阅读说明：本技术 准确区分紫海胆-中间球海胆和紫海胆上市性腺的方法 (Method for accurately distinguishing echinus-echinus intermedius and gonads of echinus on market ) 是由 常亚青 湛垚垚 宋坚 孙景贤 赵谭军 于 2021-08-16 设计创作，主要内容包括：本发明公开一种准确区分紫海胆-中间球海胆和紫海胆上市性腺的方法,是以15种小分子代谢物的相对含量标准区间值为标志区分紫海胆-中间球海胆杂交种上市性腺与紫海胆上市性腺,首先取待区分的海胆上市性腺并经过前期处理制成样本,通过液相色谱-质谱联用分析仪对样本进行小分子代谢物检测,进而取样本中15种小分子代谢物的相对含量均位于标准区间值的海胆上市性腺为紫海胆-中间球海胆杂交种的性腺。可以准确地将紫海胆-中间球海胆杂交种上市性腺与紫海胆上市性腺进行区分,准确率达到100%,有助于紫海胆-中间球海胆和紫海胆上市性腺的质量分级以及定价。(The invention discloses a method for accurately distinguishing a purple sea urchin-Strongylocentrotus intermedius and a gonad of the purple sea urchin in the market, which is characterized in that a standard interval value of relative contents of 15 small molecule metabolites is used as a mark to distinguish the gonad of the purple sea urchin-Strongylocentrotus intermedius hybrid from the gonad of the purple sea urchin in the market, firstly, the gonad of the sea urchin in the market to be distinguished is taken and is subjected to early-stage treatment to prepare a sample, a liquid chromatography-mass spectrometry combined analyzer is used for detecting the small molecule metabolites of the sample, and further, the gonad of the sea urchins in the market, of which the relative contents of the 15 small molecule metabolites are all located at the standard interval value, is taken as the gonad of the purple sea urchins-Strongylocentrotus intermedius. The sex glands of the purple sea urchin-Strongylocentrotus intermedius hybrids which come into the market can be accurately distinguished, the accuracy rate reaches 100%, and the quality grading and pricing of the sex glands of the purple sea urchin-Strongylocentrotus intermedius and the purple sea urchin are facilitated.)

1. A method for accurately distinguishing the sex glands of purple sea urchins, intermediate ball sea urchins and purple sea urchins on the market is characterized by sequentially comprising the following steps:

a. preparing a liquid chromatography-mass spectrometry sample from the sex gland of the purple sea urchin to be distinguished and the sex gland of the middle ball sea urchin to be listed or the sex gland of the purple sea urchin to be listed;

b. performing small molecule metabolite composition analysis on the sample by using a liquid chromatography-mass spectrometry analyzer to obtain the relative content of the small molecule metabolites in the sample;

c. selecting the gonad of the sea urchin, in which the relative contents of 15 small molecule metabolites are all located in a standard interval value, in the sample as the gonad of the purple sea urchin-Strongylocentrotus intermedius, otherwise, selecting the gonad of the purple sea urchin, wherein the standard interval values of the 15 small molecule metabolites and the relative contents are as follows:

5' -adenosine monophosphate 1544935) -2683833; piperazine citrate 27169 and 34038; phenoxybenzamine hydrochloride 16285-; tripidine 13729-27718; pan amine 7160 and 18202; adenine 57401-; phosphoglycolate phosphatase (22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:2(11Z,14Z)) 125-29861; n-acetylimidazole 4389-25508; (3S) -2-oxo-3-phenylbutyric acid 2922-21405; lysoPA (22:0/0:0) 251-11093; 1-dodecyl-sn-glycerol 2995-10281; 3 beta-hydroxypregna-5-en-20-one sulfate 1075-4203; 2,2' - (3-methylcyclohexane-1, 1-diacyl) diacetic acid 345-9888; (Ara-f)3-Hyp 1452-11140; prostaglandin E2 phenyl terephthalamide 133-8733.

2. The method for accurately distinguishing the marketed gonads of echinus-Strongylocentrotus intermedius and echinus according to claim 1, characterized in that the step a is sequentially as follows:

weighing 50 +/-2 mg of marketable gonads, adding the marketable gonads into a 2 mL centrifuge tube, and adding the marketable gonads into 500 uL of methanol solution precooled at the temperature of-20 ℃ and containing 1 ppm of 2-chlorophenylalanine, wherein the mass concentration of the methanol solution is 70%; ultrasonically homogenizing for 4 times, 30 HZ for 30 s each time; homogenizing, oscillating for 5 min, and standing on ice for 15 min; centrifuging at 4 ℃ and 12000 r/min for 10min, and sucking 400 uL of supernatant into another centrifuge tube; adding 500 uL ethyl acetate/methanol solution into the precipitate of the original centrifuge tube, wherein the volume ratio of ethyl acetate to methanol is 1: 3, oscillating for 5 min, standing for 15 min on ice, centrifuging for 10min at 4 ℃ and 12000 rmp, and taking 400 uL of supernatant; mixing the two supernatants, concentrating, and drying; adding 100 uL of 70% methanol solution into the dried substance, performing ultrasonic treatment for 3min, centrifuging at 4 deg.C and 12000 r/min for 3min, and sucking 60 uL of supernatant to obtain sample for liquid chromatography-mass spectrometry.

3. The method for accurately distinguishing the marketed gonads of echinus-Strongylocentrotus intermedius and echinus according to claim 2, characterized in that the b steps are as follows:

the conditions of the liquid chromatography-mass spectrometry analyzer are as follows:

a chromatographic column: waters T3C 18 column, i.d.2.1X 100 mm, 1.8 μm;

mobile phase A: 0.04% by mass of formic acid/water; mobile phase B: pure acetonitrile containing 0.04% by mass of formic acid;

column temperature: 35 ℃; flow rate: 0.3 ml/min; sample introduction amount: 1 uL;

converting the original data of the mass spectrum off-line into an mzML format through ProteWizard, extracting peaks by adopting an XCMS program, aligning, and correcting retention time; correcting peak areas by adopting an SVR method and filtering peaks with deletion rate of more than 50% in each group of samples; correcting the screened peaks, calculating peak areas in the small molecule metabolite spectrogram to determine the relative content of the metabolites, and determining the types of the small molecule metabolites.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for accurately distinguishing echinacea purpurea, echinacea intermedia and gonads of echinacea purpurea on the market.

Background

Edible sea urchins are important components of global fishery economy, and the gonads of the edible sea urchins are precious sea products popular with consumers and are also one of traditional medicines in traditional Chinese medicine. Strongylocentrotus intermedius (Strongylocentrotus intermedius) is also called Strongylocentrotus intermedius, and is native to northern Japan and the far east coast region of Russia. Compared with other edible sea urchins, the sea urchins in the middle ball have the characteristics of good gonad color, high unsaturated fatty acid content, sweet taste and the like, are well loved by consumers in southeast Asia, have increased market demand year by year, and have formed a culture scale in Liaoning, Shandong and the like at present. Research shows that the optimal growth water temperature of the intermediate sea urchins is 15-20 ℃, and when the water temperature is increased to 23 ℃, a great amount of death of the intermediate sea urchins can be caused. Purple sea urchins (Anthocidia riscrassipina) are sea urchins naturally distributed in the southern sea area of China, and have high tolerance to high-temperature environments. Experiments prove that the gonad of the hybrid of the purple sea urchin and the intermediate sea urchin, which is generated by hybridizing the purple sea urchin (male parent) and the intermediate sea urchin (female parent), at the age of 12 months is superior to the gonad quality of the female parent in the aspects of content of unsaturated fatty acid and active substances and the like. However, the gonads of the hybrid of echinus and echinus intermedia have similar colors to those of the gonads of echinus, and are difficult to distinguish by conventional color, elasticity, plumpness and other traditional indexes, and the unsaturated fatty acids of the partial gonads of the two echinus and bioactive substances have a certain coincidence interval in relative content, so that the two gonads cannot be distinguished accurately by using the difference point of the two gonads on the nutrient components as a mark. Therefore, the quality grading, pricing and other aspects of the gonads of the purple sea urchins, the Strongylocentrotus intermedius and the purple sea urchins are influenced to a certain extent.

Disclosure of Invention

The invention aims to solve the technical problems in the prior art and provides a method for accurately distinguishing the gonads of the purple sea urchins, the intermediate ball sea urchins and the purple sea urchins on the market,

the technical solution of the invention is as follows: a method for accurately distinguishing the sexual glands of the purple sea urchins, the Strongylocentrotus intermedius and the purple sea urchins on the market sequentially comprises the following steps:

a. preparing a liquid chromatography-mass spectrometry sample from the sex gland of the purple sea urchin to be distinguished and the sex gland of the middle ball sea urchin to be listed or the sex gland of the purple sea urchin to be listed;

b. performing small molecule metabolite composition analysis on the sample by using a liquid chromatography-mass spectrometry analyzer to obtain the relative content of the small molecule metabolites in the sample;

c. selecting the gonad of the sea urchin, in which the relative contents of 15 small molecule metabolites are all located in a standard interval value, in the sample as the gonad of the purple sea urchin-Strongylocentrotus intermedius, otherwise, selecting the gonad of the purple sea urchin, wherein the standard interval values of the 15 small molecule metabolites and the relative contents are as follows:

5' -adenosine monophosphate 1544935) -2683833; piperazine citrate 27169 and 34038; phenoxybenzamine hydrochloride 16285-; tripidine 13729-27718; pan amine 7160 and 18202; adenine 57401-; phosphoglycolate phosphatase (22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:2(11Z,14Z)) 125-29861; n-acetylimidazole 4389-25508; (3S) -2-oxo-3-phenylbutyric acid 2922-21405; lysoPA (22:0/0:0) 251-11093; 1-dodecyl-sn-glycerol 2995-10281; 3 beta-hydroxypregna-5-en-20-one sulfate 1075-4203; 2,2' - (3-methylcyclohexane-1, 1-diacyl) diacetic acid 345-9888; (Ara-f)3-Hyp 1452-11140; prostaglandin E2 phenyl terephthalamide 133-8733.

The step a is as follows in sequence:

weighing 50 +/-2 mg of marketable gonads, adding the marketable gonads into a 2 mL centrifuge tube, and adding the marketable gonads into 500 uL of methanol solution precooled at the temperature of-20 ℃ and containing 1 ppm of 2-chlorophenylalanine, wherein the mass concentration of the methanol solution is 70%; ultrasonically homogenizing for 4 times, 30 HZ for 30 s each time; homogenizing, oscillating for 5 min, and standing on ice for 15 min; centrifuging at 4 ℃ and 12000 r/min for 10min, and sucking 400 uL of supernatant into another centrifuge tube; adding 500 uL ethyl acetate/methanol solution into the precipitate of the original centrifuge tube, wherein the volume ratio of ethyl acetate to methanol is 1: 3, oscillating for 5 min, standing for 15 min on ice, centrifuging for 10min at 4 ℃ and 12000 rmp, and taking 400 uL of supernatant; mixing the two supernatants, concentrating, and drying; adding 100 uL of 70% methanol solution into the dried substance, performing ultrasonic treatment for 3min, centrifuging at 4 deg.C and 12000 r/min for 3min, and sucking 60 uL of supernatant to obtain sample for liquid chromatography-mass spectrometry.

The step b is as follows:

the conditions of the liquid chromatography-mass spectrometry analyzer are as follows:

a chromatographic column: waters T3C 18 column, i.d.2.1X 100 mm, 1.8 μm;

mobile phase A: 0.04% by mass of formic acid/water; mobile phase B: pure acetonitrile containing 0.04% by mass of formic acid;

column temperature: 35 ℃; flow rate: 0.3 ml/min; sample introduction amount: 1 uL;

converting the original data of the mass spectrum off-line into an mzML format through ProteWizard, extracting peaks by adopting an XCMS program, aligning, and correcting retention time; correcting peak areas by adopting an SVR method and filtering peaks with deletion rate of more than 50% in each group of samples; correcting the screened peaks, calculating peak areas in the small molecule metabolite spectrogram to determine the relative content of the metabolites, and determining the types of the small molecule metabolites.

The invention relates to a method for distinguishing the sexual gland of a purple sea urchin-Strongylocentrotus intermedius hybrid with the sexual gland of the purple sea urchin in the market by taking the relative content standard interval value of 15 small molecule metabolites as a mark, firstly taking the sexual gland of the sea urchin to be distinguished in the market and preparing a sample through pretreatment, detecting the small molecule metabolites of the sample by a liquid chromatography-mass spectrometry combined analyzer, and further taking the sexual gland of the sea urchin in the market, of which the relative contents of the 15 small molecule metabolites are all in the standard interval value, as the sexual gland of the purple sea urchin-Strongylocentrotus intermedius hybrid. The sex glands of the purple sea urchin-Strongylocentrotus intermedius hybrids which come into the market can be accurately distinguished, the accuracy rate reaches 100%, and the quality grading and pricing of the sex glands of the purple sea urchin-Strongylocentrotus intermedius and the purple sea urchin are facilitated.

Detailed Description

The invention obtains the marker-15 small molecule metabolites and relative content standard interval values according to the following steps:

step 1: respectively selecting 60 healthy and viable adult intermediate sea urchins and purple sea urchins for conventional culture, feeding 10-20g of fresh sea tangles to each sea urchins every day during the culture, wherein the culture water body is natural seawater (the salinity is 30 +/-1 and the pH value is 8.0) at the temperature of 20 +/-1 ℃, and the water change is carried out once every day and the water temperature difference before and after the water change is not more than 1 ℃;

step 2: during the propagation period, 30 pieces of echinocandis intermedia and 30 pieces of echinocandis intermedia are induced to spawn, 5 female echinocandis with the highest spawning amount and 5 male echinocandis intermedia with the best sperm quality are selected for hybridization, and offspring hybrid echinocandis are obtained; simultaneously, 5 male and female purple sea urchins are respectively selected for selfing (the selection standard is the same as that in the previous step); feeding the two groups of offspring normally according to the step 1, culturing the offspring to 16 months of age and meeting the requirement of marketing;

and step 3: at least 6 sea urchins are randomly selected in each group, the gonads are obtained through dissection, and samples for liquid chromatography-mass spectrometry are prepared according to the following methods:

weighing 50 +/-2 mg of tube foot, adding the tube foot into a 2 mL centrifuge tube, and adding the tube foot into 500 uL of methanol solution precooled at the temperature of minus 20 ℃ and containing 1 ppm of 2-chlorophenylalanine, wherein the mass concentration of the methanol solution is 70%; ultrasonically homogenizing for 4 times, 30 HZ for 30 s each time; homogenizing, oscillating for 5 min, and standing on ice for 15 min; centrifuging at 4 ℃ and 12000 r/min for 10min, and sucking 400 uL of supernatant into another centrifuge tube; adding 500 uL ethyl acetate/methanol solution into the precipitate of the original centrifuge tube, wherein the volume ratio of ethyl acetate to methanol is 1: 3, oscillating for 5 min, standing for 15 min on ice, centrifuging for 10min at 4 ℃ and 12000 rmp, and taking 400 uL of supernatant; mixing the two supernatants, concentrating, and drying; adding 100 uL of 70% methanol solution into the dried substance, performing ultrasonic treatment for 3min, centrifuging at 4 deg.C and 12000 r/min for 3min, and collecting 60 uL of supernatant to obtain sample for liquid chromatography-mass spectrometry;

and 4, step 4: performing small molecule metabolite composition analysis on the two groups of samples by adopting a liquid chromatography-mass spectrometry analyzer (LC-MS/MS) to obtain the relative content of the small molecule metabolites in each group of samples;

a chromatographic column: waters T3C 18 column, i.d.2.1X 100 mm, 1.8 μm;

mobile phase A: 0.04% by mass of formic acid/water; mobile phase B: pure acetonitrile containing 0.04% by mass of formic acid;

column temperature: 35 ℃; flow rate: 0.3 ml/min; sample introduction amount: 1 uL;

converting the original data of the mass spectrum off-line into an mzML format through ProteWizard, extracting peaks by adopting an XCMS program, aligning, and correcting retention time; correcting peak areas by adopting an SVR method and filtering peaks with deletion rate of more than 50% in each group of samples; correcting the screened peaks, and calculating peak areas in the spectrogram of the metabolites by utilizing SVR regression analysis to determine the relative content of the metabolites; comparing the obtained product with information such as the chromatographic passing speed of a known metabolite standard, the retention time of the metabolite in the mass spectrum after secondary dissociation and the like by using a MetDNA method according to the passing speed of the metabolite in the chromatogram, the retention time of the metabolite from the start of mass spectrum sample introduction to the time of reaching the peak top and the retention time of the metabolite in the mass spectrum after secondary dissociation, and determining the metabolite type and annotating the metabolite type; finally, carrying out statistical analysis through an R program;

and 5: and (3) further screening 15 small molecule metabolites with significant difference in relative content in hybrid glands of the echinacea purpurea and the echinacea purpurea-Strongylocentrotus by combining the P-value and the difference times value (FC) of univariate analysis, and determining the relative content interval value (shown in the following table).

As can be seen from the above table, the relative content interval values of the 15 small molecule metabolites in the gonads of the purple sea urchin and the purple sea urchin-Strongylocentrotus intermedius hybrid have great difference, so that the 15 small molecule metabolites and the relative content standard interval value thereof are used as markers for distinguishing the gonads of the purple sea urchin-Strongylocentrotus intermedius hybrid on the market.

The technical solution of the invention is as follows: a method for accurately distinguishing the sexual glands of the purple sea urchins, the Strongylocentrotus intermedius and the purple sea urchins on the market sequentially comprises the following steps:

a. taking gonads of the purple sea urchins to be distinguished and the intermediate ball sea urchins to be listed or gonads of the purple sea urchins to be listed, and preparing a sample for liquid chromatography-mass spectrometry:

weighing 50 +/-2 mg of marketable gonads, adding the marketable gonads into a 2 mL centrifuge tube, and adding the marketable gonads into 500 uL of methanol solution precooled at the temperature of-20 ℃ and containing 1 ppm of 2-chlorophenylalanine, wherein the mass concentration of the methanol solution is 70%; ultrasonically homogenizing for 4 times, 30 HZ for 30 s each time; homogenizing, oscillating for 5 min, and standing on ice for 15 min; centrifuging at 4 ℃ and 12000 r/min for 10min, and sucking 400 uL of supernatant into another centrifuge tube; adding 500 uL ethyl acetate/methanol solution into the precipitate of the original centrifuge tube, wherein the volume ratio of ethyl acetate to methanol is 1: 3, oscillating for 5 min, standing for 15 min on ice, centrifuging for 10min at 4 ℃ and 12000 rmp, and taking 400 uL of supernatant; mixing the two supernatants, concentrating, and drying; adding 100 uL of 70% methanol solution into the dried substance, performing ultrasonic treatment for 3min, centrifuging at 4 deg.C and 12000 r/min for 3min, and sucking 60 uL of supernatant to obtain sample for liquid chromatography-mass spectrometry.

b. Carrying out small molecule metabolite composition analysis on the sample by adopting a liquid chromatogram-mass spectrum combined analyzer to obtain the relative content of the small molecule metabolites in the sample:

the conditions of the liquid chromatography-mass spectrometry analyzer are as follows:

a chromatographic column: waters T3C 18 column, i.d.2.1X 100 mm, 1.8 μm;

mobile phase A: 0.04% by mass of formic acid/water; mobile phase B: pure acetonitrile containing 0.04% by mass of formic acid;

column temperature: 35 ℃; flow rate: 0.3 ml/min; sample introduction amount: 1 uL;

converting the original data of the mass spectrum off-line into an mzML format through ProteWizard, extracting peaks by adopting an XCMS program, aligning, and correcting retention time; correcting peak areas by adopting an SVR method and filtering peaks with deletion rate of more than 50% in each group of samples; correcting the screened peaks, and calculating peak areas in the spectrogram of the metabolites by utilizing SVR regression analysis to determine the relative content of the metabolites; comparing the obtained product with information such as the chromatographic passing speed of a known metabolite standard, the retention time of the metabolite in the mass spectrum after secondary dissociation and the like by using a MetDNA method according to the passing speed of the metabolite in the chromatogram, the retention time of the metabolite from the start of mass spectrum sample introduction to the time of reaching the peak top and the retention time of the metabolite in the mass spectrum after secondary dissociation, and determining the metabolite type and annotating the metabolite type;

c. selecting the gonad of the sea urchin, in which the relative contents of 15 small molecule metabolites are all located in a standard interval value, in the sample as the gonad of the purple sea urchin-Strongylocentrotus intermedius, otherwise, selecting the gonad of the purple sea urchin, wherein the standard interval values of the 15 small molecule metabolites and the relative contents are as follows:

5' -adenosine monophosphate 1544935) -2683833; piperazine citrate 27169 and 34038; phenoxybenzamine hydrochloride 16285-; tripidine 13729-27718; pan amine 7160 and 18202; adenine 57401-; phosphoglycolate phosphatase (22:6(4Z,7Z,10Z,13Z,16Z,19Z)/20:2(11Z,14Z)) 125-29861; n-acetylimidazole 4389-25508; (3S) -2-oxo-3-phenylbutyric acid 2922-21405; lysoPA (22:0/0:0) 251-11093; 1-dodecyl-sn-glycerol 2995-10281; 3 beta-hydroxypregna-5-en-20-one sulfate 1075-4203; 2,2' - (3-methylcyclohexane-1, 1-diacyl) diacetic acid 345-9888; (Ara-f)3-Hyp 1452-11140; prostaglandin E2 phenyl terephthalamide 133-8733.

Experimental example:

a. taking 6 purple-middle hybrid sea urchins with the shape of purple sea urchins and the specification of more than 16 months old and meeting the marketing requirement and 6 purple sea urchins individuals, respectively dissecting to obtain gonad tissues and labeling, and randomly disordering the sequence until the gonad tissues can not be distinguished to serve as sea urchins to be distinguished;

b. preparing the obtained gonadal tissue into a sample for liquid chromatography-mass spectrometry;

c. carrying out metabonomic analysis on each sample according to the method of the embodiment of the invention, taking the sea urchins with the relative contents of 15 small molecular metabolites all in a standard interval value as gonads for identifying purple sea urchins-Strongylocentrotus intermedius hybrids, and taking the rest of the sea urchins gonads as the gonads,

d. the comparison result with the label shows that the method for distinguishing the purple sea urchins, the Strongylocentrotus intermedius and the gonads on which the purple sea urchins appear in the market has the accuracy of 100 percent.