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    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G01N2021/786—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour with auxiliary heating for reaction
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
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    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/067—Preparation by reaction, e.g. derivatising the sample

Definitions

  • the invention belongs to the field of food chemistry and food additives, and in particular relates to a method for preparing an intermediate through the Maillard reaction of a reduced glutathione indicating amino acid.
  • Maillard reaction also known as non-enzymatic browning, is widely present in food processing and storage, and directly affects the flavor, taste, color and nutritional value of food. It is one of the important research hotspots in the flavor and fragrance industry. At present, the complete Maillard reaction product is mainly used in the food field, but the final product of the reaction is extremely unstable, and flavor substances are easily lost during high-temperature processing, and the application range is limited.
  • amino compounds and The intermediates formed by the reaction of carbonyl compounds Amadori rearrangement products (ARP) and Heyns rearrangement products (HRP) are two important types of flavor precursors.
  • cysteine tracer method to prepare Maillard reaction intermediates in aqueous phase is a new method proposed in recent years, which solves the above problems to a certain extent, but cysteine has poor water solubility and is not completely dissolved. Will lead to deviations in the results, and its mechanism as a tracer is not clear. Therefore, in order to overcome the shortcomings of the existing preparation technology, find a more suitable new tracer and study the theory and technical system of ARP and HRP's low-temperature water phase controllable preparation theory and technical system urgently need to be completed.
  • High performance liquid chromatography is usually used to detect the amount of ARP or HRP produced, but This method requires the first preparation, purification and characterization of standard products, and the batch preparation of Maillard reaction intermediates has many difficulties to be solved urgently.
  • the preparation method of the intermediate disclosed in the present invention has simple operation, feasibility and universality, and research has confirmed that the determined preparation conditions are completely consistent with the results of the high-performance liquid chromatography detection method.
  • cysteine is used as the color inhibition and intermediate tracer of Maillard products, but the solubility of cysteine is poor, and it takes time to dissolve after adding the solution Over time, the time and cost required for production increase.
  • the different concentration of cysteine added in the system has a significant impact on the color of the final Maillard product. Therefore, once the cysteine is not completely dissolved, the concentration in the system is not uniform, and the interaction with the intermediate is not sufficient, which is likely to cause the final stage There are errors in the browning value, and the optimal preparation conditions are difficult to determine.
  • the present invention uses reduced glutathione as the tracer, and its solubility in aqueous solution is far greater than that of cysteine, which greatly saves production time and is beneficial to industrial production. In addition, it can more accurately trace intermediates. Optimal production conditions effectively overcome the deficiency of using cysteine as a tracer.
  • the tracer method is a new method for preparing Maillard reaction intermediates.
  • the mechanism of using cysteine as a tracer has not been clearly reported, and theoretically, different tracers
  • the mechanism of action is also different.
  • the present invention is based on the effect of tracing Maillard reaction intermediates of reduced glutathione, and further explores the mechanism by which the substance can be used as a tracer.
  • the study found that the oxidation product of reduced glutathione, namely oxidized glutathione, showed a gradual increase in the browning value during the high temperature stage as the reaction time of the first stage increased. Shows that the substance does not have the effect of tracing intermediates.
  • the main difference in the structure of reduced and oxidized glutathione is the presence or absence of free sulfhydryl groups. Based on this, it can be inferred that the free sulfhydryl groups in reduced glutathione are the key factor that can be used as a tracer.
  • the present invention finds that the sulfhydryl group of reduced glutathione can interact with the degradation products of the intermediate through the combined technology of liquid chromatography and liquid chromatography-mass spectrometry, which can effectively inhibit the subsequent Maillard cascade reaction and reduce the subsequent products such as : Glyoxal, pyruvaldehyde, furfural and other characteristic substances of Maillard reaction are produced, which can change the path of Maillard reaction, thereby reducing the formation of melanoid, and obtaining light-colored flavor flavors, which are produced in large quantities in the intermediate
  • the final product obtained by adding reduced glutathione has the lightest color, which is consistent with the liquid chromatography verification results.
  • reduced glutathione can not only be used as an intermediate tracer, but also as a browning inhibitor.
  • reduced glutathione and its Maillard products have a mellow taste, and can give people a thick, rich, round and balanced taste experience, while cysteine has no such effect. Therefore, if reduced glutathione is compounded with an intermediate as a flavor precursor, the subsequent thermal processing can not only form the characteristic flavor of Maillard reaction, but also provide a mellow flavor to satisfy consumers' high taste richness. It is required to reduce the use of salt, sugar and other seasonings, and meet the requirements of healthy diet proposed by modern people.
  • the optimal preparation conditions of intermediates can be determined by measuring the degree of browning of the Maillard reaction product in the second stage of the water phase. It is a technology that can control the preparation of intermediates in the water phase.
  • the key and important breakthrough has overcome the main drawbacks of organic solvent preparation technology.
  • the entire analysis and measurement work can be completed only by using a spectrophotometer, with simple method, safe operation, low cost and high feasibility. Using this method to prepare Maillard reaction intermediates in a green, safe, directional and efficient manner in the water phase will enable the large-scale production of ARP and HRP, which has strong practical application value.
  • FIG. 1 is a graph showing the relationship between the absorbance value of the Maillard reaction solution at the second stage of variable temperature and the Maillard reaction time of the first stage after dilution in Example 1 of the present invention
  • Example 2 is a mass spectrum of ARP prepared in Example 1 of the present invention.
  • FIG 3 shows the ARP proton nuclear magnetic resonance spectrum (a) and the carbon nuclear magnetic resonance spectrum (b) prepared in Example 1;
  • Fig. 5 is a graph of the ARP liquid chromatography-mass spectrometry characterization results obtained in Example 2 of the present invention, wherein (a) is a total ion current chromatogram, and (b) is a mass spectrum;
  • Example 6 is a graph showing the relationship between the absorbance value of the Maillard reaction solution at the second stage of variable temperature and the Maillard reaction time in the first stage after dilution in Example 3 of the present invention
  • FIG. 7 is a graph of the ARP liquid chromatography-mass spectrometry characterization results obtained in Example 3 of the present invention, wherein (a) is a total ion current chromatogram, and (b) is a mass spectrum;
  • Example 8 is a graph showing the relationship between the absorbance value of the Maillard reaction solution at the second stage of variable temperature and the Maillard reaction time of the first stage after dilution in Example 4 of the present invention
  • FIG. 9 is a mass spectrum of HRP prepared in Example 4 of the present invention.
  • FIG 10 is a graph showing the relationship between the ARP concentration generated in the cysteine and xylose system in Comparative Example 1 and different low-temperature reaction times.
  • the intermediate is prepared at the selected temperature and optimal time, further concentrated at low temperature, separated and purified by a hydrogen-type cation exchange resin to obtain the pure intermediate (ARP) of the glycine-ribose system, and then freeze-dried to obtain a solid sample.
  • ARP pure intermediate
  • the obtained solid was dissolved in water and analyzed by high performance liquid chromatography-mass spectrometry analysis technology, and the total ion current chromatogram and mass spectrum were obtained as shown in FIG. 7.
  • the intermediate is prepared at the selected temperature and optimal time, further concentrated at low temperature, separated and purified by hydrogen type cation exchange resin to obtain the pure intermediate of proline-fructose system (HRP), and then freeze-dried to obtain a solid sample.
  • HRP proline-fructose system
  • the obtained solid was dissolved in water and analyzed by mass spectrometry technology, and a mass spectrum was obtained as shown in FIG. 9.
  • the experimental water in the foregoing examples and comparative examples is distilled water, aldose or ketose and amino acids are food grade, the chemical reagents used in the high performance liquid chromatography-mass spectrometry analysis experiment are chromatographically pure, and the remaining chemical reagents are analytically pure.
  • the detection conditions of high performance liquid chromatography were: chromatographic column CSH C18, mobile phase of acetonitrile and 0.1% formic acid water, flow rate of 0.3mL/min, gradient elution, column temperature of 45°C.
  • the conditions of mass spectrometry analysis are as follows: adopt ESI+ mode, detector voltage 1.8kV, capillary voltage 3.5kV, cone voltage 20V, extraction voltage 7V.
  • the electron source temperature and the desolvent gas temperature are 100°C and 400°C, respectively, the gas flow rate is 700L/h, and the cone gas flow rate is 50L/h.
  • the sample is scanned in the range of m/z 20 ⁇ 1000 mass-to-composite ratio, the scanning time is 1s, and the scanning time delay is 0.1s.
  • the separated pure intermediate was dissolved in D2O, and the intermediate was analyzed with a nuclear magnetic resonance instrument.
  • the test temperature was 298K.

Abstract

一种通过还原型谷胱甘肽指示氨基酸美拉德反应制备中间体的方法，采用二阶段变温反应，在低温反应不同时间后添加还原型谷胱甘肽，基于还原型谷胱甘肽与中间体降解产物相互作用可有效抑制后续美拉德反应，从而减少呈色物质生成的发现，对比高温阶段美拉德产物的褐变情况，色泽抑制效果最佳的反应时间即为中间体最佳制备条件，在此条件下水相低温制备中间体。方法采用水溶性较好的还原型谷胱甘肽作为示踪剂，可减少生产时间，并能提升示踪的准确性。在水相中绿色安全制备中间体，使其规模化生产成为可能，可实现食品加工风味受控形成，能增强消费者的成就感和愉悦感，具有较强的实际应用价值。

Description

一种通过还原型谷胱甘肽指示氨基酸美拉德反应制备中间体的方法 技术领域
本发明属于食品化学和食品添加剂领域，尤其涉及一种通过还原型谷胱甘肽指示氨基酸美拉德反应制备中间体的方法。
 背景技术
消费者对于食品的期望随着社会的进步和时代的变迁不断趋于层次化与多样化，食品的饱腹功能已不能满足大众的基本需求，人们对感官愉悦的天然创新产品的期望日益增加。如今食品行业呈现健康、多样和方便的发展趋势，其中加工风味受控形成技术是食品风味调控技术重要的创新方向，利用这种技术，可根据加工食品或餐饮需求由适宜的风味前体预制调味半成品，产品本身尚未形成完整的风味，但在后续热加工过程中能迅速产生新鲜、预期的理想风味。美拉德反应又称非酶褐变，广泛存在于食品加工和储藏过程中，直接影响着食品的风味、滋味、色泽和营养价值，是香精香料行业重要的研究热点之一。目前在食品领域中应用的主要是完全美拉德反应产物，但该反应的终产物极其不稳定，高温加工过程中风味物质容易损失，应用范围有限，而美拉德反应初级阶段，氨基化合物和羰基化合物反应形成的中间体：Amadori重排产物（ARP）和Heyns重排产物（HRP）是两类重要的风味前体物。这两类中间体低温下具有较稳定的理化性质，但在加热条件下保留较高的反应活性，极易完成后续美拉德反应，产生挥发性风味物质，因此在食品烹饪和加工过程中比完全美拉德反应产物具有更加广阔的应用前景。
关于中间体（ARP和HRP）的制备已有一定的研究，大多在甲醇等有机溶剂中合成，这些方法污染严重、成本较高，仅适用于理论研究，无法满足规模生产需要。当今社会提倡的可持续发展理念意指经济、社会、资源和环境保护协调发展，因此在工业生产中必须以绿色、低碳、环保、可持续发展的理念为宗旨,统筹经济成本与生产安全。水相高温美拉德反应虽能降低成本，但高温下，氨基酸和还原糖会迅速的进行一系列的级联反应，反应进程无法控制，从而难以制备得到中间体。采用半胱氨酸示踪法水相制备美拉德反应中间体是一种近年来提出的新方法，从一定程度上解决了以上的问题，但半胱氨酸水溶性较差，不完全溶解会导致结果的偏差，且其作为示踪剂的机理还未明确。因此，要克服现有制备技术的缺点，寻找一种更加合适的新型示踪剂并研究ARP和HRP的低温水相可控制备理论和技术体系亟待完成。
 技术问题
在此处键入技术问题描述段落。
 技术解决方案
在此处键入技术解决方案描述段落。
 有益效果
（1）采用理化性质稳定的氨基酸美拉德反应中间体制备预制调味料，解决了完全美拉德反应产物高温加工过程中易损失的问题，从整体风味上提升加工食品的品质，能够解决现有完全美拉德反应香精产物风味易散失的缺点，促进我国食品工业转型升级，为消费者提供美食的同时增强其成就感和愉悦感。由于美拉德反应路径复杂，产物众多，目前对于美拉德反应中间体，特别是新型中间体的制备和检测仍存在一定的困难，通常采用高效液相色谱检测ARP或HRP的生成量，但这种方法需要首先制备、纯化和表征标准品，美拉德反应中间体的批量制备就存在众多困难亟待解决。本发明所公开的中间体制备方法，操作简单、具有可行性和普适性，而且研究证实，其确定的制备条件与高效液相色谱检测法结果完全相符。
（2）现有公开的技术中，仅有采用半胱氨酸作为美拉德产物色泽抑制和中间体示踪剂的报道，但半胱氨酸溶解性较差，加入溶液后溶解所需时间久，生产所需时间成本提高。体系中半胱氨酸的添加浓度不同对最终美拉德产物色泽有显著的影响，因此一旦半胱氨酸未能全部溶解，体系中浓度不一，与中间体作用不够充分，容易造成最终阶段褐变值存在误差，最佳制备条件难以确定。而本发明采用还原型谷胱甘肽作为示踪剂，其在水溶液中的溶解性远远大于半胱氨酸，大大节约了生产时间，利于工业化生产，此外能够更加准确地示踪中间体最佳生成条件，有效克服了以半胱氨酸作为示踪剂的不足。
（3）示踪法作为一种制备美拉德反应中间体的新方法，以半胱氨酸作为示踪剂的机理还未有明确报道，且从理论上而言，不同的示踪剂的作用机理也存在一定区别。本发明基于还原型谷胱甘肽具有示踪美拉德反应中间体的效果，进一步探究了该物质能作为示踪剂的机理。采用相同的二阶段美拉德反应，研究发现还原型谷胱甘肽的氧化产物即氧化型谷胱甘肽随着第一阶段反应时间的增加，高温阶段的褐变值呈现逐渐增加的趋势，表明该物质不具有示踪中间体的效果。还原型和氧化型谷胱甘肽在结构上最主要的差异是游离巯基的存在与否，据此可以推断还原型谷胱甘肽中的游离巯基是其能作为示踪剂的关键因素。此外，本发明通过液相色谱和液相色谱-质谱联用技术发现还原型谷胱甘肽的巯基能与中间体降解产物相互作用，可有效抑制后续美拉德级联反应，减少后续产物如：乙二醛、丙酮醛、糠醛等美拉德反应特征性物质的产生，改变美拉德反应的路径，从而减少类黑精的形成，得到浅色型风味香精，并且在中间体正好大量生成的时候添加还原型谷胱甘肽得到的最终产物色泽最浅，与液相色谱验证结果一致。由此可见，还原型谷胱甘肽不但能够作为中间体的示踪剂，也可作为一种褐变抑制剂。基于已有的报道，还原型谷胱甘肽及其美拉德产物均具有醇厚味，而能给人以厚重、丰富、圆润、平衡的味觉感受，而半胱氨酸并没有该效果。因此，若将还原型谷胱甘肽与中间体复配一起作为风味前体，通过后续热加工不仅能形成美拉德反应特征风味，而且能够提供醇厚味，满足消费者对味觉丰富性的高要求，达到减少食盐、糖等调味料的使用量，符合现代人们所提出的健康饮食要求。
（4）以还原型谷胱甘肽作为新型示踪剂，通过测定水相二阶段美拉德反应产物的褐变程度来确定中间体最佳制备条件，是水相可控制备中间体的技术关键和重要突破，克服了有机溶剂制备技术的主要弊端。此外，仅采用分光光度计即可完成整个分析测定工作，方法简单、操作安全、成本低廉、可行性高。利用该方法在水相中绿色安全、定向高效制备美拉德反应中间体，将使ARP和HRP的规模化生产成为可能，具有较强的实际应用价值。
 附图说明
图1为本发明实施例1所述稀释后第二阶段变温美拉德反应液吸光度值与第一阶段美拉德反应时间的关系曲线图；
图2为本发明实施例1中制备所得ARP的质谱图；
图3为实施例1中制备的ARP核磁共振氢谱图（a）和核磁共振碳谱图（b）；
图4为本发明实施例2所述稀释后第二阶段变温美拉德反应液吸光度值与第一阶段美拉德反应时间的关系曲线图；
图5为本发明实施例2中制备所得ARP液相色谱质谱联用表征结果图，其中（a）为总离子流色谱图，（b）为质谱图；
图6为本发明中实施例3所述稀释后第二阶段变温美拉德反应液吸光度值与第一阶段美拉德反应时间的关系曲线图；
图7为本发明实施例3中制备所得ARP液相色谱质谱联用表征结果图，其中（a）为总离子流色谱图，（b）为质谱图；
图8为本发明中实施例4所述稀释后第二阶段变温美拉德反应液吸光度值与第一阶段美拉德反应时间的关系曲线图；
图9为本发明实施例4中制备所得HRP的质谱图；
图10为比较例1中半胱氨酸和木糖体系中生成的ARP浓度与不同低温反应时间的关系曲线图。
 本发明的最佳实施方式
在此处键入本发明的最佳实施方式描述段落。
 本发明的实施方式
下面结合附图和实施例，对本发明进行具体描述。
实施例1
（1）将17.8kg丙氨酸和60kg木糖溶解于1000 kg水中，调节混合溶液pH至8.0，在80℃水浴条件下反应，分别在40、60、80、100、120min取样180L，置于冰浴中冷却终止反应；
（2）分别在上述所得到的五份反应液中加入1.8kg还原型谷胱甘肽，并重新调节反应液pH至8.0，转移至耐温耐压瓶，然后升温至120℃，二阶段高温美拉德反应60min，置于冰浴中冷却终止反应，得到变温美拉德反应液；
（3）分别将各份变温美拉德反应溶液稀释5倍，测定在波长420nm下的吸光度值，根据吸光度值和步骤（1）中的相应低温反应时间绘制曲线图，结果如图1所示。由图1可知，变温美拉德反应液吸光度值低点对应的反应时间为80min，即为最佳色泽抑制效果，由此可确定在第一反应阶段80℃条件下的最适反应时间为80min。
在所选温度及最适时间下制备中间体，进一步经低温浓缩，经氢型阳离子交换树脂分离纯化后即可得到丙氨酸-木糖体系纯品中间体（ARP），后经冷冻干燥得到固体样品。将所得固体溶于水后通过质谱分析技术对其进行分析，得到质谱图如图2所示。通过核磁共振对其进行结构表征，得到核磁共振的谱图如图3所示。
实施例2
（1）将8kg半胱氨酸和19.8kg木糖溶解于800kg水中，调节混合溶液pH至7.5，在100℃水浴条件下反应，分别在10、20、30、40、50、60min取样130L，置于冰浴中冷却终止反应；
（2）分别在上述所得到的六份反应液中加入1.3kg还原型谷胱甘肽，并重新调节反应液pH至7.5，转移至耐温耐压瓶，然后升温至130℃，二阶段高温美拉德反应90min，置于冰浴中冷却终止反应，得到变温美拉德反应液；
（3）分别将各份变温美拉德反应溶液稀释2倍，测定在波长420nm下的吸光度值，根据吸光值和步骤（1）中的相应低温反应时间绘制曲线图，结果如图4所示。由图4可知，变温美拉德反应液吸光度值低点对应的反应时间为40min，即为最佳色泽抑制效果，由此可确定在第一反应阶段100℃条件下的最适反应时间为40min。
在所选温度及最适时间下制备中间体，进一步经低温浓缩，经氢型阳离子交换树脂分离纯化后即可得到半胱氨酸-木糖体系纯品中间体（ARP），后经冷冻干燥得到固体样品。将所得固体溶于水后通过高效液相色谱质谱联用分析技术对其进行分析，得到总离子流色谱图和质谱图，如图5所示。
实施例3
（1）将15kg甘氨酸和60kg核糖溶解于1000kg水中，调节混合溶液pH至6.0，在90℃水浴条件下反应，分别在20、40、60、80、100、120min取样150L，置于冰浴中冷却终止反应；
（2）分别在上述所得到的六份反应液中加入3kg还原型谷胱甘肽，并重新调节反应液pH至6.0，转移至耐温耐压瓶，然后升温至110℃，二阶段高温美拉德反应120min，置于冰浴中冷却终止反应，得到变温美拉德反应液；
（3）分别将各份变温美拉德反应溶液稀释50倍，测定在波长420nm下的吸光度值，根据吸光值和步骤（1）中的相应低温反应时间绘制曲线图，结果如图6所示。由图6可知，变温美拉德反应液吸光度值低点对应的反应时间为60min，即为最佳色泽抑制效果，由此可确定在第一反应阶段90℃条件下的最适反应时间为60min。
在所选温度及最适时间下制备中间体，进一步经低温浓缩，经氢型阳离子交换树脂分离纯化后即可得到甘氨酸-核糖体系纯品中间体（ARP），后经冷冻干燥得到固体样品。将所得固体溶于水后通过高效液相色谱质谱联用分析技术对其进行分析，得到总离子流色谱图和质谱图如图7所示。
实施例4
（1）将20kg脯氨酸和20kg果糖溶解于400kg水中，调节混合溶液pH至7.0，在100℃水浴条件下反应，分别在100、120、140、160、180min取样80L，置于冰浴中冷却终止反应；
（2）分别在上述所得到的五份反应液中加入20.0kg还原型谷胱甘肽，并重新调节反应液pH至7.0，转移至耐温耐压瓶，然后升温至120℃，二阶段高温美拉德反应180min，置于冰浴中冷却终止反应，得到变温美拉德反应液；
（3）分别将各份变温美拉德反应溶液稀释2倍，测定在波长420nm下的吸光度值，根据吸光值和步骤（1）中的相应低温反应时间绘制曲线图，结果如图8所示。由图8可知，变温美拉德反应液吸光度值低点对应的反应时间为140min，即为最佳色泽抑制效果，由此可确定在第一反应阶段100℃条件下的最适反应时间为140min。
在所选温度及最适时间下制备中间体，进一步经低温浓缩，经氢型阳离子交换树脂分离纯化后即可得到脯氨酸-果糖体系纯品中间体（HRP），后经冷冻干燥得到固体样品。将所得固体溶于水后通过质谱分析技术对其进行分析，得到质谱图如图9所示。
比较例1
将8kg半胱氨酸和19.8kg木糖溶解于800kg水中，调节混合溶液pH至7.5，在100℃水浴条件下反应，不同时间后取少量样品置于冰浴中冷却终止反应，用高效液相色谱测定不同反应时间中间体含量的变化规律，结果如图10所示。
由图10可知，低温反应阶段的初期，体系中中间体的累积量逐渐增加，40min后中间体的含量趋于稳定，这与图4中色泽最低点相对应，即半胱氨酸-木糖体系中，中间体大量生成的时间是40min。
上述实施例和比较例中的实验用水为蒸馏水，醛糖或酮糖和氨基酸为食品级，高效液相色谱-质谱分析实验所用化学试剂为色谱纯，其余化学试剂均为分析纯。高效液相色谱检测条件为：色谱柱CSH C18，流动相为乙腈与0.1%甲酸水，流速为0.3mL/min，采用梯度洗脱，柱温为45℃。质谱分析的条件如下：采用ESI+模式，检测器电压1.8kV，毛细管电压3.5kV，锥孔电压20V，提取电压7V。电子源温度和脱溶剂气温度分别为100°C和400°C，气流量700L/h，锥孔气体流量50L/h。在m/z 20～1000的质合比范围内对样品进行扫描，扫描时间为1s，扫描时间延迟0.1s。将分离得到的纯品中间体溶解在D2O中，采用核磁共振仪对中间体进行分析，测试温度为298K。
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Claims (3)

  1. 一种通过还原型谷胱甘肽指示氨基酸美拉德反应制备中间体的方法，其特征在于，所述方法包括如下步骤：
    （1）取氨基酸和醛糖或酮糖，加水溶解，调节混合溶液pH至6~8；原料用量以重量份数计；所述氨基酸10份；醛糖或酮糖10~40份；水200~1000份；
    （2）将步骤（1）中所得溶液置于80~100℃恒温循环水浴中进行第一阶段美拉德反应，在反应10~180min过程中间隔10~20min依次取等体积样品5~8份，将取出的样品立即置于冰浴中冷却，终止反应得第一阶段美拉德反应液；
    （3）在步骤（2）中所得每份样品中分别加入等量的还原型谷胱甘肽，混匀后重新调节溶液pH至6~8，之后转移至耐温耐压瓶中进行第二阶段高温美拉德反应，相同温度下反应60~180min，置于冰浴中冷却，终止反应分别得到第二阶段变温美拉德反应液；所述反应温度为110~130℃；
    （4）将步骤（3）中所得第二阶段变温美拉德反应液分别进行稀释，测定稀释后各份第二阶段变温美拉德反应液在波长420nm下的吸光度值，将所得吸光度值与步骤（2）中的相应反应时间绘制曲线图，根据吸光度值最低点对应的反应时间，确定在相应反应条件下的最适反应时间；
    （5）重复步骤（1）的操作：取氨基酸和醛糖或酮糖，加水溶解，调节混合溶液pH至6~8；原料用量以重量份数计；
    （6）将步骤（5）中所得溶液置于80~100℃水浴中恒温循环进行第一阶段美拉德反应，反应时间为步骤（4）中所述最适反应时间，反应结束后立即置于冰浴中冷却，终止反应得第一阶段美拉德反应液；
    （7）将步骤（6）中所得第一阶段变温美拉德反应液减压低温浓缩，除去80%-90%水后用阳离子交换树脂分离纯化得到纯品中间体ARP或HRP；
    步骤（1）中所述氨基酸为丙氨酸、甘氨酸、半胱氨酸、脯氨酸中的一种或多种；
    步骤（1）中所述醛糖或酮糖为核糖、木糖、果糖中的一种或多种；
    步骤（3）中所述还原型谷胱甘肽添加量为步骤（2）中所取等体积溶液的1%~2.5%，w/v；
    步骤（7）中所述减压低温浓缩时控制温度为20~30℃，真空度为0.025~0.05MPa。
  2. 根据权利要求1所述的方法，其特征在于，步骤（2）、（3）中所述冷却时间为10~30min，反应液冷却至10℃以下即为终止反应。
  3. 根据权利要求1所述的方法，其特征在于，步骤（4）中所述第二阶段变温美拉德反应液用蒸馏水进行稀释，稀释倍数为2~50倍。
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CN112535271B (zh) * 2020-12-03 2023-12-08 安徽强旺生物工程有限公司 一种通过美拉德中间体制备减盐增鲜调味料的方法
CN113475630A (zh) * 2020-12-31 2021-10-08 江苏翼邦生物技术有限公司 一种用美拉德反应中间体制备猫用风味剂的方法
CN113735934B (zh) * 2021-09-14 2023-08-08 江南大学 一种选择性制备肽美拉德反应中间体和糖肽交联物的方法
CN115261111A (zh) * 2022-08-18 2022-11-01 合肥工业大学 一种破壁自源浓香型芝麻油的生产方法
CN116098259B (zh) * 2023-01-16 2023-11-28 江南大学 一种抑制含山梨醇-甘氨酸的无色体系黄变的方法

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