CN101865888A - Method for rapidly detecting nutrient content of plant by extracting - Google Patents
Method for rapidly detecting nutrient content of plant by extracting Download PDFInfo
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Abstract
The invention provides a method for rapidly detecting the nutrient content of a plant, in particular the nutrient content of a soybean polygene polymerization breeding filial generation. The method comprises the following steps of: extracting primary samples of substances, such as a water-soluble substance, namely, isoflavone and fat-soluble substances, namely, vitamin E, xanthophyll, carotene and the like at the same time; and performing a high performance liquid chromatography (HPLC) two-step analysis method to realize synchronous analysis and detection of various functional nutrients through primary samples. Therefore, the method has the advantages of accelerating the selection process of breeding generations, cultivating a new soybean variety with a high added value, shortening detection time, reducing the investments on manpower, material resources and financial resources and reducing the pollution of wastes to the environment.
Description
Technical field
The present invention relates to the rapid extraction detection method of the functional nutrient component content of a kind of plant nutrient ingredient, especially soybean, be particularly related to a kind of functional nutrient composition that can extract soybean multiple gene polymerization breeding cross offspring simultaneously and rapidly, and by measuring ability that HPLC had, it is carried out qualitative, quantitative methods, can screen its filial generation fast thus.
Background technology
Many vegetable seedss, especially soya seeds not only is rich in the vegetable protein and the fatty acid of high-quality, and contains multiple functional nutrient compositions such as isoflavones, vitamin E.Especially in recent years, along with growth in the living standard, the consumer is risen sharply to the demand of functional nutrient compositions such as isoflavones, vitamin E.But, in the existing soybean varieties of having bred popularization, still there is not the comprehensive agronomy proterties good, all high kinds of functional nutrient component content such as isoflavones, vitamin E, the processing and utilization that has greatly influenced soybean itself is worth, and has reduced the deep processing economic benefit of enterprises.
Utilize soybean gene convergent cross breeding technique, gene with functional nutrient component contents such as control isoflavones, vitamin E, xenthophylls, be aggregated in the same genome, thereby cultivate all high new soybean varieties of isoflavones, vitamin E and lutein content, improve the soybean comprehensive utilization value.Existing vitamin E (Tocopherol) extraction and analysis technology has multiple, and the past is used Emmerie-Engel method (EE method), fluorescence method, thin layer chromatography (TLC method) etc. always.In recent years, continuous improvement along with high performance liquid chromatography (HPLC) technology, utilize the liquid-phase chromatographic analysis technology can carry out simple qualitative and quantitative analysis (DellaPenna to four isomers α-Tocopherol (alpha-tocopherol), β-Tocopherol (betatocopherol), γ-Tocopherol (Gamma-Tocopherol) and the δ-Tocopherol (Delta-Tocopherol) of vitamin E substantially, 2005, Ujiieet al., 2005).Same as a kind of xenthophylls of carotenoid (Lutein: a kind of strong anti-oxidation material, the prevention of cataract, veteran form macular degeneration disease) (Monma et al., 1994, Kanamaru et al., 2006), and isoflavones (Isoflavone) the extraction and analysis technology of estrogen sample material, high performance liquid chromatography (HPLC) commonly used in recent years (Wang Chune etc. that carry out qualitative and quantitative analysis, 1998, Wang 1994).
These existing vitamin Es, xenthophylls and isoflavones analytical technology are being examined under the not limited prerequisite of sample number, quality, all can carry out seed vitamin E, xenthophylls and isoflavones to soya seeds and carry out qualitative and quantitative analysis.But classic method needs the quantity of sample or quality many, and extraction step is loaded down with trivial details, and the expense that wastes time and energy reagent is big for environment pollution.Problem is that the functional nutrient component content of every the seed of filial generation behind gene pyramiding detects aspect the choice, because the analysis and detection technology of these existing isoflavones, vitamin E and xenthophylls, all be analytic system independently separately, 1 sub-sampling can only be supported the choice of above-mentioned a kind of nutritional labeling.Wonder the content of above-mentioned two or three composition of the filial generation seed of each preciousness, must at twice or three times sampling be (each 〉=as 20mg), to carry out the qualitative and quantitative analysis of HPLC respectively respectively.And the quality of every seed of hybrid generation is less, and the proterties that seed has all has nothing in common with each other, if utilize prior art to it repeatedly behind the sample analysis, even if therefrom select the filial generation seed that meets breeding objective, because seed is by repeatedly sampling excision, behind the remainder planting seed, can't finish eventually the next generation and breed and breed, and then can't realize the breeding objective of expecting because of can not normally emerging.
With vitamin E and xenthophylls hybridization pyramiding breeding offspring is example, all less because of having parent's 100-grain weight of homovitamin E (maternal 14-16g/100 grain) and high lutein (6-8g/100 grain) content, hybridization single seed quality from generation to generation in early stage, have only about 60-100mg, after twice sample analysis screening, even if obtain the two high offsprings of vitamin E and lutein content, also can be lost offspring's reproduction ability by too much excision because of seed self, and then can't realize the breeding objective (Wang et al., 2007) of expecting.
And, when detecting the content of vitamin E in the prematurity soybean cotyledon etc., because of a lot of compositions in the seed are in the formation phase or are in the presoma state that desire detects composition, when detecting with traditional method, can detect the inter-adhesive peak of a lot of the unknowns, at all can't qualitative objective peak, the more accurately content of quantitative objective composition.Limited a lot of enzyme expression of gene parsings in the prematurity soybean cotyledon, and the exploitation of associated gene mark.
Therefore, in soybean (plant) multiple gene polymerization crossbreeding offspring selection process, press for a kind of and carry out " primary sample, but the technology of multiple functional nutrient composition simultaneous extraction, detection ".This problem is in case solution for soybean quality improvement breeding work person has solved " breeding bottleneck ", is fields such as cereal, food inspection also not only, and the detection method of providing convenience has reduced the detection cost, has reduced person property's waste and environmental pollution.
Summary of the invention
In order to solve in the individual selection process of soybean function nutritional labeling gene pyramiding breeding cross offspring, the same grain seed that exists in the same generation, the plurality of target composition that contains, can't carry out the difficult problem of quantitative detecting analysis simultaneously, the object of the present invention is to provide a kind of extraction detection method, to realize " primary sample, but multiple functional nutrient composition simultaneous extraction, detection ", and then accelerate the breeding offspring and select process, cultivate the high added value new soybean varieties; Also in order to shorten detection time, reduce the input of human and material resources, financial resources simultaneously, reduce the pollution that detects discarded object environment.
The present invention has realized above-mentioned purpose by the improvement to existing vitamin E, xenthophylls and isoflavones extractive technique and HPLC analytical technology, and the technical scheme of employing is as follows:
The invention provides a kind of plant, concrete rapid extraction and detection method as nutrient components of soybean content, this method is specially adapted to the rapid extraction and the detection of soybean multiple gene polymerization crossbreeding offspring nutrient composition content, also can be used for measuring vitamin E, isoflavones, xenthophylls, carrotene, chlorophyll a in the food, component contents such as b, this method comprises the steps:
1, the primary sample of carotenoid material such as water-soluble substances isoflavones and liposoluble substance vitamin E, xenthophylls is extracted simultaneously, the preparation sample;
2, HPLC two-step analysis method.
After wherein said extraction was carotenoid material stripping such as the vitamin E, xenthophylls in the vegetable seeds micro-example that desire is detected with the normal hexane reagent under the deepfreeze condition, residue was used ethanol reagent stripping isoflavones again.
Preferably, described plant is a hybrid soybean, soybean multiple gene polymerization crossbreeding offspring particularly, and described low temperature is 0-4 ℃; Described normal hexane is a chemically pure reagent, and its consumption is 250-500 μ l; Described seed micro-example is 5-10mg, the broken end of preferred seed; Described ethanol is 70-80% ethanol, and its consumption is 250-500 μ l.
Preferably, contain the β-A Piaohuluobusuquan of 0.25 μ g/ml and 3 μ g/ml tocols in the described normal hexane reagent as the internal standard thing.
Preferably, described extraction is a seed that desire is detected by grain with the blade of cutting a sheet of paper, cut broken end at the cotyledon position that the positive of hilum is tossed about and add centrifuge tube, add the described normal hexane reagent that contains β-A Piaohuluobusuquan and tocol internal standard simultaneously, oscillator vibration 10-20 is after second, 4 ℃ following ultrasonic echography 8-10 minute, after the refrigeration of shading is afterwards left standstill 15-30 minute, centrifugal 10 minutes, get supernatant 300-400 μ l and obtain carotenoid HPLC analytic samples such as vitamin E, xenthophylls; Get precipitation again and add 70-80% ethanol reagent, oscillator vibrates 10-20 second, ultrasonic echography 8-10 minute, after room temperature leaves standstill 15-30 minute afterwards, centrifugal 10 minutes, gets supernatant 300-400 μ l and obtains isoflavones HPLC analytic sample.
Wherein said HPLC two-step analysis method comprises 1) HPLC of carotenoid such as vitamin E and xenthophylls detect and 2) the HPLC detection of isoflavones.
1) HPLC of carotenoid such as vitamin E and xenthophylls (Hitachi LaChrom Elite, HitachiHigh-Technologies Crop., Japan) testing conditions is: use the reverse post of ODS-3, column temperature is 40 ℃, UV detecting device wavelength is available between 200-900nm, and sample size is 25-30 μ l, and mobile phase A is 75% methanol solution, mobile phase B is an ethyl acetate, and the volume ratio of mobile phase A and mobile phase B is 50-60: 50-40; Adopt the linear gradient method to detect, flow velocity is set at 0.8ml/min.
Wherein detecting vitamin E is 292-295nm with the UV wavelength, and detecting xenthophylls is 445-450nm with the UV wavelength, detects chlorophyll and beta carotene 430nm wavelength, and described mobile phase agents useful for same is the liquid chromatography special use, and uses ultrasonic degas.
2) HPLC of isoflavones (Hitachi LaChrom Elite, Hitachi High-TechnologiesCrop., Japan) testing conditions is: use the reverse post of ODS-3, column temperature: 40 ℃, UV detecting device wavelength can be used at 200-400nm, and sample size 5 μ l, mobile phase A are acetonitrile (interior interpolation final concentration is 0.1% acetate), mobile phase B is deionized water (interior interpolation final concentration is 0.1% acetate), and the volume ratio of mobile phase A and mobile phase B is 20-50: 80-50; Adopt the linear gradient method to detect flow velocity 0.8ml/min.
Wherein detecting isoflavones is 254nm with the UV wavelength, and described mobile phase agents useful for same and water are the liquid chromatography special use, and use ultrasonic degas.
Detection method provided by the invention has following technique effect:
1, a microsampling (5-10mg) only just can carry out the detection of multiple composition simultaneously.
2, the normal growth that does not influence the hybrid generation after the sampling is grown.
3, the fast quantification method after the improvement is compared with existing method, analysis result does not have significant difference, promptly in gene pyramiding breeding offspring selection process, the alternative existing method of the inventive method solves the difficult problem of its " primary sample can detect the plurality of target composition " that can't finish.
4, pre-treatment and HPLC analyze quick, easy, easy row, save time, laborsaving, cost-saving.
5, can carry out qualitative, quantitative resolution to all compositions of vitamin E in the prematurity soybean cotyledon preferably.
6, owing to extract vitamin E, xenthophylls and isoflavones agents useful for same, need 4 kinds of reagent to add up to 5 parts of consumptions by 3 kinds of compositions of original extraction, be reduced to 3 kinds of compositions of present extraction and only need 2 kinds of reagent, on kind, just reduced 60%, that reduces in quantity is just more, the quality that existing method need take by weighing sample will exceed this method more than 2 times at least, the minimizing of waste liquid discharge rate only just can reduce 60% in the extraction stage, there are 3 times original analyses to be reduced to 2 times in the HPLC analysis phase, just can reduce 30%, not only greatly reduce analysis cost but also greatly reduced the pollution of environment.
Description of drawings
Fig. 1 carries out the simultaneous extraction analysis process figure of compositions such as vitamin E, xenthophylls, isoflavones for adopting the inventive method.
Fig. 2 carries out carotenoid HPLC Synchronization Analysis sharp side figure such as the vitamin E of hybrid generation (or prematurity soybean) cotyledon, xenthophylls, chlorophyll for adopting the inventive method.
Fig. 3 analyzes sharp side figure for the HPLC of the vitamin E of existing method.
Fig. 4 analyzes sharp side figure for the carotenoid HPLC such as xenthophylls of existing method.
Fig. 5 is that each component HPLC of isoflavones of the inventive method analyzes sharp side figure.
Fig. 6 analyzes sharp side figure for each component HPLC of isoflavones of existing method.
1-xenthophylls wherein, 2-tocol, 3-Delta-Tocopherol, 4-β-A Piaohuluobusuquan, 5-Gamma-Tocopherol, the 6-chlorophyll b, 7-alpha-tocopherol, 8-chlorophyll a, 9-beta carotene, the 10-daidzin, 11-genistin, 12-malonyl daidzin, 13-malonyl genistin.
Embodiment
The invention provides a kind of plant, refer to soybean multiple gene polymerization breeding cross offspring's nutrient composition content detection method especially, comprise the steps:
At first, the primary sample of carotenoid materials such as water-soluble substances isoflavones and liposoluble substance vitamin E, xenthophylls is extracted simultaneously, the preparation sample.After being the carotenoid material strippings such as vitamin E, xenthophylls in the vegetable seeds micro-example that desire is detected with the normal hexane reagent under the deepfreeze condition, residue is used ethanol reagent stripping isoflavones again.
Adopt HPLC two-step analysis method then, comprise 1) HPLC of carotenoid such as vitamin E and xenthophylls detect and 2) the HPLC detection of isoflavones.
Below be explanation of nouns of the present invention and ellipsis:
Vitamin E: formal name used at school, tocopherol, English name, Tocopherol.4 kinds of isomerss are arranged: alpha-tocopherol (α-Tocopherol in soybean cotyledon, slightly: α-Toc, biologically active is the strongest), betatocopherol (β-Tocopherol, slightly: β-Toc), Gamma-Tocopherol (γ-Tocopherol, slightly: γ-Toc), Delta-Tocopherol (δ-Tocopherol, slightly: δ-Toc).A kind of as antioxidant has softening blood vessel, prophylaxis of cancer, cardiovascular and cerebrovascular disease, and strengthen effect such as human immunologic function, soybean is the main raw material that extracts vitamin E.
Tocol (Tocol): as internal standard, it is quantitative to be used for vitamin E.
Trans β-A Piao-carotenal (β-Apo-8 '-carotenal) (trans), be used for the quantitative internal standard of carotenoid.
Isoflavones quantitatively, because of there not being suitable internal standard product, generally adopt the external perimysium reference method.
Internal standard method (internal standard method): when measuring certain component content, being suitable for of finding is dissolved in it in sample extracting solution, detected synchronously in company with test sample, and when HPLC detects, its appearance time had better not to detect composition overlapping with desire, and promptly overlapping area is big more, and quantitative error is also big more.
External perimysium reference method (external standard method): when measuring certain component content, owing to can not find the reagent that is suitable for doing internal standard, the external standard methods that adopt more, it is not dissolved in the sample extracting solution, when sample detection, as one independently sample detect, according to the area (or peak height) at the peak of the area (or peak height) at the concentration of standard model of preparation and peak and test sample, the content of calculating unknown sample.
Xenthophylls, English name: Lutein.Be a kind of of carotenoid, constitute one of two big pigments of eye retina macula lutea with luteole jointly.Mainly be present in the yellowish green vegetables, as: spinach, purple leaf romaine lettuce, pumpkin etc.In recent years,, be used to prevent senile macular degeneration disease (ADM), cataract, cardiovascular and cerebrovascular disease, functions such as protection skin as a kind of powerful antioxidant of new rule.Though its content in the cultivated soybean is lower, through our investigation, its content in some wild soybean cotyledon imports to this merit in the cultivated soybean not second to vegetable content, will greatly improve the soybean added value, strengthens its physiological hygiene function.But because the seed of wild soybean is very little, also very little with the seed of the cultivated soybean hybridization back early generation, existing detection method can't be applicable to the quality-improving breeding practice.
Isoflavones, English name Isoflavone (following Iso slightly), main nine kinds of isoflavone glucosides in soybean, daidzin (Daidzin) wherein, genistin (Genistin), the content of malonyl daidzin (Malonyl-daidzin) and malonyl genistin (Malonyl-genistin) is maximum.Have the activity of regulating estrogen, enzyme that inhibition is relevant with cancer, elimination activity oxygen base has effects such as antioxidation and protect against osteoporosis.
Filial generation, the descendants that different two or more parents (parents) post-coitum of genetic background produces has parents' feature concurrently.
The multiple gene polymerization breeding: multiple gene polymerization is exactly by hybridization or biotechnology means in brief, will be dispersed in ideal basis in different individualities, kind or the strain because of being aggregated in the same genome.
High performance liquid chromatograph: English name: High-performance liquid chromatography, abbreviation: HPLC.High resolving power, high sensitivity, speed are fast because it has, chromatographic column can be recycled, and flow out advantages such as component easily collecting, thereby are widely applied to biological chemistry, food analysis, medical research, environmental analysis, inorganic analysis etc.
Other notes of abridging: the abbreviation of C-concentration c ontent, the abbreviation of A-area area, the abbreviation of s-standard model standard, the abbreviation of t-tocopherol tocopherol, the abbreviation of 1-xenthophylls lutein, the abbreviation of i-isoflavones isoflavone.
The invention will be further described below in conjunction with embodiment, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.
The extraction functionality nutritional labeling
As shown in Figure 1, hybrid soybean seeds of the maturation that desire is detected by grain with the blade of cutting a sheet of paper, toss about at the positive of hilum and to cut broken end at all (not injure plumule, radicle, plumular axis is as the criterion) (detect under the situation of prematurity soybean cotyledon, with the prematurity soybean cotyledon through the freeze drier freeze drying of routine after 24 hours, grinding powdered) 10mg adds the eppendorf centrifuge tube of 1.5ml, the ice-cold normal hexane reagent 500 μ l that add the β-A Piaohuluobusuquan contain 0.25 μ g/ml (β-Apo-8 '-carotenal (trans)) and 3 μ g/ml tocol (Tocol) internal standards simultaneously, 10-20 is after second in the oscillator vibration, 4 ℃ following ultrasonic echography 8-10 minute, after the refrigeration of shading is afterwards left standstill 15-30 minute, 13,000 rev/min centrifugal 10 minutes, get supernatant 300-400 μ l and carry out HPLC and analyze vitamin E, carotenoid such as xenthophylls; Get precipitation and add 75% ethanol reagent, 500 μ l, oscillator vibration 10-20 second, ultrasonic echography 8-10 minute, after room temperature leaves standstill 15-30 minute afterwards, 13,000 rev/mins centrifugal 10 minutes, get supernatant 300-400 μ l and carry out HPLC and analyze isoflavones.
The HPLC analytic approach
When the HPLC quantitative test, different because of analyzing the used moving phase of isoflavones with the former, HPLC needs when analyzing finish for twice: for the first time: the synchronous quantitative test of liposoluble substance vitamin E and xenthophylls (carotenoids all can), for the second time: the quantitative test of water-soluble substances isoflavones.
1) the HPLC analytical approach of carotenoid such as vitamin E and xenthophylls is as follows: the special-purpose bottle of HPLC that the supernatant of 300-400 μ l will be housed is put into the automatic sampling apparatus (auto-sampler) of HPLC.HPLC (Hitachi LaChrom Elite, Hitachi High-TechnologiesCrop., Japan) condition enactment: use the reverse post (4.6 * 250mm of ODS-3,5 μ m, GL Science, Japan), column temperature: 40 ℃ of .UV detecting devices should possess 2 wave bands (wavelength is available between 200-900nm), wherein detecting vitamin E is 292-295nm with the UV wavelength, and detecting xenthophylls is 445-450nm with the UV wavelength, detects chlorophyll and beta carotene 430nm wavelength.Mobile phase A is 75% the special-purpose methyl alcohol of high performance liquid chromatography, is mixed with 75% methanol solution then with the special-purpose deionized water of high performance liquid chromatography, and uses ultrasonic degas; Mobile phase B is the special-purpose ethyl acetate of high performance liquid chromatography.Sample size 25-30 μ l, the flow process that adopts the linear gradient method to detect sees Table 1.
The nutritional labeling HPLC (Hitachi) of the vitamin E of table 1. prematurity soybean cotyledon and xenthophylls etc. is ladder synchronously
Degree analysis process flow process
Annotate: the reverse post of Inertsil ODS-3 (4.6 * 250mm, 5 μ m, GL Science, Japan), 40 ℃
A:75% methyl alcohol, B: ethyl acetate
As table 1 and shown in Figure 2, in the time of 0-12 minute, the score of mobile phase A is from 60% 50% (corresponding B then rises to 50% from 40%) when dropping to 12 minutes of beginning, and corresponding wavelength is 450nm, flow velocity is set at 0.8ml/min, and xenthophylls detects in the section between can be at this moment.In the time of 12-24 minute, the score of mobile phase A remains on 50%, and wavelength remains on 295nm, and flow velocity does not become 0.8ml/min, and tocol was detected when the order that detects composition was 18-20 minute; In the time of 22-23 minute, Delta-Tocopherol is detected; In the time of 24-26 minute, the score of mobile phase A still remains on 50%, and wavelength becomes 450nm, and flow velocity remains on 0.8ml/min, and detecting composition is β-A Piaohuluobusuquan; In the time of 26-28 minute, wavelength becomes 295nm, and other are constant, can detect Gamma-Tocopherol; 28-29 minute, the score of mobile phase A was 60%, and wavelength is 430nm, and the constant 0.8ml/min of flow velocity can detect chlorophyll b; 28-30 minute wavelength is 295nm, and other are also constant, can detect alpha-tocopherol; In the time of 30-35 minute, mobile phase A score is 50%, wavelength 430nm, and other are constant, can detect chlorophyll a; In the time of 35-40 minute, mobile phase A rises to 60%, and wavelength becomes 430nm, and other are constant, can detect beta carotene.
Which kind of composition is above method can detect according to actual needs, just set according to the testing conditions of above-mentioned certain composition; Unwanted composition just can not set corresponding with it condition, and then this composition just can not be detected.
(2) the HPLC detection method of isoflavones:
The special-purpose bottle of HPLC that the supernatant of 300-400 μ l is housed is put into the automatic sampling apparatus (auto-sampler) of HPLC.HPLC (Hitachi LaChrom Elite, Hitachi High-TechnologiesCrop., Japan) condition enactment: use the reverse post (4.6 * 250mm of ODS-3,5 μ m, GL Science, Japan), column temperature: it is just passable that 40 ℃ of .UV detecting devices should possess 1 wave band (200-400nm).Sample size 5 μ l, mobile phase A are the special-purpose acetonitrile (interior interpolation final concentration is 0.1% acetate) of high performance liquid chromatography, will outgas before using, and mobile phase B is the special-purpose deionized water (interior interpolation final concentration is 0.1% acetate) of high performance liquid chromatography, will outgas before using.Whole process wavelength keeps 254nm, and flow velocity 0.8ml/min is constant, and the flow process that adopts the linear gradient method to detect sees Table 2.
Table 2. isoflavones HPLC (Hitachi) synchronous gradient analysis process flow process
Annotate: the reverse post of Inertsil ODS-3 (4.6 * 250mm, 5 μ m, GL Science, Japan), 40 ℃
A: the acetate of acetonitrile+0.1%, B: the acetate of deionized water+0.1%
As table 2 and shown in Figure 5,0-10 minute, the score of mobile phase A can detect daidzin (Daidzin) from 20% 30% (corresponding B then drops to 70% from 80%) when being raised to 10 minutes of beginning in the time of 6-7 minute, can detect genistin (Genistin) in the time of 7-10 minute; In the time of 10-15 minute, the score of mobile phase A rises to 40% from 30%, can detect malonyl daidzin (Malonyl-daidzin) in the time of 12-15 minute; In the time of 15-20 minute, mobile phase A rises to 50% from 40%, can detect malonyl genistin (Malonyl-genistin) in the time of 16-18 minute; 20-25 minute, mobile phase A kept 50% always, and wavelength is 254nm always, and flow velocity is 0.8ml/min, made the stable and readiness when returning to pattern detection initial of HPLC detected state, to guarantee the continuity of test sample, accuracy.
The existing detection method of Comparative Examples 1 vitamin E
Extracting method: take by weighing powdered soybean 50mg, join the 10ml test tube, 80% the ethanol that contains 3 μ g/ml tocol (Tocol) internal standards that adds 1ml, 10-20 is after second in the oscillator vibration, ultrasonic echography is 15 minutes under the room temperature, add 2ml then and contain the normal hexane saturated solution that faces benzenetriol, oscillator vibrates 10-20 second strongly, after shading is left standstill 30 minutes, 5 speed, 2,500 rev/mins, centrifugal 10 minutes, get upper strata normal hexane liquid 600 μ l, the special-purpose bottle of the HPLC that packs into carries out the vitamin E analysis (Ujiie et al., 2005) of HPLC.
The HPLC analytical approach: the special-purpose bottle of HPLC that the supernatant of 600 μ l will be housed is put into the automatic sampling apparatus (auto-sampler) of HPLC.HPLC (Hitachi LaChrom Elite, HitachiHigh-Technologies Crop., Japan) condition enactment: use the reverse post of ODS-3: (3.0 * 250mm, GLScience, Japan), column temperature: 40 ℃. flow velocity 0.5ml/min, UV wavelength 295nm, sample size 20 μ l mobile phases are acetonitrile: methyl alcohol=75: 25v/v).As shown in Figure 3, at 10-13 minute, Delta-Tocopherol detected, and at 14-16 minute, Gamma-Tocopherol was detected, and at 17-19 minute, alpha-tocopherol was detected (Ujiie et al., 2005).The existing detection method of Comparative Examples 2 xenthophylls
Extracting method: take by weighing powdered soybean 25mg, join 1.5ml eppendorf centrifuge tube, the acetone ethanol mixed liquor that contains 0.5 μ g/ml β-Apo-carotenal internal standard of adding 0.5ml (1: 1, v/v), 10-20 is after second in the oscillator vibration, ultrasonic echography is 15 minutes under the room temperature, after shading is left standstill 30 minutes, 13,000 rev/min centrifugal 10 minutes, get upper strata normal hexane liquid 300-400 μ l, the special-purpose bottle of the HPLC that packs into, carry out the xenthophylls analysis (Kanamaru et al., 2006) of HPLC.
The HPLC analytical approach: the special-purpose bottle of HPLC that the supernatant of 300-400 μ l will be housed is put into the automatic sampling apparatus (auto-sampler) of HPLC.HPLC (Hitachi LaChrom Elite, HitachiHigh-Technologies Crop., Japan) condition enactment: use the reverse post of ODS-3: (4.6 * 250mm, GLScience, Japan), column temperature: 40 ℃. flow velocity 0.8ml/min, UV detecting device wave band 400-700nm adopts the linear gradient elution method, sample size 30 μ l, mobile phase A is an acetonitrile, and mobile phase B is an ethanol, when 0-5min, mobile phase A:75% (mobile phase B is 25%), when 5-15min, mobile phase A drops to 25%, and keeps 25% to 20min.As shown in Figure 4, when 0-10min, wavelength remains on 447nm, during 5-7min, xenthophylls is detected, when 8-10min, internal standard β-Apo-carotenal is detected, when 13-20min, wavelength changes 430nm into, chlorophyll b, chlorophyll a and beta carotene are detected (Kanamaruet al., 2006) in succession.
The existing detection method of Comparative Examples 3 isoflavones
Extracting method: take by weighing powdered soybean 100mg, join the 10ml test tube, add 70-80% ethanol 2-4ml, 10-20 is after second in the oscillator vibration, and ultrasonic echography is 15 minutes under the room temperature, after shading is left standstill 30 minutes, 5 speed 2,500 rev/mins after centrifugal 10 minutes, get supernatant and get the eppendorf centrifuge tube that 1ml moves into 1.5ml, 13,000 rev/min, centrifugal 5 minutes, the supernatant 400-500 μ l special-purpose bottle of HPLC of packing into carried out the isoflavones analysis of HPLC.
HPLC analytical standard method:
(250x30mm, 5pm) analytical column, sample size are 5 μ l, and mobile phase is made up of A (0.1% aqueous acetic acid V/V) and B (0.1% acetic acid second eyeball solution V/V), adopt gradient elution to adopt YMCODS.AM-RP.Gradient is: 0～60mni, B are by 10% linear increment to 60%, and be back with 90%B wash-out 3min, last 10%B balance 10min.The ultraviolet detection wavelength is 254nm.Elution speed 0.65ml/min, 40 ℃ of column temperatures.As shown in Figure 6: daidzin when 14-17min (Daidzin) is detected, and genistin when 24-25min (genistin) is detected, and the malonyl daidzin is detected during 26-27, and the malonyl genistin is detected when 34-36min.Genistein is detected when 53-55min.The peak that Fig. 6 detects is the peak of standard substance, rather than the peak that detects from soybean cotyledon, so the peak face is good-looking, genistein is lower at the general content of soya seeds, and small sample difficulty detects.
Result's contrast
The performance index that the method detection of employing embodiment 1 is main and the contrast of prior art are as shown in table 3
The new and old analytical approach comparison sheets of carotenoid such as table 3. vitamin E, isoflavones and xenthophylls
Can draw as drawing a conclusion by table 3:
1, the inventive method is compared with existing method, and existing methods analyst above-mentioned three components must need 3 weighings, 3 extractings, and 3 times HPLC analyzes, and the inventive method only needs 1 weighing, and 2 extractings, 2 times HPLC analyzes.
2, the inventive method can reduce by two kinds of chemical reagent on used extract kind.Promptly existing method detects above-mentioned several composition, needs ethanol, methyl alcohol, normal hexane and 4 kinds of chemical reagent of acetone.And the inventive method only needs normal hexane and two kinds of reagent of ethanol.
3, the inventive method at specimen in use qualitatively, comparable at least existing method reduces 90mg.Promptly existing method will be finished the detection of above-mentioned several compositions, and adding up to needs with more than the sample size 90mg, and the inventive method only needs 5mg to get final product.
4, the inventive method can be the researcher 50-60% that saves time, and reduces the human and material resources input; Save the input of reagent cost about 50% minimizing financial resources; Reduce about discharging of waste liquid 30-60%, reduce environmental pollution.
The computing method of functional nutrient component content
According to the typical curve that records the concentration known standard model in advance, content and peak area are extremely significant linear positive correlation, ask the content of unknown sample:
Suppose: the concentration of known standard sample: C ' (μ g/ml), vitamin E standard model concentration C ' t, xenthophylls C ' l, isoflavones C ' i, corresponding peak area is As, then the peak area of vitamin E standard model is A ' ts, xenthophylls A ' ls, isoflavones A ' is; Unknown sample content is C, and content of vitamin E is Ct, and corresponding peak area is At, lutein content Cl, and corresponding peak area is Al, isoflavone content Ci, corresponding peak area is Ai, the dry matter of sample is W (mg).
Ct(μg/100mg)＝(C’t?×At)×100/(W×A’ts)
Cl(μg/100mg)＝(C’l×Al)×100/(W×A’ls)
Ci(μg/100mg)＝(C’i×Ai)×100/(W×A’is)
(F3 can obtain more seed from generation to generation at present for vitamin E that the method detection of employing embodiment 1 filters out and all high filial generation of lutein content, the bottleneck of breeding is that F2 is for seed) system designator code be 02-16,02-19,04-6,05-12,05-27, by comparing with existing method, there is not significant difference, the expression new method can be used (table 4), utilizes this new method to common strain/kind (as diplomatic relations 06-241,06-528 equally, 06-1012) detection of carrying out isoflavone content is compared with existing method, does not have significant difference (table 5).
The measurement result of table 4. xenthophylls and vitamin E relatively
Unit: mg/100g
Annotate: numerical value in the table, the mean+/-standard error of 3 repetitions.
The new and old analytic approach experimental result of table 5. isoflavones relatively
Annotate: numerical value in the table, the mean+/-standard error of 3 repetitions.
The comparison of each component content of the filial generation that draws according to described computing method, content and the aging method of the new analytical approach after the improvement be less than difference significantly as can be seen, promptly optimizes new method after easy and can be used for filial generation is carried out check and analysis and do not influence result's authenticity.
Claims (9)
1. the rapid extraction detection method of a nutrient content of plant comprises the steps:
The primary sample of carotenoid materials such as water-soluble substances isoflavones and liposoluble substance vitamin E, xenthophylls is extracted simultaneously, the preparation sample;
HPLC two-step analysis method.
2. detection method according to claim 1, after wherein said extraction was carotenoid material stripping such as the vitamin E, xenthophylls in the vegetable seeds micro-example that desire is detected with the normal hexane reagent under the deepfreeze condition, residue was used ethanol reagent stripping isoflavones again.
3. detection method according to claim 2, wherein said low temperature are 0-4 ℃; Described normal hexane is a chemically pure reagent, and its consumption is 250-500 μ l; Described vegetable seeds is a hybrid soybean, and described micro-example is 5-10mg, the broken end of preferred seed; Described ethanol is 70-80% ethanol, and its consumption is 250-500 μ l.
4. detection method according to claim 2 contains the β-A Piaohuluobusuquan of 0.25 μ g/ml and 3 μ g/ml tocols as the internal standard thing in the wherein said normal hexane reagent.
5. detection method according to claim 2, wherein said extraction is a seed that desire is detected by grain with the blade of cutting a sheet of paper, cut broken end at the cotyledon position that the positive of hilum is tossed about and add centrifuge tube, add the described normal hexane reagent that contains β-A Piaohuluobusuquan and tocol internal standard simultaneously, 10-20 is after second in the oscillator vibration, 4 ℃ following ultrasonic echography 8-10 minute, after the refrigeration of shading is afterwards left standstill 15-30 minute, centrifugal 10 minutes, get supernatant 300-400 μ l and obtain carotenoid HPLC analytic samples such as vitamin E, xenthophylls; Get the ethanol reagent that precipitation adds 70-80% again, oscillator vibrates 10-20 second, ultrasonic echography 8-10 minute, after room temperature leaves standstill 15-30 minute afterwards, centrifugal 10 minutes, gets supernatant 300-400 μ l and obtains isoflavones HPLC analytic sample.
6. detection method according to claim 1, wherein said HPLC two-step analysis method comprise that the HPLC of carotenoid such as vitamin E and xenthophylls detects and the HPLC of isoflavones detects.
7. detection method according to claim 6, the HPLC testing conditions of carotenoid such as described vitamin E and xenthophylls is: use the reverse post of ODS-3, column temperature is 40 ℃, UV detecting device wavelength is available between 200-900nm, sample size is 25-30 μ l, mobile phase A is 75% methanol solution, and mobile phase B is an ethyl acetate, and the volume ratio of mobile phase A and mobile phase B is 50-60: 50-40; Adopt the linear gradient method to detect, flow velocity is set at 0.8ml/min;
Preferred detection vitamin E is 292-295nm with the UV wavelength, and detecting xenthophylls is 445-450nm with the UV wavelength, detects chlorophyll and beta carotene 430nm wavelength, and described mobile phase agents useful for same is the liquid chromatography special use, and uses ultrasonic degas.
8. detection method according to claim 6, the HPLC testing conditions of described isoflavones is: use the reverse post of ODS-3, column temperature: 40 ℃, UV detecting device wavelength can be used at 200-400nm, sample size 5 μ l, mobile phase A is acetonitrile (interior interpolation final concentration is 0.1% acetate), and mobile phase B is deionized water (interior interpolation final concentration is 0.1% acetate), and the volume ratio of mobile phase A and mobile phase B is 20-50: 80-50; Adopt the linear gradient method to detect flow velocity 0.8ml/min;
Preferred detection isoflavones is 254nm with the UV wavelength, and described mobile phase agents useful for same and water are the liquid chromatography special use, and use ultrasonic degas.
9. the application of the described detection method of claim 1-8 is used for soybean multiple gene polymerization breeding cross offspring's fast detecting of functional nutrient composition and the screening of filial generation.
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