Natural Deep Eutectic Solvents for Solubility and Selective Fractionation of Bioactive Low Molecular Weight Carbohydrates.

Natural deep eutectic solvents (NADESs) have been shown to be selective and environmentally friendly solvents for the extraction of bioactive compounds. However, studies on the solubility of low-molecular-weight carbohydrates (LMWCs) in NADESs are scarce. In this work, new solubility data of LMWCs in NADESs are provided and a new approach based on the use of these solvents for the efficient fractionation of bioactive carbohydrates was explored for the first time. Several mono- and disaccharides and three NADESs based on choline chloride (ChCl) and different donors (2-ethylene glycol (EtG), glycerol (Gly) and ethanedioic acid dihydrate (Eth)) were considered. While the degradation of carbohydrates, mainly ketoses, was detected with ChCl:Eth due to its acidic nature, ChCl:EtG and ChCl:Gly were found to be useful alternatives for selectively separating bioactive ketoses and their corresponding aldoses (e.g., lactulose/lactose and tagatose/galactose) present in equimolar binary mixtures. In addition, the usefulness of ChCl:EtG for the selective enrichment of lactulose to be used as food ingredient or nutraceutical was proven (from a 25% in the reaction mixture to a 56% in the purified sample). NADESs could be used for the selective fractionation of value-added carbohydrates from interfering sugars for several applications, including food science, engineering or pharmaceuticals.

Quality Evaluation of Dietary Supplements for Weight Loss Based on Garcinia cambogia.

Food supplements of plant origin for weight control are increasingly being demanded by consumers as a way to promote good health. Among them, those based on Garcinia cambogia (GCFS) are widely commercialized considering their bioactive properties, mainly due to (-)-hydroxycitric acid ((-)-HCA). However, recently, controversy has arisen over their safety; thus, further research and continuous monitoring of their composition is required. Hence, in this work, a multi-analytical approach was followed to determine not only (-)-HCA but also other constituents of 18 GCFS, which could be used as quality markers to detect fraudulent practices in these samples. Discrepancies between the declared (-)-HCA content and that experimentally determined were detected by LC-UV in 33% of the samples. Moreover, GC-MS analyses of GCFS allowed the detection of different compounds not present in G. cambogia fruits and not declared on supplement labels, probably related to heat exposure or to the addition of excipients or other extracts. This multi-analytical methodology is shown to be advantageous to address different fraudulent practices affecting the quality of these supplements.

Development of a multianalytical strategy for detection of frauds in Coleus forskohlii supplements.

A new multianalytical methodology based on gas chromatography (GC) and liquid chromatography (LC) coupled to mass spectrometry (MS) has been proposed to evaluate frauds affecting the composition of Coleus forskohlii root supplements (FKS). After optimization and validation of chromatographic methods, 24 FKS were analyzed. Forskolin, their main bioactive component, was only found in 50% of the FKS evaluated (in the 0.032-17.1% range), with 27% of these supplements showing concentrations of this bioactive lower than those declared in their labels. Application of this methodology also proved to be successful for the detection of frauds regarding the replacement of C. forskohlii by other vegetable sources (green tea, soy leaves and a plant of the Berberidaceae family) in 17% of supplements analyzed. A study on stability of forskolin under accelerated conditions allowed to rule out its degradation as responsible for the lack of this bioactive or other natural constituents in 25% of FKS evaluated. It can be concluded that the multianalytical methodology here developed is an advantageous alternative to address the wide diversity of frauds affecting these supplements.

A multi-analytical strategy for evaluation of quality and authenticity of artichoke food supplements for overweight control.

Despite the widespread use of artichoke-based food supplements for obesity control (FSOC), studies on evaluation of the quality/authenticity of these commercial products are scarce. To that aim, a new multi-analytical strategy, based on the use of gas chromatography coupled to mass spectrometry (GC-MS) and high performance liquid chromatography coupled to ultraviolet and mass spectrometry detection (HPLC-UV-MS), in combination with chemometrics, has been developed. Twenty-one artichoke FSOC and different bract and leaf extracts (used as reference samples) were analysed. Sugars, inositols, caffeoylquinic acids, dicaffeoylquinic acids, flavonoids and their glycosides were detected in reference samples and in most artichoke FSOC. Low concentrations of bioactives, and the presence of other compounds probably related to heat treatment during manufacturing (difructosyl anhydrides, 3-deoxyglucosone), or to the addition of caloric additives (maltose, maltotriose) or non-declared plants (e.g. pinitol, disaccharides, silybin derivatives) were also detected in some FSOC by either GC-MS or HPLC-UV-MS. Application of Principal Component Analysis to the combined GC-MS + HPLC-UV data matrix, proved that this multi-analytical strategy provides advantages over single analytical techniques for the detection of the wide variety of fraudulent practices affecting authenticity of artichoke FSOC and for assessment of their quality.

Microwave Assisted Extraction of Bioactive Carbohydrates from Different Morphological Parts of Alfalfa (Medicago sativa L.).

Despite the nutritional properties of alfalfa, its production is mainly for animal feed and it is undervalued as a food source. In this study, the valorization of alfalfa as a potential source of bioactive carbohydrates [inositols, α-galactooligosaccharides (α-GOS)] is presented. A Box-Behnken experimental design was used to optimize the extraction of these carbohydrates from leaves, stems, and seeds of alfalfa by solid-liquid extraction (SLE) and microwave-assisted extraction (MAE). Optimal extraction temperatures were similar for both treatments (40 °C leaves, 80 °C seeds); however, SLE required longer times (32.5 and 60 min vs. 5 min). In general, under similar extraction conditions, MAE provided higher yields of inositols (up to twice) and α-GOS (up to 7 times); hence, MAE was selected for their extraction from 13 alfalfa samples. Pinitol was the most abundant inositol of leaves and stems (24.2-31.0 mg·g-1 and 15.5-22.5 mg·g-1, respectively) while seed extracts were rich in α-GOS, mainly in stachyose (48.8-84.7 mg·g-1). In addition, inositols and α-GOS concentrations of lyophilized MAE extracts were stable for up to 26 days at 50 °C. These findings demonstrate that alfalfa is a valuable source of bioactive carbohydrates and MAE a promising alternative technique to obtain functional extracts.

Separation of di- and trisaccharide mixtures by comprehensive two-dimensional liquid chromatography. Application to prebiotic oligosaccharides.

Carbohydrates are one of the most important ingredients in foods. They are normally present as complex mixtures with different glycosidic linkages, monomeric units and degrees of polymerization. This structural heterogeneity impairs their comprehensive characterization and requires the use of analytical techniques with high resolving power and sensitivity. The use of chromatographic techniques, especially liquid chromatography (LC), has been extremely helpful for the analysis of carbohydrates. However, in many cases, the use of monodimensional LC is not enough to resolve these complex mixtures; then, the use of techniques with a higher resolving power, as multidimensional LC, could be a good alternative. To the best of our knowledge, our findings are pioneer in applying online LC × LC for the analysis of carbohydrate mixtures. For this purpose, different conditions such as stationary phases (BEH amide, C18 and PGC columns) and chromatographic conditions for the separation of di- and trisaccharide mixtures were optimized. The BEH amide × C18 combination was selected for the LC × LC analysis of carbohydrate standards with different degrees of polymerization, linkages and monomeric units. In order to allow their proper UV detection, carbohydrates were previously derivatized using p-aminobenzoic ethyl ester. This method also resulted to be successful for the separation of commercial prebiotic mixtures of galacto-oligosaccharides and gentio-oligosaccharides. This is the first time that LC × LC has been applied for the separation of bioactive carbohydrate mixtures and it could be considered as a powerful analytical technique for the characterization of other oligosaccharide complex mixtures.

Extraction and characterization of low molecular weight bioactive carbohydrates from mung bean (Vigna radiata).

Due to the great interest in obtaining natural bioactive carbohydrates to be used as functional ingredients, a selective microwave assisted extraction (MAE) method was optimized to ensure the exhaustive extraction of inositols and α-galactooligosaccharides (α-GOS) from mung bean. Thereafter, a comprehensive characterization of these compounds was carried out by gas chromatography coupled to mass spectrometry (GC-MS). Apart from free inositols and α-GOS, several glycosyl-methyl-scyllo-inositols and glycosyl-inositols were detected for the first time in this legume. Under optimized MAE conditions (0.5 g dry sample, 2 cycles of 3 min, 50 °C, 10 mL 50:50 ethanol:water, v:v), bioactive carbohydrates yields were similar to those found using solid-liquid extraction (SLE), but with shorter analytical times. Concentrations of bioactive carbohydrates in MAE extracts from samples of different geographical origins ranged between 74.1 and 104.2 mg.g-1 dry sample. MAE was proved a good alternative to SLE to obtain extracts enriched in bioactive carbohydrates.

A new method for microwave assisted ethanolic extraction of Mentha rotundifolia bioactive terpenoids.

A new microwave-assisted extraction (MAE) method using ethanol as solvent has been optimized by means of a Box-Behnken experimental design for the enhanced extraction of bioactive terpenoids from Mentha rotundifolia leaves; 100°C, 5 min, 1.125 g dry sample: 10 mL solvent and a single extraction cycle were selected as optimal conditions. Improved performance of MAE method in terms of extraction yield and/or reproducibility over conventional solid-liquid extraction and ultrasound assisted extraction was also previously assessed. A comprehensive characterization of MAE extracts was carried out by GC-MS. A total of 46 compounds, mostly terpenoids, were identified; piperitenone oxide and piperitenone were the major compounds determined. Several neophytadiene isomers were also detected for the first time in MAE extracts. Different procedures (solid-phase extraction and activated charcoal (AC) treatment) were also evaluated for clean-up of MAE extracts, with AC providing the highest enrichment in bioactive terpenoids. Finally, the MAE method here developed is shown as a green, fast, efficient and reproducible liquid extraction methodology to obtain M. rotundifolia bioactive extracts for further application, among others, as food preservatives.

Enzymatic Synthesis and Structural Characterization of Theanderose through Transfructosylation Reaction Catalyzed by Levansucrase from Bacillus subtilis CECT 39.

This work addresses the high-yield and fast enzymatic production of theanderose, a naturally occurring carbohydrate, also known as isomaltosucrose, whose chemical structure determined by NMR is α-d-glucopyranosyl-(1 → 6)-α-d-glucopyranosyl-(1 → 2)-ß-d-fructofuranose. The ability of isomaltose to act as an acceptor in the Bacillus subtilis CECT 39 levansucrase-catalyzed transfructosylation reaction to efficiently produce theanderose in the presence of sucrose as a donor is described by using four different sucrose:isomaltose concentration ratios. The maximum theanderose concentration ranged from 122.4 to 130.4 g L-1, was obtained after only 1 h and at a moderate temperature (37 °C), leading to high productivity (109.7-130.4 g L-1h-1) and yield (up to 37.3%) values. The enzymatic synthesis was highly regiospecific, since no other detectable acceptor reaction products were formed. The development of efficient and cost-effective procedures for the biosynthesis of unexplored but appealing oligosaccharides as potential sweeteners, such as theanderose, could help to expand its potential applications which are currently limited by their low availability.

Assessment of Maillard reaction evolution, prebiotic carbohydrates, antioxidant activity and α-amylase inhibition in pulse flours.

In this paper, the quality of bean, chickpea, fava beans, lentil and pea flours from Algeria has been evaluated. Maillard reaction (MR) indicators, modifications in the carbohydrate and protein fractions, antioxidant activity and α-amylase inhibitor of raw, toasted and stored samples were evaluated. Fava beans, beans and peas showed higher content of raffinose family oligosaccharides while chickpeas and lentils showed higher polyol content. Toasting and storage caused slightly change in pulse quality; MR showed slight losses of lysine but increased antioxidant activity. Moreover, inhibition of α-amylase was slightly augmented during processing; this could increase the undigested carbohydrates that reach the colon, modulating the glycemic response. These results point out the suitability of these flours for preparing high-quality foodstuffs intended for a wide spectrum of the population, including hyperglycemic and gluten intolerant individuals.

Characterization of post-translationally modified peptides by hydrophilic interaction and reverse phase liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry.

This work explores the use of both hydrophilic interaction liquid chromatography (HILIC) and reverse phase liquid chromatography (RPLC) for the separation and subsequent characterization of bovine caseinomacropeptide (CMP) phosphopeptides and O-glycopeptides using a quadrupole-time-of-flight (QTOF) mass spectrometer with electrospray ionization. Two neutral, ethylene bridged hybrid (BEH) amide and polyhydroxyethyl aspartamide (PHEA), and a zwitterionic, sulfobetaine (ZIC), stationary phases were used for the HILIC mode, whilst an octadecylsilane (C18) stationary phase was employed for the RPLC separation. Overall, developed HILIC-QTOF method using the ZIC or BEH amide stationary phases resulted to be the most efficient methods to separate and characterize post-translationally modified (PTM) peptides without the need of any previous fractionation or derivatization step. The separation of phosphopeptides and differently sialylated O-glycopeptides in the ZIC stationary phase was dominated by an electrostatic repulsion interaction mechanism between the negatively charged phosphate groups or sialic acid moieties and the negatively charged terminal sulfonate group of the stationary phase, whereas the separation of either non-modified peptides or neutral O-glycopeptides both free of basic amino acids was based on a partitioning mechanism. In neutral amide columns, the separation was mainly dominated by hydrophilic partitioning, leading to a higher retention of the post-translationally modified peptides than the unmodified counterparts due to the hydrophilicity provided by the phosphate groups and/or O-glycans. As a consequence, HILIC-ESI-QTOF MS operating in the positive ion mode is a powerful tool for the characterization of underivatized O-glycopeptides and phosphopeptides.

Hydrolyzed caseinomacropeptide conjugated galactooligosaccharides support the growth and enhance the bile tolerance in Lactobacillus strains.

In this study bioactive caseinomacropeptide was conjugated with prebiotic galactooligosaccharides (hCMP:GOS) by Maillard reaction to synthesize value added prebiotic compounds to Lactobacillus strains. Growth study showed the ability of hCMP:GOS to serve as a sole carbon source for Lactobacillus strains. A significant amount of acetate and lactate was detected in cell free culture supernatant by HPLC. It demonstrated the ability of Lactobacillus strains to ferment the hCMP:GOS as a carbon source. In addition, hCMP:GOS grown Lactobacillus cells exhibited enhanced bile tolerance and retained 90% viability. Overall results of this study indicate that the hCMP conjugated GOS can be potential multipurpose prebiotic substrates to enhance the growth and bile tolerance in Lactobacillus strains and serve as a fermentable substrate to produce beneficial metabolites in the host.

Evaluation of different operation modes of high performance liquid chromatography for the analysis of complex mixtures of neutral oligosaccharides.

Chromatographic methods based on different HPLC operation modes, reverse phase (RP), high performance anion exchange chromatography (HPAEC), graphitized carbon chromatography (GCC) and hydrophilic interaction liquid chromatography (HILIC), have been developed and compared for the analysis of complex mixtures of neutral oligosaccharides with functional properties. Whereas GCC gave the best chromatographic separation of isomeric oligosaccharides with the same molecular weight (R(s) values in the range 1.0-4.0 and 2.4-5.6 for tetra- and pentasaccharides, respectively), HILIC provided the best results for mixtures including oligosaccharides of different degrees of polymerization (R(s) values of maltooligosaccharides between 3.4 and 6.2). Validation of the HILIC LC-MS method proved its utility for the analysis of oligosaccharide mixtures with functional properties: relative standard deviations lower than 10%, LOD's and LOQ's in the range 12.7-130.2 ng mL(-1) and 39.3-402.2 ng mL(-1), respectively, and linearity up to 10-20 µg mL(-1). Quantitative data for fructooligosaccharides, gentiooligosaccharides and dextransucrase cellobiose acceptor oligosaccharides were obtained by using this method.

Determination of free inositols and other low molecular weight carbohydrates in vegetables.

Different low molecular weight carbohydrates including saccharides, polyalcohols, sugar acids, and glycosides have been identified and quantified in different edible vegetables from Asteraceae, Amarantaceae, Amarylidaceae, Brassicaceae, Dioscoreaceae, and Solanaceae families by gas chromatography-mass spectrometry. Apart from glucose, fructose, and sucrose, other saccharides such as sedoheptulose in chicory, spinach, cabbage, purple yam, eggplant, radish, and oak leaf lettuce, rutinose in eggplant skin, and a glycosyl-inositol in spinach have been identified. chiro-Inositol was found in all vegetables of the Asteraceae family (3.1-32.6 mg 100 g(-1)), whereas scyllo-inositol was detected in those of purple yam, eggplant, artichoke, chicory, escarole, and endive (traces-23.2 mg 100 g(-1)). α-Galactosides, kestose, glucaric acid, and glycosyl-glycerols were also identified and quantified in some of the analyzed vegetables. Considering the bioactivity of most of these compounds, mainly chicory leaves, artichokes, lettuces, and purple yam could constitute beneficial sources for human health.

Separation of disaccharides by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry. Application to honey analysis.

A new method based on comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-ToF MS) has been developed for the first time for the analysis of complex mixtures of disaccharides previously converted to their trimethylsilyl oximes (TMSO). Among the different experimental parameters considered for optimization, both the column set combination and the dimensions of the second-dimension column were found to be the most significant with regard to the complete resolution of structurally similar disaccharides. Application of the optimized method to honey analysis allowed the separation of most of the honey disaccharides previously described in the literature. Furthermore, 12 other unknown disaccharides have been separated by this method and characterized from their mass spectral data.

Development of a new method using HILIC-tandem mass spectrometry for the characterization of O-sialoglycopeptides from proteolytically digested caseinomacropeptide.

This work addresses the optimization of HILIC (hydrophilic interaction liquid chromatography)-ESI-MS(n) conditions for the comprehensive characterization of O-glycopeptides from proteolytically digested caseinomacropeptide. O-Glycopeptides were satisfactorily analysed on a zwitterionic HILIC column based on their glycan structure and amino acid sequence. The contribution of ionic interactions to the retention of charged glycopeptides was found to be substantial. Thus, O-glycopeptides carrying neutral glycans were more retained than O-sialoglycopeptides because negatively charged sialic acid residues were electrostatically repelled by the stationary phase. In addition, glycopeptides differing only in the position of the linkage of the sialic acid moiety could be separated. The same chromatographic behaviour was observed for model systems constituted by a synthetic tetrapeptide covalently conjugated to neutral and sialylated carbohydrates. Subsequent detection of caseinomacropeptide O-glycopeptides was carried out on an electrospray ion trap tandem mass spectrometer at both positive and negative ionization modes. MS(2) fragmentation at positive ionization mode was valid for determining the glycan structure as the resulting main fragments corresponded to Y(n)-type ions derived from sequential glycosidic bond fragmentation, whilst the fragmentation of the peptide structure was preferably obtained through the formation of b(n)-type ions at the MS(3) stage, allowing the complete structure elucidation of the peptidic chain. Overall, the developed method allowed the identification and characterization of 41 O-glycopeptides covering all the known glycosylation sites without any previous enrichment step. These results point out that HILIC coupled to multistage MS procedures can be a powerful technique for future glycoproteomic applications.

Carbohydrate composition of high-fructose corn syrups (HFCS) used for bee feeding: effect on honey composition.

In this study, the carbohydrate composition of high-fructose corn syrups (HFCS) from commercial manufacturers as well as from beekeepers was characterized by GC-MS. Sucrose syrups (SS) were also included in this work for comparison. Fructosyl-fructoses and some unknown carbohydrates, which could correspond to fructosyl-glucoses, have been detected in HFCS for the first time, whereas SS were mainly characterized by the high contents of sucrose. Hydroxymethylfurfural (HMF) content of samples supplied by beekeepers was much more variable; the mean level of HMF was 64.61 ppm (+/-16.92 ppm, 95% CI ranging from 26.91 to 102.31 ppm). Syrups were used to feed caged bees and the resulting honeys produced were analyzed in order to determine their influence in carbohydrate composition. Fructosyl-fructoses were mainly detected in honeys from bees fed with HFCS, but not from those honeys coming from free-flying bees or bees fed with SS.

Characterization of O-trimethylsilyl oximes of trisaccharides by gas chromatography-mass spectrometry.

Gas chromatography (GC) data (linear retention indices and relative areas) and mass spectra (most representative m/z fragments) of 12 reducing trisaccharides as trimethylsilyl oximes (TMSO) and four non-reducing as trimethylsilyl (TMS) ethers have been described for the first time and related to their structural features. Some trends have been observed: earlier elution of non-reducing compounds and fructotrioses; aldotrioses bearing the reducing end with link in position 6 showing the highest retention. Abundance of several fragment ions and their ratios were useful for trisaccharide characterization; some of these features seem to be useful for the characterization of new trisaccharides.

Mass spectrometric characterization of glycated beta-lactoglobulin peptides derived from galacto-oligosaccharides surviving the in vitro gastrointestinal digestion.

A mass spectrometric study has been carried out to elucidate the structures of glycated peptides obtained after in vitro gastrointestinal digestion of bovine beta-lactoglobulin (beta-LG) glycated with prebiotic galacto-oligosaccharides (GOS). The digests of both native and glycated beta-LG were analyzed by MALDI-MS, LC-ESI-MS, and LC-ESI-MS/MS. MALDI-MS profiles showed marked differences mainly related to the lower intensity of ions corresponding to the digest of glycated beta-LG. Overall, 58 and 23 unglycated peptides covering 97% and 63% of the mature beta-LG sequence could be identified in the digests of native and glycated samples, respectively. The LC-ESI-MS analyses corroborated the MALDI-MS results regarding the unglycated peptides but they also enabled an extensive investigation into the digest of glycated beta-LG. Thus, a total of 19 peptides glycated with GOS from two to seven hexose units could be identified. The tandem mass spectra of glycated peptides were mostly characterized by two neutral losses of 1026/1056, 864/894, 702/732, 540/570, 378/408, and 216/246 u, corresponding to the formation of the furylium ion and its subsequent "CHOH" loss, indicative of the peptide glycation with hepta-, hexa-, penta-, tetra-, tri-, and disaccharides, respectively. Also, other minor ionic species containing the furylium ring linked to different galactose units could be also detected, showing the diversity of the fragmentation pattern of peptides glycated with larger size carbohydrates. Finally, the putative GOS glycation sites could be determined at the NH(2)-terminal Leu residue and at Lys residues located in positions 14, 47, 75, 77, 83, 91, 100, 135, and 138.

Simultaneous analysis of lysine, Nepsilon-carboxymethyllysine and lysinoalanine from proteins.

Protein quality was assayed by simultaneous measurement of lysine (Lys), carboxymethyllysine (CML) and lysinoalanine (LAL). GC-FID analysis of N-tert-butyl dimethylsilyl (tBDMSi) derivatives of these amino acids was undertaken. tBDMSi derivates were separated on a CP-SIL 5CB commercially fused silica capillary column (25 m x 0.25 mm i.d., 0.25 microm film thickness) employing a thermal gradient programmed from 200 to 300 degrees C. The identity of tBDMSi derivatives of Lys, CML and LAL was established by GC-MS while FID detection was employed for quantification. Analytical parameters such as linearity (lysine 350-4200 microM, LAL 3-81 microM, CML 16-172 microM), precision (1-13% variation coefficients), accuracy (85-108% average recovery) and limits of detection (lysine 0.4 mg/100 g protein, LAL 5.0 mg/100 g protein, CML 3.4 mg/100 g protein) and quantification (lysine 1.4 mg/100g protein, LAL 15.2 mg/100 g protein, CML 11.2 mg/100 g protein) were determined for validation of the analytical approach. Model systems and real foods have been studied. Kinetic of CML formation from different food proteins (BSA, soy protein, casein and gluten) was performed employing model systems. Carboxymethylation rate depended on the source of protein. Maillard reaction progressed to advanced stages damaging the protein quality of stored infant foods, soy drinks, boiled eggs and dry powdered crepes. CML values ranged from 62 to 440 mg/100 g protein were measured. LAL was also formed during boiling eggs (21-68 mg/100g protein) indicating additional damage by crosslinking reaction. In agreement, lysine content was affected by both food processing and storage.