Construction of an enzymatic route using a food-grade recombinant Bacillus subtilis for the production and purification of epilactose from lactose

Abstract

Lactose is a main by-product in the cheese industry. Many attempts have been made to convert the lactose to high value-added products, including epilactose. Epilactose is a valuable prebiotic and can be epimerized from lactose with cellobiose 2-epimerase (CEase). The objective of the present work was to construct a food-grade recombinant Bacillus subtilis that produces CEase from Thermoanaerobacterium saccharolyticum. The CEase was expressed in B. subtilis without antibiotic resistance genes. After fermentation, the maximum volumetric activity of the fermented broth was more than 7 U/mL. The activity of the recombinant B. subtilis was increased by up to 3.7 fold after ethanol permeabilization. Then, 66.9 ± 0.7 g/L of epilactose was produced from 300 g/L of whey powder solution in 1 h with 13.3 U/mL of permeabilized biocatalyst. In addition, an enzymatic route including degradation of the lactose, yeast fermentation, and cation exchange chromatography was described to further purify the produced epilactose from lactose. Finally, epilactose with a purity >98% was produced from 300 g/L of lactose with a yield of 24.0%. In conclusion, neither antibiotics nor pathogenic bacteria were used throughout the epilactose production and purification procedure.

Identification of a thermostable cellobiose 2‐epimerase from Caldicellulosiruptor sp. Rt8.B8 and production of epilactose using Bacillus subtilis

“…The yeast cell treated epilactose/glucose/galactose mixed syrup showed nearly 100% elimination on glucose and galactose while epilactose was not metabolized. This was in accordance with the conclusion that epilactose was a non‐digestible food ingredient 40 …”

“…As a comparison, Chen et al . obtained 88.5 g L −1 epilactose from 300 g L −1 lactose, which was catalyzed by ethanol permeabilized Bacillus subtilis 1A751 whole cells 40 . Saburi et al .…”

“…As a comparison, Chen et al obtained 88.5 g L −1 epilactose from 300 g L −1 lactose, which was catalyzed by ethanol permeabilized Bacillus subtilis 1A751 whole cells. 40 Saburi et al obtained 53 g L −1 epilactose from 200 g L −1 lactose in 48 h using a mesophilic enzyme RaCE. Separation of epilactose from lactose was difficult.…”

“…Chen et al used ⊎-galactosidase and achieved a higher hydrolysis ratio of lactose as wells as limited loss of epilactose. 40 However, the microbial origin of the ⊎-galactosidase was not published. To solve this problem, we screened several industrial ⊎-galactosidase and proved that BsGal was the most suitable candidate.…”

Trends in lactose-derived bioactives: synthesis and purification

“…For instance, Krewinkel et al [53] reported that cellobiose 2-epimerase from the mesophiles Flavobacterium johnsoniae and Pedobacter heparinus are able to catalyze the synthesis of epilactose with a reaction yield around 33%, without formation of side product. More recently, Chen et al [54] produced epilactose from cheese whey (300 g/L) using the cellobiose 2-epimerase from Thermoanaerobacterium saccharolyticum expressed in Bacillus subtilis. Then, they hydrolyzed the remaining lactose with the β-galactosidase form Bifidobacterium bifidum and removed the monosaccharides using selective fermentation with Saccharomyces cerevisiae.…”