CN108241035A - The new method of rich protein quality sample and its assay in a kind of oil samples from trench - Google Patents
The new method of rich protein quality sample and its assay in a kind of oil samples from trench Download PDFInfo
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Abstract
The present invention provides a kind of method of rich protein quality sample and its assay in oil samples from trench.It is screened and is verified by the method to different grease enrichment proteins, establish the preprocess method of a rich protein from gutter oil grease；On the basis of Bradford methods measure protein content, a kind of assay method that can be suitble to measure protein content in trench oil samples is established to improve the solubility of fat-soluble protein using tetrahydrofuran for solvent.
Description
Invention field
The present invention relates to the new method of rich protein quality sample and its assay in a kind of oil samples from trench, belong to point
Analysis chemistry and food analysis crossing domain.
Background technology
In recent years, it is continuously increased in the food-safety problem event of China, the research in relation to " gutter oil " causes extensively
Concern.Gutter oil refers to all kinds of poor oils, as cook after edible oil and repeatedly frying oil [Zhan H.L., Xi J.F.,
Zhao K., et al.Food Control, 2016,67：114-118].Gutter oil is recycling, the process of bleaching and deodorization
Often contain bacterium, heavy metal and harmful chemical, can directly seriously endanger public health when being used as edible oil
[Ramani K., Karthikeyan S., Boopathy R., et al.Process Biochemistry, 2012,47：435-
445].Therefore, the accurate quickly authentication technique undoubtedly controllably diffusion of ditch oil, improves food security, and protection conscientiously disappears
The person of expense interests [He J., Xu W.T., Shang Y., et al.Food Control, 2013,31：71-79].
In fact, it is extremely difficult work to distinguish edible oil and gutter oil, due to the two, not only appearance is similar, mainly into
Point also roughly the same, main component is all myristin.Physical and chemical index such as acid value, hard fat, heavy metal etc.,
It has been difficult to distinguish gutter oil and edible oil [Maggio R.M., Cerretani L., Chiavaro in current situation
E., et al.Food Control, 2010,21,890-895].Due to containing certain protein in grease, in different greases
Kinds of protein and content are also different, especially fat-soluble albumen, therefore, utilize the protein kind in gutter oil and edible oil
Class and content difference are accurately and reliably methods both to differentiate.How but micro egg is detached and detected from grease matrix
White matter component
First, the pretreatment of oil sample is one of key technology, successfully establishes one kind and egg is extracted from oil sample
The necessary condition that white matter method should meet is that processing step is simple, takes interference that is small and can removing grease matrix completely.Mesh
Before, method of protein is extracted from oil sample it has been reported that being broadly divided into aqueous solvent extraction and organic solvent sinks
Shallow lake method.As using 0.1M NaCl, 10% (v/v) glycerine is contained, the 50mM Tris-HCl buffer extractions of pH 7.5 eat olive
Protein [Georgalaki M.D., Sotiroudis T.G., Xenakis A.Journal of the in olive oil
American Oil Chemists ' Society, 1998,75：155-159].It is extracted through 50mM PBS, centrifugation, dialysis obtains
Protein [Zitouni N., Errahali Y., Metche M., et al.Journal of Allergy in sunflower oil
And Clinical Immunology, 2000,106：962-967].The characteristics of water-soluble extracting process is simple and practicable, but is extracted
Taken amount is too low.Protein in soybean oil can be extracted effectively from grease using petroleum ether as solvent, but it is organic molten to study discovery
Agent extracting process is complicated for operation, and takes pre-treatment step [the Olszewski A., Pons that cannot function as ideal analysis method
L., Moute ' te ' F., et al.Clinical and Experimental Allergy, 1998,28：850-859].In order to
Protein can be quickly and effectively extracted from grease, is had also been developed in the method extraction olive oil of acetone precipitation-filter paper filtering
Protein [Hidalgo F.J., Alaiz M., Zamora R.Analytical Chemistry, 2001,73：698-
702].This method is a kind of easy to operate, takes short method, but simultaneously it has also been found that this method is when using filter paper to filter, residual
Some greases is stayed to influence subsequent measurements.
Further, since without or with extremely micro protein in general grease, the measure of content is also very tired
Difficult work.At present, the assay method of common protein content has a Lowry methods in grease, bicinchoninic acid (BCA) method,
Bradford methods etc..Wherein Lowry methods and BCA are owned by France in chemical method, and Bradford is owned by France in dye binding method.But by
In the above method primarily directed to water-soluble albumen, and for the measure of fat-soluble protein content, obtain
Result be difficult accurate.For this purpose, methods of the Hidalgo et al. using acetone precipitation, obtained protein or polypeptide is hydrolyzed, so
Afterwards with amino acid analysis come quantitative protein content.This is the measure of protein in the edible oil that unique chemical process is developed
It is relatively accurate that method, wherein amino acid analysis method measure protein content.Relative to Lowry methods and Bradford methods
It says, the grease in the albumen of precipitation is noiseless to measuring, and obtained result is accurate, but its hydrolysis and measure operating process are compared
Complexity also restricts its extensive use.
Invention content
The present invention will be screened and be verified to the method for different grease enrichment proteins, establish one from gutter oil
The preprocess method of rich protein；On the basis of Bradford methods measure protein content, liposoluble is improved with different solvents
Property protein solubility, find out it is a kind of can be suitble to measure trench oil samples in protein content method.
In order to realize above-mentioned task, the present invention takes solution technical solution as follows：
1st, the new method of rich protein quality sample and its assay in a kind of oil samples from trench, it is characterised in that including
Following steps：
(1) a kind of preprocess method of the rich protein from different greases；
(2) efficient reversed phase liquid chromatography is to the analysis from grease rich protein sample；
(3) SDS-15%PAGE is to the analysis from grease rich protein sample；
(4) the Bradford methods of protein content determination in grease are improved；
2nd, the method for rich protein quality sample and its assay in the oil samples according to claim 1 from trench,
It is characterized in that：In step (1), 40-50g oil samples are weighed in 250mL beakers, place 1-2 hours at 18 DEG C, then
4 DEG C, 80-100mL acetone are added in into beaker, after stirring evenly, which in 4 DEG C of decentralizations is set to 0 .5-1 hours, uses Bu Shi
Funnel filters mixed liquor, and used filter paper is Whatman NO.1 filter paper (diameter 85mm, 11 μm of aperture) during suction filtration, will be filtered
Filter paper afterwards is put into beaker, adds in 5-15mL tetrahydrofurans, and then which is put into another beaker by concussion, add in 5-
15mL dioxane, concussion merge extracting solution twice, are dried up with nitrogen, the albumen being enriched in grease.
3rd, the method for rich protein quality sample and its assay in the oil samples according to claim 1 from trench,
It is characterized in that：In step (1), select organic filter membrane (0.45 μm) of Whatman filter paper and polyvinylidene fluoride material united
Method retains protein, is trapped in filter membrane sample and is washed 3~5 times using the acetone of refrigeration, first removes the oil remained on filter membrane
Then fat is selected tetrahydrofuran to be dissolved as solvent and is precipitated, then dried up with nitrogen, obtains solid protein sample.
4th, the method for rich protein quality sample and its assay in the oil samples according to claim 1 from trench,
It is characterized in that：In step (2), 50-100mL rich solids protein samples are divided into two parts, add in 70% tetrahydrofuran solution
Dissolving, a copy of it add in the lysozyme soln of 10-20 μ L 1.0mg/mL standards, through centrifugal treating, supernatant are taken to be used for
HPRPLC is analyzed.Chromatographic condition is Shimadzu VP-ODS C18Chromatographic column (4.6 × 150mm, 5 μm), mobile phase A (contain for pure water
0.1%TFA), B is acetonitrile (containing 0.1%TFA), and under the conditions of flow velocity 1.0mL/min, wavelength 280nm, the sample for taking 50 μ L is straight
It taps on sample to the chromatographic column for having used 95%A equilibrated, it is 100% then to carry out 30min linear gradient elutions to Mobile phase B,
And continue to elute 10min.
5th, the method for rich protein quality sample and its assay in the oil samples according to claim 1 from trench,
It is characterized in that：In step (3), rich solids protein sample is separately added into the loading buffer 50-100 μ L of electrophoresis, and concussion is mixed
Close uniform, 3~5min boiled in boiling water, after centrifugation can loading, coomassie brilliant blue R_250 dyeing, decoloration are swept with dual wavelength thin layer
Retouch instrument analytical electrophoresis band.
6th, the method for rich protein quality sample and its assay in the oil samples according to claim 1 from trench,
It is characterized in that：In step (4), 50-100mL rich solids protein sample adds in the dissolving of 70% tetrahydrofuran (THF) solution.With
Bovine serum albumin(BSA) (BSA) is standard, utilizes protein content in Bradford method determination samples.Improve protein content determination
Bradford methods, i.e., the solubility that tetrahydrofuran promotes fat-soluble protein is added in solution is measured, this method is 1~60
It is in good linear relationship (R in μ g ranges2=0.9880), high sensitivity.
7th, the method for rich protein quality sample and its assay in the oil samples according to claim 1 from trench,
It is characterized in that：In step (4), the Bradford methods of protein content determination are improved to protein content in eight kinds of oil samples
It measures and finds, the protein content in five kinds of commercially available edible oils is between 0.9~1.3ppm, and protein contains in three kinds of gutter oils
Amount is between 3.5~4.9ppm, 3~5 times of about normal edible oil.
The advantage of the invention is that：(1) it can be very good protein in processing grease using the method for organic filter membrane-suction filtration
Sample, compared with ultrafiltration centrifugal process, Whatman filter paper filtration methods, this method rate of filtration is fast, and 100mL oil samples 10min is just
Can processing complete, and by the use of tetrahydrofuran dissolved as solvent when without grease remain and the interference such as fiber residue, it is easy to operate, be one
The good oil sample preprocess method of kind.(2) it is using the Bradford methods of improved protein content determination, i.e., molten in measure
The solubility that tetrahydrofuran promotes fat-soluble protein is added in liquid, the content that not only can be used to measure fat-soluble protein should
Method linear relationship is good, and high sensitivity can be as the new method for differentiating detection " gutter oil ".
Description of the drawings
The filter device of Fig. 1 oil samples enrichment.
A in figure：Filter device figure；B：Normal edible oil；C：Gutter oil
The uv absorption spectra of Fig. 2 Bradford methods
Specific embodiment
The invention will be further described by the following examples：
Embodiment
(1) protein in organic filter membrane-suction method enrichment grease
50g oil samples are taken in 250mL beakers, 60min is placed at 20 DEG C, 4 DEG C, 100mL is then added in into beaker
The mixed liquor after stirring evenly, is placed 40min by acetone at 4 DEG C, takes out, the mixed liquor is filtered with solvent filtration apparatus, is taken out
Filter membrane used is 0.45 μm of organic system filter membrane during filter, after mixed liquor has filtered, then with 4 DEG C of acetone is washed the filter membrane 4~5 times
Filter membrane after washing is removed, is put into small beaker, add in 5mL tetrahydrofurans, ultrasound to remove remaining grease by (each 10mL)
3~4min takes out filter membrane, and it is the protein example (Fig. 1) being enriched with that extracting solution is dried up with nitrogen.
(2) efficiently analysis of the reversed phase liquid chromatography to protein in enrichment oil sample
It is divided into two parts using organic filter membrane-suction method processing 50mL oil samples, adds in the dissolving of 70% tetrahydrofuran solution,
A copy of it adds in the lysozyme soln of 10 μ L 1.0mg/mL standards, through centrifugal treating, supernatant is taken to be analyzed for HPRPLC.
Chromatographic condition：Shimadzu VP-ODS C18 chromatographic columns (4.6 × 150mm, 5 μm), mobile phase A (contain 0.1% for pure water
TFA), B is acetonitrile (containing 0.1%TFA), under the conditions of flow velocity 1.0mL/min, wavelength 280nm, takes the sample direct injected of 50 μ L
To the chromatographic column for having used 95%A equilibrated, 30min linear gradient elutions are then carried out, until Mobile phase B is 100%, and are held
Continuous elution 10min, makes chromatography column regeneration, finally chromatographic system is balanced with 100%A again, to form a complete chromatographic process.
(3) it is enriched with the measure of protein content in grease
Organic filter membrane-suction method processing 50mL oil samples are added in into the dissolving of 70% tetrahydrofuran (THF) solution, are added
Enter the ultra-violet absorption spectrum (Fig. 2) of BSA standard solution of the BSA standard solution of tetrahydrofuran with not adding tetrahydrofuran, confirm most
Big wavelength is 595nm.Using Bradford methods, with bovine serum albumin(BSA) (BSA) for standard, the concentration c (μ g/mL) of BSA is horizontal stroke
Coordinate, the absorbance A at 595nm are ordinate, draw standard curve, obtain equation of linear regression.
Claims (7)
1. the new method of rich protein quality sample and its assay in a kind of oil samples from trench, it is characterised in that including following
Step：
(1) a kind of preprocess method of the rich protein from different greases；
(2) efficient reversed phase liquid chromatography is to the analysis from grease rich protein sample；
(3) SDS-15%PAGE is to the analysis from grease rich protein sample；
(4) it is enriched with the measure of protein content in grease.
2. the method for rich protein quality sample and its assay in the oil samples according to claim 1 from trench, special
Sign is：In step (1), 40-50g oil samples are weighed in 250mL beakers, are placed 1-2 hours at 18 DEG C, then to burning
4 DEG C, 80-100mL acetone are added in cup, after stirring evenly, which in 4 DEG C of decentralizations is set to 0 .5-1 hours, uses Buchner funnel
Mixed liquor is filtered, used filter paper is Whatman NO.1 filter paper (diameter 85mm, 11 μm of aperture) during suction filtration, will be filtered
Filter paper is put into beaker, adds in 5-15mL tetrahydrofurans, and then which is put into another beaker by concussion, add in 5-15mL
Dioxane, concussion merge extracting solution twice, are dried up with nitrogen, the albumen being enriched in grease.
3. the method for rich protein quality sample and its assay in the oil samples according to claim 1 from trench, special
Sign is：In step (1), organic filter membrane (0.45 μm) united method of Whatman filter paper and polyvinylidene fluoride material is selected
Protein is retained, filter membrane sample is trapped in and is washed 3~5 times using the acetone of refrigeration, first remove the grease remained on filter membrane, so
Tetrahydrofuran is selected to dissolve as solvent afterwards to precipitate, then dried up with nitrogen, obtains solid protein sample.
4. the method for rich protein quality sample and its assay in the oil samples according to claim 1 from trench, special
Sign is：In step (2), 50-100mL rich solids protein samples are divided into two parts, add in the dissolving of 70% tetrahydrofuran solution,
A copy of it adds in the lysozyme soln of 10-20 μ L 1.0mg/mL standards, through centrifugal treating, takes supernatant for HPRPLC points
Analysis.Chromatographic condition is Shimadzu VP-ODS C18Chromatographic column (4.6 × 150mm, 5 μm), mobile phase A are pure water (contain 0.1%TFA), B
For acetonitrile (contain 0.1%TFA), under the conditions of flow velocity 1.0mL/min, wavelength 280nm, the sample direct injected of 50 μ L is taken to having used
In chromatographic column equilibrated 95%A, it is 100% then to carry out 30min linear gradient elutions to Mobile phase B, and continues to elute
10min。
5. the method for rich protein quality sample and its assay in the oil samples according to claim 1 from trench, special
Sign is：In step (3), rich solids protein sample is separately added into the load sample buffering 50-100 μ L of electrophoresis, and concussion is uniformly mixed,
3~5min is boiled in boiling water, after centrifugation can loading, coomassie brilliant blue R_250 dyeing, decoloration, with dual-wavelength lamellar scanning instrument point
Analyse electrophoretic band.
6. the method for rich protein quality sample and its assay in the oil samples according to claim 1 from trench, special
Sign is：In step (4), 50-100mL rich solids protein sample adds in the dissolving of 70% tetrahydrofuran (THF) solution.With ox blood
Pure albumen (BSA) is standard, utilizes protein content in Bradford method determination samples.Improve protein content determination
Bradford methods add in the solubility that tetrahydrofuran promotes fat-soluble protein that is, in solution is measured, and this method is in 1~60 μ g
In the range of be in good linear relationship (R2=0.9880), high sensitivity.
7. the method for rich protein quality sample and its assay in the oil samples according to claim 1 from trench, special
Sign is：In step (4), the Bradford methods of protein content determination are improved to protein content determination in eight kinds of oil samples
It was found that the protein content in five kinds of commercially available edible oils is between 0.9~1.3ppm, and protein content exists in three kinds of gutter oils
Between 3.5~4.9ppm, 3~5 times of about normal edible oil.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11073261B1 (en) | 2020-06-23 | 2021-07-27 | Lamues Light Enterprise Co., Ltd | String lights |
| US11391421B2 (en) | 2019-11-21 | 2022-07-19 | Lamues Light Enterprise Co., Ltd | String lighting and methods of assembly |
Citations (3)
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|---|---|---|---|---|
| CN102858988A (en) * | 2010-04-06 | 2013-01-02 | 赫里开发公司 | Sequential solvent extraction of oil and proteinaceous material from oleaginous material by using solvents of decreasing polarity |
| CN103748104A (en) * | 2011-04-06 | 2014-04-23 | 赫里开发公司 | Extraction of proteins from algae |
| US20160081976A1 (en) * | 2014-09-18 | 2016-03-24 | Virun, Inc. | Formulations of water-soluble derivatives of vitamin e and soft gel compositions, concentrates and powders containing same |
-
2016
- 2016-12-23 CN CN201611213592.0A patent/CN108241035B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102858988A (en) * | 2010-04-06 | 2013-01-02 | 赫里开发公司 | Sequential solvent extraction of oil and proteinaceous material from oleaginous material by using solvents of decreasing polarity |
| CN103748104A (en) * | 2011-04-06 | 2014-04-23 | 赫里开发公司 | Extraction of proteins from algae |
| US20160081976A1 (en) * | 2014-09-18 | 2016-03-24 | Virun, Inc. | Formulations of water-soluble derivatives of vitamin e and soft gel compositions, concentrates and powders containing same |
Non-Patent Citations (4)
| Title |
|---|
| FRANCISCO J. HIDALGO ET AL: "Determination of Peptides and Proteins in Fats and Oils", 《ANAL. CHEM.》 * |
| 姜春宁等: "油松胚珠蛋白质提取分离技术的优化 ", 《北京林业大学学报》 * |
| 彭立凤: "有机溶剂对酶催化活性和选择性的影响", 《化学进展》 * |
| 赵春杰 主编: "《药学专业知识（一）》", 30 April 2012 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11391421B2 (en) | 2019-11-21 | 2022-07-19 | Lamues Light Enterprise Co., Ltd | String lighting and methods of assembly |
| US11073261B1 (en) | 2020-06-23 | 2021-07-27 | Lamues Light Enterprise Co., Ltd | String lights |
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