SLE Single-Step Purification and HPLC Isolation Method for Sterols and Triterpenic Dialcohols Analysis from Olive Oil.

The unsaponifiable fraction of oils and fats constitutes a very small fraction but it is an essential part of the healthy properties of some specific oils. It is a complex fraction formed by a large number of minor compounds and it is a source of information to characterize and authenticate the oil sample. Specially, the composition of sterols of any oil or fat is a distinctive feature of itself and, therefore, it has become a useful tool for detecting contaminants and adulterants in oils. A new supported liquid extraction (SLE) technique for the analysis and characterization of the unsaponifiable fraction of fats and oils is proposed. The SLE system includes, as a stationary phase, a combination of adsorbent materials which allow a highly purified unsaponifiable matter ready to be isolated by high performance liquid chromatography (HPLC) and quantified by gas chromatography (GC). This method ensures the removal of fatty acids, avoiding possible interferences and making the analysis of sterols and triterpenic dialcohols easier. The procedure uses a small sample size (0.2 g), reduces the volume of solvents and reagents, and reduces the handling of samples subjected to analytical control. All this is achieved without losing either precision-a relative standard deviation of each compound lower than the reference value (≤16.4%)-or recovery, being for all compounds higher than 88.00%. Therefore, this new technique represents a significant economic and time saving in business control laboratories, a larger productivity and enhancement of working safety.

Formation of Lipid and Protein Oxidation Products during In Vitro Gastrointestinal Digestion of Dry-Cured Loins with Different Contents of Nitrate/Nitrite Added.

The effect of nitrate/nitrite (0, 37.5, 75, and 150 mg/kg) in the dry-cured loin formulation on the formation of lipid and protein oxidation products during in vitro digestion was evaluated. Dry-cured loins formulated with nitrate/nitrite resulted in significantly less lipid and protein oxidation than uncured loins before and after simulated digestion. Compared to loins added with 0 mg/kg nitrate/nitrite, dry-cured loins with 37.5, 75, and 150 mg/kg contained a significantly lower content of conjugated dienes, malondialdehyde, carbonyls, and non-heme iron, and higher amounts of nitrosylmioglobin and thiols. During in vitro digestion, the content of conjugated dienes, malondialdehyde, and carbonyls increased, while thiol content decreased, indicating the development of lipid and protein oxidative processes. At the end of the intestinal phase, the 75 mg/kg digests had a significantly higher content of conjugated dienes, while no differences were found among the other digests. During the in vitro intestinal phase (180 and 240 min), nitrate/nitrite curing resulted in significantly lower malondialdehyde concentrations in the 37.5, 75, and 150 mg/kg loin digests than in the uncured loin digests. No significant differences were observed at the end of the intestinal digestion phase between the cured loin digests. Digests of dried loins without nitrate/nitrite addition showed higher carbonyl contents than the nitrate/nitrite cured counterparts. The loss of thiols was significantly higher in loin digests without added nitrate/nitrite than in loin digests with different amounts of curing salts. The addition of 37.5 mg/kg nitrate/nitrite in the cured loin formulation prevents the formation of lipid peroxidation products and carbonyls from protein oxidation and thiol loss during digestion.

Effect of season, feeding, and anatomical region on the triacylglycerol profile of Iberian pig fat.

The work studies the effects of season, feeding type, and anatomical region on the Iberian pig fat triacylglycerol (TAG) profiles, considered as compositional data (CoDa). The analysis consisted of applying exploratory tools in the simplex and standard multivariate techniques to data transformed into the Euclidean space (ilr coordinates). Compositional biplot showed differences in TAG containing palmitic (P) and oleic (O) acids between the 2005 and 2003/2004 seasons but not within these. PPP (clr variance, 0.139), OLL (0.095), PPS (0.075), POPo + PLP (0.074), and PSS (0.629) showed high CoDa variability among treatments. The ANOVA analysis found significant (p ≤ 0.05) effects of season, feeding type, and anatomical region on pig fat TAG profile, but only that for 2005 season was well predicted (97.5%) by discriminant analysis (DA). Overall, season was more influential on the Iberian pig fat TAG profile than montanera length and sampling region, which effects were not significant for some statistical techniques.

Cardiotrophin-1 contributes to metabolic adaptations through the regulation of lipid metabolism and to the fasting-induced fatty acid mobilization.

It is becoming clear that several human pathologies are caused by altered metabolic adaptations. During liver development, there are physiological changes, from the predominant utilization of glucose (fetal life) to the use of lipids (postnatal life). Fasting is another physiological stress that elicits well-known metabolic adjustments. We have reported the metabolic properties of cardiotrophin-1 (CT-1), a member of the interleukin-6 family of cytokines. Here, we aimed at analyzing the role of CT-1 in response to these metabolic changes. We used different in vivo models. Furthermore, a differential study was carried out with wild-type and CT-1 null mice in fed (ad libitum) and food-restricted conditions. We demonstrated that Ct-1 is a metabolic gene induced in the liver via PPARα in response to lipids in mice (neonates- and food-restricted adults). We found that Ct-1 mRNA expression in white adipose tissue directly involved PPARα and PPARγ. Finally, the physiological role of CT-1 in fasting is confirmed by the impaired food restriction-induced adipose tissue lipid mobilization in CT-1 null mice. Our findings support a previously unrecognized physiological role of CT-1 in metabolic adaptations, through the regulation of lipid metabolism and contributes to fasting-induced free fatty acid mobilization.

Differentiation between Ripening Stages of Iberian Dry-Cured Ham According to the Free Amino Acids Content.

In this paper, the differentiation of three ripening stages, postsalting, drying, and cellar, of Iberian dry-cured ham has been carried out according to their free amino acids contents. Eighteen L-amino acids, alanine, 2-aminobutanoic acid, aspartic acid, cysteine, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine have been determined by gas chromatography with derivatization with N,O-bis(trimethylsilyl)-trifluoroacetamide. Gas chromatography-mass spectrometry was used to confirm the presence of the eighteen amino acids in the ham samples, and gas chromatography using a DB-17HT column and flame ionization detector was used for quantitative determination. Extraction with a mixture methanol-acetonitrile has been carried out, achieving recoveries in the range 52-164%. Methimazole was used as internal standard. Limits of detection ranged between 7.0 and 611.7 mg·kg-1. Free amino acids have been used as chemical descriptors to differentiate between the ripening stages. Principal component analysis and linear discriminant analysis have been used as chemometric techniques, achieving complete differentiation between the ripening stages. Alanine, tyrosine, glutamine, proline, 2-aminobutanoic acid, cysteine, and valine were the most differentiating amino acids.

Assessing the effect of season, montanera length and sampling location on Iberian pig fat by compositional data analysis and standard multivariate statistics.

The work studies the effect of season, montanera length, and sampling location on the Iberian pig fat, using compositional data (CoDa) analysis and standard statistics. CoDa variation array and logratios involving C18:3: (C18:3/C17:1), (C18:3/C20:0), and (C18:3/18:0) as well as the ilr balances (coordinates), based on C18:3, C17:1, and C20:0, showed the highest variances. Discriminant Analysis (DA) led to similar (conventional/coordinates) correct assignations regarding seasons (69/70%), montanera length (71/70%) and sampling location (68/67%). Re-analyzing the subcomposition of only the major fatty acids (FAs), led to slightly poorer results; therefore, the removed FAs might play a role in segregation. Results are in line with those from other authors and could indicate a partial capacity of pigs to control their FA profiles. Overall, CoDa analysis provided useful information on data variability, the effects of factors and, after conversion into coordinates, allowed applying standard statistics while being respectful with the compositional sampling space.

Valorization of Tomato Processing by-Products: Fatty Acid Extraction and Production of Bio-Based Materials.

A method consisting of the alkaline hydrolysis of tomato pomace by-products has been optimized to obtain a mixture of unsaturated and polyhydroxylated fatty acids as well as a non-hydrolysable secondary residue. Reaction rates and the activation energy of the hydrolysis were calculated to reduce costs associated with chemicals and energy consumption. Lipid and non-hydrolysable fractions were chemically (infrared (IR) spectroscopy, gas chromatography/mass spectrometry (GC-MS)) and thermally (differential scanning calorimetry (DSC), thermogravimetric analysis (TGA)) characterized. In addition, the fatty acid mixture was used to produce cutin-based polyesters. Freestanding films were prepared by non-catalyzed melt-polycondensation and characterized by Attenuated Total Reflected-Fourier Transform Infrared (ATR-FTIR) spectroscopy, solid-state nuclear magnetic resonance (NMR), DSC, TGA, Water Contact Angles (WCA), and tensile tests. These bio-based polymers were hydrophobic, insoluble, infusible, and thermally stable, their physical properties being tunable by controlling the presence of unsaturated fatty acids and oxygen in the reaction. The participation of an oxidative crosslinking side reaction is proposed to be responsible for such modifications.

A new SPE/GC-fid method for the determination of cholesterol oxidation products. Application to subcutaneous fat from Iberian dry-cured ham.

A new method for the isolation and analysis of cholesterol oxidation products (COPs) using solid phase extraction (SPE) and silica columns was developed using gas chromatography-flame ion detection (GC-FID). The method comprises of saponification and liquid-liquid extraction of the unsaponifiable fraction prior to the isolation and derivatization of the COPs to trimethylsilyl ethers. The COPs used in this study are cholestane-5α-6α-epoxide, cholestane-3ß-5α-6ß-triol, 25-hydroxycholesterol and 5-cholesten-3ß-ol-7-one. In order to identify the COPs fraction a GC-ion-trap-mass spectrometry experiment were conducted using authentic standards to verify the presence of the COPs. The method was effective at rapidly separating the COPs (25 min run). Calibration curves were linear with the LODs and LOQs bellow 0.03 and 0.07 mgkg(-1) for all cases, respectively. This methodology gave a total recovery for every compound that was used in the study. Betulin was used as an internal standard to monitor the recovery. The method was validated with a standard mixture of COPs. The method has been applied to characterize the COP fraction of subcutaneous fat from Iberian dry-cured ham. Cholestane-5α-6α-epoxide, cholestane-3ß-5α-6ß-triol, 25-hydroxycholesterol and 5-cholesten-3ß-ol-7-one have been identified for the first time in these samples.

Feasibility of use of fatty acid and triacylglycerol profiles for the authentication of commercial labelling in Iberian dry-cured sausages.

In the present study, fatty acid and triacylglycerol profiles were used to evaluate the possibility of authenticating Iberian dry-cured sausages according to their label specifications. 42 Commercial brand 'chorizo' and 39 commercial brand 'salchichón' sausages from Iberian pigs were purchased. 36 Samples were labelled Bellota and 45 bore the generic Ibérico label. In the market, Bellota is considered to be a better class than the generic Ibérico since products with the Bellota label are manufactured with high quality fat obtained from extensively reared pigs fed on acorns and pasture. Analyses of fatty acids and triacylglycerols were carried out by gas chromatography and a flame ion detector. A CP-SIL 88 column (highly substituted cyanopropyl phase; 50 m × 0.25 mm i.d., 0.2 µm film thickness) (Varian, Palo Alto, USA) was used for fatty acid analysis and a fused silica capillary DB-17HT column (50% phenyl-50% methylpolysiloxane; 30 m × 0.25 mm i.d., 0.15 µm film thickness) was used for triacylglycerols. Twelve fatty acids and 16 triacylglycerols were identified. Various discriminant models (linear quadratic discriminant analyses, logistic regression and support vector machines) were trained to predict the sample class (Bellota or Ibérico). These models included fatty acids and triacylglycerols separately and combined fatty acid and triacylglycerol profiles. The number of correctly classified samples according to discriminant analyses can be considered low (lower than 65%). The greatest discriminant rate was obtained when triacylglycerol profiles were included in the model, whilst using a combination of fatty acid and triacylglycerol profiles did not improve the rate of correct assignation. The values that represent the reliability of prediction of the samples according to the label specification were higher for the Ibérico class than for the Bellota class. In fact, quadratic and Support Vector Machine discriminate analyses were not able to assign the Bellota class (0%) when combined fatty acids and triacylglycerols were included in the model. The use of fatty acid and triacylglycerol profiles to discriminate Iberian dry-cured sausages in the market according to their labelling information is unclear. In order to ensure the genuineness of Iberian dry-cured sausages in the market, identification of fatty acid and triacylglycerol profiles should be combined with the application of quality standard traceability techniques.

Subcutaneous fat triacylglycerols profile from Iberian pigs as a tool to differentiate between intensive and extensive fattening systems.

Triacylglycerols of subcutaneous fat of Iberian pigs reared on two different feeding systems, extensive and intensive, have been determined by gas chromatography with a flame ionization detector. Analyses were performed on a column coated with a bonded stationary phase (50% phenyl-50% methylpolysiloxane) with hydrogen as the carrier gas. Lipids were extracted by melting the subcutaneous fat in a microwave oven and then filtering and dissolving in hexane. A total amount of 1995 samples from several campaigns were considered. Palmitoyl-stearyl-oleoyl glycerol and palmitoyl-dioleoyl glycerol were the most abundant triacylglycerols found in the samples. A study on the discriminating power of the triacylglycerols to differentiate samples according to the pig feeding system was performed. By using the triacylglycerols as chemical descriptors, principal component analysis, linear discriminant analysis, and soft independent modeling of class analogy were applied. Dioleoyl-linoleoyl glycerol and oleoyl-dilinoleoyl glycerol were the most discriminating variables. Variable-variable plots of these two glycerols allow separation of the samples according to their content.

A new high-performance liquid chromatographic method with evaporative light scattering detector for the analysis of phospholipids. Application to Iberian pig subcutaneous fat.

A new method for the analysis of phospholipids by normal-phase HPLC is described using a silica column. Addition of ammonia and triethylamine to a gradient based on chloroform/methanol/water promoted a good and rapid separation of phospholipid classes (20 min run). The use of an evaporative light scattering detector permitted an accurate analysis of a mixture of phospholipids. Calibration curves were linear within different range for each phospholipid class. The LOD and LOQ obtained were below 0.03 and 0.05 mg kg⁻¹ for all cases, respectively. Besides, a new method for the separation of phospholipids from total lipids before HPLC analysis by a solid-phase extraction (SPE) with Si cartridges has been developed. This methodology gave a good recovery ranging from 97 to 117%. The method was validated with a standard mixture of phospholipids. This method has been applied to characterize the phospholipid fraction of subcutaneous fat from Iberian pig. Cardiolipin, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and sphingomyelin have been described for first time in these samples. The fatty acid composition of the different phospholipid classes and their HPLC electrospray ionization mass spectrometry have been used for characterizing the molecular species present in each one.

Volatile hydrocarbon profile of Iberian dry-cured hams. A possible tool for authentication of hams according to the fattening diet.

The aims of this work were to carry out a comprehensive study of the volatile hydrocarbons of 34 Iberian dry-cured hams and to evaluate the efficiency of these compounds for discriminating hams according to the fattening system: "Montanera" (B) and "Cebo" (C). The samples of hams were obtained by mincing the semimembranosus and semitendinosus muscles from slices of dry-cured ham. The analyses were carried out by gas chromatography-mass spectrometry with a polar capillary column and after a previous extraction by Purge and Trap method. Forty-three volatile hydrocarbons were identified, 26 of them for the first time in Iberian dry-cured ham. Only five compounds showed significant differences between the two types of hams. Among the 33 volatile hydrocarbons, 22 of them allowed a complete discrimination of the two groups of hams according the fattening system.

Authentication of fattening diet of goat kid according to their fatty acid profile from perirenal fat.

Fatty acids of forty-two samples of perirenal fat of goat kids reared on three different feeding systems: goat milk (B), milk replacer (R) and milk-based starter (F) have been analyzed by Gas Chromatography flame ionization detector. The lipids were extracted by melting of perirenal fat in a microwave oven. The fat was then filtered and dissolved in hexane. This analysis was performed on a column (100 m x 0.25 mm i.d. and 0.25 microm film thickness) coated with a polar stationary phase HP-88 and flame ionization detector was used. Hydrogen (25 psi inlet constant pressure) was used as carrier gas. Programmed temperature was kept at 175 degrees C and held isothermally for 10 min, and was then raised to 205 degrees C at a rate of 3 degrees C/min and held isothermally for 10 min. By using the fatty acids as chemical descriptors, pattern recognition techniques were applied to differentiate between goat milk, milk replacer and milk-based starter fattening diet of goat kid. C18:2 and C18:3 acids were found to be the most differentiating variables.

Determination of ent-kaurene in subcutaneous fat of Iberian pigs by gas chromatography multi-stage mass spectrometry with the aim to differentiate between intensive and extensive fattening systems.

This work presents a gas chromatography multi-stage mass spectrometry (GC-MS(3)) method for the determination of ent-kaurene in subcutaneous fat of Iberian pig, present in adipose tissue of animals due to pasture ingestion (extensive fattening system). The method comprises a saponification and a liquid-liquid extraction of the unsaponifiable fraction, followed by an isolation of the hydrocarbon fraction by high performance liquid chromatography (HPLC) and analysis by GC-MS(3) (ion trap) with electronic ionization. The GC-MS(3) analysis allows the isolation and characterization of specific fragments from the original (MS(1)) molecular structure, and particularly, those fragments originated from the precursor ion (m/z=229) characteristic of ent-kaurene. The MS/MS product fragment m/z=213 is used as a further precursor fragment giving rise to a MS(3) spectrum specific for ent-kaurene. The limit of detection of the MS(3) technique is lower than 0.2 microg kg(-1) and a linear regression has been found between 0.2 and 112 microg kg(-1). This method is applicable for the determination of the fattening system of the Iberian pig.

Changes in the fatty acid and triacylglycerol profiles in the subcutaneous fat of Iberian ham during the dry-curing process.

In this study, we have evaluated the changes that occur in the profiles of total fatty acids and triacylglycerols during the dry-curing process (730 days) of Iberian ham. The subcutaneous adipose tissues of six hams obtained from three Iberian pigs fed on acorns were analyzed periodically during the processing time (from the raw to the dry-cured samples), including postsalting, drying, and ripening stages. The environmental conditions were also registered. The curing process significantly decreased (p < 0.01) the relative percentages of total polyunsaturated fatty acids, including C18:2n-6 and C18:3n-3 and, therefore, significantly increased (p < 0.05) the level of monounsaturated fatty acids. The triglycerides containing 0-2 double bonds showed an increase during the curing process. On the contrary, the more unsaturated ones (3-5 double bonds) suffered a significant decrease. We have postulated that these changes could also be due to polymerization and oxidation reactions that affect the triacylglycerols and besides the fatty acids. In general, most fatty acids and triacylglycerols reversed the trend by about 500-600 days of processing.

Changes in the concentrations of free fatty acid, monoacylglycerol, and diacylglycerol in the subcutaneous fat of Iberian ham during the dry-curing process.

Changes in diacylglycerols, monoacylglycerols, and free fatty acid composition of subcutaneous fat of six Iberian hams during the dry-cured process were investigated. In addition, an analytical method for simultaneous quantification of diacylglycerols, monoacylglycerols, and free fatty acid by solid-phase extraction-gas chromatography was developed. The different molecular species of free fatty acids, monoacylglycerols, and diacylglycerols and 1,2- and 1,3-isomers of diacylglycerols have been described for the first time in this type of sample. A logarithmic increase of the 1,3-diacylglycerol profile throughout the processing time has been found, reaching a balance value of 62% around 500 days. The formation of diacylglycerol isomers takes place, although the 1,3-/1,2-diacylglycerol ratio increases during the process to 1.65 due to isomerization of the 1,2-form toward the 1,3-form. The profiles of monoacyl- and diacylglycerols and free fatty acids follow the same trend. The experimental values of free fatty acid are greater than theoretical prediction, probably due to phospholipid and monoacylglycerol hydrolysis.

Contenido en acidos grasos trans de las margarinas: evolución en las últimas décadas y tendencias actuales / Margarine's trans fatty acid composition: modifications during the last decades and new trends

El proceso de hidrogenación llevado a cabo en la industria para obtener margarinas conlleva la formación de una cierta cantidad de isómeros trans. El consumo de este alimento así como la cantidad de estos isómeros que contiene, han sido motivo de gran controversia en las últimas décadas. Mientras que en la década de los ochenta, se consideraba saludable el consumo de margarinas, en la década de los noventa numerosos estudios indicaron que el 18:1t, incrementaba las concentraciones de LDL-colesterol y reducía los niveles de HDL-colesterol, además de asociar su ingesta con un incremento del riesgo de sufrir enfermedad coronaria. La creciente publicidad sobre los efectos desfavorables de los ácidos grasos trans parece haber influenciado a los productores de margarinas, a reducir la cantidad de estos isómeros en sus productos. Mientras tanto, Estados Unidos ha adoptado una legislación para especificar su contenido en el etiquetado de los alimentos y en Europa, Dinamarca ha limitado los niveles máximos permitidos en los alimentos

[Margarine's trans-fatty acid composition: modifications during the last decades and new trends]. / Contenido en ácidos grasos trans de las margarinas: evolución en las últimas décadas y tendencias actuales.

Trans fatty acids isomers are formed during the hydrogenation process used in the food industry to harden oils. In the last decades there has been a great controversy about the consumption of margarine due to the levels of trans fatty acids they contain. While in the eighties consumption of margarines was considered healthy, during the nineties several studies indicated that consumption of 18:1t increased LDL-cholesterol levels and decreased HDL-cholesterol level, and was related with an increased risk of coronary heart disease. The publicity about the unfavourable effects of trans fatty acid consumption seems to have influenced margarine producers to reduce the trans fatty acid content of margarines. Meanwhile USA has adopted a new legislation about trans fatty acid labelling. In Europe, Dinamarca has limited the maximum level of trans fatty acids allowed in food products.

Multivariate characterization of the fatty acid profile of spanish cookies and bakery products.

The fatty acid compositions of 39 brands of cookies and bakery products were analyzed with special attention to the their trans-fatty acid content. The average contents (percent w/w of the total fatty acids detected) for the different nutritional fractions were as follows: saturated fatty acids (SFA), 49.43% (SD = 17.04); monounsaturated fatty acids (MUFA), 32.87% (SD = 10.94); polyunsaturated fatty acids (PUFA), 12.48% (SD = 11.29); and trans-fatty acids (TFA), 5.20% (SD = 9.30), the mean fat content being 22.7% (SD = 7.4). C18:1t was the predominant trans isomer found in all of the analyzed samples (mean = 4.04, SD = 8.46). Factor analysis performed on the fatty acid profile allowed a meaningful classification of the samples according to the main source of fat employed in their elaboration. Four factors that explained 75% of the total variance were retained. According to the results obtained, the fat used to elaborate the samples was mainly composed of vegetable and animal fat, and only in a few samples were partially hydrogenated vegetable oils used as the main source of fat.