Analysis of trans–cis fatty acids in fatty acid steryl esters isolated from soybean oil deodoriser distillate

The characterisation of the isolated natural product from soybean oil deodoriser distillate (SODD) has gained increasing importance due to health concerns. The purpose of the present work was to characterise fatty acids steryl esters (FASEs) composition and to determine the trans fatty acids (TFAs) contents in FASEs obtained from SODD by using simple and accurate method. Isolation of FASEs with saturated fatty acids moieties from FASEs mixture was also developed. It was found that the fatty acids composition of the isolated FASEs were 31.74% palmitate acid (C16:0), 9.99% stearic acid (C18:0), 0.46% elaidic acid (C18:1 trans-9), 27.51% oleic acid (C18:1 cis-9), 0.19% linoleaidic acid (C18:2 cis-9, trans-12), and 29.19% linoleic acid (C18:2 cis-9, cis-12). TFAs, which account for about 0.65% of the total fatty acids, were detected in the isolated FASEs. Palmitate and stearate steryl esters isolated from SODD are promising natural product because they are free of trans-isomers.     

An Improved Silver Ion HPLC Combined with Capillary Gas Chromatography of cis/trans Fatty Acids in Alimentary Fats and Human Milk Fat

A technique of analysis is described using a silver ion-loaded HPLC (Ag⁺/HPLC) column, gradient elution, and a laser light scattering detector followed by capillary gas chromatography (GC) for the determination of the fatty acid methyl ester (FAME) composition of rapeseed oil, the respective hydrogenated fat, some dietary fats, and human milk fat. With the use of Ag⁺/HPLC, FAMEs were separated into the following classes: saturated, trans-monounsaturated, cis-monounsaturated, trans–trans-di-unsaturated, trans–cis-di-unsaturated, cis–cis-di-unsaturated, and higher unsaturated FAMEs. Within each class, partial separation of positional isomers was also possible. Fractions of the eluate containing trans and cis C18:1 were separately collected and re-analyzed by GC in order to quantitate the positional isomers eight trans and nine cis in rapeseed oil and in human milk fat. The profiles of eight trans positional isomers in human milk fat and in hydrogenated rapeseed oil were alike but entirely different from that of six C18:1 trans isomers in bovine milk fat. Out of nine C18:1 cis isomers found in hydrogenated fat, only C18:1-9c and C18:1-11c were found in both human and bovine milk fat which may indicate some discrimination which occurs for certain isomers in fatty acid metabolism.     

Conjugated alpha-linolenic acid isomers in bovine milk and muscle

Conjugated linolenic acids (CLnA) are octadecatrienoic fatty acid isomers with at least 2 conjugated double bonds. Various CLnA isomers occur naturally, and some isomers could be formed by ruminants from dietary α-linolenic acid. Ruminant biohydrogenation of polyunsaturated fatty acids gives rise to the formation of numerous metabolites having conjugated or nonconjugated structures. The objectives of this study were to identify and characterize CLnA isomers in milk fat and muscle lipid extracts from cattle fed a high-forage diet. The analysis of total fatty acid methyl esters revealed levels of total CLnA of 0.39% in a single milk lipid extract and 0.34% in a single muscle lipid extract. Fatty acid methyl esters were fractionated by argentation thin-layer chromatography. A fraction containing dienoic fatty acids as well as CLnA isomers was isolated and analyzed. The double bond positions of CLnA isomers (cis-9, trans-11, cis-15 and cis-9, trans-13, cis-15 18:3) were confirmed by mass spectrometry of their 4,4-dimethyloxazoline derivatives. Mass spectra of the cis-9, trans-13, cis-15 18:3 isomer was characterized by an intense ion at m/z 236 attributable to the formation of 2 stabilized allylic radical fragments, whereas this intense ion corresponding to the stabilized radical fragments was located at m/z 262 for the cis-9, trans-11, cis-15 18:3 isomer. The gap of 12 amu between m/z 250 and 262 confirmed the occurrence of a double bond in position Δ¹³. Configuration of the double bonds of standards having similar mass spectra and gas-liquid chromatographic retention times was confirmed by ¹H nuclear magnetic resonance. We also showed that both CLnA isomers were found in the muscle lipid extract, whereas only the cis-9, trans-11, cis-15 18:3 isomer was identified in the milk lipid extract. This study appears to be the first to identify 2 CLnA isomers in bovine muscle lipid extract.     

Effect of plant oils and camelina expeller on milk fatty acid composition in lactating cows fed diets based on red clover silage

Five multiparous Finnish Ayrshire cows fed red clover silage-based diets were used in a 5 × 5 Latin square with 21-d experimental periods to evaluate the effects of various plant oils or camelina expeller on animal performance and milk fatty acid composition. Treatments consisted of 5 concentrate supplements containing no additional lipid (control), or 29 g/kg of lipid from rapeseed oil (RO), sunflower-seed oil (SFO), camelina-seed oil (CO), or camelina expeller (CE). Cows were offered red clover silage ad libitum and 12 kg/d of experimental concentrates. Treatments had no effect on silage or total dry matter intake, whole-tract digestibility coefficients, milk yield, or milk composition. Plant oils in the diet decreased short- and medium-chain saturated fatty acid (6:0-16:0) concentrations, including odd- and branched-chain fatty acids and enhanced milk fat 18:0 and 18-carbon unsaturated fatty acid content. Increases in the relative proportions of cis 18:1, trans 18:1, nonconjugated 18:2, conjugated linoleic acid (CLA), and polyunsaturated fatty acids in milk fat were dependent on the fatty acid composition of oils in the diet. Rapeseed oil in the diet was associated with the enrichment of trans 18:1 (Δ4, 6, 7, 8, and 9), cis-9 18:1, and trans-7,cis-9 CLA, SFO resulted in the highest concentrations of trans-5, trans-10, and trans-11 18:1, Δ9,11 CLA, Δ10,12 CLA, and 18:2n-6, whereas CO enhanced trans-13-16 18:1, Δ11,15 18:2, Δ12,15 18:2, cis-9,trans-13 18:2, Δ11,13 CLA, Δ12,14 CLA, Δ13,15 CLA, Δ9,11,15 18:3, and 18:3n-3. Relative to CO, CE resulted in lower 18:0 and cis-9 18:1 concentrations and higher proportions of trans-10 18:1, trans-11 18:1, cis-9,trans-11 CLA, cis-9,trans-13 18:2, and trans-11,cis-15 18:2. Comparison of milk fat composition responses to CO and CE suggest that the biohydrogenation of unsaturated 18-carbon fatty acids to 18:0 in the rumen was less complete for camelina lipid supplied as an expeller than as free oil. In conclusion, moderate amounts of plant oils in diets based on red clover silage had no adverse effects on silage dry matter intake, nutrient digestion, or milk production, but altered milk fat composition, with changes characterized as a decrease in saturated fatty acids, an increase in trans fatty acids, and enrichment of specific unsaturated fatty acids depending on the fatty acid composition of lipid supplements.     

Exclusive quantification of methyl-branched fatty acids and minor 18:1-isomers in foodstuff by GC/MS in the SIM mode using 10,11-dichloroundecanoic acid and fatty acid ethyl esters as internal standards

Minor fatty acids (iso- and anteiso-fatty acids, vaccenic acid, elaidic acid) in foodstuff (seafood, milk, and dairy products) were analyzed by gas chromatography with electron ionisation mass spectrometry in the selected ion monitoring mode (GC/EI-MS-SIM). For this purpose, lipids were obtained by accelerated solvent extraction (ASE) and the fatty acid constituents were converted into methyl esters. Instead of the determination of the relative contribution of the minor fatty acids to the sum of all fatty acids detected (the so-called 100% method), we exclusively quantified the minor fatty acids, which was possible by using two types of internal standards (IS-1 and IS-2). For recovery checks during the extraction and/or the transesterification step we added the novel 10,11-dichloroundecanoic acid (DC-11:0) as IS-1. DC-11:0, which has never been detected in foodstuff, was synthesized by electrophilic addition of chlorine to 10-undecenoic acid (11:1n-10). The novel IS eluted in the range of 23:0 from the polar GC column used and showed the same properties as fatty acids in foodstuff during sample preparation. Recovery rate of DC-11:0 was generally >96% in the various samples analyzed. Ethyl esters (FAEE) of a12:0, a14:0, a15:0, a16:0, a17:0, and a18:0 (IS-2) were added to both the external standard (a quantitative mixture of methyl esters of methyl-branched fatty acids and 18:1n-9trans) in order to determine their response factors relative to FAEE and to the food samples. With this technique, (only) methyl-branched fatty acids (MBFAs) as well as vaccenic acid (18:1n-7) and elaidic acid (18:1n-9trans) were quantified in a range of dairy products (including twelve cheeses) and seafood. All samples were analyzed in triplicates, and good standard deviations (concentrations 0.002–5 g/100 g; standard deviations 0.00–0.03) were obtained in all cases. MBFAs were detected in all samples analyzed. The highest content of MBFAs (3.0 g/100 g) was determined in red-smear of romadur cheese. In all except two cheeses, i17:0 was the most abundant MBFA. The highest amount of 18:1n-9trans was found in feta (2.84 g/100 g) whereas 0.03 g/100 g in big eye snapper (Pricanthus tayenus) marked the lowest record of this minor fatty acid. Seal oil contained the highest amount of 18:1n-7 with 5.00 g/100 g, whereas emmental cheese was the sample with the lowest content of this monoenoic fatty acid. The combination of suitable IS and a sensitive GC/EI-MS-SIM method proved to be well suited for the quantification of minor fatty acids in foodstuff. When only a set of fatty acids is going to be analyzed, this method is less time consuming compared to “100% methods” and less prone to false results due to the higher selectivity of GC/MS compared to GC in combination with flame ionisation detection (GC/FID).     

Effect of induction of subacute ruminal acidosis on milk fat profile and rumen parameters

High-concentrate diets can lead to subacute ruminal acidosis and are known to result in changes of the ruminal fermentation pattern and mammary secretion of fatty acids. The objective of this paper is to describe modifications in milk fatty acid proportions, particularly odd- and branched-chain fatty acids and rumen biohydrogenation intermediates, associated with rumen parameters during a 6-wk subacute ruminal acidosis induction protocol with 12 ruminally fistulated multiparous cows. The protocol involved a weekly gradual replacement of a standard dairy concentrate with a wheat-based concentrate (610 g of wheat/kg of concentrate) during the first 5 wk and an increase in the total amount of concentrate in wk 6. Before the end of induction wk 6, cows were switched to a control diet because 7 cows showed signs of sickness. The pH was measured continuously by an indwelling pH probe. Milk and rumen samples were taken on d 2 and 7 of each week. Data were analyzed using a linear mixed model and by principal component analysis. A pH decrease occurred after the first concentrate switch but rumen parameters returned to the original values and remained stable until wk 5. In wk 5 and 6, rumen pH values were indicative of increasing acidotic conditions. After switching to the control diet in wk 6, rumen pH values rapidly achieved normal values. Odd- and branched-chain fatty acids and C18:1 trans-10 increased with increasing amount of concentrate in the diet, whereas C18:1 trans-11 decreased. Four fatty acids [C18:1 trans-10, C15:0 and C17:0+C17:1 cis-9 (negative loadings), and iso C14:0 (positive loading)] largely correlated with the first principal component (PC1), with cows spread along the PC1 axis. The first 4 wk of the induction experiment showed variation across the second principal component (PC2) only, with high loadings of anteiso C13:0 (negative loading) and C18:2 cis-9,trans-11 and C18:1 trans-11 (positive loadings). Weeks 5 and 6 deviated from PC2 and tended toward the negative PC1 axis. A discriminant analysis using a stepwise approach indicated the main fatty acids discriminating between the control and acidotic samples as iso C13:0, iso C16:0, and C18:2 cis-9,trans-11 rather than milk fat content or C18:1 trans-10, which have been used before as indicators of acidosis. This shows that specific milk fatty acids have potential in discriminating acidotic cases.     

Occurrence of C16:1 isomers in milk fats from ewes fed with different dietary lipid supplements

The trans as well as the cis C16:1 isomer profiles were established in 43 ewe milk fats supplemented with different dietary lipid sources representative of the variety of unsaturated fatty acids found in nature such as olive, sunflower, linseed and fish oils. Fractionation by silver-ion solid phase extraction facilitated a rapid separation of the trans, cis and saturated FA before gas chromatography analysis took place. C16:1 isomers with a double bond in positions 7, 9 and 13 in the cis group and 8 and 9 in the trans fraction were the most abundant. Dietary lipid supplementation produced a noticeable increase in the total trans C16:1 content and elevated correlations were observed between trans-8 C16:1 and trans-10 C18:1 as well as trans-9 C16:1 and trans-11 C18:1. These results support the idea that altering the trans C18:1 profile affects trans C16:1 isomer composition consistent with the β-oxidation products from the trans C18:1 isomers.     

Fatty acids patterns in camel,moose, cow and human milk as determined with GC/MS after silver ion solid phase extraction

Silver ion solid phase extraction (Ag⁺-SPE) followed by GC/EI–MS operated in the selected ion monitoring (SIM) mode was used for the thorough analysis and comparison of fatty acids from bulk milk samples of farmed camel, moose, and cow along with one human milk sample. The GC/EI–MS–SIM mode took advantage of different time windows and enabled the unequivocal assignment of the number of double bonds. GC/MS was also used for structure verification. Ag⁺-SPE was of particular importance for the separation of cis- and trans-isomers. By the combination of both techniques we were able to determine the amounts of 29 saturated fatty acids (including up to eleven branched-chain fatty acids and the scarcely described 11-cyclohexylundecanoic acid which was detected in all samples), 16 cis- and 23 trans-monoenoic fatty acids as well as 19 polyunsaturated fatty acids including the conjugated linoleic acid (CLA) 18:2(9,11tr). Characteristic differences were observed in that human milk differed from the animal milks by its lower trans-fatty acid and branched-chain fatty acid content. In this set of samples, the branched-chain fatty acid content was highest in camel milk while moose milk contained the highest proportion of 18:1(9), 18:3(9,12,15), and 22:6(4,7,10,13,16,19). Although only one bulk sample was available for the animal milk the amounts determined fitted well with published data. Thus, this approach might be helpful to establish a more comprehensive data base for statistic evaluation.     

Quantification of trans fatty acid content in French fries of local food service retailers using attenuated total reflection – Fourier transform infrared spectroscopy

The contents of trans fatty acids in French fries served at the local food service retailers in Honolulu were determined by simple Fourier transform infrared (FTIR) spectroscopic technique without the pretreatment of fatty acid extraction. A horizontal attenuated total reflection (ATR) crystal made of zinc selenide (ZnSe) was used to obtain FTIR spectra of French fries with and without fatty acid extraction. Residual oil films obtained by pressing French fries directly on the ATR crystal surface without removal of any solid particles or air bubbles were scanned for the spectral measurement. The calibration set consisted of triolein (C18:1, 9-cis) and trielaidin (C18:1, 9-trans) mixed in varying ratios. All spectral data were averaged and converted into GRAMS format. The peak heights of each spectrum at 966 cm⁻¹ were found to be linearly correlated with the contents of trans fatty acids in the validation set (n = 8, R² = 0.9835). The developed calibration model was validated by comparing the results obtained from the ATR-FTIR with Mojonnier extraction and gas chromatography–mass spectrometry (GC–MS).     

Stearoyl-coenzyme A desaturase gene polymorphism and milk fatty acid composition in Italian Holsteins

Milk fatty acid composition is a parameter of great interest for evaluation of nutritional quality of milk. Stearoyl-CoA desaturase (SCD) is a key enzyme in mammary lipid metabolism because it is able to add a double bond in the cis Δ⁹-position in a large spectrum of medium- and long-chain fatty acids. A polymorphism with 2 alleles (A and V) in the fifth exon of the SCD gene has been reported. The effect of SCD genotype on individual milk fatty acid composition and on cis-9 unsaturated/saturated fatty acid ratios of 297 Holstein Italian Friesian cows was investigated in this paper. The SCD genotypes were determined by using a single strand conformation polymorphism method. Relative frequencies of SCD genotypes were 27, 60, and 13% for AA, AV, and VV, respectively. Milk of AA cows had a greater content of cis-9 C18:1 and total monounsaturated fatty acids and a higher C14:1/C14 ratio than did milk of VV cows. The relative contribution of SCD genotype to variation of monounsaturated fatty acids, cis-9 C18:1, and cis-9 C14:1 was 5, 4, and 7.7%, respectively. No significant differences were detected between SCD genotypes in the milk content of cis-9, trans-11 C18:2. Results of the present work provide some indication of an association between SCD locus and the fatty acid profile in the examined sample of Italian Holsteins, thus suggesting a possible role of this gene in the genetic variation of milk nutritional properties.     

Short communication: Discrimination between retail bovine milks with different fat contents using chemometrics and fatty acid profiling

We used a multivariate chemometric approach to differentiate or associate retail bovine milks with different fat contents and non-dairy beverages, using fatty acid profiles and statistical analysis. We collected samples of bovine milk (whole, semi-skim, and skim; n = 62) and non-dairy beverages (n = 27), and we analyzed them using gas-liquid chromatography. Principal component analysis of the fatty acid data yielded 3 significant principal components, which accounted for 72% of the total variance in the data set. Principal component 1 was related to saturated fatty acids (C4:0, C6:0, C8:0, C12:0, C14:0, C17:0, and C18:0) and monounsaturated fatty acids (C14:1 cis-9, C16:1 cis-9, C17:1 cis-9, and C18:1 trans-11); whole milk samples were clearly differentiated from the rest using this principal component. Principal component 2 differentiated semi-skim milk samples by n-3 fatty acid content (C20:3n-3, C20:5n-3, and C22:6n-3). Principal component 3 was related to C18:2 trans-9,trans-12 and C20:4n-6, and its lower scores were observed in skim milk and non-dairy beverages. A cluster analysis yielded 3 groups: group 1 consisted of only whole milk samples, group 2 was represented mainly by semi-skim milks, and group 3 included skim milk and non-dairy beverages. Overall, the present study showed that a multivariate chemometric approach is a useful tool for differentiating or associating retail bovine milks and non-dairy beverages using their fatty acid profile.     

Conjugated linoleic acid content and isomer distribution during ripening in three varieties of cheeses protected with designation of origin

Cheeses have been identified as important sources of conjugated linoleic acid (CLA), a mixture of positional and geometric isomers with potential anticarcinogenic activity and other beneficial properties. The objectives of this study were to examine the effects of ripening on the overall CLA content as well as on the isomers profile using GC and Ag⁺-HPLC. Three Spanish cheeses Protected with Designation of Origin (Mahón, Manchego and Cabrales) were manufactured in different cheesemaking plants and monitored at different times during the ripening period. Total CLA content varied from 3 to 9 mg/g of total fatty acids and rumenic acid (9-cis, 11-trans C18:2, RA) represented more than 75% of total CLA. After RA, 7–9 (cis/trans plus trans/cis), 11-trans, 13-trans and 11-trans, 13-cis C18:2 were the main CLA isomers. The results achieved confirm that the effect of ripening on the total CLA concentration and isomer distribution was negligible.     

Effect of abomasal infusions of a mixture of octadecenoic acids on milk fat synthesis in lactating cows

Diets causing milk fat depression (MFD) are known to alter ruminal lipid metabolism leading to the formation of specific biohydrogenation intermediates that exert antilipogenic effects. Several isomers of conjugated linoleic acid (CLA), namely trans-10, cis-12 CLA, cis-10, trans-12 CLA, and trans-9, cis-11 CLA, inhibit mammary lipogenesis in the lactating cow, but ruminal outflow of these biohydrogenation intermediates does not account entirely for the reductions in milk fat synthesis during diet-induced MFD. Milk fat trans-10 18:1 concentrations are consistently increased on diets that cause MFD, suggesting a possible role in the regulation of milk fat secretion. Three rumen-fistulated cows in mid lactation were used in a 3 × 3 Latin square to evaluate the effects of a mixture of 18:1 fatty acid methyl esters (FAME) on milk fat synthesis. Experimental treatments consisted of abomasal infusions of ethanol (control), 6 g/d of trans-10, cis-12 CLA (positive control; CLA), or 247 g/d of a mixture of 18:1 FAME containing (% fatty acids) cis-9 (9.45), cis-12 (3.35), trans-10 (37.3), trans-11 (37.4), and trans-12 (2.66) as major isomers (T181 treatment). Administration of the T181 treatment supplied 92.1 g/d of trans-10 18:1. Infusions were conducted over a 5-d period with a 9-d interval between treatments. Treatments had no effect on dry matter intake, milk yield, or milk protein. Relative to the control, abomasal infusion of T181 and trans-10, cis-12 CLA treatments reduced milk fat secretion by 19.5 and 41.5%, respectively. Even though a direct cause and effect on mammary lipogenesis could not be established, comparisons with published data and considerations of the relative abundance of constituent FAME in treatment T181 implicated trans-10 18:1 as the isomer responsible. In conclusion, current data suggest that trans-10 18:1 potentially exerts antilipogenic effects and may contribute to the reduction in milk fat synthesis during diet-induced MFD in the lactating cow.     

Diet and plasma evaluation of the main isomers of conjugated linoleic acid and trans-fatty acids in a population sample from Mediterranean north-east Spain

The aim of the present study was to describe the dietary pattern of a representative sample of 516 adult participants (203 men and 313 women) from Catalonia, a Spanish Mediterranean region, to assess their current dietary and plasma levels of trans C18:1, the major trans-fatty acid (TFA), and cis-9, trans-11 CLA, and trans-10, cis-12 CLA, the two major conjugated linoleic acid (CLA) isomers, and to evaluate their correlation with several cardiovascular disease risk factors. The population was a random sample derived from the Catalan Nutrition Survey. Plasma levels of the CLA isomers were determined in a subsample of 100 volunteers. The Catalan diet seemed to maintain some traits of the ‘traditional’ Mediterranean diet, although other components were lost. The dietary intakes of saturated fatty acids (SFA), TFA, cis-9, trans-11 CLA, and trans-10, cis-12 CLA were 12.3%, 0.84% (2.0 g/d), 0.030% (71.5 mg/d), and 0.0015% (3.4 mg/d) of the energy intake, respectively. Trans C18:1 accounted for 0.19% of the total plasma fatty acids, while the sum of cis-9, trans-11and trans-10, cis-12 CLA isomers represented about 0.09% of the plasma fatty acids. Trans C18:1 isomers correlated significantly with the intake of French fries and pastries, while cis-9, trans-11 CLA significantly correlated with the intake of dairy products and ruminant meat. None of the cardiovascular disease risk factors were found to be associated with the plasma levels of TFA or CLA. The results of this study suggest that monounsaturated fatty acids (MUFA) are the main dietary fat source in the Catalan population, due to their regular olive oil consumption. Moreover, plasma levels of the main TFA and CLA suggest that the Catalan diet is not at present strongly influenced by the occidental dietary patterns. However, a reduction of the intake of SFA in the Catalan population should be recommended.     

Isomeric distribution and rapid determination of trans-octadecenoic acids in German brands of partially hydrogenated edible fats

The determination of trans fatty acids (TFA), in particular trans-octadecenoic acids, in edible fats is of current interest, since just in the last years a variety of negative physiological effects has been related to TFA. A main source of C18:1 trans fatty acids are partially hydrogenated fats. Besides the total content of trans-octadecenoic acids, their isomeric distribution seems to be even more important, as fats of different origin, e.g. partially hydrogenated fats and ruminant fats, possibly show different physiological properties. In this study, 46 margarines and 16 shortenings and cooking fats, purchased in August of 1994, were analyzed for trans-octadecenoic acid isomers by a two-step method (Ag-TLC/HRGC). The mean relative isomeric distributions (g/100 g TFA) of both groups determined with a 100 m-column were comparable, with Δ9 and Δ10 being the main isomers. By repeated analysis of 15 brands between August of 1994 and January of 1996, the mean total C18:1 TFA content was found to have decreased in these margarines (n = 8) from 9.58% to 4.62% but not in shortenings/cooking fats (n = 7; 11.62% to 11.92%). The relative isomeric distribution was not affected in both groups. To avoid the problem of overlaps between cis-and trans-C18:1 isomers with GC analysis, formulae for the rapid determination of total C18:1 TFA contents in margarines and shortenings/cooking fats from direct GC data were statistically derived. Thus, applying these formulae and considering the mean relative distributions, absolute contents of all individual isomers of trans-octadecenoic acids can rapidly be determined from direct GC.     

Evaluating the conjugated linoleic acid and trans 18:1 isomers in milk fat of dairy cows fed increasing amounts of sunflower oil and a constant level of fish oil

The objective was to evaluate different levels of sun-flower oil (SFO) in dairy rations to increase vaccenic (trans-11-18:1) and rumenic acids (cis-9,trans-11-18:2) in milk fat, and assess the content and composition of other trans-octadecenoic (trans-18:1) and conjugated linoleic acids (CLA) isomers. Eighty lactating Holstein cows were fed control diets for 4 wk and then placed on 4 diets for 38 d; milk fat was analyzed after 10 and 38 d. The treatments were: control, 1.5% SFO plus 0.5% fish oil (FO), 3% SFO plus 0.5% FO, and 4.5% SFO plus 0.5% FO. The forage-to-concentrate ratio was 50:50 and consisted of barley/alfalfa/hay silage and corn/barley grain concentrate. There were no differences in milk production. Supplementation of SFO/FO reduced milk fat compared with respective pretreatment periods, but milk protein and lactose levels were not affected. There was a linear decrease in all short- and medium-chain saturated fatty acids (SFA) in milk fat after 10 d (25.5, 24.1, 20.2, and 16.7%) and a corresponding linear increase in total trans-18:1 (5.2, 9.1, 14.1, and 21.3%) and total CLA (0.7, 1.9, 2.4, and 3.9%). The other FA in milk fat were not affected. Separation of trans-18:1 isomers was achieved by combination of gas chromatography (GC; 100-m highly polar capillary column) and prior separation of trans FA by silver ion-thin layer chromatography followed by GC. The CLA isomers were resolved by a combination of GC and silver ion-HPLC. The trans-11- and trans-10-18:1 isomers accounted for ∼50% of the total trans-18:1 increase when SFO/FO diets were fed. On continued feeding to 38 d, trans-11-18:1 increased with 1.5% SFO/FO, stayed the same with 3%, and declined with 4.5% SFO/FO. Rumenic acid showed a similar pattern on continued feeding as trans-11-18:2; levels increased to 0.43, 1.5, 1.9, and 3.4% at 10 d and to 0.42, 2.15, 2.09, and 2.78% at 38 d. Rumenic acid was the major CLA isomer in all 4 diets: 66, 77, 78 and 85%. The CLA isomers trans-7,cis-9-, trans-9,cis-11-, trans-10,cis-12-, trans-11,trans-13-, and trans-9,trans-11-/trans-10,trans-12-18:2 also increased from 0.18 (control) to 0.52% (4.5% SFO/FO). Milk fat produced from 3% SFO/FO appeared most promising: trans-11-18:1 and cis-9,trans-11-18:2 increased 4.5-fold, total SFA reduced 18%, and moderate levels of trans-10-18:1 (3.2%), other trans-18:1 (6.6%) and CLA isomers (0.5%) were observed, and that composition remained unchanged to 38 d. The 4.5% SFO/FO diet produced higher levels of trans-11-18:1 and cis-9,trans-11-18:2, a 28% reduction in SFA, and similar levels of other trans-18:1 (9.2%) and CLA isomers (0.52%), but the higher levels of trans-11-18:1 and cis-9,trans-11-18:2 were not sustained. A stable milk fat quality was achieved by feeding moderate amounts of SFO (3% of DM) in the presence of 0.5% FO that had 4% vaccenic and 2% rumenic acids.     

A simple method for the analysis of trans fatty acid with GC–MS and AT-Silar-90 capillary column

The study was aimed to evaluate a simplified gas chromatography method based on the AOAC method 996.06 to analyze the trans fat content in food samples. The gas chromatograph was equipped with mass spectrometer and Alltech AT^™-Silar-90 capillary column. Ten kinds of the trans fatty acid standard were separated completely from the cis standard and the chemical composition of the peaks was verified by using the mass spectrum. Under the optimized conditions, the recovery rate for triheptadecanoin was 99.0%, the correlation coefficients of trans fatty acid calibration curve was 0.9998 or higher. It demonstrated that the methylation and hexane extraction procedures used in this method was effective and the result was consistent. The major fatty acids found in the shortening sample were 16:0, 18:0, trans-18:1, cis-18:1, cis-18:2, and cis-18:3. The total trans fat content in the sample was 283.6 ± 18.2 mg/g. The current method was more convenient. It is adequate for the routine analysis of trans fat content in food products with low free fatty acid content.     

Inhibition of ∆(9)-desaturase activity with sterculic acid: Effect on the endogenous synthesis of cis-9 18:1 and cis-9, trans-11 18:2 in dairy sheep

This study was conducted in lactating ewes to examine the involvement of ?⁹-desaturase in mammary lipogenesis, especially in the endogenous synthesis of cis-9, trans-11 18:2 and cis-9 18:1, because no information on this matter was available for dairy sheep. With this aim, 6 Assaf ewes were monitored in a 15-d experiment, which included a 5-d pretreatment period, a 5-d treatment period, and a 5-d posttreatment period. During the treatment period, ewes received 0.5 g/d of sterculic acid (a cyclopropene fatty acid that inhibits ?⁹-desaturase), delivered intravenously in 4 equal doses at 6-h intervals. Animals were fed pasture to supply mainly α-linolenic acid and minimize the amount of milk cis-9, trans-11 18:2 of ruminal origin. Sterculic acid administration was calculated to inhibit ?⁹-desaturase by 70% based on the milk content of cis-9 14:1. This inhibition resulted in decreases in the milk content of the enzyme products (e.g., cis-9 10:1, cis-9 14:1, cis-9 16:1, cis-9 18:1, and cis-9, trans-11 18:2) and increases in its substrates (e.g., 14:0, 18:0, and trans-11 18:1), as well as in reductions in the desaturase indexes. Some other milk fatty acids, further to previously reported products or substrates of ?⁹-desaturase (e.g., cis-15 18:1 and cis-9, cis-15 18:2, or trans-11, trans-15 18:2, and cis-9, trans-11, trans-15 18:3), were also affected by sterculic acid administration. Endogenous synthesis was the major source of cis-9 18:1 and cis-9, trans-11 18:2, accounting for 63 and 74% of its content in milk fat, respectively. To our knowledge, the present study provides the first estimates of endogenous synthesis of these 2 bioactive fatty acids in ovine milk fat.     

Fatty acid profile of goat milk in diets supplemented with chia seed (Salvia hispanica L.)

Chia seed (Salvia hispanica L.) is the greatest known plant source of n-3 α-linolenic acid. The present study evaluated the effects of 3 inclusion levels of chia seed [zero (control); low, 2.7% (CLow); and high, 5.5% (CHigh)] in diets of dairy goats on milk yield and fatty acid profile. Nine Saanen dairy goats in the last third of lactation period, live weight 38 ± 8.7 kg, housed in metabolic cages, were fed iso-proteic and iso-energetic (160 g of crude protein/d and 11 MJ of metabolizable energy/d) diets. Gas chromatography was used to analyze fatty acid profile and total conjugated linoleic acid (CLA). Silver ion HPLC was used to analyze the isomeric profile of CLA. The results were subjected to variance analysis using a Latin square design repeated 3 × 3. The CHigh treatment was higher for dry matter, neutral detergent fiber, and acid detergent fiber intake compared with CLow and control diets. Digestibility was not affected by the inclusion of chia seeds. The CHigh diet improved N intake with respect to the control and CLow diet. Milk yield and chemical composition were not affected by the treatment. The milk fatty acid profile of C18:0, C18:1, C18:2, and C:20 was higher for CHigh than the other treatments. The in vitro gas production (mL of gas/g of dry matter) was lower in CHigh than the control diet. In conclusion, the addition of chia seeds at the CHigh level in dairy goat diets negatively affected in vitro rumen fermentation, but increased the milk fatty acid profile of C18:0, C18:1n-9 cis, and C:20, monounsaturated fatty acids, and polyunsaturated fatty acids. The total CLA content increased from 0.33 to 0.73% with the supplementation of chia to the diet, as well as the isomers cis-9,trans-11, trans-7,cis-9, trans-11,cis-13, and trans-12,trans-14.     

Effect of fish oil and sunflower oil on rumen fermentation characteristics and fatty acid composition of digesta in ewes fed a high concentrate diet

Studies in ruminants have shown that supplementing the diet with a mixture of fish oil (FO) and sunflower oil (SO) enhances the concentration of cis-9, trans-11 conjugated linoleic acid (CLA), 20:5 n-3, and 22:6 n-3 in milk because of alterations in ruminal biohydrogenation, but the intermediates formed under these conditions are not well characterized. Five ewes fitted with rumen cannula and fed a high concentrate diet were used to examine the effect of a mixture (30 g/kg of DM) of FO and SO (1:2, wt/wt) on temporal changes in rumen fermentation characteristics and the relative abundance of biohydrogenation intermediates in ruminal digesta collected after 0, 3, and 10 d on diet. Appearance and identification of biohydrogenation intermediates was determined based on complementary gas-liquid chromatography and Ag+-HPLC analysis of fatty acid methyl esters and gas chromatography-mass spectrometry analysis of corresponding 4,4-dimethyloxazoline derivatives. Inclusion of FO and SO in the diet had no effect on rumen pH, volatile fatty acid concentrations, or nutrient digestion, but altered the fatty acid composition of ruminal digesta, changes that were characterized by time-dependent decreases in 18:0 and 18:2 n-6 and the accumulation of trans 16:1, trans 18:1, 10-O-18:0, and trans 18:2. Lipid supplements enhanced the proportion of 20:5 n-3 and 22:6 n-3 in digesta and resulted in numerical increases in cis-9, trans-11 conjugated linoleic acid concentrations, but decreased the relative abundance of trans-10, cis-12 conjugated linoleic acid. Furthermore, detailed analysis revealed the appearance of several unique 20:1, 20:2, 22:1, 22:3, and 22:4 products in ruminal digesta that accumulated over time, providing the first indications of 20 and 22 carbon fatty acid intermediates formed during the biohydrogenation of long-chain unsaturated fatty acids in sheep. In conclusion, FO and SO in a high concentrate diet caused a time-dependent inhibition of the complete biohydrogenation of 16 and 18 carbon unsaturated fatty acids, and resulted in the accumulation of trans 16:1, trans 18:1, and trans 18:2, 20, and 22 carbon metabolites in ruminal digesta of sheep, with no evidence of a shift in ruminal biohydrogenation pathways toward trans-10 18:1 formation.