Tehran University of Medical Sciences Journal of Food Safety and Hygiene 2476-3241 8 3 2022 10 25 170 182 Folake Afolabi University of Ibadan, Ibadan, Nigeria. Oluwaseyi Peter Adewale University of Ibadan, Ibadan, Nigeria. Stella Mojisola Adeyemo Obafemi Awolowo University, Osun State, Nigeria. 2022 05 12 2022 10 15 β-galactosidase is an enzyme that converts lactose into glucose and galactose. It alleviates the issue associated with intolerance to lactose and pollution caused as a result of milk by-products (whey). This study aimed to isolate and select yeast strains that can produce β-galactosidase from fresh milk, yoghurt and locally made cheese. A total of 115 yeasts were isolated from the samples, 9 yeast isolates had the ability to produce β-galactosidase and three (3) were selected for further analysis. β-galactosidase producers were screened using Ortho-nitrophenyl-β-D-galactopyranoside (ONPG) assay. Kluyveromyces marxianus strain SLDY – 005 produced the highest β-galactosidase. It showed maximum enzyme activity (277U/mL) at a temperature of 30oC, pH 5.5, after an incubation period of 36 h. Glucose decreased β-galactosidase production while yeast extract and urea were considered appropriate nitrogen sources for the best enzyme synthesis. Crude β-galactosidase produced by Kluyveromyces marxianus SLDY – 005 was purified. The partially purified enzyme after dialysis showed a specific activity of 165.12 U/mL and had a purification fold of 6.02 and yield of 45.91 %. The purified enzyme had an optimum temperature of 40oC and a pH 6.0. Kluyveromyces marxianus strain isolated from local cheese is a potential candidate for the production of β-galactosidase and could be used to combat the problem of lactose intolerance. https://jfsh.tums.ac.ir/index.php/jfsh/article/view/379 https://jfsh.tums.ac.ir/index.php/jfsh/article/download/379/197