CN108241035B - A new method for enrichment of protein samples from waste oil samples and their content determination - Google Patents

Abstract

The invention provides a protein enrichment sample from a waste oil sample and a method for measuring the content of the protein enrichment sample. Screening and verifying different methods for enriching proteins in oil and fat to establish a pretreatment method for enriching proteins from illegal cooking oil and fat; on the basis of measuring the protein content by the Bradford method, tetrahydrofuran is used as a solvent to improve the solubility of fat-soluble protein, and a measuring method which can be suitable for measuring the protein content in the illegal cooking oil sample is established.

Description

A new method for enrichment of protein samples from waste oil samples and their content determination

Field of Invention

The invention relates to a new method for enriching protein samples from waste oil samples and determining their content, belonging to the cross field of analytical chemistry and food analysis.

Background technique

In recent years, incidents of food safety issues in China have continued to increase, and research on "gutter oil" has attracted widespread attention. Waste oil generally refers to all kinds of inferior oils, such as cooking oil and repeated frying oil [Zhan H.L., Xi J.F., Zhao K., et al. Food Control, 2016, 67: 114-118]. Waste oil often contains bacteria, heavy metals, and harmful chemicals during recycling, bleaching, and deodorization, which directly and seriously endanger public health when used as edible oil [Ramani K., Karthikeyan S., Boopathy R., et al. al. Process Biochemistry, 2012, 47: 435-445]. Therefore, accurate and rapid identification technology can undoubtedly control the spread of waste oil, improve food safety, and effectively protect the interests of consumers [He J., Xu W.T., Shang Y., et al. Food Control, 2013, 31: 71-79 ].

In fact, it is very difficult to distinguish between edible oil and waste oil, because the two are not only similar in appearance, but also have roughly the same main components, the main components of which are all glycerol trimyristate. Physical and chemical indicators such as acid value, solid fat, heavy metals, etc., have been difficult to distinguish between waste oil and edible oil in the current situation [Maggio R.M., Cerretani L., Chiavaro E., et al. Food Control, 2010, 21, 890-895] . Since oil contains a certain amount of protein, the type and content of protein in different oils are also different, especially fat-soluble protein. Therefore, it is an accurate and reliable method to use the difference in type and content of protein in waste oil and edible oil to identify the two. But how to separate and detect trace protein components from lipid matrices?

First, the pretreatment of oil samples is one of the key technologies. The necessary conditions to successfully establish a method for protein extraction from oil samples are simple processing steps, low time-consuming and complete removal of the interference of the oil matrix. At present, methods for protein extraction from oil samples have been reported in the literature, which are mainly divided into aqueous solvent extraction method and organic solvent precipitation method. For example, protein in edible olive oil was extracted with 50 mM Tris-HCl buffer containing 0.1 M NaCl, 10% (v/v) glycerol, pH 7.5 [Georgalaki M.D., Sotiroudis T.G., Xenakis A. Journal of the American Oil Chemists' Society, 1998 , 75: 155-159]. The protein in sunflower oil was obtained by extraction with 50 mM PBS, centrifugation and dialysis [Zitouni N., Errahali Y., Metche M., et al. Journal of Allergy and Clinical Immunology, 2000, 106:962-967]. The characteristics of water-soluble extraction methods are simple and easy, but the extraction amount is too low. The use of petroleum ether as a solvent can effectively extract the protein in soybean oil from oil, but the study found that the organic solvent extraction method is complicated and time-consuming and cannot be used as an ideal pretreatment step for the analysis method [Olszewski A., Pons L., Moute'te 'F., et al. Clinical and Experimental Allergy, 1998, 28:850-859]. In order to quickly and efficiently extract proteins from oils, an acetone precipitation-filter paper filtration method was also developed to extract proteins from olive oil [Hidalgo F.J., Alaiz M., Zamora R. Analytical Chemistry, 2001, 73: 698-702]. This method is a simple and time-consuming method, but at the same time, it is also found that when using filter paper for filtration, some residual oil will affect the subsequent determination.

In addition, since general fats and oils do not contain or contain extremely small amounts of protein, the determination of their content is also very difficult. At present, the commonly used methods for the determination of protein content in oils and fats include the Lowry method, the bicinchoninic acid (BCA) method, and the Bradford method. Among them, the Lowry method and the BCA method belong to the chemical method, and the Bradford method belongs to the dye binding method. However, since the above methods are mainly aimed at water-soluble proteins, it is difficult to obtain accurate results for the determination of the content of fat-soluble proteins. To this end, Hidalgo et al. used acetone precipitation to hydrolyze the resulting protein or polypeptide, and then used amino acid analysis to quantify the protein content. This is the only chemical process developed method for the determination of protein in edible oils, in which the amino acid analysis method is relatively accurate. Compared with the Lowry method and the Bradford method, the fat in the precipitated protein does not interfere with the determination, and the obtained results are accurate, but the hydrolysis and determination operations are complicated, which also restricts its wide application.

SUMMARY OF THE INVENTION

The present invention will screen and verify different methods for enriching proteins from oil, and establish a pretreatment method for enriching proteins from waste oil; based on the determination of protein content by Bradford method, different solvents are used to improve the concentration of lipid-soluble proteins. Solubility, find a method that can be suitable for the determination of protein content in waste oil samples.

In order to realize the above-mentioned tasks, the present invention adopts the following technical solutions:

1. A novel method for enriching protein sample and content thereof from waste oil sample, is characterized in that comprising the following steps:

(1) a pretreatment method for enriching protein from different oils;

(2) Analysis of protein samples enriched from oil by high-performance reversed-phase liquid chromatography;

(3) SDS-15% PAGE analysis of protein samples enriched from lipids;

(4) Improving the Bradford method for the determination of protein content in oils and fats;

2. The method for enriching protein samples from waste oil samples and assaying their content according to claim 1, characterized in that: in step (1), take 40-50g of oil and fat samples in a 250mL beaker, and at 18° C. Place the mixture at 4°C for 1-2 hours, then add 4°C and 80-100mL acetone into the beaker, stir evenly, place the mixture at 4°C for 0.5-1 hour, and filter the mixture with a Buchner funnel. The filter paper used is Whatman NO.1 filter paper (diameter 85mm, pore size 11μm), put the filter paper after suction filtration into a beaker, add 5-15mL tetrahydrofuran, shake, then put the filter paper into another beaker, add 5 -15mL dioxane, shake, combine the two extracts, dry with nitrogen, and enrich the protein in the oil.

3. The method for enriching protein samples and their content from waste oil samples according to claim 1, characterized in that: in step (1), select Whatman filter paper and an organic filter membrane (0.45 m) of polyvinylidene fluoride material. μm) combined method to intercept protein, and the intercepted sample on the filter membrane was washed with refrigerated acetone for 3 to 5 times, first to remove the grease remaining on the filter membrane, then use tetrahydrofuran as a solvent to dissolve the precipitate, and then dry it with nitrogen to obtain solid protein sample.

4. The method for enriching protein samples and their content from waste oil samples according to claim 1, wherein in step (2), 50-100mL enriched solid protein samples are divided into two parts, and 70 % tetrahydrofuran solution was dissolved, and 10-20 μL of 1.0 mg/mL standard lysozyme solution was added to one portion, and after centrifugation, the supernatant was taken for HPLC analysis. Chromatographic conditions were Shimadzu VP-ODS C ₁₈ column (4.6×150mm, 5μm), mobile phase A was pure water (containing 0.1% TFA), B was acetonitrile (containing 0.1% TFA), at a flow rate of 1.0 mL/min, Under the condition of wavelength of 280nm, take 50μL of sample and inject it directly to the chromatographic column that has been equilibrated with 95% A, and then carry out 30min linear gradient elution until the mobile phase B is 100%, and continue to elute for 10min.

5. The method for enriching protein samples from waste oil samples and assaying their content according to claim 1, characterized in that: in step (3), the enriched solid protein samples are respectively added to the sample loading buffer of electrophoresis for 50- 100 μL, shaken and mixed evenly, boiled in boiling water for 3-5 min, centrifuged and then loaded, stained and decolorized with Coomassie brilliant blue R-250, and analyzed electrophoresis bands with a dual-wavelength thin-layer scanner.

6. The method for enriching protein samples from waste oil samples and determining their content according to claim 1, wherein in step (4), 50-100 mL of enriched solid protein samples are added with 70% tetrahydrofuran (THF) The solution dissolves. With bovine serum albumin (BSA) as the standard, the protein content in the samples was determined by Bradford method. The improved Bradford method for protein content determination, that is, adding tetrahydrofuran to the determination solution to promote the solubility of fat-soluble proteins, has a good linear relationship (R ² =0.9880) in the range of 1-60 μg, and has high sensitivity.

7. The method for enriching protein samples from waste oil samples and assaying their content according to claim 1, characterized in that: in step (4), the Bradford method for improving the assay of protein content is used to measure the protein content in eight kinds of oil samples. It was found that the protein content of the five commercially available edible oils was between 0.9 and 1.3 ppm, while the protein content of the three kinds of waste oils was between 3.5 and 4.9 ppm, which was about 3 to 5 times that of normal edible oils.

The advantages of the present invention are: (1) The method of organic membrane-suction filtration can well process protein samples in oil and fat. Compared with ultrafiltration centrifugation and Whatman filter paper filtration, this method has a fast filtration speed, and it takes 10 minutes for 100 mL of oil and fat samples. The treatment can be completed, and there is no interference such as oil residue and fiber residue when tetrahydrofuran is used as a solvent for dissolving. The operation is simple, and it is a good pretreatment method for oil samples. (2) Using the improved Bradford method for protein content determination, that is, adding tetrahydrofuran to the determination solution to promote the solubility of fat-soluble proteins, which can not only be used to determine the content of fat-soluble proteins, this method has a good linear relationship and high sensitivity and can be used as a differential detection " A new method for waste oil".

Description of drawings

Figure 1. Filter device for enrichment of grease samples.

In the picture, A: filter device diagram; B: normal edible oil; C: waste oil

Figure 2 UV absorption spectrum of Bradford method

Detailed ways

The present invention is further described below by embodiment:

Example

(1) Organic membrane-suction filtration method to enrich protein in oil

Take 50g of oil and fat sample in a 250mL beaker, put it at 20°C for 60min, then add 4°C and 100mL of acetone to the beaker, stir evenly, place the mixture at 4°C for 40min, take it out, and use a solvent filter device to suction filter the mixture. For the mixed solution, the filter membrane used in suction filtration is a 0.45 μm organic filter membrane. After the mixed solution is suction filtered, the filter membrane is washed with 4°C acetone for 4 to 5 times (10 mL each time) to remove residual oil. After washing, remove the filter membrane, put it into a small beaker, add 5 mL of tetrahydrofuran, sonicate for 3-4 min, take out the filter membrane, and dry the extract with nitrogen to obtain the enriched protein sample (Figure 1).

(2) Analysis of protein in enriched oil samples by high performance reversed-phase liquid chromatography

Utilize organic membrane-suction filtration to process 50mL oil sample and divide it into two parts, add 70% tetrahydrofuran solution to dissolve, and add 10μL 1.0mg/mL standard lysozyme solution to one part, after centrifugation, take the supernatant for HPRPLC analysis .

Chromatographic conditions: Shimadzu VP-ODS C18 column (4.6×150mm, 5μm), mobile phase A is pure water (containing 0.1% TFA), B is acetonitrile (containing 0.1% TFA), flow rate 1.0mL/min, wavelength Under the condition of 280nm, take 50μL of the sample and inject it directly to the chromatographic column that has been equilibrated with 95% A, and then carry out linear gradient elution for 30 minutes until the mobile phase B is 100%, and continue to elute for 10 minutes to regenerate the chromatographic column. , and finally equilibrate the chromatographic system with 100% A to form a complete chromatographic process.

(3) Determination of protein content in enriched oil

The organic filter membrane-suction filtration method was used to treat 50 mL of oil and fat samples and add 70% tetrahydrofuran (THF) solution to dissolve, to obtain the ultraviolet absorption spectra of the BSA standard solution with tetrahydrofuran and the BSA standard solution without tetrahydrofuran (Fig. 2). Confirm that the maximum wavelength is 595nm. Using the Bradford method, with bovine serum albumin (BSA) as the standard, BSA concentration c (μg/mL) as the abscissa, and absorbance A at 595 nm as the ordinate, a standard curve was drawn to obtain a linear regression equation.

Claims (4)

1. a method for enriching protein sample from waste oil sample, is characterized in that comprising the following steps: (1) a pretreatment method for enriching proteins from different oils; (2) Analysis of protein samples enriched from oil by high-performance reversed-phase liquid chromatography; (3) SDS-15% PAGE analysis of protein samples enriched from lipids; (4) Determination of protein content in enriched oil; In step (2), 50-100 mL of the enriched solid protein sample is divided into two parts, and 70% tetrahydrofuran solution is added to dissolve, and 10-20 μL of 1.0 mg/mL standard lysozyme solution is added to one part, and the supernatant is taken after centrifugation. The liquid was used for HPLC analysis; the chromatographic conditions were Shimadzu VP-ODS C18 chromatographic column, the mobile phase A was pure water, the mobile phase A also contained 0.1% TFA, and B was acetonitrile, and the mobile phase B also contained 0.1% TFA. %TFA, at a flow rate of 1.0 mL/min and a wavelength of 280 nm, take 50 μL of the sample and directly inject it onto the chromatographic column that has been equilibrated with 95% A, and then carry out 30 min linear gradient elution until the mobile phase B is 100%, And continue to elute for 10min. 2. the method for enriching protein sample from waste oil sample according to claim 1, is characterized in that: in step (1), take by weighing 40-50g grease sample in 250mL beaker, place 1- After 2 hours, add 4°C, 80-100mL acetone to the beaker, stir evenly, place the mixture at 4°C for 0.5-1 hour, and filter the mixture with a Buchner funnel. The filter paper used in the suction filtration Whatman No.1 filter paper, diameter 85mm, pore size 11μm, put the filter paper after suction filtration into a beaker, add 5-15mL tetrahydrofuran, shake, then put the filter paper into another beaker, add 5-15mL dioxane Ring, shake, combine the two extracts, dry with nitrogen, and enrich the protein in the oil. 3. the method for enriching protein samples from waste oil samples according to claim 1, is characterized in that: in step (3), enrichment solid protein samples are respectively added to the sample loading buffer 50-100 μL of electrophoresis, shake and mix evenly , boiled in boiling water for 3 to 5 minutes, centrifuged to load the samples, stained and decolorized with Coomassie brilliant blue R-250, and analyzed the electrophoretic bands with a dual-wavelength thin-layer scanner. 4. the method for enriching protein sample from waste oil sample according to claim 1, is characterized in that: in step (4), 50-100mL enrichment solid protein sample adds 70% tetrahydrofuran solution to dissolve, with bovine serum albumin Protein is used as the standard, and the protein content in the sample is determined by the Bradford method; the improved Bradford method for the determination of protein content is to add tetrahydrofuran to the determination solution to promote the solubility of fat-soluble proteins. =0.9880, high sensitivity.

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