Detailed Analysis of Prebiotic Fructo- and Galacto-Oligosaccharides in the Human Small Intestine.

Galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS) are food ingredients that improve human health, but their degradation throughout the human small intestine is not well understood. We studied the breakdown kinetics of FOS and GOS in the intestines of seven healthy Dutch adults. Subjects were equipped with a catheter in the distal ileum or proximal colon and consumed 5 g of chicory-derived FOS (degree of polymerization (DP) DP2-10), and 5 g of GOS (DP2-6). Postprandially, intestinal content was frequently collected until 350 min and analyzed for mono-, di-, and oligosaccharides. FOS and GOS had recoveries of 96 ± 25% and 76 ± 28%, respectively. FOS DP ≥ 2 and GOS DP ≥ 3 abundances in the distal small intestine or proximal colon matched the consumed doses, while GOS dimers (DP2) had lower recoveries, namely 22.8 ± 11.1% for ß-D-gal-(1↔1)-α-D-glc+ß-D-gal-(1↔1)-ß-D-glc, 19.3 ± 19.1% for ß-D-gal-(1 → 2)-D-glc+ß-D-gal-(1 → 3)-D-glc, 43.7 ± 24.6% for ß-D-gal-(1 → 6)-D-gal, and 68.0 ± 38.5% for ß-D-gal-(1 → 4)-D-gal. Lactose was still present in the distal small intestine of all of the participants. To conclude, FOS DP ≥ 2 and GOS DP ≥ 3 were not degraded in the small intestine of healthy adults, while most prebiotic GOS DP2 was hydrolyzed in a structure-dependent manner. We provide evidence on the resistances of GOS with specific ß-linkages in the human intestine, supporting the development of GOS prebiotics that resist small intestine digestion.

Simplified Synthesis of Poly(ethyleneimine)-Modified Silica Particles and Their Application in Oligosaccharide Isolation Methods.

There are great challenges in the field of natural product isolation and purification and in the pharmacological study of oligosaccharide monomers. And these isolation and purification processes are still universal problems in the study of natural products (NPs), traditional Chinese medicine (TCM), omics, etc. The same polymer-modified materials designed for the special separation of oligosaccharides, named Sil-epoxy-PEI and Sil-chloropropyl-PEI, were synthesized via two different methods and characterized by scanning electron microscopy combined with energy spectrum analysis, Fourier transform infrared spectroscopy, thermogravimetric analysis, zeta potential as well as surface area analysis, etc. Several nucleotide/nucleoside molecules with different polarities and selectivities were successfully isolated in our laboratory using stainless-steel columns filled with the synthesized material. In addition, the separation of saccharide probes and oligosaccharides mixtures in water extracts of Morinda officinalis were compared in HILIC mode. The results showed that the resolution of separations for the representative analytes of the Sil-epoxy-PEI column was higher than for the Sil-chloropropyl-PEI column, and the developed stationary phase exhibited improved performance compared to hydrothermal carbon, amide columns and other HILIC materials previously reported.

Renal-Targeted Drug Delivery by Chitosan Oligosaccharide Micelles with HSA-Enriched Protein Corona for the Treatment of Ischemia/Reperfusion-Induced Acute Kidney Injury.

Renal-specific nanoparticulate drug delivery systems have shown great potential in reducing systemic side effects and improving the safety and efficacy of treatments for renal diseases. Here, stearic acid-grafted chitosan oligosaccharide (COS-SA) was synthesized as a renal-targeted carrier due to the high affinity of the 2-glucosamine moiety on COS to the megalin receptor expressed on renal proximal tubular epithelial cells. Specifically, COS-SA/CLT micelles were prepared by encapsulating celastrol (CLT) with COS-SA, and different proportions of human serum albumin (HSA) were then adsorbed onto its surface to explore the interaction between the protein corona and cationic polymeric micelles. Our results showed that a multilayered protein corona, consisting of an inner "hard" corona and an outer "soft" corona, was formed on the surface of COS-SA/CLT@HSA8, which was beneficial in preventing its recognition and phagocytosis by macrophages. The formation of HSA protein corona on COS-SA/CLT micelles also increased its accumulation in the renal tubules. Furthermore, the electropositivity of COS-SA/CLT micelles affected the conformation of adsorbed proteins to various degrees. During the adsorption process, the protein corona on the surface of COS-SA/CLT@HSA1 was partially denatured. Overall, COS-SA/CLT and COS-SA/CLT@HSA micelles demonstrated sufficient safety with renal targeting potential, providing a viable strategy for the management of ischemia/reperfusion-induced acute kidney injury.

N-Difluoroacetylhexosamine as a Substrate-Engineering Precursor for Glycosylation Reactions.

We present the application of N-difluoroacetylglucosamine (GlcNDFA) in a chemical evolution strategy to synthesize oligosaccharides. In comparison to conventional N-trifluoroacetylglucosamine, GlcNDFA exhibits superior substrate compatibility with glycosyltransferases as well as stability in aqueous environments. Using our 16-step assembly line, GlcNDFA can be used to produce homogeneous dekaparin, a heparin-like medication, with a yield of 62.2%. This underscores the significant potential of GlcNDFA as a chemical evolution precursor in the precise synthesis of structurally defined polysaccharides.

Gas-Phase Structures of Fucosylated Oligosaccharides: Alkali Metal and Halogen Influences.

Fucosylated carbohydrate antigens play critical roles in physiology and pathology with function linked to their structural details. However, the separation and structural characterization of isomeric fucosylated epitopes remain challenging analytically. Here, we report for the first time the influence of alkali metal cations (Li+, Na+, K+, Rb+, and Cs+) and halogen anions (Cl-, Br-, and I-) on the gas-phase conformational landscapes of common fucosylated trisaccharides (Lewis A, X, and H types 1 and 2) and tetrasaccharides (Lewis B and Y) using trapped ion mobility spectrometry coupled to mass spectrometry and theoretical calculations. Inspection of the mobility profiles of individual standards showed a dependence on the number of mobility bands with the oligosaccharide and the alkali metal and halogen; collision cross sections are reported for all of the observed species. Results showed that trisaccharides (Lewis A, X, and H types 1 and 2) can be best mobility resolved in the positive mode using the [M + Li]+ molecular ion form (baseline resolution r ≈ 2.88 between Lewis X and A); tetrasaccharides can be best mobility resolved in the negative mode using the [M + I]- molecular ion form (baseline separation r ≈ 1.35 between Lewis B and Y). The correlation between the number of oligosaccharide conformers as a function of the molecular ion adduct was studied using density functional theory. Theoretical calculations revealed that smaller cations can form more stable structures based on the number of coordinations, while larger cations induced greater oligosaccharide reorganizations; candidate structures are proposed to better understand the gas-phase oligosaccharide rearrangement trends. Inspection of the candidate structures suggests that the interplay between ion size/charge density and molecular structure dictated the conformational preferences and, consequently, the number of mobility bands and the mobility separation across isomers. This work provides a fundamental understanding of the gas-phase structural dynamics of fucosylated oligosaccharides and their interaction with alkali metals and halogens.

Is it possible to obtain substitutes for human milk oligosaccharides from bovine milk, goat milk, or other mammal milks?

Considering the current level of chemical and biological synthesis technology, it was a sensible selection to obtain milk oligosaccharides (MOs) from other mammals as the potential substitute for human MOs (HMOs) that possessed various structural features in the infant formula. Through a comprehensive analysis of the content, structure, and function of MOs in six distinct varieties of mammal milk, it has been shown that goat milk was the most suitable material for the preparation as a human milk substitute. Goat MOs (GMOs) had a relatively high content and diverse structural features compared to those found in other mammalian milks. The concentration of GMOs in colostrum ranged from 60 to 350 mg/L, whereas in mature milk, it ranged from 200 to 24,00 mg/L. The acidic oligosaccharides in goat milk have attracted considerable attention due to their closeness in acidic content and structural diversity with HMOs. Simultaneously, it was discovered that some structures, like N-glycolylneuraminic acid, were found to have a certain content in GMOs and served essential functional properties. Moreover, studies focused on the extraction of MOs from goat milk indicated that the production of GMOs on an industrial scale was viable. Furthermore, it is imperative to do further study on GMOs to enhance the preparation process, discover of new MOs structures and bioactivity evaluation, which will contribute to the development of both the commercial production of MOs and the goat milk industry.

Intracellular Construction of Organelle-like Compartments Facilitates Metabolic Flux in Escherichia coli.

The formation of well-designed synthetic compartments or membraneless organelles for applications in synthetic biology and cellular engineering has aroused enormous interest. However, establishing stable and robust intracellular compartments in bacteria remains a challenge. Here, we use the structured DIX domains derived from Wnt signaling pathway components, more specifically, Dvl2 and Axin1, as building blocks to generate intracellular synthetic compartments in Escherichia coli. Moreover, the aggregation behaviors and physical properties of the DIX-based compartments can be tailored by genetically embedding a specific dimeric domain into the DIX domains. Then, a pair of interacting motifs, consisting of the aforementioned dimeric domain and its corresponding binding ligand, was incorporated to modify the client recruitment pattern of the synthetic compartments. As a proof of concept, the human milk oligosaccharide lacto-N-tetraose (LNT) biosynthesis pathway was selected as a model metabolic pathway. The fermentation results demonstrated that the co-compartmentalization of sequential pathway enzymes into intracellular compartments created by DIX domain, or by the DIX domain in conjunction with interacting motifs, prominently enhanced the metabolic flux and increased LNT production. These synthetic protein compartments may provide a feasible and effective tool to develop versatile organelle-like compartments in bacteria for applications in cellular engineering and synthetic biology.

Application of an α-galactosidase from Bacteroides fragilis on structural analysis of raffinose family oligosaccharides.

Raffinose family oligosaccharides (RFOs) have diverse structures and exhibit various biological activities. When using RFOs as prebiotics, their structures need to be identified. If we first knew whether an RFO was classical or non-classical, structural identification would become much easier. Here, we cloned and expressed an α-galactosidase (BF0224) from Bacteroides fragilis which showed strict specificity for hydrolyzing α-Gal-(1 â†’ 6)-Gal linkages in RFOs. BF0224 efficiently distinguished classical from non-classical RFOs by identifying the resulting hydrolyzed oligo- and mono-saccharides with HPAEC-PAD-MS. Using this strategy, we identified a non-classical RFO from Pseudostellaria heterophylla (Miquel) Pax with DP6 (termed PHO-6), as well as a classical RFO from Lycopus lucidus Turcz. with DP7 (termed LTO-7). To characterize these RFO structures, we employed four other commercial or reported α-galactosidases in combination with NMR and methylation analysis. Using this approach, we elucidated the accurate chemical structure of PHO-6 and LTO-7. Our study provides an efficient analytical approach to structurally analyze RFOs. This enzyme-based strategy also can be applied to structural analysis of other glycans.

Oligosaccharides from Stellaria dichotoma L. var. lanceolate bind to galectin-3 and ameliorate effects of colitis.

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.

Rational modification of Neisseria meningitidis ß1,3-N-acetylglucosaminyltransferase for lacto-N-neotetraose synthesis with reduced long-chain derivatives.

Lacto-N-neotetraose (LNnT), as a neutral core structure within human milk oligosaccharides (HMOs), has garnered widespread attention due to its exceptional physiological functions. In the process of LNnT synthesis using cellular factory approaches, substrate promiscuity of glycosyltransferases leads to the production of longer oligosaccharide derivatives. Here, rational modification of ß1,3-N-acetylglucosaminyltransferase from Neisseria meningitidis (LgtA) effectively decreased the concentration of long-chain LNnT derivatives. Specifically, the optimal ß1,4-galactosyltransferase (ß1,4-GalT) was selected from seven known candidates, enabling the efficient synthesis of LNnT in Escherichia coli BL21(DE3). Furthermore, the influence of lactose concentration on the distribution patterns of LNnT and its longer derivatives was investigated. The modification of LgtA was conducted with computational assistance, involving alanine scanning based on molecular docking to identify the substrate binding pocket and implementing large steric hindrance on crucial amino acids to obstruct LNnT entry. The implementation of saturation mutagenesis at positions 223 and 228 of LgtA yielded advantageous mutant variants that did not affect LNnT synthesis while significantly reducing the production of longer oligosaccharide derivatives. The most effective mutant, N223I, reduced the molar ratio of long derivatives by nearly 70 %, showcasing promising prospects for LNnT production with diminished byproducts.

Combined pretreatment of malic acid and kraft pulping for the production of fermentable sugars and highly active lignin.

The separation and utilization of cellulose, hemicellulose, and lignin in lignocellulosic biorefineries present significant challenges. This study proposes a pretreatment method for biomass refining by combining acid and kraft pulping. Firstly, the biomass was pretreated by malic acid, resulting in the isolation of xylo-oligosaccharides (XOS) with a yield of 86.26 % with optimized conditions of 180 °C, 1 wt% concentration, 40 min. Secondly, a mixture of 12.98 wt% NaOH and 1.043 wt% Na2S is employed to achieve lignin removal efficiency up to 63.42 %. Physical refinement techniques are then applied to enhance the enzyme digestion efficiency of cellulose, resulting in an increase from 55.03 % to 91.4 % for efficient cellulose conversion. The reacted samples exhibit a lignin composition rich in ß-O-4 ether bonds, facilitating their high-value utilization. The results indicated that the combined pretreatment approach demonstrates high efficiency in separating cellulose, hemicellulose, and lignin while obtaining XOS, highly active lignin, and enzyme-digested substrates.

Galacto-oligosaccharides modified whey protein isolate ameliorates cyclophosphamide-induced immunosuppression.

The effect of whey protein isolate (WPI)- galacto-oligosaccharides (GOS)/fructo-oligosaccharides (FOS) conjugates on RAW264.7 cells, and further the effect of WPI-GOS conjugates on CTX-induced immunosuppressed mice were investigated. Compared to WPI-FOS conjugates, WPI-GOS conjugates exhibited deeper glycation extent, more pronounced structural unfolding and helix-destabilizing, and obviously improved functional indicators of RAW264.7 macrophages. In addition, WPI-GOS conjugates also repaired immune organ and intestinal barrier and increased IL-1ß and IFN-γ levels in immunosuppressed mice. The alteration of gut microbiota induced by WPI-GOS conjugates changed the serum metabolites, causing the activation of NFκB pathway, which strengthens the immune system. The activation of NFκB pathway maybe associated with the mTOR signal pathway and ABC transporters. However, the precise mechanisms by which NFκB pathway interacts with mTOR signal pathway and ABC transporters to modulate the immune response need for further research.

GA receptor targeted chitosan oligosaccharide polymer nanoparticles improve non-alcoholic fatty liver disease by inhibiting ferroptosis.

Excessive iron in the liver may exacerbate Non-alcoholic fatty liver disease (NAFLD) by increasing the risk of liver cell expansion, inflammation and fibrosis. Ferroptosis in liver cells may lead the progression of simple fatty liver degeneration to steatohepatitis (NASH). More and more studies shew that ferroptosis played a crucial role in the pathological process of NAFLD. Based on the mechanism of ferroptosis, this study first synthesized a liver targeted 18-ß-Glycyrrhetinic-acid-chitosan oligosaccharide -N-acetylcysteine polymer (GCNp), and further curcumin (Cur) was used as model drug to prepare Cur loaded nanodelivery system (GCNp-Cur NPs). The particle size of GCNp-Cur NPs was 132.5 ± 9.8 nm, PDI was 0.148 ± 0.026 and the potential was 23.8 mV. GCNp-Cur NPs can regulate the GPX4/GSH pathway, inhibit lipid peroxidation, restore cellular oxidative environment, reduce free Fe2+, improve cellular lipid metabolism and iron metabolism, thereby NPs inhibited liver cell ferroptosis through multiple pathways. Additionally, GCNp-Cur NPs could also alleviate liver tissue lipid accumulation and oxidative damage, delaying disease progression, and providing a new method and theoretical basis for the treatment of NAFLD.

Orally self-disintegrating milk protein puffs enriched with food by-products for the elderly.

Supercritical fluid extrusion (SCFX) processing was used to develop milk protein-based orally self-disintegrating puffs enriched with fruit and dairy by-products, designed specifically to cater to the needs of elderly population having swallowing issues and lactose intolerance. Lactose hydrolyzed skim milk powder (LHSMP) was also added in the formulation to mitigate lactose intolerance while LHSMP was also exploited as a precursor for the polymerization of galactose and lactose to generate galacto-oligosaccharides (GOS) in the puffs. This study for the first time took advantage of the unique features of SCFX processing for in-process GOS formation and enrichment of puffs, achieving GOS contents up to 0.48 g/30 g serving of puffs, thereby making them nutritionally superior and functionally attractive snacks. The estimated nutritional profile revealed that SCFX puffs contained higher levels of protein (16.3 g/30 g), fiber (1.6 g/30 g), phenolics and other valuable nutrients compared to the starch-rich, disintegrating Market Baby puffs.

Using a cellulose-complementary oligosaccharide as a tool to probe exposed cellulosic surfaces in cotton fibres and growing plant cell walls.

Cellulosic microfibrils in plant cell walls are largely ensheathed and probably tethered by hydrogen-bonded hemicelluloses. Ensheathing may vary developmentally as hemicelluloses are peeled to enable cell expansion. We characterised a simple method to quantify ensheathed versus naked cellulosic surfaces based on the ability to adsorb a radiolabelled 'cellulose-complementary oligosaccharide', [3H]cellopentaitol. Filter-paper (cellulose) adsorbed 40% and >80% of aqueous 5 nM [3H]cellopentaitol within ∼1 and ∼20 h respectively. When [3H]cellopentaitol was rapidly dried onto filter-paper, ∼50% of it was desorbable by water, whereas after ∼1 day annealing in aqueous medium the adsorption became too strong to be reversible in water. 'Strongly' adsorbed [3H]cellopentaitol was, however, ∼98% desorbed by 6 M NaOH, ∼50% by 0.2 M cellobiose, and ∼30% by 8 M urea, indicating a role for hydrogen-bonding reinforced by complementarity of shape. Gradual adsorption was promoted by kosmotropes (1.4 M Na2SO4 or 30% methanol), and inhibited by chaotropes (8 M urea), supporting a role for hydrogen-bonding. [3H]Cellopentaitol adsorption was strongly competed by non-radioactive cello-oligosaccharides (Cell2-6), the IC50 (half-inhibitory concentration) being highly size-dependent: Cell2, ∼70 mM; Cell3, ∼7 mM; and Cell4-6, ∼0.05 mM. Malto-oligosaccharides (400 mM) had no effect, confirming the role of complementarity. The quantity of adsorbed [3H]cellopentaitol was proportional to mass of cellulose. Of seven cottons tested, wild-type Gossypium arboreum fibres were least capable of adsorbing [3H]cellopentaitol, indicating ensheathment of their microfibrillar surfaces, confirmed by their resistance to cellulase digestion, and potentially attributable to a high glucuronoarabinoxylan content. In conclusion, [3H]cellopentaitol adsorption is a simple, sensitive and quantitative way of titrating 'naked' cellulose surfaces.

Methotrexate-Loaded Chitosan Oligosaccharide-ES2 for Targeted Cancer Therapy.

Cancer presents a significant health threat, necessitating the development of more precise, efficient, and less damaging treatment approaches. To address this challenge, we employed the 1-ethyl-(3-dimethyl aminopropyl) carbodiimide/N-hydroxy succinimide (EDC/NHS) catalytic system and utilized quaternized chitosan oligosaccharide (HTCOSC) as a drug carrier to construct a nanoparticle delivery system termed HTCOSC-cRGD-ES2-MTX (CREM). This system specifically targets integrin αvß3 on tumor cell surfaces and enables simultaneous loading of the antiangiogenic agent ES2 (IVRRADRAAVP) and the chemotherapy drug methotrexate (MTX). Due to its amphiphilic properties, CREM self-assembles into nanoparticles in aqueous solution, exhibiting an average diameter of 179.47 nm. Comparative studies demonstrated that CREM, in contrast to free ES2 and MTX-free nanoparticles (CRE), significantly suppressed the proliferation of EAhy926 endothelial cells and B16 melanoma cells in vitro, resulting in inhibition rates of 71.18 and 82.25%, respectively. Furthermore, CREM exhibited a hemolysis rate below 2%, indicating excellent in vitro antiangiogenic and antitumor activity as well as favorable blood compatibility. Additionally, both CRE and CREM demonstrated favorable tumor targeting capabilities through the specific binding action of cyclic RGD (cRGD) to integrin αvß3. Further in vivo investigations revealed that CREM induced apoptosis in tumor cells via the mitochondrial apoptotic pathway and reduced the expression of angiogenic factors such as vascular endothelial growth factor (VEGF), thereby inhibiting tumor angiogenesis. This potent antitumor effect was evident through a tumor suppression rate of 80.19%. Importantly, histopathological staining (HE staining) demonstrated the absence of significant toxic side effects of CREM on various organs compared to MTX. In conclusion, the CREM nano drug delivery system synergistically enhances the therapeutic efficacy of antiangiogenic drugs and chemotherapeutic agents, thus offering a novel targeted approach for cancer treatment.

Non-carrier immobilization of yeast cells by genipin crosslinking for the synthesis of prebiotic galactooligosaccharides from plant-derived galactose.

Galactooligosaccharides (GOS), as mimics of human milk oligosaccharides, are important prebiotics for modulating the ecological balance of intestinal microbiota. A novel carrier-free cell immobilization method was established using genipin to cross-link Kluyveromyces lactis CGMCC 2.1494, which produced ß-galactosidase, an enzyme essential for GOS synthesis. The resulting immobilized cells were characterized as stable by thermogravimetric analysis and confirmed to be crosslinked through scanning electron microscopy analysis (SEM) and Fourier transform infrared spectroscopy (FTIR). The Km and Vmax values of ß-galactosidase in immobilized cells towards o-nitrophenyl ß-D-galactoside were determined to be 3.446 mM and 2210 µmol min-1 g-1, respectively. The enzyme in the immobilized showed higher thermal and organic solvent tolerance compared to that in free cells. The immobilized cells were subsequently employed for GOS synthesis using plant-derived galactose as the substrate. The synthetic reaction conditions were optimized through both single-factor experiments and response surface methodology, resulting in a high yield of 49.1 %. Moreover, the immobilized cells showed good reusability and could be reused for at least 20 batches of GOS synthesis, with the enzyme activity remaining above 70 % at 35 °C.

Gut microbiota: A key role for human milk oligosaccharides in regulating host health early in life.

Human milk oligosaccharides (HMOs) are an evolutionarily significant advantage bestowed by mothers for facilitating the development of the infant's gut microbiota. They can avoid absorption in the stomach and small intestine, reaching the colon successfully, where they engage in close interactions with gut microbes. This process also enables HMOs to exert additional prebiotic effects, including regulating the mucus layer, promoting physical growth and brain development, as well as preventing and mitigating conditions such as NEC, allergies, and diarrhea. Here, we comprehensively review the primary ways by which gut microbiota, including Bifidobacteria and other genera, utilize HMOs, and we classify them into five central pathways. Furthermore, we emphasize the metabolic benefits of bacteria consuming HMOs, particularly the recently identified intrinsic link between HMOs and the metabolic conversion of tryptophan to indole and its derivatives. We also examine the extensive probiotic roles of HMOs and their recent research advancements, specifically concentrating on the unsummarized role of HMOs in regulating the mucus layer, where their interaction with the gut microbiota becomes crucial. Additionally, we delve into the principal tools used for functional mining of new HMOs. In conclusion, our study presents a thorough analysis of the interaction mechanism between HMOs and gut microbiota, emphasizing the cooperative utilization of HMOs by gut microbiota, and provides an overview of the subsequent probiotic effects of this interaction. This review provides new insights into the interaction of HMOs with the gut microbiota, which will inform the mechanisms by which HMOs function.

In situ imaging of LPMO action on plant tissues.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidatively cleave recalcitrant polysaccharides such as cellulose. Several studies have reported LPMO action in synergy with other carbohydrate-active enzymes (CAZymes) for the degradation of lignocellulosic biomass but direct LPMO action at the plant tissue level remains challenging to investigate. Here, we have developed a MALDI-MS imaging workflow to detect oxidised oligosaccharides released by a cellulose-active LPMO at cellular level on maize tissues. Using this workflow, we imaged LPMO action and gained insight into the spatial variation and relative abundance of oxidised and non-oxidised oligosaccharides. We reveal a targeted action of the LPMO related to the composition and organisation of plant cell walls.

Discovery and characterization of a novel poly-mannuronate preferred alginate lyase: The first member of a new polysaccharide lyase family.

Alginate is one of the most important marine colloidal polysaccharides, and its oligosaccharides have been proven to possess diverse biological functions. Alginate lyases could specifically degrade alginate and therefore serve as desirable tools for the research and development of alginate. In this report, a novel catalytic domain, which demonstrated no significant sequence similarity with all previously defined functional domains, was verified to exhibit a random endo-acting lyase activity to alginate. The action pattern analysis revealed that the heterologously expressed protein, named Aly44A, preferred to degrade polyM. Its minimum substrates and the minimum products were identified as unsaturated alginate trisaccharides and disaccharides, respectively. Based on the sequence novelty of Aly44A and its homologs, a new polysaccharide lyase family (PL44) was proposed. The discovery of the novel enzyme and polysaccharide lyase family provided a new entrance for the gene-mining and acquiring of alginate lyases, and would facilitate to the utilization of alginate and its oligosaccharides.