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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.

SUBMITTER: Zhong C

PROVIDER: S-EPMC7458430 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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

Zhong Chao C Ukowitz Christina C Domig Konrad J KJ Nidetzky Bernd B

Journal of agricultural and food chemistry 20200731 32

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 ...[more]

PMID: 32687709

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Project description:BackgroundContinuous processing with enzyme reuse is a well-known engineering strategy to enhance the efficiency of biocatalytic transformations for chemical synthesis. In one-pot multistep reactions, continuous processing offers the additional benefit of ensuring constant product quality via control of the product composition. Bottom-up production of cello-oligosaccharides (COS) involves multistep iterative β-1,4-glycosylation of glucose from sucrose catalyzed by sucrose phosphorylase from Bifidobacterium adeloscentis (BaScP), cellobiose phosphorylase from Cellulomonas uda (CuCbP) and cellodextrin phosphorylase from Clostridium cellulosi (CcCdP). Degree of polymerization (DP) control in the COS product is essential for soluble production and is implemented through balance of the oligosaccharide priming and elongation rates. A whole-cell E. coli catalyst co-expressing the phosphorylases in high yield and in the desired activity ratio, with CdP as the rate-limiting enzyme, was reported previously.ResultsFreeze-thaw permeabilized E. coli cells were immobilized in polyacrylamide (PAM) at 37-111 mg dry cells/g material. PAM particles (0.25-2.00 mm size) were characterized for COS production (~ 70 g/L) in mixed vessel with catalyst recycle and packed-bed reactor set-ups. The catalyst exhibited a dry mass-based overall activity (270 U/g; 37 mg cells/g material) lowered by ~ 40% compared to the corresponding free cells due to individual enzyme activity loss, CbP in particular, caused by the immobilization. Temperature studies revealed an operational optimum at 30 °C for stable continuous reaction (~ 1 month) in the packed bed (volume: 40 mL; height: 7.5 cm). The optimum reflects the limits of PAM catalyst structural and biological stability in combination with the requirement to control COS product solubility in order to prevent clogging of the packed bed. Using an axial flow rate of 0.75 cm- 1, the COS were produced at ~ 5.7 g/day and ≥ 95% substrate conversion (sucrose 300 mM). The product stream showed a stable composition of individual oligosaccharides up to cellohexaose, with cellobiose (48 mol%) and cellotriose (31 mol%) as the major components.ConclusionsContinuous process technology for bottom-up biocatalytic production of soluble COS is demonstrated based on PAM immobilized E. coli cells that co-express BaScP, CuCbP and CcCdP in suitable absolute and relative activities.

Project description:BackgroundA critical role for host-microbe interactions and establishment of vaccine responses has been postulated. Human milk oligosaccharides, of which 2'-fucosyllactose (2'FL) is the most prevalent, are known to alter host-associated microbial communities and play a critical role in the immunologic development of breastfed infants.ObjectivesDietary supplementation with a combination of 2'FL and prebiotic short-chain (sc) galacto-oligosaccharides (GOS) and long-chain (lc) fructo-oligosaccharides (FOS) was employed to examine human milk oligosaccharide effects on immune responsiveness, within a murine influenza vaccination model.MethodsFemale mice (6 wk old, C57Bl/6JOlaHsd) were fed either control diet (CON) or scGOS/lcFOS/2'FL-containing diet (GF2F) for 45 d. After starting dietary intervention (day 14), mice received a primary influenza vaccination (day 0) followed by a booster vaccination (day 21), after which ear challenges were conducted to measure vaccine-specific delayed type hypersensitivity (DTH). Serum immunoglobulin (Ig) levels, fecal and cecal microbial community structure, short-chain fatty acids, host intestinal gene expression and cellular responses in the mesenteric lymph nodes (MLNs) were also measured.ResultsRelative to CON, mice fed the GF2F diet had increased influenza vaccine-specific DTH responses (79.3%; P < 0.01), higher levels of both IgG1 (3.2-fold; P < 0.05) and IgG2a (1.2-fold; P < 0.05) in serum, and greater percentages of activated B cells (0.3%; P < 0.05), regulatory T cells (1.64%; P < 0.05), and T-helper 1 cells (2.2%; P < 0.05) in their MLNs. GF2F-fed mice had elevated cecal butyric (P < 0.05) and propionic (P < 0.05) acid levels relative to CON, which correlated to DTH responses (R2 = 0.22; P = 0.05 and R2 = 0.39; P < 0.01, respectively). Specific fecal microbial taxa altered in GF2F diet fed mice relative to CON were significantly correlated with the DTH response and IgG2a level increases.ConclusionsDietary GF2F improved influenza vaccine-specific T-helper 1 responses and B cell activation in MLNs and enhanced systemic IgG1 and IgG2a concentrations in mice. These immunologic changes are correlated with microbial community structure and metabolites.

Project description:Periodontal disease is a chronic inflammatory condition caused by periodontal pathogens in the gingival sulcus. Short-chain fatty acids (SCFAs) produced by causal bacteria are closely related to the onset and progression of periodontal disease and have been reported to proliferate in the periodontal sulcus of patients experiencing this pathology. In such patients, propionic acid (C3), butyric acid (C4), isobutyric acid (IC4), valeric acid (C5), isovaleric acid (IC5), and caproic acid (C6), henceforth referred to as [C3-C6], has been reported to have a detrimental effect, while acetic acid (C2) exhibits no detrimental effect. In this study, we established an inexpensive and simple enzymatic assay that can fractionate and measure these acids. The possibility of applying this technique to determine the severity of periodontal disease by adapting it to specimens collected from humans has been explored. We established an enzyme system using acetate kinase and butyrate kinase capable of measuring SCFAs in two fractions, C2 and [C3-C6]. The gingival crevicular fluid (GCF) and saliva of 10 healthy participants and 10 participants with mild and severe periodontal disease were measured using the established enzymatic method and conventional gas chromatography-mass spectrometry (GC-MS). The quantification of C2 and [C3-C6] in human GCF and saliva was well correlated when using the GC-MS method. Furthermore, both C2 and [C3-C6] in the GCF increased with disease severity. However, while no significant difference was observed between healthy participants and periodontal patients when using saliva, [C3-C6] significantly differed between mild and severe periodontal disease. The enzymatic method was able to measure C2 and [C3-C6] separately as well as using the GC-MS method. Furthermore, the C2 and [C3-C6] fractions of GCF correlated with disease severity, suggesting that this method can be applied clinically. In contrast, the quantification of C2 and [C3-C6] in saliva did not differ significantly between healthy participants and patients with periodontal disease. Future studies should focus on inflammation rather than on tissue destruction.

Dataset Information

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

Publications

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

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