High-performance Anion-exchange Chromatography Research Articles

Surface-Displayed Mannanolytic and Chitinolytic Enzymes Using Peptidoglycan Binding LysM Domains.

Using Lactiplantibacillus plantarum as a food-grade carrier to create non-GMO whole-cell biocatalysts is gaining popularity. This work evaluates the immobilization yield of a chitosanase (CsnA, 30 kDa) from Bacillus subtilis and a mannanase (ManB, 40 kDa) from B. licheniformis on the surface of L. plantarum WCFS1 using either a single LysM domain derived from the extracellular transglycosylase Lp_3014 or a double LysM domain derived from the muropeptidase Lp_2162. ManB and CsnA were fused with the LysM domains of Lp_3014 or Lp_2162, produced in Escherichia coli and anchored to the cell surface of L. plantarum. The localization of the recombinant proteins on the bacterial cell surface was successfully confirmed by Western blot and flow cytometry analysis. The highest immobilization yields (44-48%) and activities of mannanase and chitosanase on the displaying cell surface (812 and 508 U/g of dry cell weight, respectively) were obtained when using the double LysM domain of Lp_2162 as an anchor. The presence of manno-oligosaccharides or chito-oligosaccharides in the reaction mixtures containing appropriate substrates and ManB or CsnA-displaying cells was determined by high-performance anion exchange chromatography. This study indicated that non-GMO Lactiplantibacillus chitosanase- and mannanase-displaying cells could be used to produce potentially prebiotic oligosaccharides.

Extraction of Pithecellobium clypearia Benth polysaccharides by dual-frequency ultrasound-assisted extraction: Structural characterization, antioxidant, hypoglycemic and anti-hyperlipidemic activities

In this research, the extraction process of polysaccharides from Pithecellobium clypearia Benth (PCBPs) was optimized using dual-frequency ultrasound-assisted extraction (DUAE). The biological activities of PCBPs were investigated by in vitro antioxidant, hypoglycemic, and anti-hyperlipidemic assay. High-performance anion-exchange chromatography, high-performance gel permeation chromatography, SEM, UV–Vis spectroscopy, and FT-IR spectra were used to analyze the monosaccharide composition, molecular weight, microscopic morphology, and characteristic structure of PCBPs. The results showed that the maximum extraction rate of PCBPs was 9.90 ± 0.16% when the ultrasonic time was 8 min, the liquid-to-material ratio was 32 mL/g, and the ultrasonic power was 510 W. The PCBPs also possessed excellent in vitro antioxidant, hypoglycemic, and anti-hyperlipidemic activities. In addition, the average molecular weight of PCBPs was 15.07 kDa. PCBPs consisted of rhamnose, arabinose, galactose, glucose, xylose, mannose, and glucuronic acid, with the molar ratios of 11.07%, 18.54%, 48.17%, 10.44%, 4.62%, 4.96%, and 2.20%, respectively. Moreover, the results of SEM showed that PCBPs mainly showed a fine spherical mesh structure. The above studies provided a valuable theoretical basis for the subsequent in-depth study of PCBPs.

Relationship between dietary fiber content and prebiotic potential of polysaccharides from the seaweeds of the North west coast of India

The macroalgae are a sustainable bioresource that can be harnessed for their functional food and nutraceutical applications. This study characterized the biochemical composition and bioactive potential of natural biological macromolecules, such as macroalgal polysaccharides extracted using a green, aqueous extraction process. The in-vitro antioxidant and antiglycemic activity of these polysaccharides were evaluated using model, free radical and antiglycemic compounds. The prebiotic potential of macroalgal polysaccharides were analysed based on their ability to promote the growth of two potential probiotic bacteria Lactobacillus acidophilus and L. bulgaricus and suppress the growth of enteric bacteria, Escherichia coli. Among the polysaccharides studied, the brown algal polysaccharide MPS8 MPS9 and MPS10 exhibited good antioxidant, antiglycemic and prebiotic activity. Based on infrared spectroscopy, the functional groups sulfation and carboxylation were identified in potential polysaccharides. The monosaccharide composition in the bioactive polysaccharides was determined using High Performance Anion Exchange Chromatography Pulse Amperometric detector (HPAEC-PAD). These bioactive polysaccharides were fractionated using ion exchange chromatography to purify it and further characterized using gel permeation chromatography and NMR spectroscopy. The results these polysaccharides are mainly composed of fucose and glucose which is due to the fucoidan and laminarin, respectively. Such macromolecules with high dietary fiber content and bioactivity are in global demand as functional food, nutraceutical and pharmaceutical formulations.

Analysis of unsaturated alginate oligosaccharides using high-performance anion exchange chromatography coupled with mass spectrometry.

Alginate is a commercially important polysaccharide composed of mannuronic acid and its C5 differential isomer guluronic acid. Comprehensive research on alginate and alginate lyases requires efficient and precise analytical methods for alginate oligosaccharides. In this research, high-performance anion exchange chromatography (HPAEC) in parallel with pulsed amperometric detection (PAD) and mass spectrometry (MS) was applied to the analysis of oligosaccharides obtained by alginate lyase. By optimizing the chromatographic conditions including mobile phase concentration, flow rate, and elution gradient, the analysis of a single sample could be completed in 30 min. Seven unsaturated alginate oligosaccharides were separated and identified through their analysis time observed with PAD, including all structurally different unsaturated disaccharides and trisaccharides. The quantitative analysis of seven oligosaccharides was performed based on the quantitative capability of PAD. The method exhibited adequate linearity and precision parameters. All the calibration curves showed good linearity at least in the concentration range of 0.002 to 0.1 mg/mL. The HPAEC-PAD/MS method provides a general and efficient online method to analyze alginate oligosaccharides.

Use of human Caco-2 cells and HPAE-PAD for α-glucosidase assay

To measure α-glucosidase activity, rat intestinal acetone powder is commonly used as a source of α-glucosidase, and the mutarotase-glucose oxidase (GOD) methods commonly used to quantitate glucose produced by enzymatic hydrolysis of the substrates. In this study, we compared human Caco-2 cell extracts with rat intestinal acetone powder extracts. We also compared high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) with the mutarotase-GOD method. The sensitivity of HPAE-PAD was higher than that of mutarotase-GOD. The glucose concentration quantified by HPAE-PAD was similar to that quantified using the mutarotase-GOD method. In the maltase reaction, 1-deoxynojirimycin (1-DNJ) exerted a more potent inhibitory effect on human enzymes than on rat enzymes. This order was reversed during the sucrase reaction. These results suggested that the combined use of Caco-2 cell extracts and HPAE-PAD is advantageous for use in α-glucosidase-related basic research.

A novel approach to the dual sugar test for the assessment of intestinal epithelium permeability in response to exertional heat stress and nutritional intervention

An acute increase in intestinal epithelium permeability is induced by prolonged exertion in the heat, resulting in the translocation of pathogenic bacteria and endotoxins from the lumen into the circulation, causing a systemic inflammatory response and debilitating symptoms(1). Acute exercise-induced gastrointestinal syndrome mimics chronic health conditions with which an impaired intestinal barrier function is associated, including coeliac disease, inflammatory bowel disease, diabetes, Alzheimer’s and liver diseases(2). Intestinal epithelium permeability is typically assessed using a dual sugar absorption test, by administering a drink containing non-metabolisable sugars (e.g. lactulose [L] and L-rhamnose [R]) that can enter the circulation by paracellular translocation when the epithelium is compromised, and are subsequently excreted and measured cumulatively in the urine(3). We aimed to demonstrate that our recently developed ion chromatography protocol(4) can be used to accurately quantify L/R ratio in the plasma of participants exercising in hot ambient conditions and to determine the impact of nutritional intervention on intestinal epithelium permeability. Further, we hypothesised that measuring L/R in plasma collected at intervals during the post-exercise recovery period would reveal more information about intestinal permeability compared to previously published cumulative urine L/R data(3). Endurance-trained participants completed a set of randomised crossover studies, consisting of 2 h running at 60% V˙O2max in temperate, warm and hot ambient conditions (n = 8) and/or in the heat while consuming water, carbohydrate or protein (n = 9). The dual sugar solution was ingested at 90 min of exercise and blood was sampled at 0, 1, 2 and 4 h post-exercise. Plasma sugars were quantified by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and L/R ratios were compared by two-way repeated measures ANOVA with Tukey’s multiple comparisons. Plasma L/R increased immediately post-exercise in the heat (0.15 ± 0.11) compared with temperate (0.06 ± 0.04, p<0.001) and warm (0.09 ± 0.08, p<0.01) conditions, while consuming glucose before and during exercise alleviated this (0.02 ± 0.02, p<0.001), and this novel information was otherwise missed when measuring urine L/R. Consuming glucose or whey protein hydrolysate during exercise attenuated intestinal permeability from exertional heat stress throughout recovery, with the mean plasma L/R over 4 h reduced from 0.11 ± 0.05 to 0.04 ± 0.03 (p<0.001) and 0.06 ± 0.04 (p<0.01) with glucose and protein, respectively. We recommend using the dual sugar test with quantification of plasma sugars at intervals by HPAEC-PAD to maximise intestinal permeability data collection in exercise gastroenterology research and beyond, as this gives additional acute response information compared to urinary measurements. Our approach can be employed to investigate and develop personalised nutrition strategies that prevent intestinal hyperpermeability during exertional heat stress. This has implications for athlete performance and safety, and can also build upon occupational health and safety practices and inform chronic disease management.

Investigating the effect of polyphenols from nuts on human carbohydrate digestion in vitro

Recent studies have documented the importance of postprandial hyperglycaemia in the incidence of chronic diseases, including type 2 diabetes. Inhibition of digestive enzymes, including membrane-bound brush-border α-glucosidases, leads to slowed carbohydrate digestion and absorption, and reduced postprandial glycemia. Nuts are widely eaten around the world and have the potential to inhibit α-glucosidases through their content of polyphenols and other bioactive compounds. According to our recent systematic review(1), no study has investigated the inhibitory effects of nut extracts on human α-glucosidase activities. Almost all studies in this area have been conducted on yeast α-glucosidase, with only a few using rat α-glucosidase. While there is no sequence homology between yeast and human α-glucosidase, there is 74% to 78% sequence homology between rat and human α-glucosidases(1). The lack of studies on the effect of bioactive compounds from nuts on human α-glucosidases, along with the growing attention to nuts as an important component of a healthy diet with the potential to reduce the risk of chronic diseases(2), highlights the need for research to evaluate the inhibitory effect of nut extracts on human α-glucosidases. The aim of the current study is to explore the inhibitory effect of extracts from nuts on human carbohydrate digestive enzymes. Walnuts and almonds were ground and defatted with hexane, extracted in 80% (v/v) acetone, and further purified using solid-phase extraction to obtain phenolic-rich extracts. The Folin–Ciocalteu assay was used to approximate the polyphenol content of the samples. Following our recently published detailed protocol(3), cell-free extracts from human intestinal Caco-2/TC7 cells were used as a source of α-glucosidase in enzyme inhibition assays, with sucrose, maltose and isomaltose as substrates and appropriate controls. The assay products were quantified using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Glucose production in the presence of various concentrations of phenol-rich nut extracts was compared using a one-way ANOVA and half-maximal inhibitory concentration (IC50) values were calculated. The Folin–Ciocalteu data demonstrate that walnut extracts comprise a relatively high polyphenol content, with 18.1 ± 0.23 mg (epigallocatechin gallate [EGCG] equivalent) per gram of fresh weight, while almond extracts contain 0.87 ± 0.03 mg EGCG equivalent/g of fresh weight. The walnut phenolic-rich extract dose-dependently inhibited human intestinal sucrase and maltase activities (both p<0.01), with IC50 values of 1.67 mg/mL and 2.84 mg/mL, respectively. We demonstrate that phenolic-rich walnut extracts can inhibit human α-glucosidases in vitro and therefore walnuts may contribute to slowing carbohydrate digestion in humans. As such, we plan to assess the effects of walnuts on postprandial glycaemia in vivo.

Chemical characterization of polysaccharides from Gracilaria gracilis from Bizerte (Tunisia)

Polysaccharides were extracted from Gracilaria gracilis collected from Manzel Jemil Lake in Bizerte Tunisia, with two different solvents (water and NaOH 0.3 M). Different assays were performed on samples (total sugars, neutral sugars, uronic acids, anhydrogalactose, proteins, sulphates, pyruvates), followed by high performance anion-exchange chromatography (HPAEC) to observe the monosaccharide composition, high pressure size exclusion chromatography with multi-angle laser light scattering (HPSEC-MALS) to obtain the molecular mass, Fourier transform infrared spectroscopy (FTIR), and 1D and 2D nuclear magnetic resonance (NMR) to access to structural data. Results have shown that the polysaccharide extracted from Gracilaria gracilis collected from Manzel Jemil Lake in Bizerte Tunisia, is of agar type but with high molecular mass and some original structural features. Hence, the sample was found to contain 9 % of pyruvate groups and is partly sulphated at the C4 of β-d-galactose and methylated on C2 of anhydro-α-l-galactose. The polymer from G. gracilis from Bizerte thus presents a never described structure that could be interesting for further rheological or biological activities applications.

An efficient and environmentally-friendly extraction, characterization and activity prediction of polysaccharides from Rhizoma et Radix Notopterygii

Traditional hot water reflux extraction, ultrasonic-water extraction (UW), ultrasonic-natural deep eutectic solvent (NADES) extraction (U-NADES), ultrasonic-water and enzyme extraction (U-W-E) and ultrasonic-NADES and enzyme extraction (U-NADES-E) are employed for the extraction of Rhizoma et Radix Notopterygii polysaccharides (RNP), in which, the U-NADES-E has being proved as the most effective method. Response Surface Methodology (RSM) was utilized to optimize the conditions for U-NADES-E method. Using the optimal extraction conditions, the yield of RNP can be enhanced by nearly two-fold in comparison to the traditional extraction method, achieving a yield of 7.38 %, with a mere 30-min treatment and low ultrasonic power at 240 W. The RNP's composition included Rhamnose, Arabinose, Galactose, Glucose and Galacturonic Acid by high-performance anion-exchange chromatography. The polysaccharides from two different species of Rhizoma et Radix Notopterygii have also been characterized and identified. Network pharmacology and molecular docking predict that RNP may exert its effects in vivo through binding to PPARA, ACE and REN proteins, thereby potentially impacting diabetes outcomes. This study proposes a new, efficient, energy-saving and environmentally-friendly method for the extraction of RNP.

Revealing the structural changes of lignin in Chinese quince (Chaenomeles sinensis) fruit as it matures

Chinese quince fruits (Chaenomeles sinensis) contain substantial amounts of lignin; however, the exact structure of lignin remains to be investigated. In this study, milled wood lignins (Milled wood lignin (MWL)-1, MWL-2, MWL-3, MWL-4, MWL-5, and MWL-6) were extracted from fruits harvested once a month from May to October 2019 to investigate their structural evolution during fruit growth. The samples were characterized via High-performance anion exchange chromatography (HPAEC), Fourier transform–infrared spectroscopy (FT-IR), gel permeation chromatography (GPC), thermogravimetric (TGA), pyrolysis–gas chromatography/mass spectrometry (Py–GC/MS) and NMR (2D-heteronuclear single quantum coherence (HSQC) and 31P). The MWL samples in all fruit growth stages were GS-type lignin and lignin core undergoing minimal alterations during fruit development. The predominant linkage in the lignin structure was β-O-4′, followed by β-β' and β-5′. Galactose and glucose were the main monosaccharides associated with MWL. In MWL-6, the lignin exhibited the highest homogeneity and thermal stability. As the fruit matured, a gradual increase in the β-O-4′ proportion and the ratio of S/G was observed. The results provide comprehensive characterization of the cell wall lignin of quince fruit as it matures. This study could inspire innovative applications of quince fruit lignin and provide the optimal harvest time for lignin utilization.

Structural Characterization and In Vitro Antioxidant Activity of a Novel Polysaccharide from Summer-Autumn Tea.

To enhance the utilization of summer-autumn tea, a water-soluble polysaccharide (D1N1) was isolated through a series of techniques including hot water extraction, ethanol precipitation, and column chromatography. The structure of D1N1 was determined through the utilization of ultraviolet, Fourier-transform infrared, high-performance anion-exchange chromatography, gas chromatography-tandem mass spectrometry, and nuclear magnetic resonance. The results revealed that glucose was the predominant component of D1N1, accounting for 95% of its composition. Additionally, D1N1 also contained galactose, arabinose, and rhamnose. The molecular weight (Mw) of D1N1 was determined to be 224.71 kDa. The backbone of D1N1 consisted of →4)-α-D -Glcp (1→, →3,4)-α-D-Galp-(1→, →4,6)-α-D -Glcp (1→ at a molar ratio of 35:1:1, and branching at the O-3 position of →3,4)-α-D-Galp-(1→ and O-6 position of →4,6)-α-D-Glcp (1→ with α-D -Glcp (1→. In addition, the antioxidant activity of D1N1 was also evaluated. D1N1 exhibited excellent antioxidant bioactivity against the DPPH, superoxide anion radical, and ABTS+ radical. These findings provide a theoretical basis for the application of summer-autumn tea polysaccharide as a potential functional food.

Novel antioxidant peptides from sheep plasma protein hydrolysates: Purification, identification and cytoprotective effects against H2O2-induced oxidative stress.

This study sought to purify and identify antioxidant peptides from sheep (Ovis aries) plasma protein hydrolysates and assess their protective impacts on H2O2-induced Caco-2 cells. The purification process involved reversed high-performance liquid chromatography, anion-exchange chromatography, and Sephadex G-25. Three peptides, namely Trp-Glu-Glu-Pro-Ala-Met (WEEPAM), Ser-Leu-His-Phe-Met-Glu (SLHFME), and His-Cys-Thr-Thr-Phe-Met-Ile, with molecular weights of 761.84, 762.87, and 852.03Da, respectively, were identified by liquid chromatography with tandem mass spectrometry. Among the three antioxidant peptides, superoxide radical (O2 -) radical scavenging capacity of WEEPAM and SLHFME was not significantly different from glutathione (GSH) (p>0.05), while their 1,1-diphenyl-2-picrylhydrazyl radical scavenging capacity was greater than GSH (p<0.05). WEEPAM revealed increased antioxidant activity after pepsin and trypsin hydrolysis under an in vitro digestion model. In addition, WEEPAM inhibited oxidative damage in Caco-2 cells by significantly reducing reactive oxygen species accumulation, early apoptosis, malondialdehyde formation, and increasing intracellular superoxide dismutase, glutathione peroxidase, and catalase activities.

Quantitative analysis of fucose in fucoidans from Sargassum spp. in Jeju Island, South Korea using 3-methyl-1-phenyl-5-pyrazolone derivatization and RP-HPLC-UV method

Sargassum species are representative brown algae found worldwide, with 27 species native to the coast of Jeju Island in South Korea. Fucoidans are derived from brown algae and exhibit a variety of biological activities. Analysis of the monosaccharide composition is necessary for an in-depth study of fucoidans. However, quantitative analysis of fucoidans with complex structures according to species and habitat is insufficient. Furthermore, complex pre-processing is often required to analyze sugars, making it less economical. This study aimed to validate high-performance liquid chromatography (HPLC)-UV analysis based on 3-methyl-1-phenyl-5-pyrazolone (PMP) derivatization of monosaccharides. The proposed method had a short running time and could be repeated economically using UV detection. The detected PMP-fucose was confirmed to be uninterfered by comparing the analytical chromatograms of the internal standard solution. This analytical method was characterized for system suitability, precision, accuracy, linearity, limit of detection (LOD), and limit of quantification (LOQ), demonstrating satisfactory results. The quantification was verified by comparison with the quantitative results of commercial fucoidan obtained using high-performance anion-exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD). Furthermore, the antioxidant effects of algal polysaccharides from Sargassum spp. isolated on Jeju Island was evaluated using a zebrafish model. Polysaccharides from Sargassum coreanum (SCpol) and Sargassum autumnale (SApol) showed remarkable free radical scavenging activities. In addition to the results obtained in the zebrafish experiments, all species-derived polysaccharides showed antioxidant potential. In conclusion, this study demonstrates a useful analytical method using HPLC-UV and PMP derivatives for the effective determination of monosaccharide contents, including fucose, in Sargassum spp.

Quantification of Enzymatic Biofilm Removal Using the Sauerbrey Equation: Application to the Case of Pseudomonas protegens.

A methodology for the quantitative analysis of enzymatic removal of biofilms (BF) was developed, based on a quartz crystal microbalance (QCM) under stationary conditions. This was applied to the case of Pseudomonas protegens (PP) BFs, through a series of five enzymes, whose removal activity was screened using the presented methodology. The procedure is based on the following: when BFs can be modeled as rigid materials, QCM can be used as a balance under stationary conditions for determining the BFs mass reduction by enzymatic removal. For considering a BF as a rigid model, energy dissipation effects, associated with viscoelastic properties of the BF, must be negligible. Hence, a QCM system with detection of dissipation (referred to as QCM with dissipation) was used for evaluating the energy losses, which, in fact, resulted in negligible energy losses in the case of dehydrated PP BFs, validating the application of the Sauerbrey equation for the change of mass calculations. The stationary methodology reduces operating times and simplifies data analysis in comparison to dynamic approaches based on flow setups, which requires the incorporation of dissipation effects due to the liquid media. By carrying out QCM, glycosidase-type enzymes showed BF removal higher than 80% at enzyme concentration 50 ppm, reaching removal over 90% in the cases of amylase and cellulase/xylanase enzymes. The highest removal percentage produced a reduction from about 15 to 1 μg in the BF mass. Amylase enzyme was tested from below 50 to 1 ppm, reaching around 60% of removal at 1 ppm. The obtained results were supported by other instrumental techniques such as Raman spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, atomic force microscopy, high performance anion exchange chromatography, thermogravimetric analysis, and differential scanning calorimetry. The removal quantifications obtained with QCM were compared with those obtained by well-established screening techniques (UV-vis spectrophotometry using crystal violet and agar diffusion test). The proposed methodology expands the possibility of using a quartz microbalance to perform enzymatic activity screening.

Label-Free Liquid Chromatography-Mass Spectrometry Quantitation of Relative N- and O-Glycan Concentrations in Human Milk in Japan.

Human milk is abundant in carbohydrates and includes human milk oligosaccharides (HMOs) and N/O-glycans conjugated to proteins. HMO compositions and concentrations vary in individuals according to the maternal secretor status based on the fucosyltransferase 2 genotype; however, the profile of N/O-glycans remains uninvestigated because of the analytical complexity. Herein, we applied a label-free chromatography-mass spectrometry (LC-MS) technique to elucidate the variation in the composition and concentration of N/O-glycans in human milk. We used label-free LC-MS to relatively quantify 16 N-glycans and 12 O-glycans in 200 samples of Japanese human milk (1-2 months postpartum) and applied high performance anion exchange chromatography with pulsed amperometric detection to absolutely quantify the concentrations of 11 representative HMOs. Cluster analysis of the quantitative data revealed that O-glycans and several HMOs were classified according to the presence or absence of fucose linked to galactose while N-glycans were classified into a different group from O-glycans and HMOs. O-glycans and HMOs with fucose linked to galactose were more abundant in human milk from secretor mothers than from nonsecretor mothers. Thus, secretor status influenced the composition and concentration of HMOs and O-glycans but not those of N-glycans in human milk.

Separation of All Classes of Carbohydrates by HPAEC-PAD

High performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) is a potential method of choice for the analysis of carbohydrates.

Evaluation of a novel stationary phase for the separation of lactose and isomers in lactose-free products using high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD)

Lactose intolerance is a widespread condition, which prevents a large number of people from consuming dairy products as a part of their daily diet. It is estimated that an average of 65% of the global population is suffering from lactose intolerance. The global market for 'lactose-free' dairy products is rapidly growing and the criteria for 'lactose-free' labelled products are becoming stricter. To check the lactose contents in these products there is a need for fast, sensitive, and selective analytical method. A method is presented for fast and sensitive determination of lactose and its isomers using High-Performance Anion Exchange Chromatography in combination with Pulsed Amperometric Detection (HPAEC-PAD). The use of a new anion-exchange column, SweetSep™ AEX200, which is a strong anion-exchange column with highly monodisperse 5µm particles, allowed the separation of all compounds of interest in less than 8min with high resolution. A variety of dairy products were analyzed to demonstrate the versatility of the method.

Sugar Profile Method by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection in Food, Dietary Supplements, Pet Food, and Animal Feeds: Interlaboratory Validation Study, Final Action 2018.16.

A method for sugar profile analysis granted First Action 2018.16 was subjected to a multi-laboratory study. Perform a multi-laboratory study with this method to determine the performance parameters of repeatability and reproducibility against the AOAC Standard Method Performance Requirements (AOAC SMPR 2018.001) for Final Action status. Eleven laboratories from three different countries participated in the study. Each laboratory was provided practice materials for successful method setup. Each laboratory then proceeded with analysis of blind duplicates of 10 different products covering the scope of the method. Results were reported to the study directors with any modifications and assessed following the procedures of Appendix D of the AOAC Official Methods of AnalysisSM (guidelines for collaborative study procedures). The majority of results from the study met the SMPR requirements. The data is presented along with any outlying observations or modifications. The method was proven to be flexible across different instrumentation and laboratories, and the method was updated to provide further system suitability and guidelines to maintain the performance of the method across the large scope of matrixes. The results from the collaborative study supported the method for Final Action status. The Expert Review Panel reviewed and voted to move the method forward to Final Action and was followed by review from the Official Methods Board and granted approval. The method was granted Final Action Official Methods status.

Characterization of the chemical composition of different parts of Dolichos lablab L. and revelation of its anti-ulcerative colitis effects by modulating the gut microbiota and host metabolism

Ethnopharmacological relevanceUlcerative colitis (UC) is a non-specific inflammatory disease characterized by long duration and easy relapse. Dolichos lablab L. (DLL) belongs to the family Fabaceae, was listed in a famous Chinese medical classic, Compendium of Materia Medic, and described as possessing features that invigorate the spleen, alleviate dampness, provide diarrhea relief, and other effects. The DLL-dried white mature seeds (DS) and dried flower (DF), which hold significant medicinal value in China, were used in clinical prescriptions to prevent and treat UC. DS and DF have appeared in different editions of the Pharmacopoeia of the People's Republic of China from 1977 to 2020. However, their chemical composition, pharmacological effects, and mechanism of treating UC are unclear. Aim of the studyThis study aimed to characterize the chemical composition of different parts of DLL (seeds and flowers), further explore their pharmacological effects, and elaborate its underlying mechanism of treating UC. MethodsThe chemical composition of DS and DF crude polysaccharides (DSP and DFP) and ethanolic extracts (DSE and DFE) were characterized by high-performance anion-exchange chromatography (HPAEC), ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS), and gas chromatography-mass spectrometry (GC-MS). Then, based on the acute UC mice model, the pharmacodynamic effects were investigated by Western blotting, ELISA, and other methods. Finally, the 16S rRNA gene sequencing and metabonomic analysis were used to explore the regulatory effects of DS and DF on intestinal microbiota and host metabolism. ResultsDSE and DFE inhibited the oxidative stress response, reducing proinflammatory factor production and maintaining intestinal barrier integrity in UC mice. The 16S rRNA gene sequencing and metabonomic analysis revealed that DS and DF treated UC by regulating the intestinal microbiota structure and reversing the abnormal metabolism of the host. ConclusionThis study suggested that different parts of DLL (flowers and seeds) may be potential medicines for treating UC, which exert their therapeutic effects through various active ingredients and might contribute significantly to reducing the economic pressures and challenges of UC treatment worldwide.

Evaluation of transgalactosylation activity of commercial β-galactosidase from Bifidobacterium bifidum for synthesis of prebiotic oligosaccharides

Galacto-oligosaccharides (GOS) are products of transgalactosylation reaction of β-galactosidase when lactose is used as the substrate. These carbohydrates are considered prebiotics which stimulate beneficial effects to human health. In the present work, Saphera, a commercial preparation of β-galactosidase from Bifidobacterium bifidum, was biochemically characterised for production of GOS. Using o-nitrophenyl-β-D-galactopyranoside (oNPG) as the substrate, optimal activity for the enzyme was found to be at pH 6.0 and at 45°C. Ten (10) mM each of either Na+ or K+ enhanced enzyme activity by 10%, while Cu2+, Zn2+, Fe2+, and EDTA showed inhibitory effect on the enzyme activity. When incubated in 50 mM sodium phosphate and pH 6.5, the enzyme was found to have half-life time of 136 ± 6 and 2.1 ± 0.2 h at 30 and 50°C, respectively. The hydrolysis activity of the enzyme predominated when the initial lactose concentration used was 5% (w/v). When initial lactose concentration was increased to 20% (w/v), maximum GOS yield obtained was 10% (w/w) achieved at 86% lactose conversion. Analysis using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) revealed that the major oligosaccharides produced by the enzyme were 3’-galactosylglucose, 3’-galactosyllactose, 3’-galactobiose, and allolactose, thus showing that this enzyme would prefer to form β-(13)- and β-(16)-linked GOS. Despite its predominantly hydrolytic activity, Saphera synthesised prebiotic GOS which could be interesting to dairy industry.