Controllable atmosphere frying oxidation of triglyceride isotopic tracing method and system

Abstract

This invention provides a controlled atmosphere deep-frying method and system for isotope tracing of oxidized triglycerides, using H2O, D2O, or H2¹. 8 O prepares starch blocks, heats oil in a jacketed reactor, and then introduces natural or containing natural gas after vacuuming and nitrogen replacement. 18 O2 / ¹ 6 The oil was fried in O2-synthesized air and then sampled. The oil sample was analyzed by liquid chromatography-high resolution mass spectrometry (LC-HPLC-MS), and the structure was confirmed based on characteristic ions and neutral loss. [M+NH4] was then extracted. + Isotope peak clusters according to D, 18 O-incremental attribution determines and quantifies the source of oxygen and hydrogen. The system includes gas control, pressure monitoring, and sampling valves, which, together with a sealing cap and a mesh inner liner, allow for material feeding without disrupting the atmosphere. This invention solves the problems of insufficient reliability in confirming the structure of oxidized triglycerides in frying systems, difficulty in source analysis, and poor repeatability of experimental conditions in existing technologies, achieving traceable determination and quantification of the source of oxygen and hydrogen in oxidized triglycerides.

Description

Technical Field

[0001] This invention belongs to the field of food technology and relates to lipid oxidation products in frying processing systems. Specifically, it relates to a method and system for tracing the isotopes of oxidized triglycerides during controlled atmosphere frying. Background Technology

[0002] During frying, oils undergo free radical-initiated oxidation reactions under high temperatures and the influence of air / moisture, accompanied by addition and fragmentation processes, resulting in a wide variety of oxidized derivatives. Among these products, oxidized triglycerides typically have low abundance and numerous isomers, leading to overlapping chromatographic peaks and shared fragment ions in mass spectrometry. This results in insufficient reliability in structural determination and differentiation of formation pathways when relying solely on conventional chromatography-mass spectrometry. Existing technologies can detect and infer the structure of some oxidized triglycerides, but there is still a lack of directly verifiable methods for the source of oxygen atoms (e.g., from aqueous phase, gaseous oxygen, or substrate internal migration) and the contribution ratio of different sources to product formation. Therefore, it is necessary to propose a technical solution combining a controllable reaction atmosphere device and multi-source stable isotope labeling to achieve traceable analysis and relative quantitative assessment of the formation mechanism of oxidized triglycerides.

[0003] In current evaluations of oil oxidation, chemical titration methods such as peroxide value and acid value, or spectroscopic methods, are commonly used to characterize primary or secondary oxidation products. However, these methods are mostly based on redox reactions or acid-base neutralization, are easily affected by matrix interference, and usually only provide information on the macroscopic degree of oxidation, unable to distinguish specific molecular types, and even less able to provide evidence at the level of oxidation pathway and product structure.

[0004] For the analysis of polar components in frying oils, methods exist for determining total polar substances or polymeric triglycerides, such as silica gel column chromatography combined with gravimetric analysis or gel permeation / size exclusion chromatography. However, these methods primarily focus on monitoring changes in "total amount" or "major component categories," and are insufficient for reliably analyzing the molecular composition, oxidized functional group types, and isomer differences of oxidized triglyceride monomers.

[0005] In recent years, lipidomics techniques based on high-resolution mass spectrometry (LC-MS / MS) have been used for the screening and identification of oxidized triglycerides. However, triglycerides are composed of different fatty acid chain combinations, and multiple oxidation sites may be generated on the same fatty acid chain, resulting in various types of oxidized molecules. In existing technologies, the identification of oxidized lipids largely relies on the matching and inference of theoretical fragment libraries. However, due to factors such as insufficient standards, isomer interference, and incomplete fragment rules, there are still risks of misannotation and structural misjudgment in actual sample analysis, making it difficult to achieve high-confidence confirmation.

[0006] For oxidized triglycerides containing unstable groups such as peroxide groups, structural identification is even more challenging. Positional and geometric isomers of oxidized groups often coexist and exhibit similar chromatographic behaviors. In practical analysis, relying solely on empirical characteristic fragment ions and neutral loss rules can easily lead to isomer confusion or misidentification of functional group types, affecting the reliability of qualitative results.

[0007] Besides the detection and identification process itself, the experimental controllability of the frying system is also a key factor affecting the reliability of mechanistic studies. Traditional frying experiments are mostly conducted in open systems, where variables such as the interfacial area between oil and air, oxygen mass transfer rate, temperature gradient, and moisture introduction method are difficult to control precisely, resulting in insufficient reproducibility of oxidation kinetic data and product spectra. Existing technologies show that changes in the oxygen partial pressure in the reactor headspace and the gas-liquid contact method significantly affect the thermal oxidation pathway of oils, thus, constructing standardized, controllable atmosphere frying reactors is of great significance.

[0008] Meanwhile, during frying, moisture migration and oil hydrolysis are inevitable. Moisture may participate in hydrolysis as a reactant or affect free radical chain reactions. Although existing studies have attempted to discuss the impact of hydrolysis on oxidation, traditional methods still lack a direct molecular-level chain of evidence for the crucial question of whether the oxygen atoms in oxidized triglyceride molecules originate from gaseous oxygen (O2) or aqueous phase (H2O). Stable isotope tracing technology has been applied in other systems, but its application in high-temperature, multiphase, and matrix-complex frying systems, and its ability to precisely trace the source of oxygen and hydrogen at the molecular level of specific oxidized triglycerides, remains relatively rare.

[0009] In summary, existing technologies still face several challenges when dealing with complex frying systems: First, macroscopic indicators and total amount methods are insufficient for achieving hierarchical structural analysis of oxidized triglycerides; second, LC-MS / MS identification is affected by isomer interference and a lack of standards, resulting in lower confidence levels; third, the lack of standardized frying model devices with controllable atmosphere and mass transfer leads to insufficient experimental reproducibility; and fourth, the absence of a definitive method to directly trace the origin of oxygen and hydrogen in oxidized molecules limits a deeper understanding of the specific molecular mechanisms of water oxidation at high temperatures. Therefore, an integrated method and system are urgently needed to address these issues. Summary of the Invention

[0010] To address the shortcomings of existing technologies, the present invention aims to provide a controlled atmosphere deep-frying method for tracing the isotopes of oxidized triglycerides, thereby solving the technical problem that the certainty of mechanism analysis and the confidence level of identification in existing tracing methods need to be further improved.

[0011] In view of the shortcomings of the existing technology, another objective of the present invention is to provide a controllable atmosphere frying system, which solves the technical problems that the repeatability and comparability of the frying system and the stability of sequence sampling in the existing technology need to be further improved.

[0012] To solve the above-mentioned technical problems, the present invention adopts the following technical solution.

[0013] A controlled atmosphere deep-frying method for isotope tracing of oxidized triglycerides, comprising the following steps.

[0014] Step 1: Prepare a starch-based frying mold.

[0015] Starch is mixed with an aqueous medium and stirred to gelatinize, resulting in a gelatinized slurry. The gelatinized slurry is then transferred to a mold and compacted to form a block model. After the block model has been balanced and regenerated, a starch-based frying model is obtained.

[0016] In step one, the aqueous phase medium is H2O, D2O, and H2, respectively. 18 O.

[0017] Step 2: Construct a controlled atmosphere frying system.

[0018] Edible oil is added to the oil-filling liner of the controlled atmosphere frying system and heated while stirring.

[0019] Step 3: Atmosphere replacement and labeling.

[0020] The reactor body of the controlled atmosphere frying system is evacuated and replaced with inert gas. Then, the target gas is introduced into the reactor body and pressurized.

[0021] In step three, the target gases are natural air and gases containing... 18 O2 synthesis air and containing 16 O2 synthesized air.

[0022] Step 4: Deep frying cycle and sampling.

[0023] The starch-based frying model prepared in step one was added to edible oil through the feed inlet of a controlled atmosphere frying system for frying. Oil samples were collected at 0h and every 4h during the frying process, and the oil samples were transferred to a pre-vacuumed and nitrogen-purged sealed container for low-temperature storage and analysis.

[0024] Step 5: Liquid chromatography-mass spectrometry detection.

[0025] The oil sample obtained in step four was diluted and injected, and full-scan MS data were acquired using liquid chromatography-tandem mass spectrometry in positive ion mode. 1 and MS 2Data were used to obtain characteristic ion information of triglycerides and oxidized triglycerides.

[0026] Step six, structural identification.

[0027] Based on MS 1 Precision quality and MS 2 Neutral loss fragments with neutral acyl cations and / or charge retention were used to confirm the molecular composition and oxidative functional group type of oxidized triglycerides.

[0028] Step 7: Isotope peak cluster analysis and source determination.

[0029] The oxidized triglyceride [M+NH4] + Addition ion theory m / z Let M be the peak intensity I(M+i) of isotope peaks M and M+1 to M+4 within a preset quality window, calculate the relative abundance ratio I(M+i) / I(M); compare the relative abundance ratio with the control group to determine the source of oxygen and / or hydrogen in oxidized triglycerides, and output the quantitative evaluation results.

[0030] The present invention also has the following technical features.

[0031] In step one, the deuterium isotope abundance of D2O is not less than 99 atom%; the H2 18 O 18 The abundance of O isotopes is not less than 99 atoms.

[0032] In step one, the starch mentioned is potato starch.

[0033] In step one, the starch and aqueous medium are mixed at a mass ratio of 1:1.5.

[0034] In step one, the gelatinization conditions are as follows: gelatinize at 80°C and 100 rpm for 6 minutes to obtain a gelatinized slurry.

[0035] In step one, the mold is a stainless steel trough with dimensions of 10cm×1cm×1cm; the block model has dimensions of 1×1×1cm.

[0036] In step one, the condition for equilibrium recombination is to undergo equilibrium recombination at 4°C for 8 hours after sealing.

[0037] In step two, the edible oil is palm oil, and the volume of edible oil added is 70 mL.

[0038] In step two, the edible oil is heated to 180°C; the stirring is performed using magnetic stirring at 60 rpm.

[0039] In step two, the volume of the oil-filling inner liner is 100 mL.

[0040] In step two, the controlled atmosphere frying system is the controlled atmosphere frying system described below.

[0041] In step three, the inert gas is nitrogen, and the replacement is performed three times.

[0042] In step three, including 18 O2 synthesis air 18 The volume fraction of O2 is 21%, and the remainder is N2; 18 O2 18 The abundance of O isotopes is not less than 99 atoms.

[0043] In step three, the pressure is increased to 1 atmosphere.

[0044] In step four, the frying conditions are as follows: a single frying time of 3 minutes; frying is carried out in cycles of 1 hour, with a total frying time of 20 hours, and the edible oil is not changed during the total frying time.

[0045] In step four, the temperature for low-temperature preservation is -80℃.

[0046] This invention also protects a controlled atmosphere frying system, including a reactor body, a reactor cover installed on the top of the reactor body, the reactor cover being provided with a feed inlet, a sampling inlet, an air inlet, an exhaust outlet, a pressure gauge and a pressure relief valve; a sampling valve is installed on the sampling inlet; the air inlet is connected to a gas cylinder through an air inlet valve; the exhaust outlet is connected to a vacuum pump through an exhaust valve.

[0047] The feed inlet is equipped with an openable sealing cap, and the top opening of the model net bag is connected to the sealing cap; the inside of the reactor body is fitted with an oil-filling liner, and the model net bag is suspended inside the oil-filling liner.

[0048] The model net is made of stainless steel and is connected to the sealing cap by a hook; the mesh size of the model net is 2mm.

[0049] The bottom of the reactor body is equipped with a magnetic heating stirrer, and a temperature control jacket is fitted on the outer wall of the reactor body.

[0050] Compared with the prior art, the present invention has the following technical effects.

[0051] (I) The method of the present invention uses a cross-tracing strategy of "aqueous medium isotope labeling + gas phase oxygen isotope labeling" and combines it with quantitative analysis of isotope peak clusters to achieve molecular-level confirmation of the oxygen / hydrogen source in oxidized triglyceride molecules. It can distinguish the contribution of gas phase oxygen and aqueous medium in the oxidation process and improve the certainty of mechanism analysis.

[0052] (II) The method of the present invention confirms the molecular composition and oxidized functional group type of oxidized triglycerides by combining high-resolution LC-MS / MS with fragmentation rules, thereby reducing the risk of mis-annotation caused by relying solely on theoretical fragment libraries; for products related to unstable peroxide groups, the identification confidence is improved by using charge retention neutral loss characteristics.

[0053] (III) The device of the present invention achieves controllable adjustment of temperature, stirring and headspace atmosphere through a jacketed magnetic stirring reactor. Combined with vacuuming-nitrogen replacement and constant pressure ventilation process, it reduces the randomness caused by gas-liquid mass transfer and environmental fluctuations in an open system, and improves the repeatability and comparability of the frying system.

[0054] (IV) The device of the present invention achieves standardization of solid sample size, water content and feeding / removal method by using a standardized starch-based solid frying model and an integrated feeding structure of "sealed cap-mesh inner liner", thereby reducing the impact of atmospheric disturbance and secondary reaction of the sample during sampling and improving the stability of sequential sampling. Attached Figure Description

[0055] Figure 1 This is a schematic diagram of a controlled atmosphere frying system.

[0056] Figure 2 for Figure 1 A schematic diagram showing the connection between the sealing cap and the model net bag.

[0057] Figure 3 A schematic diagram of the process for traceability of isotopes of oxidized triglycerides through controlled atmosphere frying.

[0058] Figure 4 This is a schematic liquid chromatogram of triglycerides and oxidized triglyceride products in a sample from the frying process, with the retention time ranges of the triglyceride and oxidized triglyceride products indicated.

[0059] Figure 5 MS / MS fragmentation diagram and structural identification schematic of oxidized triglyceride TG(16:0_18:1_18:2)+OH.

[0060] Figure 6 MS / MS fragmentation diagram and structural identification diagram of oxidized triglyceride TG(16:0_18:2_18:2)+OOH.

[0061] Figure 7 MS / MS fragmentation diagram and structural identification diagram of oxidized triglyceride TG(18:1_18:1_18:1)+OOH.

[0062] Figure 8(a) shows the MS of oxidized triglyceride TG(16:0_18:1_18:2)+OH under the experimental conditions of natural air + H2O. 1 Comparison of signal changes in isotope peak clusters (M to M+4).

[0063] Figure 8(b) shows the oxidation of triglycerides TG(16:0_18:1_18:2)+OH in... 18 MS under experimental conditions of O synthesis air + D2O 1 Comparison of signal changes in isotope peak clusters (M to M+4).

[0064] Figure 8(c) shows the oxidation of triglycerides TG(16:0_18:1_18:2)+OH in... 16 O synthesizes air + H2 18 MS under O experimental conditions 1 Comparison of signal changes in isotope peak clusters (M to M+4).

[0065] Figure 9(a) shows the MS of oxidized triglyceride TG(18:1_18:1_18:1)+OOH under the experimental conditions of natural air + H2O. 1 Comparison of signal changes in isotope peak clusters (M to M+4).

[0066] Figure 9(b) shows the oxidation of triglycerides TG(18:1_18:1_18:1)+OOH in... 18 MS under experimental conditions of O synthesis air + D2O 1 Comparison of signal changes in isotope peak clusters (M to M+4).

[0067] Figure 9(c) shows the oxidation of triglycerides TG(18:1_18:1_18:1)+OOH in... 16 O synthesizes air + H2 18 MS under O experimental conditions 1 Comparison of signal changes in isotope peak clusters (M to M+4).

[0068] Figure 10 A comparison of the relative abundance ratio of isotopic peak clusters of oxidized triglycerides TG(16:0_18:1_18:2)+OH under three experimental conditions with the change of frying time.

[0069] Figure 11 This is a comparison of the relative abundance ratio of isotopic peak clusters of oxidized triglycerides TG(18:1_18:1_18:1)+OOH under three experimental conditions as a function of frying time.

[0070] The labels in the diagram represent the following: 1-Reaction vessel body, 2-Vessel lid, 3-Feed inlet, 4-Sampling port, 5-Gas inlet, 6-Exhaust port, 7-Pressure gauge, 8-Pressure relief valve, 9-Sampling valve, 10-Gas inlet valve, 11-Gas cylinder, 12-Exhaust valve, 13-Vacuum pump, 14-Sealing cap, 15-Model net bag, 16-Inner oil liner, 17-Magnetic heating stirrer, 18-Temperature control jacket, 19-Hook.

[0071] The specific content of the present invention will be further explained in detail below with reference to the embodiments. Detailed Implementation

[0072] It should be noted that, unless otherwise specified, all components, equipment and methods in this invention are based on components, equipment and methods known in the prior art.

[0073] This invention relates to the analysis and mechanism attribution of lipid oxidation products in frying processing systems. Specifically, it focuses on the spectroscopic characterization, isotope labeling tracking, and formation pathway analysis of oxidized triglycerides. It proposes an analytical method and system that introduces stable isotopes from multiple sources and performs data integration processing under a controlled atmosphere frying model to distinguish and relatively quantitatively assess the formation pathway and oxygen source of oxidized triglycerides.

[0074] This invention provides a controlled atmosphere frying method and system for isotopic tracing of oxidized triglycerides, which can also be called a controlled atmosphere frying system for source analysis and structural identification of oxidized triglycerides based on multi-source stable isotope tracing. H2O, D2O, or H2 is used. 18 To prepare a starch block model, heat the oil in a jacketed magnetically stirred reactor and evacuate it—after nitrogen purging, introduce natural air or air containing nitrogen. 18 O2 / 16 O2-synthesized air was used for cyclic frying with timed sampling. Oil samples were then acquired by liquid chromatography-high resolution mass spectrometry (LC-HPLC-MS). 1 / MS 2 The structure of oxidized triglycerides was confirmed based on characteristic ion / neutral loss information, and [M+NH4] was extracted. + Isotope peak clusters and according to D, 18 O-mass increment attribution determines the source of oxygen / hydrogen and performs relative quantification. The system includes gas control, pressure monitoring, and valve sampling structures, which, together with a sealed cap and mesh inner liner, allow for feeding and discharging without disrupting the atmosphere inside the vessel, improving frying repeatability and traceability reliability.

[0075] In this invention, "edible oils" refers to palm oil and other vegetable oils suitable for frying; the "frying model" is preferably a starch-based block model, the size, water content, and dispensing method of which can be adjusted according to the research objectives. The isotope-labeled aqueous medium includes H2O, D2O, and H2. 18 O; isotope-labeled gaseous media include natural air, containing... 18 O2 synthesis air and containing 16 O2 synthesized air.

[0076] This invention employs a cross-tracing strategy: using natural air + H2O as the control condition; and using a mixture containing... 18 The model prepared by combining O2 with D2O in the synthetic air was used as the first tracer condition; with a concentration of O2... 16 O2 synthesis air combination with H2 18 The model prepared with O was used as a second tracer condition to distinguish the potential sources of oxygen and hydrogen in oxidized triglycerides.

[0077] Following the above technical solutions, specific embodiments of the present invention are given below. It should be noted that the present invention is not limited to the following specific embodiments, and all equivalent modifications made based on the technical solutions of this application fall within the protection scope of the present invention.

[0078] Example 1:

[0079] This embodiment provides a controlled atmosphere frying system, such as Figure 1 As shown, the reactor includes a reactor body 1, a reactor cover 2 is installed on the top of the reactor body 1, and the reactor cover 2 is provided with a feed inlet 3, a sampling port 4, an air inlet 5, an exhaust port 6, a pressure gauge 7 and a pressure relief valve 8; a sampling valve 9 is installed on the sampling port 4; the air inlet 5 is connected to a gas cylinder 11 through an air inlet valve 10; the exhaust port 6 is connected to a vacuum pump 13 through an exhaust valve 12. like Figure 1 As shown, an openable sealing cover 14 is installed on the feed inlet 3, and the top opening of the model net bag 15 is connected to the sealing cover 14; as Figure 2 As shown, the reactor body 1 is fitted with an oil-filled inner liner 16, and the model net bag 15 is suspended inside the oil-filled inner liner 16.

[0080] In this embodiment, the reactor body 1 is designed to withstand a pressure of not less than 15 MPa.

[0081] In this embodiment, the sampling port 4 is used to take oil samples during the frying process. The inner oil container 16 is used to hold edible oil.

[0082] As a preferred embodiment, the model net 15 is made of 304 stainless steel. Figure 2As shown, the model net 15 is connected to the sealing cap 14 via hooks 19; the mesh size of the model net 15 is 2mm. In this embodiment, the model net 15 is used to hold the frying model and allows edible oil to come into contact with the frying model during the frying process, and the frying model can be lifted out of the liquid along with the sealing cap without disrupting the atmosphere inside the pot. In this embodiment, the diameter of the feed inlet 3 is 20mm, and the outer diameter of the model net 15 is less than 20mm.

[0083] As a preferred embodiment of this invention, such as Figure 1 As shown, a magnetic heating stirrer 17 is installed at the bottom of the reactor body 1, and a temperature control jacket 18 is fitted onto the outer wall of the reactor body 1. In this embodiment, the magnetic heating stirrer 17 is a commonly known magnetic heating stirrer in the art. The magnetic heating stirrer 17 is used to heat the reactor body 1 and drive the magnetic stirrer in the oil-filling inner liner 16 to magnetically stir the edible oil. The temperature control jacket 18 is a commonly known temperature control jacket in the art, and the temperature control jacket 18 is used to control the temperature of the reactor body 1.

[0084] Example 2: This embodiment provides a controlled atmosphere deep-frying method for isotopic tracing of oxidized triglycerides, such as... Figure 3 As shown, the method includes the following steps.

[0085] Step 1: Prepare a starch-based frying mold.

[0086] Starch is mixed with an aqueous medium and stirred to gelatinize, resulting in a gelatinized slurry. The gelatinized slurry is then transferred to a mold and compacted to form a block model. After the block model has been balanced and regenerated, a starch-based frying model is obtained.

[0087] In step one, the aqueous phase media are H2O, D2O, and H2, respectively. 18 The deuterium isotope abundance of O;D2O is not less than 99 atom%;H2 18 O 18 The abundance of O isotopes is not less than 99 atoms.

[0088] In step one, the starch is potato starch.

[0089] In step one, the starch and aqueous medium are mixed at a mass ratio of 1:1.5.

[0090] In step one, the gelatinization conditions are: stirring at 100 rpm for 6 minutes at 80°C to obtain a gelatinized paste.

[0091] In step one, the mold is a stainless steel trough with dimensions of 10cm×1cm×1cm; the block model has dimensions of 1×1×1cm.

[0092] In step one, the condition for equilibrium recombination is to equilibrate and recombine at 4°C for 8 hours after sealing.

[0093] Step 2: Construct a controlled atmosphere frying system.

[0094] Edible oil is added to the oil-filling liner of the controlled atmosphere frying system and heated while stirring.

[0095] In step two, the edible oil is palm oil, and the volume of edible oil added is 70 mL.

[0096] In step two, the edible oil is heated to 180°C; stirring is performed using magnetic stirring at 60 rpm.

[0097] In step two, the volume of the oil-filling inner tank is 100mL.

[0098] In step two, the controlled atmosphere frying system is the controlled atmosphere frying system given in Example 1.

[0099] Step 3: Atmosphere replacement and labeling.

[0100] The reactor body 1 is evacuated and replaced with inert gas. Then, the target gas is introduced into the reactor body 1 and pressurized.

[0101] In step three, the inert gas is nitrogen, and the replacement is performed three times.

[0102] In step three, the target gases are natural air and gases containing... 18 O2 synthesis air and containing 16 Synthetic air containing O2; 18 O2 synthesis air 18 The volume fraction of O2 is 21%, and the remainder is N2; 18 O2 18 The abundance of O isotopes is not less than 99 atoms.

[0103] In step three, the pressure is increased to 1 atmosphere.

[0104] Step 4: Deep frying cycle and sampling.

[0105] The starch-based frying model obtained in step one is added to edible oil through the feed port 3 of the controlled atmosphere frying system for frying. Oil samples are collected at 0h and every 4h during the frying process, and the oil samples are transferred to a pre-vacuumed and nitrogen-purged sealed container for low-temperature storage and analysis.

[0106] In step four, the frying conditions are as follows: a single frying time of 3 minutes; frying is repeated once every 1 hour, with a total frying time of 20 hours, and the edible oil is not changed during the total frying time.

[0107] In step four, the temperature for cryopreservation is -80℃.

[0108] Step 5: Liquid chromatography-mass spectrometry detection.

[0109] The oil sample obtained in step four was diluted and injected, and full-scan MS data were acquired using liquid chromatography-tandem mass spectrometry in positive ion mode. 1 and MS 2 Data were used to obtain characteristic ion information of triglycerides and oxidized triglycerides.

[0110] In this embodiment, the preserved oil sample was diluted to 5 ppm with isopropanol, mixed well, filtered through a 0.22 μm filter membrane, and the filtrate was injected for analysis. To facilitate comparison between different time points and different tracer conditions, the target ion peak area was normalized using total ion current (TIC).

[0111] In this embodiment, as Figure 4 As shown, after separation by liquid chromatography, the triglyceride component of the fried sample was mainly distributed in the retention time range of approximately 12.65–15.66 min, while the oxidized triglyceride component was mainly distributed in the retention time range of approximately 11.45–12.72 min. Signals within these ranges were extracted and subsequently analyzed by MS. 1 and MS 2 Confirmation can provide a data foundation for the structural identification and isotope peak cluster analysis of oxidized triglycerides.

[0112] In step five, a C18 column was used for liquid chromatography. Mobile phase A was acetonitrile / water (60:40, v / v), and mobile phase B was isopropanol / acetonitrile (90:10, v / v). The mobile phase contained 0.05%-0.2% formic acid and 5-20mM ammonium formate.

[0113] In step five, the flow rate of the liquid chromatography was 0.4 mL / min, the column temperature was 60 °C, and the injection volume was 1 μL.

[0114] In step five, the gradient elution program for liquid chromatography is as follows: 0–2.0 min, B phase maintains 5%; 2.0 min, B phase increases to 43%; 2.1 min, B phase increases to 52%; 9.0 min, B phase increases to 53%; 9.1 min, B phase increases to 75%; 18.0 min, B phase increases to 100% and maintains until 18.2 min; 18.2 min, B phase decreases to 5% and equilibrates to 20.0 min.

[0115] In step five, the liquid chromatography-tandem mass spectrometry (LC-MS / MS) system is a high-resolution mass spectrometry system known in the art, equipped with a heated electrospray ionization source operating in positive ion mode. Preferably, the high-resolution mass spectrometry system is the Orbitrap Q Exactive Plus mass spectrometry system.

[0116] In step five, mass spectrometry acquisition adopts a data-dependent acquisition mode, including one full scan MS. 1 and multiple data dependencies on MS 2 Scanning, with the full scan quality range being [missing information]. m / z 150-2000, MS 1 Resolution of 30,000-140,000, MS 2 The resolution is 10,000-35,000.

[0117] In step five, the full scan quality range m / z 150–2000; MS 1 70,000 resolution, MS 2 Resolution 17,500; step-normalized collision energy (NCE) of 15, 30 and 45. HESI parameters: spray voltage (positive ion) 3200V, capillary temperature 320℃, sheath gas 50, auxiliary gas 10, S-LensRFLevel 50; collision gas is nitrogen.

[0118] In step five, MS 2 The collision energy was normalized in a stepped manner, with normalized collision energies of 15, 30, and 45.

[0119] Step six, structural identification.

[0120] Based on MS 1 Precision quality and MS 2 Neutral loss fragments with neutral acyl cations and / or charge retention were used to confirm the molecular composition and oxidative functional group type of oxidized triglycerides.

[0121] In step six, MS of oxidized triglycerides 2 Structural confirmation includes detecting fatty acid acyl cations, diacylglycerol-like ions, and neutral loss fragments of corresponding fatty acids or oxidized fatty acids, with the combination of these fragments satisfying a preset fragment rule as the confirmation criterion.

[0122] Specifically, in this embodiment, the collected features are searched and initially screened in a database, and then [M+NH4] is used. + Channel based on precision quality (MS) 1 ) and fragment maps (MS) 2 The structure of oxidized triglycerides is confirmed based on the following criteria: the combination rules of fatty acid acyl cations, diacylglycerol-like ions, and charge-retaining neutral loss fragments; for oxidized triglycerides containing unstable functional groups such as peroxide groups (–OOH), charge-retaining neutral loss fragments are preferred for confirmation to reduce the risk of misjudgment caused by the easy cleavage of peroxide acyl ions under CID conditions.

[0123] In this embodiment, based on the aforementioned screening and confirmation rules, the identifiable oxidized triglycerides in the fried samples are summarized, and their identification information is shown in Table 1. Table 1 lists the theoretical m / z, retention time, adduct ion type, oxidation type, and neutral loss ion and characteristic identification ion information of each oxidized triglyceride.

[0124] Table 1 Summary of Identification Information of Oxidized Triglycerides in Fried Samples

[0125] As a specific example in this embodiment, Figure 5 The MS / MS fragmentation and structural identification of TG(16:0_18:1_18:2)+OH are illustrated. Figure 6 A schematic diagram of MS / MS fragmentation and structural identification of TG(16:0_18:2_18:2)+OOH is given; Figure 7 MS / MS fragmentation and structural identification of TG(18:1_18:1_18:1)+OOH are presented. It should be understood that the above molecules are only representative examples, and the types of oxidized triglyceride molecules that can be identified in actual samples are not limited to these.

[0126] Step 7: Isotope peak cluster analysis and source determination.

[0127] The oxidized triglyceride [M+NH4] + Addition ion theory m / z Let M be the peak intensity I(M+i) of isotope peaks M and M+1 to M+4 within a preset quality window, calculate the relative abundance ratio I(M+i) / I(M); compare the relative abundance ratio with the control group to determine the source of oxygen and / or hydrogen in oxidized triglycerides, and output the quantitative evaluation results.

[0128] In step seven, the monosubstitution mass increment used is Δm(D) = +1.006277Da and Δm( 18 O) = +2.004245Da, and the peak clusters from M+1 to M+4 are attributed based on the said quality increment.

[0129] In step seven, the basis for determining the source of oxygen is: if it contains... 18 If, under the conditions of O2 synthesis air, I(M+2) / I(M) and / or I(M+4) / I(M) exceed the preset threshold relative to the control group data, then the oxygen is determined to originate from gaseous oxygen. In step seven, the preset threshold is that I(M+2) / I(M) and / or I(M+4) / I(M) is more than 10% higher than the control group data.

[0130] In step seven, the basis for determining the hydrogen source is: if, under the condition of using D2O as the aqueous phase medium, I(M+1) / I(M) exceeds a preset threshold relative to the control group, then the hydrogen source is determined to be related to the aqueous phase; if, under the condition of using D2O as the aqueous phase medium, I(M+1) / I(M) exceeds a preset threshold relative to the control group, then the hydrogen source is determined to be related to the aqueous phase. 18 If, under O-labeled conditions, the ratio of I(M+2) / I(M) exceeds a preset threshold relative to the control group, the source of oxygen is determined.

[0131] Specifically, this embodiment uses the confirmed oxidized triglyceride [M+NH4]. + Addition ion theory m / z Let M be the peak intensity I(M+i) (i=1–4) of isotopes M and M+1 to M+4 within a mass window of ±5 ppm. Calculate the relative abundance ratio I(M+i) / I(M). The monosubstituted mass increment is Δm(D) = +1.006277 Da and Δm( 18 O) = +2.004245Da, based on which the contribution of the M+1 to M+4 peak clusters is determined.

[0132] In this embodiment, TG(16:0_18:1_18:2)+OH is specifically used as an example, and its MS under three conditions is analyzed. 1 The variation of isotopic peak clusters (M to M+4) is shown in Figures 8(a) to 8(c), where Figure 8(a) is under natural air + H2O conditions, and Figure 8(b) is under conditions containing H2O. 18 O2 synthesis of air + D2O conditions, Figure 8(c) shows the conditions for the synthesis of air + D2O. 16 O2 combines with air to form H2 18 O conditions; taking TG(18:1_18:1_18:1)+OOH as an example, its MS under the above three conditions 1 The variation of isotopic peak clusters is shown in Figures 9(a) to 9(c). Figure 9(a) shows the conditions under natural air + H2O, and Figure 9(b) shows the conditions under the conditions containing H2O. 18 O2 synthesis of air + D2O conditions, Figure 9(c) shows the conditions containing O2 and D2O. 16 O2 combines with air to form H2 18 Condition O. By comparing the relative intensity distribution of peaks M to M+4 under different tracer conditions, the relationship between D and... 18 The effect of O labeling on isotope peak clusters.

[0133] In this embodiment, specifically, the ratios of I(M+2) / I(M) and / or I(M+4) / I(M) under tracer conditions are compared with those under control conditions (natural air + H2O): when containing 18When the ratios I(M+2) / I(M) and / or I(M+4) / I(M) under O2 synthesis in air conditions increase by more than a preset threshold compared to the control conditions, the oxygen is determined to originate from gaseous oxygen; in this application example, the threshold is set to increase by more than 10%. A similar rule is used to determine the source of hydrogen: when the ratios I(M+1) / I(M) under D2O aqueous medium conditions increase by more than a preset threshold compared to the control conditions, the hydrogen source is determined to be related to the aqueous phase.

[0134] Specifically, in this embodiment, Figure 10 The results show the comparison of the relative abundance ratio of isotopic peak clusters of TG(16:0_18:1_18:2)+OH under three conditions with the change of frying time. Figure 11 The results comparing the relative abundance ratios of isotopic peak clusters of TG(18:1_18:1_18:1)+OOH under three conditions with varying frying time are presented. Under the control condition (natural air + H2O), the isotopic ratios generally remain at a relatively stable level; under the condition containing... 18 Under O2 synthesis air + D2O conditions, the ratio of m+2 / m to m+4 / m of TG(16:0_18:1_18:2)+OH increases significantly with frying time, and reaches a high plateau in the later stage (e.g., m+4 / m increases from about 1 to about 7, see details). Figure 10 ); while containing 16 O2 combines with air to form H2 18 Under O conditions, the above ratios also show an increasing trend, but the magnitudes differ. By comparing and analyzing the time series changes of m+1 / m, m+2 / m, m+3 / m, and m+4 / m, the relative contributions of gaseous oxygen and aqueous medium in the formation of oxidized triglycerides can be determined, and relative quantitative assessment results can be output.

[0135] In this embodiment, specifically for each confirmed oxidized triglyceride, its [M+NH4] is used. + The following information is output for the target ion: oxidation type, theoretical value. m / z The data includes retention time, isotope peak cluster ratio I(M+i) / I(M) (i=1–4), and their variation relative to control conditions; and a source determination label is generated based on threshold rules. The source determination label includes at least: when containing... 18 When I(M+2) / I(M) and / or I(M+4) / I(M) exceed the threshold under O2 conditions, it is marked as "gas-phase oxygen source". 18 Under O conditions, when the ratio exceeds the threshold, it is marked as "aqueous oxygen source"; when both conditions are met, it is marked as "mixed source". Hydrogen source is marked according to the I(M+1) / I(M) exceeding the threshold under D2O conditions. The relative quantitative index uses the peak area normalized to the total ion current and its change over time for comparative analysis between different conditions.

[0136] In summary, this invention provides a method and system for traceability of isotopes of oxidized triglycerides obtained by controlled atmosphere frying, using H2O, D2O, or H2¹. 8 O prepares starch blocks, heats oil in a jacketed reactor, and then introduces natural or containing natural gas after vacuuming and nitrogen replacement. 18 O2 / ¹ 6 The oil was fried in O2-synthesized air and then sampled. The oil sample was analyzed by liquid chromatography-high resolution mass spectrometry (LC-HPLC-MS), and the structure was confirmed based on characteristic ions and neutral loss. [M+NH4] was then extracted. + Isotope peak clusters according to D, 18 O-incremental attribution determines and quantifies the source of oxygen and hydrogen. The system includes gas control, pressure monitoring, and sampling valves, which, together with a sealing cap and a mesh inner liner, allow for material feeding without disrupting the atmosphere. This invention solves the problems of insufficient reliability in confirming the structure of oxidized triglycerides in frying systems, difficulty in source analysis, and poor repeatability of experimental conditions in existing technologies, achieving traceable determination and quantification of the source of oxygen and hydrogen in oxidized triglycerides.

Claims

1. A method for traceability of isotopes of oxidized triglycerides obtained by controlled atmosphere frying, characterized in that, The method includes the following steps: Step 1: Prepare a starch-based frying model: Starch is mixed with an aqueous medium and stirred to gelatinize to obtain a gelatinized slurry. The gelatinized slurry is transferred to a mold and compacted to form a block model. After the block model is balanced and regenerated, a starch-based fried model is obtained. In step one, the aqueous phase medium is H2O, D2O, and H2, respectively. 18 O; Step 2: Construct a controlled atmosphere frying system: Edible oil is added to the oil-filling liner of the controlled atmosphere frying system and heated while stirring. Step 3, Atmosphere Replacement and Marking: The reactor body (1) of the controlled atmosphere frying system is evacuated and replaced with inert gas. Then, the target gas is introduced into the reactor body (1) and pressurized. In step three, the target gases are natural air and gases containing... 18 O2 synthesis air and containing 16 O2 synthesized air; Step 4, Deep-frying cycle and sampling: The starch-based frying model prepared in step one is added to edible oil through the feed port (3) of the controlled atmosphere frying system. Oil samples are collected at 0h and every 4h during the frying process. The oil samples are then transferred to a pre-vacuumed and nitrogen-purged sealed container and stored at low temperature for testing. Step 5, Liquid Chromatography-Mass Spectrometry Detection: The oil sample obtained in step four was diluted and injected, and full-scan MS data were acquired using liquid chromatography-tandem mass spectrometry in positive ion mode. 1 and MS 2 Data were used to obtain characteristic ion information of triglycerides and oxidized triglycerides; Step Six, Structural Identification: Based on MS 1 Precision quality and MS 2 Neutral loss fragments with retained acyl cations and / or charge were used to confirm the molecular composition and oxidative functional group type of oxidized triglycerides. Step 7: Isotope peak cluster analysis and source determination: The oxidized triglyceride [M+NH4] + Addition ion theory m / z Let M be the peak intensity I(M+i) of isotope peaks M and M+1 to M+4 within a preset quality window, calculate the relative abundance ratio I(M+i) / I(M); compare the relative abundance ratio with the control group to determine the source of oxygen and / or hydrogen in oxidized triglycerides, and output the quantitative evaluation results.

2. The controlled atmosphere deep-frying oxidized triglyceride isotope tracing method as described in claim 1, characterized in that, In step one, the deuterium isotope abundance of D2O is not less than 99 atom%; the H2 18 O 18 O isotope abundance not less than 99% In step one, the starch mentioned is potato starch; In step one, the starch and aqueous medium are mixed at a mass ratio of 1:1.

5. In step one, the gelatinization conditions are as follows: gelatinize at 80°C and 100 rpm for 6 minutes to obtain a gelatinized paste. In step one, the mold is a stainless steel trough with dimensions of 10cm×1cm×1cm; the block model has dimensions of 1×1×1cm. In step one, the condition for equilibrium recombination is to undergo equilibrium recombination at 4°C for 8 hours after sealing.

3. The controlled atmosphere deep-frying oxidized triglyceride isotope tracing method as described in claim 1, characterized in that, In step two, the controlled atmosphere frying system includes a reactor body (1), and a lid (2) is installed on the top of the reactor body (1). The lid (2) is characterized by having a feed inlet (3), a sampling port (4), an air inlet (5), an exhaust port (6), a pressure gauge (7), and a pressure relief valve (8); a sampling valve (9) is installed on the sampling port (4); the air inlet (5) is connected to a gas cylinder (11) through an air inlet valve (10); and the exhaust port (6) is connected to a vacuum pump (13) through an exhaust valve (12). The feed inlet (3) is equipped with an openable sealing cover (14), and the top opening of the model net bag (15) is connected to the sealing cover (14); the reactor body (1) is fitted with an oil-filling inner liner (16), and the model net bag (15) is suspended inside the oil-filling inner liner (16).

4. The controlled atmosphere deep-frying oxidized triglyceride isotope tracing method as described in claim 3, characterized in that, In step two, the model net bag (15) is made of stainless steel 304 material. The model net bag (15) is connected to the sealing cover (14) through the hook (19). The mesh size of the model net bag (15) is 2mm. A magnetic heating stirrer (17) is provided at the bottom of the reactor body (1). A temperature control jacket (18) is fitted on the outer side wall of the reactor body (1).

5. The controlled atmosphere deep-frying oxidized triglyceride isotope tracing method as described in claim 1, characterized in that, In step two, the edible oil is palm oil, and the volume of edible oil added is 70 mL; In step two, the edible oil is heated to 180°C; the stirring is performed using magnetic stirring at 60 rpm. In step two, the volume of the oil-filling inner liner is 100 mL.

6. The controlled atmosphere deep-frying oxidized triglyceride isotope tracing method as described in claim 1, characterized in that, In step three, the inert gas is nitrogen, and the replacement is performed three times. In step three, including 18 O2 synthesis air 18 The volume fraction of O2 is 21%, and the remainder is N2; 18 O2 18 O isotope abundance not less than 99% In step three, the pressure is increased to 1 atmosphere.

7. The controlled atmosphere deep-frying oxidized triglyceride isotope tracing method as described in claim 1, characterized in that, In step four, the frying conditions are as follows: a single frying time of 3 minutes; frying is carried out in cycles of 1 hour, with a total frying time of 20 hours, and the edible oil is not changed during the total frying time; In step four, the temperature for low-temperature preservation is -80℃.

8. A controlled atmosphere frying system, comprising a reactor body (1), wherein a reactor lid (2) is mounted on the top of the reactor body (1), characterized in that, The vessel lid (2) is provided with a feed inlet (3), a sampling port (4), an air inlet (5), an exhaust port (6), a pressure gauge (7), and a pressure relief valve (8); a sampling valve (9) is installed on the sampling port (4); the air inlet (5) is connected to the gas cylinder (11) through an air inlet valve (10); the exhaust port (6) is connected to the vacuum pump (13) through an exhaust valve (12); The feed inlet (3) is equipped with an openable sealing cover (14), and the top opening of the model net bag (15) is connected to the sealing cover (14); the reactor body (1) is fitted with an oil-filling inner liner (16), and the model net bag (15) is suspended inside the oil-filling inner liner (16).

9. The controlled atmosphere frying system as described in claim 8, characterized in that, The model net bag (15) is made of stainless steel 304 and is connected to the sealing cap (14) by a hook (19); the mesh size of the model net bag (15) is 2mm.

10. The controlled atmosphere frying system as described in claim 8, characterized in that, The bottom of the reactor body (1) is equipped with a magnetic heating stirrer (17), and a temperature control jacket (18) is fitted on the outer side wall of the reactor body (1).

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