Detection and analysis method for carotenoid and degradation product thereof

Abstract

The invention belongs to the technical field of compound detection, and particularly relates to a method for detecting and analyzing carotenoid and degradation products thereof. In order to rapidly, accurately and comprehensively monitor, detect and analyze carotenoid and degradation products thereof, the invention provides a method for detecting and analyzing carotenoid and degradation products thereof, which combines liquid chromatography-mass spectrometry with dynamic headspace to detect and analyze carotenoid and degradation products thereof which are different in concentration of a carotenoid reaction solution with time change. The types and the contents of substances in the reaction liquid are determined. According to the method, specific chromatography, mass spectrometry and dynamic headspace parameters are set to comprehensively monitor and detect the carotenoid and the degradation products thereof. Experimental results show that the variety and content of the carotenoid degradation product can be qualitatively and quantitatively obtained through the method, and a new way is provided for generation of the carotenoid degradation product; and more technical supports are provided for deeply researching a carotenoid degradation mechanism and improving the aroma quality of wine.

Description

Technical Field

[0001] The present invention belongs to the technical field of compound detection, and in particular relates to a detection and analysis method for carotenoids and degradation products thereof. Background Art

[0002] Carotenoids are a class of fat-soluble pigments widely found in nature. They belong to the terpene family of compounds and primarily include carotenes and xanthophylls. Carotenes include β-carotene, α-carotene, and lycopene. As important precursors of aroma and flavor compounds, carotenoids play a key role in numerous industries. Their degradation products, including retinoids, pigments, plant hormones, and aroma compounds, are substances with important functions in organisms. In the winemaking process, grains are rich in carotenoids, and the degraded isoprenoid compounds produced by their degradation, such as β-ionone and β-cyclocitral, have a profound impact on the aroma quality of wine.

[0003] At present, there are many ways to degrade carotenoids, such as physical degradation, chemical degradation and biological degradation. However, different degradation methods lead to different locations of chemical bond breakage of carotenoids, and the rates, types and contents of degradation products are significantly different. For example, the degradation products of β-carotene after the action of potassium permanganate oxidant include 8', 10', 12', 14', 15-carotenal, apo-carotene substances and carotene epoxides; and the products of β-carotene after ozone oxidation-NaBH4 reduction are mainly β-ionone, 2,3-epoxy-β-ionone, 3-oxy-β-ionone, dihydroactinolactone and 2,6,6-trimethylcyclohexene-1-acetaldehyde and other aroma substances; for example, high temperature can accelerate the degradation of carotenoids.

[0004] Therefore, in-depth research on the degradation mechanism of carotenoids and rapid, accurate and comprehensive monitoring, detection and analysis of carotenoid degradation products are of great significance for analyzing the origin and formation process of wine aroma and improving the aroma quality of wine. Summary of the Invention

[0005] In order to quickly, accurately and comprehensively monitor and detect and analyze carotenoids and their degradation products, the present invention provides a detection and analysis method for carotenoids and their degradation products.

[0006] To achieve the above application objectives, the technical solutions adopted in this application are as follows:

[0007] In a first aspect, the present invention provides a method for detecting and analyzing carotenoids and their degradation products, comprising the following steps:

[0008] Liquid chromatography-mass spectrometry (LC-MS) was combined with dynamic headspace (DHS) analysis to detect and analyze the types and contents of carotenoid reaction solutions containing different concentrations of trifluoroacetic acid (TFA) over time.

[0009] The volume concentration of TFA in the reaction solution is 0% to 10%.

[0010] The time point is 0 to 3 days after the carotenoid solution is prepared or the carotenoid reaction solution to which TFA is added is prepared.

[0011] Wherein, the carotenoids include at least one of β-carotene, α-carotene or lycopene.

[0012] The carotenoid degradation products include at least one of 13Z-β-carotene, 9Z-β-carotene, 10-apo-β-carotenal, 12-apo-β-carotenal, 8-apo-β-carotenal, 2(4H)-actinolactone, 5,6-epoxy-β-ionone, trans-β-ionone, β-cyclocitral or α-ionone.

[0013] Furthermore, the LC-MS is composed of a liquid chromatography Vanquish Flex Binary and an Orbitrap Exploris120 connected in series.

[0014] The LC-MS detection parameters and conditions are as follows:

[0015] Liquid phase conditions:

[0016] Chromatographic column: YMC C30 Carotenoid (150 mm × 2.1 × 2.6 μm);

[0017] Mobile phase A: methanol, acetonitrile and water; mobile phase B: methyl tert-butyl ether;

[0018] Column temperature: 20°C;

[0019] Injection volume: 1 μL;

[0020] Flow rate: 0.2 mL / min;

[0021] Gradient elution: 0-2 min, 0% B; 2-15 min, 41% B; 15-18 min, 80% B; 18-22 min, 100% B.

[0022] Mass spectrometry conditions:

[0023] Ion source: APCI;

[0024] Scan mode: Full MS-SIM MS;

[0025] Ion transfer tube temperature: 300°C;

[0026] Ion source heating temperature: 350°C;

[0027] Sheath gas flow: 45Arb;

[0028] Auxiliary gas flow: 5Arb;

[0029] Scan range: 40-1400;

[0030] First level resolution 12000:

[0031] SIM resolution: 60000.

[0032] Furthermore, the DHS parameters and detection conditions are as follows:

[0033] DHS conditions:

[0034] During sample pretreatment, accurately measure 50 μL of the reaction solution. Incubate at 35°C for 20 minutes on an oscillator at 800 rpm, then extract at 100 mL / min for 60 minutes. The hydrazine trapping medium is Tenax-TA. The purge gas is N2 (≥99.999%), at a temperature of 20°C, a flow rate of 20 mL / min, and a purge time of 8 minutes.

[0035] Thermal desorption and cold injection conditions:

[0036] Thermal desorption adopted the solvent evacuation mode, and the transfer line temperature was set to 260°C; initial temperature: 25°C; hold for 0.2 min; increase to 240°C at 300°C / min and hold for 6 min; cold injection port: initial temperature 40°C, equilibrate for 0.5 min, increase to 240°C at 10°C / s and hold for 10 min.

[0037] Furthermore, after the volatile substances are enriched by DHS, gas chromatography-mass spectrometry (GC-MS) is also used to detect and analyze them.

[0038] Preferably, the GC-MS detection parameters and conditions are as follows:

[0039] Chromatographic column: DB-WAX capillary column;

[0040] Heating program: initial temperature 40°C, hold for 2 min; increase the temperature to 250°C at 4°C / min, hold for 15 min;

[0041] Inlet temperature: 40°C;

[0042] Detector temperature: 250°C;

[0043] Carrier gas: He;

[0044] Flow rate: 1 mL / min, splitless.

[0045] Mass spectrometry detection conditions:

[0046] The electron impact ionization source was EI, the electron energy was 70 eV, the transfer line temperature was 230 °C, the ion source temperature was maintained at 230 °C, the quadrupole temperature was set to 150 °C, the scan mode was full scan, the scan range was m / z 35-350, and the solvent delay time was 2 min.

[0047] In a second aspect, the present invention further provides a method for accelerating the degradation of carotenoids and the generation of their degradation products, which comprises adding TFA at a volume concentration of 1 to 10% to a carotenoid solution.

[0048] Furthermore, the degradation products include at least one of 13Z-β-carotene, 9Z-β-carotene, 10-apo-β-carotenal, 12-apo-β-carotenal, 8-apo-β-carotenal, 2(4H)-actinolactone, 5,6-epoxy-β-ionone, trans-β-ionone, β-cyclocitral or α-ionone.

[0049] Beneficial Effects: This method combines LC-MS, DHS, and GC-MS analytical methods, enabling comprehensive monitoring and detection of carotenoids and their degradation products by setting specific chromatographic, mass spectrometric, and DHS parameters. Experimental results show that varying concentrations of TFA significantly affect the degradation, isomerization, and aldehyde formation of β-carotene, and can also alter the types and content of terpenoids. The degradation products primarily include norisoprenoid compounds such as trans-β-ionone, β-cyclocitral, and 5,6-epoxy-β-ionone. This method efficiently and rapidly obtains carotenoid degradation products, and can qualitatively and quantitatively determine their types and content, providing a new approach for the generation of carotenoid degradation products. It also enables the simultaneous and precise detection of non-volatile intermediates such as β-carotene isomers and apo-carotenal, along with volatile aroma components such as terpenes and aldehydes, providing further technical support for in-depth research into the degradation mechanisms of carotenoids and improving the aroma quality of wine. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a trend diagram of the content change of β-carotene detected by LC-MS without TFA at 0, 1, and 3 days in an embodiment of the present invention;

[0051] Figure 2This is a trend diagram of the content change of β-carotene at 0, 1, and 3 days after LC-MS detection of 1% TFA in an embodiment of the present invention;

[0052] Figure 3 This is a trend diagram of the content change of β-carotene at 0, 1, and 3 days after LC-MS detection of 10% TFA in an embodiment of the present invention;

[0053] Figure 4 This is the total ion current graph of β-carotene degradation products under natural degradation, 1% TFA, and 10% TFA on day 0 of DHS detection in an embodiment of the present invention;

[0054] Figure 5 This is the total ion current of β-carotene degradation products under natural degradation, 1% TFA, and 10% TFA on the first day of DHS detection in an embodiment of the present invention;

[0055] Figure 6 This is the total ion current graph of β-carotene degradation products under natural degradation, 1% TFA, and 10% TFA on the third day of DHS detection in an embodiment of the present invention;

[0056] Figure 7 This is a graph showing the change over time of terpenoids produced by natural degradation of β-carotene detected by DHS in an embodiment of the present invention;

[0057] Figure 8 This is a graph showing the change of terpenoids produced by β-carotene under the action of 1% TFA detected by DHS in an embodiment of the present invention over time;

[0058] Figure 9 This is a graph showing the change of terpenoids produced by β-carotene under the action of 10% TFA over time detected by DHS in an embodiment of the present invention. DETAILED DESCRIPTION

[0059] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clear, the application is further described in detail below in conjunction with the embodiments. Unless otherwise defined, all scientific and technical terms used herein have the same meanings as understood by ordinary technicians in this field.

[0060] In one embodiment of the present invention, ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) analysis combined with dynamic headspace (DHS) sampling technology and gas chromatography-mass spectrometry (GC-MS) analysis is used to comprehensively monitor the carotenoid degradation process. During the monitoring process, on the one hand, DHS technology is used to qualitatively monitor volatile substances; on the other hand, based on ultra-high performance liquid chromatography combined with quadrupole-electrostatic field orbitrap mass spectrometry, scan mode is used to quickly monitor carotenoid degradation intermediates, and by comparing with a standard database, non-volatile intermediate substances in the reaction process are accurately identified and analyzed. At the same time, the present invention has also successfully established a dynamic headspace sampling and gas chromatography-mass spectrometry method for determining volatile substances produced during the degradation of β-carotene.

[0061] In one embodiment of the present invention, UPLC-MS analysis utilizes a reversed-phase column, mobile phase A is a mixture of methanol, acetonitrile, and water in a specific ratio, and mobile phase B is methyl tert-butyl ether. Gradient elution is employed to effectively separate the target substance. Mass spectrometry is performed using an atmospheric pressure chemical ionization source, positive ion mode, and FullMS scanning mode to ensure high sensitivity and accuracy.

[0062] In one embodiment of the present invention, DHS sampling technology is combined with GC-MS analysis. This technique is not only suitable for detecting highly volatile components in complex matrices, but also exhibits excellent detection results for less volatile and less concentrated components. Furthermore, it requires a small sample volume, offers excellent reproducibility, high sensitivity, and is fully automated, enabling a comprehensive and accurate analysis of the overall profile of volatile components.

[0063] In one embodiment of the present invention, the test samples were prepared as follows: β-carotene was removed from cryopreserved water and dissolved in methyl tert-butyl ether. The reaction flask was then filled with nitrogen to create an anaerobic environment. TFA was added to the experimental group to obtain TFA-β-carotene reaction solutions with varying concentrations, which were then stirred at room temperature. A control group was stirred at room temperature under anaerobic conditions. Subsequently, at specific time points, the samples were tested using a dynamic headspace-mass spectrometry (MS / MS) method using pre-defined liquid chromatography-mass spectrometry (LC-MS) conditions to obtain accurate experimental data.

[0064] Specific examples will be listed below to explain the scheme of the present invention. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product specifications. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0065] The carotenoid standards used in the following examples were purchased from Nature Carotene, Switzerland.

[0066] Before the experiment, the samples and instruments were prepared as follows:

[0067] 1. Accurately weigh a certain amount of β-carotene standard and dissolve it in methyl tert-butyl ether (MTBE). Prepare all necessary chemical reagents, such as methanol, acetonitrile, and water in mobile phase A and MTBE in mobile phase B, ensuring that the reagent purities meet experimental requirements. Also, prepare sufficient high-purity nitrogen for purging and trap tube activation.

[0068] Instrument Commissioning: Strictly follow the instrument operating instructions to fully commission and calibrate the Vanquish Flex Binary liquid chromatography instrument, Orbitrap Exploris 120 mass spectrometer, dynamic headspace device, and gas chromatography-mass spectrometry instrument. Check all instrument performance indicators to ensure optimal operating conditions. Activate Tenax TA adsorption tubes by placing them in a nitrogen atmosphere at 300°C at a flow rate of 50 mL / min for 30 minutes. After activation, store the tubes securely for future use.

[0069] 2. Instrument detection parameters and conditions

[0070] (1) Liquid chromatography-mass spectrometry (LC-MS) instrument conditions:

[0071] The data acquisition system based on ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) detection mainly consists of a liquid chromatography Vanquish Flex Binary and an Orbitrap Exploris 120 connected in series.

[0072] Liquid phase conditions:

[0073] Chromatographic column: YMC C30 Carotenoid (150 mm × 2.1 × 2.6 μm);

[0074] Mobile phase A: methanol, acetonitrile, and water (volume ratio: 73.5:24.5:2); mobile phase B: methyl tert-butyl ether;

[0075] Column temperature: 20°C;

[0076] Injection volume: 1 μL;

[0077] Flow rate: 0.2 mL / min;

[0078] Gradient elution: 0-2 min, 0% B; 2-15 min, 41% B; 15-18 min, 80% B; 18-22 min, 100% B.

[0079] Mass spectrometry conditions:

[0080] Ion source: APCI;

[0081] Scan mode: Full MS-SIM MS;

[0082] Ion transfer tube temperature: 300°C;

[0083] Ion source heating temperature: 350°C;

[0084] Sheath gas flow: 45Arb;

[0085] Auxiliary gas flow: 5Arb;

[0086] Scan range: 40-1400;

[0087] First level resolution 12000:

[0088] SIM resolution: 60000.

[0089] (2) Dynamic headspace (DHS) experimental parameters:

[0090] DHS conditions:

[0091] During sample pretreatment, accurately measure 50 μL of the reaction solution. Incubate at 35°C for 20 minutes on an oscillator at 800 rpm, then extract at 100 mL / min for 60 minutes. The hydrazine trapping medium is Tenax-TA. The purge gas is nitrogen (N2, ≥99.999%) at a temperature of 20°C, a flow rate of 20 mL / min, and a purge time of 8 minutes.

[0092] Thermal desorption and cold injection conditions:

[0093] Thermal desorption adopted the solvent evacuation mode, and the transfer line temperature was set to 260°C; initial temperature: 25°C; hold for 0.2 min; increase to 240°C at 300°C / min and hold for 6 min; cold injection port: initial temperature 40°C, equilibrate for 0.5 min, increase to 240°C at 10°C / s and hold for 10 min.

[0094] (3) Gas chromatography-mass spectrometry (GC-MS) experimental conditions:

[0095] Chromatographic column: DB-WAX capillary column (60 m × 0.25 mm × 0.25 μm);

[0096] Heating program: initial temperature 40°C, hold for 2 min; increase the temperature to 250°C at 4°C / min, hold for 15 min;

[0097] Inlet temperature: 40°C;

[0098] Detector temperature: 250°C;

[0099] Carrier gas: high-purity helium (He, ≥99.999%);

[0100] Flow rate: 1 mL / min, splitless.

[0101] Mass spectrometry detection conditions:

[0102] The electron impact ionization source was EI, the electron energy was 70 eV, the transfer line temperature was 230 °C, the ion source temperature was maintained at 230 °C, the quadrupole temperature was set to 150 °C, the scan mode was full scan, the scan range was m / z 35-350, and the solvent delay time was 2 min.

[0103] Example 1

[0104] 1. Sample Preparation

[0105] Accurately measure appropriate amounts of β-carotene standard solution and add varying amounts of trifluoroacetic acid (TFA) to create β-carotene reaction solutions with volume concentrations of 1% TFA and 10% TFA. A β-carotene solution without TFA was also prepared as a control. Multiple replicates were run for each reaction solution to ensure the reliability and reproducibility of the experimental data.

[0106] 2. LC-MS detection of non-volatile substances

[0107] Perform the test according to the above LC-MS parameters and conditions. During the test, pay close attention to the instrument operating status to ensure the accuracy and stability of the test data.

[0108] Data Recording: The instrument automatically records the chromatograms and mass spectra of β-carotene and its degradation products at different time points (0 days, 1 day, and 3 days). The collected data is backed up in real time for subsequent analysis and processing.

[0109] 3. DHS testing for volatile substances

[0110] Sample pretreatment: Accurately weigh 50 μL of each reaction solution and transfer it to a headspace vial for oscillation. Oscillate at 800 rpm at 35°C for 20 minutes to allow the volatile components in the sample to fully release and reach gas-liquid equilibrium.

[0111] Purge and trap: The equilibrated sample bottle was connected to a dynamic headspace device and purged with high-purity nitrogen at a flow rate of 20 mL / min at 20°C for 8 min to purge the volatile components in the sample and capture them in the Tenax TA adsorption tube.

[0112] Desorption sampling: After the adsorption tube is captured, thermal desorption is performed. The adsorption tube is placed in a thermal desorption device and rapidly heated to 240°C and maintained for 6 minutes to desorb the volatile substances adsorbed on the capture tube. The desorbed volatile substances are then introduced into a gas chromatography-mass spectrometry (GC-MS) via a 260°C transfer line for analysis.

[0113] Data Collection: The instrument collects data related to volatile substances, including peak areas and retention times of typical carotenoid degradation products such as β-ionone, β-cyclocitral, and dihydroactin. Similarly, the collected data is backed up and organized in a timely manner.

[0114] 4. Data Analysis

[0115] 4.1 LC-MS detection of β-carotene content changes

[0116] (1) 0% TFA control group

[0117] Under natural degradation conditions, β-carotene undergoes slow isomerization, but the content of isomerization products is low. Figure 1 It can be seen that during the entire experimental period, the chromatographic peak morphology of β-carotene changed relatively little, and there was no significant decrease in content as obvious as in the TFA-containing group, indicating that in the absence of TFA interference, the isomerization process of β-carotene itself is slow and the content level can basically be maintained at a relatively stable level.

[0118] (2)1% TFA group

[0119] Under the action of 1% TFA, the content of β-carotene showed a gradual downward trend. Figure 2 The chromatographic peak area steadily decreased over time. While the degradation rate was relatively slow compared to the 10% TFA group, the degradation reaction continued over time. This suggests that although low-concentration TFA acts relatively slowly, it still has a significant impact on β-carotene, and over a longer period of time, it can still lead to a significant decrease in β-carotene content.

[0120] (3)10% TFA group

[0121] After adding 10% TFA, the β-carotene content showed a sharp decline. Figure 3 It can be clearly observed that the chromatographic peak intensity of β-carotene rapidly decreases during the initial reaction. For example, in the one-day test, the peak area is significantly reduced compared to the control group, indicating that a large amount of β-carotene has been degraded. This clearly demonstrates that high concentrations of TFA have an extremely strong degradative effect on β-carotene, significantly reducing its content in a short period of time and severely damaging its stability.

[0122] Under acidic conditions, the parent peak of β-carotene (all-trans, RT = 17.84 min) weakened with time ( Figure 1-Figure 3 ), while the cis-isomer peaks (e.g. 13Z-, RT = 16.13min; 9Z-, RT = 18.49min) were enhanced, proving that TFA destabilizes the double bond by protonation and induces isomerization. In the 10% TFA group, the parent peak intensity at 3d decreased to 40% of that at 0d, while the isomer peak intensity increased by 2 times ( Figure 3 ), indicating that the high concentration acidic environment significantly accelerates the isomerization reaction.

[0123] 4.2DHS detection of β-carotene degradation products

[0124] 4.2.1 Changes in terpenoid species: Figures 4-6 The experiment analyzed the dynamics of volatile degradation products of β-carotene under natural degradation, 1% TFA and 10% TFA conditions by total ion current (TIC). The results showed that time and acid concentration jointly regulated the degradation efficiency: 1% TFA significantly accelerated degradation in the short term (1 day), generating detectable volatile small molecules (such as aldehydes and ketones), with peak intensity significantly higher than natural degradation; while 10% TFA did not meet expectations at 1 day and 3 days, which may be due to excessive acidification leading to secondary decomposition or volatilization loss of the product (such as very low molecular weight substances not captured by GC-MS). And from Figure 4 、 Figure 5 and Figure 6 It can be seen that there are certain differences in the types of terpenoids detected in different systems. Terpenoids such as 2(4H)-actinolactone (2(4H)-Benzofuranone), 5,6-epoxide-β-ionone (β-lonon-5,6-epoxide), trans-β-ionone (trans-β-lonone), and β-Cyclocitral (β-Cyclocitral) were mainly detected in natural degradation; the types of substances in the products of the 10% TFA system were similar to those of the 1% TFA system, but the relative content of each substance was different. Over time (1 day and 3 days), the types of terpenoids in each group also changed. In the initial state, the degradation products of all conditions (natural degradation, 1% TFA, 10% TFA) were not obvious, indicating that the volatile substances generated by the reaction were not dominant, and the corresponding Figure 2 、 Figure 3 From the above, we can see that with the participation of TFA, the isomerization of β-carotene is dominant in the early stage.

[0125] 4.2.2 Changes in terpenoid content: Analyze the effect of different concentrations of TFA on the peak area of terpenoids. Figure 7 、 Figure 8 and Figure 9It can be seen that in the absence of TFA Figure 7 In the experiment, the peak area of terpenoids showed an overall upward trend with the extension of time, indicating that terpenoids will also undergo some changes under natural conditions, resulting in an increase in their content. Figure 8 and Figure 9 The changes in the peak areas of terpenoids are more complex. Figure 8 ), in the initial stage of the reaction (0-1 day), the peak area of some terpenoids increased rapidly, and then during the period of 1-3 days, the growth rate of the peak area slowed down, but still maintained an upward trend; 10% TFA ( Figure 9 ), the peak areas of terpenoids varied significantly between days 0 and 1, with some substances experiencing a sharp increase and others decreasing. Furthermore, the number of species decreased between days 0 and 3, exhibiting an overall trend different from that observed with 1% TFA. This suggests that TFA concentration significantly influences terpenoid content. Higher TFA concentrations may trigger more intense chemical reactions and lead to secondary decomposition or volatilization of the products, resulting in more complex and diverse changes in terpenoid content.

[0126] 4.3 Effect of TFA on β-carotene

[0127] 4.3.1 Degradation: TFA effectively degrades β-carotene, and the degradation rate is affected by its concentration. 10% TFA causes a sharp drop in β-carotene content, with the chromatographic peak intensity rapidly weakening during the initial reaction, resulting in a significant amount of β-carotene degradation within one day. At 1% TFA, β-carotene content gradually decreases, with a slower but continuous degradation rate. This degradation behavior may affect the β-carotene content in raw materials during the winemaking process, thereby altering the content of precursors to baijiu flavor compounds.

[0128] 4.3.2 Induced isomerization: The acidic environment of TFA first induces double bond isomerization of β-carotene (e.g. Figure 2 and 3 Isomerized products, 13Z-β-carotene and 9Z-β-carotene, were detected in both the 1% TFA and 10% TFA groups. The high concentration in the 10% TFA group accelerated the reaction of β-carotene molecules, resulting in a more pronounced isomerization peak early in the reaction. Despite the lower concentration in the 1% TFA group, a certain amount of isomerization products accumulated over time.

[0129] Isomerization products can also serve as intermediates, further degrading into volatile compounds (such as trans-β-ionone). However, this degradation process is influenced by TFA concentration: high TFA concentrations (10%) accelerate isomerization and degradation into smaller molecules, while low TFA concentrations (1%) tend to favor the formation of closed-ring volatile products (such as 5,6-epoxy-β-ionone). Isomerization alters the molecular structure of β-carotene, affecting its reactivity during the winemaking process and its ultimate contribution to baijiu flavor.

[0130] 4.3.3 Promoting Aldehyde Formation: Experiments detected aldehydes such as 10-apo-β-carotenal, 12-apo-β-carotenal, and 8-apo-β-carotenal, both in the presence of TFA (1% TFA group) and in the absence of TFA (control group). These aldehydes are metabolites of β-carotene oxidation and decomposition. Their chemical structures and biological activities differ from those of β-carotene and may act as new flavor substances or flavor precursors, affecting the flavor of liquor.

Claims

1. A method for detecting and analyzing carotenoids and their degradation products, characterized in that: The following steps are involved: The LC-MS and DHS analysis methods were combined to detect and analyze the types and contents of substances in the carotenoid reaction solution containing different concentrations of TFA over time.

2. The method for detecting and analyzing carotenoids and their degradation products according to claim 1, wherein: The volume concentration of TFA in the reaction solution is 0% to 10%.

3. The method for detecting and analyzing carotenoids and their degradation products according to claim 1 or 2, characterized in that: The time point is 0 to 3 days after the preparation of the carotenoid solution or the carotenoid reaction solution with the addition of TFA.

4. The method for detecting and analyzing carotenoids and their degradation products according to any one of claims 1 to 3, characterized in that: The carotenoids include at least one of β-carotene, α-carotene or lycopene; and the carotenoid degradation products include at least one of 13Z-β-carotene, 9Z-β-carotene, 10-apo-β-carotenal, 12-apo-β-carotenal, 8-apo-β-carotenal, 2(4H)-actinolactone, 5,6-epoxy-β-ionone, trans-β-ionone, β-cyclocitral or α-ionone.

5. The method for detecting and analyzing carotenoids and their degradation products according to any one of claims 1 to 4, characterized in that: The LC-MS detection parameters and conditions are as follows: (1) Liquid phase conditions: Column: YMC C30 Carotenoid; Mobile phase A: methanol, acetonitrile and water; mobile phase B: methyl tert-butyl ether; Column temperature: 20°C; Injection volume: 1 μL; Flow rate: 0.2 mL / min; Gradient elution: 0-2 min, 0% B; 2-15 min, 41% B; 15-18 min, 80% B; 18-22 min, 100% B; (2) Mass spectrometry conditions: Ion source: APCI; Scan mode: FullMS-SIM MS; Ion transfer tube temperature: 300°C; Ion source heating temperature: 350°C; Sheath gas flow: 45Arb; Auxiliary gas flow: 5Arb; Scan range: 40-1400; First level resolution 12000: SIM resolution: 60000.

6. The method for detecting and analyzing carotenoids and their degradation products according to any one of claims 1 to 5, characterized in that: The DHS parameters and test conditions are as follows: DHS conditions: During the sample pretreatment phase, the reaction solution was measured and incubated at 35°C for 20 minutes on an oscillator at 800 rpm, followed by extraction at 100 mL / min for 60 minutes. The hydrazine trapping filler was Tenax-TA. The purge gas was N2 at a purge temperature of 20°C, a purge flow rate of 20 mL / min, and a purge time of 8 minutes. Thermal desorption and cold injection conditions: Thermal desorption adopted the solvent evacuation mode, and the transfer line temperature was set to 260°C; initial temperature: 25°C; hold for 0.2 min; increase to 240°C at 300°C / min and hold for 6 min; cold injection port: initial temperature 40°C, equilibrate for 0.5 min, increase to 240°C at 10°C / s and hold for 10 min.

7. The method for detecting and analyzing carotenoids and their degradation products according to any one of claims 1 to 6, characterized in that: After the volatile substances are enriched by DHS, GC-MS is also used to detect and analyze them.

8. The method for detecting and analyzing carotenoids and their degradation products according to claim 7, wherein: The GC-MS detection parameters and conditions are as follows: Chromatographic column: DB-WAX capillary column; Heating program: initial temperature 40°C, hold for 2 min; increase the temperature to 250°C at 4°C / min, hold for 15 min; Inlet temperature: 40°C; Detector temperature: 250°C; Carrier gas: He; Flow rate: 1 mL / min, splitless; Mass spectrometry detection conditions: The electron impact ionization source was EI, the electron energy was 70 eV, the transfer line temperature was 230 °C, the ion source temperature was maintained at 230 °C, the quadrupole temperature was set to 150 °C, the scan mode was full scan, the scan range was m / z 35-350, and the solvent delay time was 2 min.

9. A method for accelerating the degradation of carotenoids and the generation of their degradation products, characterized in that: The method comprises adding TFA with a volume concentration of 1 to 10% to a carotenoid solution.

10. The method for accelerating the degradation of carotenoids and the generation of their degradation products according to claim 9, characterized in that: The degradation products include at least one of 13Z-β-carotene, 9Z-β-carotene, 10-apo-β-carotenal, 12-apo-β-carotenal, 8-apo-β-carotenal, 2(4H)-actinolactone, 5,6-epoxy-β-ionone, trans-β-ionone, β-cyclocitral or α-ionone.

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