HPAEC-PAD Analytical Evaluation of Carbohydrates Pattern for the Study of Technological Parameters Effects in Low-FODMAP Food Production.

BACKGROUND: "FODMAPs" (fermentable-oligo-, di-, monosaccharides, and polyols) are a group of fermentable carbohydrates and polyols largely diffused in food products. Despite their beneficial effects as prebiotics, people affected by irritable bowel syndrome manifest symptoms when eating these carbohydrates. A low-FODMAP diet seems to be the only possible therapy proposed for symptom management. Bakery products are a common source of FODMAPs, whose pattern and total amount can be affected by their processing. This work aims at studying some of the technological parameters that can influence the FODMAPs pattern in bakery products during the production process. METHODS: high-performance anion exchange chromatography coupled to a pulsed amperometric detector (HPAEC-PAD) was used as a highly selective system for carbohydrates evaluation analyses on flours, doughs, and crackers. These analyses were performed using two different columns, the CarboPac PA200 and CarboPac PA1, which are selective for oligosaccharide and simple sugar separation, respectively. RESULTS: emmer and hemp flours were selected to prepare doughs as they contained low oligosaccharide content. Two different mixes of ferments were used at different times of fermentation to evaluate the best conditions to achieve low-FODMAP crackers. CONCLUSION: the proposed approach allows carbohydrate evaluation during crackers processing and permits the selection of opportune conditions to obtain low-FODMAP products.

Oligomers: Hidden sources of bisphenol A from reusable food contact materials.

A recent European Regulation further restricted the use of bisphenol A in food-contact materials, reducing its limit of migration. However, all analytical systems of control are aimed at identifying and quantifying the molecules of this monomer without taking in consideration the possible presence of its oligomers, species originating from material degradation and able to follow an in-vivo hydrolysis providing bisphenol A generation. Thus, the presence of oligomers of polycarbonate deriving by unreacted species or polymer degradation can be considered a hidden source of several bisphenol A units, that remains outside the control of legislation and should be considered of high concern. This work was focused on the identification and the description of kinetics of release of different molecules migrating from polycarbonate food contact materials to simulants and to a model food sample such as melted chocolate. Analyses were performed by UHPLC system coupled to a Q-Exactive mass spectrometer. Targeted and untargeted analysis through data dependent acquisition mode allowed to detect the occurrence of several species deriving from polycarbonate, and permitted to investigate the polymer degradation pattern and explore the correlation of the recorded amounts of each product with age, hours of usage, and washing cycles of the plastic items.

Optimization of mass spectrometry acquisition parameters for determination of polycarbonate additives, degradation products, and colorants migrating from food contact materials to chocolate.

The interest towards "substances of emerging concerns" referred to objects intended to come into contact with food is recently growing. Such substances can be found in traces in simulants and in food products put in contact with plastic materials. In this context, it is important to set up analytical systems characterized by high sensitivity and to improve detection parameters to enhance signals. This work was aimed at optimizing a method based on UHPLC coupled to high resolution mass spectrometry to quantify the most common plastic additives, and able to detect the presence of polymers degradation products and coloring agents migrating from plastic re-usable containers. The optimization of mass spectrometric parameter settings for quantitative analysis of additives has been achieved by a chemometric approach, using a full factorial and d-optimal experimental designs, allowing to evaluate possible interactions between the investigated parameters. Results showed that the optimized method was characterized by improved features in terms of sensitivity respect to existing methods and was successfully applied to the analysis of a complex model food system such as chocolate put in contact with 14 polycarbonate tableware samples. A new procedure for sample pre-treatment was carried out and validated, showing high reliability. Results reported, for the first time, the presence of several molecules migrating to chocolate, in particular belonging to plastic additives, such Cyasorb UV5411, Tinuvin 234, Uvitex OB, and oligomers, whose amount was found to be correlated to age and degree of damage of the containers.

Combined effects of robot­assisted gait training and botulinum toxin type A on spastic equinus foot in patients with chronic stroke: a pilot, single blind, randomized controlled trial.

BACKGROUND: Despite the growing evidence about the use of robotic gait training in neurorehabilitation, there is a scant literature about the combined effects of this innovative technological approach and a first­line treatment for focal spasticity as botulinum toxin type A. In particular, to the best of our knowledge, no previous study evaluated if robotic gait training may enhance the antispastic effect of botulinum toxin type A. AIM: To evaluate the combined effects of robot­assisted gait training and botulinum toxin type A on spastic equinus foot in patients with chronic stroke. DESIGN: Pilot, single blind, randomized controlled trial. SETTING: University hospital. POPULATION: Twenty­two adult outpatients with spastic equinus due to chronic stroke. METHODS: Participants were randomly assigned to two groups: patients allocated to the group 1 received robot­assisted gait training (30 minutes a day for five consecutive days) after AbobotulinumtoxinA injection into the spastic calf muscles as well as patients allocated to the group 2 were only injected with AbobotulinumtoxinA into the same muscles. All patients were evaluated immediately before and one month after injection. The following outcome measures were considered: the modified Ashworth scale, the Tardieu scale and the 6-minute walking test. RESULTS: No difference was found between groups as to the modified Ashworth scale and the Tardieu scale measured at the affected ankle one month after botulinum toxin injection. A significant difference in the 6-minute walking test was noted between groups at the post­treatment evaluation (P=0.045). CONCLUSIONS: Our preliminary findings support the hypothesis that robot­assisted gait training does not enhance the effect of botulinum toxin type A on spastic equinus foot in patients with chronic stroke. CLINICAL REHABILITATION IMPACT: Our observations should be taken into account in daily clinical rehabilitation practice in order to develop effective treatment protocols based on the enhancement of antispastic drugs effect.

Correlation between different markers for the assessment of red chilli pepper powders stability during shelf-life.

Pungency and red colour of Capsicum powders deteriorate during processing and storage, resulting in a decrease in market value. Two varieties of pepper with different pungencies were monitored for capsaicinoids, colour and furosine. Aliquots were stored at room and at low temperature during one year. At low temperature all indicators were stable in both varieties, while at room temperature, redness and capsacinoids decreased significantly, while furosine increased. High correlation was found between those markers. The more pungent variety exhibited higher stability in terms of all parameters. Differences observed suggest a potential protective effect exerted by capsaicinoids on powder stability. The decrease in capsaicinoids and redness accompanied by furosine increase showed a linkage between those markers never reported before. Considering that capsaicinoids and furosine occurrence have strong impact on the nutritional profile, the findings of this work show relevant changes in the nutritional value of chilli pepper powder after storage.

Effects of alternative steeping methods on composition, antioxidant property and colour of green, black and oolong tea infusions.

Cold water steeping is reported to maximise tea health benefits, but requires long infusion time. In this work, the employment of a brief hot infusion step followed by ice addition was evaluated. The comparison of this innovative method with hot and cold steeping was investigated on green, black and oolong teas. Catechins, xanthines and gallic acid content, antioxidant power, total phenolics and colour analysis were evaluated. Hot infusion shown rapid extractive power, but relevant compound degradation. On the contrary, cold infusion extracted higher level of healthy molecules with slow kinetic. The innovative method achieved in short time similar properties of cold infusion in terms of antioxidant power. As for bioactive compounds, such as gallic acid and epigallocatechin gallate, highest values, about double than in hot infusion, were recorded for green and black teas. This steeping method may represent an alternative approach for industrial beverage preparation.

UHPLC-high-resolution mass spectrometry determination of bisphenol A and plastic additives released by polycarbonate tableware: influence of ageing and surface damage.

A new UHPLC-ESI-Orbitrap method for the identification and the quantitative determination of bisphenol A and some common additives employed in plastic manufacturing has been developed and validated. The method has been applied to evaluate the migration from 14 samples of tableware of different age and degree of surface damage, in both ethanol and isooctane (used as food simulants according to EU plastic regulation). Bisphenol A, three UV light absorbers, and one whitening agent were detected and quantified. Data were analyzed with the aim of exploring a possible correlation between bisphenol A and additives release, ageing, and surface integrity. A high correlation between age of samples, surface damage, and bisphenol A migration has been evaluated, while the release of additives was not correlated with other parameters. Obtained data showed for the first time that the release of bisphenol A seems to be more connected to ageing than to scratches and cracks occurrence. Graphical Abstract Bisphenol A and additives released by polycarbonate tableware: influence of ageing and surface damage.

Targeted and untargeted data-dependent experiments for characterization of polycarbonate food-contact plastics by ultra high performance chromatography coupled to quadrupole orbitrap tandem mass spectrometry.

Materials that come in contact with foods are potential sources of chemical food contamination. Consequently, characterization of their composition is of paramount importance considering the possible occurrence of several unknown molecules such as non-intentionally added substance (NIAS), residual monomers, degradation products, plastic additives and organic colorants. Previous studies concerning the characterization in terms of composition are focalized in the recognition of additives. To the best author's knowledge there are no scientific data about the composition of a plastic material in terms of colorants. In this work, an analytical method employing capillary ultra high-performance liquid chromatography and electrospray ionization quadrupole orbitrap high-resolution mass spectrometry (UHPLC-ESI Q-orbitrap) was exploited for characterization of polycarbonate for food contact material. Data-dependent experiments for targeted and untargeted analysis were employed after a total dissolution of polycarbonate samples and extraction of its components. The presence of common additives such as antioxidants and UV absorbers was confirmed by targeted analysis, while, the untargeted approach combined with the high mass accuracy of orbitrap technology allowed to identify for the first time some polycarbonate degradation products and the organic dyes effectively used for the coloration of plastic objects intended to come in contact with food. The present study shows the high potential of this technique in the field of material characterization aimed at food safety evaluation.

Natamycin based sol-gel antimicrobial coatings on polylactic acid films for food packaging.

In this work a comprehensive study on a new active packaging obtained by a hybrid organic-inorganic coating with antimicrobial properties was carried out. The packaging system based on polylactic acid was realised by sol-gel processing, employing tetraethoxysilane as a precursor of the inorganic phase and polyvinyl alcohol as the organic component, and incorporating natamycin as the active agent. Films with different organic-inorganic ratios (in a range between 1:19 and 1:4) were prepared, and the amount of antimycotic entrapped was found to be modulated by the sol composition, and was between 0.18 and 0.25mg/dm(2). FTIR microspectroscopic measurements were used to characterise the prepared coatings. The antifungal properties of the films were investigated against mould growth on the surface of commercial semi-soft cheese. The release of natamycin from the films to ethanol 50% (v/v) was studied by means of HPLC UV-DAD. The maximal level released was about 0.105 mg/dm(2), which is far below the value allowed by legislation.

Association between severe upper limb spasticity and brain lesion location in stroke patients.

Association between the site of brain injury and poststroke spasticity is poorly understood. The present study investigated whether lesion analysis could document brain regions associated with the development of severe upper limb poststroke spasticity. A retrospective analysis was conducted on 39 chronic stroke patients. Spasticity was assessed at the affected upper limb with the modified Ashworth scale (shoulder, elbow, wrist, and fingers). Brain lesions were traced from magnetic resonance imaging performed within the first 7 days after stroke and region of interest images were generated. The association between severe upper limb spasticity (modified Ashworth scale ≥ 2) and lesion location was determined with the voxel-based lesion-symptom mapping method implemented in MRIcro software. Colored maps representing the z statistics were generated and overlaid onto the automated anatomical labeling and the Johns Hopkins University white matter templates provided with MRIcron. Thalamic nuclei were identified with the Talairach Daemon software. Injuries to the insula, the thalamus, the basal ganglia, and white matter tracts (internal capsule, corona radiata, external capsule, and superior longitudinal fasciculus) were significantly associated with severe upper limb poststroke spasticity. Further advances in our understanding of the neural correlates of spasticity may lead to early targeted rehabilitation when key regions are damaged.

Evaluation of thermal treatment markers in wheat flour-derived products cooked in conventional and in low-emissivity ovens.

Different markers for the assessment of thermal treatment entity of food products were investigated on a model bread prepared from wheat flour. Samples were submitted to different cooking procedures by combining three different times and temperatures, and employing two different ovens: a low-emissivity oven and a conventional one. The cook value index was calculated for each sample to evaluate the entity of the thermal treatment. Furosine, maltose:maltulose ratio, colour indexes (L, a, b) have been evaluated in all samples. Furosine has been quantified by capillary electrophoresis-mass spectrometry, maltose:maltulose ratio was determined by HPAEC-PAD, colour indexes were measured by spectrophotometer method. Values for weight loss during cooking and surface temperature have also been monitored. A statistical analysis showed good correlation between the cook value index and all the parameters evaluated. Low emissivity oven shown higher performances and lower energy consumption than conventional oven.

Characterization of 12 Capsicum varieties by evaluation of their carotenoid profile and pungency determination.

In this research 12 different varieties of Capsicum cultivars belonging to three species (Capsicum chinense, Capsicum annuum, Capsicum frutescens) and of various colour, shape, and dimension have been characterised by their carotenoids and capsaicinoids content. The berries were cultivated in the region Emilia-Romagna, in Northern Italy. The native carotenoid composition was directly investigated by an HPLC-DAD-APCI-MS methodology, for the first time. In total, 52 carotenoids have been identified and considerable variation in carotenoid composition was observed among the various cultivars investigated. Among the cultivars with red colour, some Habanero, Naga morich and Sinpezon showed an high ß-carotene content, whereas Serrano, Tabasco and Jalapeno showed an high capsanthin content and the absence of ß-carotene. Habanero golden and Scotch Bonnet showed a high lutein, α-carotene and ß-carotene amounts, and Habanero orange was rich in antheraxanthin, capsanthin and zeaxanthin. Cis-cryptocapsin was present in high amount in Habanero chocolate. The qualitative and quantitative determination of the capsaicinoids, alkaloids responsible for the pungency level, has also been estimated by a validated chromatographic procedure (HPLC-DAD) after a preliminary drying step and an opportune extraction procedure. Results have also been expressed in Scoville units. Dry matter and water activity have also been established on the fresh berries. The dried peppers of each variety were then submitted to the evaluation of the total nitrogen content, measured by a Dumas system, permitting to provide information on the protein content that was found to be in the range between 7 and 16%.

Innovative analytical tools to characterize prebiotic carbohydrates of functional food interest.

Functional foods are one of the most interesting areas of research and innovation in the food industry. A functional food or functional ingredient is considered to be any food or food component that provides health benefits beyond basic nutrition. Recently, consumers have shown interest in natural bioactive compounds as functional ingredients in the diet owing to their various beneficial effects for health. Water-soluble fibers and nondigestible oligosaccharides and polysaccharides can be defined as functional food ingredients. Fructooligosaccharides (FOS) and inulin are resistant to direct metabolism by the host and reach the caecocolon, where they are used by selected groups of beneficial bacteria. Furthermore, they are able to improve physical and structural properties of food, such as hydration, oil-holding capacity, viscosity, texture, sensory characteristics, and shelf-life. This article reviews major innovative analytical developments to screen and identify FOS, inulins, and the most employed nonstarch carbohydrates added or naturally present in functional food formulations. High-performance anion-exchange chromatography with pulsed electrochemical detection (HPAEC-PED) is one of the most employed analytical techniques for the characterization of those molecules. Mass spectrometry is also of great help, in particularly matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which is able to provide extensive information regarding the molecular weight and length profiles of oligosaccharides and polysaccharides. Moreover, MALDI-TOF-MS in combination with HPAEC-PED has been shown to be of great value for the complementary information it can provide. Some other techniques, such as NMR spectroscopy, are also discussed, with relevant examples of recent applications. A number of articles have appeared in the literature in recent years regarding the analysis of inulin, FOS, and other carbohydrates of interest in the field and they are critically reviewed.

High-performance liquid chromatographic phenolic compound fingerprint for authenticity assessment of honey.

BACKGROUND: Phenolic compound profiles of 20 honeys of different botanical origin (eucalyptus, citrus, chestnut and linden) were obtained by high-performance liquid chromatography with ultraviolet detection after solid phase extraction, in order to evaluate the effectiveness of the fingerprint method for monofloral honey discrimination. RESULTS: A total of 58 peaks were detected at λ = 280 nm. Distinctive phenolic compound profiles were obtained in which both the nature and the relative amount of the detected compounds were characteristic for different botanical source honeys. In order to detect sample groupings, chromatographic peak areas were submitted to principal component analysis. Then linear discriminant analysis was carried out on the first three principal components. In addition, linear discriminant analysis was carried out on the 58 variables, allowing the selection of five variables able to discriminate honeys of different botanical origin. CONCLUSION: The chemometric evaluation of the phenolic compound profiles yielded classification models able to group honey samples according to their floral source with an excellent degree of agreement. The main advantage of the fingerprint approach with respect to traditional methods is that it does not require time-consuming identification and quantification of the analytes. The method proved to be effective for the assessment of honey authenticity.

Determination of furosine in food products by capillary zone electrophoresis-tandem mass spectrometry.

A new method for qualitative and quantitative analysis of furosine in food products by capillary electrophoresis coupled to tandem mass spectrometry (CE-MS(2) ) has been optimized and validated. In analytical conditions, the pH value of the run buffer was similar to that of the hydrolyzed samples, which helps in avoiding interactions of the analyte with silanol groups of the inner wall of the fused silica capillary. In all previous CE methods proposed in literature, no SPE treatment has been used. The method has been applied to the analysis of food products, such as flour, having low amounts of furosine. Flour samples of different origin (wheat, chestnut, lupin, einkorn, chickpeas) have been investigated. Different food products such as pasta, milk, and tigelle bread (a typical Italian unleavened bread) were also analyzed. Some of the samples have also been analyzed by reversed-phase HPLC, and when compared to CE-MS, data obtained showed good agreement. CE-MS, in comparison to HPLC, showed advantages in terms of time of analysis and cost. The validation procedure provided good values for LOD and LOQ (respectively 0.07 and 0.25 mg L(-1) ), recovery (77 and 97%), and precision investigated in terms of intraday repeatability (RSD%: 4-6% for peak areas; 1-2% for migration times) and intermediate precision (below 16% for peak areas, and below 7% for migration times). Therefore, the technique reported can be proposed as a powerful analytical method and a valid alternative to common traditional analytical techniques.

Capillary electrophoresis coupled with mass spectrometry for the evaluation of substance P enzymatic degradation by SaOS-2 human osteosarcoma.

A new analytical method for the detection and the quantitative evaluation of the undecapeptide substance P by capillary electrophoresis coupled with ion trap mass spectrometry (CE-MS) by a co-axial sheath liquid interface has been developed. Conditions of analysis employed an acidic buffer and a 60 cm fused silica capillary installed by overcoming the UV window position, thus allowing to perform the analysis in a brief time. The method has been applied to the evaluation of substance P enzymatic hydrolysis during incubation with the human osteosarcoma SaOS-2 cell line. The analysis of amino acids derived from the cleavage of substance P has been also carried out simultaneously under the same electrophoretic conditions allowing the description of a kinetic of amino acid formation, parallel with substance P disappearance. The amounts of intact substance P and of free amino acids were monitored along 600 s of incubation time. A steady decrease of substance P as function of reaction time was observed. Peptide's half-life was found to be about 4.3s, indicating an extremely fast hydrolysis in the presence of the SaOS-2 cells. Proline, phenilalanine and methionine were the predominant free amino acids recorded. Obtained results lead to hypothesize the occurrence of endopeptidases activity, followed by aminopeptidases responsible for the release of free amino acids originated after primary bond cleavage.

Validated HPAEC-PAD method for prebiotics determination in synbiotic fermented milks during shelf life.

Interest concerning functional food has been growing in recent years, and much attention has been focused on the choice of prebiotic fibers and probiotic microorganisms added to food products with the aim of improving health, producing synbiotic products. In the work reported here, an innovative analytical method performed by high-performance anion-exchange chromatography (HPAEC) with pulsed electrochemical detection has been optimized and validated for application to the study of prebiotic effects in synbiotic fermented milk prepared by addition of probiotics. The proposed method permits the evaluation of fructooligosaccharides and inulooligosaccharides with degrees of polymerization of 6-7 and 4-7, respectively. Quantitative determination was performed on oligosaccharides, whose standards are not commercially available, by employing calibration curves built by adding a known amount of the fiber used as an ingredient to the matrix. The work provides results from a parallel study on simultaneous variations of prebiotics and probiotics during the shelf life of fermented milk samples. The main advantage over time-consuming, classic enzymatic methods, whose results are limited only to average fiber content, is the possibility of dosing each carbohydrate by performing a single HPAEC run. Validation in terms of detection and quantitation limits, linearity, precision, and recovery was carried out.

Characterization and quantitation of short-chain fructooligosaccharides and inulooligosaccharides in fermented milks by high-performance anion-exchange chromatography with pulsed amperometric detection.

With the increased interest in the possible link between fructooligosaccharides (FOS) intake from functional foods and health, the need for reliable data on the individual FOS content of those foods has become very important. In this study a new high-performance anion-exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD) method is described for the selective determination of short-chain FOS and inulooligosaccharides (IOS) present in the investigated prebiotic food ingredients. Response factor of PAD was investigated in relationship with degree of polymerization and isomers. The developed HPAEC-PAD method has been applied to characterize short-chain oligosaccharides of the inulin series with DP ranging from DP3 to DP7 and IOS from DP2 to DP7. Their quantification in commercial water-soluble fibers of relevant importance as prebiotic food ingredient added in fermented milks has been performed. In order to evaluate the reliability of the proposed method, results have been compared with those obtained by the official enzymatic method. Furthermore, this new HPAEC-PAD method may contribute to clarify the real importance, prebiotic effectiveness and biological role of these carbohydrates.

Evaluation of Fructooligosaccharides and Inulins as Potentially Health Benefiting Food Ingredients by HPAEC-PED and MALDI-TOF MS.

This paper describes the complementarity of high-performance anion exchange chromatography coupled with pulsed electrochemical detection (HPAEC-PED) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) to evaluate commercial available fructans (fructooligosaccharides (FOS) and inulins), having different degrees of polymerization (DP) which are usually employed by food industry as functional ingredients either for their prebiotic properties or as a fat replacer, giving a fat-like mouth feel and texture. The developed HPAEC-PED methods are able to analyze FOS (fructans with DP 3-10) and inulins (DP ranging from 3 to 80) with a good resolution and relatively short retention times to evaluate structural differences between fructooligosaccharide and inulins and the possible presence of inulooligosaccharides as well as of branching. To characterize FOS and inulin at different degrees of polymerization and to assure correct molecular assignment, MALDI-TOF MS analysis was also investigated. The 2,5-dihydroxy benzoic acid (2,5-DHB) was found to be the best matrix for FOS analysis as Actilight and Raftilose P95 products, while 3-aminoquinoline (3-AQ) seems to be the best matrix for inulin with higher DP. The applicability of the optimized methods to the identification and determination of FOS contained in a symbiotic milk as well as a type of inulin added as functional ingredient to a cooked ham is demonstrated.

Optimization of a rapid microwave assisted extraction method for the liquid chromatography-electrospray-tandem mass spectrometry determination of isoflavonoid aglycones in soybeans.

A very fast chromatographic separation of isoflavonoids genistein, daidzein, formononetin and biochanin A was developed on a C18 high-speed column under isocratic conditions. The method was validated in terms of detection limits, quantitation limits (LOQs), linearity and precision. LOQs in 0.04-0.2 microg/g range were calculated, making feasible the determination of these compounds of nutritional concern at trace levels. Good linearity was demonstrated over three concentration orders of magnitude for each analyte (r2 0.990-1.000). The intra-day and inter-day repeatability was evaluated in terms of relative standard deviation (RSD%) at two concentration levels for each analyte (RSD% <9%). An optimization strategy was adopted to find the best conditions for the extraction of isoflavonoid aglycones from yellow soybeans using microwave-assisted extraction. The most relevant parameters resulted to be the microwave power, the extraction time and the acid concentration, optimal values being 600 W, 1 min and 12 M, respectively. When performing sample treatment on a fortified soybean sample, high recovery percentage was obtained for both compounds (94+/-8% for daidzein and 97+/-5% (n = 4) for genistein). The concentration level at which daidzein and genistein were found in the soybean sample were 1.21+/-0.15 mg/g and 2.38+/-0.09 mg/g (n=4), respectively.