CN112557361A

CN112557361A - Method for analyzing heterocyclic amine formation mechanism by using dry-hot soybean protein isolate as model

Info

Publication number: CN112557361A
Application number: CN202011389930.2A
Authority: CN
Inventors: 席俊; 陈阳; 付杨; 陈慧彬; 汪学德; 马宇翔
Original assignee: Henan University of Technology
Current assignee: Henan University of Technology
Priority date: 2020-12-02
Filing date: 2020-12-02
Publication date: 2021-03-26
Anticipated expiration: 2040-12-02
Also published as: CN112557361B

Abstract

The invention relates to a method for analyzing a heterocyclic amine forming mechanism by using dry-hot soybean protein isolate as a model, which comprises the following steps: carrying out dry heat treatment on the isolated soy protein; measuring the content of heterocyclic amine; analyzing the protein change; analyzing the secondary structure and the tertiary structure of the protein; analyzing the degree of oxidation of the protein; analyzing the degradation degree of the protein; analyzing the content change of formaldehyde and acetaldehyde in the protein pyrolysis process; the protein system was evaluated for changes in oxidation resistance. The method can simultaneously detect the correlation between the heterocyclic amine content after the dry-hot soybean protein isolate and the physicochemical property after the protein heat treatment, has complete experimental method and important academic significance, and simultaneously has guiding significance for reasonably adding the soybean protein isolate in the food industry and properly processing the food quality safety problem facing the development of foods taking the soybean protein isolate as a main raw material.

Description

Method for analyzing heterocyclic amine formation mechanism by using dry-hot soybean protein isolate as model

Technical Field

The invention relates to a method for analyzing a heterocyclic amine formation mechanism by using dry-hot soybean protein isolate as a model, belonging to the technical field of food processing.

Background

The heat transfer is an important purpose of food heat processing, and can achieve the effects of sterilization, curing, unique flavor providing and the like. It is generally considered that a high protein food containing not less than 12 g of protein per 100 g of solid food is a high protein food, and during thermal processing, many hazards including heterocyclic amines (HAAs) are generated due to changes in oxidation, degradation, aggregation, etc. of proteins at high temperatures. Toxicology experiments show that the toxicity of heterocyclic amine is far beyond that of typical carcinogens, namely aflatoxin B1, polycyclic aromatic hydrocarbon, nitrite and the like. More than 30 heterocyclic amines are currently isolated from food substrates. The international agency for research on cancer IARC classified 12 heterocyclic amines as group 2B carcinogens and IQ as group 2A carcinogens. The basis for mutagenesis of heterocyclic amines has been well characterized, and cytochrome P450 enzymes convert heterocyclic amines to hydroxylamines and then to the corresponding esters by N-acetyltransferases or sulfotransferases. These ester instabilities can spontaneously hydrolyze to produce reactive aryl nitrogen ions that covalently bind to DNA, and heterocyclic amine-DNA adducts are prone to frameshift and point mutations.

Soybean products are one of the major food products in many asian countries, especially china and japan, and after the concept of meat analogue has been proposed, the trend of using vegetable proteins such as soybean protein as an animal protein substitute is more vigorous. It has been previously reported that pyrolysis of cured soybean cakes and soy protein can produce HAAs. However, structural modification of plant proteins and chemical reactions at high temperatures are still poorly understood in terms of factors affecting HAAs formation, and the change in heterocyclic amine formation during heat treatment of proteins has not been studied systematically. The generation mechanism of the prior heterocyclic amine is mainly researched by two modes, one mode is to prepare solutions of amino acid-reducing sugar-creatine (anhydride) and the like or dry heat according to proportion, and the defects are that heterocyclic amine precursor substances such as amino acid and the like are consumed in one-dimensional direction in the heating process and are not consistent with the changes of protein decomposition, amino acid supplement and the like in the real food thermal processing process; one of the various thermal processes for using real meat products has the disadvantage that the real food system has complex ingredients and limits on exploring the mechanism of heterocyclic amine generation. Therefore, it is necessary to study the mechanism of heterocyclic amine formation using soybean protein isolate as a heterocyclic precursor.

Disclosure of Invention

In view of the deficiencies of the prior art, it is an object of the present invention to provide a method for analyzing the mechanism of heterocyclic amine formation using dry-hot soy protein isolate as a model.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for analyzing a heterocyclic amine formation mechanism by using dry-hot soy protein isolate as a model, comprising the following steps:

(1) carrying out dry heat treatment on the isolated soy protein, and measuring the concentration of soluble protein of the dry heat isolated soy protein;

(2) performing acid hydrolysis on the dry-hot soybean protein isolate to obtain heterocyclic amine; purifying heterocyclic amine by combining a solid phase extraction method, concentrating, and determining the content of the heterocyclic amine by adopting a liquid chromatography-mass spectrometer;

(3) analyzing changes in the dry-hot soy protein isolate using an SDS-PAGE gel;

(4) analyzing the secondary structure of the dry-hot soybean protein isolate by using circular dichroism spectroscopy, and analyzing the tertiary structure of the dry-hot soybean protein isolate by using a fluorescence spectrophotometer;

(5) analyzing the oxidation degree of the dry-hot soybean protein isolate by using a fluorescence spectrophotometer;

(6) analyzing the degradation degree of the dry-hot soybean protein isolate by using an ultraviolet-visible spectrophotometer;

(7) analyzing the content change of formaldehyde and acetaldehyde in the pyrolysis process of the dry-heat soybean protein isolate by using high performance liquid chromatography;

(8) the antioxidant change of the dry-hot soy protein isolate system was evaluated using scavenging PTIO free radicals.

The step (1) dry heat treatment is carried out in a forced air drying oven.

6M hydrochloric acid is selected for acid hydrolysis in the step (2); during solid phase extraction, the eluent is blown dry by nitrogen at 35 ℃.

The step (3) uses fluorescein-5-thiosemicarbazide to mark carbonyl protein to visualize the degree of protein oxidation and oxidation sites.

The analysis method in the step (4) comprises the following steps: analyzing the endogenous fluorescence spectrum by using a fluorescence spectrophotometer to obtain the maximum fluorescence intensity and the wavelength of the maximum fluorescence; the CD spectrum in the far ultraviolet region was measured using a circular dichroism spectrometer.

The analysis method in the step (5) comprises the following steps: the content of conjugated schiff base and N-formyl kynurenine was determined using a fluorescence spectrophotometer.

The analysis method in the step (6) comprises the following steps: through the reaction of ninhydrin and amino, a total amino standard curve with alanine as a standard is established, and the content change of the total amino in the dry-hot soybean protein isolate is determined.

The analysis method in the step (7) comprises the following steps: deriving formaldehyde and acetaldehyde by using 2, 4-dinitrophenylhydrazine, separating the formaldehyde and the acetaldehyde by using high performance liquid chromatography at 360nm, and quantitatively analyzing the content change of the formaldehyde and the acetaldehyde in the pyrolysis process of the dry-hot soybean protein isolate.

The analysis method in the step (8) comprises the following steps: and (3) establishing a PTIO standard curve and a Trolox PTIO free radical eliminating standard curve, and measuring the oxidation resistance of the dry-hot soybean protein isolate system.

The use of the process for the preparation of a thermally processed food product using soy protein isolate.

The invention has the beneficial effects that:

protein is the material basis of all life, is an important component of body cells, and is the main raw material for human tissue renewal and repair. The human body is supplemented with proteins by ingested plant and animal food, which have similar regular changes during heat processing. The dry-hot soybean protein isolate model established by the method has very important academic research significance for understanding and researching the generation mechanism of heterocyclic amine and the heat treatment change of protein products in the heat processing process.

The invention finds that the protein solubility is reduced, the oxidation is accelerated, the carbonyl compound is increased along with the increase of the heat treatment temperature, and the correlation is provided with the increase of the heterocyclic amine content. The method determines the highest temperature applied to the dry-hot soybean protein isolate, and the optimal experimental condition is heat treatment at 170 ℃ for 10min or heat treatment at 150 ℃ for 60min, under the condition, the content of the heterocyclic amine is rapidly increased due to the pyrolysis of the protein into the carbonyl compound-rich small molecular protein and peptide, so that the method can be used for researching the formation of the HAAs after heat treatment and the relation between the protein structure and the chemical reaction, and can also be used for screening the heterocyclic amine inhibitor.

The method for analyzing the formation mechanism of heterocyclic amine has guiding significance for reasonably using the soybean protein isolate as the main raw material or ingredient of the hot processed food, and simultaneously has important guiding significance for reasonably adding the soybean protein isolate in the food industry and properly processing the food quality safety problem facing the development of the food taking the soybean protein isolate as the main raw material.

Drawings

FIG. 1 is a standard curve for the determination of soluble protein concentration using bovine serum albumin in example 1;

FIG. 2 is a soluble protein concentration of the dry heat-treated soy protein isolate of example 1;

FIG. 3 is the content of each heterocyclic amine in the dry-heated soy protein isolate of example 2;

FIG. 4 is a gel electrophoresis of the dry-heated soy protein isolate of example 3;

wherein: a, heat treatment is carried out for 10 min; b, heat treatment is carried out for 60 min; c, carrying out heat treatment on the FTC mark for 10 min; d, carrying out FTC marking heat treatment for 60 min; e, staining the gel C by using Coomassie brilliant blue R250 after fluorescence photographing; f, staining the gel D with Coomassie brilliant blue R250 after fluorescent photographing; lane M, standard molecular weight protein; lane 1, unheated soy protein isolate;

lane

2, 90 ℃;

lane

3, 110 ℃;

lane

4, 130 ℃;

lane

5, 150 ℃;

lane

6, 170 ℃;

lane

7, 190 ℃;

FIG. 5 is a graph showing the change in the tertiary structure of the dry-heated soybean protein isolate of example 4;

FIG. 6 is a graph showing the secondary structure change of the dry-heated soybean protein isolate of example 4;

FIG. 7 is a graph showing the change in the content of conjugated Schiff base in the dry-heated soy protein isolate of example 5;

FIG. 8 is a graph showing the variation of the N-formylkynurenine content of the dry-heated soybean protein isolate of example 5;

FIG. 9 is a standard curve for amino group determination using ninhydrin in example 6;

FIG. 10 is a graph showing the change in the degree of degradation of the dry-heated soy protein isolate of example 6;

FIG. 11 is a diagram of the separation of formaldehyde and acetaldehyde standard by HPLC in example 7;

FIG. 12 is a standard curve of the HPLC quantitative analysis of formaldehyde and acetaldehyde in example 7;

FIG. 13 is a graph showing the change in the contents of formaldehyde and acetaldehyde in the dry-heated soy protein isolate of example 7;

FIG. 14 is a standard curve for PTIO, a standard curve for Trolox scavenging PTIO radicals in example 8;

FIG. 15 is a graph showing the measurement of antioxidant capacity of the dry-heated soybean protein isolate in example 8;

FIG. 16 is a graph showing the correlation analysis of experimental data of examples 1 to 8.

Detailed Description

The following examples further illustrate the embodiments of the present invention in detail.

Example 1

In order to determine the optimum condition suitable for simultaneously analyzing the physicochemical property of the protein and the generation content change of heterocyclic amine, the dry heat treatment condition of the soybean protein isolate is designed. Weighing 5g of DuPont 603 soybean protein isolate, placing in a glass culture dish with a diameter of 10cm, placing in a forced air drying oven with constant temperature of 30min, heating at 90-230 deg.C for 10min and 60min respectively, taking out the culture dish after the completion, naturally cooling, placing in a sealed bag, and storing in a refrigerator at-20 deg.C for use.

Since the color change of the dry-heated soy protein isolate (the soy protein isolate after the dry-heat treatment) is obvious, the L, a and b values of the dry-heated soy protein isolate are measured.

The value of L represents the shade (black and white), a represents the red and green, b represents the yellow and blue, where L: if the value is positive, the sample is brighter than the standard plate; if the value is negative, the sample is darker than the standard plate; a: if the value is positive, the sample is more red than the standard plate, and if the value is negative, the sample is more green than the standard plate; b: if the value is positive, the sample is more yellow than the standard plate, and if the value is negative, the sample is more blue than the standard plate.

As shown in tables 1 to 3, the results revealed that the L value of the protein powder decreased, the a value increased, and the b value increased with the increase of the heat treatment time (10min to 60min) or the heat treatment temperature (90 ℃ to 230 ℃), which indicates that melanoid formation is promoted by the Maillard reaction.

The soluble protein concentration was measured by the Bradford method using Bovine Serum Albumin (BSA) as a standard protein, and the configuration was as shown in Table 4, and the obtained standard curve is shown in FIG. 1.

When the dry-heated soy protein isolate samples were measured at the respective treatment temperatures, the mass concentration of the sample was set to 10mg/mL, the sample was sufficiently dissolved in a vortex mixer, 10. mu.L of the sample was centrifuged at 10000rpm, and the soluble protein concentration was measured, and the results are shown in FIG. 2.

As can be seen from fig. 2, as the heat treatment temperature or time of the soy protein isolate increased, the protein solubility decreased significantly.

TABLE 1L value of dry-hot soy protein isolate

TABLE 2 a-value of the dry-hot soy protein isolate

TABLE 3 b-value of the dry-hot soy protein isolate

TABLE 4 creation of standard curve of bovine serum albumin concentration

Pipe number	1	2	3	4	5	6	7	8
									1μg/μL BSA(μL)	0	10	20	40	60	80	100	120
PBS(μL)	1000	990	980	960	940	920	900	880
									BSA concentration (μ g/mL)	0	10	20	40	60	80	100	120
Coomassie brilliant blue G250(mL)	5	5	5	5	5	5	5	5

Example 2

Performing acid hydrolysis on the dry-hot soybean protein isolate obtained in the example 1 to obtain heterocyclic amine, and purifying and concentrating the heterocyclic amine by combining a solid phase extraction method; and measuring the content of each heterocyclic amine in the dry-hot soybean protein isolate by adopting a liquid chromatography-mass spectrometer, wherein the experimental result is shown in figure 3, wherein the ordinate of the figure 3 represents the content of the heterocyclic amine, and the unit is ng/g.

Accurately weighing 1g of each dry-hot soybean protein isolate sample subjected to heat treatment at 90-230 ℃ in example 1, mixing the dry-hot soybean protein isolate sample with 10mL of 6mol/L hydrochloric acid in a 10mL hydrolysis tube, hydrolyzing in an air-blast drying oven at 110 ℃ for 24h, adding water into the hydrolysate to be close to 100mL, performing suction filtration by using an organic filter membrane, and fixing the volume of the hydrolysate to be 100 mL.

Taking 10mL of the diluted hydrolysate for solid phase extraction. The solid phase extraction column was activated with 10mL of methanol and a mixture of 10mL of methanol and 5% hydrochloric acid (methanol: 5% hydrochloric acid in a volume ratio of 20: 80). The leaching uses 10mL of water, 10mL of methanol, and a mixture of ammonia water and water (the volume ratio of methanol to ammonia water to water is 25: 5: 70). A mixture of 10mL of methanol and aqueous ammonia (methanol: aqueous ammonia in a volume ratio of 95: 5) was used for elution. The eluate was blown dry at 35 ℃ with nitrogen. For detection, 1mL of a mixture of 5% formic acid and acetonitrile was used for reconstitution (5% formic acid/acetonitrile ratio 95: 5 by volume), and the mixture was passed through a 0.2 μm organic filter.

The heterocyclic amine content was determined using an LC-20 ADXR/Triple Quad 3500 LC MS. The column was XDB C18 columns (150X 2.1mm,3.5 μm), and the mobile phase A was: 5mmol/L ammonium formate aqueous solution and 0.5% formic acid, mobile phase B is: 5mmol/L ammonium formate methanol solution and 0.5% formic acid, the liquid phase condition is initial 5% B, 0 min; 60% B, 1.1 min; 80% B, 5 min; 95% B, 6 min; 95% B for 8 min; 5% B, 8.1 min; 5% B, 8.2 min; 5% B, 10 min. The multi-reaction detection scan information for each heterocyclic amine standard is shown in table 5.

As can be seen from table 5, the parent ion and the daughter ion information of each heterocyclic amine substance were used for qualitative and quantitative analysis of each heterocyclic amine substance.

As can be seen from fig. 3, the content of heterocyclic amine increases significantly with increasing temperature and time after the same heat treatment time node (170 ℃, 10min or 150 ℃, 60min), and it is more convenient to analyze the heterocyclic amine under this heat treatment condition or under the condition of more intense heating. It was also found that dry-heated soy protein isolate failed to produce all of the target heterocyclic amine species.

TABLE 5 multiple reaction detection scan information for each heterocyclic amine standard

Example 3

Changes in dry-hot soy protein isolate were analyzed using SDS-PAGE:

SDS-PAGE gels with 4% concentrated gel and 15% split gel were prepared using a Biosharp gel Rapid preparation kit. The isolated soy protein heat-treated in example 1 was dissolved in PBS, centrifuged, and the supernatant was subjected to gel electrophoresis.

Fluorescein-5-thiosemicarbazide (FTC) binds to carbonyl compounds in proteins, and thus the degree of protein oxidation and the oxidation site can be visualized using FTC to label carbonyl proteins.

The specific operation is as follows: 0.4mL of the protein of example 1 (0.5mg/mL) was mixed with 0.1mL of 5mmol/L FTC solution and incubated at 37 ℃ in the dark for 150min with gentle stirring every 30 min. The protein was then precipitated with 20% pre-cooled trichloroacetic acid. The mixture was then incubated at 4 ℃ for 10min under dark conditions and centrifuged at 12000rpm for 10min at 4 ℃. The precipitate was washed five times with a mixed solution of ethanol and ethyl acetate (ethanol: ethyl acetate volume ratio 1: 1), and finally resuspended in a 1mmol/LTris-HCl buffer solution of 80. mu. LpH: 8 in the dark at 4 ℃ and MgCl₂(5mmol/L), urea (8mol/L), NaCl (150mmol/L), and disodium ethylenediamine tetraacetic acid (100 mmol/L).

Protein loading buffer and electrophoresis buffer required for electrophoresis were configured according to the specifications of the SDS-PAGE gel kit. The concentration of the derivative protein obtained by the above procedure was determined using the Bradford assay method. The derivatized protein was mixed with the loading buffer prepared according to the kit, and separated on SDS-PAGE gel without heating. After electrophoresis, the gel was washed with pre-chilled water to remove unbound FTC in the gel background. Carbonyl protein gel images were obtained using a GEAmersham Imager 600RGB scanning gel equipped with a transparent sample pan, followed by staining with Coomassie Brilliant blue R250 solution, destaining, and scanning the gel again using Tanon 2500 to obtain protein gel electrophorograms. The results are shown in FIG. 4.

As can be seen from FIG. 4, as the heat treatment temperature increases and the time is prolonged, the high molecular weight protein band gradually disappears and the low molecular weight protein content increases (FIG. 4A, FIG. 4B), i.e., the high temperature causes the decomposition of the protein. After Coomassie brilliant blue staining, a protein band shows a weaker electrophoresis channel (figure 4E, electrophoresis channel 7; figure 4F, electrophoresis channel 6) or even shows no electrophoresis channel, the fluorescence intensity of the electrophoresis channel is highest (figure 4C, electrophoresis channel 7; figure 4D, electrophoresis channel 6), namely, high molecular weight protein (higher than 75kDa) is gradually oxidized and decomposed at 150 ℃, 10min and 130 ℃ after 60min to generate low molecular weight (lower than 25kDa) protein rich in carbonyl compounds.

Example 4

The secondary structure of the dry-hot soy protein isolate was analyzed using circular dichroism spectroscopy, and the tertiary structure of the dry-hot soy protein isolate was analyzed using a fluorescence spectrophotometer:

the dry-heat isolated soybean protein samples obtained in each temperature treatment of example 1 were diluted to 0.25mg/mL, and the CD spectrum in the far ultraviolet region (190-250nm) was analyzed for protein secondary structure changes using MOS-450/AF-CD-STP-A circular dichroism spectroscopy. The results are shown in FIG. 6.

Diluting the dry and hot soybean protein isolate sample obtained in the temperature treatment of example 1 to 0.1mg/mL, analyzing the protein endogenous fluorescence spectrum by using an Agilent Cary Eclipse fluorescence spectrophotometer, obtaining an emission spectrum of a protein solution at 300-400nm under the excitation condition of 280nm, wherein the scanning speed is 120nm/min, extracting the maximum fluorescence intensity and the wavelength of the maximum fluorescence of each sample after data is derived, and carrying out quantitative analysis, wherein the result reflects the tertiary structure of the protein. The results are shown in FIG. 5.

As can be seen from FIGS. 5 and 6, the protein structure regularly changed as the degree of heat treatment increased. The wavelength of the maximum fluorescence intensity of the protein is red-shifted, which indicates that the fluorescent group of the protein is positioned in a more hydrophilic microenvironment. Whereas a decrease in fluorescence intensity indicates that oxidation of the protein causes quenching of the fluorescent group within the protein.

Example 5

The oxidation degree of the dry-hot soy protein isolate was analyzed using a fluorescence spectrophotometer:

conjugated schiff bases and N-formyl kynurenines (NFKs) are products of protein oxidation, the content of which represents the degree of oxidation of the protein. Diluting the dry and hot soybean protein isolate sample obtained in the temperature treatment of example 1 to 0.1mg/mL, and obtaining 440nm fluorescence emission intensity of the protein solution under the excitation condition of 330nm by using an Agilent Cary Eclipse fluorescence spectrophotometer, wherein the scanning speed is 30nm/min, and the change of the fluorescence emission intensity indicates the content change of the conjugated Schiff base in the protein solution; the 460nm emission fluorescence intensity of the protein solution is obtained under the excitation condition of 350nm, the scanning speed is 30nm/min, and the change of the scanning speed indicates the content change of the N-formyl kynurenine in the protein solution. The results are shown in FIGS. 7 and 8.

As can be seen from FIGS. 7 and 8, the contents of conjugated Schiff base and N-formyl kynurenine are increased with the increase of the heat treatment temperature or time of the isolated soy protein, indicating that the oxidation degree of the protein is increased.

Example 6

The degradation degree of the dry-hot soy protein isolate was analyzed using a uv-vis spectrophotometer:

a total amino group measurement standard curve based on alanine was prepared by a specific reaction between ninhydrin and an amino group. Wherein alanine was diluted with methanol/0.1% formic acid solution (methanol to 0.1% formic acid volume ratio of 1: 1). Each solution was prepared as follows, 3% ninhydrin: 0.75g ninhydrin was metered to a 25mL volumetric flask using absolute ethanol. 1mg/mL ascorbic acid: the solvent is water. Acetic acid-sodium acetate buffer: 21.70g of anhydrous sodium acetate and 6.665mL of acetic acid were added to a constant volume of 100mL, the solutions were mixed according to Table 6, heated in a water bath (100 ℃) for 15min, rapidly cooled, then 5mL of anhydrous ethanol was added, shaken well, then a constant volume of 10mL was added, and the absorbance was measured at 570 nm. The standard curve is shown in fig. 9.

40mg of the dry and hot isolated soybean protein sample obtained in example 1 by the treatment at different temperatures was accurately weighed, dissolved in 1mL of methanol/0.1% formic acid solution (the volume ratio of methanol to 0.1% formic acid was 1: 1), centrifuged at 12000rpm to obtain 0.3mL, and the change in the total amino group content in the sample was measured. The results are shown in fig. 10, where the different lower case letters a-f in the figure indicate that the data differed significantly (P < 0.05).

As can be seen from FIG. 10, under the heat treatment condition of 10min, when the temperature is lower than 150 ℃, the total amino group content decreases with the increase of the temperature, which is related to the consumption of the amino acid reaction; when the temperature is 150 ℃ and 190 ℃, the total amino content is kept stable, and as can be seen from the combination of FIG. 4A and electrophoresis channels 5-7, the total amino content is kept constant due to the generation of a large number of amino groups by thermal decomposition of the protein under such conditions. Similarly, the heat treatment has similar rules under the condition of 60 min.

TABLE 6 determination of Total amino Standard Curve

Numbering	1	2	3	4	5	6
							1 μmol/mL alanine solution (mL)	0.00	0.05	0.10	0.20	0.30	0.40
Distilled water (mL)	0.45	0.40	0.35	0.25	0.15	0.05
							Acetic acid-sodium acetate buffer (mL)	0.50	0.50	0.50	0.50	0.50	0.50
3% ninhydrin (mL)	0.50	0.50	0.50	0.50	0.50	0.50
							Ascorbic acid (ul) 1mg/mL	50.00	50.00	50.00	50.00	50.00	50.00

Example 7

Analyzing content changes of formaldehyde and acetaldehyde in the pyrolysis process of the dry-heat soybean protein isolate by using high performance liquid chromatography:

boiling distilled water for 10min, cooling to prepare a derivative: 2.64g of sodium acetate and 0.3g of 2, 4-dinitrophenylhydrazine are weighed, dissolved using 50% aqueous acetonitrile, 1mL of glacial acetic acid are added and the volume is made to 1L. All glassware used in the experiment is preheated for 10min at 150 ℃ to reduce environmental pollution. Preparing mixed standard solution of formaldehyde and acetaldehyde with gradient concentration, mixing with 20mL of derivative in a 25mL graduated test tube, uniformly mixing, placing in an ultrasonic cleaner, and performing ultrasonic treatment at 60 ℃ for 90 min. Adding 8g ammonium sulfate into the test tube, stirring uniformly by using a glass rod, standing slightly, centrifuging for 10min at the temperature of 4 ℃ and under the condition of 5000rpm/min, and taking the upper acetonitrile phase 1. Adding 8mL of acetonitrile into the lower layer solution, washing once, centrifuging to obtain an upper layer acetonitrile phase 2, mixing with the upper layer acetonitrile phase 1, and metering to 20 mL. 1mL of acetonitrile phase was subjected to quantitative analysis by high performance liquid chromatography. Chromatographic conditions are as follows: eclipse Plus C18 (4.6X 250nm,5 μm) column at 360 nm; the mobile phase is water (A) and acetonitrile (B), 0-7min, 30-90% B, 7-10min, 90% B, 10-15min, 90-30% B, 15-30min, 30% B. The liquid phase separation and the calibration curve of the obtained standard are shown in fig. 11 and 12.

When the dry and hot soybean protein isolate samples obtained in example 1 were measured by different temperature treatments, 2g of the samples were weighed and quantitative determination of formaldehyde and acetaldehyde in the samples was carried out. The results are shown in FIG. 13.

As can be seen from fig. 13, under the condition that the duration of the heat treatment was 10min, it was observed that the formaldehyde content level increased when the heating temperature was 90 ℃ to 130 ℃, decreased when the heating temperature was 130 ℃ to 210 ℃, and increased again when the heating temperature was more than 210 ℃. However, at 60min, it was found that the formaldehyde level increased at heating temperatures of 90 ℃ to 110 ℃, decreased at heating temperatures of 110 ℃ to 190 ℃, and increased again at heating temperatures greater than 190 ℃. In two experiments with different heating temperatures, the formaldehyde content level shows the same rule, namely, the formaldehyde content level is increased firstly, then decreased and then increased, and the difference is that the turning point of the formaldehyde content change is earlier when the heat treatment is carried out for 60min than when the heat treatment is carried out for 10 min.

Under the condition that the duration of the heat treatment is 10min, when the heating temperature is 90-190 ℃, the content level of acetaldehyde increases along with the increase of the temperature, and when the heating temperature is more than 190 ℃, the content level of acetaldehyde decreases. However, at a heating temperature of 90 ℃ to 170 ℃ for a heat treatment duration of 60min, the acetaldehyde content level increases with increasing temperature, and at a heating temperature greater than 170 ℃, the acetaldehyde content level decreases. Two sets of experimental results with different heating temperatures also have similar variations.

Example 8

Evaluation of oxidative Change in Dry Heat Soy protein isolate System Using scavenging PTIO free radicals

Preparing 1mg/mL PTIO stock solution by using PBS buffer solution with the pH value of 7.4, mixing 1mL PBS according to 3mL PTIO working solution, incubating for 2h in a dark place at 37 ℃, and then measuring the absorbance value at 557nm to establish a standard curve. Because the PTIO free radical is relatively stable, and experimental results show that the PTIO free radical solution still has a good linear relation when the absorbance value is lower than 0.2. The antioxidant experiment measurement is carried out by taking 0.06mg/mL PTIO free radical solution. A1 mg/mL Trolox solution was prepared and used for scavenging PTIO free radicals as shown in Table 7 below. The resulting standard curve is shown in FIG. 14.

Example 1 measurement of antioxidant capacity of dry-hot soy protein isolate samples obtained by different temperature treatments: 40mg of sample is dissolved in 2mL of PBS to be fully dispersed, the mixture is stood for 1h at room temperature and centrifuged at 5000rpm for 20min at 4 ℃, and 1mL of the mixture is taken for system antioxidant experiment determination. The results are shown in FIG. 15.

As can be seen from FIG. 15, the two experiments of heat treatment for 10min and 60min show similar variation rules, wherein under the condition of 10min of heat treatment, the oxidation resistance value undergoes reduction (90-130 ℃), increase (130-.

TABLE 7 establishment of Trolox eliminating PTIO free radical standard curve

From a comprehensive analysis of the results of the above examples, it can be seen that the protein solubility decreased significantly with increasing heat treatment temperature or increasing heat treatment time (FIG. 2), and the high molecular weight protein (above 75kD) decomposed gradually after 150 ℃ C., 10min, or 130 ℃ C., 60min, and the low molecular weight (less than 25kD) carbonyl-rich protein increased (FIG. 4), while the increase of the conjugated Schiff base and N-formyl kynurenine (FIG. 7, FIG. 8) also indicates a gradual increase in the degree of protein oxidation, and the heterocyclic amine content was observed to increase significantly with increasing temperature and time at the same heat treatment time node (170 ℃ C., 10min, or 150 ℃ C., 60min) (FIG. 3). Thus, it is reasonable to speculate that proteins are pyrolyzed and that oxidation of low molecular weight proteins/peptides is a pathway for the formation of heterocyclic amines during thermal processing of protein foods.

It is noted that formaldehyde and acetaldehyde also have a content transition change at this time node (fig. 15), and since formaldehyde and acetaldehyde are intermediate products generated by heterocyclic amine, the content change of the formaldehyde and acetaldehyde in the heat treatment process can be combined with the content change of the heterocyclic amine, so that the formaldehyde and acetaldehyde can be presumed to participate in the formation process of the heterocyclic amine. The data from the experiment were analyzed for correlation on OriginPro 2021 (fig. 16). The result shows that formaldehyde and heterocyclic amines such as Trp-P-2, Harman, Norharman and the like show obvious negative correlation, and acetaldehyde and heterocyclic amines such as MeIQx, A alpha C, IQ and the like show obvious positive correlation; the soluble protein concentration and total amino content are significantly negatively correlated with heterocyclic amines; quenching of the intrinsic fluorescence of the protein is significantly inversely related to the formation of heterocyclic amines, but the red shift of the wavelength at which the fluorescence maximum is present is significantly positively related.

Therefore, the method for analyzing the formation mechanism of the heterocyclic amine by using the dry-hot soybean protein isolate as a model can simultaneously detect the correlation between the content of the heterocyclic amine after the dry-hot soybean protein isolate and the physicochemical property after the protein heat treatment, has complete experimental method and very important academic significance, and simultaneously has guiding significance for reasonably adding the soybean protein isolate in the food industry and properly processing the food quality safety problem in the development of foods taking the soybean protein isolate as a main raw material.

Claims

1. A method for analyzing a heterocyclic amine formation mechanism by using dry-hot soybean protein isolate as a model is characterized by comprising the following steps:

(3) analyzing changes in the dry-hot soy protein isolate using an SDS-PAGE gel;

2. The method of claim 1, wherein the step (1) dry heat treatment is performed in a forced air drying oven.

3. The method of claim 1, wherein step (2) employs 6M hydrochloric acid for acid hydrolysis; during solid phase extraction, the eluent is blown dry by nitrogen at 35 ℃.

4. The method of claim 1, wherein step (3) uses fluorescein-5-thiosemicarbazide to label carbonyl proteins to visualize the extent of protein oxidation and the site of oxidation.

5. The method of claim 1, wherein the analysis method of step (4) is: analyzing the endogenous fluorescence spectrum by using a fluorescence spectrophotometer to obtain the maximum fluorescence intensity and the wavelength of the maximum fluorescence; the CD spectrum in the far ultraviolet region was measured using a circular dichroism spectrometer.

6. The method of claim 1, wherein the analysis method of step (5) is: the content of conjugated schiff base and N-formyl kynurenine was determined using a fluorescence spectrophotometer.

7. The method of claim 1, wherein the analysis method of step (6) is: through the reaction of ninhydrin and amino, a total amino standard curve with alanine as a standard is established, and the content change of the total amino in the dry-hot soybean protein isolate is determined.

8. The method of claim 1, wherein the analysis method of step (7) is: deriving formaldehyde and acetaldehyde by using 2, 4-dinitrophenylhydrazine, separating the formaldehyde and the acetaldehyde by using high performance liquid chromatography at 360nm, and quantitatively analyzing the content change of the formaldehyde and the acetaldehyde in the pyrolysis process of the dry-hot soybean protein isolate.

9. The method of claim 1, wherein the analysis method of step (8) is: and (3) establishing a PTIO standard curve and a Trolox PTIO free radical eliminating standard curve, and measuring the oxidation resistance of the dry-hot soybean protein isolate system.

10. Use of the process of claim 1 for preparing a thermally processed food product using soy protein isolate.