Guar gum (GG) containing mainly galactomannan (GM) is one of the most used hydrocolloids in food industry. beta-Mannooligosaccharides (beta-MOS) as building blocks generated from GG are potential prebiotics, yet their structures are still unclear. Preparation of GG-derived beta-MOS and decoding the galactose substitution information of beta-MOS are an ongoing challenge. Here, a novel beta-mannanase BF0736 that belongs to the glycoside hydrolase family 26 from Bacteroides fragilis NCTC 9343 strain was cloned and applied to degrade GG for preparation of beta-MOS mixture with a yield of 81.3 +/- 2.2% (w/w; relative to GG). Eleven major galacto-mannooligosaccharides (G-MOS) with degree of polymerization (DP) 2-11 were purified from the mixture by SEC and HPAEC techniques. A new strategy for decoding the galactose substitution information of G-MOS was developed by using two-step labeling (derivatizations of anomeric site in mannose and primary hydroxyl group site in galactose) combined with tandem mass spectrometry analysis methods. Precise structures of these G-MOS were characterized, and eight oligosaccharides including 6(3)-alpha-d-galactosyl-beta-d-mannotriose, 6(1),6(3)-di-alpha-d-galactosyl-beta-d-mannotriose, 6(3),6(4)-di-alpha-d-galactosyl-beta-d-mannotetraose, 6(1),6(3),6(4)-tri-alpha-d-galactosyl-beta-d-mannotetraose, 6(3),6(4),6(5)-tri-alpha-d-galactosyl-beta-d-mannopentaose, 6(1),6(3),6(4),6(5)-tetra-alpha-d-galactosyl-beta-d-mannopentaose, 6(3),6(4),6(5),6(6)-tetra-alpha-d-galactosyl-beta-d-mannohexaose and 6(1),6(3),6(4),6(5),6(6)-penta-alpha-d-galactosyl-beta-d-mannohexaose were prepared and identified from GG for the first time, which provided critical insights into the distribution pattern of galactose in GG. Our results contribute to the utilization of GG and its derived beta-MOS in functional foods.

Even though Stellaria dichotoma L. var. lanceolate (S. dichotoma) is a well-known medicinal plant in the family Caryophyllaceae, its oligosaccharides remain unexplored in terms of their potential as bioactive agents. Here, we isolated a mixture of oligosaccharides from S. dichotoma (Yield: 12 % w/w), that are primarily non-classical raffinose family oligosaccharides (RFOs). Nine major oligosaccharides were purified and identified from the mixture, including sucrose, raffinose, 1-planteose, lychnose, stellariose, along with four new non-classical RFOs. Two of the four new oligosaccharides are linear hexose pentamers with α-galactosyl extensions on their lychnose moieties, and the other two are branched hexose hexamers with α-galactosyl extensions on their stellariose groups. Their interactions with galectin-3 (Gal-3) revealed significant binding, with the terminal galactose providing enhanced affinity for the lectin. Notably, Gal-3 residues Arg144, His158, Asn160, Arg162, Asn174, Trp181, Glu184 and Arg186 coordinate with the lychnose. In vivo studies using the dextran sulfate sodium (DSS) mouse model for colitis demonstrated the ability of these carbohydrates in mitigating ulcerative colitis (UC). Overall, our study has provided structural information and potential applications of S. dichotoma oligosaccharides, also offers new approaches for the development of medicinal oligosaccharides.

Even though Cimicifuga sp. is widely used in functional foods around the world, the content and structure of its oligosaccharides remain unclear. Here, we isolated a mixture of oligosaccharides from Cimicifuga heracleifolia Kom. rhizomes with a yield of 9.5% w/w. Twenty-six oligosaccharide monomers from the mixture were purified using optimized SEC and HILIC techniques. The oligosaccharides were identified as belonging to two groups by using HPAEC-PAD, MALDI-TOF-MS, NMR and GC-MS methylation analyses. One group belongs to sucrose and inulin type fructo-oligosaccharides (FOS) {& beta;-D-Fruf-(2 & RARR; 1)-[& beta;-D-Fruf-(2 & LRARR; 1)]n=1-12-& alpha;-D-Glcp} with a 3-14 de-gree of polymerization (DP). Oligosaccharides in the other group belong to the inulo-n-ose type FOS {& beta;-D-Fruf-(2 & RARR; 1)-[& beta;-D-Fruf-(2 & RARR; 1)]m=0-12-& beta;-D-Frup} with a DP of 2-14. This appears to be the first time that these oligo-saccharides have been purified from Cimicifuga heracleifolia Kom., thus providing useful information concerning the utilization of Cimicifuga heracleifolia Kom. in functional foods.

Linear β-manno-oligosaccharides (l-β-MOS) are widely used to investigate oligo- and poly-saccharide structures and mannanolytic enzyme activities. l-β-MOS are also being used as prebiotic agents with potential bio-active properties. In this study, we developed an efficient protocol to prepare a series of l-β-MOS by hydrolyzing cassia gum (CG) using mannanolytic enzymes (endo-1,4-β-mannanase, α-galactosidases and β-glucosidases). By using medium pressure liquid chromatography (MPLC), we purified l-β-MOS with different degrees of polymerization (DPs). HPAEC-PAD, MALDI-TOF-MS and NMR studies confirmed that these l-β-MOS species ranged from 1,4-β-d-mannobiose to 1,4-β-d-mannononaose (DP 2–9) with >95% purity. Our results provide a robust approach to preparing l-β-MOS, thus enabling l-β-MOS to be further used in the fields of chemistry, life science, and nutritional food.

Endo-1,4-β-galactanase is an indispensable tool for preparing prebiotic β-galacto-oligosaccharides (β-GOS) from pectic galactan resources. In the present study, a novel endo-1,4-β-galactanase (PoβGal53) belonging to glycoside hydrolase family 53 from Penicillium oxalicum sp. 68 was cloned and expressed in Pichia pastoris GS115. Upon purification by affinity chromatography, recombinant PoβGal53 exhibited a single band on SDS-PAGE with a molecular weight of 45.0 kDa. Using potato galactan as substrate, PoβGal53 showed optimal reaction conditions of pH 4.0, 40 °C, and was thermostable, retaining >80 % activity after incubating below 45 °C for 12 h. Significantly, PoβGal53 exhibited relatively conserved substrate specificity for (1 → 4)-β-D-galactan with an activity of 6244 ± 282 U/mg. In this regard, the enzyme is in effect the most efficient endo-1,4-β-galactanase identified to date. By using PoβGal53, β-GOS monomers were prepared from potato galactan and separated using medium pressure liquid chromatography. HPAEC-PAD, MALDI-TOF-MS and ESI-MS/MS analyses demonstrated that these β-GOS species ranged from 1,4-β-D-galactobiose to 1,4-β-D-galactooctaose (DP 2-8) with high purity. This work provides not only a highly active tool for enzymatic degradation of pectic galactan, but an efficient protocol for preparing β-GOS.

Polysaccharides from Pleurotus eryngii exhibit a variety of biological activities. Here, we obtained a homogeneous branched β-1,6-glucan (APEP-A-b) from the fruiting bodies of P. eryngii and investigated its effect on immunity and gut microbiota. Our results showed that APEP-A-b significantly increases splenic lymphocyte proliferation, NK cell activity and phagocytic capacity of peritoneal cavity phagocytes. Furthermore, we found that the proportion of CD4+ and CD8+ T cells in lamina propria are significantly increased upon APEP-A-b treatment. Additionally, APEP-A-b supplementation demonstrated pronounced changes in microbiota reflected in promotion of relative abundances of species in the Lachnospiraceae and Rikenellaceae families. Consistently, APEP-A-b significantly increased the concentration of acetic and butyric acid in cecum contents. Overall, our results suggest that β-1,6-glucan from P. eryngii might enhance immunity by modulating microbiota. These results are important for the processing and product development of P. eryngii derived polysaccharides.

Pectins are nutrient components of plants and are widely used in the food industry. In this study, one major pectin fraction (WLJP-A0.2b) with Mw of 40.6 kDa was purified from Lonicera japonica Thunb. The structural feature and antioxidant activity of it was investigated. Monosaccharide composition, Fourier transform infrared (FT-IR) spectra, enzymatic hydrolysis, and nuclear magnetic resonance (NMR) spectra analysis indicated that WLJP-A0.2b consisted of rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), and homogalacturonan (HG) domains, with mass ratio of 0.4:1.0:2.1. The RG-I domain contained highly branched α-L-1,5-arabinan, β-D-1,4-galactan and type II arabinogalactan (AG-II) side chains. The HG domain was released in the form of un-esterified and partly methyl-esterified and/or acetyl-esterified oligogalacturonides with degree of polymerization 1–8 after degradation by endo-polygalacturonase. Radical scavenging assays indicated that WLJP-A0.2b exhibited antioxidant activity through the synergistic effects of different pectin domains. Oligogalacturonides, especially de-esterified oligogalacturonides, showed better antioxidant activities than RG-II and RG-I domains. Moreover, de-esterified oligogalacturonides remarkably reduced H2O2-induced reactive oxygen species production in HEK-293T cells. These results provide useful information for screening of natural antioxidants from Lonicera japonica Thunb. and application of pectin in functional food field.

Bupleurum chinense DC, a traditional medicinal plant in China that has many pharmacological effects, contains polysaccharide as one of its active components. In this study, we isolated and structurally characterized the polysaccharide from B. chinense. Water-soluble polysaccharides (termed WBCP) were extracted from the plant and fractionated by anion-exchange and size exclusion chromatographies. From this procedure, we obtained a homogeneous acidic polysaccharide (WBCP-A2) and determined its monosaccharide composition. Analysis by FT infrared and 13C NMR spectroscopies, along with enzymatic hydrolysis, indicated that WBCP-A2 is a pectic polysaccharide, composed of rhamnogalacturonan I, rhamnogalacturonan II, highly methyl-esterified homogalacturonan (HG), and either non- or low methyl-esterified HG domains. These different fractions may be covalently linked through HG segments to form the complex pectin molecules.