CN103018185A - Method for quickly measuring collagen content - Google Patents
Method for quickly measuring collagen content Download PDFInfo
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- CN103018185A CN103018185A CN 201210513484 CN201210513484A CN103018185A CN 103018185 A CN103018185 A CN 103018185A CN 201210513484 CN201210513484 CN 201210513484 CN 201210513484 A CN201210513484 A CN 201210513484A CN 103018185 A CN103018185 A CN 103018185A
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Abstract
The invention discloses a method for quickly measuring the collagen content. By the conventional detection method, a sample can be detected after being required to be hydrolyzed by concentrated acid at high temperature of 110 to 130 DEG C for 4 to 24 hours, so that operation is dangerous, and a detection result is low in accuracy. By the detection method provided by the invention, the sample is subjected to heat preservation in a water bath of 70 DEG C for 30 minutes, and then enzymolysis is executed for 1 hour by adding alkaline protease. The method is operated quickly and easily and is high in accuracy; the working efficiency is greatly improved; any potential safety hazard is avoided; and the method has wide application and popularization value.
Description
Technical field
The present invention relates to a kind of collagen detection technique, be specifically related to a kind of method of Fast Measurement collagen content.
Background technology
Collagen is a kind of biological polymer substance, mainly is present in the animal tissue, is one of important starting material of biotech industry, and its application comprises gives birth to doctor's material, cosmetics, food, health products and scientific research.And along with the in recent years raising of people's living standard and the reinforcement of health care consciousness, the application of collagen in cosmetics, food and health products improves year by year, so the detection of collagen content seems particularly important.
The preprocess method of collagen sample mainly is acidic treatment at present, need to just can detect after 4 ~ 24 hours in the lower hydrolysis of high temperature (110 ~ 130 ℃), not only the reaction time is long, accuracy is low, and have a potential safety hazard, therefore set up a kind of simple, fast, the detection method of collagen that safety, accuracy are high is significant.
Summary of the invention
The objective of the invention is to solve prior art exist detection time long, accuracy is low and have the technical matters of potential safety hazard, and a kind of method of Fast Measurement collagen content is provided, the method is simple to operate, the result is accurate, without any potential safety hazard.The present invention relates to a kind of assay method of collagen content, its principle is to utilize collagen to be become hydroxyproline by the alkali protease enzymolysis, and hydroxyproline can be generated by the toluene-sodium-sulfonchloramide oxidation material of pyrrole ring, and this pyrrole ring material is developed the color by the dimethylamino benzaldehyde, generates red compound.Concrete operation step is as follows:
(1) preparation of hydroxyproline standard solution: draw the hydroxyproline storage liquid 10mL of 1mg/mL in the 100mL volumetric flask, with the distilled water constant volume to 100mL, concentration is 100 μ g/mL, draw respectively 3.0,6.0,9.0,12.0,15.0mL, to 100mL, be mixed with the titer that concentration is respectively 3.0,6.0,9.0,12.0,15.0 μ g/mL with the distilled water constant volume;
(2) testing sample pre-service: sample thief powder 5～20mg is in tool plug test tube, and heating adds alkali protease 5mg after 30 minutes in 70 ℃ of water-baths, and enzymolysis is after 1～3 hour, and it is for subsequent use to be settled to 100mL;
(3) blank sample: described water or damping fluid are as blank sample, and its protein concentration is 0mg/mL;
(4) draw respectively titer in the step (1) and the testing sample 1mL in the step (2), add the oxidation after 20 minutes in 25 ℃ of water-baths of 1mL citrate buffer solution and 1mL toluene-sodium-sulfonchloramide solution, add perchloric acid 1mL, in 25 ℃ of water-baths, placed 10 minutes, add Paradimethylaminobenzaldehyde 1mL, 65 ℃ of water-baths developed the color 20 minutes, surveyed absorbance after the cooling, and converse the concentration of hydroxyproline in the sample, thereby obtain the content of collagen in the sample.
The advantage such as that method of the present invention has is easy to detect, measuring speed is fast, accuracy is high and operating conditions is gentle; Method of the present invention is highly sensitive, can measure 0.05mg/mL; Degree of accuracy is high, and RSD≤2% is well suited for carrying out the fast detecting of collagen content in food, cosmetics and the health products, is with a wide range of applications.
Embodiment
Embodiment 1: the mensuration of collagen content in the collagen powder
(1) preparation of hydroxyproline standard solution: draw the hydroxyproline storage liquid 10mL of 1mg/mL in the 100mL volumetric flask, with the distilled water constant volume to 100mL, concentration is 100 μ g/mL, draw this solution 3.0,6.0,9.0,12.0,15.0mL, use respectively the distilled water constant volume to 100mL, namely get the standard solution that concentration is respectively 3.0,6.0,9.0,12.0,15.0 μ g/mL;
(2) making of typical curve: get above-mentioned titer 1mL, add the oxidation after 20 minutes in 25 ℃ of water-baths of 1mL citrate buffer solution and 1mL toluene-sodium-sulfonchloramide solution, add perchloric acid 1mL, in 25 ℃ of water-baths, placed 10 minutes, add Paradimethylaminobenzaldehyde 1mL, absorbance is surveyed in 65 ℃ of water-baths colour developing 20 minutes after the cooling, according to concerning the drawing standard curve between hydroxyproline concentration and the corresponding hydroxyproline absorbance that records;
(3) collagen content is measured in the sample: get collagen sample powder 20mg in tool plug test tube, heating adds alkali protease 5mg after 30 minutes in 70 ℃ of water-baths, behind the enzymolysis 3 hours, be settled to 100mL, get the 1mL hydrolyzate, measure according to hydroxyproline standard curve making method, the corresponding relation between the absorbance of mensuration and the hydroxyproline typical curve is determined the content of collagen in the testing sample.
Embodiment 2: the mensuration of collagen content in the fish-bone collagen protein powder
Collagen content is measured in the sample: get the about 10mg of collagen sample powder in tool plug test tube, heating adds alkali protease 5mg after 30 minutes in 70 ℃ of water-baths, behind the enzymolysis 2 hours, be settled to 100mL, get the 1mL hydrolyzate, measure according to hydroxyproline standard curve making method, the corresponding relation between the absorbance of mensuration and the hydroxyproline typical curve is determined the content of collagen in the testing sample.All the other are with embodiment 1.
Embodiment 3: the mensuration of collagen content in the health products collagen protein powder
Collagen content is measured in the sample: get the about 5mg of collagen sample powder in tool plug test tube, heating adds alkali protease 5mg after 30 minutes in 70 ℃ of water-baths, behind the enzymolysis 1 hour, be settled to 100mL, get the 1mL hydrolyzate, measure according to hydroxyproline standard curve making method, the corresponding relation between the absorbance of mensuration and the hydroxyproline typical curve is determined the content of collagen in the testing sample.All the other are with embodiment 1.
Above-described embodiment is just in order to illustrate the present invention, rather than limitation of the present invention, and all variations of making in essential scope of the present invention, remodeling, interpolation or replacement all should belong to protection scope of the present invention.
Claims (2)
1. the method for a Fast Measurement collagen content, it is characterized in that: get collagen sample powder 5～20mg in tool plug test tube, heating adds alkali protease 5mg after 30 minutes in 70 ℃ of water-baths, and enzymolysis was transferred in another test tube after 1～3 hour, be settled to 100mL, get the 1mL hydrolyzate, measure according to hydroxyproline standard curve making method, survey absorbance at 560 nm places, and converse the concentration of sample hydroxyproline, thereby obtain the content of collagen.
2. the method for a kind of Fast Measurement collagen content according to claim 1, it is characterized in that: hydroxyproline standard curve making method is: preparation concentration is respectively the hydroxyproline titer of 3.0,6.0,9.0,12.0,15.0 μ g/mL, draw respectively 1mL, add the oxidation after 20 minutes in 25 ℃ of water-baths of 1mL citrate buffer solution and 1mL toluene-sodium-sulfonchloramide solution, add perchloric acid 1mL, in 25 ℃ of water-baths, placed 10 minutes, add Paradimethylaminobenzaldehyde 1mL, 65 ℃ of water-baths developed the color 20 minutes, and 560 nm survey absorbance after the cooling.
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| CN 201210513484 CN103018185A (en) | 2012-12-05 | 2012-12-05 | Method for quickly measuring collagen content |
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| CN 201210513484 CN103018185A (en) | 2012-12-05 | 2012-12-05 | Method for quickly measuring collagen content |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103776778A (en) * | 2014-01-10 | 2014-05-07 | 华南理工大学 | Quantitative determination method in extraction process of fish collagen and application of quantitative determination method |
| 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 |
| CN106872377A (en) * | 2015-12-10 | 2017-06-20 | 中国科学院大连化学物理研究所 | Bicinchoninic acid is used for the quantitative detecting method of protein content in alginate material |
| CN108226062A (en) * | 2016-12-15 | 2018-06-29 | 江苏维赛科技生物发展有限公司 | A kind of method that the content of hydroxyproline in fish-skin is measured using spectrophotometry |
| CN110208508A (en) * | 2019-06-18 | 2019-09-06 | 余碧芝 | Determine the method for collagen peptide quality |
| CN111693472A (en) * | 2019-03-11 | 2020-09-22 | 广东博与再生医学有限公司 | Corneal hydroxyproline detection method |
| RU2735375C1 (en) * | 2020-04-03 | 2020-10-30 | Федеральное государственное бюджетное научное учреждение "Федеральный исследовательский центр фундаментальной и трансляционной медицины" (ФИЦ ФТМ) | Method of determining fractions of hydroxyproline in biological material |
-
2012
- 2012-12-05 CN CN 201210513484 patent/CN103018185A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103776778A (en) * | 2014-01-10 | 2014-05-07 | 华南理工大学 | Quantitative determination method in extraction process of fish collagen and application of quantitative determination method |
| 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 |
| CN106872377A (en) * | 2015-12-10 | 2017-06-20 | 中国科学院大连化学物理研究所 | Bicinchoninic acid is used for the quantitative detecting method of protein content in alginate material |
| CN108226062A (en) * | 2016-12-15 | 2018-06-29 | 江苏维赛科技生物发展有限公司 | A kind of method that the content of hydroxyproline in fish-skin is measured using spectrophotometry |
| CN111693472A (en) * | 2019-03-11 | 2020-09-22 | 广东博与再生医学有限公司 | Corneal hydroxyproline detection method |
| CN110208508A (en) * | 2019-06-18 | 2019-09-06 | 余碧芝 | Determine the method for collagen peptide quality |
| RU2735375C1 (en) * | 2020-04-03 | 2020-10-30 | Федеральное государственное бюджетное научное учреждение "Федеральный исследовательский центр фундаментальной и трансляционной медицины" (ФИЦ ФТМ) | Method of determining fractions of hydroxyproline in biological material |
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Application publication date: 20130403 |