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2 . 2022

Efficiency of an enzyme preparation obtained from a new mutant Bacillus subtilis-96 strain in the hydrolysis of whey and egg white proteins


Whey and hen egg white proteins are characterized by high nutritional value, but possess antigenic properties, which limit their use in the production of dietary products. Enzymatic hydrolysis decreases significantly the allergenicity of proteins. The efficiency of hydrolysis depends on the specificity of the proteases used.

The aim of this work was to determine the effectiveness of EP-96 enzyme preparation obtained from Bacillus subtilis-96 culture liquid in the hydrolysis of whey and egg white proteins in comparison with commercial bacterial proteases preparations – Alcalase, Neutrase, and Protosubtilin.

Material and methods. Whey and egg white protein concentrates were used as substrates. Commercial enzyme preparations Alcalase, Neutrase, and Protosubtilin, and an experimental sample of EP-96 preparation obtained from Bacillus subtilis-96 culture liquid were used for hydrolysis. Hydrolysis was carried out at a substrate concentration of 100 g/L for 3 h at 55 °C or for 24 h at 50 °C. After hydrolysis, the reaction mixture was incubated at 90 °C for 15 min to inactivate the enzymes. The content of peptides with a molecular weight of less than 10 kDa was determined in the obtained hydrolysates. The hydrolysis of the main allergenic proteins was assessed by the disappearance of the corresponding protein bands on the hydrolysate supernatants electrophoregrams.

Results and discussion. All the studied preparations showed high efficiency in the hydrolysis of whey proteins and provided the yield of low molecular weight peptides at the level of 18.8–22.8% after 3 h of hydrolysis and 39.4–41.6% after 24 h. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a residual amount of protein with a molecular weight of about 14 kDa, corresponding to α-lactoalbumin, after 3 h of hydrolysis when using Neutrase. The preparations containing serine protease, including EP-96, provided more intensive hydrolysis of whey proteins. In the hydrolysis of egg white protein, Neutrase showed the greatest efficiency. The efficiency of EP-96 was comparable to Neutrase both in the yield of low molecular weight peptides and in the intensity of cleavage of the main allergenic proteins. The effectiveness of preparations with predominant content of serine proteases – Alcalase and Protosubtilin was significantly lower.

Conclusion. The optimal ratio of neutral and serine proteases in the EP-96, obtained on the basis of the B. subtilis-96 strain, provided the high efficiency and its versatility in the hydrolysis of the main allergenic proteins of whey and egg white. The parameters of the hydrolysis technology using EP-96 are recommended, which provide intensive conversion of the main immunogenic proteins of whey and egg white to soluble and low molecular weight fractions (duration 3 h at a temperature of 55 °C and the proteolytic activity of the preparation is not less than 2 units per g of substrate) and an increase of subsequent ultrafiltration efficiency in the production of protein hydrolysates for foods for special dietary uses.

Keywords:whey protein; egg white; proteases; hydrolysis

Funding. The research was carried out at the expense of a subsidy for the fulfillment of the state assignment under the Program of Fundamental Scientific Research of the State Academies of Sciences for 2022–2024 (topic No. 0410-2022-0006).

Conflict of interest. The authors declare no conflict of interest.

For citation: Kostyleva E.V., Sereda A.S., Velikoretskaya I.A., Mineeva D.T., Tsurikova N.V. Efficiency of an enzyme preparation obtained from a new mutant Bacillus subtilis-96 strain in the hydrolysis of whey and egg white proteins. Voprosy pitaniia [Problems of Nutrition]. 2022; 91 (2): 72–80. DOI: (in Russian)


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Viktor A. Tutelyan
Full Member of the Russian Academy of Sciences, Doctor of Medical Sciences, Professor, Scientific Director of the Federal Research Centre of Nutrition, Biotechnology and Food Safety (Moscow, Russia)
Индустрия и медицина 2022
Medicine today

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