High Performance Anion Exchange Chromatography Coupled With Pulsed Amperometric Detection Research Articles

Maillard conjugates of whey protein and prebiotic dietary fibers: Emulsion properties, encapsulation efficiency and chemical stability after in vitro gastrointestinal digestion

The incorporation of dietary fibers into food formulations is a promising approach to developing new functional ingredients. In this study, we investigated the formation of Maillard conjugates using whey protein concentrate (WPC) combined with fructooligosaccharides (FOS) and xylooligosaccharides (XOS). These conjugates, innovatively utilized as emulsifiers for annatto seed oil, were subjected to in vitro gastrointestinal digestion to assess their chemical stability and effectiveness as encapsulating systems. Our study confirmed the occurrence of the Maillard reaction through different methods, including grafting degree (GD) assessment and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) to evaluate molecular weight variations in the proteins. The key innovation of our research lies in demonstrating the conjugation process through carbohydrate mass balance analysis using High-Performance Anion Exchange Chromatography coupled with Pulsed Amperometric Detection (HPAEC-PAD). The GD for the WPC-XOS and WPC-FOS was 46 ± 3 % and 56 ± 3 %, respectively. Our findings suggest that sugar quantification could serve as a novel approach to confirming the formation of Maillard conjugates. The emulsions stabilized by these conjugates exhibited larger oil droplet sizes compared to those stabilized by WPC alone yet maintained high encapsulation efficiency (≥ 90 %). Notably, the WPC-XOS emulsion demonstrated kinetic stability after 30 days of storage at 4°C, while the WPC-FOS emulsion showed signs of creaming after just 16 days. In vitro gastrointestinal digestion revealed that while the conjugates were susceptible to enzymatic degradation, the FOS and XOS sugars displayed gastrointestinal resistance of up to 82 % and 83 %, respectively. This study highlights the potential of WPC-XOS conjugates not only as effective emulsifiers but also as functional ingredients that enhance the nutritional and antioxidant properties of food products, presenting a novel approach to functional food development.

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.

PSIII-1 Effect of Cannabidiol Extraction on the Cell Wall Carbohydrate Composition of Hemp Inflorescences

Abstract Since the 2018 Farm Bill, US growers have been allowed to cultivate hemp (Cannabis sativa L.), prompting interest in exploring the use of hemp products in ruminant diets. Extraction of cannabidiol (CBD) oil from hemp biomass generates spent inflorescent material that could be upcycled as a feedstuff, yet little is known about its fiber composition. A recent study measured in vitro true digestibility and fermentation parameters of multiple hemp cultivars, some intact and some CBD-extracted (Altman et al., 2022). Both categories of hemp inflorescence material had similar or greater total digestible nutrients (TDN) than an alfalfa cube control, supporting hemp’s potential as a feedstuff. However, comparisons between non-extracted and CBD-extracted samples could not be made, and the fiber content was only described as neutral detergent fiber (NDF). Fiber composition can significantly impact ruminal digestibility, and in-depth characterizations of cell wall carbohydrate composition can provide further insight into the digestibility of a specific plant-based feed. Therefore, in the present study, we examined the effect of CBD oil extraction on the cell wall carbohydrate composition of the inflorescent material of two hemp cultivars. Intact (Biomass) and CBD-extracted (Spent) inflorescent material from two hemp cultivars, Mary and Zelios, were analyzed. The scell wall monosaccharides of the samples were released via two complementary acid hydrolyses, methanolysis and Saeman (sulfuric acid) hydrolysis, and separated and quantified using high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). Additionally, the carbohydrate linkage patterns were assessed by generating partially methylated alditol acetates (PMAAs). Student’s unpaired t-tests were run in GraphPad Prism, and no differences (P > 0.05) in monosaccharide concentrations based upon treatment (CBD extraction) or cultivar were found with either hydrolysis method except for the methanolysis glucose concentration between the biomass forms of the two cultivars where Mary Biomass possessed more glucose than Zelios Biomass (P <b0.001). In the Saeman hydrolysates, glucose was the dominant monosaccharide (38.75 ± 1.44 molar %), indicating a large amount of cellulose, supported by the presence of 1,4-Glcp in the linkage analysis. Linkage analysis also revealed the presence of xyloglucans and rhamnogalacturonan Type 1 pectins in all materials. The data showed no effect of CBD oil extraction on the monosaccharide composition, suggesting that the extraction process leaves the fiber components mostly intact, but there may be differences in glucose concentration (and consequently cellulose content) between cultivars of hemp. Future studies may benefit more from investigating the impact of hemp cultivar on fiber digestibility rather than impact of the CBD extraction process.

An oxygen-insensitive amperometric galactose biosensor based on galactose oxidase co-immobilized with an Os-complex modified redox polymer

Monitoring galactose levels in dairy products can help to prevent severe complications with a hereditary metabolic disease such as galactosemia, a life-threatening disease. The current state of the art requires the development of less expensive, more reliable and specific methods to determine galactose levels in food in a practical way. We report the development and optimization of an amperometric biosensor for determination of galactose in dairy products based on galactose oxidase (GaOx) co-immobilized with an osmium-complex modified redox polymer on glassy carbon electrodes. To attain the maximum catalytic currents based on mediated electron transfer, two Os-complex based polymers with different redox potentials and different enzyme:redox polymer ratios were studied. The optimized GaOx-modified electrode gave a maximum electrocatalytic response of galactose oxidation that was not affected by the presence of O2, indicating fast wiring of the enzyme by the Os-complex modified redox polymer. The biosensor that gave the best analytical parameters for galactose detection was further tested for measuring galactose concentration in lactose-containing and lactose-free milk and yogurt samples under aerobic conditions. The results obtained with the amperometric biosensor were validated by high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

Enzymatic bioconversion of beechwood xylan into the antioxidant 2′-O-α-(4-O-methyl-D-glucuronosyl)-xylobiose

Acidic xylooligosaccharides (XOS), also called aldouronics, are hetero-oligomers of xylose randomly branched with 4-O-methyl-D-glucuronic acid residues linked by α(1 → 2) bonds, which display bioactive properties. We have developed a simple and integrated method for the production of acidic XOS by enzymatic hydrolysis of a glucurono-xylan from beechwood. Among the enzymes screened, Depol 670L (a cellulolytic preparation from Trichoderma reesei) displayed the highest activity (70.3 U/mL, expressed in reducing xylose equivalents). High-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) analysis revealed the formation of a neutral fraction (corresponding to linear XOS, mainly xylose and xylobiose) and a group of more retained products (acidic XOS), which were separated using strong anion-exchange cartridges. The acidic fraction contained a major product, characterized by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and mono- and two-dimensional nuclear magnetic resonance spectroscopy (NMR) as 2′-O-α-(4-O-methyl-α-D-glucuronosyl)-xylobiose (X2_MeGlcA). Starting from 2 g of beechwood xylan, 1.5 g of total XOS were obtained, from which 225 mg (11% yield) corresponded to the aldouronic X2_MeGlcA. The acidic XOS exhibited higher antioxidant activity (measured by the ABTS·+ discoloration assay) than xylan, whilst neutral XOS displayed no antioxidant activity. This work demonstrates that it is possible to obtain a safe and natural antioxidant by enzymatic biotransformation of hardwood hemicellulose.

Quantification of naturally occurring prebiotic fiber in Italian foods

Prebiotic fiber may have a beneficial effect on the human’s microbiota. However, due to the lack of comprehensive food composition data (FCD), it is challenging to assess their dietary intake. The aim of present study is to quantify naturally occurring prebiotics, such as inulin-type fructans (ITFs), fructo-oligosacharides (FOSs), and galacto-oligosacharides (GOSs), in raw and processed Italian foods and to estimate their intake in a healthy population (N = 4154) belonging to an Italian case-control study on colorectal cancer. ITFs were determined using enzymatic method and high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). FOSs and GOSs were determined by alkaline hydrolysis and HPAEC-PAD. Garlic (25.1 g/100 g) and jerusalem artichoke (16.7 g/100 g) were the ITFs-richest foods. Jerusalem artichoke was also the richest in FOSs (4.45 g/100 g). GOSs were mainly detected in dry and canned pulses (0.01–2.04 g/100 g). In our population, the median intake of ITFs, FOSs and GOSs was 0.797, 0.187 and 0.283 g/day, respectively. Given the unclear definition of inulin due to the heterogenicity of methodologies, it was challenging to compare published prebiotic fiber’s FCD and use them in epidemiological research. Within the present work we produced novel FCD able to preliminary estimate the intake of prebiotic fiber.

Enhancement of large ring cyclodextrin production using pretreated starch by glycogen debranching enzyme from Corynebacterium glutamicum

Synthesis of large-ring cyclodextrins (LR-CDs) in any significant amount has been challenging. This study enhanced the LR-CDs production by Thermus filiformis amylomaltase (TfAM) enzyme by starch pretreatment using glycogen debranching enzyme from Corynebacterium glutamicum (CgGDE). CgGDE pretreated tapioca starch gave LR-CD conversion of 31.2 ± 2.2%, compared with LR-CDs produced from non-treated tapioca starch (16.0 ± 2.4%). CgGDE pretreatment enhanced amylose content by approximately 30%. Notably, a shorter incubation time of 1 h is sufficient for CgGDE starch pretreatment to produce high LR-CD yield, compared with 6 h required for the commercial isoamylase. High-Performance Anion Exchange Chromatography coupled with Pulsed Amperometric Detection (HPAEC-PAD) and Gel Permeable Chromatography (GPC) revealed that CgGDE is more efficient than the commercial isoamylase in debranching tapioca starch and gave lower molecular weight products. In addition, lower amount of by-products (linear oligosaccharides) were detected in cyclization reaction when using CgGDE-pretreated starch. In conclusion, CgGDE is a highly effective enzyme to promote LR-CD synthesis from starch with a shorter incubation time than the commercial isoamylase.

The Determination of Carbohydrates by High-Performance Anion-Exchange Chromatography Coupled with Pulsed Amperometric Detection (HPAEC-PAD).

Chromatography techniques are widely used to separate, identify, and quantify molecules depending on their physicochemical properties. Standard methods range from simple size exclusion to separation based on affinity or ion exchange. Here, we present a method for the direct analysis of carbohydrates in Magnaporthe oryzae using high-performance anion-exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD). The combination of HPAEC with PAD provides the highest selectivity and sensitivity with minimal sample preparation and cleanup time. Utilizing our HPAEC-PAD approach, we obtain reliable and highly reproducible determination of carbohydrates produced as osmotic stress response by M. oryzae. Thus, the method described provides a fast, precise, and comprehensive analysis of stress-dependent metabolic adjustments of carbohydrates not only relevant for M. oryzae but also applicable in other systems.

Levansucrase from Bacillus amyloliquefaciens KK9 and Its Y237S Variant Producing the High Bioactive Levan-Type Fructooligosaccharides.

Levan-typed fructooligosaccharide (LFOS), a β-2,6 linked oligofructose, displays the potential application as a prebiotic and therapeutic dietary supplement. In the present study, LFOS was synthesized using levansucrase from Bacillus amyloliquefaciens KK9 (LsKK9). The wild-type LsKK9 was cloned and expressed in E. coli, and purified by cation exchanger chromatography. Additionally, Y237S variant of LsKK9 was constructed based on sequence alignment and structural analysis to enhance the LFOS production. High-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) analysis indicated that Y237S variant efficiently produced a higher amount of short-chain LFOS than wild type. Also, the concentration of enzyme and sucrose in the reactions was optimized. Finally, prebiotic activity assay demonstrated that LFOS produced by Y237S variant had higher prebiotic activity than that of the wild-type enzyme, making the variant enzyme attractive for food biotechnology.

Structural Characterization and Chain Conformation of Water-Soluble β-Glucan from Wild Cordyceps sinensis.

Water-soluble β-d-glucan was obtained from wild Cordyceps sinensis by alkali solution and ethanol precipitation. The structure characteristics were determined using high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD), methylation combined with gas chromatography-mass spectrometry, and one-/two-dimensional nuclear magnetic resonance spectroscopy. Results showed that β-d-glucan had a structure of every seven (1→3)-β-d-Glcp backbone residues with two (1→6)-β-d-Glcp branches. Additionally, conformation properties in different solvents were investigated by static light scattering, dynamic light scattering, and HPSEC with multiple detectors. It was found that β-d-glucan in 0.5 M NaOH had a narrow unimodal distribution of hydrodynamic radius displaying a spherical coil conformation, whereas it formed severe aggregation in dimethyl sulfoxide. In 0.1 M NaNO3, β-d-glucan mainly existed as a rod-like conformation corresponding to a helical structure together with small aggregates (10%). This work added more information to the understanding of C. sinensis polysaccharides.

Processing of inulin-enriched apple juice using high-intensity ultrasound technology

The effects of high-intensity ultrasound (HIUS) processing on chemical composition of inulin-enriched apple juice were evaluated at different specific energy levels (0, 1, 4, 7 and 10 kJ/g). The functional beverage was characterized with respect to fructooligosaccharides (FOS) and total sugars content. In addition, the inulin chemical profile was evaluated. The analysis of sugars and fructo-oligosaccharides in the functional juice was performed by high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

Analysis of Monosaccharides from Arabidopsis Seed Mucilage and Whole Seeds Using HPAEC-PAD

Arabidopsis seed coat epidermal cells deposit a significant quantity of mucilage, composed of the cell wall components pectin, hemicellulose, and cellulose, into the apoplast during development. When mature seeds are hydrated, mucilage extrudes to form a gelatinous capsule around the seed. Determining the monosaccharide composition of both extruded mucilage and whole seeds is an essential technique for characterizing seed coat developmental processes and mutants with altered mucilage composition. This protocol covers growth of plants to produce seeds suitable for analysis, extraction of extruded mucilage using water and sodium carbonate (used for mutants with impaired mucilage release), and extraction of alcohol insoluble residue (AIR) from whole seeds. The prepared polysaccharides are then hydrolyzed using sulfuric acid, which hydrolyses all polysaccharides including cellulose. Sensitive and reproducible quantification of the resulting monosaccharides is achieved using high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

Structure analysis of β-glucan in barley and effects of wheat β-glucanase

In this study, β-glucan in samples of sifted flour from six barley varieties was sequentially extracted with water and NaOH obtaining three fractions: water-extractable (WE), NaOH- extractable (NaE) and residual (Res). β-Glucan isolates were incubated with lichenase and oligomers released were analysed with high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). A higher ratio of 3-O-β-cellobiosyl-D-glucose (DP3) to 3-O-β-cellotriosyl-D-glucose (DP4) is reported in WE, NaE and Res fractions in variety SLU 7 (shrunken endosperm) compared to the other varieties, suggesting a more packed β-glucan structure. Water-extractable and water-unextractable β-glucan fractions were incubated with wheat extract and effects of wheat β-glucanase on the structure was assessed after lichenase digestion and analysis with HPAEC-PAD. Findings suggest that wheat β-glucanase manifests a selective hydrolysis towards β-glucan with lower DP3/DP4 ratio and that β-glucan of SLU 7 is composed of a population with higher DP3/DP4 ratio and hence with a more tighten structure which may be more resistant to enzymic action. Overall, the results obtained are of interest to characterise barleys differing in starch and dietary fibre composition, specifically shrunken endosperm barley SLU 7 in the perspective of its inclusion in the production of β-glucan enriched foods.

The ripening disorder berry shrivel affects anthocyanin biosynthesis and sugar metabolism in Zweigelt grape berries

Main conclusionTimescale analyses suggest the berry shrivel (BS) disorder is induced before veraison with strong effects on anthocyanin biosynthesis, and minor effects on sugar transport and metabolism.Berry shrivel (BS)-affected grapes have low sugar contents, high acidity, less anthocyanins and flaccid berries. To date no pathogenic causes are known, and studies to elucidate the molecular basis leading to symptom induction and development are limited. Here we present a study on pre-symptomatic as well as symptomatic BS berries to characterize early metabolic changes, with focus on anthocyanin biosynthesis and sugars metabolism. Healthy and BS berries from six sampling time points were used (BBCH79–BBCH89). Our objectives are (1) to search for the beginning of BS-related physiological processes; (2) to search for key enzymes and sugar transporters involved in BS induction and development and (3) to understand the consequences on polyphenol biosynthesis. We employed high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) technology for sugar and polyphenol analyses, respectively. Additionally we conducted expression analyses (qPCR) of key genes and enzymatic activity assays. Our results show that BS-related processes start before veraison, as determined by slightly reduced hexose contents and reduced expression levels of a vacuolar invertase (VviGIN1), two monosaccharide transporters (VviTMT2, VviTMT3) and the anthocyanin biosynthesis (VviUFGT, VviMYBA1/2) genes. Lower amounts of delphinidin and cyanidin glycosidic forms were determined, while caftaric acid, quercetin-3-O-glucuronide and (+)-catechin were increased in BS berries. Although not all results were conclusive, especially for the sugar metabolism, our data provide important knowledge to improve the understanding of the highly complex berry shrivel ripening disorder.

Influence of corn variety, drying temperature, and moisture content at harvest on the saccharides released during an in vitro pepsin‐pancreatin digestion

High‐performance anion‐exchange chromatography coupled with pulsed amperometric detection (HPAEC‐PAD) was used to quantify and to determine the nature of saccharides released after the digestion of two corn grains submitted to different drying and harvesting protocols. Five saccharides were quantified: glucose, maltose, isomaltose, maltotriose, and glucosyl‐maltotriose. After a subsequent hydrolysis with amyloglucosidase of the released saccharides, the amount of total glucose recovered increased with the increase in drying temperature and the moisture content of grain at harvest. These results suggest that structural changes induced within corn starch granules during drying affect their hydrolysis by amylases. These changes may lead to different pathways of sugar absorption in the small intestine of monogastrics. Thus, corn grains with a similar composition may lead to different levels of metabolizable energy upon gastro‐intestinal digestion.

Neutral sugars determination in Chlorella: Use of a one-step dilute sulfuric acid hydrolysis with reduced sample size followed by HPAEC analysis

The accurate determination of the chemical composition of microalgae is essential when evaluating their potential as feedstock for the production of biofuels and biochemicals. The usual methodology for the determination of total microalgal carbohydrate content combines a two-step acid hydrolysis of the microalgal biomass with an analysis of the released monosaccharides using phenol-sulfuric acid, but the latter is a hazardous procedure. Alternatively, the sugar monomers that are present in the acid hydrolysate can be identified using high performance liquid chromatography (HPLC), whose accuracy may be compromised by compounds derived from the complex cell composition of the microalgae. Aiming to further the extant analytical procedures for determination of the neutral sugar content in microalgae of the Chlorella genus, the present study evaluated a one-step dilute sulfuric acid hydrolysis followed by the analysis of the released monosaccharides using high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). The proposed HPAEC-PAD method proved to be specific, sensitive, accurate, precise and robust. The one-step dilute sulfuric acid hydrolysis process showed an acceptable correlation with the widely used two-step technique for the determination of total neutral sugars in Chlorella, despite the use of a sixfold smaller sample size. The method was also successfully applied to Mychonastes homosphaera, another green microalga of the phylum Chlorophyta, indicating its applicability for broader use beyond the Chlorella genus. Furthermore, the combination of both methods is proposed as a useful tool to evaluate recalcitrant glucose moieties in microalgal biomass.

Effect of ultrasonic treatment on the physicochemical properties and antioxidant activities of polysaccharide from Cyclocarya paliurus

The effects of ultrasonic treatment on the physicochemical properties and antioxidant activities of the polysaccharide from Cyclocarya paliurus leaves were investigated. The physicochemical properties were detected by high-performance gel permeation chromatography (HPGPC), high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) and fourier transform infrared spectrometer (FT-IR) as well. The antioxidant activities were evaluated by the assay of DPPH, hydroxyl free radical, β-carotene-linoleic acid, respectfully. Results showed that no considerable change was observed on the structure between the polysaccharide of Cyclocarya paliurus (CP) and the ultrasonic polysaccharide of Cyclocarya paliurus (UCP) by FT-IR, and no apparent differences were found on the content of sugar, uronic acid and protein after the ultrasonic treatment, but difference appeared in the ratio of monosaccharide composition of CP (1.0:4.6:3.5:4.5) and UCP (1.0:5.9:3.9:4.4), and the antioxidant activities of CP were promoted after ultrasonic treatment.

Purification, characterization and in vitro anticoagulant activity of polysaccharides from Gentiana scabra Bunge roots

Three water-soluble polysaccharide fractions (GSP-1, GSP-2 and GSP-3) were obtained from Gentiana scabra Bunge roots by DEAE-Sepharose CL-6B and Sepharose CL-6B column chromatography. Their chemical characterizations were determined by high performance gel permeation chromatography (HPGPC), high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) and Fourier transform infrared (FT-IR) spectrometer. Moreover, their in vitro anticoagulant activities were evaluated by activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) assays. GSP-1 and GSP-2 were composed of rhamnose, arabinose, galactose, glucose and galacturonic acid, while GSP-3 consisted of rhamnose, arabinose, galactose and galacturonic acid with a weight-average molecular weight of 5.8×104Da. In comparison with the control group (saline), GSP, GSP-1, GSP-2 and GSP-3 could prolong APTT and TT, but not PT. Overall, GSP-3 exhibited potent anticoagulant activity and would be expected to be a potential source of anticoagulant.

Tyrosine-directed conjugation of large glycans to proteins via copper-free click chemistry.

We have demonstrated that the insertion of alkyne-containing bifunctional linkers into the tyrosine residues of the carrier protein, followed by the copper mediated azide-alkyne [3 + 2] cycloaddition of carbohydrates, is a robust approach for the preparation of glycoconjugates with defined glycans, carrier, and connectivity. Conjugation of Group B Streptococcus (GBS) capsular polysaccharides to streptococcal pilus protein could extend the vaccine coverage to a variety of strains. Application of our protocol to these large charged polysaccharides occurred at low yields. Herein we developed a tyrosine-directed conjugation approach based on the copper-free click chemistry of sugars modified with cyclooctynes, which enables efficient condensation of synthetic carbohydrates. Most importantly, this strategy was demonstrated to be more effective than the corresponding copper catalyzed reaction for the insertion of GBS onto the tyrosine residues of GBS pilus proteins, previously selected as vaccine antigens through the so-called reverse vaccinology. Integrity of protein epitopes in the modified proteins was ascertained by competitive ELISA, and conjugation of polysaccharide to protein was confirmed by SDS page electrophoresis and immunoblot assays. The amount of conjugated polysaccharide was estimated by high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). The described technology is particularly suitable for proteins used with the dual role of vaccine antigen and carrier for the carbohydrate haptens.