Research explores biochemical and practical aspects of recombinant bacterial β-galactosidases, including lactose-free milk, whey, and galactooligosaccharides yield.

Relevance of the project

According to the International Dairy Federation (IDF), world milk production in 2021 has reached approximately 931 million. According to the National Bureau of Statistics of Kazakhstan, raw milk production in Kazakhstan in 2022 amounted to 3 million 975 thousand tons. Whey and its derivatives are currently being used more and more in innovative ways to produce a range of food products that are higher-quality and have better health effects. They study how these products affect health and cure and prevent diseases like osteoporosis, cancer, and vascular disease. Typically, whey contains 4-6% lactose. The enzyme β-galactosidase can convert lactose into galactooligosaccharides (GOS). This prebiotic is attractive for food ingredients and supplements because specific Lactobacillaceae and Bifidobacterium families and other gut bacteria may selectively eat and digest GOSs for proliferation and activity. GOS reach the colon undamaged due to their stability and resistance to hydrolysis in the GI tract.

The scientific novelty of the project lies in the study of factors affecting hydrolase and transglycosylase activity and the study of galacto-oligosaccharide formation processes with a focus on prebiotic properties. The practical significance of the project lies in the fact that, in the end, a technology for processing milk whey to obtain specific and demanded products will be proposed. The socio-economic effect is in the development of the processing industry, thanks to which raw materials in the form of whey will be processed into more valuable products in demand in the food industry and with export-oriented potential.

Project aim

The study compares biochemical parameters of recombinant bacterial β-galactosidases and analyzes how galactooligosaccharides affect probiotic microorganism proliferation.

Expected results

Lactose-utilizing microbe isolates will be obtained. The gene organization of isolated lactose-utilizing bacteria will be determined. Bacterial recombinant -galactosidases will be isolated and purified, and biochemical parameters will be investigated. The hydrolyzing properties of the isolated enzymes will be determined and the substrate specificity of the isolated β-galactosidases will be studied on various substrates. The transglycosylase activity of the obtained β-galactosidases will be studied and the profile of galactooligosaccharide formation will be compared. The prebiotic properties of galactooligosaccharides with different degrees of polymerization will be determined.

Project  leader

Tursunbekova Annelya E. – Researcher ID: HIG-8491-2022,Scopus Author ID: 58131481100, ORCID: 0000-0002-7536-7451)

Research team members

Akishev Zhiger D. – Researcher ID: N-6206-2017, Scopus Author ID: 56674741700, ORCHID: 0000-0001-9943-1625)

Mussakhmetov Arman S. – Researcher ID: AAQ-9945-2020, ORCID: 0000-0002-6182-3487, Scopus Author ID: 57203751227

Sagynova Malika N. – ResearcherID: ABR-5877-2022, ORCID: 0000-0002-6948-6817, Scopus Author ID: 58189714400      

Maduakhasova Arina K. – Researcher ID: JNE-6827-2023, ORCHID: 0009-0005-1460-0802

Auez Madina K. – Researcher ID: GZG-7318-2022 ORCID: 0000-0001-7159-2793

Publications and security documents of the project leader and members of the research team related to the project topic

1. Kiribayeva A.K., Silayev D.V, Tursunbekova A.E., Ramankulov Y.M., Khassenov B.В. Cloning, purification and study of the biochemical properties of α-amylase from Bacillus licheniformis T5 strain // Vestnik nauki Kazahskogo agrotekhnicheskogo universiteta im. S. Sejfullina (mezhdisciplinarnyj), 2022, № 1(112) S. 181-189. doi: 10.51452/kazatu.2022.1(112).942.
2. Akishev ZH.D., Tursunbekova A.E. Hasenov B.B. Molokosvertyvayushchaya aktivnost’ rekombinantnogo verblyuzh’ego himozina // Vestnik KazNU. Seriya biologicheskaya. Tom 90 №1. 2022.S. 39-49. https://doi:10.26577/eb.2022.v90.i1.04.
3. Aktayeva S., Kiribayeva A., Makasheva D., Astrakhanov M., Tursunbekova A., Baltin K., Khassenov B. Isolation, identification and usage of Bacillus strains in microbial inhibition test in milk // Eurasian Journal of Applied Biotechnology, 2022. No. 4. P.49-57 doi: 10.11134/btp.4.2022.6 (RSCI-0,117).
4. Akishev Zh., Aktayeva S., Shamsiyeva Yu., Tursunbekova A., Kalemshariv B., Tultabayeva Т., Khassenov B. Milk-clotting activity of recombinant bovine and camel chymosin for cow’s, goat’s and ewes’ milk // Eurasian Journal of Applied Biotechnology, 2023. No. 2. P.61-68. doi: 10.11134/btp.2.2023.8 (RSCI-0,117).
5. Aktayeva S., Sarsen A., Mussakhmetov A., Kiribayeva A., Tursunbekova A., Khassenov B. Development of microbiological diffusion inhibition test for the determination of antibiotic residues in the milk // Eurasian Journal of Applied Biotechnology, 2023. No. 2. P.44-51. doi: 10.11134/btp.2.2023.6 (RSCI-0,117).
6. Akishev Zh., Abdullayeva A., Mussakhmetov A., Bekbayeva A., Tursunbekova A., Kalemshariv B., Tultabayeva Т., Khassenov B. The obtaining of the recombinant camel chymosin by submerge fermentation in the pilot bioreactor // Eurasian Journal of Applied Biotechnology, 2023. No. 1. P.48-55 doi: 10.11134/btp.1.2023.4 (RSCI-0,117).

Achieved results

2024  year

The primary screening of isolated microorganisms was carried out according to their ability to hydrolyze bromochlorindolyl-galactopyranoside, and their identification by cultural and morphological characteristics. 19 bacterial isolates were isolated on MRS agar. Screening of isolates on MRS agar with bromochlorindolyl galactopyranoside showed that 6 isolates did not exhibit X-gal hydrolase activity. The species identification of the strains was carried out using molecular genetic methods based on conserved DNA 16S rRNA sequencing loci and the proteomic profile using the MALDI-TOF (Biotyper) method. Based on the results obtained on β-galactosidase activity, 6 strains were selected for further analysis.Their gene organization was determined, and specific primers for the amplification of beta-galactosidase genes were selected.Amplified beta-galactosidase genes in bacterial plasmids were cloned and 6 bacterial strains of beta-galactosidase producers were obtained.