CN101871893A - Colorimetric method for determining free L-hydroxyproline (L-HYP) in milk powder - Google Patents
Colorimetric method for determining free L-hydroxyproline (L-HYP) in milk powder Download PDFInfo
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- CN101871893A CN101871893A CN200910049666A CN200910049666A CN101871893A CN 101871893 A CN101871893 A CN 101871893A CN 200910049666 A CN200910049666 A CN 200910049666A CN 200910049666 A CN200910049666 A CN 200910049666A CN 101871893 A CN101871893 A CN 101871893A
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Abstract
The invention relates to a colorimetric method for determining free L-hydroxyproline in milk powder, in particular to a method for rapidly determining the content of free L-hydroxyproline in the milk powder. according to relevant rules of China, the L-hydroxyproline is not allowed to be added to various foods as a food additive, but for pursuing great profits, some enterprises add the free L-HYP illegally in the milk powder production process so as to enable the protein content of products to reach the standard. However, the method for determining the free L-hydroxyproline in the milk powder is not available in China at present. In the invention, protein in the milk powder is deposited, and then the classical colorimetric method is adopted to determine the free L-HYP in the milk powder to obtain a relevant result. The precision of an experiment is between 2.6 percent and 4.9 percent, which can completely meet the demands of quantitative analysis. Moreover, the experimental operation is simple and fast, and the detection cost is low.
Description
Technical field
The present invention relates to the mensuration of free L-hydroxyproline content in the milk powder.
Background technology
L-hydroxyproline (L-HYP) is the constituent of human collagen albumen, but does not belong to 20 kinds of common amino acid." food additives use hygienic standard 2760-2007 " that China puts into effect recently do not allow it to add in the varieties of food items as food additives.But some enterprises are for profit-push, adulterate, the protein content that makes own product is up to standard and illegally added free L-HYP in producing the sharp milk-contained drink process of milk powder, deceive people to sell on market by the supervision and check of quality monitoring department, the dairy products of this poor quality will seriously injure health.The mensuration of L-HYP mainly comprises colourimetry, amino-acid analyzer method and electrophoresis etc., and is wherein commonly used with colourimetry.China is decided to be colourimetry the national standard method of " meat and meat products L-hydroxyproline content are measured " in nineteen ninety, and uses till today always.But regrettably, also do not occur at present free L-HYP in the milk powder is carried out method for measuring.
Summary of the invention
The purpose of patent of the present invention is to provide the method for free L-hydroxyproline content in a kind of fast measuring milk powder.
Patent of the present invention comprises carries out precipitation process to protein in the milk powder, adopts classical colorimetric method that wherein free L-HYP is measured the acquisition correlated results then.
As the feature of patent of the present invention, protein precipitant is 219g/L zinc acetate and 106g/L potassium ferrocyanide.
As the feature of patent of the present invention, developer is a para diaminobenzene formaldehyde.
The method that patent of the present invention is set up, the precision of experiment can satisfy the needs of quantitative test fully between 2.6%-4.9%, show that the free L-HYP that measures in the milk powder with the method is feasible.In addition, experimental implementation is simple, quick and to detect cost low.
Embodiment
Be ready to key instrument, comprise ultraviolet-visible pectrophotometer, electric-heated thermostatic water bath and electronic balance.It is stand-by to take by weighing the some grams of L-HYP standard items.Protein precipitant comprises 219g/L zinc acetate (take by weighing the 219g zinc acetate, add the 30ml glacial acetic acid, water-soluble and be settled to 1000ml) and 106g/L potassium ferrocyanide (it is water-soluble to take by weighing the 106g potassium ferrocyanide, and is settled to 1000mL).Buffer solution (pH 6): take by weighing the 50.0g citric acid, 26.3g NaOH and 146.1g sodium acetate, dissolved in distilled water is settled to 1000mL.Add 200mL distilled water and 300mL n-propanol again, mixing is preserved.Toluene-sodium-sulfonchloramide solution (this solution matching while using): take by weighing 1.41g toluene-sodium-sulfonchloramide dissolving 10mL distilled water, add 10mL n-propanol and 80mLpH6 damping fluid successively.Developer (this solution matching while using): take by weighing 10.09 para diaminobenzene formaldehyde, the 35mL high chloro acid dissolution slowly adds people 65mL isopropyl alcohol behind the mixing.
Preparation L-HYP standard stock solution (500 μ g/mL): accurately take by weighing L-HYP standard items 0.2500g, add 1 1mol/L dissolving with hydrochloric acid, distilled water is settled to 500mL, and 4 ℃ of refrigerators are preserved.Typical curve work mother liquor preparation (matching while using): draw 500 μ g/mL L-HYP standard stock solutions 0,0.5,1,2,4,6,8mL to 100mL volumetric flask respectively, be settled to 100mL, obtain the L-HYP typical curve work mother liquor that concentration is respectively 0,2.5,5,10,20,30,40 μ g/mL.With distilled water is blank, draws L-HYP standard operation mother liquor 1mL, adds toluene-sodium-sulfonchloramide 2mL solution, after fully room temperature condition reacts 20min down behind the mixing, add developer 2mL, in 60 ℃ of constant temperature water baths reaction 20min, be settled to 25ml after being cooled to room temperature rapidly, 558nm measures absorbance.With L-HYP concentration is horizontal ordinate, and absorbance is an ordinate drawing standard curve, obtains regression equation and related coefficient.
Accurately take by weighing 20g milk powder (being accurate to 0.0001g) in the 250mL volumetric flask, the limit is rotated the volumetric flask limit and is added potassium ferrocyanide solution 20ml, after shaking up, adds zinc acetate solution 20ml again, with precipitating proteins.Then, add water to scale, mixing, place half an hour after, discard upper strata fat, clear liquid filters with filter paper or absorbent cotton, discards filtrate 5～10ml just, filtrate for later use.From the liquid to be measured for preparing, draw 2mL in 25mL tool plug test tube.Draw liquid 1mL to be measured, add toluene-sodium-sulfonchloramide 2mL solution, after fully room temperature condition reacts 20min down behind the mixing, add developer 2mL, after 60 ℃ of constant temperature water baths react 20min, are cooled to room temperature rapidly, be settled to 25ml, with distilled water is blank, and 558nm measures absorbance.Each sample is done 3 repetitions.Absorbance is obtained hydroxyproline content for people's regression equation.
Claims (3)
1. free L-hydroxyproline in the colorimetric method for determining milk powder comprises protein in the milk powder is carried out precipitation process, obtains correlated results thereby adopt classical colorimetric method that wherein free L-HYP is measured then.
2. free L-hydroxyproline in the colorimetric method for determining milk powder according to claim 1, it is characterized in that: protein precipitant is 219g/L zinc acetate and 106g/L potassium ferrocyanide.
3. free L-hydroxyproline in the colorimetric method for determining milk powder according to claim 1, it is characterized in that: developer is a para diaminobenzene formaldehyde.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN200910049666A CN101871893A (en) | 2009-04-21 | 2009-04-21 | Colorimetric method for determining free L-hydroxyproline (L-HYP) in milk powder |
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| CN200910049666A CN101871893A (en) | 2009-04-21 | 2009-04-21 | Colorimetric method for determining free L-hydroxyproline (L-HYP) in milk powder |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101900679A (en) * | 2010-07-05 | 2010-12-01 | 浙江大学 | Method for rapidly detecting proline content of rape leaf |
| CN102636608A (en) * | 2012-04-10 | 2012-08-15 | 广东东泰乳业有限公司 | Method for detecting content of fructooligosaccharide in infant formula milk rice flour |
| CN102998306A (en) * | 2012-05-16 | 2013-03-27 | 江西天佳实业有限公司 | Detection method for gamma-aminobutyric acid containing calcium carbonate |
| CN103512880A (en) * | 2013-09-18 | 2014-01-15 | 山东大学齐鲁医院 | Joint detection kit for sialic acid and hydroxyproline |
| CN104730070A (en) * | 2015-03-19 | 2015-06-24 | 邢海霞 | Agent and method for jointly detecting sialic acid and hydroxyproline |
| CN104792707A (en) * | 2015-04-24 | 2015-07-22 | 江苏省理化测试中心 | Portable tester for leather protein milk and testing method of tester |
| CN105203478A (en) * | 2015-09-15 | 2015-12-30 | 陕西省食品药品检验所 | Fast detection method of leather-hydrolyzed protein L-hydroxyproline |
| CN105572069A (en) * | 2016-01-19 | 2016-05-11 | 江南大学 | Rapid detection method of cis-3-hydroxyL-proline in fermentation liquor |
| CN105651716A (en) * | 2016-02-06 | 2016-06-08 | 渭南市华隆畜牧有限公司 | Rapid ELISA (enzyme-linked immunosorbent assay) method for leather hydrolyzed protein L-hydroxyproline in raw milk |
| CN109765211A (en) * | 2019-01-18 | 2019-05-17 | 河北省食品检验研究院 | The rapid detection method of residual sulfur dioxide in a kind of natural plant extracts |
| CN110530856A (en) * | 2019-07-22 | 2019-12-03 | 福建省国鼎检测技术有限公司 | A kind of detection method of protein content in foodstuff |
-
2009
- 2009-04-21 CN CN200910049666A patent/CN101871893A/en active Pending
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101900679A (en) * | 2010-07-05 | 2010-12-01 | 浙江大学 | Method for rapidly detecting proline content of rape leaf |
| CN101900679B (en) * | 2010-07-05 | 2012-06-27 | 浙江大学 | Method for rapidly detecting proline content of rape leaf |
| CN102636608A (en) * | 2012-04-10 | 2012-08-15 | 广东东泰乳业有限公司 | Method for detecting content of fructooligosaccharide in infant formula milk rice flour |
| CN102998306A (en) * | 2012-05-16 | 2013-03-27 | 江西天佳实业有限公司 | Detection method for gamma-aminobutyric acid containing calcium carbonate |
| CN103512880A (en) * | 2013-09-18 | 2014-01-15 | 山东大学齐鲁医院 | Joint detection kit for sialic acid and hydroxyproline |
| CN103512880B (en) * | 2013-09-18 | 2014-06-11 | 山东大学齐鲁医院 | Joint detection kit for sialic acid and hydroxyproline |
| CN104730070A (en) * | 2015-03-19 | 2015-06-24 | 邢海霞 | Agent and method for jointly detecting sialic acid and hydroxyproline |
| CN104792707A (en) * | 2015-04-24 | 2015-07-22 | 江苏省理化测试中心 | Portable tester for leather protein milk and testing method of tester |
| CN105203478A (en) * | 2015-09-15 | 2015-12-30 | 陕西省食品药品检验所 | Fast detection method of leather-hydrolyzed protein L-hydroxyproline |
| CN105572069A (en) * | 2016-01-19 | 2016-05-11 | 江南大学 | Rapid detection method of cis-3-hydroxyL-proline in fermentation liquor |
| CN105651716A (en) * | 2016-02-06 | 2016-06-08 | 渭南市华隆畜牧有限公司 | Rapid ELISA (enzyme-linked immunosorbent assay) method for leather hydrolyzed protein L-hydroxyproline in raw milk |
| CN109765211A (en) * | 2019-01-18 | 2019-05-17 | 河北省食品检验研究院 | The rapid detection method of residual sulfur dioxide in a kind of natural plant extracts |
| CN110530856A (en) * | 2019-07-22 | 2019-12-03 | 福建省国鼎检测技术有限公司 | A kind of detection method of protein content in foodstuff |
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Application publication date: 20101027 |