Proteins in food or feed ingredients often change in structure and solubility due to heat treatment or chemical reaction during the machining process, resulting in poor solubility.

Protein detection methods disclosed in the national standards mainly include three kinds: Kjeldahl method, spectrophotometry and combustion method. Biuret method, BCA method, Coomassie blue method, Lowry method and ultraviolet method are also often used in biochemical laboratory. The details are as follows:

Table 1. Protein detection methods

Therefore, there is an urgent need to develop an assay that can ensure the accuracy of the measurement results, and also has a short measurement time and a wide range of applicable measurement targets (especially, can be directly used for measuring the protein content in samples containing poorly soluble proteins).

The researchers [1] developed a new method including the following steps:

(1) Reagent A: Dissolving linear sodium alkyl sulfate and sodium hydroxide in water to a molar concentration of 45~100 mM and 150~300 mM, respectively; Reagent B: copper sulphate or hydrated copper sulfate, sodium tartrate and sodium hydroxide soluble in water to molar concentrations of 5~10 mM, 15~30 mM and 150~750 mM;

(2) Samples Preparation: using protein-free water or buffer as a blank sample; dissolving bovine serum albumin in water or 3-morpholinepropanesulfonic acid (MOPS) buffer and adjusting the concentration of the protein, thereby obtaining a series of solutions having a protein concentration ranging from 0.2 mg/mL to 30 mg/mL as a standard protein sample; dispersing the protein-containing material to be tested in water or buffer, and optionally by dilution processing to prepare a sample to be tested;

(3) mixing reagent A with a blank sample, a standard protein sample, and a sample to be tested, respectively, to obtain a corresponding reagent A mixture, wherein the protein content in the reagent A mixture of the sample to be tested meets the linear range requirement. And each reagent A mixture is heated in a water bath at a temperature of 90~100 °C, and then cool to room temperature;

(4) mixing each reagent A mixture with reagent B to obtain a corresponding reagent B mixture, and heating each reagent B mixture in a water bath at a temperature of 50~70 °C, and cool to room temperature;

(5) Filtering each of the reagent B mixtures, using the reagent B mixture of the cooled blank sample as a reference, and measuring the reagent B mixture of the cooled standard protein sample and the reagent B of the sample to be tested at 540 nm using a spectrophotometer.

(6) According to the protein content of the quasi-protein sample, the relationship between the protein content and the absorbance is obtained by linear fitting, and the protein content in the sample to be tested is calculated.

The protein detection method requires a shorter detection time, increases the solubility of insoluble proteins, has a wide range of applications, accurate measurement results, wide linear range, high sensitivity, high precision and good reproducibility. In addition, the test method is compatible with high-throughput measurement accessories and equipment, and can quickly detect large quantities of samples, making it more suitable for large-scale industrial processes.

Reference

Protein detection method and protein detection kit. CN105987884B, 2015-03-03.

Edited by Suzhou Yacoo Science Co., Ltd.