CN102507775A - Method for detecting content of xanthophyll in soy-based food - Google Patents
Method for detecting content of xanthophyll in soy-based food Download PDFInfo
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- CN102507775A CN102507775A CN2011103371156A CN201110337115A CN102507775A CN 102507775 A CN102507775 A CN 102507775A CN 2011103371156 A CN2011103371156 A CN 2011103371156A CN 201110337115 A CN201110337115 A CN 201110337115A CN 102507775 A CN102507775 A CN 102507775A
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Abstract
The invention relates to a method for detecting the content of xanthophyll in soy-based food. The method includes the working steps: treating samples by accurately weighing 1.0g of samples, being accurate to 0.0001g, placing the samples into a 50mL polypropylene centrifugal tube with a stopper, adding 20mL of water at the temperature of 45-50 DEG C, adding 0.1g of amylase, blending the samples, the water and the amylase by means of whirling, placing the mixture into an incubator at the temperature of 50-60 DEG C, culturing the mixture for 30 minutes, taking out the mixture, cooling the mixture to the temperature below 30 DEG C by means of ice-bath, adding 20mL of petroleum ether under the condition of keeping in a dark place, performing ice-bath ultrasonic extraction for 10 minutes, centrifuging for 5 minutes at the speed of 5000r/min, accurately transferring 10mL of supernate into a 15mL centrifugal tube, flushing nitrogen at the temperature below 30 DEG C until drying the mixture, constantly dissolving the mixture to 1mL with acetone solution containing 0.1% of BHT (butylated hydroxytoluene), blending the solution by means of whirling and filtering the solution with an organic filter membrane of 0.2 micrometer; determining by means of HPLC (high performance liquid chromatography) and quantifying by an external standard method; and performing quantitative analysis by drawing standard curves, determining test solution and finally computing by the aid of a formula. The method for detecting the content of the phytoxanthin in the soy-based food has the advantages of convenience, rapidness, accuracy and the like.
Description
Technical field
The present invention relates to a kind of detection method of content that contains xenthophylls in the beans base food.
Background technology
Xenthophylls is amphiblestroid main pigment composition, and the blue light of filtration and oxidation resistant effect are arranged, and is the important nutrient of helping eye development.Because the irradiation of light, short light (blue light) is very big to amphiblestroid injury.Crystalline lens when the baby comes from is thorough relatively clearly, zero-two years old section, and the penetrable crystalline lens of the blue light of about 70-80% arrives retina.Xenthophylls has played barrier action for the tender eyes of infant children, so there is the people to liken xenthophylls to " stealthy sunglasses ".Because human body self can not synthesize xenthophylls, so must replenish a certain amount of xenthophylls to baby every day.
Just added xenthophylls in the new beans base product of releasing of company at present, the assay for xenthophylls in the beans base food does not have corresponding detecting method as yet in the GB.Therefore, the detection method of content of xenthophylls is suitable for the required xenthophylls beans base class food that contains of infant growth most significance is arranged mixing in the exploitation beans base food.
Summary of the invention
The object of the present invention is to provide a kind of detection method of content that contains xenthophylls in the beans base food.Be intended to convenient, fast, detect the content of xenthophylls in the beans base food exactly.
The present invention, its principle is: starch-containing in the beans base food, behind amylase enzymolysis, utilize sherwood oil to extract earlier, it is heavily molten with acetone to concentrate the back, measures through UV-detector, and external standard method is quantitative.
The present invention comprises following job step:
(1) reagent is prepared, and agents useful for same comprises:
(a) methyl alcohol: chromatographically pure;
(b) methyl tert-butyl ether: chromatographically pure;
(c) acetone: chromatographically pure;
(d) DBPC 2,6 ditertiary butyl p cresol (BHT): purity >=99%;
(e) methylene chloride: chromatographically pure;
(f) sherwood oil: analyze pure;
(g) diastase: enzyme activity >=1.5U/mg;
(h) xenthophylls standard items: purity >=96.2%;
(i) acetone soln of 0.1%BHT: take by weighing 0.500gBHT in the 500mL volumetric flask, be dissolved in water and be settled to scale;
(j) standard reserving solution 100 μ g/mL: accurately take by weighing 10mg xenthophylls standard items, with methylene chloride dissolving and be settled to 100mL, in-80 ℃ of refrigerators, keep in Dark Place, storage life is 3 months;
(k) standard intermediate liquid 20 μ g/mL: accurately pipette the 2mL standard reserving solution, with methanol constant volume to 10mL;
(l) standard series working fluid: the acetone soln to contain 0.1%BHT is diluted to 0.4 μ g/mL, 0.8 μ g/mL, 1.6 μ g/mL, 2.0 μ g/mL, 2.5 μ g/mL respectively with the standard intermediate liquid;
(m) water: ultrapure water;
(2) instrument and equipment, instrument and equipment comprises:
(a) high performance liquid chromatograph: Tianjin, island LC-20AT pump, SPD-20A UV-detector;
(b) electronic balance: be accurate to 0.0001 g;
(c) Extraction by Ultrasound device;
(d) turbine mixer;
(e) Nitrogen evaporator;
(f) supercentrifuge;
(3) sample preparation: accurately take by weighing sample 1.0g, be accurate to 0.0001 g, place 50mL tool plug polypropylene centrifuge tube, add 20mL45-50 ℃ of water; Add 0.1g diastase again, the vortex mixing is put in the 50-60 ℃ of incubator and is cultivated 30min; Take out ice bath and be cooled to below 30 ℃, under the lucifuge condition, add the 20mL sherwood oil, ice-bath ultrasonic is extracted 10min; Ultrasonic procedure need be changed ice cube, guarantees that whole leaching process temperature is no more than 30 ℃, takes out to be cooled to below 10 ℃; In the centrifugal 5min of 5000r/min, accurately pipette the 10mL supernatant in a 15mL centrifuge tube, nitrogen below 30 ℃ blows to doing; Surely dissolve to 1mL with the acetone soln that contains 0.1%BHT, the vortex mixing is crossed the organic filter membrane of 0.2 μ m; Supply HPLC to measure, external standard method is quantitative;
(4) chromatographic condition, chromatographic condition comprises:
(a) chromatographic column: YMC
TMCarotenoid (250 mm * 4.6mm, 5 μ m) or suitable person;
(b) sample size: 20 μ L;
(c) flow velocity: 1.0mL/min;
(d) UV-detector wavelength: 445nm;
(e) column temperature: 25 ℃;
(f) moving phase: 85% methyl alcohol-15% methyl tert-butyl ether;
(5) quantitative test:
(a) typical curve is drawn: the standard series working fluid being injected high performance liquid chromatograph respectively, measure corresponding peak area, is horizontal ordinate with the peak area, and standard operation liquid concentration is ordinate drawing standard curve;
(b) test solution is measured: the appearance liquid that will prepare injects high performance liquid chromatograph, records corresponding peak area, with typical curve sample is carried out quantitatively recording test solution concentration;
(c) calculate, computing formula is: X=C * V
1* 100/ (m * V
2), in the formula:
X: the content of xenthophylls in the sample, unit are μ g/100g;
C: by the test solution concentration that typical curve obtains, unit is μ g/mL;
V
1: add the volume of sherwood oil, unit is mL;
V
2: pipette the volume of supernatant, unit is mL;
M: sample mass, unit are g.
The present invention is on the basis of " xenthophylls is measured in the milk powder " (GB/T 23209-2008), to improve.(1) in the infant food, the check of beans base food and milk powder has many similarities.But because of containing starch in the beans base, need enzymolysis and in the milk powder extraction agent of xenthophylls be acetone, acetone and water dissolve each other, after the extraction can't with water stratification (enzymolysis process need add water), also can't blow concentrated by nitrogen at last.This method adopts sherwood oil to extract, and extraction back layering is obvious, is beneficial to pipetting of supernatant and blows concentrated with nitrogen; (2) for the xenthophylls in the beans base is fully extracted, this method adopts ultrasonic Extraction to replace oscillation extraction.And because of xenthophylls to thermoae instability, so the whole ultrasonic leaching process adopts the ice bath cooling, thereby reduce the loss in the leaching process to greatest extent; (3) adopt methyl alcohol and methyl tert-butyl ether to carry out gradient elution among the GB/T 23209-2008, whole gradient is covered needs 15 minutes.The present invention adopts isocratic elution, as moving phase, can go out peak about 7.3 minutes with 85% methyl alcohol and 15% methyl tert-butyl ether, and every sample needle is only covered and needed 9 minutes.
The present invention, the content of detection xenthophylls has advantages such as convenient, fast, accurate in beans base food.Be suitable for most the required xenthophylls beans base class food that contains of infant growth significance is arranged mixing.
Description of drawings
Fig. 1 is the lutein content canonical plotting.Among the figure, ordinate is a standard operation liquid concentration, and horizontal ordinate is the corresponding peak area of standard operation liquid.
Embodiment
A kind of detection method of content that contains xenthophylls in the beans base food comprises following job step:
(1) reagent is prepared, and agents useful for same comprises:
(a) methyl alcohol: chromatographically pure;
(b) methyl tert-butyl ether: chromatographically pure;
(c) acetone: chromatographically pure;
(d) DBPC 2,6 ditertiary butyl p cresol (BHT): purity >=99%;
(e) methylene chloride: chromatographically pure;
(f) sherwood oil: analyze pure;
(g) diastase: enzyme activity >=1.5U/mg;
(h) xenthophylls standard items: purity >=96.2%;
(i) acetone soln of 0.1%BHT: take by weighing 0.500gBHT in the 500mL volumetric flask, be dissolved in water and be settled to scale;
(j) standard reserving solution 100 μ g/mL: accurately take by weighing 10mg xenthophylls standard items, with methylene chloride dissolving and be settled to 100mL, in-80 ℃ of refrigerators, keep in Dark Place, storage life is 3 months;
(k) standard intermediate liquid 20 μ g/mL: accurately pipette the 2mL standard reserving solution, with methanol constant volume to 10mL;
(l) standard series working fluid: the acetone soln to contain 0.1%BHT is diluted to 0.4 μ g/mL, 0.8 μ g/mL, 1.6 μ g/mL, 2.0 μ g/mL, 2.5 μ g/mL respectively with the standard intermediate liquid;
(m) water: ultrapure water;
(2) instrument and equipment, instrument and equipment comprises:
(a) high performance liquid chromatograph: Tianjin, island LC-20AT pump, SPD-20A UV-detector;
(b) electronic balance: be accurate to 0.0001 g;
(c) Extraction by Ultrasound device;
(d) turbine mixer;
(e) Nitrogen evaporator;
(f) supercentrifuge;
(3) chromatographic condition, chromatographic condition comprises:
(a) chromatographic column: YMC
TMCarotenoid (250 mm * 4.6mm, 5 μ m) or suitable person;
(b) sample size: 20 μ L;
(c) flow velocity: 1.0mL/min;
(d) UV-detector wavelength: 445nm;
(e) column temperature: 25 ℃;
(f) moving phase: 85% methyl alcohol-15% methyl tert-butyl ether;
(4) typical curve is drawn:
Standard series working fluid 0.4 μ g/mL, 0.8 μ g/mL, 1.6 μ g/mL, 2.0 μ g/mL and 2.5 μ g/mL are injected high performance liquid chromatograph respectively; Record corresponding peak area and be respectively 16618.7,31541.1,60045.9,74646.1,93660.2; With the peak area is horizontal ordinate; Standard operation liquid concentration is ordinate drawing standard curve, and is as shown in Figure 1.When utilizing high performance liquid chromatograph to carry out determination and analysis, can directly read the numerical value of peak area;
(5) sample preparation: accurately take by weighing sample 1.0g, be accurate to 0.0001 g, place 50mL tool plug polypropylene centrifuge tube, add 20mL45-50 ℃ of water; Add 0.1g diastase again, the vortex mixing is put in the 50-60 ℃ of incubator and is cultivated 30min, takes out ice bath and is cooled to below 30 ℃; Under the lucifuge condition, add the 20mL sherwood oil, ice-bath ultrasonic is extracted 10min, and ultrasonic procedure need be changed ice cube, guarantees that whole leaching process temperature is no more than 30 ℃; Taking-up is cooled to below 10 ℃, in the centrifugal 5min of 5000r/min, accurately pipette the 10mL supernatant in a 15mL centrifuge tube (for the high sample of fat content, because of fat floating in the ether layer; Influence supernatant and pipette, visual concrete condition suitably reduces moves liquid measure), nitrogen below 30 ℃ blows to doing; Surely dissolve to 1mL with the acetone soln that contains 0.1%BHT, the vortex mixing is crossed the organic filter membrane of 0.2 μ m; Supply HPLC to measure, external standard method is quantitative;
(6) quantitative test: present embodiment is to the detection of lutein content in baby's beans base food of certain batch; Extract sample 1 and sample 2 respectively; Measure the content of xenthophylls in sample 1 and the sample 2 respectively; Calculate its mean value again, if deviation≤10% is qualified for detecting, if deviation>=10% o'clock must detect again;
The appearance of sample 1 heavily is 1.0005g; According to the appearance liquid that (3) sample treatment obtains sample 1, appearance liquid is injected high performance liquid chromatograph, record corresponding peak area; With typical curve sample is carried out quantitative test, obtaining kind concentration of liquid xenthophylls is 0.402 μ g/mL; Because when sample preparation, adding the volume of sherwood oil is 20mL; The volume that pipettes supernatant is 10mL; Therefore by computing formula X=C * V
1* 100/ (m * V
2) content that can calculate xenthophylls in the sample is 0.402 * 20 * 100/ (1.0005 * 10)=80.4 (μ g/100g);
The appearance of sample 2 heavily is 1.0016g; According to the appearance liquid that (3) sample treatment obtains sample 1, appearance liquid is injected high performance liquid chromatograph, record corresponding peak area; With typical curve sample is carried out quantitative test, obtaining kind concentration of liquid xenthophylls is 0.425 μ g/mL; Because when sample preparation, adding the volume of sherwood oil is 20mL; The volume that pipettes supernatant is 10mL; Therefore by computing formula X=C * V
1* 100/ (m * V
2) content that can calculate xenthophylls in the sample is 0.425 * 20 * 100/ (1.0016 * 10)=84.9 (μ g/100g);
The calculating of mean value: (80.4+84.9)/2=82.6 (μ g/100g);
The calculating of precision: [(84.9-80.4)/82.6] * 100%=5.45%.
Conclusion:Present embodiment detects this batch baby beans base food, and the content of xenthophylls is 82.6 μ g/100g in the sample, and twice is detected precision is 5.45%.
Claims (1)
1. detection method of content that contains xenthophylls in the beans base food is characterized in that comprising following job step:
(1) reagent is prepared, and agents useful for same comprises:
(a) methyl alcohol: chromatographically pure;
(b) methyl tert-butyl ether: chromatographically pure;
(c) acetone: chromatographically pure;
(d) DBPC 2,6 ditertiary butyl p cresol (BHT): purity >=99%;
(e) methylene chloride: chromatographically pure;
(f) sherwood oil: analyze pure;
(g) diastase: enzyme activity >=1.5U/mg;
(h) xenthophylls standard items: purity >=96.2%;
(i) acetone soln of 0.1%BHT: take by weighing 0.500gBHT in the 500mL volumetric flask, be dissolved in water and be settled to scale;
(j) standard reserving solution 100 μ g/mL: accurately take by weighing 10mg xenthophylls standard items, with methylene chloride dissolving and be settled to 100mL, in-80 ℃ of refrigerators, keep in Dark Place, storage life is 3 months;
(k) standard intermediate liquid 20 μ g/mL: accurately pipette the 2mL standard reserving solution, with methanol constant volume to 10mL;
(l) standard series working fluid: the acetone soln to contain 0.1%BHT is diluted to 0.4 μ g/mL, 0.8 μ g/mL, 1.6 μ g/mL, 2.0 μ g/mL, 2.5 μ g/mL respectively with the standard intermediate liquid;
(m) water: ultrapure water;
(2) instrument and equipment, instrument and equipment comprises:
(a) high performance liquid chromatograph: Tianjin, island LC-20AT pump, SPD-20A UV-detector;
(b) electronic balance: be accurate to 0.0001 g;
(c) Extraction by Ultrasound device;
(d) turbine mixer;
(e) Nitrogen evaporator;
(f) supercentrifuge;
(3) sample preparation: accurately take by weighing sample 1.0g, be accurate to 0.0001 g, place 50mL tool plug polypropylene centrifuge tube, add 20mL45-50 ℃ of water; Add 0.1g diastase again, the vortex mixing is put in the 50-60 ℃ of incubator and is cultivated 30min; Take out ice bath and be cooled to below 30 ℃, under the lucifuge condition, add the 20mL sherwood oil, ice-bath ultrasonic is extracted 10min; Ultrasonic procedure need be changed ice cube, guarantees that whole leaching process temperature is no more than 30 ℃, takes out to be cooled to below 10 ℃; In the centrifugal 5min of 5000r/min, accurately pipette the 10mL supernatant in a 15mL centrifuge tube, nitrogen below 30 ℃ blows to doing; Surely dissolve to 1mL with the acetone soln that contains 0.1%BHT, the vortex mixing is crossed the organic filter membrane of 0.2 μ m; Supply HPLC to measure, external standard method is quantitative;
(4) chromatographic condition, chromatographic condition comprises:
(a) chromatographic column: YMC
TMCarotenoid (250 mm * 4.6mm, 5 μ m) or suitable person;
(b) sample size: 20 μ L;
(c) flow velocity: 1.0mL/min;
(d) UV-detector wavelength: 445nm;
(e) column temperature: 25 ℃;
(f) moving phase: 85% methyl alcohol-15% methyl tert-butyl ether;
(5) quantitative test:
(a) typical curve is drawn: the standard series working fluid being injected high performance liquid chromatograph respectively, measure corresponding peak area, is horizontal ordinate with the peak area, and standard operation liquid concentration is ordinate drawing standard curve;
(b) test solution is measured: the appearance liquid that will prepare injects high performance liquid chromatograph, records corresponding peak area, with typical curve sample is carried out quantitatively recording test solution concentration;
(c) calculate, computing formula is: X=C * V
1* 100/ (m * V
2), in the formula:
X: the content of xenthophylls in the sample, unit are μ g/100g;
C: by the test solution concentration that typical curve obtains, unit is μ g/mL;
V
1: add the volume of sherwood oil, unit is mL;
V
2: pipette the volume of supernatant, unit is mL;
M: sample mass, unit are g.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103175934A (en) * | 2013-03-05 | 2013-06-26 | 福建省农业科学院作物研究所 | Strawberry carotenoid component content determination method |
| CN104749293A (en) * | 2015-04-21 | 2015-07-01 | 江苏省农业科学院 | Method for efficiently extracting carotenoids in yellow peach fruits and determining carotenoids in yellow peach fruits by liquid phase |
| CN106198818A (en) * | 2016-08-13 | 2016-12-07 | 广州白云山汉方现代药业有限公司 | A kind of quality determining method of xanthophyll preparation |
| CN107478738A (en) * | 2017-08-01 | 2017-12-15 | 内蒙古蒙牛乳业（集团）股份有限公司 | The method for detecting dairy products Lutein |
| CN110879257A (en) * | 2019-10-28 | 2020-03-13 | 江苏康缘药业股份有限公司 | Method for determining xanthophyll component |
| CN114778742A (en) * | 2022-05-14 | 2022-07-22 | 重庆市食品药品检验检测研究院 | Method for determining high-efficiency and high-precision dehydroacetic acid in puffed food |
| CN115078567A (en) * | 2022-05-19 | 2022-09-20 | 江苏艾兰得营养品有限公司 | Determination method of lutein ester and zeaxanthin ester |
-
2011
- 2011-10-31 CN CN2011103371156A patent/CN102507775A/en active Pending
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103175934A (en) * | 2013-03-05 | 2013-06-26 | 福建省农业科学院作物研究所 | Strawberry carotenoid component content determination method |
| CN104749293A (en) * | 2015-04-21 | 2015-07-01 | 江苏省农业科学院 | Method for efficiently extracting carotenoids in yellow peach fruits and determining carotenoids in yellow peach fruits by liquid phase |
| CN106198818A (en) * | 2016-08-13 | 2016-12-07 | 广州白云山汉方现代药业有限公司 | A kind of quality determining method of xanthophyll preparation |
| CN107478738A (en) * | 2017-08-01 | 2017-12-15 | 内蒙古蒙牛乳业（集团）股份有限公司 | The method for detecting dairy products Lutein |
| CN110879257A (en) * | 2019-10-28 | 2020-03-13 | 江苏康缘药业股份有限公司 | Method for determining xanthophyll component |
| CN114778742A (en) * | 2022-05-14 | 2022-07-22 | 重庆市食品药品检验检测研究院 | Method for determining high-efficiency and high-precision dehydroacetic acid in puffed food |
| CN115078567A (en) * | 2022-05-19 | 2022-09-20 | 江苏艾兰得营养品有限公司 | Determination method of lutein ester and zeaxanthin ester |
| CN115078567B (en) * | 2022-05-19 | 2023-10-24 | 江苏艾兰得营养品有限公司 | Method for determining lutein ester and zeaxanthin ester |
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Application publication date: 20120620 |