Study on enzymatic hydrolysis of soybean β-conglycinin using alkaline protease from Bacillus subtilis ACCC 01746 and antigenicity of its hydrolysates

Abstract

Due to its beneficial health effects, the use of soybean protein has shown a continuous increase, but concerns regarding the allergenicity of soybean antigenic protein have also increased. This study aimed to evaluate the hydrolytic effects of a non-commercial alkaline protease isolated from the Bacillus subtilis ACCC 01746 on soybean β-conglycinin and the allergenicity of its hydrolysates. Alkaline protease of the strain was separated by precipitation method of organic solvents, and the β-conglycinin was separated by alkali-solution and acid-isolation and purified by use of gel column. Using the degree of hydrolysis (DH) and inhibition rate as evaluation indexes, the enzymatic hydrolysis parameters of β-conglycinin was optimized by single factor and L₉(3⁴) orthogonal tests, so as to explore the effect of the protease on the hydrolysis degree and the antigenicity of β-conglycinin hydrolysates. The results showed that the native enzyme existed as an 18.3 kDa monomer with a 430 U/g maximum activity. The purity of β-conglycinin was 84.8%. The single-factor test results showed that DH showed the oppostie trendency with the inhibition rate, and the increase of protein concentration caused monotone increasing and monotone decreasing of the inhibition rate and the DH, and the optimal protein concentration was 30 mg/mL. The optimization results showed that pH had the largest impacts on both DH and the inhibition rate, followed by enzyme dosage, hydrolysis temperature and hydrolysis time. Under the optimum hydrolysis conditions of protein concentration 30 mg/mL, enzyme dosage 0.7%, hydrolysis time 40 min, temperature 55 °C and pH 8.5, the DH reached the highest of 76.28%, and the inhibition rate was the lowest of 27.03%, which was reduced greatly compared with that before optimization. These results suggested that alkaline protease appeared to show a relatively high effeciency in lowering soybean allergenicity, making it possible to produce low-allergenicity soybean protein.