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.

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.

Redox differences between rat neonatal and adult cardiomyocytes under hypoxia.

It is generally accepted that oxidative stress plays a key role in the development of ischemia-reperfusion injury in ischemic heart disease. However, the mechanisms how reactive oxygen species trigger cellular damage are not fully understood. Our study investigates redox state and highly reactive substances within neonatal and adult cardiomyocytes under hypoxia conditions. We have found that hypoxia induced an increase in H2O2 production in adult cardiomyocytes, while neonatal cardiomyocytes experienced a decrease in H2O2 levels. This finding correlates with our observation of the difference between the electron transport chain (ETC) properties and mitochondria amount in adult and neonatal cells. We demonstrated that in adult cardiomyocytes hypoxia caused the significant increase in the ETC loading with electrons compared to normoxia. On the contrary, in neonatal cardiomyocytes ETC loading with electrons was similar under both normoxic and hypoxic conditions that could be due to ETC non-functional state and the absence of the electrons transfer to O2 under normoxia. In addition to the variations in H2O2 production, we also noted consistent pH dynamics under hypoxic conditions. Notably, the pH levels exhibited a similar decrease in both cell types, thus, acidosis is a more universal cellular response to hypoxia. We also demonstrated that the amount of mitochondria and the levels of cardiac isoforms of troponin I, troponin T, myoglobin and GAPDH were significantly higher in adult cardiomyocytes compared to neonatal ones. Remarkably, we found out that under hypoxia, the levels of cardiac isoforms of troponin T, myoglobin, and GAPDH were elevated in adult cardiomyocytes, while their level in neonatal cells remained unchanged. Obtained data contribute to the understanding of the mechanisms of neonatal cardiomyocytes' resistance to hypoxia and the ability to maintain the metabolic homeostasis in contrast to adult ones.

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.

Ultrastructural Abnormalities in Induced Pluripotent Stem Cell-Derived Neural Stem Cells and Neurons of Two Cohen Syndrome Patients.

Cohen syndrome is an autosomal recessive disorder caused by VPS13B (COH1) gene mutations. This syndrome is significantly underdiagnosed and is characterized by intellectual disability, microcephaly, autistic symptoms, hypotension, myopia, retinal dystrophy, neutropenia, and obesity. VPS13B regulates intracellular membrane transport and supports the Golgi apparatus structure, which is critical for neuron formation. We generated induced pluripotent stem cells from two patients with pronounced manifestations of Cohen syndrome and differentiated them into neural stem cells and neurons. Using transmission electron microscopy, we documented multiple new ultrastructural changes associated with Cohen syndrome in the neuronal cells. We discovered considerable disturbances in the structure of some organelles: Golgi apparatus fragmentation and swelling, endoplasmic reticulum structural reorganization, mitochondrial defects, and the accumulation of large autophagosomes with undigested contents. These abnormalities underline the ultrastructural similarity of Cohen syndrome to many neurodegenerative diseases. The cell models that we developed based on patient-specific induced pluripotent stem cells can serve to uncover not only neurodegenerative processes, but the causes of intellectual disability in general.

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.

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.

The Effect of Dietary Phospholipids on the Ultrastructure and Function of Intestinal Epithelial Cells.

Dietary composition substantially determines human health and affects complex diseases, including obesity, inflammation and cancer. Thus, food supplements have been widely used to accommodate dietary composition to the needs of individuals. Among the promising supplements are dietary phospholipids (PLs) that are commonly found as natural food ingredients and as emulsifier additives. The aim of the present study was to evaluate the effect of major PLs found as food supplements on the morphology of intestinal epithelial cells upon short-term and long-term high-dose feeding in mice. In the present report, the effect of short-term and long-term high dietary PL content was studied in terms of intestinal health and leaky gut syndrome in male mice. We used transmission electron microscopy to evaluate endothelial morphology at the ultrastructural level. We found mitochondrial damage and lipid droplet accumulation in the intracristal space, which rendered mitochondria more sensitive to respiratory uncoupling as shown by a mitochondrial respiration assessment in the intestinal crypts. However, this mitochondrial damage was insufficient to induce intestinal permeability. We propose that high-dose PL treatment impairs mitochondrial morphology and acts through extensive membrane utilization via the mitochondria. The data suggest that PL supplementation should be used with precaution in individuals with mitochondrial disorders.

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.

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.

Tropism of Extracellular Vesicles and Cell-Derived Nanovesicles to Normal and Cancer Cells: New Perspectives in Tumor-Targeted Nucleic Acid Delivery.

The main advantage of extracellular vesicles (EVs) as a drug carrier system is their low immunogenicity and internalization by mammalian cells. EVs are often considered a cell-specific delivery system, but the production of preparative amounts of EVs for therapeutic applications is challenging due to their laborious isolation and purification procedures. Alternatively, mimetic vesicles prepared from the cellular plasma membrane can be used in the same way as natural EVs. For example, a cytoskeleton-destabilizing agent, such as cytochalasin B, allows the preparation of membrane vesicles by a series of centrifugations. Here, we prepared cytochalasin-B-inducible nanovesicles (CINVs) of various cellular origins and studied their tropism in different mammalian cells. We observed that CINVs derived from human endometrial mesenchymal stem cells exhibited an enhanced affinity to epithelial cancer cells compared to myeloid, lymphoid or neuroblastoma cancer cells. The dendritic cell-derived CINVs were taken up by all studied cell lines with a similar efficiency that differed from the behavior of DC-derived EVs. The ability of cancer cells to internalize CINVs was mainly determined by the properties of recipient cells, and the cellular origin of CINVs was less important. In addition, receptor-mediated interactions were shown to be necessary for the efficient uptake of CINVs. We found that CINVs, derived from late apoptotic/necrotic cells (aCINVs) are internalized by in myelogenous (K562) 10-fold more efficiently than CINVs, and interact much less efficiently with melanocytic (B16) or epithelial (KB-3-1) cancer cells. Finally, we found that CINVs caused a temporal and reversible drop of the rate of cell division, which restored to the level of control cells with a 24 h delay.

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.

Ultrastructural heterogeneity of the mitochondrial population in rat embryonic and induced pluripotent stem cells.

Even though rats are popular model animals, the ultrastructure of their pluripotent cells, that is, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), remains unexplored, although fine structure of pluripotent stem cells of mice and humans and its changes during differentiation have been investigated well. In the present study, we carried out ultrastructural and morphometric analyses of three lines of rat ESCs and two lines of rat iPSCs. The rat pluripotent stem cells were found to have the main typical morphological features of pluripotent cells: large nuclei of irregular or nearly round shape, scanty cytoplasm with few membrane organelles, and a poorly developed Golgi apparatus and endoplasmic reticulum. The cytoplasm of the rat pluripotent cells contains clusters of glycogen, previously described in human ESCs. To identify possible differences between rat ESCs and iPSCs, we performed a morphometric analysis of cell parameters. The mean area of cells and nuclei, the nuclear/cytoplasmic ratio, distributions of glycogen and diversity of mitochondria showed marked variations among the lines of rat pluripotent stem cells and were more pronounced than variations between rat ESCs and iPSCs as separate types of pluripotent stem cells. We noted morphological heterogeneity of the mitochondrial population in the rat pluripotent stem cells. The cells contained three types of mitochondria differing in the structure of cristae and in matrix density, and our morphometric analysis revealed differences in cristae structure.

A Novel Stoichio-Kinetic Model for the DPPH• Assay: The Importance of the Side Reaction and Application to Complex Mixtures.

The 2,2-diphenyl-1-picrylhydrazyl (DPPH•) assay is widely used to determine the antioxidant activity of food products and extracts. However, the common DPPH• protocol uses a two-point measurement and does not give information about the kinetics of the reaction. A novel stoichio-kinetic model applied in this study monitors the consumption of DPPH• by common antioxidants following the second order reaction. The fitting of such decay yields the rate constant k1, which describes the main reaction between antioxidants and DPPH•, and the rate constant k2, which is attributed to a slower side reaction considering the products generated between the transient radicals (AO•) and another molecule of DPPH•. The model was first applied to antioxidant standards. Sinapic acid, Trolox and ascorbic and chlorogenic acids did not show any side reaction. Instead gallic, ferulic and caffeic acids achieved the best fitting with k2. The products of the side reaction for these compounds were confirmed and identified with high-resolution mass spectrometry. Finally, the kinetic model was applied to evaluate the antioxidant activity of eight herbal extracts. This study suggests a new kinetic approach to standardize the common DPPH• assay for the determination of antioxidant activity.

The Effect of Microfluidization Pressure and Tocopherol Content on the Retention of Vitamin A in Oil-In-Water Emulsions.

This work investigates the oxidative stability of vitamin A encapsulated in oil-in-water emulsions, which were prepared by using a microfluidizer. All emulsions were prepared with a fixed content of vitamin A (525 µM), corn oil (10%), water (90%), and whey protein (2%), but varying two main factors: the microfluidizer pressure (10, 50, 100, 200 MPa) and the amount of α-tocopherol (0, 0.25, 0.50, 1.00 mg/g). The content of vitamin A before and after the microfluidization process, and during the subsequent five weeks of storage at 40 °C were determined by HPLC-DAD. The results of the analysis of variance performed either on the data obtained before and after the microfluidization process or during the storage showed that the highest stability of vitamin A was obtained with the highest content of α-tocopherol and with an applied pressure between 100 and 200 MPa. The highest stability was explained by the smaller particle size of the resulting oil droplets. However, high pressures (200 MPa) showed a negative effect on vitamin A retention. These results could be useful for future formulations of retinoids.