Use of Chemical Mutagenesis for the Isolation of Food Grade β-Galactosidase Overproducing Mutants of Bifidobacteria, Lactobacilli and Streptococcus thermophilus

Abstract

A classical chemical mutagenesis protocol was evaluated for increasing β-galactosidase production by probiotic bacteria to improve their potential to treat symptoms of lactose malabsorption in humans. Two Bifidobacterium species (B. breve and B. longum) and one strain each of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus were tested by a single exposure to two chemical mutagens, ethyl methanesulfonate (EMS) and N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). To screen for β-galactosidase (β-gal) overproducing mutants, optimized EMS and MNNGmutant pots for each strain were plated on BHI agar containing 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal). Colonies that exhibited a blue color were selected for quantitative β-gal activities using the o-nitrophenyl-β-galactoside (ONPG) assay. Seventy-five mutants were obtained out of more than 2million colonies screened and showed increased β-galactosidase activities compared with the wild-type strains. EMS gave a higher frequency of β-gal overproducing mutants than MNNG for three of the four strains, S. thermophilus, B. breve, and B. longum, whereas the frequency of L. delbrueckii ssp. bulgaricus β-gal mutants was similar with both mutagens. The highest β-gal increases, when induced during growth in lactose, for mutants of each culture were 137% for L. delbrueckii ssp. bulgaricus; 104% for S. thermophilus; 70% for B. breve; and 222% for B. longum mutants. This food-grade classical approach has the ability to moderately increase β-gal concentrations in probiotic cultures to improve their potential for treating the symptoms of lactose malabsorption in humans.