UPLC-ESI-TOF MS Profiling Discriminates Biomarkers in Authentic and Adulterated Italian Samples of Saffron (Crocus sativus L.).

Italian saffron (Crocus sativus L.) is gaining visibility due to its high quality and difference in growing area. In this study, the metabolite composition and quality of Italian saffron samples purchased from local producers and supermarkets were investigated using an untargeted metabolomics approach using UPLC-ESI-TOF MS with simultaneous acquisition of low- and high-collision energy mass spectrometry (MSe). Unsupervised statistical method (PCA) highlighted significant differences in the metabolomes, even if not related to the geographical origin. OPLS-DA revealed 9(S)-,10-(S)-,13-(S)-tri-hydroxy-11-(E)-octadecenoic acid as the most decisive compound to distinguish supermarket saffron, while oxidized crocins represented the most valuable markers to further describe the quality of saffron, even in locally produced samples. Known adulterations with paprika and turmeric were detected at a limit of 10%, and the increasing signals of cyclocurcumin was a significant biomarker for turmeric contamination. The results were underlined with conventional and kinetic antioxidant assays.

Real-time monitoring of vegetable oils photo-oxidation kinetics using differential photocalorimetry.

This study introduced differential photocalorimetry (DPC) as a method for real-time monitoring of the photo-oxidation kinetics of vegetable oils. DPC measures the heat flow generated during the oxidation of oils upon light exposure. Experiments conducted with stripped linseed oil (SLSO), an oil depleted from its natural antioxidants, showed no induction time (τ). Conversely, spiking SLSO with increasing concentrations of trans-ferulic acid resulted in an induction time (τ) proportional to the antioxidant concentration (R2 = 0.99). A comparative study among different vegetable oils revealed that rice bran oil exhibited the highest resistant to photo-oxidation, followed by corn, soybean, and sunflower oils. The results are discussed in terms of sample oxidizability and antioxidant efficiency (A.E.), and validated through high-performance liquid chromatography with diode array detection (HPLC-DAD). Furthermore, the measured heat flow enabled the determination of the rates of inhibited (Rinh) and uninhibited (Runi) periods, as well as the rate constant of propagation (kp) and inhibition (kinh) reactions.

Structuring vegetable oils through enzymatic glycerolysis for water-in-oil emulsions.

Enzymatic glycerolysis is a biotechnological process for structuring vegetable oils. This study investigates the kinetics of glycerolysis of peanut oil and explores the potential of the resulting structured oil to enhance the physical stability of water-in-oil emulsions. Using a 1:1 glycerol-to-oil molar ratio and 4 % lipase B from Candida antarctica as a catalyst, the reaction was conducted at 65 °C with stirring at 400 rpm. Acylglyceride fractions changes were quantified through NMR and DSC. Fat crystal formation was observed using scanning electron microscopy. The results revealed a first-order decay pattern, converting triglycerides into monoacylglycerides and diacylglycerides in less than 16 h. Subsequently, water-in-oil emulsions prepared with glycerolized oil showed augmented stability through multiple light scattering techniques and visual assessment. The structured oils effectively delayed phase separation, highlighting the potential of glycerolysis in developing vegetable oil-based emulsions with improved functional properties and reduced saturated fatty acid content.

A Kinetic Approach to Oxygen Radical Absorbance Capacity (ORAC): Restoring Order to the Antioxidant Activity of Hydroxycinnamic Acids and Fruit Juices.

This study introduces a kinetic model that significantly improves the interpretation of the oxygen radical absorbance capacity (ORAC) assay. Our model accurately simulates and fits the bleaching kinetics of fluorescein in the presence of various antioxidants, achieving high correlation values (R2 > 0.99) with the experimental data. The fit to the experimental data is achieved by optimizing two rate constants, k5 and k6. The k5 value reflects the reactivity of antioxidants toward scavenging peroxyl radicals, whereas k6 measures the ability of antioxidants to regenerate oxidized fluorescein. These parameters (1) allow the detailed classification of cinnamic acids based on their structure-activity relationships, (2) provide insights into the interaction of alkoxyl radicals with fluorescein, and (3) account for the regeneration of fluorescein radicals by antioxidants. The application of the model to different antioxidants and fruit extracts reveals significant deviations from the results of traditional ORAC tests based on the area under the curve (AUC) approach. For example, lemon juice, rich in 'fast' antioxidants such as ascorbic acid, shows a high k5 value, in contrast to its low AUC values. This finding underscores the limitations of the AUC approach and highlights the advantages of our kinetic model in understanding antioxidative dynamics in food systems. This study presents a comprehensive, quantitative, mechanism-oriented approach to assessing antioxidant reactivity, demonstrating a significant improvement in ORAC assay applications.

Determination of free and bound antioxidants in Kamut® wheat by HPLC with triple detector (DAD-CAD-MS).

Kamut® wheat (Triticum turgidum ssp. turanicum), an ancient, underutilized cereal, offers potential health benefits due to its phenolic compounds. This study aimed to investigate the antioxidant potential of Kamut® wheat's free and bound phenolic extracts using an HPLC system equipped with three detectors. The bound extracts, released after alkaline hydrolysis, exhibited higher total phenolic and flavonoid content compared to the free extracts (p < 0.05). The total antioxidant capacity of bound extracts was six-fold greater than in free extracts (p < 0.05). The main antioxidants in free extracts were tyrosine, phenylalanine, tryptophan, and apigenin. In bound extracts, ferulic acid, its dimers and trimer were present. Kamut® wheat exhibited a source of dietary antioxidants and should be considered a potential ingredient for the development of functional foods. Also, the HPLC-triple detector system is effective for in-depth profiling of antioxidant compounds, paving the way for future research on similar grains.

Establishing authenticity of hay milk: Detection of silage feeding through cyclopropane fatty acids analysis using 1H NMR spectroscopy.

Cyclopropane fatty acids (CPFAs) serve as indicators of silage feeding, verifying the authenticity of hay milk where silage feeding is forbidden. In this study, the authenticity of hay milk was determined by detecting CPFAs using proton nuclear magnetic resonance (1H NMR) spectroscopy. 245 milk samples were collected in South Tyrol (Italy), categorized as follows: 98 from grass silage-fed cows, 98 from maize silage-fed cows, and 49 authentic hay milk. The limit of detection of CPFAs was 12 µM, corresponding to 70 mg/kg of freeze-dried milk. The CPFAs were absent in all of the hay milk samples, verifying their authenticity. In contrast, 97 % of maize silage and 77 % of grass silage samples exhibited distinct CPFAs signals. These findings were further corroborated by gas chromatography-mass detector (GC-MS) analysis. The study highlights 1H NMR as a robust, and rapid technique for hay milk authentication, supporting alpine dairy production and increasing consumer trust in food authenticity.

Early detection of acrolein precursors in vegetable oils by using proton transfer reaction - mass spectrometry.

Acrolein is a toxic volatile compound derived from oxidative processes, that can be formed in foods during storage and cooking. This study employs proton transfer reaction mass spectrometry (PTR-MS) to detect acrolein precursors in vegetable oils by focusing on the m/z (mass-to-charge ratio) 57. To this purpose, hempseed, sesame, walnut, olive and linseed oils were stored for 168 h at 60 °C in presence of 2,2'-azobis(2-metilpropionitrile) (3 mM) radicals initiator. The evolution of m/z 57 by PTR-MS was also compared with traditional lipid oxidation indicators such as peroxide value, conjugated diene, oxygen consumption and, isothermal calorimetry. The obtained results were explained by the fatty acid composition and antioxidant capacity of the oils. Hempseed fresh oil presented a very low total volatile organic compounds (VOCs) intensity (5.6 kncps). Nonetheless, after storage the intensity increased ∼70 times. A principal component analysis (PCA) confirmed the potential of m/z 57 to differentiate fresh versus rancid hempseed oil sample. During an autoxidation experiment oils high in linolenic and linoleic acids showed higher m/z 57 emissions and shorter induction times: linseed oil (38 h) > walnut oil (47 h) > hempseed oil (80 h). The m/z 57 emission presented a high correlation coefficient with the total VOC signal (r > 0.95), conjugated dienes and headspace oxygen consumption. A PCA analysis showed a complete separation of the fresh oils on the first component (most significant) with the exception of olive oil. Walnut, hempseed and linseed oil were placed on the extreme right nearby total VOCs and m/z 57. The results obtained highlight the potential of PTR-MS for the early detection of oil autoxidation, serving as a quality control tool for potential acrolein precursor emissions, thereby enhancing food safety in the industry.

Applications of Solution NMR Spectroscopy in Quality Assessment and Authentication of Bovine Milk.

Nuclear magnetic resonance (NMR) spectroscopy is emerging as a promising technique for the analysis of bovine milk, primarily due to its non-destructive nature, minimal sample preparation requirements, and comprehensive approach to untargeted milk analysis. These inherent strengths of NMR make it a formidable complementary tool to mass spectrometry-based techniques in milk metabolomic studies. This review aims to provide a comprehensive overview of the applications of NMR techniques in the quality assessment and authentication of bovine milk. It will focus on the experimental setup and data processing techniques that contribute to achieving accurate and highly reproducible results. The review will also highlight key studies that have utilized commonly used NMR methodologies in milk analysis, covering a wide range of application fields. These applications include determining milk animal species and feeding regimes, as well as assessing milk nutritional quality and authenticity. By providing an overview of the diverse applications of NMR in milk analysis, this review aims to demonstrate the versatility and significance of NMR spectroscopy as an invaluable tool for milk and dairy metabolomics research and hence, for assessing the quality and authenticity of bovine milk.

Apples and Apple By-Products: Antioxidant Properties and Food Applications.

In recent years, there has been a growing interest in utilizing natural antioxidants as alternatives to synthetic additives in food products. Apples and apple by-products have gained attention as a potential source of natural antioxidants due to their rich phenolic content. However, the extraction techniques applied for the recovery of phenolic compounds need to be chosen carefully. Studies show that ultrasound-assisted extraction is the most promising technique. High yields of phenolic compounds with antioxidant properties have been obtained by applying ultrasound on both apples and their by-products. Promising results have also been reported for green technologies such as supercritical fluid extraction, especially when a co-solvent is used. Once extracted, recent studies also indicate the feasibility of using these compounds in food products and packaging materials. The present review aims to provide a comprehensive overview of the antioxidant properties of apples and apple by-products, their extraction techniques, and potential applications in food products because of their antioxidant or nutritional properties. The findings reported here highlight the proper utilization of apples and their by-products in food to reduce the detrimental effect on the environment and provide a positive impact on the economy.

LC-Q-TOF-MS/MS data of phyllobilins in apple peels cv. 'Gala' during ripening under shelf-life conditions.

During senescence and ripening, higher plants degrade the green pigment chlorophyll to linear tetrapyrrols, referred to as phyllobilins (PBs). This dataset provides chromatograms and mass spectral data of PBs acquired from methanolic extracts of cv. Gala apple peels at five different shelf life (SL) stages. Data were obtained using a (ultra-high) pressure liquid chromatograph (UHPLC) coupled to high resolution quadrupole-time of flight-mass spectrometer (HRMS-Q-TOF). A data-dependent inclusion list (IL) ex professo containing all known masses of PBs was applied to address PBs, and fragmentation patterns were studied to confirm their identity operating a MS2 method. Mass accuracy was set to 5 ppm for parent ion peaks, this parameter was adopted as inclusion criterium. The detection of PBs' appearance during ripening can be helpful for assessing the quality and maturity of the apples.

A kinetic-based stopped-flow DPPH• method.

The reaction kinetics of antioxidants with free radicals is crucial to screen their functionality. However, studying antioxidant-radical interactions is very challenging for fast electron-donor substances, such as ascorbic acid, because the reaction ends in a few seconds. Accordingly, this work proposes a rapid and sensitive method for the determination of the absolute rate constant of the reaction between fast antioxidants and DPPH•. The method consists of a stopped-flow spectrophotometric system, which monitors the decay of DPPH• during its interaction with antioxidants. A kinetic-based reaction mechanism fits the experimental data. Kinetic parameters include a second order kinetics (k1) and, depending on the type of antioxidant, a side reaction (k2). Ascorbic acid was the fastest antioxidant (k1 = 21,100 ± 570 M-1 s-1) in comparison with other eleven phenols, showing k1 values from 45 to 3070 M-1 s-1. Compounds like catechin, epicatechin, quercetin, rutin, and tannic, ellagic and syringic acids presented a side reaction (k2 from 15 to 60 M-1 s-1). Among seven fruit juices, strawberry was the fastest, while red plum the slowest. Overall, the proposed kinetic-based DPPH• method is simple, rapid, and suitable for studying the activity and capacity of different molecules, and food samples rich in fast antioxidants, like fruit juices.

Chlorophyll breakdown during fruit ripening: Qualitative analysis of phyllobilins in the peel of apples (Malus domestica Borkh.) cv. 'Gala' during different shelf life stages.

Chlorophyll (Chl) degradation is an important process of fruit ripening, mediated by the pheophorbide a oxygenase/phyllobilin (PaO/PB) pathway. Chl is catabolized to linear tetrapyrrolic phyllobilins that resemble the bile pigments. Determining the optimal harvest date is of pivotal importance for the fruit industry. Among the physiological changes at the onset of ripening and during shelf life, the breakdown of Chl appears as a promising indicator for fruit maturity. In addition, PBs are a frequently overlooked component of the human diet with antioxidant and antiproliferative activities. However, their relevance for a healthy diet and their potential as ripening indicator in fruit cannot be evaluated yet, as details on emergence and composition of PBs in fruit are yet elusive. Using apple (Malus × domestica Borkh.) cv. 'Gala' as a model fruit, the Chl content and the emergence and identity of PBs were analyzed during a controlled shelf life period using UV/Vis spectroscopy and ultra-high pressure liquid chromatography coupled to high resolution quadrupole-time of flight-mass spectrometry (UHPLC-Q-TOF-MS). An in-house database with chromatographic and MS data from 51 PBs, revealed ten chlorophyll catabolites, including five NCCs, one YCC, and four DNCCs (including a previously unknown one). PBs were identified with increasing abundance and diversity from the onset of Chl degradation, suggesting a potential role as ripening indicators.

Analysis of Phenolic Compounds in Food by Coulometric Array Detector: A Review.

Phenolic compounds are an important group of organic molecules with high radical scavenging, antimicrobial, anti-inflammatory, and antioxidant properties. The emerging interest in phenolic compounds in food products has led to the development of various analytical techniques for their detection and characterization. Among them, the coulometric array detector is a sensitive, selective, and precise method for the analysis of polyphenols. This review discusses the principle of this method and recent advances in its development, as well as trends in its application for the analysis of phenolic compounds in food products, such as fruits, cereals, beverages, herbs, and spices.

Antioxidant Capacity, Vitamin C and Polyphenol Profile Evaluation of a Capsicum chinense By-Product Extract Obtained by Ultrasound Using Eutectic Solvent.

Habanero pepper leaves and stems (by-products) have been traditionally considered waste; however, bioactive compounds such as polyphenols, vitamin C and carotenoids have been identified that can be used for formulation of nutraceuticals or functional foods. Furthermore, the extraction of these bioactive compounds by using environmentally friendly methods and solvents is desirable. Thus, the aim of this study was to assess the antioxidant capacity, total polyphenol content (TPC), the phenolic profile and vitamin C content in extracts obtained from by-products (stems and leaves) of two varieties (Mayapan and Jaguar) of habanero pepper by ultrasound-assisted extraction (UAE) using natural deep eutectic solvents (NADES). The results showed that NADES leads to extracts with significantly higher TPC, higher concentrations of individual polyphenols (gallic acid, protocatechuic acid, chlorogenic acid, cinnamic acid, coumaric acid), vitamin C and, finally, higher antioxidant capacity (9.55 ± 0.02 eq mg Trolox/g DM) than UAE extraction performed with methanol as the solvent. The association of individual polyphenols with NADES was confirmed by principal component analysis (PCA). Overall, NADES is an innovative and promising "green" extraction technique that can be applied successfully for the extraction of phenolic compounds from habanero pepper by-products.

Oxidizability of Oils Recovered from Olive Seeds by Isothermal Calorimetry.

This work aims to apply isothermal calorimetry for the determination of the oxidative stability of bulk oils by deriving kinetic and thermodynamic parameters. The method consists of measuring the heat flow produced during the oxidation of the oils in the presence of oxygen. To this purpose, an oil was recovered from olive seeds, the solid waste derived from the transformation of olives, by using two different technologies: supercritical carbon dioxide and mechanical press. The oxidative stability of both extracted oils was then compared with commercial sunflower, soybean, corn, and rice oils. The kinetic and thermodynamic parameters, obtained from the analysis of isothermal calorimetry traces at 60 °C, allowed the calculation of the oxidizability index obtaining the following ranking: olive seeds by supercritical carbon dioxide (3.55 ± 0.4 × 10−3 (s/M)0.5) > sunflower (3.42 ± 0.8 × 10−3 (s/M)0.5) > olive seeds by mechanical press (3.07 ± 0.3 × 10−3 (s/M)0.5) > soybean (2.44 ± 0.6 × 10−3 (s/M)0.5) > corn (1.11 ± 0.4 × 10−3 (s/M)0.5) > rice oils (0.98 ± 0.4 × 10−3 (s/M)0.5). The results were then supported with the analysis of total phenolic content, antioxidant activity, fatty acid profile, and peroxide values. Overall, the findings of the present study support the use of isothermal calorimetry as a direct and non-invasive technique for determining the oxidizability of bulk oils.

Determination of Peak Purity in HPLC by Coupling Coulometric Array Detection and Two-Dimensional Correlation Analysis.

This work aims to evaluate the purity of chromatographic peaks by a two-dimensional correlation (2D-corr) analysis. Such an analysis leads to two contour plots: synchronous and asynchronous. The synchronous contour plot provides information on the number of peaks present in the chromatogram. The asynchronous contour plot reveals the presence of overlapping species on each peak. The utility of 2D-corr analysis was demonstrated by the chromatographic analysis of Capsicum chili extracts obtained by HPLC coupled with a coulometric array of sixteen detectors. Thanks to 16 electrochemical sensors, each poised at increasing potentials, the resulting 2D-corr analysis revealed the presence of at least three species on the peak located at a retention time of 0.93 min. Mass spectrometry (MS) analysis was used to analyze the coeluting species, which were identified as: quinic acid (3.593 min), ascorbic acid (3.943 min), and phenylalanine (4.229 min). Overall, this work supports the use of 2D-corr analysis to reveal the presence of overlapping compounds and, thus, verify the signal purity of chromatographic peaks.

In Vivo Anti-Inflammatory Effect, Antioxidant Activity, and Polyphenolic Content of Extracts from Capsicum chinense By-Products.

By-products of Capsicum chinense Jacq., var Jaguar could be a source of bioactive compounds. Therefore, we evaluated the anti-inflammatory effect, antioxidant activity, and their relationship with the polyphenol content of extracts of habanero pepper by-products obtained from plants grown on black or red soils of Yucatán, Mexico. Moreover, the impact of the type of extraction on their activities was evaluated. The dry by-product extracts were obtained by maceration (ME), Soxhlet (SOX), and supercritical fluid extraction (SFE). Afterward, the in vivo anti-inflammatory effect (TPA-induced ear inflammation) and the in vitro antioxidant activity (ABTS) were evaluated. Finally, the polyphenolic content was quantified by Ultra-Performance Liquid Chromatography (UPLC), and its correlation with both bioactivities was analyzed. The results showed that the SFE extract of stems of plants grown on red soil yielded the highest anti-inflammatory effect (66.1 ± 3.1%), while the extracts obtained by ME and SOX had the highest antioxidant activity (2.80 ± 0.0052 mM Trolox equivalent) and polyphenol content (3280 ± 15.59 mg·100 g-1 dry basis), respectively. A negative correlation between the anti-inflammatory effect, the antioxidant activity, and the polyphenolic content was found. Overall, the present study proposed C. chinense by-products as a valuable source of compounds with anti-inflammatory effect and antioxidant activity.

Extrusion cooking of protein-based products: potentials and challenges.

Extrusion cooking is receiving increasing attention as technology applied for the production of protein-based products. Researchers in this field showed that proteins from several sources are barely consumed because of their poor functionality and lack of acceptability related to the presence of some antinutritional factors. In this regard, extrusion is becoming of key importance thanks to its ability to improve protein functional properties. Based on this remarkable advantage, several studies have been published so far providing evidence of the enhanced functional, physicochemical and sensory properties of protein-based extruded products. The objective of the present review is to give a detailed overview of the potential of extrusion for the production of protein-based products. More specifically, the work describes all the studies published so far on vegetable and animal proteins including those recently released applying the technology on insect proteins. The aspects related to the functional properties of the extrudates together with the quality changes occurring during the process are also described to highlight the potential of the technology for future applications.

Authenticity of Hay Milk vs. Milk from Maize or Grass Silage by Lipid Analysis.

Hay milk is a traditional dairy product recently launched on the market. It is protected as "traditional specialty guaranteed" (TSG) and subjected to strict regulations. One of the most important restrictions is that the cow's feed ration must be free from silage. There is the need for analytical methods that can discriminate milk obtained from a feeding regime including silage. This study proposes two analytical approaches to assess the authenticity of hay milk. Hay milk and milk from cows fed either with maize or grass silage were analyzed by targeted GC-MS for cyclopropane fatty acid (dihydrosterculic acid, DHSA) detection, since this fatty acid is strictly related to the bacterial strains found in silage, and by HPLC-HRMS. The presence of DHSA was correlated to the presence of maize silage in the feed, whereas it was ambiguous with grass silage. HPLC-HRMS analysis resulted in the identification of 14 triacylglycerol biomarkers in milk. With the use of these biomarkers and multivariate statistical analysis, we were able to predict the use of maize and grass silage in the cow's diet with 100% recognition. Our findings suggest that the use of analytical approaches based on HRMS is a viable authentication method for hay milk.

Multiple Light Scattering Measurements for Online Monitoring of Milk Fermentation.

The present paper investigates the use of multiple light scattering for the monitoring of milk fermentation. The experiments were performed on milk fermented with different starter concentrations (0.05% to 4.5% (w/w) at temperatures from 36 to 44 °C and in the presence of antibiotics at concentrations up to 100 µg/kg. The fermentation was monitored continuously by using a multiple light scattering technique and simultaneously by a pH meter, a rheometer and a texture analyzer. The backscattering signal recorded by multiple light scattering measurements was correlated with the changes in pH, rheological parameters and firmness of the samples along the fermentation. A gelation time of 120 min was obtained when the highest concentration of starter (4.5%, w/w) and incubation temperature of 44 °C were used. These results were confirmed by the pH, rheological and texture monitoring. The analysis of backscattering spectra allowed the detection of the effect of antibiotic on the gel formation even at low concentrations (1.3 µg/kg). Overall, the results highlighted the advantages of using a multiple light scattering technique as quality control tool for online monitoring of milk fermentation.