Engineering lactococci to reduce human lactose intolerance; biotechnology and risk/benefit assessment toward the gut microbiota of lactose-intolerants

β-Galactosidase, known as lactase, plays a crucial role in the dairy sector by hydrolyzing lactose into glucose and galactose. Traditionally, β-galactosidases have been sourced from Aspergillus spp. and Kluyveromyces spp., though bacterial sources like Bifidobacterium and Lactobacillus offer advantages such as higher activity, stability, and easier fermentation. However, enzyme extraction remains costly due to its intracellular production, prompting interest in using whole cells for lactose hydrolysis. Advances in recombinant DNA technology have allowed the creation of engineered strains expressing β-galactosidases from diverse sources, expanding their industrial applications. This study aimed to develop an engineered Lactococcus cremoris strain for reducing milk lactose using a whole-cell approach, bypassing the need for enzyme purification. Recombinant strains expressing bifidobacterial β-galactosidase were constructed and tested for their ability to hydrolyze lactose in UHT (Ultra High Temperature) milk. The effects on the gut microbiota of lactose-intolerant adults were assessed through an in vitro Risk/Benefit Assessment (RBA), combining INFOGEST® simulation of digestion and MICODE simulation of colonic fermentation. Results showed that L. cremoris strain 559, which secretes the enzyme into the supernatant, was more efficient at lactose removal than L. cremoris strain 558, which attaches the enzyme to the cell wall. Strain 559 also exhibited higher stability and performance, making it a better candidate for industrial lactose reduction. In vitro microbiota analysis revealed that milk treated with strain 559 promoted the growth of beneficial taxa and increased beneficial metabolites, such as short-chain fatty acids.