Search

BACKGROUND Phosphatidylserine (PS) externalization out of the membrane facilitates the eryptotic erythrocytes (EE) binding to endothelial cells (EC), potentially leading to atherosclerosis. Thus, the levels of eryptosis and interactions of EE-EC in hypercholesterolemic patients, either non-medicated or medicated, compared with healthy subjects were studied. METHODS A total of 56 subjects clustered into three groups: (control (n = 20), hypercholesterolemic non-treated (HCNT) (n = 15), and statin-treated (HCT) (n = 21)) were enrolled in this cross-sectional study. Biochemical parameters were determined with validated and standard methods. PS exposure was estimated from annexin-V-binding, cell volume from forward scatter (FSC), and GSH from CMFDA fluorescence by flow cytometry. The erythrocyte-EC adhesion assay was performed by using the parallel-plate flow chamber technique. RESULTS Higher PS externalization and adhesion of erythrocytes to EC (P

The consumption of milk-based fruit beverages enriched with plant sterols (PSs) has previously showed a cholesterol-lowering effect in postmenopausal women [1]. [...]

Unabsorbed cholesterol, along with that of bile secretions and flaked colon cells, can be metabolized by colonic microbiota. The generated metabolites have been proposed as promoters of colorectal cancer (CRC). In this study, the cytotoxicity (MTT assay) of the main commercially available cholesterol-derived metabolites (coprostanol, cholestanol, coprostanone, and cholestenone) on human colon cancer (Caco-2) and non-tumor (CCD-18Co) cells was evaluated at different physiologically relevant concentrations (9.4–300 µM) and incubation times (24–72 h). In general, the metabolites that most reduced cell viability were coprostanone (54–85% in Caco-2 and 20–81% in CCD- 18Co) and cholestenone (17–91% in Caco-2 and 14–81% in CCD-18Co). These two metabolites are the most hydrophobic, thus reflecting a possible relationship between hydrophobicity and cytotoxicity. Moreover, cholestenone (IC50 at 72 h: 5 ± 1 µg/mL) should be considered cytotoxic on CCD- 18Co cells (non-tumor cells) since it shows an IC50 close to the one considered toxic (

Combining natural products as co-adjuvants in 5-fluorouracil (5-FU) chemotherapy might enhance the effectiveness of 5-FU by avoiding a high dosage and/or reducing treatment times. We explored the anticancer efficacy of the phytosterols (PS) at concentrations achievable in the human colon, as well as their potential as sensitizing agents of human colon cancer cells (Caco-2 and HT-29) to 5-FU treatment. Cells proliferation, combination index, cell cycle, apoptosis, caspases activation, ROS production, and ΔΨm were determined. Co-treatment (PS+5-FU) had an antiproliferative additive effect, and moreover, in general a significantly improved efficacy was observed on cell cycle arrest at S phase, apoptosis induction and increase in caspases activation compared to 5-FU alone, while no significant effects were observed on ROS levels and ΔΨm. Our results suggest, for the first time, the ability of PS to sensitize colon cancer cells to 5-FU chemotherapy and warrant further in vivo studies to confirm our preclinical findings.

Sterols in foods are susceptible to oxidation to form oxysterols. It is interesting for consumer health to know real intake and the possible associated adverse effects associated to oxysterols. This study measured oxysterol formation and bioaccessibility (BA) in a plant sterol (PS)-enriched milk-based fruit beverage with milk fat globule membrane (MFGM) added at 0, 3 and 6 months of storage at room temperature. The same cholesterol (COPs) and phytosterol oxidation products (POPs) (exclusively from β-sitosterol) (7α/β-hydroxy, α/β-epoxy, triol and 7-keto) were detected in the beverage and its bioaccessible fraction. Total COPs and POPs contents were maintained during storage, and their BA ranged between 58 and 80% and 45–49%, respectively, without significant differences throughout storage. β-Sitosterol showed a lower mean oxidation percentage (0.028%) than cholesterol (1.24%), but the estimated POPs intake (0.5 mg/day) was two-fold higher than that of COPs (0.25 mg/day) from 250 g of beverage. These results show that the presence of milk fat and MFGM in the formulation of this beverage did not imply an increase in the contents of oxysterols and their BA. Thus, the beverage is suitable as a PS-enriched food matrix for the length of its shelf-life, and its consumption appears to be safe for consumers.

The hypocholesterolemic effect and the modification of serum biomarkers of a dietary plant sterol (PS) intake, cholesterol precursors and cytokines after the consumption of milk-based fruit beverages with a milk fat globule membrane were evaluated by a randomized, double-blind, crossover, multiple dose bioavailability study. Postmenopausal women (n = 38) consumed daily 250 mL of a beverage with or without 2 g of PS added during 6 weeks in each of the study periods. With the intake of the PS-added beverage, significant decreases (mg dL-1) in serum total cholesterol (pre-treatment: 220.0 ± 27.8 vs. post-treatment: 212.9 ± 25.8; p < 0.05) and LDL-cholesterol (129.4 ± 28.5 vs. 121.7 ± 24.4; p < 0.05) were detected. The cholesterol precursor lathosterol (11.2%), markers of the dietary PS intake (campesterol 43.1% and β-sitosterol 32.5%), and anti-inflammatory IL-10 cytokine (22.5%) increased significantly, with a concomitant significant reduction in pro-inflammatory IL-1β (6.7%). No variations in HDL-cholesterol, other sterols (desmosterol and stigmasterol) or cytokines (IL-6, IL-8, IL-12p70 and TNF-α) were detected. These results indicated that this kind of PS-enriched milk-based fruit beverage is suitable during the period of clinical intervention, and its consumption may be an adequate way to improve PS functionality since a significant reduction in cholesterol levels has been observed. Therefore, the intake of this beverage could contribute to reduce the risk of cardiovascular disease also obtaining a beneficial effect on the serum inflammatory status in postmenopausal women.

Plant sterols (PS) (phytosterols and phytostanols) are bioactive compounds of all vegetable foods where can be found as free sterol alcohols and as conjugates. These latter forms have been less studied, although they may have potential beneficial effects, whereas some PS have several approved health claims, especially in lowering blood cholesterol levels. In this context, it is necessary to establish analytical methods for food authentication, assessing PS health benefits and unfolding future applications. Several extraction methodologies have been employed for the extraction of PS from food matrixes (usually solvent extraction and saponification) although solid-phase extraction, solid-phase microextraction or enzymes have also been applied. In addition, the determination of PS may also need a subsequent saponification, purification or enrichment step before their determination. Gas chromatography after derivatization has historically been the method of choice for analysis, although liquid chromatography simplifies the analysis. Furthermore, although various detectors have been employed in analysing PS, mass spectrometry can address many of the challenges observed in other methodologies. Therefore, it has been widely used in conjunction with other separation techniques due to its sensitivity and selectivity by the application of tandem mass spectrometry. This review provides an overall and critical picture of the current methodologies used for the analysis of PS in foods, particularly focused on the individual applications and limitations.

The impact of a plant sterol-enriched beverage on the sterol metabolism, organic acid production and microbiota composition was evaluated by means of a dynamic gastrointestinal and colonic fermentation model. After one week of fermentation, an absence of sterol metabolites was reported, in accordance with the lack of microbiota related to their metabolism. Although total organic acid content was lower in the ascending colon (AC) compared to the transversal (TC) and descending colon (DC) (28–57 mmol/L vs. 55-87 and 44–64 mmol/L, respectively), its increments, with respect to the initial value, were higher (2-fold vs. 1.6- and 1.5-fold). Increments of acetate, butyrate and propionate were observed in all colon vessels, whereas lactate production was only observed in the AC at the first hours of fermentation. Results showed a progressive increase in microbial species richness and evenness from the AC to the DC. Beverage fermentation also modulated certain members of the microbiota: Bifidobacterium (within 24 h) and Megasphaera genera were stimulated, and Mitsuokella was reduced in AC, while the growth of Klebsiella genus was promoted and Bacteroidetes phylum decreased in TC and DC. These results show that the beverage modulates the microbial community and activity, which could be physiologically relevant to the host.

Gangliosides (GGs) are important bioactive compounds that offer beneficial anti-infective, anti-inflammatory, and neuronal development effects. The present work reviews the techniques used to deter...

International audience; Oat is rich in a wide range of phytochemicals with various physico-chemical, colloidal and interfacial properties. These characteristics are likely to influence human lipid metabolism and the subsequent effect on health following oat consumption. The aim of this work was to investigate the impact of oat materials varying in complexity on the lipolysis process. The composition, structure and digestibility of different lipid systems (emulsions, oil bodies and oil enriched in phytosterols) were determined. The surface activities of phytosterols were examined using the pendant drop technique. Differences in lipid digestibility of the oat oil emulsions and the oil bodies were clearly seen. Also, the digestion of sunflower oil was reduced proportionally to the concentration of phytosterols present. This may be due to their interfacial properties as demonstrated by the pendant drop experiments. This work highlights the importance of considering the overall structure of the system studied and not only its composition.

Beverages are by far the most active functional food category, since they are excellent means for delivering nutrients and bioactive compounds such as plant sterols (PS). These phytochemicals are used as functional food ingredients due to their well-known cholesterol-lowering activity. The PS-enriched milk-based fruit beverages seem appropriate for complying with health recommendations (restrictions of saturated fatty acids, cholesterol, and energy value), as good sources of other bioactive compounds (polyphenols, carotenoids, and vitamins), and as a good option for obtaining the recommended daily amount of PS in subjects with moderate hypercholesterolemia. This chapter focuses on the development of functional PS-enriched milk-based fruit beverages considering: (i) PS ingredient selection; (ii) bioaccessibility: influence of matrix modification; (iii) bioavailability; and (iv) bioactivity. This integrated approach could serve as an example of the unequivocal demonstration of the efficacy of these functional beverages in promoting health benefits.

International audience; Serum concentrations of lathosterol, the plant sterols campesterol and sitosterol and the cholesterol metabolite 5alpha-cholestanol are widely used as surrogate markers of cholesterol synthesis and absorption, respectively. Increasing numbers of laboratories utilize a broad spectrum of well-established and recently developed methods for the determination of cholesterol and non-cholesterol sterols (NCS). In order to evaluate the quality of these measurements and to identify possible sources of analytical errors our group initiated the first international survey for cholesterol and NCS. The cholesterol and NCS survey was structured as a two-part survey which took place in the years 2013 and 2014. The first survey part was designed as descriptive, providing information about the variation of reported results from different laboratories. A set of two lyophilized pooled sera (A and B) was sent to twenty laboratories specialized in chromatographic lipid analysis. The different sterols were quantified either by gas chromatography-flame ionization detection, gas chromatography- or liquid chromatography-mass selective detection. The participants were requested to determine cholesterol and NCS concentrations in the provided samples as part of their normal laboratory routine. The second part was designed as interventional survey. Twenty-two laboratories agreed to participate and received again two different lyophilized pooled sera (C and D). In contrast to the first international survey, each participant received standard stock solutions with defined concentrations of cholesterol and NCS. The participants were requested to use diluted calibration solutions from the provided standard stock solutions for quantification of cholesterol and NCS. In both surveys, each laboratory used its own internal standard (5alpha-cholestane, epicoprostanol or deuterium labelled sterols). Main outcome of the survey was, that unacceptably high interlaboratory variations for cholesterol and NCS concentrations are reported, even when the individual laboratories used the same calibration material. We discuss different sources of errors and recommend all laboratories analysing cholesterol and NCS to participate in regular quality control programs.

This document is he Accepted Manuscript version of a Published Work that appeared in final form in Journal of Agricultural and Food Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.jafc.9b04796, A beverage enriched with plant sterols (1 g/100 mL) and galactooligosaccharides (1.8 g/100 mL) was subjected to a dynamic gastrointestinal and colonic fermentation process to evaluate the effect on sterol metabolism, organic acid production, and microbiota composition. Production of sterol metabolites (coprostanol, methylcoprostanol, ethylcoprostenol, ethylcoprostanol, and sitostenone) was observed in the transverse colon (TC) and descending colon (DC) vessels in general, from 24 and 48 h, respectively. Microbial activity was assessed through the production of organic acids, mainly acetate in all colon vessels, lactate in the AC, and butyrate and propionate in the TC and DC. A higher diversity in the microbial community was found in the TC and DC, in accordance with a higher sterol metabolism and organic acid production. Although the prebiotic effect of galactooligosaccharides was not detected, changes in microbiota composition (an increase in the Parabacteroides genus and the Synergistaceae and Lachnospiraceae families) indicated an enhancement of sterol metabolism.

Human milk (HM) is the exclusive food during the first 4–6 months of an infant's life. Breastfeeding has been related to significant health benefits for infants, and hence it is of interest to study the bioactive compounds present in HM, such as sterols (cholesterol being the most abundant). The aim of this study was to determine the contents of sterols (cholesterol, desmosterol, lathosterol, lanosterol, campesterol, stigmasterol and β-sitosterol) in 10 pools of colostrum, transitional milk, and 1, 3 and 6 month HM obtained from Spanish volunteers from two different geographical areas (coastal and central) and to estimate the intake and bioaccessibility (BA) of sterols in order to ascertain the fate of sterols after digestion. The results showed that the total sterol contents decreased to half the initial level during lactation (24–11 mg per 100 mL) and was significantly higher in samples from the coastal area. Total and animal sterol intakes were between 200 and 400 times higher than plant sterol intakes and were significantly higher in samples from the coastal area. However, no statistically significant differences were found in cholesterol and plant sterol intakes between areas. The BA of total sterols ranged from 45% to 69% and was higher in the first month, which coinciding with the highest fat content of milk. In conclusion, the sterol content varies depending on the lactation stage and the geographical area, and the BA of sterols can be positively affected by a higher lipid content. All these data may contribute to the development of infant formulas that are more similar to HM in terms of composition and behaviour after digestion, according to the lactation stage involved.

This study validates a gas chromatography (GC) method for determining the sterol profile of human milk (HM) and compares it with an enzymatic-spectrophotometric (E-S) method. Good linearity ( r > 0.97) and low limits of detection and quantification were obtained with the GC method (

The effect of the addition of galactooligosaccharides (GOS) on sterol bioaccessibility in three plant sterol (PS)-enriched milk-based fruit beverages (without GOS addition (MfB) and with 2.5 g (MfB-G2) and 5.0 g (MfB-G5) GOS per 250 mL) was evaluated after micellar gastrointestinal digestion. Cholesterol bioaccessibility was very similar among beverages, though a slight significant increase (from 80% to 85%) was observed by the addition of 5.0 g GOS. The addition of GOS did not affect total PS bioaccessibility (≈37%). Based on the results obtained after micellar digestion, it has been demonstrated that these beverages could be a suitable food matrix for simultaneous enrichment with PS and GOS. The harmonized in vitro digestion model INFOGEST was applied to the MfB beverage, but the cholesterol content could not be quantified due to its contribution of bile salts. Hence, it was proposed: (i) a change in porcine bile salt concentration from 10 mM to 1.4 mM (in order to compare with micellar digestion); or (ii) a change of bile salt origin (bovine instead of porcine), maintaining physiological concentration (10 mM, INFOGEST condition). Both options allowed cholesterol quantification, with bioaccessibilities of 62% (reduction of bile salts) and 38% (replacement of the bile salt source), whereas plant sterol bioaccessibilities were 22% and 14%, respectively. Therefore, the change of bile salt origin maintaining INFOGEST concentration is proposed as a method to evaluate sterol (cholesterol and PS) bioaccessibility in these beverages, demonstrating the need for the selection of appropriate conditions of the INFOGEST harmonized method according to the food matrix and compounds to be determined.

The design of infant formulas (IFs) seeks to resemble human milk (HM) composition and functionality. The fat sources used usually comprise vegetable oil blends to mimic the fatty acid composition of HM and introduce changes in the animal/plant sterol ratio. In contrast, the use of milk fat globule membrane (MFGM)-rich ingredients could improve this aspect by increasing the ratio. The present study evaluates the bioaccessibility (BA) of sterols (cholesterol, desmosterol, brassicasterol, campesterol, stigmasterol, and β-sitosterol) in three IFs (with or without MFGM) using an in vitro digestion method simulating infant conditions. Analytical parameters confirmed the suitability of the method for all of these sterols. Results showed the presence of MFGM to increase cholesterol content (6-7 vs 2 mg/100 mL), this being the most bioaccessible sterol in the IFs. Although the BA of cholesterol was reduced in MFGM-enriched IF (65.6-80.4% vs 99.7%), the intake of bioaccessible cholesterol from these IFs was higher.

This study for the first time compares the in vivo (response in serum from individuals of an interventional study) and in vitro effects (bioaccessibility determined from simulated gastrointestinal digestion) of two plant sterol (PS)-enriched milk-based fruit beverages (with and without tangerine fruit juice rich in β-cryptoxanthin (β-Cx)) in order to assess their suitability as PS-enriched food matrixes, and the possible interactions between the two bioactive compounds (β-Cx and PS) that might affect their absorption. The randomized, double-blind, crossover study (performed in 36 postmenopausal women) showed the β-sitosterol and campesterol serum concentrations after four weeks of consumption of the beverages (1.5 g PS/day) to increase significantly (ranging 1.6–2.4 µg/mL and 0.3–0.4 µg/mL, respectively), thereby confirming suitability as matrices for PS-enrichment. The results therefore indicated similar behavior of PS supplied by these beverages and evaluated through in vitro or in vivo assays, independently of whether the samples contained β-Cx or not.

Sterols are components present in the fat fraction of infant formulas (IFs). Their characterization is therefore of interest, though there are no official reference methods for their analysis in these matrices.Aim: To validate a gas chromatographic method with flame ionization detection for the determination of animal (cholesterol and desmosterol) and plant sterols (brassicasterol, campesterol, stigmasterol, β-sitosterol and sitostanol) found in IFs.All correlation coefficients obtained for the calibration curves of sterols studied were >0.99. Limits of detection (

In this study the quality characteristics and content of healthy minor components of four crude canola oils as an effect of different oil extraction method (solvent extraction, hot pressing, and cold pressing) were studied. Cold-pressed canola oils had lower concentrations of FFA, PV, p-AV and chlorophylls than solvent-extracted, and hot-pressed canola oils. Oils obtained via the different extraction methods had different fatty acid profiles as well as dissimilar amounts of tocopherols, phytosterols, and polyphenols. The amount of total tocopherols in solvent-extracted canola oil was 493 mg/kg compared to 388 mg/kg for hot-pressed canola oil. The tocopherol content for two other cold-pressed and one other RBD canola oil was 366, 354, and 327 mg/kg, respectively. Solvent-extracted canola oil exhibited the highest free phytosterol content (178 mg/100 g), while RBD canola oil only had 129 mg/100 g of free phytosterols. While cold-pressed canola oil had the lowest amount of polyphenols, traditional refining resulted in almost complete removal of polyphenols from canola oil.

Five plant sterol (PS)-enriched dairy products from the Spanish market were characterised for fatty acids (FA), volatile compounds and lipid stability. The ingredients used for PS enrichment by the food industry may come from different sources, thus influencing the composition and stability of the lipid fraction. In this study, the FA profile proved highly variable among samples, not agreeing with the nutritional labelling of the products. The volatile profile was characterised. A total of 55 volatile compounds were identified in the samples by GC–MS. Concentrations of hexanal (from 1.1 to 7.5 ng/g), nonanal (from 0.9 to 1.2 ng/g) and decane (from n.d. to 11.9 ng/g) indicated a low lipid oxidation extent. Peroxide value, ranking between 0.7 and 3.2 meq of active oxygen per kilogram of fat, except sample with fish oil, where peroxide value was 7.7 meq of active oxygen per kilogram of fat, and thiobarbituric acid reactive species, from 2 to 21 μg malondialdehyde (MDA)/g of fat, were in the lower limits of the ranges found in the literature.

The minimal refining method described in the present study made it possible to neutralize crude canola oil with Ca(OH)2, MgO, and Na2SiO3 as alternatives to NaOH. After citric acid degumming, about 98 % of the phosphorous content was removed from crude oil. The free fatty acid content after minimal neutralization with Ca(OH)2 decreased from 0.50 to 0.03 %. Other quality parameters, such as peroxide value, anisidine value, and chlorophyll content, after traditional and minimal neutralization were within industrial acceptable levels. The use of Trisyl silica and Magnesol R60 made it feasible to remove the hot-water washing step and decreased the amount of residual soap to

The intake of fruits and vegetables is associated with reduced incidence of many chronic diseases. These foods contain phytochemicals that often possess antioxidant and free radical scavenging capacity and show anti-inflammatory action, which are also the basis of other bioactivities and health benefits, such as anticancer, anti-aging, and protective action for cardiovascular diseases, diabetes mellitus, obesity and neurodegenerative disorders. Many factors can be included in the etiopathogenesis of all of these multifactorial diseases that involve oxidative stress, inflammation and/or cell death processes, oxysterols, i.e. cholesterol oxidation products (COPs) as well as phytosterol oxidation products (POPs), among others. These oxidized lipids result from either spontaneous and/or enzymatic oxidation of cholesterol/phytosterols on the steroid nucleus or on the side chain and their critical roles in the pathophysiology of the abovementioned diseases has become increasingly evident. In this context, many studies investigated the potential of dietary phytochemicals (polyphenols, carotenoids and vitamins C and E, among others) to protect against oxysterol toxicity in various cell models mimicking pathophysiological conditions. This review, summarizing the mechanisms involved in the chemopreventive effect of phytochemicals against the injury by oxysterols may constitute a step forward to consider the importance of preventive strategies on a nutritional point of view to decrease the burden of many age-related chronic diseases.

Apple juices and purees represent categories widely consumed by whole population and above all children. Patulin (PAT) is a mycotoxin known for its acute and chronic effects in animals. Several studies indicate there is a risk associated to the PAT intake, through the consumption of purees and apple juices. In this study, apple juice and puree were prepared and artificially contaminated with PAT at 50 μg/kg and submitted to a thermal treatment simulating pasteurization to evaluate PAT’s reduction. In a second phase of the work, apple products samples ( n = 7) included juices, nectars and purees belonging to different commercial brands were collected, artificially contaminated with PAT at 50 μg/L (limit established for PAT in juices) and 25 μg/kg (limit established for PAT in purees), digested with an in vitro gastrointestinal protocol and bioaccessibility values (%) were calculated. After thermal treatment, the PAT’s loss evidenced in purees and juices was of 1.41 ± 0.52% and 62.62 ± 2.53% respectively. Related to the bioaccessibility data, two juices with pulp showed values of 70.89 ± 4.93 and 67.30 ± 10.76%; two purees showed levels of 58.15 ± 5.50 and 55.69 ± 4.73%, whereas nectar and two clarified juice showed percentages of 38.88 ± 2.42, 28.59 ± 0.46 and 25.28 ± 0.61%, respectively.

Two different plant sterol (PS) sources (free PS from tall oil and esterified PS from vegetable oils) were used for manufacturing two types of functional beverages (fruit and milk-based fruit beverages), and their PS and phytosterol oxidation product (POP) contents were determined. Gas chromatography–tandem mass spectrometry (GC–MS/MS) was used for identification and gas chromatography–flame ionization detection (GC–FID) for quantitation purposes. Brassicasterol, campesterol, campestanol, stigmasterol, β-sitosterol and sitostanol were the quantified PS, conforming a profile in order with current legislation. The relative percentages of PS differed according to the enrichment source involved, though the enrichment levels (g/100 g beverage) were of the same order (1.77 from tall oil and 1.84 from vegetable oils). Only POPs from β-sitosterol (the prevalent PS in the analyzed beverages) were detected — the predominant representative being 7β-hydroxysitosterol (39–58.5% of total POP content). The following POPs were quantified: 7α-hydroxy, β-epoxy, α-epoxy, and 7-ketositosterol, yielding a total POP content ranging between 42.9 and 57.4 mg/100 g of PS. No statistically significant differences (p > 0.05) in total and individual POP content according to the source of PS were found. The mean β-sitosterol oxidation percentage was

Plant sterols (PS) stability, antioxidant parameters, and color were studied during 6 months of storage at 4, 24, and 37 °C in three PS-enriched functional beverages. Beverages were skimmed milk with fruit juice and PS (MFJPS), fruit juice and PS (FJPS), and skimmed milk with PS (MPS). No loss in total PS content occurred during storage observing the same values at any given storage time point. Total carotenoids decreased 36% with storage at two months and then remained stable. Total polyphenols showed fluctuations throughout the storage, remaining stable at 6 months and reaching initial values. The antioxidant capacity (TEAC method) increased 18% at 6 months, and there was an increase in color over time and temperature, probably due to Maillard reaction compound formation. The increase in total antioxidant capacity might have helped PS maintenance throughout storage, these beverages being a good PS source even after 6 months of storage.

Determination of the main phytosterols (Ps, β-sitosterol and campesterol) and cholesterol precursors (desmosterol and lathosterol) in human serum using a simple GC-FID method has been validated. Direct saponification, without lipid extraction, sterols extraction, and further derivatization was applied to samples prior to GC analysis. To evaluate the method, a pool of serum samples from eight healthy women was used. Good linearity (r>0.99) was found in the assay range: β-sitosterol (0.99–17.82 µg/mL), campesterol (0.14–10.8 µg/mL), desmosterol (0.17–2.6 µg/mL), and lathosterol (0.6–5.97 µg/mL). Limits of detection (ng/mL) were: 86 (β-sitosterol), 42 (campesterol), 4 (desmosterol), and 44 (lathosterol). Accuracy, estimated by recovery assays (%), were: 113 (β-sitosterol), 114 (campesterol), 111 (desmosterol), and 102 (lathosterol). Within and between precision values (%), expressed as the relative SD (RSD), were: 2.6 and 8.1 (β-sitosterol), 1.6 and 7.2 (campesterol), 2.1 and 7.9 (desmosterol), and 4.1 and 5.8 (lathosterol), respectively. The developed methodology allowed fast (1-day analysis) and reliable quantification of sterols in serum, required a small volume of sample and reduced use of solvents. It therefore could be used in clinical assays for the determination of serum sterols, as in evaluating the pharmacological response to lipid-lowering agents, and in assessing biological responses to Ps-enriched diets. Practical applications: This methodology allows fast and reliable quantification of sterols in serum, requiring a small volume of sample and reduced use of solvents. It can be used as a routine method for the quantification of phytosterols and cholesterol precursors in clinical assays, and it is also suitable for monitoring biological responses to health-promoting phytosterol-enriched diets.

The content of plant sterol (PS) and their oxidation products (POPs) in eight ingredients used to enrich functional foods was studied. A gas chromatographic (GC) technique with mass-spectrometric detection was used for identification, while GC with a flame ionization detector (GC-FID) was used for quantification. β-Sitosterol was the most abundant phytosterol, and the main POPs found were derived from this compound (7α/β-hydroxysitosterol, 7-ketositosterol, and sitostanetriol). The total amount of POPs found in the ingredients ranged from 29.03 to 110.02 μg/100 g PS. The β-sitosterol oxidation rates ranged from 10 to 50 μg β-sitosterol oxides/100 g of β-sitosterol. In view of this low rate of oxidation in the ingredients tested, it can be concluded that the PS remain stable in these ingredients. Significant correlations (p0.01) were found between total oxysitosterols versus β-sitosterol contents (R(2) = 86.5%) and between total POPs and total PS (R(2) = 81.6%).

The bioaccessibility (BA) of total and individual plant sterols (PS) of four commercial PS-enriched fermented milk beverages (designated as A to D) was evaluated using in vitro gastrointestinal digestion including the formation of mixed micelles. The fat content of the samples ranged from 1.1 to 2.2% (w/w), and PS enrichment was between 1.5 and 2.9% (w/w). β-Sitosterol, contained in all samples, was higher in samples A and B (around 80% of total PS). The campesterol content was C (22%) > A (7%) > B (5%). Sitostanol was the most abundant in sample D (85%). Stigmasterol was only present in sample C (33%). The greatest BA percentage for total PS corresponded to samples A and B (16–17%), followed by sample D (11%) and sample C (9%). The total BA was not related to the protein, lipid or PS content of the beverages, whereas samples with higher carbohydrates and fiber contents showed lower BA. The BA of the individual PS differed according to the sample considered, and was not related to the PS profile of the sample, thus indicating strong dependency upon the matrix (PS ingredient and other components). Although in vivo studies should be carried out to better assess the functionality of PS in functional foods such as enriched fermented milk beverages, our in vitro study is a useful preliminary contribution to evaluation of the efficacy of these products.

Calcium plays an important role in prevention of chronic diseases and in critical biological functions; however, not only must the calcium content in foods be known, but also its bioavailability. This chapter discusses the usefulness of Caco-2 culture cells as an in vitro method for evaluating calcium bioavailability in foods. Structural and functional characteristics of this cell culture, beside some methodological aspects of Caco-2 cell assays, and calcium-absorption mechanisms are reported. In particular, the effect of food components upon the bioavailability of calcium, as well as its influence upon iron absorption is discussed in this chapter.

Several in vitro methods have been developed to simulate the physiological conditions of the human gastrointestinal digestion, the simplest being the static methods. The following chapter clarifies the concepts of bioaccessibility and dialyzability, and describes the conditions (pH, enzymes, agitation, etc.) to be applied in oral, gastric and intestinal phases when assessing a food component (nutrient, bioactive or toxin) or a food product, in a single or multi-phase model. The advantages and disadvantages of the static models vs. dynamic and in vivo models are discussed, and a review of specific conditions applied on nutrients (minerals, vitamins, proteins, fatty acids, etc.) and bioactive compounds (carotenoids, plant sterols, etc.) from recent studies is provided. Currently, it must be considered that, although the static digestion conditions must be adapted according to the component or food sample to be studied, a harmonization and standardization of the models are needed in order to establish suitable correlations among in vitro and in vivo assays, as it has been defined for some food components (carotenoids, proteins and minerals).

The effect of storage (time and temperature) on the evolution of pentanal, hexanal, heptanal and pentane as volatile lipid oxidation products in two liquid ready-to-eat milk-cereal-based infant foods was studied. An SPME-GC method was used to this effect. Samples were stored for 9 months at 25, 30 and 37 °C and tested eight times during this period. Freshly produced infant foods contained pentanal, hexanal and heptanal (mean values: 10.71, 71.5 and 1.2 μg/kg, respectively), which decreased during the first 3 months of storage, although from the fourth month onwards no significant differences among storage times were found. Aldehyde content was inversely proportional to storage temperature. Pentane content was directly proportional to storage temperature and increased (19.9 μg/kg at zero time) over all months of storage up to 43.1 μg/kg.

Cholesterol can undergo oxidation through enzymatic or chemical mechanisms, generating a wide range of oxidation products (COPs) with adverse biological effects. COPs are characterized by different functional groups and are produced in different ratios or amounts, depending on the treatment and storage conditions. To follow the cholesterol oxidation process, 7-ketocholesterol (7-KC) has been often used as an oxidation marker in both model and food systems, since it is easily formed and is one of the most representative ring COPs. However, 7-KC does not always rise with increasing time/temperature conditions, especially in complex systems and high-protein or extensively processed foods. The following review provides a critical picture of the utilization of 7-KC as a cholesterol oxidation marker in model and food systems, focusing on the possible causes and effects of the different behaviours and trends, as well as on the advantages and disadvantages of using 7-KC when the extent of cholesterol oxidation is to be assessed.

Background and aim Post-menopausal women are at higher risk of cardiovascular disease and bone demineralization. Phytosterols (PS) may be used for hypercholesterolemia in some groups and β-cryptoxanthin (β-Cx) displays a unique anabolic effect on bone. Our aim was to assess the changes in cardiovascular and bone turnover markers from the oral intake of β-Cx and PS in post-menopausal women. Methods and results A randomized, double-blind, crossover study with β-Cx (0.75 mg/day) and PS (1.5 g/day), single and combined, was performed in 38 postmenopausal women. Diet was supplemented with 1 × 250 mL milk-based fruit drink/day for 4 weeks with a wash-out period of 4-weeks in between. Serum β-Cx and PS were determined by UPLC and CG-FID respectively. Outcome variables included markers of bone turnover and cardiovascular risk. Biological effect was assessed by paired t test and generalized estimating equations analysis that included the previous treatment, the order of intervention and the interactions. The intake of beverages containing β-Cx and PS brought about a significant increase in serum levels of β-Cx, β-sitosterol and campesterol. Intervention caused changes in almost all the markers while the order, previous treatment and the interaction did not reach statistical significance. Only the intake of the beverage containing β-Cx plus PS brought about significant decreases in total cholesterol, c-HDL, c-LDL and bone turnover markers. Conclusions β-Cx improves the cholesterol-lowering effect of PS when supplied simultaneously and this combination may also be beneficial in reducing risk of osteoporosis. Clinical Trial Registry ClinicalTrials.gov number NCT01074723.

This study evaluates the bioaccessibility (percentage of soluble compound available for absorption) of plant sterols (PS) and their oxides (phytosterol oxidation products, POPs) after simulated gastrointestinal digestion in fruit (Fb), milk (M) and fruit-based milk beverages with (FbM a ) or without (FbM b ) tangerine juice. In beverages and their bioaccessible fraction (BF), campesterol, campestanol, stigmasterol, β-sitosterol and sitostanol were detected. Bioaccessibility of total PS ranged between 2.62 and 6.48%, FbM b yielding the highest value, followed by FbM a > Fb > M. Campesterol/campestanol were the most bioaccessible PS. Only oxides of β-sitosterol were detected in beverages and BF (7α-hydroxy, 7β-hydroxy, β-epoxy, α-epoxy, sitostanetriol and 7-ksitosterol). Bioaccessibility of total POPs ranged between 19.08 and 49.29%, sitostanetriol yielding the highest value. Bioaccessibility of POPs was higher than that of PS, suggesting different patterns of solubility for these compounds.

The kinetics of ascorbic acid (AA) degradation in a fruit-based beikost product added with AA were determined after storage at 4, 25, 37 and 50 °C during 4, 8, 12, 16 and 32 weeks in plastic polypropylene/ethylenevinyl alcohol vacuum packaging. It was confirmed that AA degradation followed an Arrhenius first-order kinetics, with an activation energy of 20.11 ± 0.33 kcal mol−1. No AA losses at 4 °C were recorded during the entire storage period. In contrast, a time - and temperature - dependent decrease (p < 0.05) in AA was observed at the other tested temperatures the degradation rate decreasing from 50 °C to 25 °C, as expected. AA percentage retention at the end of storage ranged between 6.4% (50 °C/16 weeks) and 100.9% (4 °C/32 weeks).

Amino Acids, Peptides, and Proteins Essential Amino Acids M. C. Aristoy and F. Toldra Glutamine and Taurine C. G. Prosser and S. M. Rutherfurd Bioactive Peptides M. Miguel, B. Hernandez-Ledesma, R. Lopez-Fandino, and I. Recio Glutathione O. Demirkol and N. Ercal l-Carnitine J. Demarquoy, C. Rigault, and F. Le Borgne Creatine L. Mora and F. Toldra Analysis of Bioactive Peptides and Proteins P. Rupa and Y. Mine Vitamins Vitamin K X. Fu and S. L. Booth Methods for the Simultaneous Quantitative Analysis of Water-Soluble Vitamins in Food Products O. Heudi Tocopherols, Tocotrienols, and Their Bioactive Analogs S. Pacifico, M. Scognamiglio, B. D'Abrosca, P. Monaco, and A. Fiorentino Vitamin C O. Martin-Belloso, I. Odriozola-Serrano, and R. Soliva-Fortuny Vitamin D J. Jakobsen and R. B. Japelt Terpenes Hydrocarbon Carotenoids M. da Graca Dias Oxycarotenoids (Xanthophylls) D. Giuffrida, P. Dugo, P. Donato, G. Dugo, and L. Mondello Phenolic Compounds Flavonoids: Flavonols, Flavones, and Flavanones F. Ferreres and F. A. Tomas-Barberan Flavan--Ols and Proanthocyanidins S. Guyot Stilbenes and Resveratrol M. do Rosario Bronze, C. M. M. Duarte, and A. Matias Flavonoids: Anthocyanins V. Cheynier, C. Gomez, and A. Ageorges Isoflavones G. G. C. Kuhnle Hydrolyzable Tannins: Gallotannins Ellagitannins, and Ellagic Acid M. Jourdes, L. Pouysegu, S. Quideau, F. Mattivi, P. Truchado, and F. A. Tomas-Barberan Analysis of Chlorogenic Acids and Other Hydroxycinnamates in Food, Plants, and Pharmacokinetic Studies N. Kuhnert, H. Karakose, and R. Jaiswal Fibers and Polysaccharides Analytical Methodologies of Chitosan in Functional Foods Y. Liu, Y. Bai, and H. Liu Insoluble Dietary Fiber L. P. da Silva and M. Walter Fructans Including Inulin M. M. Jacob and S. G. Prapulla Pectin Extraction, Gelation, and Sources A. Rascon-Chu, V. U. Orona, A. Sanchez, and E. Carvajal-Millan Cyclodextrins K. Martina and G. Cravotto Probiotics The Selection and Evaluation of Probiotics G. Perez Martinez, C. Bauerl, and M. C. Collado Amores Phytoestrogens and Hormones Anise Oil L. M. L. Nollet Occurrence and Analysis of Melatonin in Food Plants M. Iriti, S. Vitalini, M. Rossoni, and F. Faoro Tetrapyrroles and Alkaloids Chlorophylls, Chlorophyll-Related Molecules, and Open-Chain Tetrapyrroles B. Schoefs Minerals and Trace Elements Minerals G. Garcia-Llatas, A. Alegria, R. Barbera, M. J. Lagarda, and R. Farre Lipid Compounds Omega and Omega Fatty Acids K. D. Stark Conjugated Linoleic Acid M. A. de la Fuente and M. Juarez Lecithin M. Leon-Camacho and M. Narvaez-Rivas Sterols H Saoussem Stanols H. Saoussem Analytical Methods for the Determination of alpha-Lipoic Acid, Dihydrolipoic Acid, and Lipoyllysine in Dietary Supplements and Foodstuffs G. Sontag and H. Schwartz Alliin and Allicin L. M. L. Nollet Sweeteners Methods of Analysis of Acesulfame-K and Aspartame G.-P. Nikoleli, A. G. Asimakopoulos, and D. P. Nikolelis Methods of Analysis of Saccharin A. G. Asimakopoulos, G.-P. Nikoleli, N. S. Thomaidis, and D. P. Nikolelis Salt Replacers and Taste Modifying Compounds Sodium Replacers M. Reig, M. Armenteros, M-C. Aristoy, and F. Toldra Triterpene Glycosides M. Ganzera Index

The effect of storage on sterol oxidation of ready-to-eat infant foods was evaluated. Two different flavor (honey -LH- or fruits-LF-) liquid infant foods, prepared with milk and cereals, were stored for 0, 2, 4, 7 and 9 months, at 25ºC. Sterol oxidation products (SOP) were extracted by cold saponification, purified by silica solid-phase and analyzed by gas chromatography (GC) and GC-mass spectrometry. -sitosterol was the most representative sterol, followed by cholesterol and campesterol. No significant differences in the total and single SOP content (0.8-1 mg/kg of product) were observed with respect to storage time and type of sample; the main SOP found was 7-ketositosterol (< 0.2 mg/kg of product). The extent of stigmasterol oxidation (2.9%) was higher than that of cholesterol (1.9%) and -sitosterol (1.4%). The type and quality of raw materials, as well as the processing conditions, seem to greatly influence SOP formation and accumulation in infant foods.

The determination of two secondary lipid oxidation compounds (hexanal and pentane) in liquid infant foods using a headspace solid-phase microextraction gas chromatographic (HS-SPME-GC) method has been developed and validated. The HS-SPME analytical conditions (fibre position, equilibration and sampling times) were selected. The analytical parameters of the method (linearity: hexanal from 2.48 to 84.78 ng/g, pentane from 6.21 to 79.55 ng/g; precision: hexanal 2.87%, pentane 2.343.46%; recovery: hexanal 106.60%, pentane 95.39%; detection limit: hexanal 3.63 ng and pentane 4.2 ng) demonstrate the usefulness of the method. Once optimized, the method was applied to liquid infant foods based on milk and cereals, and to powdered adapted and follow-up milk-based infant formulas (IF), stored for four and seven months. In all cases the hexanal content was higher in IF than in milk-cereal based infant foods. No pentane was found in IF.

Fat oxidation during storage of baby foods gives rise to primary and secondary oxidation products. Among the latter, aldehydes are particularly important from a toxicological point of view and in relation to the organoleptic alteration. In baby foods one of the main volatile compounds is hexanal, resulting from the oxidation of linoleic acid. Hexanal content can be used as an indicator of lipid oxidation in these products.