A Combined Gas and Liquid Chromatographic Approach for Quality Evaluation of Saffron-Based Food Supplements.

Considering the interest in the bioactive properties of saffron (Crocus sativus L.), as well as its limited production and high price, saffron-based food supplements (SFS) are highly susceptible to adulteration. However, their complex composition and the wide variety of potential fraudulent practices make the comprehensive assessment of SFS quality a challenging task that has been scarcely addressed. To that aim, a new multianalytical strategy based on gas chromatography coupled to mass spectrometry (GC-MS) and liquid chromatography with diode array detection coupled to mass spectrometry (HPLC-DAD-MS) was developed and validated in order to detect different frauds affecting SFS. Dried saffron stigmas and a commercial standardized saffron extract (affron®) were selected as reference samples (RS) to obtain an authenticity profile, which was further used to evaluate the quality of 17 SFS. Up to 17 crocins and crocetins, 5 kaempferol glycosides, picrocrocin (determined for the first time by GC-MS), safranal, furanone and isophorone-related compounds were determined in RS. Safranal and crocins were identified in all SFS except for one sample. However, discrepancies with the content declared were detected in 65% of the cases. Moreover, this multianalytical methodology also allowed identifying undeclared additives and the non-declared addition of vegetable sources other than saffron.

Fingerprint of Characteristic Saffron Compounds as Novel Standardization of Commercial Crocus sativus Extracts.

Food supplements based on saffron (Crocus sativus L.) dried stigma extracts are widely consumed due to their multiple bioactive properties. Saffron extract (SE) standardization is of crucial importance, as it determines the reproducibility of the product quality and is essential for the evaluation of its bioactive effect and safety. Although SEs are commonly standardized considering their safranal content, the lack of specificity of the official methods may give inaccurate measurements. In addition to the development of more precise methodologies, the evaluation of alternative saffron components, such as crocins and picrocrocin, for standardization purposes would also be of interest. Thus, in this study, qualitative and quantitative information regarding picrocrocin and crocin isomers of different commercial saffron extracts was first obtained by a validated methodology using liquid chromatography (HPLC) coupled to diode array (DAD) and mass spectrometer (MS) detectors. Principal component analysis (PCA) was applied to gain insight into the compositional variability and natural grouping of SE. These studies suggested the potential use of the relative content of crocin isomers and trans-/cis-crocins and trans-4 GG/picrocrocin ratios as novel criteria for SE standardization. Their reproducibility and stability under controlled storage conditions for 36 months was demonstrated in a commercial standardized SE (affron®).

Development of a microwave-assisted extraction method for the recovery of bioactive inositols from lettuce (Lactuca sativa) byproducts.

A microwave-assisted extraction (MAE) method was developed for the extraction of bioactive inositols (D-chiro- and myo-inositols) from lettuce (Lactuca sativa) leaves as a strategy for the revalorization of these agrofood residues. Gas chromatography-mass spectrometry was selected for the simultaneous determination of inositols and sugars (glucose, fructose, and sucrose) in these samples. A Box-Behnken experimental design was used to maximize the extraction of inositols based on the results of single factor tests. Optimal conditions of the extraction process were as follows: liquid-to-solid ratio of 100:1 v/w, 40°C, 30 min extraction time, 20:80 ethanol:water (v/v), and one extraction cycle. When compared with conventional solid-liquid extraction (SLE), MAE was found to be more effective for the extraction of target bioactive carbohydrates (MAE 5.42 mg/g dry sample versus SLE 4.01 mg/g dry sample). Then, MAE methodology was applied to the extraction of inositols from L. sativa leaves of different varieties (var. longifolia, var. capitata and var. crispa). D-chiro- and myo-inositol contents varied between 0.57-7.15 and 0.83-3.48 mg/g dry sample, respectively. Interfering sugars were removed from the extracts using a biotechnological procedure based on the use of Saccharomyces cerevisiae for 24 h. The developed methodology was a good alternative to classical procedures to obtain extracts enriched in inositols from lettuce residues, which could be of interest for the agrofood industry.

Evaluation of different ionic liquid stationary phases for the analysis of carbohydrates by gas chromatography-mass spectrometry.

Five commercial ionic liquid (IL) columns have been evaluated for the first time for the gas chromatography-mass spectrometry (GC-MS) analysis of low molecular weight carbohydrate (LMWC) standards (mono-, di-, and trisaccharides, inositols, and iminosugars). A previous derivatization step was necessary to convert the LMWCs into their volatile and stable derivatives. Compared with conventional GC stationary phases, such as HP-1 and Supelcowax® 10, IL columns have shown a different selectivity in the separation of target compounds. Among the IL columns, only SLB™-IL82 allowed the elution of all the LMWCs studied. Its performance in terms of peak width and asymmetry, evaluated under different oven temperature conditions, was shown to be dependent on the carbohydrate class considered. As an example of application, a SLB™-IL82 column was successfully used to separate the complex mixtures of LMWCs in hyacinth and mulberry extracts. This column is an interesting alternative to the conventional stationary phases used in the GC analysis of LMWCs in real-world samples. Graphical abstract.

Changes in Caprine Milk Oligosaccharides at Different Lactation Stages Analyzed by High Performance Liquid Chromatography Coupled to Mass Spectrometry.

Changes of the abundance of caprine milk oligosaccharides (CMO) at different lactation stages have been evaluated by hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC-Q MS) and nanoflow liquid chromatography-quadrupole-time-of-flight mass spectrometry (nano-LC-Chip-QTOF MS). Eight major oligosaccharides (OS) were quantified at different lactation stages by HILIC-Q MS, while the use of nano-LC-Chip-QToF MS allowed expanding the study to forty-nine different OS by monitoring neutral non- and fucosylated species, as well as acidic species containing not only N-acetyl-neuraminic acid or N-glycolyl-neuraminic acid residues but also the combination of both sialic acids. Overall, the most abundant OS decreased with lactation time, whereas different trends were observed for minor OS. 6'-Sialyl-lactose was the most abundant acidic OS while galactosyl-lactose isomers were identified as the most abundant neutral OS. This is the first time that a comprehensive study regarding the changes of the abundance of CMO, both neutral and acidic, at different lactation stages is carried out.

Synthesis, optimization and structural characterization of a chitosan-glucose derivative obtained by the Maillard reaction.

Chitosan (Chit) was submitted to the Maillard reaction (MR) by co-heating a solution with glucose (Glc). Different reaction conditions as temperature (40, 60 and 80 °C), Glc concentration (0.5%, 1%, and 2%, w/v), and reaction time (72, 52 and 24h) were evaluated. Assessment of the reaction extent was monitored by measuring changes in UV absorbance, browning and fluorescence. Under the best conditions, 2% (w/v) of Chit, 2% (w/v) of Glc at 60°C and 32 h of reaction time, a chitosan-glucose (Chit-Glc) derivative was purified and submitted to structural characterization to confirm its formation. Analysis of its molecular weight (MW) and the degree of substitution (DS) was carried out by HPLC-Size Exclusion Chromatography (SEC) and a colloid titration method, respectively. FT-IR and (1)H NMR were also used to analyze the functional groups and evaluate the introduction of Glc into the Chit molecule. According to our objectives, the results obtained in this work allowed to better understand the key parameters influencing the MR with Chit as well as to confirm the successful introduction of Glc into the Chit molecule obtaining a Chit-Glc derivative with a DS of 64.76 ± 4.40% and a MW of 210.37 kDa.

Improving Properties of a Novel ß-Galactosidase from Lactobacillus plantarum by Covalent Immobilization.

A novel ß-galactosidase from Lactobacillus plantarum (LPG) was over-expressed in E. coli and purified via a single chromatographic step by using lowly activated IMAC (immobilized metal for affinity chromatography) supports. The pure enzyme exhibited a high hydrolytic activity of 491 IU/mL towards o-nitrophenyl ß-D-galactopyranoside. This value was conserved in the presence of different divalent cations and was quite resistant to the inhibition effects of different carbohydrates. The pure multimeric enzyme was stabilized by multipoint and multisubunit covalent attachment on glyoxyl-agarose. The glyoxyl-LPG immobilized preparation was over 20-fold more stable than the soluble enzyme or the one-point CNBr-LPG immobilized preparation at 50 °C. This ß-galactosidase was successfully used in the hydrolysis of lactose and lactulose and formation of different oligosaccharides was detected. High production of galacto-oligosaccharides (35%) and oligosaccharides derived from lactulose (30%) was found and, for the first time, a new oligosaccharide derived from lactulose, tentatively identified as 3'-galactosyl lactulose, has been described.

Identification and determination of 3-deoxyglucosone and glucosone in carbohydrate-rich foods.

BACKGROUND: α-Dicarbonyl compounds (α-DCs) such as 3-deoxyglucosone (3-DG) and glucosone are markers of both Maillard and degradation reactions of sugars and also of certain enzymatic processes. However, quantitation of these compounds is not straightforward when more abundant carbohydrates are present in real samples. Therefore in this work a GC/MS method was developed to separate monosaccharides, 3-DG and glucosone and applied to analyze them in carbohydrate-rich food products. Difructose anhydrides (DFAs), known markers of sugar degradation, were also determined. The effect of time and temperature in the production and storage of these compounds was also evaluated. RESULTS: Under optimized conditions, good separation between monosaccharides and α-DCs was achieved. Must syrups showed the highest concentrations of 3-DG and glucosone (average values 9.2 and 5.8 mg g(-1) respectively). Coffee substitutes based on carob, chicory and blends showed the highest content of DFAs. Heating and storage assays proved that production of 3-DG was influenced by temperature, while glucosone was more affected by storage time. CONCLUSION: The proposed method allows the rapid quantitation of 3-DG and glucosone along with carbohydrates and DFAs in different food products, which is essential to determine their degradation level. Moreover, the α-DC content in several foods is reported for the first time.

Enzymatic generation of chitooligosaccharides from chitosan using soluble and immobilized glycosyltransferase (Branchzyme).

Chitooligosaccharides possessing remarkable biological properties can be obtained by enzymatic hydrolysis of chitin. In this work, the chitosanase activity of soluble and immobilized glycosyltransferase (Branchzyme) toward chitosan and biochemical characterization are described for the first time. This enzyme was found to be homotetrameric with a molecular weight of 256 kDa, an isoelectric point of 5.3, and an optimal temperature range of between 50 and 60 °C. It was covalently immobilized to glutaraldehyde-agarose with protein and activity immobilization yields of 67% and 17%, respectively. Immobilization improved enzyme stability, increasing its half-life 5-fold, and allowed enzyme reuse for at least 25 consecutive cycles. The chitosanase activity of Branchzyme on chitosan was similar for the soluble and immobilized forms. The reaction mixture was constituted by chitooligosaccharides with degrees of polymerization of between 2 and 20, with a higher concentration having degrees of polymerization of 3-8.

Optimisation of a biotechnological procedure for selective fractionation of bioactive inositols in edible legume extracts.

BACKGROUND: Currently, disorders such as diabetes mellitus, obesity or atherosclerosis are recognised as major global health problems. The use of inositols for treating these illnesses has attracted considerable attention and their extraction from natural sources presents added value as they are considered bioactive ingredients in the food industry. Legumes are natural and rich sources of inositols; however, the co-existence of other low molecular weight carbohydrates (LMWCs) in their extracts, which interfere in their bioactivity, might constitute an important drawback, thereby making their removal essential. RESULTS: LMWCs, including inositols, methyl-inositols and glycosyl-inositols of different legume extracts, were determined by GC-MS; the presence of bornesitol (2.35 mg g(-1) ) and lathyritol (0.27 mg g(-1) ) were reported for the first time in grass peas. The use of Saccharomyces cerevisiae for the selective removal of interfering carbohydrates was optimised. Incubation time (3-40 h) was highly dependent on the composition of the legume considered; inositol contents were generally stable along the treatment. CONCLUSION: Removal of interfering LMWCs from inositol-enriched extracts was successfully achieved using a clean and easily scalable fractionation methodology. This biotechnological procedure not only represents high interest for the production of bioactive food ingredients but for applications in other research areas.

Evaluation of oligosaccharide synthesis from lactose and lactulose using ß-galactosidases from Kluyveromyces isolated from artisanal cheeses.

The ß-galactosidase activity of 15 Kluyveromyces strains isolated from cheese belonging to Kluyveromyces lactis and Kluyveromyces marxianus species was tested for the production of oligosaccharides derived from lactose (GOS) and lactulose (OsLu). All Kluyveromyces crude cell extracts (CEEs) produced GOS, such as 6-galactobiose and 3'-, 4'-, and 6'-galactosyl-lactose. At 4 h of reaction, the main trisaccharide formed was 6'-galactosyl-lactose (20 g/100 g of total carbohydrates). The formation of OsLu was also observed by all CEEs tested, with 6-galactobiose, 6'-galactosyl-lactulose, and 1-galactosyl-lactulose being found in all of the reaction mixtures. The synthesis of trisaccharides predominated over other oligosaccharides. K. marxianus strain O3 produced the highest yields of GOS and OsLu after 4 h of reaction, reaching 42 g/100 g of total carbohydrates (corresponding to 80% lactose hydrolysis) and 45 g/100 g of total carbohydrates (corresponding to 87% lactulose hydrolysis), respectively. Therefore, the present study contributes to a better insight into dairy Kluyveromyces ß-galactosidases and shows the feasibility of these enzymes to transglycosylate lactose and lactulose, producing high yields of prebiotic oligosaccharides.

Effect of dextransucrase cellobiose acceptor products on the growth of human gut bacteria.

The selective fermentation by human gut bacteria of gluco-oligosaccharides obtained from the reaction between the glucosyl group of sucrose and cellobiose, catalyzed by dextransucrases (DSR) from Leuconostoc mesenteroides , has been evaluated. Oligosaccharides were fractionated according to their molecular weight, and their effect on the growth of different bacterial groups was studied. To determine the structure (position and configuration of glycosidic linkages)-function relationship, their properties were compared to those of DSR maltose acceptor products (DSRMal) and of recognized prebiotic carbohydrates (fructo-oligosaccharides, FOS). Cellobiose acceptor products (DSRCel) showed bifidogenic properties similar to those of FOS. However, no significant differences related to molecular weight or isomeric configurations were found for DSRCel and DSRMal products.

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.

Analysis of cyclitols in different Quercus species by gas chromatography-mass spectrometry.

BACKGROUND: The carbohydrate profile of some woods used for aging wines and spirits has been recently studied using a pressurized liquid extraction method, the main differences found being related to cyclitol content. The aim of this study was to perform a detailed study of these compounds in woods of different Quercus species in order to identify two unknown compounds which appeared in the extracts and to verify whether the obtained profile was homogeneous for other Quercus species. RESULTS: Besides the known monosaccharides and five cyclitols previously described, three deoxy-inositols (epi-, vibo- and scyllo-quercitol) were identified. The presence of these eight cyclitols was confirmed in all subgenera and species of Quercus analyzed, allowing a characteristic cyclitol profile. CONCLUSIONS: Three deoxy-inositols (quercitols) have been identified in the carbohydrate profile of oak wood. All examined Quercus species displayed a common profile consisting of four inositols and four quercitols, which represent a good dataset for characterization of this genus.