High-performance Anion-exchange Chromatography With Pulsed Amperometric Detection Research Articles

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.

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.

Complete composition analysis of polysaccharides based on HPAEC-PAD coupled with quantitative analysis of multi-components by single marker.

Monosaccharide compositions analysis (MCA) is indispensable for structural characterisations and structure-activity relationships of plant polysaccharides. To develop a concise and direct MCA method, we established a quantitative analysis of the multi-monosaccharaides by single marker (QAMS) by high-performance anion-exchange chromatography with pulsed-amperometric detection (HPAEC-PAD) method. A stable and reproducible HPAEC-PAD method for simultaneous determination of aldoses, ketoses and uronic acids (i.e., l-arabinose, d-xylose, d-ribose, l-rhamnose, d-fucose, d-mannose, d-glucose, d-galactose, d-fructose, d-glucuronic acid and d-galacturonic acid) was established by systematic optimisation of stationary phases, column temperatures and elution programmes. On this basis, the QAMS method was proposed through comprehensive investigations of relative correction factor (RCF) variations under different influencing factors, for example, sample concentrations, flow rates, and column temperatures. Using rhamnose as an internal reference standard, the contents of the other monosaccharidecomponents in polysaccharides from Panax quinquefolium L. and Achyranthes bidentata Bl. samples were simultaneously determined by QAMS, and there was no significant difference between the results from the QAMS and external standard method (t test, P > 0.520). In addition, a MCA fingerprinting of 30 batches of P. quinquefolium polysaccharide was established by HPAEC-PAD, and six common peaks were assigned and determined. The established HPAEC-PAD-QAMS method was successfully applied to the MCA of polysaccharides from P. quinquefolium and A. bidentata after optimisation of hydrolysis conditions. HPAEC-PAD-QAMS was proposed and established for MCA of plant polysaccharides for the first time.

Acid hydrolysis conditions for quantification of meningococcal X polysaccharide in a pentavalent vaccine using HPAEC-PAD/ESI-MS

A selective and sensitive method was evaluated for quantitation of meningococcal X (Men X) polysaccharide in pentavalent meningococcal A, C, W, Y and X conjugate vaccine using different acid hydrolysis conditions like HCl, TFA, HF, HF-TFA, and HF–HCl. High-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using CarboPac PA10 column was used to identify the hydrolyzed products based on retention time and its comparison with monosaccharide standards. Complete release of glucosamine (GlcN) from Men X in monovalent bulk and pentavalent vaccine samples was achieved using HF hydrolysis at 80 °C for 2 h. The Men X HF-hydrolyzed polysaccharide to glucosamine along with the reference standard was identified using collision-induced dissociation (CID) electrospray mass spectroscopy and the MS/MS fragments of m/z 162, m/z 144 and m/z 84. Meningococcal polysaccharide concentration was determined with a correlation coefficient r2 >0.99 using polysaccharide reference standard. The serogroups A, W, and Y were converted to their monosaccharides units and quantified using this method however, milder acid hydrolysis 0.1 M HCl 80 °C 2 h for release of sialic acid for Men C polysaccharide was found to be more suitable. These methods will provide necessary tools and prove to be beneficial to laboratories developing new saccharide-based vaccine combinations.

Plasma measurements of the dual sugar test reveal carbohydrate immediately alleviates intestinal permeability caused by exertional heat stress.

The aim of this set of randomised cross-over studies was to determine the impact of progressive heat exposure and carbohydrate or protein feeding during exertional stress on small intestine permeability using a dual sugar test. In our previous work, and typically in the field, recovery of lactulose and l-rhamnose is measured cumulatively in urine. This follow-up study exploits our novel high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) protocol to accurately quantify the sugars in plasma. Endurance-trained participants completed experimental trial A (ET-A; n=8), consisting of 2h running at 60% in temperate, warm and hot ambient conditions, and/or experimental trial B (ET-B; n=9), consisting of 2h running at 60% in the heat while consuming water, carbohydrate or protein. Blood samples were collected and plasma lactulose (L) and l-rhamnose (R) appearance, after dual sugar solution ingestion at 90min of exercise, was quantified by HPAEC-PAD to measure plasma L/R and reveal new information about intestinal permeability immediately post-exercise and during recovery. In ET-A, plasma L/R increased immediately post-exercise in hot compared with temperate and warm conditions, while, in ET-B, carbohydrate alleviated this, and this information was otherwise missed when measuring urine L/R. Consuming carbohydrate or protein before and during exercise attenuated small intestine permeability throughout recovery from exertional heat stress. We recommend using the dual sugar test with quantification of plasma sugars by HPAEC-PAD at intervals to maximise intestinal permeability data collection in exercise gastroenterology research, as this gives additional information compared to urinary measurements. KEY POINTS: Intestinal permeability is typically assessed using a dual sugar 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 measured in urine. We demonstrate that our recently developed ion chromatography protocol can be used to accurately quantify the L/R ratio in plasma, and that measuring L/R in plasma collected at intervals during the post-exercise recovery period reveals novel acute response information compared to measuring 5-h cumulative urine L/R. We confirm that exercising in hot ambient conditions increases intestinal epithelial permeability immediately after exercise, while consuming carbohydrate or protein immediately before and during exercise attenuates this. We recommend using our dual sugar absorption test protocol to maximise intestinal epithelial permeability data collection in exercise gastroenterology research and beyond.

Effects and Stress-Relieving Mechanisms of Dark Tea Polysaccharide in Human HaCaT Keratinocytes and SZ95 Sebocytes.

A new skincare application scenario for dark tea, a unique and post-fermented tea popular in the health food industry, was developed in this paper. The effects of dark tea polysaccharide (DTP) on stress-induced skin problems and its mechanism of action were investigated by modeling cortisone-induced stress injury in human HaCaT keratinocytes and SZ95 sebaceous gland cells. The results showed a reduced cortisol conversion induced by cortisone under the action of DTP with a concentration of 200 μg/mL, probably by inhibiting the expression of the HSD11B1 enzyme. DTP was also able to suppress the cortisone-induced elevation of lipid levels in SZ95 sebocytes at this concentration. In addition, the composition and structure of DTP were verified by ultrafiltration, ultraviolet-visible spectrophotometry (UV-VIS), high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and infrared spectroscopy. In brief, DTP has a unique and significant stress-relieving effect, which provides new ideas for the development of new ingredients for the skin care industry.

Method for the Determination of 2'-Fucosyllactose (2'-FL), 3-Fucosyllactose (3-FL), 6'-Sialyllactose (6'-SL), 3'-Sialyllactose (3'-SL), Lacto-N-Tetraose (LNT), and Lacto-N-neoTetraose (LNnT) by High-Performance Anion-Exchange Chromatography With Pulsed Amperometric Detection (HPAEC-PAD): First Action 2022.04.

A method for simultaneous determination of six human milk oligosaccharides (HMOs) is described. The HMOs include 2'-fucosyllactose (2'-FL, CAS number 41263-94-9), 3-fucosyllactose (3-FL, CAS number 41312-47-4), 6'-sialyllactose (6'-SL, CAS number 35890-39-2), 3'-sialyllactose (3'-SL, CAS number 35890-38-1), lacto-N-tetraose (LNT, CAS number 14116-68-8), and lacto-N-neotetraose (LNnT, CAS number 13007-32-4). The method was designed to comply with the respective Standard Method Performance Requirements (SMPR®; seeTable1), respectively. The method is valid for six HMOs in infant formula and adult nutritional matrixes, including samples with intact protein, protein hydrolysates, elemental formulations free of intact protein, and rice flour over the ranges defined in the SMPR (seeTable2). The method is not valid for determination of difucosyllactose (DFL/DiFL). For most samples, reconstitution with water followed by filtration. For products containing interferences (fructans and maltodextrins), hydrolysis with enzymes is used. After preparation, samples are analyzed using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The method provides for separation of six HMOs and other carbohydrates commonly found in infant formula and adult nutritional products (e.g., lactose, sucrose, and GOS). This study includes data from multiple matrixes evaluated by multiple laboratories globally. RSDr ranged from 0.0068 to 4.8% RSDr, and spike recovery results ranged from 89.4 to 109%. Calibration fit optimal with a quadratic curve; alternately linear fit showed no statistically significant impact to data (when correlation passes). This method was reviewed by the AOAC SPIFAN Expert Review Panel (ERP) and determined to meet the SMPRs for the six noted HMOs. The method was granted First Action Official MethodsSM status.

Profiling of cool-season forage arabinoxylans via a validated HPAEC-PAD method.

Cool-season pasture grasses contain arabinoxylans (AX) as their major cell wall hemicellulosic polysaccharide. AX structural differences may influence enzymatic degradability, but this relationship has not been fully explored in the AX from the vegetative tissues of cool-season forages, primarily because only limited AX structural characterization has been performed in pasture grasses. Structural profiling of forage AX is a necessary foundation for future work assessing enzymatic degradability and may also be useful for assessing forage quality and suitability for ruminant feed. The main objective of this study was to optimize and validate a high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) method for the simultaneous quantification of 10 endoxylanase-released xylooligosaccharides (XOS) and arabinoxylan oligosaccharides (AXOS) in cool-season forage cell wall material. The following analytical parameters were determined or optimized: chromatographic separation and retention time (RT), internal standard suitability, working concentration range (CR), limit of detection (LOD), limit of quantification (LOQ), relative response factor (RRF), and quadratic calibration curves. The developed method was used to profile the AX structure of four cool-season grasses commonly grown in pastures (timothy, Phleum pratense L.; perennial ryegrass, Lolium perenne L.; tall fescue, Schedonorus arundinaceus (Schreb.) Dumort.; and Kentucky bluegrass, Poa pratensis L.). In addition, the cell wall monosaccharide and ester-linked hydroxycinnamic acid contents were determined for each grass. The developed method revealed unique structural aspects of the AX structure of these forage grass samples that complemented the results of the cell wall monosaccharide analysis. For example, xylotriose, representing an unsubstituted portion of the AX polysaccharide backbone, was the most abundantly-released oligosaccharide in all the species. Perennial rye samples tended to have greater amounts of released oligosaccharides compared to the other species. This method is ideally suited to monitor structural changes of AX in forages as a result of plant breeding, pasture management, and fermentation of plant material.

Early development of infant gut microbiota in relation to breastfeeding and human milk oligosaccharides.

Infant gut microbiota composition is influenced by various factors early in life. Here, we investigate associations between infant gut microbiome development, infant age, breastfeeding duration, and human milk oligosaccharides (HMO) composition in breastmilk. A total of 94 mother-infant pairs were recruited as part of the Cambridge Baby Growth and Breastfeeding Study (CBGS-BF) (Cambridge, UK). Infant stool samples (n = 337) were collected at 2 week, 6 week, 3 month, and 6 month of age. The 16S rRNA V3-V4 rRNA region was sequenced using MiSeq Illumina to determine microbiota composition and diversity. Mother's hindmilk samples were collected at birth, 2 week, 6 week, 3 month, and 6 month postpartum. Concentrations of five neutral [2'FL, 3'FL, lacto-N-fucopentaose 1 (LNFP1), LNnT, LNT] and two acidic (3'SL, and 6'SL) HMOs were measured in all milk samples using High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). We explored the associations between infant gut microbiome parameters and age, duration of exclusive breastfeeding (EBF), and levels of individual HMOs. Bifidobacterium was the most abundant genus in infant stool at all-time points, irrespective of breastfeeding duration, with an overall mean relative abundance of 70%. The relative abundance of B. bifidum in stool from infants who were breastfed for longer than 6 months was significantly higher compared to the infant breastfed up to 3 months (p = 0.0285). Alpha-diversity (both Shannon and ASV-level Richness) of infant gut microbiota showed a biphasic change with infant age, decreasing from 2 weeks until 3 months and then increasing until 6 months of age. Bifidobacterium relative abundance was associated with higher concentrations of 2'FL and LNFP1 in breastmilk across all time-points (p = 0.049 and 0.017, respectively), with trends toward a higher abundance of B. longum species. No significant association with Bifidobacterium was found for breastmilk LNnT, 3'SL, and 6'SL levels. Our study is in line with previous data demonstrating that EBF duration in the first months of life impacts infant gut microbiota composition. The observed links between specific HMOs in breastmilk and bacteria in infant stool provide evidence of how mother's milk affects infant microbiome development.

Production and characterization of manno-oligosaccharides from hydrolysis of mannan substrates by recombinant beta-mannanase from Bacillus licheniformis (Weigmann) Chester DSM 13

Mannooligosaccharides (MOS) is a prebiotic produced by the hydrolysis of mannans and promotes gut health. One route in producing MOS is via enzymatic method, which employs microbial enzyme (e.g., β-mannanase) for mannan hydrolysis. In this work, we investigated the stability and propensity of β-mannanase from Bacillus licheniformis (BlManB) overexpressed in Lactiplantibacillus plantarum WCSF1 towards β-MOS production using several mannan sources. We also determined the fermentation conditions for optimal enzyme production in the food-grade system. Maximal crude enzyme yield was obtained (17.3 ± 1.2 kU per liter of fermentation broth with specific activity of 194 U/mg protein) by maintaining the pH at 6–6.5 at fermentation. Crude BlManB is stable at storage conditions of -20°C and 4°C. The crude overexpressed enzyme was also stable and active at conversion conditions (30 °C and 37 °C) in the presence of 2 mM dithiothreitol (DTT) . Analysis by high-performance anionexchange chromatography with pulsed amperometric detection (HPAEC-PAD) indicated that the crude enzyme produced known linear MOS of 235.88 mg/g using locust bean gum, 121.63 mg/g using copra meal, 71.71 mg/g using guar gum, 21.33 mg/g using spent coffee ground, and 86.76 mg/g using konjac glucomannan. The study has also shown that crude BlManB overexpressed in L. plantarum WCSF1 could be efficiently used to produce food-grade MOS.

Identification of carbohydrate in Polygonatum sibiricum: fructo-oligosaccharide was a major component

Abstract Polygonatum sibiricum, famous for its physiological activities, is a popular Chinese food and a traditional Chinese herb containing many carbohydrates as the main component. However, except for most reported polysaccharides, other detailed compositions of carbohydrates are still not clear. To verify the controversial existence of starch and investigate other components, especially oligosaccharides, we used iodine–potassium iodide colorimetric detection and enzymatic hydrolysis to determine starch. Then, oligosaccharides were analyzed by thin-layer chromatography, gel permeation chromatography, high-performance anion exchange chromatography with pulsed amperometric detection, and hydrophilic interaction chromatography–electrospray tandem mass spectrometry. The results showed that the rhizome of P. sibiricum lacked starch, and fructo-oligosaccharides were the main component, accounting for approximately 28.95%. Oligosaccharides with degrees of polymerization above 10 were the most abundant components. This study clearly illustrated the unknown carbohydrate components of the Polygonatum rhizome, promoting its functional value with new evidence.

Measuring key human carbohydrate digestive enzyme activities using high-performance anion-exchange chromatography with pulsed amperometric detection.

Carbohydrate digestion in the mammalian gastrointestinal tract is catalyzed by α-amylases and α-glucosidases to produce monosaccharides for absorption. Inhibition of these enzymes is the major activity of the drugs acarbose and miglitol, which are used to manage diabetes. Furthermore, delaying carbohydrate digestion via inhibition of α-amylases and α-glucosidases is an effective strategy to blunt blood glucose spikes, a major risk factor for developing metabolic diseases. Here, we present an in vitro protocol developed to accurately and specifically assess the activity of α-amylases and α-glucosidases, including sucrase, maltase and isomaltase. The assay is especially suitable for measuring inhibition by compounds, drugs and extracts, with minimal interference from impurities or endogenous components, because the substrates and digestive products in the enzyme activity assays are quantified directly by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). Multiple enzyme sources can be used, but here we present the protocol using commercially available human α-amylase to assess starch hydrolysis with maltoheptaose as the substrate, and with brush border sucrase-isomaltase (with maltase, sucrase and isomaltase activities) derived from differentiated human intestinal Caco-2(/TC7) cells to assess hydrolysis of disaccharides. The wet-lab assay takes ~2-5 h depending on the number of samples, and the HPAE-PAD analysis takes 35 min per sample. A full dataset therefore takes 1-3 d and allows detection of subtle changes in enzyme activity with high sensitivity and reliability.

Polysaccharides from tropical green seaweed Chaetomorpha antennina induces non-specific immune responses and improves antioxidative activities in common carp (Cyprinus carpio) leukocyte culture cell line

Polysaccharides from seaweeds are promising biomolecules that could sustainably improve the health status of cultured fish species. In this study, water-soluble polysaccharides extracted from Chaetomorpha antennina were investigated to determine their physicochemical characteristics and biological activities. C. antennina water-soluble polysaccharides (CAWP) comprised mainly of carbohydrate (69.0 ± 2.3 %), sulphate (12.6 ± 0.5 %), uronic acid (8.8 ± 0.1 %) and protein (3.5 ± 0.2 %). High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) analysis revealed that CAWP contained mostly arabinose (17.1 ± 2.3 mg/L), galactose (9.3 ± 1.0 mg/L), glucose (5.1 ± 0.6 mg/L), xylose/mannose (1.4 ± 0.6 mg/L) and a small proportion of fucose (0.3 ± 0.1 mg/L). CAWP, with intrinsic viscosity of 0.66 dL/g and estimated molecular weight of 318 kDa, were non-toxic to Carp leukocyte culture (CLC) cell line at concentrations ≤25 μg/mL. CAWP at concentrations of 12.5 and 25 μg/mL stimulated CLC to release significant amount of nitric oxide (NO), reactive oxygen species (ROS) and augmented phagocytic activity. Intracellular antioxidant activities indicated that CAWP did not induce oxidative damage in CLC but could improve superoxide dismutase activity (SOD), total glutathione (tGSH) and decrease malondialdehyde (MDA) level. The findings from our study provide a foundation for the applicability of water-soluble polysaccharides from C. antennina in the aquaculture industry as a potent immunomodulatory agent. • Chaetomorpha antennina is a rich source of sulphated polysaccharides. • Induction of oxidative burst response in Carp leucocytes • Phagocytosis of Red Zymosan® bioparticles by Carp leucocytes • Improved intracellular antioxidant activities

Unveiling the compositional remodelling of Arbutus unedo L. fruits during ripening

• The architecture of the cell wall of strawberry tree ( Arbutus unedo L.) fruits, during four ripening stages, is herein elucidated for the first time. • Glucose and xylose are the major neutral sugars in the cell wall, similarly to other Ericaceae. • A. unedo fruits are unusually rich in lignin. • Multiple structures of aggregated sclereids are observed in the fruits, thus unveiling their peculiar gritty texture. • Sclereids are the source of high levels of xylose and lignin. Arbutus unedo is a Mediterranean shrub or tree that produces edible fruits with promising applications for the food industry and nutraceutical applications. A deep understanding of the cell wall structure and modifications of its components during fruit softening would benefit the development of new food products and retail of fresh fruits. Aiming to enhance the valorization of A. unedo fruits, its cell wall was characterized, for the first time, during ripening. In this study, four distinct ripening stages (green, yellow, orange and red) were considered. Several preliminary quality parameters were determined. The nature of the compounds making part of the cell walls were elucidated through Fourier‑transform infrared spectroscopy in attenuated total reflectance mode (FTIR-ATR) and spectral datasets were analyzed using unsupervised principal component analysis (PCA). High-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was employed to assess monosaccharide composition, and lignin measurements were performed following the acetyl bromide soluble lignin methodology. In addition, scanning electron microscopy (SEM) analysis allowed to observe some relevant aspects of the microstructure of fruits. Significant modifications on the composition of these fruits were mostly found by the end of the ripening process when the fruits reached the red-ripe stage. Glucose, xylose and arabinose were determined as the major neutral cell wall sugars. Furthermore, the microscopy assessment unveiled the abundance of aggregated sclereids, and lignin measurements showed that A. unedo fruits are highly lignified. The richness in lignin and monosaccharides, such as glucose and xylose, may suggest the role displayed by that polyphenol and hemicelluloses like xylans and xyloglucans, which is supported by FTIR-ATR spectral assignments. It is hypothesised that the high amounts of xylose and lignin herein determined are highly associated with the sclereids. Relevant data were also obtained for the fundamental understanding of the ripening of other Ericaceae fruits and other berries.

Fructooligosaccharides as molecular markers of geographic origin, growing region, genetic background and prebiotic potential in strawberries: A TLC, HPAEC-PAD and FTIR study

Strawberries are one of the most consumed fruits worldwide. Scientific research has revealed unique nutraceutical properties, mainly steaming from their content of polyphenols and fructooligosaccharides (FOS), well known prebiotics. Here, we report the fingerprint (chemical composition) of strawberries as molecular markers of geographical origin, growing region, and genetic background, after analyzing different strawberry varieties grown and collected in Mexico and Canada by thin layer chromatography (TLC), high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), and Fourier-transform infrared spectroscopy – principal component analysis (FTIR-PCA). The most abundant carbohydrates in all cases were glucose and fructose. The highest levels of sucrose were observed in Canada-grown strawberries, while Mexico-grown varieties presented the highest FOS abundance. There were clear and distinct patterns correlating FOS isomers determined by HPAEC-PAD and geographic origin. FTIR-PCA second derivative spectra also allowed the classification and differentiation of all strawberry varieties grown in both countries. We suggest that carbohydrates fingerprinting could be associated to specific environmental growing conditions, and not only to genetic makeup.

Molecular size distribution assessment of Haemophilus influenzae vaccine containing lactose by HPAEC-PAD and colorimetric assays

Molecular size distribution of Haemophilus influenzae type b (Hib) conjugate vaccine is an important indicator for its immunogenicity and stability. Molecular size distribution was evaluated by High-Performance Protein Chromatography on Sepharose CL-4B column, and fractions were pooled. The use of high flow rate, incorporation of a calibration standard with the injected buffer and pooling method yielded a superior assay compared to conventional pharmacopeial method. The pools were analyzed for determination of distribution coefficient (KD) of 0.2 and 0.5 using two validated techniques: High Performance Anion Exchange Chromatography with pulsed amperometric detection (HPAEC-PAD) for ribitol determination and an optimized colorimetric assay for phosphorus determination. Linearity was achieved over range of 0.10–10.0 μg/mL and 1.0–8.0 μg/mL with LOD of 0.03 and 0.28 μg/mL for HPAEC and colorimetric assays, respectively. The developed assays were successfully applied in quality control monitoring of Hib conjugate vaccine. The optimized colorimetric method had shortened the analysis time to 25 min compared to 3.5 h for the European pharmacopeial assay by modifying the burning process. HPAEC stability results revealed 40% decrease in MSD after stressed storage conditions. The proposed assays offer a reliable and economic platform for monitoring the quality attributes of Hib for biopharma industry.

Quantitative analysis of fructans in functional foods by high-performance anion-exchange chromatography with pulsed amperometric detection

This study describes a method for determining fructans in functional foods by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The sample was first extracted with hot water. After hydrolyzed by a mixture of sucrase/β-amylase/pullulanase/maltase, the extract solution was treated with sodium borohydride to remove completely free fructose and glucose in the sample. The reaction conditions with sodium borohydride were investigated: 5 mg/mL of sodium borohydride was used, and the reaction time was 20 min. Fructans were then hydrolyzed by endo- and exo-inulinase to completely form fructose and glucose. The content of fructans was calculated based on the amount of fructose and glucose formed after fructans were hydrolyzed. The method was developed and validated on four different food matrices: milk powder, milk liquid, hard capsule, and soft capsule. The results showed that this method has good specificity and linearity (R2 > 0.99). This method also showed good accuracy and precision, with recovery values ranged between 95.0 and 103.6%, while RSD (%) values were 0.45–3.59%. The method also had a low limit of quantification and limit of detection that can quantify food samples with a small amount of fructans. The validated method has been successfully applied to determine the content of fructans in functional foods, and this method provides a valuable tool to evaluate the quality of functional food products.

An improved method for galactosyl oligosaccharide characterization

Due to beneficial effects of galactosyl oligosaccharides (GOS) on digestive and immune health, their characterization has become increasingly important. This is especially so as GOS are synthesized enzymatically and contain oligosaccharides of different sizes and linkages. High performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) is widely used for GOS characterization. With its high resolving power, it can separate structural isomers. Here we present a significant improvement to currently used methods. Our approach combines high resolution HPAE separation on a CarboPac PA300 column with 4 µm particle size with PAD and Orbitrap mass spectrometry (MS) detections to provide in-depth information on GOS composition. Oligosaccharide resolution, especially in the disaccharide region, is significantly improved and can be routinely achieved. Improvement in technology to remove sodium before MS results in minimal peak dispersion, allowing GOS degrees of polymerization 2 to 6 to be identified based on mass spectra obtained from intact oligosaccharides and confirmed using fragmentation patterns observed in MS/MS data. Combining HPAE with MS led to identification of 28 oligosaccharides in a commercial GOS sample. We attempted to correlate oligosaccharide structure with observed elution behavior. To our knowledge this is first such attempt and can form a basis for a comprehensive structure vs HPAE elution behavior database.