Impact of a Plant Sterol- and Galactooligosaccharide-Enriched Beverage on Colonic Metabolism and Gut Microbiota Composition Using an In Vitro Dynamic Model.

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.

First international descriptive and interventional survey for cholesterol and non-cholesterol sterol determination by gas- and liquid-chromatography-Urgent need for harmonisation of analytical methods.

Serum concentrations of lathosterol, the plant sterols campesterol and sitosterol and the cholesterol metabolite 5α-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 (5α-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.

Impact of colonic fermentation on sterols after the intake of a plant sterol-enriched beverage: A randomized, double-blind crossover trial.

BACKGROUND: Cholesterol microbial transformation has been widely studied using in vitro fermentation assays, but less information is available on the biotransformation of plant sterols (PS). The excretion percentage of animal sterols (AS) (67-73%) is considerably greater than that of PS (27-33%) in feces from healthy humans following a Western diet. However, a lower content of AS in feces from subjects following a vegetarian, vegan or low-fat animal diet has been seen when compared to omnivorous subjects. Although only one human study has reported fecal sterol excretion after the consumption of PS-enriched food (8.6 g PS/day), it was found that the target group showed an increase in the excretion of cholesterol and a 57% decrease in its metabolites compared to the control group. OBJECTIVE: Evaluation of the impact of a PS-enriched milk based fruit beverage intake on fecal sterol excretion and the microbial conversion of sterols in postmenopausal women with mild hypercholesterolemia. METHODS: Forty postmenopausal women participated in a randomized, double-blind, crossover study with two beverages, with a PS-enriched (2 g PS/day) or without. The women were divided in two groups: 20 women consumed the PS-enriched beverage and the other 20 women consumed a placebo (without PS) beverage for 6 weeks. After a four-week washout period, the type of beverage was exchanged and consumed for another 6 weeks. Feces were collected at the start (0 and 10 weeks) and end of each intervention period (6 and 16 weeks), and fecal sterols were determined by capillary gas chromatography with mass spectrometry. RESULTS: The intake of the PS-enriched beverage modified the fecal sterol excretion profile. A significant increase mainly in PS and their metabolites versus the placebo intervention period was observed. Although the same effect was not observed in the case of AS, a tendency towards increased cholesterol and decreased coprostanol (the main metabolite of cholesterol) was recorded after PS-enriched beverage intake versus placebo. Furthermore, the PS-enriched beverage also modified the microbial conversion of sterols. In this context, an important decrease in the conversion percentage of cholesterol in 16 women (between 11% and 50%) and of sitosterol in 24 women (between 15% and 61%) was observed. CONCLUSIONS: The results obtained suggest that the microbiota could preferably use PS as a substrate, when present in a greater proportion compared with cholesterol. Besides, a lower sitosterol and cholesterol conversion trend would mean that intake of the PS-enriched beverage could modulate the metabolic activity of the gut microbiota. Therefore, further studies on the impact of PS-enriched foods upon gut microbiota modulation are needed. Clinical Trial Registry Number: NCT02065024 listed on the NIH website: ClinicalTrials.gov. Clinical Trial Registry Name: Food Matrix and Genetic Variability as Determinants of Bioavailability and Biological Effects of Beta-cryptoxanthin and Phytosterols (foodmagenpol). The full trial protocol is available upon request to the corresponding author.

Oat and lipolysis: Food matrix effect.

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.

Sterols in human milk during lactation: bioaccessibility and estimated intakes.

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 ß-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.

International descriptive and interventional survey for oxycholesterol determination by gas- and liquid-chromatographic methods.

Increasing numbers of laboratories develop new methods based on gas-liquid and high-performance liquid chromatography to determine serum concentrations of oxygenated cholesterol metabolites such as 7α-, 24(S)-, and 27-hydroxycholesterol. We initiated a first international descriptive oxycholesterol (OCS) survey in 2013 and a second interventional survey 2014 in order to compare levels of OCS reported by different laboratories and to define possible sources of analytical errors. In 2013 a set of two lyophilized serum pools (A and B) was sent to nine laboratories in different countries for OCS measurement utilizing their own standard stock solutions. In 2014 eleven laboratories were requested to determine OCS concentrations in lyophilized pooled sera (C and D) utilizing the same provided standard stock solutions of OCS. The participating laboratories submitted results obtained after capillary gas-liquid chromatography-mass selective detection with either epicoprostanol or deuterium labelled sterols as internal standards and high-performance liquid chromatography with mass selective detection and deuterated OCS as internal standard. Each participant received a clear overview of the results in form of Youden-Plots and basic statistical evaluation in its used unit. The coefficients of variation of the concentrations obtained by all laboratories using their individual methods were 58.5-73.3% (survey 1), 56.8-60.3% (survey 2); 36.2-35.8% (survey 1), 56.6-59.8, (survey 2); 61.1-197.7% (survey 1), 47.2-74.2% (survey 2) for 24(S)-, 27-, and 7α-hydroxycholesterol, respectively. We are surprised by the very great differences between the laboratories, even under conditions when the same standards were used. The values of OCS's must be evaluated in relation to the analytical technique used, the efficiency of the ample separation and the nature of the internal standard used. Quantification of the calibration solution and inappropriate internal standards could be identified as major causes for the high variance in the reported results from the different laboratories. A harmonisation of analytical standard methods is highly needed.

Cholesterol Content in Human Milk during Lactation: A Comparative Study of Enzymatic and Chromatographic Methods.

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 (<1.8 and <6 µg/100 g of HM, respectively). Suitable intra- and interassay precisions (all <18%) and satisfactory recovery percentages (80-109%) were obtained for both methods. In addition, both methodologies were used to assess cholesterol evolution in HM during lactation, showing a 50% decrease at 6 months versus colostrum. The E-S method overestimated cholesterol content by <20% versus the GC method. The results indicate that both methods may be used by the industry and in research to better understand the differences between the sterol profiles of infant formulas and HM.

The impact of galactooligosaccharides on the bioaccessibility of sterols in a plant sterol-enriched beverage: adaptation of the harmonized INFOGEST digestion 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.

Sterols in Infant Formulas: A Bioaccessibility Study.

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 ß-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.

Determination of Fecal Sterols Following a Diet with and without Plant Sterols.

The aim of this study was to develop a method for neutral fecal sterols determination in subjects receiving a normal diet with or without a plant sterols-enriched beverage using gas chromatography-mass spectrometry (GC/MS). Sample preparation conditions (homogenization of lyophilized feces with water) were evaluated. Sterol determination required direct hot saponification, unsaponifiable extraction with hexane, and the formation of trimethylsilyl (TMS) ether derivatives. The method allows the quantification of cholesterol, plant sterols and their metabolites (coprostanol, coprostanone, cholestanol, cholestanone, methylcoprostanol, methylcoprostanone, ethylcoprostenol, stigmastenol, ethylcoprostanol and ethylcoprostanone). Good linearity was obtained (r > 0.96) and interference was only observed for coprostanone, where the standard addition method proved necessary for quantification. The limits of detection (LOD) ranged from 0.10 to 3.88 µg/g dry feces and the limits of quantitation (LOQ) from 0.34 to 12.94 µg/g dry feces. Intra- and inter-assay precision (RSD %) were 0.9-9.2 and 2.1-11.3, respectively. Accuracy, expressed as percentage recovery (80-119%) was obtained for all determined sterols.

Sterols in infant formulas: validation of a gas chromatographic method.

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, ß-sitosterol and sitostanol) found in IFs. All correlation coefficients obtained for the calibration curves of sterols studied were >0.99. Limits of detection (<1 µg/100 mL) and quantification (<4 µg/100 mL) are suitable for sterols determination in IFs. The within-assay precision ranged from 1.6% to 8.8%, while the between-assay precision was <10% for most of sterols. Accuracy was satisfactory and was calculated by recovery assays (ranging 93-108%). The analytical parameters obtained showed the suitability of the proposed method for the determination of sterols in IFs.

Evaluation of Sialic Acid in Infant Feeding: Contents and Bioavailability.

Sialic acid (Sia) contents and bioaccessibility (BA) in human milk (HM) and infant formulas (IFs) were determined, and Sia intakes by infants between 0 and 6 months of age were evaluated. Total Sia contents in HM decreased during lactation from 136.14 to 24.47 mg/100 mL. The total Sia contents in IFs (13.15-25.78 mg/100 mL) were lower than in HM and were not related to the addition of ingredients acting as sources of Sia in their formulation. The Sia intakes derived from IF consumption were lower than in HM, and only one IF reached the intakes provided by HM from the age of 2 months. Despite the lower total Sia content in IFs, the BA of Sia in IFs (88.08-92.96%) was significantly greater than in mature HM (72.51%) and similar to that found in colostrum (96.43%). However, the Sia contents in the available soluble fraction of IFs did not reach those provided by HM.

Impact of Lipid Components and Emulsifiers on Plant Sterols Bioaccessibility from Milk-Based Fruit Beverages.

Sterol bioaccessibility (BA) of three plant sterol (PS)-enriched milk-based fruit beverages (MFb) with different fat contents (1.1-2.4%), lipid sources (animal or vegetable), and without or with emulsifiers (whey proteins enriched with milk fat globule membrane (MFGM) or soy lecithin) was evaluated after simulated gastrointestinal digestion. The BA of total PS followed the order 31.4% (MFbM containing milk fat and whey proteins enriched with MFGM) = 28.2% (MFbO containing extra virgin olive oil and soy lecithin) > 8.7% (MFb without fat addition). Total and individual PS content in the bioaccessible fractions followed the order MFbM > MFbO > MFb. Consequently, formulation with MFGM is proposed in beverages of this kind to ensure optimum bioavailability of PS. Our results suggest that the BA of PS is influenced by the type and quantity of fat and the emulsifier type involved.

Bioaccessibility study of plant sterol-enriched fermented milks.

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). ß-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.

DETERMINATION OF CHOLESTEROL IN HUMAN MILK: AN ALTERNATIVE TO CHROMATOGRAPHIC METHODS.

INTRODUCTION: human milk (HM) is considered the best option for feeding healthy infants. Cholesterol (CHOL) is important for proper development of the nervous system, and for hormone and vitamin synthesis in growing infants. Breastfeeding and dietary CHOL intake during infancy have been suggested to affect blood lipid levels and the risk of cardiovascular disease in adulthood. Gas chromatography is the technique most widely used to determine CHOL in HM. Chromatographic methods are specific for the determination of CHOL and other sterols present in HM, but are extremely time consuming, and the costs and equipment requirements mean that they are not accessible to all laboratories. AIM: the present study describes the optimization and validation of an enzymatic-spectrophotometric method for CHOL determination in mature HM. METHOD: determination of CHOL involves fat extraction with chloroform:methanol, hot saponification and extraction of the unsaponifiable fraction with diethyl ether. CHOL was determined by an enzymatic method in which the concentration of the lutidine dye formed is stoichiometric to the amount of CHOL, and is measured by the increase in light absorbance at 405 nm. RESULTS: human milk fat (mg/mL) (27.5 ± 1.3) and CHOL (0.113 ± 0.004) in analyzed HM were within the ranges reported by others authors. Analytical parameters of the proposed method were assessed: The precision values (%) (expressed as the relative standard deviation (RSD)) were: 3.5 and 6.7 for intra- and inter-day, respectively. Accuracy, estimated by recovery assays, was 110 ± 1.6%. CONCLUSION: the validated enzymatic-spectrophotometric method for determining CHOL in HM constitutes an alternative for fast and simple analysis of CHOL with equipment requirements accessible to any laboratory.

Introducción: la leche humana (HM) se considera el modo óptimo de alimentación en lactantes sanos. El colesterol (CHOL) es importante para el correcto desarrollo del sistema nervioso y la síntesis de hormonas y vitaminas en el crecimiento del lactante. Se ha constatado que la lactancia materna y la ingesta dietética de CHOL durante la infancia influye en los niveles de lípidos en sangre, así como en el riesgo de enfermedad cardiovascular en la edad adulta. La técnica más utilizada para determinar el CHOL en HM es la cromatografía de gases. Los métodos cromatográficos son específicos para la determinación del CHOL y otros esteroles presentes en la HM, pero el elevado tiempo consumido, los costes y la necesidad de una instrumentación específica hacen que no sea accesible para cualquier laboratorio. Objetivo: el presente estudio describe la optimización y validación de un método enzimático-espectrofotométrico para la determinación del CHOL en HM madura. Métodos: la determinación del CHOL requiere una extracción lipídica con cloroformo:metanol, saponificación en caliente y extracción del insaponificable con dietil éter. El CHOL fue determinado por un método enzimático en el que la concentración de lutidina formada es estequiométrica a la cantidad de CHOL, y se mide a 405 nm. Resultados: la cantidad de grasa (mg/mL) (27,5 ± 1,3) y de CHOL (0,113 ± 0,004) en la HM analizada se halla en el intervalo indicado por otros autores. Se evalúan parámetros analíticos del método propuesto: la precisión (expresada como desviación estándar relativa en %) fue de 3,5 y 6,7 para intra- e interdía, respectivamente. La exactitud, estimada mediante ensayos de recuperación, fue de 110 ± 1,6%. Conclusión: el método enzimático-espectrofotométrico validado para determinar el CHOL en HM constituye una alternativa para el análisis rápido y sencillo de CHOL empleando equipos accesibles para cualquier laboratorio.

Determination of cholesterol in human milk: an alternative to chromatographic methods / Determinación del colesterol en leche humana: una alternativa a los métodos cromatográficos

Introduction: human milk (HM) is considered the best option for feeding healthy infants. Cholesterol (CHOL) is important for proper development of the nervous system, and for hormone and vitamin synthesis in growing infants. Breastfeeding and dietary CHOL intake during infancy have been suggested to affect blood lipid levels and the risk of cardiovascular disease in adulthood. Gas chromatography is the technique most widely used to determine CHOL in HM. Chromatographic methods are specific for the determination of CHOL and other sterols present in HM, but are extremely time consuming, and the costs and equipment requirements mean that they are not accessible to all laboratories. Aim: the present study describes the optimization and validation of an enzymatic-spectrophotometric method for CHOL determination in mature HM. Method: determination of CHOL involves fat extraction with chloroform:methanol, hot saponification and extraction of the unsaponifiable fraction with diethyl ether. CHOL was determined by an enzymatic method in which the concentration of the lutidine dye formed is stoichiometric to the amount of CHOL, and is measured by the increase in light absorbance at 405 nm. Results: human milk fat (mg/mL) (27.5 ± 1.3) and CHOL (0.113 ± 0.004) in analyzed HM were within the ranges reported by others authors. Analytical parameters of the proposed method were assessed: The precision values (%) (expressed as the relative standard deviation (RSD)) were: 3.5 and 6.7 for intra- and inter-day, respectively. Accuracy, estimated by recovery assays, was 110 ± 1.6%. Conclusion: the validated enzymatic-spectrophotometric method for determining CHOL in HM constitutes an alternative for fast and simple analysis of CHOL with equipment requirements accessible to any laboratory (AU)

Introducción: la leche humana (HM) se considera el modo óptimo de alimentación en lactantes sanos. El colesterol (CHOL) es importante para el correcto desarrollo del sistema nervioso y la síntesis de hormonas y vitaminas en el crecimiento del lactante. Se ha constatado que la lactancia materna y la ingesta dietética de CHOL durante la infancia influye en los niveles de lípidos en sangre, así como en el riesgo de enfermedad cardiovascular en la edad adulta. La técnica más utilizada para determinar el CHOL en HM es la cromatografía de gases. Los métodos cromatográficos son específicos para la determinación del CHOL y otros esteroles presentes en la HM, pero el elevado tiempo consumido, los costes y la necesidad de una instrumentación específica hacen que no sea accesible para cualquier laboratorio. Objetivo: el presente estudio describe la optimización y validación de un método enzimático-espectrofotométrico para la determinación del CHOL en HM madura. Métodos: la determinación del CHOL requiere una extracción lipídica con cloroformo:metanol, saponificación en caliente y extracción del insaponificable con dietil éter. El CHOL fue determinado por un método enzimático en el que la concentración de lutidina formada es estequiométrica a la cantidad de CHOL, y se mide a 405 nm. Resultados: la cantidad de grasa (mg/mL) (27,5 ± 1,3) y de CHOL (0,113 ± 0,004) en la HM analizada se halla en el intervalo indicado por otros autores. Se evalúan parámetros analíticos del método propuesto: la precisión (expresada como desviación estándar relativa en %) fue de 3,5 y 6,7 para intra- e interdía, respectivamente. La exactitud, estimada mediante ensayos de recuperación, fue de 110 ± 1,6%. Conclusión: el método enzimático-espectrofotométrico validado para determinar el CHOL en HM constituye una alternativa para el análisis rápido y sencillo de CHOL empleando equipos accesibles para cualquier laboratorio (AU)

Sterol Composition in Infant Formulas and Estimated Intake.

Sterol contents in infant formulas (IFs) from the European market were determined, and their intakes by infants between 0 and 6 months were evaluated. Total animal sterols (mg/100 mL) ranged from 1.71 to 5.46, cholesterol being the main animal sterol (1.46-5.1). In general, cholesterol and desmosterol were lower than the human milk (HM) values indicated by other authors. Total plant sterol (mg/100 mL) ranged from 3.1 to 5.0. ß-Sitosterol, the most abundant phytosterol, ranged from 1.82 to 3.01, followed by campesterol (0.72-1.15), stigmasterol (0.27-0.53), and brassicasterol (0.14-0.28). Cholesterol intake (mg/day) ranged from 9 to 51 and plant sterol intake (mg/day) from 19 to 50. The sterol profile of IFs is highly dependent on the type and quantity of fats used in their formula. The use of bovine milk fat and milk fat globule membrane in the IFs can approximate the profile of animal sterols to those found in HM, though cholesterol intakes in breastfed infants are still higher than in formula-fed infants.

Plant sterol oxides in functional beverages: influence of matrix and storage.

Three plant sterol (PS)-enriched beverages, milk based fruit juice (MFJPS), fruit juice (FJPS) and milk beverage (MPS), were stored at 4, 24, or 37 °C and analysed at regular time intervals of 2 months until 6 months. PS stability was analysed from the production of phytosterol oxidation products (POPs). The ß-sitosterol oxides (7α/7ß-hydroxy, ß/α-epoxy, triol, and 7-keto) and campesterol oxides (ß/α-epoxy, and 7-keto) were detected in all beverages and at all storage times and temperatures. Total POP contents followed the order MPS≫FJPS>MFJPS. In general, the beverages showed low PS oxidation levels (<0.17%). Predictive models of POP content versus storage time were established. These models explain total POP content by over 75% and individual POP content by over 50%. We propose 7-ketositosterol and 7-ketocampesterol as PS oxidation markers during storage of beverages of this kind.

7-Ketocholesterol as marker of cholesterol oxidation in model and food systems: when and how.

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.

Plant sterols and antioxidant parameters in enriched beverages: storage stability.

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.