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No simple methods are used for the quantitative analysis of the protease activity in colored food up till now. Thus, this study aims to establish a new and simple method for the quantitative detection of protease activity, especially in colored food. The detection accuracy, detection limit, and repeatability of the casein plate method were analyzed. Then, the application of the casein plate method in sample detection and recovery was further evaluated. The results showed that the casein plate method for the quantitative detection of protease activity has high accuracy, high precision, and low detection limit. The recoveries of eight kinds of colored samples were in the range of 92.26–97.84%, and the relative standard deviation (RSD) was in the range of 3.56–10.88%. The results of the casein plate method exhibited high accuracy. This indicated that the method was suitable for the detection of colored samples. The casein plate method for the quantitative detection of protease activity is simple. The newly constructed casein plate method has broad potential application value in food industry, especially for the detection of dark food.
Proteinase is a kind of enzyme for protein hydrolysis. It is a group of large and complex enzymes with highly specific protein hydrolysis. It features high specificity to biological molecules and has wide application value in the fields of food, medicine, and detergents. 1 They are involved in the selective proteolysis of various specific substrates, embryonic development, bone and organ tissue repair, neuron growth, immune and inflammatory cell regulation, angiogenesis and apoptosis, and other biological processes. 2−5
The analysis of protease activity is an important step in the research and application of protease 6 development and validation of a simple titration method for studying the heat-activated; endogenous protease in arrow tooth flounder fillet mince is described. Protease activity has been detected by the fluorescence method, 7,8 but detection based on calorimetry, 9 mass spectrometry (MS), 10 immunoassay, 11 electrophoresis, 12,13 amperometry, 14 optics, 15,16 and so on.
Fluorescence-conjugated polyelectrolytes, suspended ionic sulfonates, and carboxyl groups are used to detect protease activity. 7 The fluorescence method exhibits an obvious mechanism and entails a simple operation. However, given the need for special instruments, certain operational requirements must be met by experimenters; hence, this method is not commonly employed. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has the advantages of simplicity, high throughput, and sensitivity and thus possesses great application potential. However, this method is not commonly applied because of its high instrument and operational requirements. D1 protease activity has been determined by capillary zone electrophoresis, 12 but this method cannot detect the activities of other proteases. Kilian et al. 17 proposed an organic derivatization method using silicon-based nanoporous photonic crystals to immobilize polypeptides for the detection of proteases in solutions. The minimum detection concentration of this method is 3.7 × 10 –10 mol·L –1 . Although this method has a low detection limit and yields accurate detection results, it requires specific nanomaterials, which are usually expensive; hence, it is also not commonly used.
Mahmoud et al. 18 proposed a method for the electrochemical detection of human immunodeficiency virus type I protease by surface modification with ferrocene (Fc) peptide with a detection limit of 8 × 10 –11 mol·L –1 . This method also has a low detection limit and achieves accurate detection but requires electrodes, nanomaterials, and specific electrochemical workstations. It also entails high operational requirements from the experimenters. Serim et al. 19 described the use of probe technology to detect protease activity. At present, the colorimetric method is commonly used to detect protease activity. Common protease activities are quantitatively expressed by detecting either the decrease in substrate protein concentration or the increase in product-free amino acid or polypeptide concentration before and after enzymatic hydrolysis. Ultraviolet spectrophotometer and biuret reagent are used in the former, while ninhydrin colorimetry, trinitrobenzene sulfonic acid, and Folin-phenol reagent are used in the latter. In the determination of protease activity using an ultraviolet spectrophotometer, 20 the protease hydrolyzes the casein substrate at a certain temperature and pH, the enzyme reaction is terminated by the added trichloroacetic acid, and the unhydrolyzed casein is precipitated. The activity of the casein can be determined by ultraviolet spectrophotometry. However, this method suffers from disadvantages such as unsuitability for colored sample detection, difficulty of operation, and proneness to errors.
The Coomassie Brilliant Blue method 21 is also used to detect protease activity. In this method, the Coomassie Brilliant Blue dye binds to the protein under acidic conditions. The dye binds to basic amino acids (especially arginine) and aromatic amino acid residues in protein so that the maximum absorption peak of the dye increases from 465 to 595 nm and the color of the solution changes from brown to black. Absorption is determined at 595 nm. This method is fast, simple, stable, and highly sensitive, but its linear relationship becomes increasingly deviated with the increase in protein content. The reaction can be disturbed by a strong alkaline buffer, sodium dodecyl sulfate, and other substances. 22 In the ninhydrin chromogenic method, 23−25 protease activity can be determined by adding protease to the solution containing protease to decompose for a period of time; the content of the produced α-amino acid is then determined. However, the results of this method are quite erroneous because of the slow reaction, side reactions, unstable hydrolysis, oxidation and photolysis, and color interference. 26
However, there is no simple method suitable for detecting protease activity in dark samples. This is mainly because the commonly used spectrophotometry is seriously disturbed by color.
When the protease activity in real samples is measured, many samples are dark in color, and the extracted enzyme solution is even darker in color. For example, when a sample’s color is extremely dark, it presents a blue-black color after a reaction. For light-colored samples, the complete color of the reaction is dark blue or light blue. Hence, a new method is needed to eliminate the interference of color and accurately determine the enzyme activity of dark samples. The issue is whether a simple way may be used to achieve high accuracy and precise analysis that is unaffected by the sample color?
In the present work, we aimed to establish a new and simple method for the quantitative detection of protease activity, especially in colored samples. Commercial trypsin was diluted and cultured in different solutions. The measured results were used as standard curves. The activity of the samples was calculated according to the size of the hydrolysis circle of the samples. The feasibility of using the casein plate method in the quantitative analysis of protease activity was discussed.
