Production of lactulose‐containing sweet milk and yoghurt utilising cellobiose 2‐epimerase from Caldicellulosiruptor bescii

Background Lactulose is a semisynthetic, nondigestible disaccharide that is well‐established in pharmaceutical applications; however, its exploitation in fluid milk and yoghurt matrices remains largely unexplored. This study evaluated the feasibility of enzymatically converting lactose into lactulose within these matrices using recombinant cellobiose 2‐epimerase derived from Caldicellulosiruptor bescii . Methods The cellobiose 2‐epimerase gene was cloned into pET‐22b(+) and heterologous expressed in Escherichia coli BL21(DE3). The purified enzyme (47 kDa) was biochemically characterised for thermal (65–75°C) and pH (6.0–8.0) stability. ‘Sweet milk’ was generated by supplementing raw milk with 1.6 × 10 −8 kat mL −1 cellobiose 2‐epimerase and incubating at 75°C for 1 h. Yoghurt was subsequently manufactured using selected Streptococcus thermophilus and Lactiplantibacillus plantarum strains that metabolise lactose but not lactulose. Carbohydrate concentrations were quantified in triplicate by high‐performance liquid chromatography. Major Findings Cellobiose 2‐epimerase exhibited maximal activity at 75°C and pH 7.0, retaining >95% residual activity under these conditions. Post‐treatment, milk contained 21.68 g L −1 lactose and 8.72 g L −1 lactulose, corresponding to a 29% conversion efficiency. Fermentation with compatible starter cultures reduced lactose to 15.46 g L −1 while preserving 6.62 g L −1 lactulose in the final yoghurt. Scientific and Industrial Implications The integrated bioprocess described herein provides a scalable, single‐step strategy to produce low‐lactose, lactulose‐enriched dairy products tailored for lactose‐intolerant consumers. The thermostable cellobiose 2‐epimerase and compatible starter strains offer immediate potential for industrial translation.