Short-Chain Cello-oligosaccharides: Intensification and Scale-up of Their Enzymatic Production and Selective Growth Promotion among Probiotic Bacteria

Abstract

Short-chain cello-oligosaccharides (COS; degree of polymerization, DP ≤ 6) are promising water-soluble dietary fibers. An efficient approach to their bottom-up synthesis is from sucrose and glucose using glycoside phosphorylases. Here, we show the intensification and scale up (20 mL; gram scale) of COS production to 93 g/L product and in 82 mol % yield from sucrose (0.5 M). The COS were comprised of DP 3 (33 wt %), DP 4 (34 wt %), DP 5 (24 wt %), and DP 6 (9 wt %) and involved minimal loss (≤10 mol %) to insoluble fractions. After isolation (≥95% purity; ≥90% yield), the COS were examined for growth promotion of probiotic strains. Benchmarked against inulin, trans-galacto-oligosaccharides, and cellobiose, COS showed up to 4.1-fold stimulation of cell density for Clostridium butyricum , Lactococcus lactis subsp. lactis , Lactobacillus paracasei subsp. paracasei , and Lactobacillus rhamnosus but were less efficient with Bifidobacterium sp. This study shows the COS as selectively functional carbohydrates with prebiotic potential and demonstrates their efficient enzymatic production.

β-Glucan phosphorylases in carbohydrate synthesis

“…Cellulose or insoluble β-1,4-glucan is the major polysaccharide found in nature with an essential role as a structural polymer and material, while soluble cellodextrins of DP ≤ 6 have promising applications as nutritional ingredients, excipients in medicines, and texturizers in cosmetics (Sakamoto et al 2017;Putta et al 2018;Brucher and Häßler 2019;. Such soluble cellodextrins were proven to be useful dietary fibers and prebiotics (Otsuka et al 2004;Pokusaeva et al 2011) that stimulate the growth of a number of healthy human gut bacteria more efficiently than inulin, trans-galacto-oligosaccharides, and cellobiose alone (Zhong et al 2020b).…”

“…Finally, an efficient synthesis of 93 g/L of soluble cellodextrins was demonstrated. The final product consisted of DP3, DP4, DP5, and DP6 with a distribution of 33, 34, 24, and 9 wt%, respectively, a purity of over 95%, and a yield of 88% (Zhong et al 2020b ). We previously described the synthesis of predominantly cellotriose from both glucose and cellobiose by using a cellobiose phosphorylase variant (Ubiparip et al 2020 ).…”

Kinetic modeling of phosphorylase-catalyzed iterative β-1,4-glycosylation for degree of polymerization-controlled synthesis of soluble cello-oligosaccharides

“…Due to mass action of phosphate present in excess over αGlc1- P in the cell, phosphorolysis is the preferred direction of reaction in vivo [ 10 ]. However, the CdP reaction in reverse direction under ex vivo conditions can provide an interesting route for the bottom-up synthesis of COS. Iterative β-1,4-glycosylation of cellobiose from αGlc1- P was shown in several studies [ 6 – 8 , 11 , 12 ] and it was recently demonstrated for the enzymatic production of COS at ~ 100 g/L final concentration [ 13 ]. The COS isolated from the process were shown to stimulate the growth of certain probiotic bacteria (e.g., Clostridium butyricum ; Lactococcus lactis subsp.…”

“…The COS isolated from the process were shown to stimulate the growth of certain probiotic bacteria (e.g., Clostridium butyricum ; Lactococcus lactis subsp. lactis ), suggesting that they could be interesting dietary fibers providing a selective prebiotic effect [ 13 ]. Based on the emerging evidence on possible applications of COS for food and feed use [ 14 ], there is considerable interest in the intensification of the CdP-catalyzed conversion for the development of an efficient biocatalytic production.…”

One‐Pot Synthesis of Asymmetrically Difunctionalized Oligomaltosides by Cyclodextrin Ring Opening

“…Oligosaccharides have many interests in food and health fields, [1][2][3] but also, more recently, in materials science and nanotechnologies for more extensive applications. [4][5][6] Obtaining pure oligosaccharides both from natural sources, [7] and by multistep oligosaccharide synthesis, [8] remains challenging.…”