N‐3 fatty acids for human nutrition: stability considerations

Currently there is great interest in dietary n‐3 fatty acids to promote health. The food industry aims to produce food products enriched in α‐linolenic acid (Ln), eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) to reduce some of the physiological effects of linoleic acid (L), the major polyunsaturated fatty acid in our diet. However, the goal is hampered by the susceptibility of the n‐3 fatty acids to oxidation. As a result the sensory and nutritional quality of such foods deteriorates. Lipid scientists therefore have to find a way to stabilise these fatty acids. Innovative technologies to protect n‐3 polyunsaturates using antioxidants, adequate preparation, refining and packaging of the oil are needed. In this paper we review the inherent stability and the stabilisation of these nutritionally valuable polyunsaturated fatty acids.     

Dietary C18:1 trans fatty acids increase conjugated linoleic acid in adipose tissue of pigs

The effect of dietary C18:1 trans fatty acids on back fat composition in pigs was investigated with special emphasis on conjugated linoleic acids (CLA). A total of 12 × 4 siblings of Large White and Swiss Landrace breed were housed in groups and fattened from 22 to 103 kg live weight. Pigs were fed a control diet (barley, wheat, soybean meal) or experimental diets which consisted of the control diet with a 5% replacement of olein or stearin fractions of pork fat, or partially hydrogenated fat. The hydrogenated fat was rich in C18:1 trans fatty acids but contained only negligible amounts of CLA. In contrast olein and stearin fractions contained far less C18:1 trans fatty acids but some CLA. In the control diet no C18:1 trans fatty acids and only traces of CLA were detected. The partially hydrogenated fat led to the highest CLA content in back fat (0.44%). Intermediate amounts of CLA were measured in pigs fed the fractionated pork fat (0.22/0.23%). In pigs fed the control diet, also small amounts of CLA were detected. The results indicate that CLA may be produced by endogenous Δ9‐desaturation out of dietary trans vaccenic acid in pigs.     

Synthesis of regioisomerically pure triacylglycerols containing n‐3 very long‐chain polyunsaturated fatty acids

There is growing scientific evidence that consumption of n‐3 very long‐chain polyunsaturated fatty acids (n‐3 VLC‐PUFA) helps in brain and eye development, and protects against a range of common degenerative diseases. This has provided the impetus to the scientists to develop new and renewable sources for these important fatty acids so that the food industry is able to produce and market products fortified with n‐3 VLC‐PUFA. The bioactive efficacy and stability of food products containing n‐3 VLC‐PUFA may be determined not only by the amount of n‐3 VLC‐PUFA present but also by the positional distribution of these acids within the triacylglycerol (TAG) molecules (regiopurity). Studies of the effects of positional distribution on the functionality of n‐3 VLC‐PUFA containing oils have been hampered by a general lack of pure TAG regioisomers for experimentation. This paper reviews methods that have been used for the synthesis of TAG regioisomers containing n‐3 VLC‐PUFA, with special reference to those in which one n‐3 VLC‐PUFA occurs in combination with two long‐chain saturated acids.     

Fatty fish intake,n‐3 fatty acids and self‐rated health in middle‐aged adults

Background – Since n‐3 fatty acids, abundant in fatty fish, may improve health, we raised the question whether self‐reported intake frequency of fatty fish (FF) might be related to the percentage of n‐3 fatty acids in serum phospholipids (PL‐n‐3), and also to self‐rated health (H). Design – The study followed a cross‐sectional design. Methods – In the cross‐sectional Oslo Health Study, PL‐n‐3 were determined in 121 middle‐aged ethnic Norwegians and 102 immigrants from the Indian subcontinent and correlated with FF and H. Logistic regression was used to study the relationship between PL‐n‐3 and H (dichotomized, i.e. Poor vs. Good health). Results – FF correlated positively with PL20:5n‐3 (PL‐EPA, r = 0.467, p <0.001) and PL22:6n‐3 (PL‐DHA, r = 0.499, p <0.001), and negatively with PL20:4n‐6 (PL‐AA, r = –0.350, p = 0.001). H was positively associated with PL‐EPA (r = 0.321, p <0.001) and PL‐DHA (r = 0.275; p <0.001), but negatively with PL‐AA (r = –0.220, p = 0.001). The odds ratio for reporting Poor vs. Good health was significantly higher in subjects with low levels of PL‐EPA (OR = 1.49; 95% confidence interval = 1.17–1.89, p = 0.001), persisting after adjusting for sex, physical activity, ethnicity and length of education. Conclusion – The intake frequency of fatty fish is related to n‐3 fatty acids in the serum phospholipids, and to self‐rated health.     

Duodenal availability of conjugated linoleic acids after supplementation to dairy cow diets

The objective of the present study was to investigate the effects of a lipid‐encapsulated CLA preparation on rumen metabolism and the actual post‐ruminal bioavailability of the applied CLA isomers. In the rumen, the CLA supplementation modified the molar proportions of VFA. In period CLA‐1 the rumen fermentation shifted toward more butyric acid at the expense of acetic acid. The highest CLA supplementation resulted in increased amounts of isobutyric, isovaleric, and valeric acid. The apparent ruminal digestibility of starch increased in period CLA‐2. The ruminal protein degradation was higher after CLA supplementation, while the efficiency of the use of the RDP for microbial protein synthesis declined. The duodenal flow of trans‐10,cis‐12 CLA amounted to 16 and 5% of the intake in periods CLA‐1 and CLA‐2, respectively. The transfer of trans‐10,cis‐12 CLA from duodenum into milk was 36 and 48% in periods CLA‐1 and CLA‐2, respectively. Overall, the observed effects of the supplementation of lipid‐encapsulated CLA on the parameters of rumen metabolism were negligible. The actual low post‐ruminal bioavailability of trans‐10,cis‐12 CLA suggest that most of the applied fat supplement was biohydrogenated.     

Effects of conjugated linoleic acids on protein to fat proportions,fatty acids,and plasma lipids in broilers

In a performance trial, broiler chickens received 29 g per kg feed of a preparation containing 70% linoleic acid (LA) in the control treatment and another preparation containing approximately the same amount of conjugated linoleic acids (CLA) in the experimental treatment. Diets of CLA treatment contained 18 g CLA per kg feed. The CLA preparation contained the isomers cis‐9,trans‐11 and trans‐10,cis‐12 at a proportion 1:1, other CLA isomers were quantitively negligible. Performance parameters (weight gain and feed conversion ratio over a 42 day period) were not significantly influenced by CLA intake. However, fat content of liver, breast, and leg muscles was reduced and protein contents in liver and leg muscles were elevated significantly. Fat to protein ratios in the main edible parts were shifted in favour of protein in CLA treated animals. In all analysed tissue lipids the content of saturated fatty acids was increased and that of monounsaturated fatty acids was decreased significantly. At the same time CLA was incorporated in tissue lipids effectively reaching more than 10 g per 100 g of total fatty acids. With regard to isomers the cis‐9,trans‐11 isomer was found in higher concentrations in tissue lipid fractions compared to the trans‐10,cis‐12 isomer. It was concluded that nutrient repartitioning due to CLA intake described for other species is also valid for broilers. Using appropriate feeding strategies it is possible to produce CLA enriched food from broilers.     

Effect of grass vs. concentrate feeding on the fatty acid profile of different fat depots in lambs

The aim of this study was to produce high‐quality meat from lambs under different feeding conditions, as measured by the accumulation of n‐3 fatty acids and conjugated linoleic acids (CLA) in muscle and subcutaneous fat. In total, 13 male crossbred lambs (Black Head×Gotland), each at 24 kg live weight, were divided into two feeding groups. Lambs were kept either on pasture (pasture grazing, n = 6) or in the stable (concentrate feeding, n = 7). The linolenic acid (C18:3n‐3) contained in the grass was absorbed and deposited into the different lipid classes of muscle and subcutaneous fat. The proportion of total n‐3 fatty acids in the different lipids of grazing lambs was significantly (p = 0.05) higher compared to that in concentrate‐fed lambs. The n‐6/n‐3 ratio (mean ± SEM) in muscle of grazing lambs was 1.2 ± 0.09 in contrast to 2.3 ± 0.09 (p = 0.05) of the animals kept in the stable. In subcutaneous fat, this ratio was 0.9 ± 0.2 in lambs kept on pasture versus 3.5 ± 0.2 (p = 0.05) after indoor keeping. The relative concentration of C18:1trans‐11 in total muscle lipids, phospholipids, triacylglycerols and subcutaneous fat was significantly increased by grass feeding compared to concentrate feeding. Significant influences of feeding were shown for saturated fatty acids. In concentrate‐fed lambs, a lower content of saturated fatty acids was detected. The proportion of CLAcis‐9,trans‐11 (1.9 ± 0.2% vs. 1.1 ± 0.1% in muscle, 2.5 ± 0.2% vs. 1.4 ± 0.2% in subcutaneous fat, 0.7 ± 0.04% vs. 0.4 ± 0.04% in phospholipids) in lambs was significantly (p = 0.05) higher after grazing than after concentrate feeding, respectively.     

Quantum‐chemical treatment of the linoleic acid molecule and two of its conjugated isomers

The structural, vibrational and electronic properties of the linoleic acid molecule and two of its conjugated isomers were investigated theoretically by performing the molecular mechanics (MM+ force field), the semi‐empirical self‐consistent‐field molecular‐orbital (PM3), and the density functional theory (B3LYP) calculations. The geometries of the considered molecules were optimized; the vibrational dynamics and the electronic properties were calculated in their ground states in the gas phase. It was found that the excess charge accumulated on hydrogen atoms bonded to double‐bonded carbon atoms is relatively small, which may cause these hydrogen atoms to be easily abstracted.     

Thraustochytrids as an alternative source of omega‐3 fatty acids,carotenoids and enzymes

Currently the most common microalgae used for commercial production of omega‐3 fatty acids are marine derived, particularly from family members of Thraustochytriaceae and Crypthecodiniaceae. Thraustochytrids are marine heterotrophic fungi like microorganisms known to produce several commercially interesting biotechnological compounds including omega‐3 fatty acids such as docosahexaenoic acid (DHA), docosapentaenoic acid (DPA) and eicosapentaenoic acid (EPA), carotenoids, sterols, exopolysaccharides and enzymes. Therefore, exploring the potential of thraustochytrids has much to offer to the commercial production of bioactive compounds. In response to growing demand for omega‐3 fatty acids, various isolation, fermentation and lipid recovery strategies have been developed in recent years.     

Lipase/acyltransferase‐catalysed interesterification of fat blends containing n‐3 polyunsaturated fatty acids

The lipase/acyltransferase from Candida parapsilosis is an original biocatalyst that preferentially catalyses alcoholysis over hydrolysis in biphasic aqueous/organic media. In this study, the performance of the immobilised biocatalyst in the interesterification in solvent‐free media of fat blends rich in n‐3 polyunsaturated fatty acids (n‐3 PUFA) was investigated. The interesterification activity of this biocatalyst at a water activity (a_w) of 0.97 was similar to that of commercial immobilised lipases at a_w values lower than 0.5. Thus, the biocatalyst was further used at an a_w of 0.97. Response surface modelling of interesterification was carried out as a function of medium formulation, reaction temperature (55–75 °C) and time (30–120 min). Reaction media were blends of palm stearin (PS), palm kernel oil and triacylglycerols (TAG) rich in n‐3 PUFA (“EPAX 4510TG”; EPAX AS, Norway). The best results in terms of decrease in solid fat content were observed for longer reaction time (>80 min), lower temperature (55–65 °C), higher “EPAX 4510TG” content and lower PS concentration. Reactions at higher temperature led to final interesterified fat blends with lower free fatty acid contents. TAG with high equivalent carbon number (ECN) were consumed while acylglycerols of lower ECN were produced.     

The production of n‐3 long‐chain polyunsaturated fatty acids in transgenic plants

Long‐chain polyunsaturated fatty acids (LC‐PUFA) now have a proven role in human health and nutrition, including the n‐3 forms normally found in fish oils. Unfortunately, global fish stocks are now more than ever subject to over‐fishing and environmental pollution, indicating the need for an alternative source of fish oils. Recent efforts have focussed on the production of LC‐PUFA in transgenic plants to provide a sustainable and clean source of fish oils. The current progress in this area is considered, as well as the bottlenecks that remain to be overcome.     

Quantitative determination of conjugated linoleic acid isomers in beef fat

The amounts of 14 conjugated linoleic acid (CLA) isomers (t12t14, t11t13, t10t12, t9t11, t8t10, t7t9, t6t8; 12,14 c/t, t11c13, c11t13, t10c12, 9,11 c/t, t8c10, t7c9‐18:2) in 20 beef samples were determined by triple‐column silver‐ion high‐performance liquid chromatography (Ag⁺‐HPLC). Quantitation was performed using an external CLA reference standard consisting of cis9,trans11‐18:2,trans9,trans11‐18:2 and cis9,cis11‐18: 2. Linearity was checked as being r > 0.9999 between 0.02 × 10^‐3 to 2 mg/ml. The determination limit (5‐fold signal/noise ratio) of the CLA reference was estimated to be 0.25, 0.50, 1.0 ng/injection for the cis/trans, trans,trans and cis,cis isomers, respectively. As expected, cis9,trans11‐18:2 was the predominant isomer (1.95 ± 0.54 mg/g fat) in beef, followed by trans7,cis9‐18:2 (0.19 ± 0.04 mg/g fat); cis,cis isomers were below the determination limit in most beef samples. Total CLA amounts determined by Ag⁺‐HPLC were compared to total CLAs determined by gas chromatography (GC, 100 m CPSil^TM 88 column). The amounts obtained by GC were generally higher than those determined by Ag⁺ ‐HPLC due to co‐eluting compounds.     

Modelling lipase‐catalysed transesterification of fats containing n‐3 fatty acids monitored by their solid fat content

Transesterification of fat blends rich in n‐3 polyunsaturated fatty acids (n‐3 PUFA), catalysed by a commercial immobilised thermostable lipase from Thermomyces lanuginosa, was carried out batch‐wise. Experiments were performed, following central composite rotatable designs (CCRDs) as a function of reaction time, temperature and media formulation. Mixtures of palm stearin, palm kernel oil and a commercial concentrate of triacylglycerols rich in n‐3 PUFA (“EPAX 2050TG” in CCRD‐1 and “EPAX 4510TG” in CCRD‐2) were used. The time‐course of transesterification was indirectly followed by the solid fat content (SFC) values of the blend at 10 °C, 20 °C, 30 °C and 35 °C. A decrease in all SFC values of the blends at 10 °C, 20 °C, 30 °C and 35°C was observed upon transesterification. The SFC_{10 °C} and SFC_{20 °C} of transesterified blends varied between 18 and 48 and SFC_{35 °C} between 6 and 24. These values fulfil the technological requirements for the production of margarines. Under our conditions, lipid oxidation may be neglected. However, the accumulation up to 8.3% free fatty acids in reaction media is a problem to overcome. The development of response surface models, describing both the final SFC value and the SFC decrease, will allow predicting results for novel proportions of fats and oils and/or a novel combination time‐temperature.     

Oxidative stability of conjugated linoleic acids relative to other polyunsaturated fatty acids

Contrary to current opinion, conjugated linoleic acids (CLA) as a mixture of several isomers have been previously shown to function as prooxidants in the form of free fatty acids and methyl esters in heated canola oil. Furthermore, CLA oxidizes considerably faster than linoleic acid. However, stability of CLA relative to other polyunsaturated fatty acids remains undetermined. The present study was therefore undertaken to examine the relative oxidation rate of CLA compared with that of linolenic acid (LNA), arachidonic acid (AA), and docosahexaenoic acid (DHA) in air at 90°C. CLA, both in the form of free fatty acids and triacylglycerols, were extremely unstable to the same extent as DHA, but they oxidized considerably faster than LNA and AA. The mechanism by which CLA were readily decomposed was probably due to formation of the unstable free-radical intermediate.     

Bis‐methylene‐interrupted octadecadienoic fatty acids in Bulgarian bovine butter fats

Preparative silver nitrate thin‐layer chromatography of fatty acid methyl esters and gas chromatography‐mass spectrometry of 4,4‐dimethyloxazoline derivatives were applied in combination to analyze the cis,cis octadecadienoic (18:2) fraction in commercial samples of bovine butter fat in Bulgaria. The main component, 9,12–18:2 was accompanied by a series of bis‐methylene‐interrupted isomers, cis,cis‐5,9‐; 6,10‐; 9,13‐ and 11,15–18:2, which were unambiguously identified by their specific mass spectra. The latter are characterized by the presence of very intensive diagnostic fragments which help to correctly locate the position of the double bond system in the carbon chain. The cis,cis‐5,9–18:2 isomer was found for the first time in butter fat, and the 6,10–18:2 isomer has not been described as yet.     

Microbial conversion of linoleic and linolenic acids to unsaturated hydroxy fatty acids

The conversion of oleic acid to 10-hydroxystearic acid with resting cells ofNocardia cholesterolicum (NRRL 5767) has been previously reported. These same microorganisms also convert linoleic and linolenic acids to 10-hydroxy-12c-octadecenoic and 10-hydroxy-12c,15c-octadecadienoic acids, respectively. The reaction occurs best at 35°C and a pH of 6.5. Under optimum conditions, 75–80% of the unsaturated fatty acid substrate is converted to the corresponding hydroxy acid. The hydroxy products were characterized by gas chromatography, gas chromatographymass spectrometry, nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy. Other microorganisms that successfully converted these substrates include another strain ofNocardia cholesterolicum (NRRL 5768) andNocardia sp. (NRRL 5636). Presented at the 82nd Annual meeting of the American Oil Chemists’ Society, Chicago, IL, May 12–15, 1991.