Synthesis of novel lactose-derived oligosaccharides by two-step enzymatic reactions and structural characterization of the oligomers

• Published in: Food research international Vol. 208; p. 116177
• Main Authors: Hu, Hangyu, Hu, Gege, Zhang, Min, Ren, Qingxia, Aziz, Tariq, Sarwar, Abid, Alasmari, Abdullah F., Yang, Zhennai, Yang, Zhang
• Format: Journal Article
• Language: English
• Published: Canada Elsevier Ltd 01.05.2025
• Subjects: beta-Galactosidase - metabolism; Fermentation; food research; galactose; Galactose - metabolism; galactosides; genes; glucose; glucosides; Inulosucrase; Isomerization; Kluyveromyces - metabolism; Kluyveromyces marxianus var. lactis; Lactobacillus - enzymology; Lactobacillus - genetics; Lactobacillus gasseri; Lactose; Lactose - chemistry; Lactose - metabolism; Oligosaccharides; Oligosaccharides - biosynthesis; Oligosaccharides - chemical synthesis; Oligosaccharides - chemistry; polymerization; Prebiotics; spectroscopy; Sucrose; Sucrose - metabolism; Transfructosylation; β-Galactosidase; Sucrose; DMSO; LS; HH-COSY; DP; TFA; Transfructosylation; MFOS; ESI; β-Galactosidase; HPLC; GC–MS; Isomerization; HMBC; GOS; IS; Oligosaccharides; FOS; NEA; SDS-PAGE; Lactose; NMR; Inulosucrase; IPTG; PMAA; UPLC-MS/MS; GRAS; HSQC; LFOS; RFOS
• ISSN: 0963-9969; 1873-7145; 1873-7145
• DOI: 10.1016/j.foodres.2025.116177

Exploitation of the added value of lactose by enzymatic synthesis of novel lactose-derived oligosaccharides has been a subject of increasing research interests. In this study, a strategy of two-step enzymatic reactions was designed to first synthesize galacto-oligosaccharide (GOS) from lactose by β-galactosidase, and then the novel oligosaccharides were synthesized by inulosucrase (IS) using the obtained GOS and sucrose as the substrates. The recombinant IS was initially expressed by employing the IS gene from Lactobacillus gasseri DSM 20604. The appropriate synthesis conditions by the IS were determined as: reaction time of 12 h, the concentration of IS at 25 U/mL, GOS to sucrose ratio of 1:2, and the total substrate concentration of 500 g/L. Purification of the synthesized oligosaccharides in the reaction mixture was done by fermentation with Kluyveromyces lactis CICC 1773 to remove the carbohydrate by-products, and HPLC on an oligosaccharide column. The purified oligosaccharides, namely, Oligo-1, Oligo-2, and Oligo-3 were determined for their monosaccharide components to be galactose and glucose in the molar ratios of 0.230:0.770, 0.522:0.478 and 0.637:0.363, respectively. These three oligosaccharides had relative molecular masses of 666.18, 990.26, and 1314.43, corresponding to the degree of polymerization (DP) value of 4, DP6, and DP8, respectively. NMR spectroscopic studies of the oligosaccharides revealed a common branched structure consisting of three glucosides, one of which was linked with one (Oligo-1), three (Oligo-2) and five galactosides (Oligo-3), respectively. These lactose-derived oligosaccharides with the structural patterns that were not reported earlier represented novel potential prebiotic agents for use in functional foods. [Display omitted] •Novel lactose-derived oligosaccharides (DP 4, 6, and 8) were synthesized by a two-step enzymatic method.•Inulosucrase gene from Lactobacillus gasseri DSM 20604 was expressed in Escherichia coli BL21(DE3).•Purification of oligosaccharides was done by yeast fermentation to remove carbohydrate by-products and HPLC.•The synthesized oligosaccharides with a specific branched structure were not reported earlier.