Trans unsaturated fatty acids : a study of methodology and levels in New Zealand food fats including milkfat : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Biochemistry at Massey University, New Zealand

Trans fatty acids (TFAs) occur naturally in small amounts in foods such as milk, butter and tallow as a result of biohydrogenation by ruminant gut microflora. They are formed in much larger quantities during chemical hydrogenation of fats and oils. The relationship between dietary TFAs and blood cholesterol has been investigated over the last 30 years with equivocal results because of methodological limitations, including difficulties of quantifying the consumption of TFAs. The present study was conducted to investigate the methodologies used to quantify TFAs in fat samples. Two methodologies, based on infrared spectrophotometry and argentation-thin layer chromatography/gas chromatography (Ag-TLC/GC), were optimised for TFA quantification. Improvements in the infrared methods were made using a calibration standard made up with two non-trans components (stearin and olein) in order to mimic the fatty acid background in the samples. Further improvements were made using a spectral subtraction technique where the non-trans background spectrum was subtracted from the sample spectrum using Fourier-transform infrared spectrophotometry software. Results from the improved infrared methods were compared with TFA measurements by the more detailed Ag-TLC/GC method. The spectral subtraction technique for the methyl ester samples produced results that were closest to those of the Ag-TLC/GC method. This Ag-TLC/GC method gives information about the individual trans isomers (C18:1 trans positional isomers and C18:2 and C18:3 trans isomers) that is not available by infrared. The present study was also conducted to determine, as accurately as possible, the TFA content in 18 manufactured foods commonly available in New Zealand using the TFA methods mentioned above. The TFA contents in some of the foods determined by the Ag-TLC/GC method were, margarine (15.43-15.57%), butter (6.58%), milk (5.26-6.03%), meat patties (3.42%), plain sweet biscuits (3.65%) and white bread (4.41 %). Using these TFA data and the food consumption data from a Life in New Zealand (Horwarth et al., 1991), the estimated TFA intakes in the average New Zealand diet were approximately 3.99 and 5.75 g/person/day for females and males respectively. These figures were similar to or lower than those estimated for Northern Hemisphere countries. The predominant TFA isomer in the New Zealand diet was identified as the C18:1 Δ11t positional isomer (30-33%). Further studies were made on the total TFA content in New Zealand milkfat. These studies indicated that the total TFA levels in New Zealand milkfat were influenced by seasonal variations, with the highest TFA content recorded in spring (September, 6.7%) and the lowest in summer (January, 5.3%). The C18:1 Δ11t isomer was found to be the predominant isomer in milkfat, making up 49-60% of the total TFA. Similar ranges were observed for several overseas butter samples. However, major differences were observed with the distribution of the C18:1 trans positional isomers. These differences are currently suspected to be influenced by the feed and animal husbandry methods used in some Northern Hemisphere countries, where cows are mainly grain fed in the winter months. The seasonal variation of TFAs in New Zealand butter and possible effects of feed and animal husbandry methods on the C18:1 trans positional isomer distribution are important factors that the New Zealand dairy industry could exploit for the production of low trans milkfat and/or other dairy products in which the levels of specific trans isomers implicated to be "harmful" to humans could be minimised. Margarines display a trans isomer distribution that is quite distinct from that of butter. Unlike milkfat where the predominant trans isomer is C18:1 Δ11t, in margarines and hydrogenated fats and oils the positional isomers show a normal distribution around the C18:1 Δ10t-11t isomers. The predominant isomers for the margarines analysed in this study were Δ9t-Δ12t (90%) with the polyunsaturated C18:2 and C18:3 trans making up less than 2%. The distinct distribution of C18:1 trans positional isomers could serve as an additional tool for the identification of animal or hydrogenated vegetable oils used in food fats

Chytridiomycosis of marine diatoms : the role of stress physiology and resistance in parasite-host recognition and accumulation of defense molecules

Little is known about the role of chemotaxis in the location and attachment of chytrid zoospores to potential diatom hosts. Hypothesizing that environmental stress parameters affect parasite-host recognition, four chytrid-diatom tandem cultures (Chytridium sp./Navicula sp., Rhizophydium type I/Nitzschia sp., Rhizophydium type IIa/Rhizosolenia sp., Rhizophydium type IIb/Chaetoceros sp.) were used to test the chemotaxis of chytrid zoospores and the presence of potential defense molecules in a non-contact-co-culturing approach. As potential triggers in the chemotaxis experiments, standards of eight carbohydrates, six amino acids, five fatty acids, and three compounds known as compatible solutes were used in individual and mixed solutions, respectively. In all tested cases, the whole-cell extracts of the light-stressed (continuous light exposure combined with 6 h UV radiation) hosts attracted the highest numbers of zoospores (86%), followed by the combined carbohydrate standard solution (76%), while all other compounds acted as weak triggers only. The results of the phytochemical screening, using biomass and supernatant extracts of susceptible and resistant host-diatom cultures, indicated in most of the tested extracts the presence of polyunsaturated fatty acids, phenols, and aldehydes, whereas the bioactivity screenings showed that the zoospores of the chytrid parasites were only significantly affected by the ethanolic supernatant extract of the resistant hosts

Selective enrichment of n−3 polyunsaturated fatty acids with C18–C20 acyl chain length from sardine oil using Pseudomonas fluorescens MTCC 2421 lipase

An extracellular lipase purified from Pseudomonas fluorescens MTCC 2421 was used to enrich sardine oil triglycerides with eicosapentaenoic acid (20:5 n−3) and linolenic acid (18:3 n−3) to 35.28% and 8.25%, respectively, after 6 h of hydrolysis. The corresponding n−6 fatty acids (18:2 n−6 and 20:4 n−6) exhibit a reduction (54.93% and 50%, respectively). Structure–bioactivity relationship analyses revealed that the lower hydrophobic (log P values) constants of 18:3 n−3 and 20:5 n−3 (5.65 and 5.85, respectively) result in their higher hydrolytic resistance towards lipase, leading to their enrichment in the triglyceride fraction after lipase-catalysed hydrolysis. Lipase-catalysed hydrolysis of sardine oil for 6 h followed by urea fractionation at 4 °C with methanol provided free fatty acids containing 42.50% 20:5 n−3 and 10.31% 18:3 n−3, respectively. Argentation neutral alumina column chromatography, using n-hexane/ethylacetate (2:1, v/v) resulted in 20:5 n−3 of high purity (83.62%), while 18:3 n−3 was found to be eluted with n-hexane/dichloromethane (4:1, v/v) as eluting solvent with a final purity of 75.31%

Preparation of eicosapentaenoic acid concentrates from sardine oil by Bacillus circulans lipase

An extracellular lipase extracted from Bacillus circulans, isolated from marine macro alga, Turbinaria conoides, was used to prepre n-3 polyunsaturated fatty acid(PUFA) concentrates from sardine oil trygliceride

Conjugated linolenic acid (CLnA), conjugated linoleic acid (CLA) and other biohydrogenation intermediates in plasma and milk fat of cows fed raw or extruded linseed

Thirty lactating dairy cows were used in a 333 Latin-square design to investigate the effects of a raw or extruded blend of linseed and wheat bran (70:30) on plasma and milk fatty-acids (FA). Linseed diets, containing 16.6% linseed blend on a dry-matter basis, decreased milk yield and protein percentage. They decreased the proportions of FA with less than 18 carbons in plasma and milk and resulted in cis-9, cis-12, cis-15 18:3 proportions that were more than three and four times higher in plasma and milk, respectively, whereas cis-9, cis-12 18:2 proportions were decreased by 10–15%. The cis-9, trans-11, cis-15 18:3 isomer of conjugated linolenic acid was not detected in the milk of control cows, but was over 0.15% of total FA in the milk fat of linseed-supplemented cows. Similarly, linseed increased plasma and milk proportions of all biohydrogenation (BH) intermediates in plasma and milk, including the main isomer of conjugated linoleic acid cis-9, trans-11 18:2, except trans-4 18:1 and cis-11, trans-15 18:2 in plasma lipids. In milk fat, compared with raw linseed, extruded linseed further reduced 6:0–16:0 even-chain FA, did not significantly affect the proportions of 18:0, cis-9 18:1 and cis-9, cis-12 18:2, tended to increase cis-9, cis-12, cis-15 18:3, and resulted in an additional increase in the proportions of most BH intermediates. It was concluded that linseed addition can improve the proportion of conjugated linoleic and linolenic acids, and that extrusion further increases the proportions of intermediates of ruminal BH in milk fat

Effects of preconditioning and extrusion of linseed on the ruminal biohydrogenation of fatty acids. 2. In vitro and in situ studies

The extent and/or intermediates of ruminal biohydrogenation (BH) of fatty acids (FA) were investigated in vitro and in situ using a raw, pre-conditioned or extruded blend of linseed and wheat bran (70:30). The duration of in vitro incubations were 2, 4, 8, 16 and 24 h, with 5 replicates. In situ studies used 3 dry ruminally fistulated Holstein cows in a 3 × 3 Latin square design, with 3 weeks adaptation to the linseed form. The diet contained 20% (DM basis) of the linseed based blend. The duration of in situ incubations were 2, 4, 8, 16, 24 and 48 h. BH was much slower in situ than in vitro, resulting in a much lower effective disappearance of C18:2 and C18:3. Moreover, the in situ technique suggested that the technological pre-treatment of linseed did not affect C18:2 and C18:3 rate of BH, whereas reduced rates were observed in vitro. After 8 h of in vitro incubation and onwards, proportions of cis-9,trans-11C18:2 were the highest with extruded linseed. The BH of FA from linseed resulted in the appearance of great proportions of trans-10+11 to trans-16C18:1 intermediates. Extrusion increased the proportions of trans-10+11C18:1 both in vitro and in situ and proportions or trans-C18:1 were higher in situ than in vitro. Compared to previous in vivo results with the same material, the in situ method provided poor estimates of BH rates and intermediates

Deletion of a mycobacterial gene encoding a reductase leads to an altered cell wall containing β-oxo-mycolic acid analogues, and the accumulation of longchain ketones related to mycolic acids.

Mycolic acids are essential components of the mycobacterial cell wall. In this study we show that a gene encoding a reductase involved in the final step of mycolic acid biosynthesis can be deleted in Mycobacterium smegmatis without affecting cell viability. Deletion of MSMEG4722 (ortholog of Mycobacterium tuberculosis Rv2509) altered culture characteristics and antibiotic sensitivity. The ΔMSMEG4722 strain synthesized α-alkyl, β-oxo intermediates of mycolic acids which were found esterified to cell wall-arabinogalactan. While the precursors could not be isolated directly due to their inherent instability during base-treatment, their presence was established by prior reduction of the β-oxo group by sodium borohydride. Interestingly, the mutant also accumulated unsaturated ketones, similar to tuberculenone from M. tuberculosis, which were shunt products derived from spontaneous decarboxylation of α-alkyl, β-oxo fatty acid precursors of mycolic acids

Dietary Omega-3 Oil Supplementation To Increase Omega-3 Polyunsaturated Fatty Acids In The Red Tilapia(Oreochromis Hybrid) And Catfish (Clarias Gariepinus)

A study was carried out to determine the essential polyunsaturated fatty acid (PUFA) profiles of the red tilapia (Oreochromis hybrid) and catfish (Clarias gariepinus) and an attempt was made to increase the omega-3 polyunsaturated fatty acids by dietary omega -3 oil supplementation. The fatty acid profiles of commercially farmed adult fish were determined using standard extraction, fatty acid methylation and gas liquid chromatographic procedures and the fatty acid concentration of dried fish tissues was expressed in absolute amounts (mg/g) as a measure of the actual fatty acid content in the fish tissues. The levels of total fatty acids, SFA, UFA, monoenes, total n-6, total n-3, 18:2n-6 and 18:3n-3 were higher in the catfish compared to the red tilapia. The higher (17-20% of the total fatty acids) n-6 PUFA found in both fish compared to n-3 PUFA (1.0 – 9.5 %) was characteristic for freshwater fish. The absolute amounts of total n-6 and n-3 PUFA increased as the age of the fish increased for both species of fish when measured from 10 to 75 days of age although they decreased when expressed as a percentage of total fatty acids. The absolute amounts of total n-6 PUFA in the red tilapia increased from 10.0 ± 0.6 mg/g at 10 days to 26.6 ± 2.4 mg/g at 75 days of age. The absolute amounts of total n-3 PUFA in the red tilapia increased from 3.6 ± 0.2 mg/g at 10 days to 9.4 ± 0.3 mg/g at 75 days of age. Similarly, the absolute amounts of total n-6 PUFA in the catfish increased from 15.1 ±1.0 mg/g at 10 days to 36.5 ± 2.5 mg/g at 75 days of age whilst the absolute amounts of total n-3 PUFA increased from 8.1 ± 0.2 mg/g at 10 days to 21.8 ± 1.5 mg/g at 75 days of age. The results were suggestive of a combined effect of accumulation, desaturation and elongation and oxidation of the PUFA in the fish tissues. The Δ6 desaturase enzyme activity in the liver microsomes was measured in six of each species of fish employing radiolabelled linoleic acid [1-14C] and argentation thin layer chromatography. Desaturase activities were detected in both species but the activity in the red tilapia (3.55 + 0.2%; 1.19 + 0.1 pmol/min/mg protein) was higher, although not significant (p>0.05), than the catfish (3.07 ± 0.2%; 1.02 + 0.1 pmol/min/mg protein). The antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and the lipid peroxidation value of malonaldehyde (MDA) were measured in ten of each species of fish. The antioxidant activity was higher in the catfish whilst the lipid peroxidation value was higher in the red tilapia. The activity of SOD (1.54 ± 0.1 U/g) and GSH-Px (0.37 ± 0.1 U/g) in the red tilapia was lower than the activity of SOD (2.48 ± 0.1 U/g) and GSH-Px (1.18 ± 0.1 U/g) in the catfish. The MDA values were 21.39 ± 0.5 nmol/g for the red tilapia compared to 19.15 ± 0.2 nmol/g for the catfish. The dietary omega-3 oil supplementation trial was carried out for eight weeks where the fish was reared in glass aquariums in under three dietary treatments, in triplicate. The treatment diets were the control diet CON (with no oil added), or diets containing added 10% or 20% flaxseed oil (10% FLAX or 20% FLAX) or added 10% or 20% cod-liver oil (10% COD, 20% COD). The desirable n-3 PUFA were not increased by the 10% FLAX or 10% COD diets where the total n-3 PUFA concentrations in the red tilapia were 3.2 + 0.1 mg/g (CON), 2.6 ± 0.2 mg/g (10% FLAX) and 3.4 ± 0.2 mg/g (10% COD). The n-3 PUFA concentrations in the catfish were 7.1 ± 0.4 mg/g (CON), 6.4 ± 0.1 mg/g (10% FLAX) and 6.4 ± 0.4 mg/g (10%COD). However, the n-3 PUFA concentrations were significantly increased (p<0.05) when fed the 20% FLAX and 20% COD diets. In the red tilapia the n-3 PUFA concentrations were 3.4 ± 0.1 mg/g (CON), 4.7 ± 0.1 mg/g (20% FLAX) and 3.8 ± 0.2 mg/g (20% COD). The n-3 PUFA concentrations in the catfish were 6.5 ± 0.3 mg/g (CON), 8.5 ± 0.6 mg/g (20% FLAX) and 9.0 ± 0.6 mg/g (20% COD). However high mortality rates up to 60% were encountered when the 20% FLAX and 20% COD diets were used suggesting that the levels of the oils used in these diets were toxic to the fish. Histological examinations carried out at post-mortem confirmed the toxicological condition by the occurrence of several histopathological lesions in the liver, kidney and small intestine. In conclusion, the essential PUFA profiles of the red tilapia which has a herbivorous mode of nutrition and the catfish which is more omnivorous, with different desaturase and oxidative enzyme activities are somewhat different, where the former represents a better source of desirable essential PUFA to the human consumer. The concentrations of the desirable essential PUFA in both fishes can be increased by modifying their diets to contain added oils such as flaxseed or cod-liver oil but the percentage of the added oils have to be between 10-20% of the diet to avoid toxicity and high mortalities

A complete evaluation of the antioxidant and antimicrobial potential of Glycine max

Vegetable soybean is rich in phytochemicals beneficial to the human being and is therefore considered a neutraceutical or a functional food crop. Soybean has antioxidative activity and protects tissues from oxidative stress-induced injury. Although isoflavones present in soy are believed to be major components responsible for the antioxidative activity, a recent study showed that anthocyanins present in black soybean had strong antioxidative potential.The present study focuses on both the antioxidant and antimicrobial potential of Glycine max