Method for inhibiting formation of heterocyclic amine in roasted mutton and maintaining flavor by using exogenous sulfur-containing amino acid

Abstract

The invention discloses a method for inhibiting formation of heterocyclic amine in roasted mutton and maintaining flavor by exogenous sulfur-containing amino acid, and belongs to the technical field of food processing.The method comprises the following steps that basic marinade is added into an aqueous solution of sulfur-containing amino acid to obtain marinade, the marinade is evenly smeared on the surface of diced meat, and the diced meat is placed in a sealed bag to be refrigerated and pickled; and after pickling is finished, baking is performed. According to the method, the specific influence of the exogenous sulfur-containing amino acid on the Maillard reaction path is researched, so that the effective inhibition of the heterocyclic amine and the good maintenance of the flavor are realized, and the effect of collaborative optimization is achieved. The method not only has significant new health value, but also has process feasibility and economic high benefits, and contributes to an innovative solution to the field of meat processing.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and particularly relates to a method for inhibiting the formation of heterocyclic amines in roasted mutton using exogenous sulfur-containing amino acids and maintaining the flavor. Background Art

[0002] At present, there have been some studies and applications aimed at controlling the formation of heterocyclic amines (HCAs) during high-temperature processing of meat products (including mutton).

[0003] First, the generation of heterocyclic amines is controlled by using exogenous substances as inhibitors. These can be categorized into three types based on their mechanism of action: 1. Antioxidants: These inhibit the formation of heterocyclic amines by blocking free radical chain reactions at multiple targets and stages. For example, antioxidants prepared by adding tea polyphenols, licorice extract, rosmarinic acid, and ultraviolet-irradiated gallic acid have reduced the generation of heterocyclic amines to a certain extent. Furthermore, Qian Zhang et al. used sea buckthorn flavonoids, which are rich in polyphenols and vitamins, to enhance their antioxidant activity and effectively inhibit the generation of heterocyclic amines. However, synthetic antioxidants are potentially toxic, and excessive intake may cause liver damage or cancer. Natural antioxidants are expensive and have poor thermal stability, making them difficult to preserve under high-temperature baking conditions. 2. Emulsifiers: Xin Wan et al. prepared a garlic diallyl disulfide (DAD) nanoemulsion that significantly reduced the levels of three HCAs (MeIQx, PhIP, and Harman) in roasted pork. Shiyi Zhu et al., through molecular docking, found that some emulsifier molecules can partially bind to the intermediates formed by PhIP, Norharman, and Harman, and can also bind water in meat products, thereby reducing the diffusion of precursors to the surface and inhibiting the production of heterocyclic amines. Although emulsifiers have a good inhibitory potential for total heterocyclic amine production, they can also promote the production of some potentially carcinogenic heterocyclic amines, and the reaction mechanism is currently unclear. 3. Natural plant extracts: Researchers have successfully extracted natural hesperidinoids using ultrasound-assisted heating and reflux technology. Compared to the uncertain inhibitory effect of polyphenols on heterocyclic amines and their negative impact on food taste, this method can stably and efficiently reduce the content of heterocyclic amines in roasted chicken. Natural extracts have advantages in terms of environmental friendliness and inhibitory efficiency, but their specific inhibitory mechanisms for HCAs remain unclear. Industrial application also faces challenges such as process complexity, safety verification, and scope of application. In addition, some plant extracts may have strong special flavors (such as bitterness and herbal flavor). If added in excess, they can easily mask or change the original characteristic aroma of roasted lamb, causing a negative impact on the overall flavor quality of the product and limiting its wide application.

[0004] Secondly, optimizing processing techniques is also a key measure for controlling HCA formation. High temperatures significantly promote the formation of HCAs. Modern technologies have been shown to reduce HCA formation by lowering the baking temperature, shortening the baking time, or modifying the heating method (such as microwave pretreatment combined with baking). However, adjusting these process parameters often fails to perfectly achieve a balanced balance of adequate product cooking, a good crust, and the desired caramelized flavor and texture that consumers desire. Alternatively, pretreatment methods such as marinating can be employed. For example, lamb can be pretreated with marinades containing specific ingredients (such as nitrites, ascorbic acid, sugars, and certain amino acids). While nitrites can effectively inhibit the formation of certain types of HCAs, their use as a food additive is subject to strict regulatory restrictions. Other marinade ingredients have varying inhibitory effects on HCAs, and their mechanisms of action are complex. They sometimes introduce new flavor compounds, but these flavors do not always contribute positively to the typical aroma of roasted lamb and may even produce unintended flavor changes.

[0005] Current research primarily focuses on the inhibitory effects of a single or limited number of additives on HCAs, or explores their single impact on flavor. However, research on substances that can simultaneously effectively inhibit HCA production and synergistically promote the formation of the characteristic aroma of roasted lamb, as well as their complex interaction mechanisms, is insufficient. Consequently, the market lacks a comprehensive technical solution that can systematically and synergistically address the two key issues of excessive heterocyclic amines and insufficient characteristic aroma in high-temperature roasted lamb. The Maillard reaction is a key chemical pathway that produces the characteristic aroma and attractive color of roasted lamb, and its products include a variety of important flavor compounds, including sulfur-containing compounds, pyrazines, and furans. However, the conditions for the Maillard reaction (such as high temperature, long heating time, suitable pH, and the presence of reducing sugars and amino compounds) are also conducive to the high-volume production of HCAs. A major challenge facing existing technologies is how to effectively promote the Maillard reaction to produce pleasant flavor compounds while inhibiting or blocking the heterocyclic amine formation pathway. Summary of the Invention

[0006] The present invention aims to provide a method for inhibiting heterocyclic amine formation and preserving the flavor of roasted lamb using exogenous sulfur-containing amino acids. This study explores the mechanism of action of sulfur-containing amino acids in inhibiting the formation of heterocyclic amines (HCAs) in roasted lamb and simultaneously investigates their effects on enhancing the meaty flavor. By optimizing the experimental design, the optimal amount of amino acids added was determined to maximize the inhibition of HCA formation while effectively preserving and enhancing the meaty flavor. Based on these findings, the present invention proposes a method for inhibiting HCA formation and preserving the flavor of roasted lamb using exogenous sulfur-containing amino acids.

[0007] A method for inhibiting the formation of heterocyclic amines in roasted mutton by using exogenous sulfur-containing amino acids and maintaining the flavor, comprising the following steps: adding a basic marinade to an aqueous solution of the sulfur-containing amino acids to obtain a marinade, evenly applying the marinade to the surface of diced meat, placing the diced meat in a sealed bag for refrigerated marination, and roasting the diced meat after the marinating is completed.

[0008] This invention leverages the compositional properties of sulfur-containing amino acids, leveraging the sulfhydryl groups in their molecular structures, to achieve the dual effects of heterocyclic amine inhibition and flavor enhancement in roasted lamb. This reduction occurs primarily through three mechanisms: competitive inhibition of precursors such as glucose and phenylalanine from participating in the Maillard reaction; chelation of metal ions to block free radical chain reactions; and direct binding to heterocyclic amine precursor intermediates to form stable complexes.

[0009] The aroma of barbecue is the result of the synergistic effects of multiple substances, primarily heterocyclic compounds (pyrazines, furans, etc.) produced by the Maillard reaction and aldehydes and ketones from the carbonization of fat. These volatile organic compounds contribute to the distinctive burnt, meaty, and sweet aromas of barbecue. Pyrazine flavor compounds, characterized by low flavor thresholds and high aroma permeability, are characteristic of barbecue aromas. These compounds include pyrazine, 2-methylpyrazine, 2,5-dimethylpyrazine, 2-ethylpyrazine, 2-ethyl-5-methylpyrazine, and 3-ethyl-2,5-dimethylpyrazine. Gas chromatography-mass spectrometry (GC-MS) analysis in this study confirmed that the addition of amino acids increases the total amount of pyrazine flavor compounds in a concentration-dependent manner. The levels of vinylpyrazine, 2,5-dimethylpyrazine, 2-methylpyrazine, and pyrazine are positively correlated with the concentration of sulfur-containing amino acids, while 2-ethylpyrazine is less affected by amino acids.

[0010] In a study of the inhibitory effect of heterocyclic amine formation in a Maillard simulated barbecue model system using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), the present invention found that the addition of different amino acids had a significant regulatory effect on the formation of PhIP (2-amino-1-methyl-6-phenylimidazole [4,5-b] pyridine), which has the highest content in barbecue products, and IQ (2-amino-3,4-dimethylimidazole [4,5-f] quinoline), which has the strongest toxicity.

[0011] Furthermore, the present invention uses HPLC-MS / MS technology to measure highly reactive short-chain dicarbonyl compounds (DCs), key precursors for the production of flavor and harmful substances. A method for the simultaneous detection of four carbonyl compounds (glyoxal, methylglyoxal, 2,3-butanedione, and 3-deoxyglucuronide) has been established. By monitoring the dynamic changes in DCs, it is possible to infer how exogenously added sulfur-containing amino acids regulate the balance between harmful substances and flavor compounds through competitive reaction pathways.

[0012] Furthermore, the sulfur-containing amino acid is L-cysteine.

[0013] Furthermore, the usage ratio of the sulfur-containing amino acid to water is (4-20) mg:1 mL.

[0014] Furthermore, the ratio of the sulfur-containing amino acid to water is 20 mg:1 mL.

[0015] Furthermore, the basic marinade comprises salt, monosodium glutamate and potato starch.

[0016] Furthermore, the usage ratio of the salt, MSG, potato starch powder and water is 5g:2g:8g:10mL.

[0017] Furthermore, 5 mL of the marinade is applied to every 500 g of diced meat.

[0018] Furthermore, the temperature of the refrigerated pickling is 4° C. and the time is 1-2 hours.

[0019] Furthermore, the specific operation of baking is: cut the marinated diced meat and spread it flat on tin foil, preheat the oven to 180℃ for 10 minutes, spread the tin foil flat on the baking tray, bake it in the upper and lower fire mode for 10 minutes, then turn it over and continue baking for 10 minutes, take it out and let it stand at room temperature for 3 minutes.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] By studying the specific effects of exogenous sulfur-containing amino acids on the Maillard reaction pathway, this method effectively inhibits heterocyclic amines while maintaining flavor, achieving a synergistic optimization effect. This method not only offers significant new health benefits but also combines technological feasibility with high economic efficiency, contributing an innovative solution to the meat processing industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 The effect of different amounts of exogenous L-cysteine ​​added on PhIP in Example 1;

[0024] Figure 2 The effect of different amounts of exogenous L-cysteine ​​added on IQ in Example 1;

[0025] Figure 3The effect of different amounts of exogenous L-cysteine ​​added on pyrazine flavor substances in Example 2;

[0026] Figure 4 The effect of different amounts of exogenous L-cysteine ​​added on 2,3-butanedione in Example 3;

[0027] Figure 5 The effect of different amounts of exogenous L-cysteine ​​added on glyoxal in Example 3;

[0028] Figure 6 The effect of different amounts of exogenous L-cysteine ​​added on methylglyoxal in Example 3;

[0029] Figure 7 The effect of different amounts of exogenous L-cysteine ​​added on 3-deoxyglucosone in Example 3;

[0030] Figure 8 This is the effect of different amounts of exogenous L-cysteine ​​added to the actual roasted lamb system in Application Example 1 on PhIP;

[0031] Figure 9 The effect of different amounts of exogenous L-cysteine ​​added to the actual roasted lamb system in Application Example 1 on IQ;

[0032] Figure 10 This is the effect of different amounts of exogenous L-cysteine ​​added to the actual roasted lamb system in Application Example 1 on pyrazine flavor substances. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] The room temperature in the present invention refers to 25±2°C.

[0039] Example 1

[0040] The inhibitory effect of sulfur-containing amino acids on heterocyclic amines (PhIP and IQ) was investigated by constructing a Maillard reaction model system. In this example, the sulfur-containing amino acid was L-cysteine, and the extraction column was an Oasis MCX 3CC (60 mg) solid phase extraction column. The specific operation was as follows:

[0041] To a 20 mL pressure bottle containing 10 mL of diethylene glycol aqueous solution (86 v / v%), 0.3 mmol phenylalanine, 0.2 mmol creatinine, and 0.15 mmol glucose were added. After alternating magnetic stirring and ultrasonic mixing and dissolution, a uniform Maillard reaction model system was formed. The pH of the reaction system was adjusted to 6.5 with 0.1 mol / L K2HPO4 / KH2PO4. The reaction solution was always kept at room temperature during the operation. Five parallel preparations were made, and different masses of sulfur-containing amino acids (0 mg, 20 mg, 40 mg, 100 mg, and 200 mg) were added. After heating at 230°C for 30 min, the pressure bottle was immediately removed and placed in an ice-water bath to cool to terminate the reaction. The sample was then transferred to a 50 mL centrifuge tube and stored below -20°C for the determination of heterocyclic amines in the Maillard simulation system.

[0042] In a 50 mL centrifuge tube, 5 mL of the above reaction solution and 5 mL of sodium hydroxide aqueous solution (2 mol / L) were added in sequence. After vortex mixing, 20 mL of ethyl acetate was added and extraction was performed by ultrasonication for 15 min. The extraction process was repeated twice. The mixture was allowed to stand for 10 min to allow for separation of layers. The supernatant was used for solid phase extraction.

[0043] First, activate the extraction column by sequentially adding 6 mL of methanol, 6 mL of distilled water, and 6 mL of ethyl acetate. Then, inject 10 mL of the supernatant into the solid-phase extraction column. After loading, add 6 mL of hydrochloric acid (0.1 mol / L) and methanol for elution. Finally, replace with ammoniated methanol (methanol: ammonia water = 19:1, v / v, mL) for elution. Dry the eluate with nitrogen at 50°C. After complete drying, reconstitute it with 400 μL of methanol, vortex mix, and filter through a 0.22 μm organic filter for analysis.

[0044] The present invention uses an Acquity ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer to determine the content of a target compound, and separates the target compound using an Acquity BEH C18 chromatographic column (100 mm×2.1 mm, 1.7 μm). The column temperature is set at 35° C., the liquid phase flow rate is 0.3000 mL / min, a 5 mmol ammonium acetate-water solution is used as mobile phase A, and acetonitrile is used as mobile phase B for separation of heterocyclic amines. The injection volume is 2 μL, and the elution gradient is: 0.0-0.1 min: 10% B; 0.1-7 min: 60% B; 7-10 min: 100% B; 10-11 min: 10% B; 11-11.10 min: 10% B.

[0045] Data were collected using a triple quadrupole mass spectrometer with multiple reaction monitoring (MRM) in positive ion mode (ESI+) using an electrospray ionization source. The ESI+ parameters were as follows: curtain pressure: 2.05×10 5 Pa; ion spray voltage: 5500 V; atomization temperature: 550 ° C; atomization pressure: 3.79 × 10 5 Pa, auxiliary gas pressure: 4.14×10 5 Pa, ion source Gas1: 55°C; ion source Gas2: 55°C.

[0046] The mass-to-charge ratio of the parent-daughter ion pair of PhIP was 225.0→210.1 for qualitative analysis and 225.0→140.2 for quantitative analysis. The fragmentor voltage was 120 V, the collision energy was 30 eV for qualitative analysis and 40 eV for quantitative analysis, and the retention time was 3.02±0.5 min.

[0047] The mass-to-charge ratio of the parent and daughter ion pairs of IQ was 199.1→184.0.0 for qualitative analysis and 199.1→156.9 for quantitative analysis. The fragmentor voltage was 100 V, the collision energy was 38 eV for qualitative analysis and 46 eV for quantitative analysis, and the retention time was 1.08±0.5 min.

[0048] Figure 1 The effect of different amounts of exogenous L-cysteine ​​added on PhIP in Example 1; Figure 2 The figure shows the effects of different amounts of exogenous L-cysteine ​​added on IQ in Example 1.

[0049] from Figure 1 It can be seen from the results that when the addition amount was 100 mg, the amount of PhIP generated was 7.65 ng / mL, which was 81.3% lower (P<0.05) than the blank group without sulfur-containing amino acids (the amount of PhIP generated was 40.93 ng / mL). When the addition amount was increased to 200 mg, the inhibition rate of PhIP did not increase significantly, indicating that the inhibitory effect of L-cysteine ​​on PhIP may have a dose saturation effect. Figure 2 As can be seen in the results, L-cysteine ​​also significantly inhibits IQ production. For example, when 200 mg of L-cysteine ​​was added, the inhibition rate on IQ reached 61.9% (the IQ production in the blank group was 3.23 ng / mL, and when 200 mg of L-cysteine ​​was added, the IQ production was 1.23 ng / mL). The inhibitory mechanism of L-cysteine ​​on different heterocyclic amines is related to its molecular structure and reaction pathway selectivity.

[0050] Example 2

[0051] The promoting effect of sulfur-containing amino acids on volatile flavor substances (pyrazine) was investigated by constructing a Maillard reaction model system. In this embodiment, the sulfur-containing amino acid was L-cysteine. The specific operation was as follows:

[0052] To a 20 mL pressure bottle containing 10 mL of diethylene glycol aqueous solution (86 v / v%), 0.3 mmol phenylalanine, 0.2 mmol creatinine, and 0.15 mmol glucose were added. After alternating magnetic stirring and ultrasonic mixing and dissolution, a uniform Maillard reaction model system was formed. The pH of the reaction system was adjusted to 6.5 with 0.1 mol / L K2HPO4 / KH2PO4. The reaction solution was always kept at room temperature during the operation. Five parallel preparations were added with different masses of sulfur-containing amino acids (0 mg, 20 mg, 40 mg, 100 mg, and 200 mg). The mixture was heated at 230°C for 30 min, then the pressure bottle was immediately removed and placed in an ice-water bath to cool to stop the reaction. The volatile flavor compounds in the Maillard reaction model system were analyzed.

[0053] Volatile flavor components were extracted using headspace solid-phase microextraction (HS-SPME) using an SPME fiber (50 / 30 μm DVB / CAR / BOXEN-PDMS, Supelco). 5 mL of the reaction solution was placed in a 15 mL headspace vial, and 50 μL of 2-methyl-3-heptanone (200 ng / mL) as an internal standard was added and mixed. The vial was sealed with a PTFE / BYTL septum and allowed to stand for 15 minutes. The headspace vial containing the sample solution was placed in a constant temperature water bath (70°C) and equilibrated for 3 minutes. To prevent contamination of the SPME fiber with volatile components from air, the gas chromatograph inlet was set to 250°C. The extraction fiber was inserted and conditioned at high temperature for 30 minutes. After removing interference, the extraction fiber was suspended above the sample to adsorb the volatile components. After adsorption was complete, the extraction fiber was retracted and transferred to the front inlet of the gas chromatograph for 3 minutes of desorption.

[0054] A DB-5MS capillary column (30 m × 0.25 mm × 0.25 μm) was used. The temperature program was as follows: initial oven temperature at 40°C, held for 2 minutes, then increased at a rate of 10°C / min to 100°C, held for 5 minutes, then increased at the same rate to 220°C, held for 15 minutes, and finally increased at a rate of 20°C / min to 270°C. Splitless mode was used, with an inlet temperature of 270°C and a pressure of 7.0 psi. High-purity helium was used as the carrier gas at a constant flow rate of 1 mL / min. The SPME fiber was desorbed at the inlet at 250°C for 3 minutes.

[0055] GC-MS was performed with an electron impact energy of 70 eV and an EI+ ionization mode. The mass spectrometer was operated in full scan mode (SCAN) with a mass range of 30–450 m / z and a solvent delay of 3 min. The ion source temperature was 260°C, and the transfer line temperature was 280°C.

[0056] The quantitative ion and reference ion of pyrazine were 80.1 and 53.0, respectively, with a retention time of 3.048 ± 0.5 min;

[0057] The quantitative ion and reference ion of 2-methylpyrazine were 94.1 and 67.0, respectively, with a retention time of 4.501 ± 0.5 min;

[0058] The quantitative ion and reference ion of 2-ethylpyrazine were 107.1 and 80.1, respectively, with a retention time of 6.150 ± 0.5 min;

[0059] The quantitative ion and reference ion of 2,5-dimethylpyrazine were 108.1 and 42.0, respectively, with a retention time of 6.157 ± 0.5 min;

[0060] The quantitative ion and reference ion of vinylpyrazine were 106.1 and 52.1, respectively, with a retention time of 6.444 ± 0.5 min;

[0061] Figure 3 The effect of different amounts of exogenous L-cysteine ​​added on pyrazine flavor substances in Example 2; Figure 3 It can be seen that as the amount of L-cysteine ​​added increases, the amount of pyrazine flavor substances produced also increases.

[0062] Example 3

[0063] The effect of sulfur-containing amino acids on carbonyl compounds (glyoxal, methylglyoxal, 2,3-butanedione, and 3-deoxyglucosone) was investigated by constructing a Maillard reaction model system. In this example, the sulfur-containing amino acid was L-cysteine, and the specific operation was as follows:

[0064] To a 20 mL pressure bottle containing 10 mL of diethylene glycol aqueous solution (86 v / v%), 0.3 mmol of phenylalanine, 0.2 mmol of creatinine, and 0.15 mmol of glucose were added. After alternating magnetic stirring and ultrasonic mixing and dissolution, a uniform Maillard reaction model system was formed. The pH of the reaction system was adjusted to 6.5 using 0.1 mol / L K2HPO4 / KH2PO4. During the operation, the reaction solution was always kept at room temperature. Different amounts of sulfur-containing amino acids (0 mg, 20 mg, 40 mg, 100 mg, and 200 mg) were added. After heating at 230°C for 30 min, the pressure bottle was immediately removed and cooled in an ice-water bath to stop the reaction. The α-dicarbonyl compounds in the Maillard reaction model system were analyzed.

[0065] Prepare DNPH (dinitrophenylhydrazone) derivatization solution by weighing 65 mg of DNPH and dissolving it in 3 mL of hydrochloric acid (1 mol / L). Transfer the solution to a 50 mL volumetric flask and dilute to the mark with acetonitrile. Store at room temperature and protect from light. Dilute the reaction solution of the above Maillard reaction model system (please provide the dilution factor for a 10-fold dilution) to obtain a reaction dilution solution. Take 800 μL of the reaction dilution solution, add 50 μL of DNPH derivatization solution, and then add 120 μL of hydrochloric acid (1 mol / L). Vortex evenly, store at room temperature and protect from light for 5 h. After the derivatization reaction is complete, filter through a 0.22 μm organic filter membrane and analyze by UPLC-MS / MS.

[0066] The target compounds were determined using an Acquity ultra-high performance liquid chromatography coupled with a triple quadrupole mass spectrometer. Separation of the target compounds was performed on an Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 μm). The column temperature was set at 35°C and the liquid phase flow rate was 0.350 mL / min. Mobile phase A was 0.2% formic acid in water, and mobile phase B was acetonitrile. The elution gradient was set as follows: 0.0–0.1 min: 10% B; 0.1–7 min: 60% B; 7–10 min: 100% B; 10–11 min: 10% B; and 11–11.10 min: 10% B.

[0067] Data were collected using a triple quadrupole mass spectrometer with multiple reaction monitoring (MRM) in negative ion mode (ESI-) using an electrospray ionization source. The ESI parameters were as follows: a curtain gas pressure of 2.05 × 10 5 Pa, ion spray voltage 5500 V; atomization temperature 550 ° C; atomization pressure 3.79 × 10 5 Pa, auxiliary gas pressure 4.14×10 5 Pa, ion source Gas1: 55℃, ion source Gas2: 55℃.

[0068] The mass-to-charge ratio of the parent and daughter ion pairs of glyoxal was 417.0→234.0 for qualitative analysis and 417.0→182.0 for quantitative analysis. The fragmentor voltage was 40 V, the collision energy was 20 eV for qualitative analysis and 30 eV for quantitative analysis, and the retention time was 7.206±0.5 min.

[0069] The mass-to-charge ratio of the parent and daughter ion pairs of methylglyoxal was 431.1→248.0 for qualitative analysis and 431.1→182.0 for quantitative analysis. The fragmentor voltage was 50 V, the collision energy was 20 eV for qualitative analysis and 30 eV for quantitative analysis, and the retention time was 8.120±0.5 min.

[0070] The mass-to-charge ratio of the parent and daughter ion pairs of 2,3-butanedione was 264.8→181.0 for qualitative analysis and 264.8→109.0 for quantitative analysis. The fragmentor voltage was 60 V, the collision energy was 30 eV for qualitative analysis and 40 eV for quantitative analysis, and the retention time was 7.206±0.5 min.

[0071] The mass-to-charge ratio of the parent-daughter ion pair of 3-deoxyglucosone was 521.3→431.1 for qualitative analysis and 521.3→182.0 for quantitative analysis. The fragmentation voltage was 70 V, the collision energy was 20 eV for qualitative analysis and 30 eV for quantitative analysis, and the retention time was 7.206±0.5 min.

[0072] Figure 4 The effect of different amounts of exogenous L-cysteine ​​added on 2,3-butanedione in Example 3; Figure 5 The effect of different amounts of exogenous L-cysteine ​​added on glyoxal in Example 3; Figure 6 The effect of different amounts of exogenous L-cysteine ​​added on methylglyoxal in Example 3; Figure 7 The figure shows the effect of different amounts of exogenous L-cysteine ​​added on 3-deoxyglucosone in Example 3.

[0073] according to Figure 4 It can be seen that the addition of exogenous L-cysteine ​​will aggravate the production of 2,3-butanedione to a certain extent; Figure 5 It can be seen that as the amount of exogenous L-cysteine ​​added increases, the amount of glyoxal generated decreases; according to Figure 6 It can be seen that as the amount of exogenous L-cysteine ​​added increases, the amount of methylglyoxal generated decreases; according to Figure 7 It can be seen that the addition of exogenous L-cysteine ​​promotes the formation of 3-deoxyglucosone to a certain extent. The reason why the addition of exogenous L-cysteine ​​has different effects on the formation of different carbonyl compounds is that the carboxyl (-COOH) and sulfhydryl (-SH) groups of cysteine ​​can lower the pH value of the system. The acidic environment promotes the formation of 3-DG through the 1,2-enolization pathway of the Amadori product. In addition, the sulfhydryl (-SH) group of cysteine ​​has strong nucleophilicity and easily undergoes addition reactions with the carbonyl groups of glyoxal and methylglyoxal to form stable sulfur-containing heterocyclic compounds, thereby consuming free dicarbonyl compounds.

[0074] Application Example 1 Practical Application of Sulfur-Containing Amino Acids in Roast Lamb

[0075] (1) Raw material pretreatment

[0076] Cut the lamb into triangular pieces (about 1.5cm on each side);

[0077] (2) Sulfur-containing amino acid treatment group

[0078] L-cysteine ​​was selected as the sulfur-containing amino acid, and five gradient concentrations of 0 mg (blank control), 20 mg, 40 mg, 100 mg, and 200 mg were set, respectively, dissolved in an equal amount of marinade (5 mL of water), and basic marinade (5 g of salt, 2 g of monosodium glutamate, and 8 g of potato starch) were added to obtain marinade;

[0079] (3) Pickling process

[0080] Spread 5 mL of the above marinade evenly on the surface of 500 g of diced meat, place it in a sealed bag and refrigerate (4°C) for 1 hour, turning it over twice during the marinade to ensure uniform penetration.

[0081] (4) Baking operation

[0082] After marinating, accurately weigh 10g of diced meat from each treatment group (three replicates, totaling 30g) and spread it flat on tin foil. Preheat the oven to 180°C for 10 minutes. Place the tin foil flat on a baking sheet and bake for 10 minutes using both top and bottom heat. Turn the tin foil over and continue baking for another 10 minutes. Remove the tin foil and let it rest at room temperature for 3 minutes.

[0083] (5) Detection of heterocyclic amines

[0084] Pretreatment: 2 g of the roasted lamb was ground using a grinder. The sample was accurately weighed and mixed with 5 mL of 2 mol / L sodium hydroxide solution. After homogenization for 1 min, 14 g of diatomaceous earth was added and stirred thoroughly. 20 mL of ethyl acetate was added to the mixture, and ultrasonic extraction was performed for 30 min. This extraction was repeated twice to obtain an extract. 10 mL of the extract was loaded onto a Waters Oasis MCX solid-phase extraction cartridge pre-activated with 6 mL of methanol, 6 mL of water, and 6 mL of ethyl acetate, respectively. The cartridge was then rinsed with 6 mL of 0.1 mol / L hydrochloric acid and 6 mL of methanol, respectively, and eluted with 6 mL of ammoniacal methanol (methanol:ammonia water = 19:1, v / v, mL). The resulting eluate was dried with nitrogen and reconstituted with 400 μL of methanol. The reconstituted solution was filtered through a 0.22 μm filter membrane and then loaded for analysis.

[0085] The detection method is the same as in Example 1;

[0086] Figure 8 The effect of different amounts of exogenous L-cysteine ​​added to the actual roasted lamb system in Application Example 1 on PhIP was studied. Figure 8 It can be seen that as the amount of exogenous L-cysteine ​​added increases, the amount of PhIP generated decreases. Figure 1 The differences in trends are primarily due to the complexity and multi-component interactions of the actual baking system, as well as the difficulty in extracting PhIP from real samples. In the simulated system, the reaction pathway is simple, cysteine ​​thermal decomposition is minimal, and its inhibitory effect is primarily dependent on the stoichiometric ratio.

[0087] Figure 9 The effect of different amounts of exogenous L-cysteine ​​added to the actual roasted lamb system in Application Example 1 on IQ; Figure 9 It can be seen that as the amount of exogenous L-cysteine ​​added increases, the amount of IQ generated decreases.

[0088] (6) Flavor substance detection

[0089] 5 g of sample was placed in a 15 mL headspace vial, 50 μL of 2-methyl-3-heptanone (200 ng / mL) as internal standard was added and mixed, the vial was sealed and equilibrated in a 70 °C water bath for 3 min, and headspace extraction was performed using an activated (250 °C for 20 min) extraction needle for 30 min. The extraction needle was transferred to the front inlet of the gas chromatography-mass spectrometer and analyzed for 3 min.

[0090] The detection method is the same as in Example 2.

[0091] Figure 10 The effect of different amounts of exogenous L-cysteine ​​added to the actual roasted lamb system in Application Example 1 on the pyrazine flavor substances was studied. Figure 10 It can be seen from the results that in the actual roasted lamb system, with the increase of the amount of exogenous L-cysteine ​​added, the content of pyrazine flavor substances is significantly improved.

[0092] Through the above experiments, it can be found that applying sulfur-containing amino acids to roasted lamb can significantly inhibit the formation of heterocyclic amines while maintaining the original barbecue aroma, thereby reducing the risk of cancer and improving food safety.

[0093] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for inhibiting the formation of heterocyclic amines in roasted lamb and maintaining the flavor by using exogenous sulfur-containing amino acids, characterized in that: The following steps are involved: A basic marinade is added to an aqueous solution of sulfur-containing amino acids to obtain a marinade, the marinade is evenly spread on the surface of the diced meat, the meat is placed in a sealed bag and refrigerated for marination, and the meat is roasted after the marination is completed.

2. The method of claim 1, wherein the exogenous sulfur-containing amino acid is used to inhibit the formation of heterocyclic amines in roasted lamb and maintain the flavor. The sulfur-containing amino acid is L-cysteine.

3. The method of suppressing the formation of heterocyclic amines in roasted mutton and maintaining the flavor by using exogenous sulfur-containing amino acids according to claim 2, characterized in that: The ratio of the sulfur-containing amino acid to water in the sulfur-containing amino acid aqueous solution is (4-20) mg:1 mL.

4. The method of suppressing the formation of heterocyclic amines in roasted mutton and maintaining the flavor by using exogenous sulfur-containing amino acids according to claim 3, characterized in that: The ratio of the sulfur-containing amino acid to water in the sulfur-containing amino acid aqueous solution is 20 mg:1 mL.

5. The method of suppressing the formation of heterocyclic amines in roasted mutton and maintaining the flavor by using exogenous sulfur-containing amino acids according to claim 1, characterized in that: The basic marinade comprises salt, monosodium glutamate and potato starch.

6. The method of suppressing the formation of heterocyclic amines in roasted mutton and maintaining the flavor by using exogenous sulfur-containing amino acids according to claim 5, characterized in that: The usage ratio of the salt, monosodium glutamate, potato starch powder and water is 5g:2g:8g:10mL.

7. The method of claim 1 wherein the exogenous sulfur-containing amino acid is used to inhibit the formation of heterocyclic amines in roasted mutton and maintain the flavor, wherein: Apply 5 mL of the marinade to every 500 g of diced meat.

8. The method of claim 1, wherein the exogenous sulfur-containing amino acid is used to inhibit the formation of heterocyclic amines in roasted lamb and maintain the flavor, wherein the refrigerated pickling temperature is 4°C and the pickling time is 1-2 hours.

9. The method of claim 1 wherein the exogenous sulfur-containing amino acid is used to inhibit the formation of heterocyclic amines in roasted lamb and maintain the flavor, wherein: The specific operation of the baking is: cut the marinated diced meat and spread it flat on tin foil, preheat the oven to 180°C for 10 minutes, spread the tin foil flat on the baking tray, bake in the upper and lower fire mode for 10 minutes, then turn it over and continue baking for 10 minutes, take it out and let it stand at room temperature for 3 minutes.

Images