阅读说明：本技术 一种快速对番茄果实中花青素进行种类解析和含量测定的方法 (Method for rapidly carrying out species analysis and content determination on anthocyanin in tomato fruits ) 是由 王海敬 崔霞 邱正坤 周震 于 2020-06-29 设计创作，主要内容包括：本发明公开了一种快速对番茄果实中花青素进行种类解析和含量测定的方法。本发明使用甲醇/甲酸(9:1,v/v)为溶剂,加入内标后,对番茄果实中花青素进行液液萃取,4℃提取12h后,离心取上清,过膜后,对花青素的质谱信息和光谱信息进行同时采集,对番茄果实中花青素进行定量,并进行结构解析。该检测方法前处理过程简易,检测结果精确,不需要繁琐的前处理提纯步骤。该发明可用于对番茄果实中花青素进行定性定量分析。(The invention discloses a method for rapidly carrying out species analysis and content determination on anthocyanin in tomato fruits. The method comprises the steps of using methanol/formic acid (9:1, v/v) as a solvent, adding an internal standard, carrying out liquid-liquid extraction on anthocyanin in tomato fruits, extracting at 4 ℃ for 12 hours, centrifuging to obtain a supernatant, carrying out membrane filtration, simultaneously collecting mass spectrum information and spectrum information of anthocyanin, quantifying the anthocyanin in the tomato fruits, and carrying out structure analysis. The detection method has the advantages of simple pretreatment process, accurate detection result and no need of complicated pretreatment and purification steps. The method can be used for carrying out qualitative and quantitative analysis on anthocyanin in tomato fruits.)

1. A method for carrying out species analysis and content determination on anthocyanin in tomato fruits comprises the following steps:

1) freezing and grinding the raw materials, and extracting with an extractant a to obtain an extracting solution a; centrifuging, collecting supernatant, blowing with nitrogen, adding extractant b for redissolving, filtering, and collecting filtrate to obtain extract b;

the raw materials are separated tomato peel and tomato pulp;

2) analyzing the anthocyanin species of the extracting solution b by using liquid chromatography and mass spectrometry;

3) and (4) carrying out quantitative analysis on the content of different kinds of anthocyanin obtained by the extracting solution b, and completing the kind analysis and the content determination of the anthocyanin in the tomato fruits.

2. A method for performing species analysis on anthocyanin in tomato fruits comprises the following steps: step 1) and step 2) as recited in any one of claims 1 and 4-8.

3. A method for measuring the content of anthocyanin in tomato fruits comprises the following steps: the steps 1) -3) of any one of claims 1 and 4-8.

4. The method of claim 1, wherein: in the freezing step in the step 1), the freezing mode is liquid nitrogen freezing;

in the extraction step, the extractant a is a solution containing an internal standard; the internal standard is paeoniflorin 3-glucoside;

in the extractant a, a solvent is a mixed solution consisting of methanol and formic acid; the volume ratio of the methanol to the formic acid is 7-9: 1; specifically 9: 1;

in the extractant a, the concentration of an internal standard is 1-5 mug/ml; specifically 3.75 mug/ml;

the dosage ratio of the extractant a to the raw materials is specifically 0.2-0.8 g: 1-4 ml; specifically, 0.4 g: 4 ml;

in the extraction step, the temperature is-20 ℃ to 5 ℃; in particular to 4 ℃; the time is 10-14 h; in particular 12 h;

the extractant b is a mixed solution consisting of methanol and formic acid; the volume ratio of the methanol to the formic acid is 7-9: 1; specifically 9: 1; the volume ratio of the extractant b to the supernatant is as follows: 0.5 to 2; specifically 1: 2; the specific dosage of the supernatant is 2 ml;

in the centrifugation step, the rotation speed is 2500-; specifically 3900 rpm; the time is 18-22 min; specifically 20 min;

in the filtering step, the aperture of the filter membrane is 0.22 μm.

5. The method according to claim 1 or 4, characterized in that: in the step 2), the conditions of the liquid chromatography detection are as follows:

type of column: ACQUITY UPLC CSH C18;

chromatographic column parameters: 2.1mm × 100mm i.d.,1.7 μm;

mobile phase A: a mixed solution of acetonitrile and formic acid with a volume ratio of 95: 5;

mobile phase B: a mixed solution consisting of water and formic acid with a volume ratio of 95: 5;

the gradient elution procedure was: the initial volume proportion of the mobile phase A is 2.5%, the mobile phase A is increased from 2.5% to 10% within 5min, then is increased from 10% to 25% within 15min, is kept for 5min, and then is changed from 25% to 2.5% within 5 min;

flow rate: 0.15 ml/min;

column temperature: 25 ℃;

autosampler temperature: 20 ℃;

sample introduction volume: 1 μ l.

6. The method of claim 1, 4 or 5, wherein: in the step 2), the mass spectrometry detection conditions are as follows:

the mass spectrum is in a positive ion mode;

an ion source: an ESI source;

capillary voltage: 2.0 kV;

ion source temperature: 100 ℃;

desolventizing gas temperature: 300 ℃;

desolventizing air flow rate: 800L/h;

taper hole airflow: 50L/h:

mass spectrum acquisition mode: fast DDA mode;

analysis mode: a resolution mode;

the collection range of the parent ions is as follows: 900m/z to 1000 m/z;

the collection range of the daughter ions is as follows: 50m/z to 1000 m/z;

the collision energy range is 6V-80V;

spectral conditions: the wavelength range of the diode array detector is 190nm-800 nm.

7. The method according to any one of claims 1 to 6, wherein: in the step 2), the types of the obtained anthocyanidins include delphinidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside, petunidin-3- (caffeic acid acyloxy) -rutinoside-5-glucoside, delphinidin-3- (trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside, delphinidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside, petunidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside, petunidin-3- (trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside, and the like, Petunidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside, malvidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside, malvidin-3- (trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside, malvidin-3- (feruloyloxy) -rutinoside-5-glucoside, petunidin-3- (trans-p-coumaric acyloxy-rhamnoside) -glucoside-5-glucoside and malvidin-3- (p-methoxy-trans-p-coumaric acyloxy) -rutinoside-5-glucoside;

wherein the UV retention time of delphinidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside is 11.41 min; the MS retention time is 11.55 min;

the UV retention time of the petunidin-3- (caffeic acid acyloxy) -rutinoside-5-glucoside is 11.71 min; the MS retention time is 11.83 min;

the UV retention time of delphinidin-3- (trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside is 11.86 min; the MS retention time is 11.97 min;

the UV retention time of the delphinidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside is 12.45 min; the MS retention time is 12.58 min;

the UV retention time of the petunidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside is 13.01 min; the MS retention time is 13.16 min;

the UV retention time of the petunidin-3- (trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside is 13.35 min; the MS retention time is 13.52 min;

the UV retention time of the petunidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside is 13.95 min; the MS retention time is 14.11 min;

the UV retention time of the malvidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside is 14.61 min; the MS retention time is 14.77 min;

the UV retention time of the malvidin-3- (trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside is 14.99 min; the MS retention time is 15.08 min;

the UV retention time of the malvidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside is 15.33 min; the MS retention time is 15.68 min;

the UV retention time of the petunidin-3- (trans-p-coumaric acid acyloxy-rhamnoside) -glucoside-5-glucoside is 15.73 min; the MS retention time is 15.90 min;

the UV retention time of the malvidin-3- (p-methoxy-trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside is 16.07 min; the MS retention time was 16.18 min.

8. The method according to any one of claims 1 to 7, wherein: in the quantitative analysis in the step 3), the quantitative method is to perform semi-quantitative analysis on the internal standard equivalent; specifically, the peak area of the ultraviolet absorption chromatogram of the anthocyanin at 535nm is used for quantification.

9. The method according to any one of claims 1 to 8, wherein: in the step 3), the linear range is 1-1000 mug/mL;

the standard curve is y, 221.941 x-346.284; wherein y is the peak area of the internal standard substance; x is the concentration value of the internal standard substance, and the unit is mu g/mL;

limit of quantitation < 1. mu.g/mL.

10. The method according to any one of claims 1 to 9, wherein: the anthocyanidin is selected from delphinidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside, petunidin-3- (caffeic acid acyloxy) -rutinoside-5-glucoside, delphinidin-3- (trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside, delphinidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside, petunidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside, petunidin-3- (trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside, and pharmaceutically acceptable salts thereof, Petunidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside, malvidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside, malvidin-3- (trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside, at least one of malvidin-3- (feruloyloxy) -rutinoside-5-glucoside, petunidin-3- (trans-p-coumaric acyloxy-rhamnoside) -glucoside-5-glucoside and malvidin-3- (p-methoxy-trans-p-coumaric acyloxy) -rutinoside-5-glucoside;

specifically, the compound is at least one selected from the group consisting of petunidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside, malvidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside, petunidin-3- (trans-p-coumaric acid acyloxy-rhamnoside) -glucoside-5-glucoside and malvidin-3- (p-methoxy-trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside.

Technical Field

The invention relates to a method for rapidly carrying out species analysis and content determination on anthocyanin in tomato fruits.

Background

Because of the oxygen radical absorption capacity, anthocyanin is a substance beneficial to human health and also has medicinal value. In plants, anthocyanins can improve the ability of plants to resist stress. Therefore, the identification, purification and structural analysis of anthocyanins have been the focus of research. The structural mother nucleus of anthocyanidin is 2-phenylbenzopyran cation, and mainly comprises cyanidin (cyanidin), pelargonidin (pelargonidin), delphinidin (delphinidin), peonidin (peonidin), petuniadin (petuniadin) and malvidin (malvidin). The structural analysis of anthocyanins is important, but the analysis is difficult because of their high structural similarity.

Anthocyanins are synthesized via flavonoid metabolic pathways, as shown in figure 1. Under the action of a series of enzymes, phenylalanine synthesizes anthocyanin, including PAL and phenylalanine ammonia lyase; CA4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate-coa ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone-3-hydroxylase; FLS, flavonol synthase; f3 'H, flavonoid 3' -hydroxylase; f3 '5' H, flavonoid-3 ',5' -hydroxylase; OMT1, flavone 3' -O-methyltransferase 1; DFR, flavanonol reductase, and the like. Finally, the anthocyanin is formed through glycosylation modification and acylation modification and then is transported to a vacuole for storage.

Anthocyanins in tomato are biosynthesized under light induction. At present, there are 9 anthocyanins reported in tomato. Wherein petunidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside is the main component of the tomato anthocyanin. Tomato is a very popular fruit in the market, but its fruit anthocyanin content is not optimized. Therefore, it is necessary to optimize the content of anthocyanins in tomato by studying the mechanism of anthocyanins synthesis. Although researchers have attempted to increase the anthocyanin content in tomato fruits, precise regulation is difficult. Among them, one important reason is that the detailed structure of anthocyanins synthesized in tomatoes has not been completely resolved. Structural modifications of anthocyanins include hydroxylations, methylations, glycosylations and acylations. Hydroxylation and methylation are direct modifications of the anthocyanin structure by hydroxyl and methyl groups. Glycosylation is the modification of the hydroxyl group of the 2-phenylbenzopyran cation by a monosaccharide, disaccharide, or trisaccharide. The acylation modification is to further add coumaric acid, caffeic acid, ferulic acid, etc. to the hydroxyl group of the glycoside after glycosylation modification. These structural modifications increase the stability of anthocyanins.

The pretreatment and detection processes have important influence on the rapid quantification of anthocyanin and the accurate analysis of anthocyanin structure. In previous studies, samples were mostly processed by lyophilization. Due to the longer lyophilization time, some of the anthocyanin is lost during this process. The anthocyanin is purified by column chromatography or solid phase extraction. Column chromatography requires a special column, and the experimental technical requirements are high. The solid phase extraction method requires special equipment and purification columns, is expensive, and requires steps such as activation, equilibration, sample loading, washing, elution, and the like. Due to the long process flow, less anthocyanins, such as malvidin-like anthocyanins, may be lost during purification. To ensure that there is sufficient anthocyanin for detection after pretreatment, a large sample, such as 1kg of tomato peel, needs to be taken. Meanwhile, a large amount of solvent is also used for achieving the purpose of complete extraction. In addition, the addition of acid to the extraction solvent increases the stability of anthocyanins, but mineral acids may lead to degradation of the sample, for example hydrochloric acid may lead to hydrolysis of glycosidic bonds.

Disclosure of Invention

The invention aims to provide a method for rapidly carrying out species analysis and content measurement on anthocyanin in tomato fruits.

The invention provides a method for carrying out species analysis and content determination on anthocyanin in tomato fruits, which comprises the following steps:

1) freezing and grinding the raw materials, and extracting with an extractant a to obtain an extracting solution a; centrifuging, collecting supernatant, blowing with nitrogen, adding extractant b for redissolving, filtering, and collecting filtrate to obtain extract b;

the raw materials are separated tomato peel and tomato pulp;

2) analyzing the anthocyanin species of the extracting solution b by using liquid chromatography and mass spectrometry;

3) and (4) carrying out quantitative analysis on the content of different kinds of anthocyanin obtained by the extracting solution b, and completing the kind analysis and the content determination of the anthocyanin in the tomato fruits.

The invention also provides a method for carrying out species analysis on anthocyanin in tomato fruits, which comprises the following steps: step 1) and step 2) of the aforementioned method.

The invention also provides a method for measuring the content of anthocyanin in tomato fruits, which comprises the following steps: step 1) -step 3) of the aforementioned method.

In the freezing step in the step 1) of the method, the freezing mode is liquid nitrogen freezing;

in the extraction step, the extractant a is a solution containing an internal standard; the internal standard is paeoniflorin 3-glucoside (namely peonidin-3-glucoside chloride or chloridized paeoniflorin-3-O-glucoside);

in the extractant a, a solvent is a mixed solution consisting of methanol and formic acid; the volume ratio of the methanol to the formic acid is 7-9: 1; specifically 9: 1;

in the extractant a, the concentration of an internal standard is 1-5 mug/ml; specifically 3.75 mug/ml;

the dosage ratio of the extractant a to the raw materials is specifically 0.2-0.8 g: 1-4 ml; specifically, 0.4 g: 4 ml;

in the extraction step, the temperature is-20 ℃ to 5 ℃; in particular to 4 ℃; the time is 10-14 h; in particular 12 h;

the extractant b is a mixed solution consisting of methanol and formic acid; the volume ratio of the methanol to the formic acid is 7-9: 1; specifically 9: 1; the volume ratio of the extractant b to the supernatant is as follows: 0.5 to 2; specifically 1: 2; the specific dosage of the supernatant is 2 ml;

in the centrifugation step, the rotation speed is 2500-; specifically 3900 rpm; the time is 18-22 min; specifically 20 min;

in the filtering step, the aperture of the filter membrane is 0.22 μm.

In the step 2), the conditions of the liquid chromatography detection are as follows:

type of column: ACQUITY UPLC CSH C18;

chromatographic column parameters: 2.1mm × 100mm i.d.,1.7 μm;

mobile phase A: a mixed solution of acetonitrile and formic acid with a volume ratio of 95: 5;

mobile phase B: a mixed solution consisting of water and formic acid with a volume ratio of 95: 5;

the gradient elution procedure was: the initial volume proportion of the mobile phase A is 2.5%, the mobile phase A is increased from 2.5% to 10% within 5min, then is increased from 10% to 25% within 15min, is kept for 5min, and then is changed from 25% to 2.5% within 5 min;

flow rate: 0.15 ml/min;

column temperature: 25 ℃;

autosampler temperature: 20 ℃;

sample introduction volume: 1 μ l.

In the step 2), the mass spectrometry detection conditions are as follows:

the mass spectrum is in a positive ion mode;

an ion source: an ESI source;

capillary voltage: 2.0 kV;

ion source temperature: 100 ℃;

desolventizing gas temperature: 300 ℃;

desolventizing air flow rate: 800L/h;

taper hole airflow: 50L/h:

mass spectrum acquisition mode: fast DDA mode;

analysis mode: a resolution mode;

the collection range of the parent ions is as follows: 900m/z to 1000 m/z;

the collection range of the daughter ions is as follows: 50m/z to 1000 m/z;

selecting at most 5 daughter ions for qualitative analysis;

the collision energy range is 6V-80V;

spectral conditions: the wavelength range of the diode array detector is 190nm-800 nm.

In the step 2), the types of the obtained anthocyanidins include delphinidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside, petunidin-3- (caffeic acid acyloxy) -rutinoside-5-glucoside, delphinidin-3- (trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside, delphinidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside, petunidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside, petunidin-3- (trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside, and the like, Petunidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside, malvidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside, malvidin-3- (trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside, malvidin-3- (feruloyloxy) -rutinoside-5-glucoside, petunidin-3- (trans-p-coumaric acyloxy-rhamnoside) -glucoside-5-glucoside and malvidin-3- (p-methoxy-trans-p-coumaric acyloxy) -rutinoside-5-glucoside;

wherein the UV retention time of delphinidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside is 11.41 min; the MS retention time is 11.55 min;

the UV retention time of the petunidin-3- (caffeic acid acyloxy) -rutinoside-5-glucoside is 11.71 min; the MS retention time is 11.83 min;

the UV retention time of delphinidin-3- (trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside is 11.86 min; the MS retention time is 11.97 min;

the UV retention time of the delphinidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside is 12.45 min; the MS retention time is 12.58 min;

the UV retention time of the petunidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside is 13.01 min; the MS retention time is 13.16 min;

the UV retention time of the petunidin-3- (trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside is 13.35 min; the MS retention time is 13.52 min;

the UV retention time of the petunidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside is 13.95 min; the MS retention time is 14.11 min;

the UV retention time of the malvidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside is 14.61 min; the MS retention time is 14.77 min;

the UV retention time of the malvidin-3- (trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside is 14.99 min; the MS retention time is 15.08 min;

the UV retention time of the malvidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside is 15.33 min; the MS retention time is 15.68 min;

the UV retention time of the petunidin-3- (trans-p-coumaric acid acyloxy-rhamnoside) -glucoside-5-glucoside is 15.73 min; the MS retention time is 15.90 min;

the UV retention time of the malvidin-3- (p-methoxy-trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside is 16.07 min; the MS retention time was 16.18 min.

In the quantitative analysis in the step 3), the quantitative method is to perform semi-quantitative analysis on the internal standard equivalent; specifically, the peak area of the ultraviolet absorption chromatogram of the anthocyanin at 535nm is used for quantification.

In the step 3), the standard curve is y-221.941 x-346.284; wherein y is the peak area of the internal standard substance; x is the concentration value of the internal standard substance and the unit is mu g/mL;

the linear range is 1-1000 mug/mL;

limit of quantitation < 1. mu.g/mL.

Specifically, the anthocyanidin is selected from delphinidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside, petunidin-3- (caffeic acid acyloxy) -rutinoside-5-glucoside, delphinidin-3- (trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside, delphinidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside, petunidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside, petunidin-3- (trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside, beta-gamma-glucosidase, beta-glucosidase, beta-, Petunidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside, malvidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside, malvidin-3- (trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside, at least one of malvidin-3- (feruloyloxy) -rutinoside-5-glucoside, petunidin-3- (trans-p-coumaric acyloxy-rhamnoside) -glucoside-5-glucoside and malvidin-3- (p-methoxy-trans-p-coumaric acyloxy) -rutinoside-5-glucoside;

more specifically, the compound is at least one selected from the group consisting of petunidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside, malvidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside, petunidin-3- (trans-p-coumaric acid acyloxy-rhamnoside) -glucoside-5-glucoside and malvidin-3- (p-methoxy-trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside.

The tomatoes described in the present invention are various known species of tomatoes; specifically, the extract can be indigo tomato.

The invention establishes a method for quickly quantifying anthocyanin in tomato fruits, and simultaneously accurately analyzes the substance structure of the anthocyanin. The method comprises the steps of using methanol/formic acid (9:1, v/v) as a solvent, adding an internal standard, carrying out liquid-liquid extraction on anthocyanin in tomato fruits, extracting at 4 ℃ for 12 hours, centrifuging to obtain a supernatant, carrying out membrane filtration, simultaneously collecting mass spectrum information and spectrum information of anthocyanin, quantifying the anthocyanin in the tomato fruits, and carrying out structure analysis. The method has the advantages of small sample amount, less solvent, environmental friendliness and high extraction rate; the pretreatment process is simple, the detection result is accurate, and complicated pretreatment and purification steps are not needed. The study was carried out using indigo tomato as experimental material. The research results show that 12 anthocyanins are detected in the indigo tomato, and 4 anthocyanins are not reported. The research provides a good experimental basis for further improvement of anthocyanin in tomatoes. The method can be used for carrying out qualitative and quantitative analysis on the anthocyanin in the tomato fruits.

Drawings

FIG. 1 is a biosynthetic pathway for anthocyanin in fruits. PAL, phenylalanine ammonia lyase; CA4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate-coa ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone-3-hydroxylase; FLS, flavonol synthase; f3 'H, flavonoid 3' -hydroxylase; f3 '5' H, flavonoid-3 ',5' -hydroxylase; OMT1, flavone 3' -O-methyltransferase 1; DFR, flavanonol reductase.

FIG. 2 is a photograph of fruit red ripeness stage of indigo-blue tomato. This figure shows the fruit on the plant (a), top view of the fruit (b), bottom view of the fruit (c), after peel removal (d) and cross-sectional view (e). The scale bar of the picture is 2 cm.

Fig. 3 is a chromatogram of the pericarp (a) and the pulp (b) of the indigo tomato fruit collected by liquid chromatography-time-of-flight mass spectrometry. In addition, the ultraviolet spectrogram of the anthocyanin is also collected in the experiment. The ultraviolet spectra of anthocyanins at retention times of 13.35min (c) and 14.99min (d) are shown in the figure. The experiment also collects chromatograms of the pericarp (e) and the pulp (f) of the indigo tomato fruit at 535nm of the ultraviolet absorption peak.

FIG. 4 shows the secondary mass spectra of 12 anthocyanidins (from panel a to panel i).

Detailed Description

The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The instrument comprises the following steps: UPLC-QTOF/PDA (Waters ACQUITY UPLC I-Class-Xevo G2-XS QTOF/PDAeLambda Detector)

Indigo tomato: purchased from Johnny's Selected feeds (http:// www.johnnyseeds.com /);

balance: purchased from Sartorius.