Mannotriose

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.

Recognized for its bioactive compounds, açaí has become a functional food, but it has a low pulp yield, and the seeds are the main waste. This study investigates the potential of açaí seeds (Euterpe oleracea Mart.) to produce mannooligosaccharides (MOS) through enzymatic hydrolysis. Using response surface methodology (RSM), the research optimizes MOS extraction while minimizing mannose production and reducing processing time, achieving MOS production of about 10 g/L, a value within the range of similar investigations. The RSM quadratic models establish correlations between MOS production (M2-M5) and enzymatic hydrolysis conditions, with R2 values ranging from 0.6136 to 0.9031. These models are used to emphasize MOS performance (M2-M5) while reducing mannose production, which also promotes profitability by reducing time. Experimental validation agrees with model predictions, highlighting optimal conditions near 40 °C, intermediate enzyme loading, and basic pH that effectively promotes MOS generation on mannose within an accelerated processing time frame. With predictions of experimental results within a margin of error of < 9%, the validity of the models was acceptable. This research contributes to the advancement of the understanding of the enzymatic hydrolysis of açaí seeds, which is a step toward the sustainable use of resources with a focus on process engineering aspects.

Pine nut skin (Pinus pinea L.) is a poorly explored industrial byproduct with potential to be utilized as a food ingredient. Subcritical water extraction (SWE), an eco-friendly extraction technique with higher efficiency than hot-water extraction (HWE), was studied to evaluate its suitability in producing extracts rich in soluble fiber and phenolic compounds. For this, a factorial design was developed considering temperature (120-180°C), time (2-10 min), and mass/volume ratio (1-3 g/60 mL) under microwave irradiation. This design aimed to maximize the extraction of carbohydrates, while achieving the highest content of phenolic compounds and antioxidant activity. SWE produced higher yields (2.6-fold in relation to HWE) of extracts rich in polysaccharides, determined by methylation analysis, oligosaccharides, determined by GC-qMS as alditol acetates, and phenolic compounds, determined by HPLC-ESI-MS. SWE increased the recovery of pectic oligosaccharides (10-fold) and xyloglucans (2-fold), and allowed to recover pectic polysaccharides, type II arabinogalactans and insoluble-bound phenolic compounds. Mono-, oligo- and polysaccharides were not hydrolyzed during in vitro gastrointestinal digestion, showing potential prebiotic functionality. Although phenolic compounds suffered a 23% (gallic acid equivalents) decrease, phenolic acids and aldehydes were released or conserved upon intestinal phase. These results highlight the potential of PNS valorization as functional food ingredients through the subcritical water solubilization of polysaccharides and phenolic compounds.

In this work, an enzyme cocktail with β-mannanase as the main activity was immobilized on epoxy resin foams filled with fibers from annatto capsules. The catalytic system was characterized by SEM, FTIR, and a mechanical crush resistance test. The behavior of the pH and temperature for the hydrolysis of the locust bean gum were also studied. With the same substrate and with respect to the free enzyme, the immobilized enzyme showed an activity retention of 79.61%. Its operational stability in ten reuse cycles did not show any statistically significant loss of activity. This catalytic system was used to study the preferential release of MOS of two to five degrees of polymerization from mannan present in dried and ground açaí seeds, which were not subjected to any other pretreatment. Using an experimental response surface design, the predicted quadratic models for the M2-M5 MOS content were obtained and they fit well with the experimental data, predicting a production range between 0.435 and 20 g/L of MOS (M2-M5). In addition, the production reached about 12 g/L under the optimized conditions. These results indicate that the used foamed epoxy resin supports and immobilization methodology are suitable for catalyzing the hydrolysis of mannan from açaí seeds.

Currently, the production of manno-oligosaccharides (MOS) from guar gum faces challenges of low oligosaccharide enzymatic hydrolysis yield and complicated steps in separation and purification. In this work, a potential strategy to address these issues was explored. By combining citric acid pretreatment (300 mM, 130°C, 1 h) with β-mannanase hydrolysis, an impressive MOS yield of 61.8% from guar gum (10%, w/v) was achieved. The key success lay in the optimizing conditions that completely degraded other galactomannans into monosaccharides, which could be easily removable through Saccharomyces cerevisiae fermentation (without additional nutrients). Following ion exchange chromatography for desalination, and concluding with spray drying, 4.57 g of solid MOS with a purity of 90% was obtained from 10 g of guar gum. This method offers a streamlined and effective pathway for obtaining high-yield and high-purity MOS from guar gum by combining citric acid pretreatment and enzymatic hydrolysis.

Mycotoxins are the secondary fungal metabolites generally produced by wide range of fungi including aflatoxins (AF), ochratoxin A (OTA), fumonisins (FB), zearalenone (ZEN), and deoxynivalenol (DON). Nowadays, they are main concern to food and agricultural commodities due to undesirable health and socio-economic effect. This investigation was designed to synthesized microcapsules loaded the bioactive compounds of date seed and evaluated its inhibitory activities in mice received mold-contaminated diet. The finding revealed that the developed microcapsule is homogenous and mostly spherical with size of 2.58 µm with acceptable PDI of 0.21. The main phytochemical has been confirmed by HPLC analysis were xylose, fructose, mannose, glucose and galactose with the respective values of 41.95, 2.24, 5.27 and 0.169 percent. The invivo analyses manifested that the mice received date seed microcapsules significantly (p < 0.05) improved the average daily weight gain, feed intake, liver enzymes (ALT, ALP and AST) and lipid peroxidation values compare to mice group received mycotoxin-contaminated diet. Furthermore, encapsulation date seed bioactive compounds notably up-regulated the expression of GPx, SOD, IFN-γ and IL-2 genes while down-regulated the iNOS gene. Consequently, the novel microcapsules loaded date seed is suggested to considered as a promising mycotoxin inhibitor.

β-Mannan is abundant in the human diet and in hemicellulose derived from softwood. Linear or galactose-substituted β-mannan-oligosaccharides (MOS/GMOSs) derived from β-mannan are considered emerging prebiotics that could stimulate health-associated gut microbiota. However, the underlying mechanisms are not yet resolved. Therefore, this study investigated the cross-feeding and metabolic interactions between Bifidobacterium adolescentis ATCC 15703, an acetate producer, and Roseburia hominis A2-183 DSMZ 16839, a butyrate producer, during utilization of MOS/GMOSs. Cocultivation studies suggest that both strains coexist due to differential MOS/GMOS utilization, along with the cross-feeding of acetate from B. adolescentis E194a to R. hominis A2-183. The data suggest that R. hominis A2-183 efficiently utilizes MOS/GMOS in mono- and cocultivation. Notably, we observed the transcriptional upregulation of certain genes within a dedicated MOS/GMOS utilization locus (RhMosUL), and an exo-oligomannosidase (RhMan113A) gene located distally in the R. hominis A2-183 genome. Significantly, biochemical analysis of β-1,4 mannan-oligosaccharide phosphorylase (RhMOP130A), α-galactosidase (RhGal36A), and exo-oligomannosidase (RhMan113A) suggested their potential synergistic role in the initial utilization of MOS/GMOSs. Thus, our results enhance the understanding of MOS/GMOS utilization by potential health-promoting human gut microbiota and highlight the role of cross-feeding and metabolic interactions between two secondary mannan degraders inhabiting the same ecological niche in the gut.

The study of specific glycan uptake and metabolism is an effective tool in aiding with the continued unravelling of the complexities in the human gut microbiome. To this aim fluorescent labelling of glycans may provide a powerful route towards this target. Here, we successfully used the fluorescent label 2-aminobenzamide (2-AB) to monitor and study microbial degradation of labelled glycans. Both single strain and co-cultured fermentations of microbes from the common human-gut derived Bacteroides genus, are able to grow when supplemented with 2-AB labelled glycans of different monosaccharide composition, degrees of acetylation and polymerization. Utilizing a multifaceted approach that combines chromatography, mass spectrometry, microscopy and flow cytometry techniques, it is possible to better understand the metabolism of labelled glycans in both supernatants and at a single cell level. We envisage this combination of complementary techniques will help further the understanding of substrate specificity and the role it plays within microbial communities.

Both β-mannanases and β-mannosidases are required for mannan-backbone degradation into mannose. In this study, two β-mannosidases of glycoside hydrolase (GH) families 2 (BtMan2A) and 5 (CmMan5A) were evaluated for their substrate specificities and galactomannan binding ability. BtMan2A preferred short manno-oligomers, while CmMan5A preferred longer ones; DP >2, and galactomannans. BtMan2A displayed irreversible galactomannan binding, which was pH-dependent, with higher binding observed at low pH, while CmMan5A had limited binding. Docking and molecular dynamics (MD) simulations showed that BtMan2A galactomannan binding was stronger under acidic conditions (-8.4 kcal/mol) than in a neutral environment (-7.6 kcal/mol), and the galactomannan ligand was more unstable under neutral conditions than acidic conditions. Qualitative surface plasmon resonance (SPR) experimentally confirmed the reduced binding capacity of BtMan2A at pH 7. Finally, synergistic β-mannanase to β-mannosidase (BtMan2A or CmMan5A) ratios required for maximal galactomannan hydrolysis were determined. All CcManA to CmMan5A combinations were synergistic (≈1.2-fold), while combinations of CcManA with BtMan2A (≈1.0-fold) yielded no hydrolysis improvement. In conclusion, the low specific activity of BtMan2A towards long and galactose-containing oligomers and its non-catalytic galactomannan binding ability led to no synergy with the mannanase, making GH2 mannosidases ineffective for use in cocktails for mannan degradation.

This study aimed to produce manno-oligosaccharides (MOS) from Gleditsia microphylla galactomannans (GMG) using CO₂ and solid acid (Amberlyst-35) in subcritical water. The optimal condition for MOS preparation was 3 MPa CO₂, 0.1 g/g solid acid (relative to GMG) at 150°C for 40 min. The maximum MOS yield with a degree of polymerization from 2 to 4 (M₂-M₄) was 52.19%, which doubled the yield of MOS compared to either using solely CO₂ or solid acid. Solid acid showed excellent performance in producing MOS under subcritical H₂O-CO₂ condition, due to the enhanced mass transfer efficiency and increased H⁺ concentration in the reaction system. The solid acid can be easily separated and reused. Comparing with traditional methods used to produce MOS, this approach has many merits such as higher galactomannan hydrolysis efficiency (largely reduced time and higher MOS yield), purer M₂-M₄ product, lower costs, and more environmental-friendly.