Screening method of pitaya anthocyanin metabolite based on full-targeted metabonomics and application of screening method

Abstract

The invention relates to the technical field of analysis and detection, in particular to a screening method of pitaya anthocyanin metabolite based on full-targeted metabonomics and application thereof.The method comprises the steps that pericarp and pulp of four pitaya varieties in the green ripe stage, the coloring stage and the mature stage are selected as raw materials, and samples are obtained after treatment; the method comprises the following steps: respectively extracting effective components from samples, and carrying out liquid chromatography-mass spectrometry detection on an extracting solution; detecting the obtained original data, performing anthocyanin substance difference analysis by using an OPLS-DA method, and screening out Plt; 0.05, VIPgt; according to the method, on the basis of a full-targeting metabonomics method, firstly, six types of 23 anthocyanin substances are identified through liquid chromatography-mass spectrometry, then the anthocyanin differential metabolites are obtained through an OPLS-DA method, the relative content of the differential metabolites has significant difference among the four varieties, the dragon fruit varieties can be effectively identified, and the identification accuracy of the dragon fruit varieties is improved. And the screening of the anthocyanidin differential metabolites of different colored pitaya varieties provides an important basis for a molecular mechanism of anthocyanidin formation.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a method for screening pitaya anthocyanin metabolites based on fully targeted metabolomics and an application thereof. Background Art

[0002] Pitaya, also known as red dragon fruit, fairy honey fruit, lover fruit, etc., belongs to the genus Hylocereus of the Cactaceae family. It is mainly distributed in tropical and subtropical regions of China. Because its whole body is a treasure and has nutritional, medicinal and economic value, it has gradually become one of the tropical fruits that are valued and paid attention to. It has broad application prospects in the functional food, medicine and cosmetics industries. Pitaya is rich in betacyanin and anthocyanin; Anthocyanin is a natural water-soluble pigment that is usually found in the stems, leaves, flowers, fruits, and seeds of plants. There are very few free anthocyanins in nature because the physical and chemical properties of anthocyanins themselves are extremely unstable. Usually, glycosyl bonds and glycosyl molecules condense to form organic matter in the form of anthocyanins. Currently, research on plant anthocyanin metabolic pathways is mostly focused on model plants such as Arabidopsis, tomato, and rice. As a representative of tropical fruits, there is little research on the distribution of anthocyanin content between the flesh and peel of different varieties of pitaya, the key anthocyanin substances of different varieties, and the molecular mechanism of the formation of anthocyanin metabolites. Summary of the invention

[0003] The main purpose of the present invention is to provide a method for screening anthocyanin metabolites of pitaya based on fully targeted metabolomics and its application.

[0004] To achieve the above object, the technical solution adopted by the present invention is: A method for screening pitaya anthocyanin metabolites based on fully targeted metabolomics, comprising the following steps: S1: The peels and pulps of four dragon fruit varieties, namely Baiyulong, Dahong 2, Qingyulong and Yanwoguo, at the green-ripening stage, color change stage and ripening stage were selected as raw materials, and samples were obtained after processing; S2: extracting effective components from the obtained samples respectively, and obtaining extracts for liquid chromatography-mass spectrometry detection; S3: The original data obtained were detected and the OPLS-DA method was used to perform differential analysis of anthocyanin substances, and the variables with P < 0.05, VIP > 1.0 were screened out as differential metabolites of anthocyanin.

[0005] Furthermore, the method utilizes a fully targeted metabolomics approach to identify a total of 23 anthocyanidin substances in 6 categories, wherein the 6 categories of anthocyanidin substances are cyanidin, delphinidin, malvidin, pelargonidin, petunidin and peonidin.

[0006] Further, the differential metabolites of anthocyanidins in the peel include 6-hydroxydelphinidin 3-glucoside, delphinidin 3-galactoside, delphinidin 3-sophoroside, luteolin 5,7-diglucoside, pelargonidin 3-robinoside, peonidin 3-rutinoside, and morning glory pigment 3-O-β-D-pyranoglucopyranoside, and the differential metabolites of anthocyanidins in the pulp include 6-hydroxydelphinidin 3-glucoside, delphinidin 3-galactoside, delphinidin 3-sophoroside, luteolin 5,7-diglucoside, pelargonidin 3-robinoside, peonidin 3-rutinoside, and morning glory pigment 3-O-β-D-pyranoglucopyranoside. The common anthocyanin metabolites in the peel and pulp are 6-hydroxydelphinidin 3-glucoside, delphinidin 3-sophoroside, luteolin 5,7-diglucoside, pelargonidin 3-sophoroside and peonyside 3-rutinoside, the unique anthocyanin metabolites in the peel are delphinidin 3-galactoside and morning glory pigment 3-O-β-D-pyranoglucoside; the unique anthocyanin metabolite in the pulp is morning glory pigment 3-rutinoside-5-glucoside.

[0007] Furthermore, the processing of step S1 is specifically as follows: 10 fruits with the same maturity period are selected for each sample, the peel is first washed with distilled water and dried with a paper towel, the peel of the upper, middle and lower parts of each fruit without spots and lesions is taken respectively, chopped and mixed, quick-frozen with liquid nitrogen for 1 minute, and finally placed in a -80°C ultra-low temperature refrigerator, after taking the peel, the middle part of the fruit is cut, the upper, middle and lower parts of each flesh are taken respectively, chopped and mixed, quick-frozen with liquid nitrogen for 1 minute, and finally placed in a -80°C ultra-low temperature refrigerator.

[0008] Furthermore, the extraction of the active ingredient in step S2 is specifically as follows: weigh the sample into a 2mL centrifuge tube, add 1000μL of a 60% methanol solution by mass, wherein the methanol solution contains 0.1mol / L hydrochloric acid and 0.1% disodium ethylenediaminetetraacetic acid by mass, vortex oscillate for 60s, then add 2 steel beads, put into a tissue grinder, grind at 50Hz for 120s, ultrasonically oscillate at 40°C for 10min at an ultrasonic power of 72W, and finally centrifuge at 12000rpm at 4°C for 10min, take the supernatant and filter it with a 0.22μm membrane, add the filtrate into the detection bottle, and wait for detection.

[0009] Furthermore, the chromatographic detection conditions of step S2 are: The liquid phase uses the Thermo Vanquish ultra-high performance liquid phase system; The chromatographic column was a Waters ACQUITY UPLC BEHC18 1.7 µm 2.1 × 100 mm, and the column temperature was 50 °C; In positive ion mode, mobile phase A was an aqueous solution containing 1% formic acid by volume, and mobile phase B was a formic acid methanol solution containing 1% formic acid by volume. The flow rate was 0.25 mL / min and the injection volume was 2 μL. The conditions of gradient elution were: 0.0-2.0 min, 5% B; 2.0-15.0 min, 5%-70% B; 15.0-15.1 min, 70%-95% B; 15.1-18.0 min, 95% B; 18.0-20.0 min, 95%-5% B; 20.0-25.0 min, 5% B.

[0010] Furthermore, the mass spectrometry detection conditions in step S2 are: A Thermo Q Exactive mass spectrometer was used, and the ionization mode was electrospray ionization positive mode. In the positive ion mode, the spray voltage was 3.5 kV, the sheath gas was 47 arb, the auxiliary gas was 15 arb, the capillary temperature was 320 °C, and the first-level full scan was performed with a resolution of 70,000. The first-level ion scan range was 150-1300 m / z, and HCD was used for secondary fragmentation. The collision energies were 10 eV, 50 eV, and 60 eV, and the second-level resolution was 17500. The first 4 ions of the acquisition signal were fragmented, and dynamic exclusion was used to remove unnecessary MS / MS information.

[0011] The present invention also provides an application of the anthocyanin metabolite in identifying dragon fruit varieties.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The present invention takes anthocyanins in pitaya as the research object, takes four varieties of pitaya, namely Baiyulong, Dahong No. 2, Yanwoguo and Qingyulong as raw materials, and based on a fully targeted metabolomics method, uses liquid chromatography-mass spectrometry to identify 23 anthocyanin substances in six categories, and then obtains anthocyanin differential metabolites through an OPLS-DA method. The relative contents of the differential metabolites are significantly different between the peel and the flesh of the four varieties, so that technicians in the field can intuitively identify and judge the varieties of pitaya. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Different colored pitaya varieties selected for the present invention; Figure 2 The anthocyanin substance classification diagram of different colored pitaya varieties of the present invention and the quantity and proportion of the classification; Figure 3 The PCA score diagram of anthocyanin substances of different colored pitaya varieties of the present invention; Figure 4 It is a classification heat map of anthocyanin substances of different colored pitaya varieties of the present invention; Figure 5 The Venn diagram of the difference in anthocyanin metabolites in the peel and pulp of different colored pitaya varieties of the present invention; Figure 6 This is an Upset diagram of the difference in anthocyanin metabolites in the peel and pulp of different colored pitaya varieties of the present invention; Figure 7 This is a diagram showing the abundance of seven differential metabolites in the pitaya peel of the present invention in four varieties; Figure 8 This is a diagram showing the abundance of six differential metabolites in the pitaya pulp of the present invention in four varieties. DETAILED DESCRIPTION

[0014] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Example 1

[0015] 1. Sample Collection and Preparation like Figure 1 As shown, the peels and pulps of four dragon fruit varieties, Baiyulong, Dahong No. 2, Qingyulong, and Yanwoguo, at the green-ripe stage, color change stage, and ripe stage were selected as raw materials. Ten fruits with the same ripening stage were selected for each sample. The peels were first washed with distilled water and dried with a paper towel. The peels of the upper, middle, and lower parts of each fruit without spots or lesions were respectively taken, chopped and mixed, quick-frozen with liquid nitrogen for 1 min, and finally put into a -80°C ultra-low temperature refrigerator. After taking the peels, the fruit was first cut from the middle part, and the upper, middle, and lower parts of each pulp were respectively taken, chopped and mixed, quick-frozen with liquid nitrogen for 1 min, and finally put into a -80°C ultra-low temperature refrigerator. After processing, the samples were obtained; 2. Extraction of active ingredients from samples Weigh the sample in a 2mL centrifuge tube, add 1000μL of a 60% methanol solution containing 0.1mol / L hydrochloric acid and 0.1% disodium ethylenediaminetetraacetic acid, vortex for 60s, then add 2 steel beads, put it in a tissue grinder, grind it at 50Hz for 120s, ultrasonically oscillate at 72W for 10min at 40℃, and finally centrifuge it at 12000rpm for 10min at 4℃. Take the supernatant and filter it with a 0.22μm membrane. Add the filtrate to the test bottle and wait for detection.

[0016] 3. Liquid chromatography-mass spectrometry detection 3.1 Chromatographic conditions The liquid phase was analyzed using a Thermo Vanquish (Thermo Fisher Scientific, USA) ultra-high performance liquid phase system, using an ACQUITY UPLC® BEHC18 (2.1 × 100 mm, 1.7 µm) (Waters, Milford, MA, USA) column, with a column temperature of 50 °C, in positive ion mode, mobile phase A was an aqueous solution containing 1% formic acid by volume, mobile phase B was a formic acid methanol solution containing 1% formic acid by volume, the flow rate was 0.25 mL / min, and the injection volume was 2 μL; The gradient elution conditions were: 0.0-2.0 min, 5% B; 2.0-15.0 min, 5%-70% B; 15.0-15.1 min, 70%-95% B; 15.1-18.0 min, 95% B; 18.0-20.0 min, 95%-5% B; 20.0-25.0 min, 5% B.

[0017] 3.2 Mass spectrometry conditions Thermo Q Exactive mass spectrometer (Thermo Fisher Scientific, USA), electrospray ion source (ESI). The positive ion spray voltage was 3.50 kV, the sheath gas was 47 arb, and the auxiliary gas was 15 arb. The capillary temperature was 320 °C, and the first-level full scan was performed at a resolution of 70000. The first-level ion scan range was 150-1300 m / z, and HCD was used for secondary fragmentation. The collision energy was 10 eV, 50 eV, and 60 eV, and the second-level resolution was 17500. The first 4 ions of the acquisition signal were fragmented, and dynamic exclusion was used to remove unnecessary MS / MS information.

[0018] Data processing and analysis 4.1 Anthocyanin Composition Through the full targeted metabolomics detection and analysis, a total of 23 anthocyanidins of 6 categories were identified in the 4 pitaya varieties, as shown in Table 1. The 6 categories of anthocyanidins are cyanidin, delphinidin, malvidin, pelargonidin, petunidin and peonidin. The results showed that delphinidin and petunidin are the most abundant anthocyanidins in pitaya, accounting for 26.09% of the six categories of anthocyanidins respectively. The principal component analysis showed that the three varieties of Baiyulong, Dahong No. 2 and Qingyulong are more closely related, while the bird's nest fruit is more distantly related to the other three varieties, such as Figure 3In addition, a cluster heat map of the four varieties was constructed based on the metabolomics data, and the results showed that the accumulation patterns of anthocyanins in the four varieties were significantly different, such as Figure 4 As shown, the bird's nest fruit in particular was very different from the other three varieties, which further demonstrated that the accumulation pattern of anthocyanins was highly species-dependent.

[0019] Table 1 Anthocyanin substances identified

[0020]

[0021] 4.2 Screening of differential metabolites of anthocyanins Based on the identified anthocyanin substances, the OPLS-DA method was used to analyze the differences in anthocyanin substances. The screening conditions were: P < 0.05, VIP > 1.0. In order to show the dependence of anthocyanin metabolites between peel and pulp of different varieties, a Venn diagram of anthocyanin metabolites was constructed ( Figure 5 ) and Upset graph ( Figure 6 ); The peels of four pitaya varieties were analyzed and seven anthocyanin metabolites were found, as shown in Table 2; by comparing the flesh of four varieties, six anthocyanin metabolites were found, as shown in Table 3; among them, five anthocyanins, including 6-hydroxydelphinidin 3-glucoside, delphinidin 3-sophoroside, luteolin 5,7-diglucoside, pelargonidin 3-sophoroside, and peonyside 3-rutinoside, are the common anthocyanin metabolites in peel and flesh, and the unique anthocyanin metabolites in peel are delphinidin 3-galactoside and morning glory pigment 3-O-β-D-pyranoglucoside; the unique anthocyanin metabolite in flesh is morning glory pigment 3-rutinoside-5-glucoside. The abundance of the five common anthocyanin metabolites in the peel and pulp was similar, that is, those that were abundant in the peel were also relatively abundant in the pulp. For example, 6-hydroxydelphinidin 3-glucoside was high in the peel of the Baiyulong variety and low in the peel of the bird's nest fruit ( Figure 7 ), its content in the pulp is also high in Baiyulong and low in bird's nest fruit ( Figure 8 ). In the peel of bird's nest fruit, the content of morning glory pigment 3-O-β-D-pyranoglucoside is relatively high, while in the fruit of bird's nest fruit, the content of morning glory pigment 3-rutinoside-5-glucoside is relatively high, which indicates that bird's nest fruit is rich in cyanidin substances, and cyanidin is related to its unique color.

[0022] Table 2 Differential metabolites of anthocyanins in peel

[0023] Table 3 Differential metabolites of anthocyanins in fruit pulp

[0024] It should be understood that the preferred specific embodiments of the present invention can be modified and varied by ordinary technicians in the field without creative work. Therefore, any technical solution that can be obtained by technicians in the field of technology through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A method for screening anthocyanin metabolites of pitaya based on fully targeted metabolomics, characterized in that: The following steps are involved: S1: The peels and pulps of four dragon fruit varieties, namely Baiyulong, Dahong 2, Qingyulong and Yanwoguo, at the green-ripening stage, color change stage and ripening stage were selected as raw materials, and samples were obtained after processing; S2: extracting effective components from the obtained samples respectively, and obtaining extracts for liquid chromatography-mass spectrometry detection; S3: The original data obtained were detected and the OPLS-DA method was used to perform differential analysis of anthocyanin substances, and the variables with P < 0.05, VIP > 1.0 were screened out as differential metabolites of anthocyanin.

2. According to claim 1, a method for screening anthocyanin metabolites of pitaya based on fully targeted metabolomics is characterized in that the method utilizes a fully targeted metabolomics method to identify a total of 23 anthocyanin substances in 6 categories, and the 6 categories of anthocyanin substances are cyanidin, delphinidin, malvidin, pelargonidin, petunidin and peonidin.

3. The method for screening anthocyanin metabolites of pitaya based on fully targeted metabolomics according to claim 1, characterized in that: The differential metabolites of anthocyanidins in the peel include 6-hydroxydelphinidin 3-glucoside, delphinidin 3-galactoside, delphinidin 3-sophoroside, luteolin 5,7-diglucoside, pelargonidin 3-robiniabiside, peonidin 3-rutinoside, and morning glory pigment 3-O-β-D-pyranoglucopyranoside, and the differential metabolites of anthocyanidins in the pulp include 6-hydroxydelphinidin 3-glucoside, delphinidin 3-sophoroside, luteolin 5,7-diglucoside, pelargonidin 3-robiniabiside, peonidin 3-rutinoside, and morning glory pigment 3-O-β-D-pyranoglucopyranoside. , morning glory pigment 3-rutinoside-5-glucoside, the common anthocyanin differential metabolites in the peel and pulp are 6-hydroxydelphinidin 3-glucoside, delphinidin 3-sophoroside, luteolin 5,7-diglucoside, pelargonidin 3-sophoroside and paeoniflorin 3-rutinoside, the unique anthocyanin differential metabolites in the peel are delphinidin 3-galactoside and morning glory pigment 3-O-β-D-pyranoglucopyranoside; the unique anthocyanin differential metabolite in the pulp is morning glory pigment 3-rutinoside-5-glucoside.

4. The method for screening anthocyanin metabolites of pitaya based on fully targeted metabolomics according to claim 1, characterized in that: The processing of step S1 is specifically as follows: 10 fruits with the same maturity period are selected for each sample, the peel is first washed with distilled water and dried with a paper towel, the peel of the upper, middle and lower parts of each fruit without spots and lesions are respectively taken, chopped and mixed, quick-frozen with liquid nitrogen for 1 minute, and finally placed in a -80°C ultra-low temperature refrigerator, after taking the peel, the middle part of the fruit is cut open, the upper, middle and lower parts of each flesh are respectively taken, chopped and mixed, quick-frozen with liquid nitrogen for 1 minute, and finally placed in a -80°C ultra-low temperature refrigerator.

5. The method for screening anthocyanin metabolites of pitaya based on fully targeted metabolomics according to claim 1, characterized in that: The effective ingredient extraction in step S2 is specifically as follows: weigh the sample into a 2mL centrifuge tube, add 1000μL of a 60% methanol solution by mass, wherein the methanol solution contains 0.1mol / L hydrochloric acid and 0.1% disodium ethylenediaminetetraacetic acid by mass, vortex oscillate for 60s, then add 2 steel beads, put into a tissue grinder, grind at 50Hz for 120s, ultrasonically oscillate at 40°C for 10min with an ultrasonic power of 72W, and finally centrifuge at 12000rpm at 4°C for 10min, take the supernatant and filter it with a 0.22μm membrane, add the filtrate into the detection bottle, and wait for detection.

6. The method for screening anthocyanin metabolites of pitaya based on fully targeted metabolomics according to claim 1, characterized in that: The chromatographic detection conditions of step S2 are: The liquid phase uses the Thermo Vanquish ultra-high performance liquid phase system; The chromatographic column was a Waters ACQUITY UPLC BEHC18 1.7 µm 2.1 × 100 mm, and the column temperature was 50 °C; In positive ion mode, mobile phase A was an aqueous solution containing 1% formic acid by volume, and mobile phase B was a formic acid methanol solution containing 1% formic acid by volume. The flow rate was 0.25 mL / min, and the injection volume was 2 μL. The conditions of gradient elution were: 0.0-2.0 min, 5% B; 2.0-15.0 min, 5%-70% B; 15.0-15.1min, 70%-95%B; 15.1-18.0min, 95%B; 18.0-20.0min, 95%-5%B; 20.0-25.0min, 5%B.

7. The method for screening anthocyanin metabolites of pitaya based on fully targeted metabolomics according to claim 1, characterized in that: The mass spectrometry detection conditions in step S2 are: A Thermo Q Exactive mass spectrometer was used, and the ionization mode was electrospray ionization positive mode. In the positive ion mode, the spray voltage was 3.5 kV, the sheath gas was 47 arb, the auxiliary gas was 15 arb, the capillary temperature was 320 °C, and the first-level full scan was performed with a resolution of 70,000. The first-level ion scan range was 150-1300 m / z, and HCD was used for secondary fragmentation. The collision energies were 10 eV, 50 eV, and 60 eV, and the second-level resolution was 17500. The first 4 ions of the acquisition signal were fragmented, and dynamic exclusion was used to remove unnecessary MS / MS information.

8. Use of the anthocyanin metabolite according to claim 2 or 3 in identifying pitaya varieties.

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