CN102778436B - Method for breeding freshly eaten corn with eye health-care effect - Google Patents
Method for breeding freshly eaten corn with eye health-care effect Download PDFInfo
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- CN102778436B CN102778436B CN201110427526.4A CN201110427526A CN102778436B CN 102778436 B CN102778436 B CN 102778436B CN 201110427526 A CN201110427526 A CN 201110427526A CN 102778436 B CN102778436 B CN 102778436B
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Abstract
The invention relates to a method for breeding freshly eaten corn with an eye health-care effect and aims to overcome the defect that the rubixanthine content of most of corn is reduced gradually because the corn rubixanthine in most of the current freshly eaten corn is low and the breeding method of the corn is not emphasized on breeding the corn rubixanthine. The method comprises the following steps of taking an inbred line of the freshly eaten corn as an initial material, and selecting the corn freshly eaten corn which can stably express and has over 8mg/kg of rubixanthine by ultraviolet-visible spectrophotometry and high-performance liquid chromatography, or taking the corn material rich in corn rubixanthine gene as a donor, and performing hybridization, backcross or somaclonal variation to obtain the inbred line or a hybrid variety of the corn rich in the corn rubixanthine finally.
Description
Technical field
The present invention relates to the selection of corn, particularly relate to a kind of selection with the fresh type corn of eye health effect.
Background technology
Along with the continuous aggravation of social astogeny and people's excess eye-using situation, the common eye illness of the elderly such as visual impairment, macula retinae pathology, cataract, DRP, muscae volitantes, glaucoma will progressively generalize and become younger, and the long-term lacking human body self that has its source in of its material base can not synthesize, " eye gold "---the zeaxanthin that must derive from meals.
Zeaxanthin (Zeaxanthin, 3,3 '-dihydroxy-beta carotene) be isoprene unsaturated compounds, belong to the one of carotenoid, molecular formula is C40H56O2, and occurring in nature normal and beta carotene, xenthophylls etc. coexist, and are extensively present in each tissue of human body, but its distribution in eyes is the most outstanding and most characteristic, and namely the macular area that retinal centre is responsible for meticulous eyesight, vision the sharpest is the position that zeaxanthin concentration is the highest.
Over nearly 30 years; a large amount of pathological research shows; zeaxanthin to amphiblestroid growth and maturation significant; there is good protective effect to vision, particularly in prevention, senile macular degeneration (AMD), the generation etc. that strengthens immunologic function, reduce cardiovascular disease incidence rate and reduce cancer, there is unique physiological function.Meanwhile, there is significant positive correlation between the intake of meals zeaxanthin and macular pigment optical density.
At present, people have recognized another macular pigment be present in equally in retina---the importance of xenthophylls, but the attention of zeaxanthin is also nowhere near.Research and analyse and show, in adult human retina macula lutea, the ratio of zeaxanthin and xenthophylls is 214: 1, and neonatal macula lutea is not yet formed, and therefore wherein lutein content is higher than zeaxanthin, and both ratios are 1149: 1, similar with peripheral retina.The amphiblestroid growth of baby and ripe with this in period xenthophylls and the ratio of zeaxanthin change and keep close synchronous, illustrate zeaxanthin to amphiblestroid growth and maturation significant.
In recent years, American-European many national approveds are using zeaxanthin as food coloring, and the companies such as Roche Group of Switzerland, Dutch DSM have relevant healthcare and treatment product is released.But in today that food industry is maked rapid progress, the health perception of people improves constantly, and it is also proposed new standard to food security.Although synthetic dyestuff is bright in colour, stabilizing member is good, cheap, because of problems such as synthesis toxicity, people are worried and fear; Meanwhile, natural green plant pigment safety non-toxic, and its distinctive nutritive value and physiologically active, be loved by the people.Be rich in the food of functional components, not only there is nutrition and delicious function, also have good health regulatory function.
But relative to xenthophylls, the source of zeaxanthin is more limited.All xenthophylls is rich in much fruit, leafy vegetable, zeaxanthin is main enrichment in the minority vegetable and fruits such as corn, matrimony vine, persimmon then, and if its content can be improved, not only by zeaxanthin required in the added body of ordinary meal source, delay, prevent and adjuvant treatment of diseases, improve school-ager, the elderly, visual fatigue crowd and the eye health level of crowd by light pollution, and significantly can increase the added value of agricultural product, promote increasing peasant income and Agricultural Transformation upgrading.
Up to now, there is not yet about being improved it by breeding technique in grain and vegetable crop containing quantifier elimination report.
Summary of the invention
The object of the invention is to for the zeaxanthin in current most of fresh type corn few, the selection of corn there is no and lays particular emphasis on selecting and breeding corn xanthin, cause the zeaxanthin of current most of corn to become the defects such as downtrending, a kind of selection having the fresh type corn of eye health effect is provided.
Above-mentioned technical matters of the present invention is implemented by the following technical programs: a kind of selection with the fresh type corn of eye health effect.It is parent material that the method comprises with fresh edible maize selfing, screen the corn variety of more than the zeaxanthin 8mg/kg that a class Absorbable organic halogens is expressed, or with being rich in the corn material of zeaxanthin gene for donor, through hybridization, backcross or somaclonal variation, final corn inbred line or the cenospecies must being rich in zeaxanthin.Utilize the fresh edible maize being rich in zeaxanthin of the method seed selection, zeaxanthin doubles above than common fresh edible maize kind, can zeaxanthin effectively in added body particularly needed for eyes after edible, delays and prevents disease of eye, alleviating asthenopia, safeguards health.Wherein fresh edible maize mainly comprises corn and waxy corn.
There is a selection for the fresh type corn of eye health effect, comprise the following steps:
1) collecting and plant seed outward appearance for yellow or lurid corn, being suitable for peeling entire kernel period of eating raw and with pocket knife along stitching rip cutting in seed, choosing endosperm is all yellow or lurid endosperm; Then extract saponified for subsequent use;
2) ultraviolet-visible spectrophotometry is adopted, detecting step 1) total carotinoid content in the fresh seed that screens; The method adopts determined wavelength to be 454nm; Retain the seed of total carotinoid more than content 10mg/kg;
3) step 2 is planted) seed that screens, adopt high performance liquid chromatography (HPLC method) to carry out quantitative test to zeaxanthin in its fresh corn seed; Retain the material of more than zeaxanthin 8mg/kg;
Step 4), continue plantation and expand seed selected by numerous step 3), the corn strain that screening economical character is good, coordinate force is strong, or with being rich in the corn kernel of zeaxanthin gene for donor, combo is carried out through the strain of hybridizing, backcrossing or somaclonal variation obtaining, the strain that screening general performance is good, the final corn inbred line being rich in zeaxanthin or the cenospecies obtaining inheritance stability.
Corn kernel in described step 1) is strain or the self-mating system of corn conventional variety or hybrid parents or germplasm resource or mutagenesis generation.
Saponified forming steps in described step 1) is as follows:
A, get 20g corn kernel and grind, add acetone extract, suction filtration, repeatedly several times, until extracting liquid colourless;
B, merging extract, be evaporated to dry; Time concentrated, temperature is lower than 40 degree;
C, dissolve dry acetone crude extract with a small amount of methyl alcohol, then add isopyknic 30%(volume ratio) methyl alcohol-KOH saponification more than 4 hours, obtain saponification liquor;
D, reduced pressure concentration saponification liquor, to minimum volume, add mixed liquor (1:1=V/V) extraction than configuration of a small amount of ethyl acetate and normal hexane equal-volume, re-extract repeatedly, until extract is colourless;
E, combining extraction liquid, alkali lye when washing away saponification with distilled water in separating funnel, gets upper layer of extraction liquid and is evaporated to minimum volume, obtain Carotenoids Extracts;
F, transfer in the centrifuge tube of 1.5ml by the Carotenoids Extracts concentrated, do remaining solvent with N2 air-blowing ,-20 DEG C save backup;
G, every sample are in triplicate.
Ultraviolet-visible spectrophotometry is specially: described in take 0.1-0.2g saponified, grind, add 5ml normal hexane: acetone=3:2 (volume ratio), lixiviate 16 hours under dark condition, middle shake is several times; Then 1ml 20%(volume ratio is added) KOH-methyl alcohol, reacts one hour under dark condition; Add 3ml normal hexane and 1ml 10wt% metabisulfite solution again, its static layering of mixing relief, upper strata yellow liquid is the total carotinoid solution slightly carried; Take blank as contrast, measure at ultraviolet-visible spectrophotometer 454nm place.
Because ultraviolet-visible spectrophotometer wavelength is integer, according to Fig. 1 and Fig. 2, xenthophylls measured value when 460nm reduces, and zeaxanthin is substantially similar near 454nm, therefore gets 454nm for measuring wavelength.
Described efficient liquid phase detection method is specially: detect the efficient liquid phase detection system adopting Waters, wherein detecting device is 2998 type diode array detector, chromatographic column is Spherisorb ODS2(5 μm, 250 × 4.6mm), mobile phase is acetonitrile: methyl alcohol: methylene chloride: normal hexane=85:5:5:5(volume ratio), flow velocity 1ml/min; Sample 1ml mobile phase dissolves, sample introduction after the filter membrane of mistake 0.45 μm, sample size 10 μ l.
Standard model zeaxanthin and xenthophylls dilute five concentration gradients respectively, production standard curve.
In sum, the present invention compared with prior art tool have the following advantages:
With the fresh edible maize being rich in zeaxanthin that the present invention cultivates, can:
1, Accurate Determining zeaxanthin need utilize HPLC method, and the present invention carries out a large amount of screening in early stage owing to adopting spectrophotometric method, reduces cost of determination, and can be rich in extension application in the seed selection of zeaxanthin material other crops such as paddy rice.
2, the general wavelength measuring total carotinoid is 446nm, and the spectrophotometry that the present invention adopts detects with the zeaxanthin characteristic absorption peak of 453 ~ 455nm, can effectively select the kind being rich in zeaxanthin, strengthens detection efficiency in early stage.
3, the present invention with the fresh edible maize being more and more subject to people in recent years and liking for effective carrier, carry out the research and development of being rich in zeaxanthin beneficial eye function corn in the world first, closely meet world market demand, effectively can supplement human body zeaxanthin, from the generation of meals source prevention presbyopia, cataract and senile macular degeneration, be conducive to eye health and health.
4, by the popularizing planting of this functional type corn, greatly can increase economic efficiency, promote increasing peasant income and Agricultural Transformation upgrading.
Accompanying drawing explanation
Fig. 1 is xenthophylls spectrogram.
Fig. 2 is zeaxanthin spectrogram.
Embodiment
Below in conjunction with embodiment, the present invention is further described.
Embodiment 1:
Zeaxanthin is up to the functional form fresh edible maize selection of 9.3mg/kg:
With Sweet corn variety " Hua Zhen " for parent material, through 3 generation selfed breeding, get Elite inbred " magnificent 36-9 " dry seeds of water cut 13.5%, with the gamma-ray irradiation process of cobalt-60, exposure dose is routine dose 200Gy.Live mutagenesis seed in the present age, the mixed seed receiving mutagenesis generation plant.Mixed planting mutagenesis two generation population material.Within about 21 days, peel entire kernel in pollination and stitch rip cutting with pocket knife along in seed, choosing endosperm is all yellow or lurid material.Extract saponified, adopt ultraviolet-visible spectrophotometry to detect total carotinoid content in the fresh corn seed of all material respectively.Retain the material of total carotinoid more than content 10mg/kg and plant respectively by strain, adopting HPLC method to carry out quantitative test to zeaxanthin in its fresh corn seed respectively.Detect the efficient liquid phase detection system adopting Waters, wherein detecting device is 2998 type diode array detector, and chromatographic column is Spherisorb ODS2(5 μm, 250 × 4.6mm), mobile phase is acetonitrile: methyl alcohol: methylene chloride: normal hexane=85:5:5:5, flow velocity 1ml/min.Sample 1ml mobile phase dissolves, sample introduction after the filter membrane of mistake 0.45 μm, sample size 10 μ l.Retain the material of more than zeaxanthin 8mg/kg.The expression stability simultaneously observing this mutant character controls with heredity, economical character, quality characteristic, disease resistance and hybrid vigour, through repeated screening, eliminate, final a zeaxanthin, than contrast increase by more than 120%, has excellent economical character, the fresh edible maize strain that disease-resistant and hybrid vigour is strong.
Saponified forming steps is as follows:
A, get 20g corn kernel and grind, add acetone extract, suction filtration, repeatedly several times, until extracting liquid colourless;
B, merging extract, be evaporated to dry; Time concentrated, temperature is lower than 40 degree;
C, dissolve dry acetone crude extract with a small amount of methyl alcohol, then add isopyknic 30%(volume ratio) methyl alcohol-KOH saponification more than 4 hours, obtain saponification liquor;
D, reduced pressure concentration saponification liquor, to minimum volume, add mixed liquor (1:1=V/V) extraction than configuration of a small amount of ethyl acetate and normal hexane equal-volume, re-extract repeatedly, until extract is colourless;
E, combining extraction liquid, alkali lye when washing away saponification with distilled water in separating funnel, gets upper layer of extraction liquid and is evaporated to minimum volume, obtain Carotenoids Extracts;
F, transfer in the centrifuge tube of 1.5ml by the Carotenoids Extracts concentrated, do remaining solvent with N2 air-blowing ,-20 DEG C save backup;
G, every sample are in triplicate.
Ultraviolet-visible spectrophotometry is specially: take 0.1-0.2g saponified, grinds, and adds 5ml normal hexane: acetone=3:2 (volume ratio), lixiviate 16 hours under dark condition, middle shake is several times; Then 1ml 20%(volume ratio is added) KOH-methyl alcohol, reacts one hour under dark condition; Add 3ml normal hexane and 1ml 10wt% metabisulfite solution again, its static layering of mixing relief, upper strata yellow liquid is the total carotinoid solution slightly carried; Take blank as contrast, measure at ultraviolet-visible spectrophotometer 454nm place.Because ultraviolet-visible spectrophotometer wavelength is integer, according to Fig. 1 and Fig. 2, xenthophylls measured value when 460nm reduces, and zeaxanthin is substantially similar near 454nm, therefore gets 454nm for measuring wavelength.
Efficient liquid phase detection method is specially: detect the efficient liquid phase detection system adopting Waters, wherein detecting device is 2998 type diode array detector, chromatographic column is Spherisorb ODS2(5 μm, 250 × 4.6mm), mobile phase is acetonitrile: methyl alcohol: methylene chloride: normal hexane=85:5:5:5(volume ratio), flow velocity 1ml/min; Sample 1ml mobile phase dissolves, sample introduction after the filter membrane of mistake 0.45 μm, sample size 10 μ l.Standard model zeaxanthin and xenthophylls dilute five concentration gradients respectively, production standard curve.
Ultraviolet-visible spectrophotometry is Preliminary Determination, and this mode experimental cost is low, then carries out meticulous mensuration by efficient liquid phase detection method.Efficient liquid phase detection method use cost is higher, after ultraviolet-visible spectrophotometry is tentatively got rid of and is not suitable for seed, then is undertaken measuring both saving expense by efficient liquid phase detection method, turn improves efficiency, be convenient to promote the use of.
Claims (5)
1. there is a selection for the fresh type corn of eye health effect, comprise the following steps:
(1) collecting and plant seed outward appearance for yellow or lurid corn, being suitable for peeling entire kernel period of eating raw and with pocket knife along stitching rip cutting in seed, choosing endosperm is all yellow or lurid endosperm; Then extract saponified for subsequent use;
(2) adopt ultraviolet-visible spectrophotometry, the total carotinoid content in the fresh seed that detecting step is (1) screened; Retain the seed of total carotinoid more than content 10mg/kg; Be specially: take 0.1-0.2g saponified, grind, add 5ml normal hexane: acetone=3 ︰ 2 volume ratio, lixiviate 16 hours under dark condition, middle shake several times; Then add 1ml 20% volume ratio KOH-methyl alcohol, react one hour under dark condition; Add 3ml normal hexane and 1ml 10wt% metabisulfite solution again, its static layering of mixing relief, upper strata yellow liquid is the total carotinoid solution slightly carried; Take blank as contrast, measure at ultraviolet-visible spectrophotometer 454nm place;
(3) plant the seed that (2) step screens, adopt high performance liquid chromatography (HPLC method) to carry out quantitative test to zeaxanthin in its fresh corn seed; Retain the material of more than zeaxanthin 8mg/kg.
2. the selection with the fresh type corn of eye health effect according to claim 1, it is characterized in that, further comprises step (4), continue plantation and expand numerous step (3) selected seed, the corn strain that screening economical character is good, coordinate force is strong, or with being rich in the corn kernel of zeaxanthin gene for donor, combo is carried out through the strain of hybridizing, backcrossing or somaclonal variation obtaining, the strain that screening general performance is good, the final corn inbred line being rich in zeaxanthin or the cenospecies obtaining inheritance stability.
3. the selection with the fresh type corn of eye health effect according to claim 1, is characterized in that, described step (1) in corn kernel be the strain that produces of corn conventional variety or hybrid parents or germplasm resource or mutagenesis or self-mating system.
4. the selection with the fresh type corn of eye health effect according to claim 1, is characterized in that, described step (1) in saponified forming steps as follows:
A, get 20g corn kernel and grind, add acetone extract, suction filtration, repeatedly several times, until extracting liquid colourless;
B, merging extract, be evaporated to dry; Time concentrated, temperature is lower than 40 degree;
C, dissolve dry acetone crude extract with a small amount of methyl alcohol, then add isopyknic 30% vol/vol methanol-KOH saponification more than 4 hours, obtain saponification liquor;
D, reduced pressure concentration saponification liquor, to minimum volume, add a small amount of ethyl acetate and normal hexane equal-volume and extract than the mixed liquor 1 ︰ 1=V/V of configuration, re-extract repeatedly, until extract is colourless;
E, combining extraction liquid, alkali lye when washing away saponification with distilled water in separating funnel, gets upper layer of extraction liquid and is evaporated to minimum volume, obtain Carotenoids Extracts;
F, transfer in the centrifuge tube of 1.5ml by the Carotenoids Extracts concentrated, do remaining solvent with N2 air-blowing ,-20 DEG C save backup;
G, every sample are in triplicate.
5. the selection with the fresh type corn of eye health effect according to claim 1, it is characterized in that, described efficient liquid phase detection method is specially: detect the efficient liquid phase detection system adopting Waters, wherein detecting device is 2998 type diode array detector, chromatographic column is Spherisorb ODS2:5 μm, 250 × 4.6mm, mobile phase is Yi Jing ︰ Jia Chun ︰ bis-Lv Jia Wan ︰ normal hexane=85 ︰ 5 ︰ 5 ︰ 5 volume ratio, flow velocity 1ml/min; Sample 1ml mobile phase dissolves, sample introduction after the filter membrane of mistake 0.45 μm, sample size 10 μ l.
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| CN104297407B (en) * | 2014-10-23 | 2016-01-20 | 中国农业科学院作物科学研究所 | The Ultra Performance Liquid Chromatography assay method of carotenoid content in wheat |
| CN106577267B (en) * | 2016-12-23 | 2018-07-20 | 天津农学院 | The gas chromatography mass spectrometry rapid screening method of corn inbred line combination selection |
| CN108680683B (en) * | 2018-04-17 | 2020-12-15 | 中国农业科学院郑州果树研究所 | Mobile phase formula for detecting carotenoid in fruits by liquid chromatography and HPLC (high performance liquid chromatography) method for detecting carotenoid in fruits |
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