Improving the technology for obtaining an ingredient with probiotic properties using a new complex proteolytic enzyme preparation

• Elena V. Kuksova
• Elena V. Kostyleva
• Anna S. Sereda
• Anastasia A. Toloknova
• Elizaveta A. Fursova
• Galina S. Volkova

Abstract

The development of technologies for producing bacterial concentrates and enzyme preparations using domestic microbial strains is an urgent task. The use of whey protein hydrolysates as components of nutrient media for probiotic bacteria consortia for the cultivation of lactic acid and bifidobacteria makes it possible to improve and develop innovative processes for obtaining bacterial concentrates with the required functional properties for the production of dietary supplements. A consortium of probiotic microorganisms (lactic acid and bifidobacteria) was created in the All-Russian Scientific Research Institute of Food Biotechnology as a starter culture for specialized dairy products. Using strain Aspergillus oryzae 21-154 LAP a new complex enzyme preparation with a laboratory name Protoorizin LAP has been obtained providing the extensive hydrolysis of protein substrates.

The purpose of the research was to evaluate the possibility of using the new domestic proteolytic enzyme preparation Protoorizin LAP in preparing whey-based nutrient media for culturing a consortium of probiotic microorganisms to obtain bacterial concentrates.

Material and methods. The object of the research was a symbiotic consortium, including lactic acid bacteria strains (Lactobacillus delbrueckii ssp. bulgaricus Д-16, Lactobacillus plantarum 578/25, Lactobacillus helveticus 842(D)-2, Lactococcus lactis subsp. lactis М-12, Streptococcus thermophilus В-92) and bifidobacteria (Bifidobacterium longum Б-2). Unclarified curd whey and whey protein concentrate were taken as the nutrient medium basis. The media were treated with β-galactosidase to reduce the lactose content. In order to hydrolyze proteins, the control culture medium was treated with commercial preparations: serine protease – Alcalase^® 2.4 L and leucine aminopeptidase – Flavourzyme^® 1000 L. In the experimental medium, two imported preparations were replaced with a laboratory sample of the enzyme preparation Protoorizin LAP. In the prepared nutrient media, the content of amine nitrogen, free amino acids and soluble protein was determined, and electrophoretic analysis of proteins and peptides was carried out. The consortium growth was monitored by the content of dry substances and reducing sugars, by active and titratable acidity, as well as by microscopy. The number of viable cells of lactic acid bacteria and bifidobacteria at the end of fermentation and in the resulting bacterial concentrates were determined by sieving on the appropriate selective agar media using an automatic colony counter.

Results. The effectiveness of Protoorizin LAP in the hydrolysis of whey proteins significantly exceeded the result of the combined action of Alcalase^® 2.4 L and Flavourzyme^® 1000 L both in terms of reducing the undigested protein content, including immunogenic fractions, and in terms of the yield of soluble protein, amine nitrogen and amino acids. The nutrient media obtained using proteases ensured good growth and development of the probiotic consortium. Due to the high content of free amino acids, the dynamics of carbohydrate consumption, titratable acidity, and the number of viable cells were higher in the medium obtained using Protoorizin LAP than when using commercial preparations. At the same time, a high titer of probiotic strains and good cultural and morphological characteristics were obtained on all media. The experimental preparation Protoorizin LAP provided the increase in viability of bacterial cells after lyophilization.

Conclusion. The technological method that include application of the new proteolytic preparation Protoorizin LAP in preparing nutrient media based on whey proteins was developed. The method can be used in the technology of producing bacterial concentrates at the stage of culturing of the created lactic acid and bifidobacteria consortium. The bacterial concentrate can be recommended as a recipe ingredient in the manufacture of dietary supplements or foods for special dietary uses containing probiotics.

Keywords: whey protein; hydrolysis; enzyme preparations4 probiotics; consortium; bacterial concentrates

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