Proteins are important components that make up the body's cell tissue. The protein in food is the only source of nitrogen in the human body and has an irreplaceable role of sugars and fats. Proteins are closely related to nutrient metabolism, cell structure, enzymes, hormones, viruses, immunity, substance movement and genetics, and their separation, qualitative and quantitative analysis are biochemistry and other biological subjects, food inspection, clinical testing, diagnosis of diseases, and biological drugs. The most important work in separation purification and quality inspection. With the continuous improvement of analytical methods, the determination of protein content in foods is also progressing in an accurate and rapid manner.
1 Food Protein Detection Methods
At present, there are many methods for determining the protein content of foods, which can generally be divided into indirect methods and direct methods. The indirect method is a method for estimating the protein content by determining the nitrogen content of a protein in a sample; the direct method is a method for directly determining the protein content based on the physical and chemical properties of the protein. Over the years, methods for determining the protein content have been developed by utilizing the major properties of proteins (such as nitrogen content, peptide bonds, refractive index, etc.) and the specific amino acid residues (such as aromatic groups, acidic groups, and basic groups) contained in proteins. There are mainly Kjeldahl (international classical methods), spectrophotometry, and titration methods. The instrument used is Kjeldahl, also known as a protein analyzer.
1.1 Kjeldahl method
The most commonly used method for protein determination is the Kjeldahl method, which is one of the most accurate and easiest methods for determining the total organic nitrogen in a sample. It is a statutory standard test method at home and abroad. Kjeldahl determination of protein is divided into sample digestion, distillation, absorption and titration process. Under the action of the catalyst, the sample is decontaminated with concentrated sulfuric acid to destroy the organics, convert protein nitrogen and other organic nitrogen therein into ammonia nitrogen, and then combine with sulfuric acid to produce ammonium sulfate, and add strong alkali to perform distillation to make ammonia escape, using boric acid. After absorption, acid titration was used to measure the nitrogen content. The result was multiplied by a conversion factor to calculate the crude protein content. However, this method has a long digestion time and produces poisonous and harmful gases. Therefore, it must be done in a fume hood. Zong Liuxiang and others improved this traditional method by using Se powder as a catalyst for digestion without the use of a fume hood, thus achieving environmental digestion and improving digestion efficiency.
1.2 Photometry
The spectrophotometric method is classified into coomassie brilliant blue G-250 method, biuret method, Folin phenol method (Lowry method), BCA method and ultraviolet absorption method.
1.2.1 Coomassie brilliant blue G-250 method
Coomassie Brilliant Blue G-250 is very rapid and stable with protein binding. The reaction is equilibrated in about 2 min. The conjugate can be stable at room temperature within 1 h. The Coomassie Brilliant Blue G-250 method is the most sensitive method for protein determination with a minimum protein detection of 1 μg, which is about 4 times higher than that of the Folin phenol method. This is due to the color change caused by the combination of proteins and dyes. Very large, protein-dye complexes have a higher extinction coefficient, so the light absorption changes with protein concentration much more than the Lowry method. The method is simple, easy to operate, uses fewer reagents, and is easy to formulate with color reagents; less interfering substances, such as sugars, buffer reducing agents, and complexing agents do not affect color development.
1.2.2 Biuret method
The biuret method is similar to the color reaction produced by albumin and red protein and is not affected by temperature. It can quickly determine the protein content, but its sensitivity is low. The measurement range is 1-20 mg protein, which is suitable for the determination of protein content with low precision requirements. , Commonly used for the determination of cereal protein content. Trimethylolaminomethane and some amino acids, EDTA, etc. interfere with the reaction. Li Ning compared Kjeldahl, Coomassie brilliant blue, and biuret, and concluded that Kjeldahl, biuret, and Coomassie brilliant blue all measure all The protein content of 5 kinds of samples, among which Kjeldahl method is a commonly used method for analyzing the nitrogen content of organic compounds, is a classical method for protein determination, suitable for a wide range of samples and used for accurate testing of results; biuret method The test procedure with Coomassie brilliant blue staining method is simple and rapid, and it is used in tests that can accurately match the standard protein solution without requiring high accuracy. The above two methods can be selected based on the nitrogen content.
1.2.3 Folin phenol method
The Folin phenol method is the development of the biuret method, which combines the reaction of biuret reagents and phenol reagents with proteins. The Folin phenol method is widely used for the determination of water-soluble protein content. The sensitivity of the Folin phenol method is 100 times higher than that of the biuret method, and the peptide bond chromogenic effect is enhanced, reducing deviations due to protein types. This method can be superimposed due to the two-step color reaction and its sensitivity is particularly high. Therefore, it is suitable for the determination of 20-250 μg trace protein, and the minimum detectable protein mass is 5 μg.
1.2.4 BCA Method
The BCA protein detection reagent is currently superior to the Lowry method for detecting total protein content. The method is fast, sensitive, stable, and reliable. The coefficient of variation is very small for different types of proteins, and the detectable protein mass is 0.5 μg. , is one of the most sensitive protein detection reagents currently known.
1.2.5 UV absorption method
The UV absorption method is simple, sensitive, and fast, and does not consume samples. Low-level salts do not interfere with its determination. The disadvantage of this method is that there is a certain amount of error in determining the protein that has a large difference between tyrosine and tryptophan in the standard protein. The UV absorption method is suitable for testing colorless samples. For colored samples, pretreatment is required and this method is applicable after eliminating color interference. In addition, the nucleic acid also has strong absorption in the ultraviolet region, but it can be eliminated by correction.
In the determination of protein, the photometric method is most widely applied and widely applicable. The research on this type of method has been deeper and has been continuously developed. Organic dyes combined with spectrophotometric method for the determination of proteins are easy to operate, relatively sensitive, and the general laboratory conditions can meet the detection requirements. In recent years, research and application of such methods at home and abroad have been paid more attention. The basic principle of this type of method is that in the acidic medium, the peptide bond of the protein and the N-terminal amino group are protonated into cations. Due to the charge, the protein binds with the anionic dye and precipitates or changes the light absorption characteristics of the bound dye, and the color of the dye decreases or changes. The degree to determine the protein content. Coomassie Brilliant Blue G-250 dye has been widely used; dyes such as orange red G, bromcresol green, ethidium bromide, bromophenol blue, acid magenta, etc. have also been used for protein determination; Azoquinone K, Arsenazo III. Determination of Proteins by Azoquinone M, Nitrosulfophenol C, Nitrosulfophenol S, Chlorophenol K, Chlorosulfonol S, Eosin S, ChromoAcetic Acid 2C, Chromotropic 2B, Eosin Y, etc. Spectrophotometry studies have been reported.
In recent years, the use of metal ions and organic dyes to form complex systems, combined with the method of spectrophotometric determination of protein content has been developed. When a metal ion meets an organic dye containing -OH or C=O, the lone pair of electrons in the oxygen atom can smoothly enter the hybrid orbital to form a stable coordination system. Under acidic conditions, the system encounters asymmetrically structured proteins. When the molecules are polarized, they are electrostatically combined to form new macromolecules that change the broad-spectrum properties of the original system and enable quantitative determination of the protein content. Such methods have the advantages of high sensitivity, wide linear range, less interference ions, simple operation, rapidity, and suitability for routine applications. The development of metal ions-organic dyes combined with spectrophotometric determination of protein content is rapidly evolving, such as Cu (II-chlorosulphonol S complexes, Cu (II)-arsenazo K, etc. Fluorimetric methods for the quantitative determination of proteins in recent years The new method is gradually emerging, and uses the phenomenon that the fluorescence intensity of the reactants increases with the increase of the protein concentration to perform protein determination, and has a narrow linear range and high detection limit of the working curve. Pyrimidine Orange, Ruthenium S, Ruthenium Y and Brilliant Cresol Blue.
1.3 Titration
1.3.1 Formaldehyde titration (indicator titration)
The method saves reagents, has a fast analysis speed, and is easy to operate. Liu Yulan et al. determined the protein content of cottonseed cake, soybean, peanut, wheat, corn and faba bean by formaldehyde titration method and Kjeldahl method respectively. The results were basically the same, with a deviation less than 0.20.
1.3.2 pH Titration
The pH titration method refers to the method of calculating the concentration or content of the analyte according to the amount of titrant and the relevant formula when the pH is titrated to a certain pH value. It is the latest method of titrating very weak acids and bases directly in aqueous solution. The method must determine the appropriate pH. Zhang Dalun et al. used a glass electrode as the indicator electrode and a saturated calomel electrode as the reference electrode to determine the protein content of soybean and peanut. The results were consistent with the results of the traditional Kjeldahl method.
1.3.3 Improved titration of formaldehyde
Ma Meifan and Huang Xiaodong combined formaldehyde titration with pH titration. According to the principle of the formaldehyde method, the end point of the titration was indicated by replacing the indicator with a pH meter. When the ammonium ion reacts with formaldehyde rapidly, the same amount of acid is released, the generated acid is titrated with a sodium hydroxide standard solution, the potentiometric indicator is used to indicate the stoichiometric point, and the protein content is calculated based on the consumption of the sodium hydroxide standard solution. When using this method to determine protein, the test solution must be neutral, and the pH of the solution must be strictly controlled during neutralization to avoid errors caused by ammonium salt decomposition. HCHO can be oxidized by air to formic acid and should be prepared before use. The results obtained by this method are not significantly different from the Kjeldahl method.
2 Conclusion
Among the most commonly used assays for these proteins, the most classic is the Kjeldahl method. However, this method requires an automatic or semi-automatic Kjeldahl tester. These instruments are expensive and require specialized reagents, making them difficult to use. universal. Some scholars have now improved their research. In the scientific research work, the user can select appropriate, more commonly used methods for determining the protein according to the specific circumstances.