Application of diacylglycerol trimethyl homoserine in inspection of storage quality of rice

Abstract

The invention relates to the technical field of analysis and detection, in particular to application of diacylglycerol trimethyl homoserine in inspection of storage quality of rice. Lipiomics detection finds that diacylglycerol trimethyl homoserine can be used as a biomarker and is used for rapidly, accurately and intuitively detecting the storage quality change condition of rice only by comparing the substance concentration difference multiples of the diacylglycerol trimethyl homoserine before and after the rice is stored; wide application prospects are realized.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to application of diacylglycerol trimethyl homoserine in testing the storage quality of rice. Background Art

[0002] The consumption characteristics of rice determine that it must be stored for a certain period of time before it can be sold, and polished rice also faces shelf life storage issues during the sales process. During storage, the physical and chemical properties and cooking characteristics of rice undergo many changes, which have a significant impact on the smell, appearance structure, taste, texture and overall taste quality of rice. Taste quality is an important basis for consumers to choose rice products. Therefore, detecting whether the quality of rice has deteriorated during storage is of great significance to maintaining the excellent quality of rice and reducing food losses.

[0003] Lipidomics is a high-throughput analytical method that can identify new lipid components, systematically analyze lipid metabolism and related metabolic pathways in biological tissues, organs and cells, and elucidate related biological activity processes and mechanisms. Currently, lipid composition profile analysis has been used for food processing, storage, quality, nutrition and health. The lipid content and fatty acid composition of rice are closely related to the taste quality. Lipidomics is mostly used to analyze the changes in brown rice lipid metabolism during storage. After long-term storage, brown rice lipids will be greatly reduced and degraded into low molecular weight substances such as free fatty acids, aldehydes, and ketones, becoming rancid (unpleasant odor), resulting in a decrease in taste quality. Wang et al. found that palmitoleic acid, cholesterol, linoleic acid and lauric acid are the four key metabolites of lipid metabolism during brown rice storage. During storage, TCA cycle, linoleic acid and α-linolenic acid are unique metabolic pathways of japonica rice. Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) gradually decrease during accelerated aging. However, there are relatively few studies on lipids during polished rice storage, and identifying the relationship between specific lipids and polished rice quality traits is of great significance for polished rice quality evaluation. The lipid substance - diacylglycerol trimethylhomoserine (DGTS) has been identified as a betaine lipid in lower organisms such as algae, and is involved in cell membrane formation under low-phosphorus conditions. Currently, there are no reports of its identification in higher plants, and its relationship with polished rice quality is also unknown. Since rice quality grading depends on the physical and sensory properties of polished rice, there is an urgent need to fully understand the important role of lipids in polished rice, and discovering lipid markers that can be used to test rice storage quality has become a technical problem that needs to be solved urgently in this field. Summary of the invention

[0004] The present invention uses lipidomics detection to find that diacylglycerol trimethyl homoserine can be used as a biomarker to detect changes in the storage quality of rice. Based on this, the following technical solution is proposed.

[0005] Firstly, the present invention provides the application of diacylglycerol trimethyl homoserine in testing the storage quality of rice.

[0006] Preferably, the change in rice quality is determined by detecting the change in the content of diacylglycerol trimethylhomoserine in the rice before and after storage.

[0007] Preferably, when the content of diacylglycerol trimethylhomoserine in the rice after storage is reduced by 0.19 to 0.5 times compared with the content before storage, it indicates that the quality of the rice has deteriorated.

[0008] Preferably, the diacylglycerol trimethylhomoserine includes at least one of DGTS (18:1_20:4), DGTS (18:1_20:5), DGTS (18:2_20:5), DGTS (18:1_18:3), DGTS (16:0_20:4), DGTS (16:0_20:5), and DGTS (18:2_18:3).

[0009] Preferably, diacylglycerol trimethylhomoserine is analyzed and detected by UPLC-MS / MS.

[0010] Preferably, the UPLC-MS / MS detection conditions include: the chromatographic column is Thermo Accucore TM 30 columns, the chromatographic column specifications are 2.6 μm, 2.1 mm×100 mm, and the column temperature is below 45°C; mobile phase A is a mixture of acetonitrile, formic acid, ammonium formate and water. In mobile phase A, the volume ratio of acetonitrile to water is (1.4-1.6):1, the volume percentage of formic acid in mobile phase A is 0.08%-0.12%, and the concentration of ammonium formate is 8-12 mmol / L; mobile phase B is a mixture of acetonitrile, formic acid, ammonium formate and isopropanol. In mobile phase B, the volume ratio of isopropanol to acetonitrile is (8.8-9.2):1, the volume percentage of formic acid in mobile phase A is 0.08%-0.12%, and the concentration of ammonium formate is 8-12 mmol / L.

[0011] Preferably, the detection conditions of the UPLC-MS / MS also include: a gradient elution program as follows:

[0012] During gradient elution, the sum of the volume percentages of mobile phase A and mobile phase B is 100%.

[0013] Preferably, the detection conditions of the UPLC-MS / MS also include: a flow rate of 0.35 mL / min or less.

[0014] Preferably, the detection conditions of the UPLC-MS / MS also include: an injection volume of 1 to 5 μL.

[0015] Preferably, the detection conditions of the UPLC-MS / MS also include: an injector temperature of 2-6°C.

[0016] Preferably, the detection conditions of the UPLC-MS / MS further include: an ESI ion source, and performing mass spectrometry analysis in a multiple reaction monitoring (MRM) mode.

[0017] Preferably, the detection conditions of the UPLC-MS / MS also include: the sample pretreatment method is as follows: the polished rice sample is freeze-dried and ground, the sample is thawed, a lipid extract is added to the sample, the sample is mixed and shaken, and then water is added to shake and then allowed to stand, the lipid reconstitution solution is added after centrifugation and concentration, and the lipid reconstitution solution is used for UPLC-MS / MS analysis and detection after centrifugation; the lipid extract is a mixture of methyl tert-butyl ether and methanol (preferably, the volume ratio of methyl tert-butyl ether to methanol is 2-4:1), and the lipid reconstitution solution is a mixture of acetonitrile and isopropanol (preferably, the volume ratio of acetonitrile to isopropanol is 0.8-1.2:1).

[0018] In addition, the present invention also provides the use of diacylglycerol trimethyl homoserine in preparing a reagent or a kit for testing the storage quality of rice.

[0019] Preferably, the storage is carried out under natural storage conditions at room temperature.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses lipidomics detection to discover that diacylglycerol trimethyl homoserine can be used as a biomarker for quickly, accurately and intuitively testing the storage quality changes of rice. It only requires comparing the difference multiples of the substance concentration of diacylglycerol trimethyl homoserine before and after rice storage, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the OPLS-DA score graph before and after rice storage.

[0022] Figure 2 This is a graph of the VIP values ​​and fold changes of all differential lipids before and after rice storage.

[0023] Figure 3 It is the DGTS violin plot before and after rice storage.

[0024] Figure 4 It is the ROC analysis chart. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0027] Example 1 Screening of rice storage quality markers 1. Sample collection The rice varieties Jiyuanxiang No. 1 (first accumulated temperature zone) and Wuyoudao No. 4 (first accumulated temperature zone) planted in the adaptation zone were collected and managed according to the local planting methods. A total of 100 kg of each rice was collected and transported to the laboratory and stored in a constant temperature and humidity environment (temperature = 25°C, humidity <10%) for one week to ensure that the moisture content of all samples is uniform (about 14%). The newly ground rice samples were collected in 50 ml cryovials and stored in a -80°C refrigerator for later use.

[0028] 2. Storage, processing and taste quality analysis Samples were collected according to GB / T 3543.2-1995 and international seed inspection rules, and all these varieties were divided into polished rice, brown rice, and paddy, and stored at room temperature (about 25°C) and low temperature (about 10°C) from November 15, 2022 (0 days) to June 3, 2023 (200 days). The intervals ranged from 40 days to 200 days, with an interval of 20 days. Taste quality was measured by a rice taste analyzer (STA1B–CN, Satake, Japan). All measurements were averaged using triplicate values ​​for data analysis. Samples for lipidomics analysis were quickly frozen with liquid nitrogen and stored at -80°C, with 3 replicates per variety.

[0029] As can be seen from Table 1, the taste quality of different varieties under different storage conditions and temperatures began to decline after 140 days, which proves that 140 days is the key threshold for the decline in taste quality after storage.

[0030] Table 1 Analysis of the taste quality of different rice varieties under different storage conditions and states

[0031] 3. Metabolome pretreatment of samples The polished rice samples stored for 0 days and 140 days were freeze-dried and ground. After the samples were thawed, 0.020 ± 0.001 g of the ground dry sample was weighed and added to the corresponding 2 mL centrifuge tube, and then a steel ball (inner diameter of about 4 mm) was placed. 1 mL of lipid extraction solution (methyl tert-butyl ether: methanol = 3:1, V / V) was added, and the mixture was shaken at room temperature at 2500 r / min for 15 min. 300 μL of ultrapure water was added, shaken for 1 min, and placed in a refrigerator at 4°C for 10 min. Centrifuged at 12000 r / min for 10 min at 4°C, 200 μL of the supernatant was transferred to the corresponding 1.5 mL centrifuge tube, and concentrated at 20°C for 2 h until completely dry. 200 μL of lipid reconstitution solution (acetonitrile: isopropanol = 1:1, V / V) was added, vortexed for 3 min, and centrifuged at 12000 r / min for 3 min at 4°C. 120 μL of supernatant was transferred to a glass-lined tube in an injection vial for UPLC-MS / MS analysis.

[0032] 4. Lipidomics analysis of rice samples Chromatographic conditions parameters: 1) Chromatographic column: Thermo Accucore TM 30 columns (2.6 μm, 2.1 mm×100 mm id); 2) Mobile phase: Phase A, acetonitrile / water (60 / 40, V / V) (containing 0.1% formic acid, 10 mmol / L ammonium formate); Phase B, acetonitrile / isopropanol (10 / 90, V / V) (containing 0.1% formic acid, 10 mmol / L ammonium formate); 3) Gradient elution program: 0 min for A / B ratio of 80:20 (V / V), 2 min for 70:30 (V / V), 4 min for 40:60 (V / V), 9 min for 15:85 (V / V), 14 min for 10:90 (V / V), 15.5 min for 5:95 (V / V), 17.3 min for 5:95 (V / V), 17.5 min for 80:20 (V / V), and 20 min for 80:20 (V / V); 4) Flow rate: 0.35 ml / min; column temperature: 45°C; injection volume: 2 μL.

[0033] Mass spectrometer parameter settings: Ultra Performance Liquid Chromatography (UPLC) TMAD, https: / / sciex.com.cn / ) and tandem mass spectrometry (MS / MS) (QTRAP® 6500+, https: / / sciex.com.cn / ) were used for mass spectrometry analysis in multiple reaction monitoring (MRM) mode.

[0034] The source parameters were as follows: electrospray ionization (ESI) temperature 500 °C, mass spectrometer voltage 5500 V in positive ion mode, mass spectrometer voltage -4500 V in negative ion mode, ion source gas 1 (GS1) 45 psi, gas 2 (GS2) 55 psi, curtain gas (CUR) 35 psi. In the triple quadrupole, each ion pair was scanned and detected according to the optimized declustering potential (DP) and collision energy (CE).

[0035] 5. Data processing All mass spectrometry data acquisition and quantitative analysis of target compounds were completed using SCIEX Analyst WorkStation Software (Version 1.6.3). MSconventer software was used to convert the mass spectra into TXT format, and then peak enhancement and annotation were performed.

[0036] 6. Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) Orthogonal partial least squares discriminant analysis (OPLS-DA) is used to filter out signals that are irrelevant to the model classification, i.e., orthogonal signals, and to establish a reliable OPLS-DA model. The model quality parameters are: R 2 Y(cum)=0.945,Q 2 (cum) = 0.731, the model quality is good. The OPLS-DA score is shown in the figure Figure 1 As shown in the figure. After filtering out the noise signals irrelevant to the classification, the two groups of samples have good lipid spectrum separation on PC1 (i.e. t[1]P), that is, the two groups of samples are on the positive and negative sides of the principal component (PC1, i.e. t[1]P). The variability in the control group is significantly greater than that in the storage group, which is manifested by greater discreteness between samples in the control group.

[0037] 7. Differential lipids and their structural identification Since irrelevant orthogonal signals are filtered out, the differential lipids obtained are more reliable. The VIP (Variable Importance in the Projection) value of the first principal component of the OPLS-DA model (threshold>1) was used, combined with the t-test. p The differentially expressed lipids were found by univariate statistical analysis (Hypothesis testing) and fold change (FC) analysis, and lipids were screened by fold change ≥ 2 and fold change ≤ 0.5. The qualitative methods for differential lipids were: searching the NIST commercial database (comparing mass spectra and chromatographic retention time RT or retention index RI), and determining by comparison with standard material data.

[0038] The VIP values ​​and fold changes of all differential lipids before and after rice storage are shown in the figure. Figure 2 As shown. Therefore, the above method can separate the differences between the storage group and the control group with high sensitivity and specificity, and the number of lipids reached 21 (see Table 2), confirming the metabolic differences between the storage group and the control group, which can be used to detect the deterioration of rice storage taste quality. In future tests, only the above lipidomics tests and OPLS-DA analysis are required for the 21 lipids in the table. Violin plots are drawn for the first 21 differential lipids with the largest VIP values, as shown in Figure 3 As shown, it is used to display the data distribution and its probability density. The results show that these 21 lipid substances can distinguish the grouped samples, indicating that these differential lipids can be used as potential biomarkers for rice quality analysis before and after storage.

[0039] Table 2 Differential lipids between the storage group and the control group On the basis of the above scheme, biomarkers that can represent effective storage are further screened out from the above-mentioned differential lipids to further simplify the detection procedure while ensuring the accuracy of the detection.

[0040] 8. ROC (Receiver Operating Characteristic) curve analysis and biomarker identification of peak value storage after lipidomics UPLC-MS / MS analysis The ROC curve is drawn with (1-specificity) as the horizontal axis and sensitivity as the vertical axis. ROC curve analysis links the sensitivity and specificity of a test and is a comprehensive and scientific method for evaluating test items. The larger the area under the curve (AUC), the greater the diagnostic value. When AUC > 0.9, it means that the test has a higher accuracy.

[0041] The AUC values ​​of different differential lipids are shown in Table 3, and the ROC analysis of diacylglycerol trimethyl homoserine is shown in Figure 4 As shown in the figure, in the storage group, the AUC of diacylglycerol trimethyl homoserine (DGTS) was 0.910-0.958 after ROC analysis, and the AUC values ​​of other differential lipids were lower than DGTS. Therefore, selecting diacylglycerol trimethyl homoserine as a rice storage quality marker has a high accuracy.

[0042] Table 3 AUC values ​​of differential lipids

[0043] Example 2 Validation of markers In order to further verify the reliability of diacylglycerol trimethyl homoserine as a rice storage quality marker, this example selected other varieties of polished rice for verification, and the steps were as follows: Suijing 18 (second accumulated temperature zone) and Longjing 31 (third accumulated temperature zone) were selected as validation set samples and managed according to local planting methods. A total of 100 kg of each rice was collected and transported to the laboratory and stored in a constant temperature and humidity environment (temperature = 25 ° C, humidity <10%) for one week to ensure that the moisture content of all samples was uniform (about 14%). The newly ground polished rice samples were collected in 50 ml cryovials and stored in a -80 ° C refrigerator for standby use. After the polished rice samples were stored at room temperature for 140 days, the taste quality analysis method of Example 1 determined that the quality had deteriorated. The pretreatment method of Example 1 and the UPLC-MS / MS analysis method were used to detect the substance concentration of diacylglycerol trimethyl homoserine in the samples before and after polished rice storage, and the fold change was calculated. The test results of the fold change of diacylglycerol trimethyl homoserine before and after the deterioration of Longjing 31 and Suijing 18 after 140 days of storage are shown in Table 4.

[0044] Table 4 Verification results

[0045] It can be seen that diacylglycerol trimethylhomoserine has a high reliability as a rice storage quality marker for judging the quality changes of polished rice after storage.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of diacylglycerol trimethylhomoserine in testing rice storage quality.

2. The use according to claim 1, characterized in that: The quality changes of rice can be determined by detecting the changes in the content of diacylglycerol trimethylhomoserine in rice before and after storage.

3. The use according to claim 2, characterized in that: When the content of diacylglycerol trimethylhomoserine in rice after storage is reduced by 0.19~0.5 times compared with the content before storage, it indicates that the quality of rice has deteriorated.

4. The use according to claim 1, characterized in that: The diacylglycerol trimethylhomoserine includes at least one of DGTS (18:1_20:4), DGTS (18:1_20:5), DGTS (18:2_20:5), DGTS (18:1_18:3), DGTS (16:0_20:4), DGTS (16:0_20:5), and DGTS (18:2_18:3).

5. The use according to claim 1, characterized in that: Diacylglycerol trimethylhomoserine was detected by UPLC-MS / MS analysis.

6. The use according to claim 5, characterized in that: The UPLC-MS / MS detection conditions include: the chromatographic column is Thermo Accucore TM 30 columns, the chromatographic column specifications are 2.6 μm, 2.1 mm×100 mm, and the column temperature is below 45°C; mobile phase A is a mixture of acetonitrile, formic acid, ammonium formate and water. In mobile phase A, the volume ratio of acetonitrile to water is (1.4-1.6):1, the volume percentage of formic acid in mobile phase A is 0.08%-0.12%, and the concentration of ammonium formate is 8-12 mmol / L; mobile phase B is a mixture of acetonitrile, formic acid, ammonium formate and isopropanol. In mobile phase B, the volume ratio of isopropanol to acetonitrile is (8.8-9.2):1, the volume percentage of formic acid in mobile phase A is 0.08%-0.12%, and the concentration of ammonium formate is 8-12 mmol / L.

7. The use according to claim 6, characterized in that: The detection conditions of the UPLC-MS / MS also include: the gradient elution program is as follows: During gradient elution, the sum of the volume percentages of mobile phase A and mobile phase B is 100%.

8. The use according to claim 6, characterized in that: The detection conditions of the UPLC-MS / MS also include: a flow rate of less than 0.35 mL / min; and / or an injection volume of 1 to 5 μL.

9. The use according to claim 6, characterized in that: The detection conditions of the UPLC-MS / MS also include: the sample pretreatment method is as follows: the polished rice sample is freeze-dried and ground, the lipid extract is added to the sample after thawing, the sample is mixed and shaken, and then water is added to shake and then allowed to stand, centrifugation and concentration are followed by adding the lipid reconstitution solution, and the lipid reconstitution solution is used for UPLC-MS / MS analysis and detection after centrifugation; the lipid extract is a mixture of methyl tert-butyl ether and methanol, and the lipid reconstitution solution is a mixture of acetonitrile and isopropanol.

10. Use of diacylglycerol trimethyl homoserine in preparing a reagent or a kit for testing the storage quality of rice.

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