This thesis investigated the effects of omega-3 supplementation (2.0 g/day EPA + 1.0 g/day DHA) for 12 weeks on human skeletal muscle sarcolemmal and mitochondrial membrane fatty acid (FA) composition and whole body energy expenditure in young healthy males. Supplementation resulted in significant incorporation of EPA and DHA into sarcolemmal and mitochondrial membranes, with an increase in total unsaturation of mitochondrial membranes. The incorporation profile of the sarcolemma and mitochondria differed, with the mitochondria mimicking changes in whole muscle. There were no changes in the protein content of mitochondrial and selected proteins involved in energy metabolism, except for a significant increase in the long form of UCP3. Despite changes in membrane FA compositions, there were no changes in whole body substrate oxidation at rest or during exercise. These data demonstrate that omega-3 supplementation for 12 weeks altered the FA composition of sarcolemmal and mitochondrial membranes in human skeletal muscle.

Fatty acid components on yeast plasma membrane were critical in maintaining proper cell activity during bioethanol fermentation. The alteration of fatty acid composition on yeast plasma membrane was recognized as an adaptive response to several environmental stress including osmotic pressure, ethanol inhibition and nutrients limit. These stresses were exacerbated under very-high-gravity condition in which excessive fermentable sugar was provided in feedstock. Controlling redox potential was proved beneficial in improving yeast performance under very-high-gravity condition. Fatty acid synthesis and desaturation pathways involved dissolved oxygen as well as balance between NAD+/NADH and NADP+/NADPH which could be influenced by the regulation of redox potential in media. In this study, fatty acid composition profiles under different glucose concentrations and different redox potential control level were examined. Its connection with yeast cell growth, ethanol productivity and other metabolites’ concentrations were studied as well to reveal any causal correlation between redox potential control, membrane fatty acid composition and yeast activity. Two glucose concentrations used in this study were 200 g/L and 300 g/L which represented normal and very high gravity respectively in bioethanol fermentation. In 300 g/L fermentation, three redox conditions were adopted while two different redox conditions were used in 200 g/L fermentation. Biomass concentration, ethanol productivity and fatty acid composition were observed to be affected by both gravity and ORP control strategy. Final biomass concentrations were 4.302 g/L in 200 g/L glucose with no ORP control condition and 7.658 in 200 g/L glucose with ORP controlled at -100 mV condition. In 300 g/L glucose fermentation, final biomass concentrations were 3.400 g/L for no ORP control, 4.953 g/L for -150 mV ORP control and 5.260 for -100 mV ORP control. Ethanol productivities were 2.574 g/Lh for 200 g/L glucose without ORP control and 3.780 g/Lh for 200 g/L glucose with -100 mV ORP control. In 300 g/L glucose fermentation, ethanol productivity decreased to 1.584 g/Lh when no ORP control was imposed. ORP control at -150 mV could improve the ethanol productivity to 1.693 g/Lh while -100 mV ORP control was able to further enhance the ethanol productivity to 1.829 g/Lh. Fatty acid composition was observed to shift to more saturated components when no ORP control was applied. Such trend of saturation was increased by higher gravity condition. ORP control was shown to change this tendency to saturation and help restore fatty acid components on plasma membrane to a more balanced distribution.

In this project, the eggs of tench (Tinca tinca) were used for the evaluation of changes in fatty acid (FA) composition and lipid classes during in vitro ova ageing until the occurrence of over-ripening. Stripped ova of 6 females were collected separately and stored in sterile cell culture plates in an incubator at 20 °C for up to 24 hours post stripping (HPS). Stored ova were fertilized at 0, 2, 4, 6, 8, 10 and 24 HPS. Egg eyeing and hatching rates were assessed as indices of egg quality. Upon fertilization at each HPS, samples were taken separately frozen in liquid nitrogen and stored at -80°C till further analyses. Differences in the FA and lipid composition and embryo survival rates at the different fertilization times post-stripping were evaluated. The lipid fraction was extracted from the oocytes according to Hara and Radin (1978). The lipid content of the samples was determined gravimetrically from total extracted lipid. The FA were then analysed with a gas chromatograph equipped with a flame ionisation detector and PVT injector. The peaks were identified by comparing their retention times with those of authentic standards. Major lipid classes and the phospholipid classes were separated according to (Olsen and Henderson, 1989). Quantitative analysis of the separated lipid- and phospholipid classes was done by scanning the plates after derivatisation with a CAMAG TLC Scanner 3 (Camag, Switzerland). Identification of the lipid classes was performed by comparison with authentic standards applied on the same plate. In present study no significant changes were observed in lipid content and composition during storage of eggs until over-ripening. As well FA composition did not change over time indicating another reason of deterioration of oocyte quality than the proposed lipid oxidation. However individual fishes in this study showed much different fertilization rates that corresponded with their levels of cholesterol and triacylglycerols in minor degree.

This thesis includes two main parts: I. Evaluation of techniques for oil (total lipid) quantification of chia and soybean seeds. This study evaluated ten different methods of seed oil quantification, including some methods that have not been applied to oilseeds before. The main aim of this study was to find one or more techniques that are easy, inexpensive, safe and fast with a small amount of ground seeds. The Soxhlet method was used as a standard to compared between techniques of oil quantification. The oil extraction by the Soxhlet method was evaluated with two solvents petroleum ether and acetone. There is not a statistically significant difference between petroleum ether and acetone solvents. No significant differences for the amount of oil recovered via the Soxhlet method were found between Medium Moisture Content (MMC > 10%) and Low Moisture Content (LMC < 4.0 %). The Folch technique provided higher percentages of oil extraction than Bligh and Dyer and hexane-isopropanol techniques. There is not a statistically significant difference (P =0.0844) between Soxhlet method and Folch method but less than the Soxhlet method. A supercritical fluid extraction (HCH) method provided a lower yield of oil extraction compared with the Soxhlet method for three varieties of bias samples. A Direct Transesterification (DT) method with LMC and MMC provided a statistically significant difference than the Soxhlet method. The DT with LMC produced higher yield than DT with MMC between samples but lower than the Soxhlet method. A Double Direct Transesterification (DDT) following Griffiths protocol provided more accurate results with the stir bar technique than sonication technique. 11 oilseeds bias samples (10 bias samples of soybean and one of chia) covering an oil content range of 15.4 to 32.6% showed, there is a significant difference between the Soxhlet and DDTG method and high oil quantification found with DDTG. A Double Direct Transesterification following Qiao et al. (2015) provided similar oil extraction to the Griffiths et al. (2010) method and also there is a significant difference between the Soxhlet and DDTQ method and higher oil recovered with DDTQ. The Bead Beating Extraction (BBE) protocol showed, there is a statistically significant difference ( P< 0.001) than the Soxhlet method. The BBE provided high oil quantification comparing with the Soxhlet method. The BBE provided the best results since it is the easiest, cheapest and fastest oil quantification method. A Nile red fluorescence technique yield no clear results. II. Characterization of oil and protein in mutant chia (Salvia hispanica L.) seeds. The objectives of this study were to (1) measure heritability in chia plants of levels of oil and protein content and seed yield and (2) Compare differences between two locations over two years for oil and protein percentages in chia and seeds yield. A population of 180 M3 mutant individual chia plants was harvested and for which forty M4 chia seeds were planted based on seed composition characteristics, with six plants representing each characteristic (high and lower oil, protein, and density and high yield). The forty M4 chia seeds were planted, with two replications for two locations, have been chosen in Kentucky one on Spindletop farm and the other in Quicksand farm. The M5 progeny seeds from plants grown in Spindletop and Quicksand contained significantly (P < 0.05) more protein than did seeds from the M4 parents. There were significant differences (P < 0.05) between locations where Quicksand obtain higher oil content than Spindletop. There was no significant difference between M5 parents seeds and M4 progeny seeds and higher yield kg/ha at Quicksand than Spindletop.

Soybean [Glycine max (L.) Merr.] is the world’s most widely grown protein/oilseed crop and provides about 70% of global protein meal and 53% of vegetable oil in the United States. Soybean seed oil contains five major fatty acids, from which palmitic acid and stearic acid are two saturated fatty acids, oleic acid improves oxidative stability and linolenic acid is an essential fatty acid for human health. Soybean seed protein and oil are two important quality indices for soybean germplasm breeding. Soluble carbohydrates present in soybean meal provide metabolizable energy in livestock feed. To develop soybean germplasm with improved seed composition traits, it is important to discover novel source of seed fatty acid, protein, and carbohydrates traits. This dissertation aims to develop novel functional genomic technology coupled with an integrated approach for facilitating molecular soybean breeding. In this study, the first objective is to develop a high-throughput TILLING (Targeting Induced Local Lesions IN Genomes) by Target Capture Sequencing (TbyTCS) technology to improve the efficiency of discovering mutations in soybean. The robustness of this technology underlies the high yield of true mutations in genes controlling complex traits in soybean. Soybean mutagenized lines with modified fatty acids composition have been successfully developed to meet the different needs of end users. Altered fatty acids phenotypes have been associated with induced mutations in 3-ketoacyl-acyl carrier protein (ACP) synthase II (GmKASII), Delta-9-stearoyl-acyl carrier protein desaturase (GmSACPD), omega-6 fatty acid desaturase 2 (GmFAD2), and omega-3 fatty acid desaturase (GmFAD3) genes identified through TbyTCS. The second objective is to characterize the soybean acyl-ACP thioesterase gene family through a comprehensive analysis. The additional members have been discovered belonging to 16:0-ACP fatty acid thioesterase (GmFATB) gene family. The mutations at oleoyl-ACP fatty acid thioesterase (GmFATA1A) have been revealed to result in the high seed oleic acid content. The novel alleles of GmFATB genes have also been identified to confer low palmitic acid and high oleic acid phenotypes in soybean seeds. The third objective is to assess the phenotypic variations and correlation among seed composition traits in mutagenized soybean populations. Correlation analyses have been conducted among soybean carbohydrates, protein, and oil content of soybean mutagenized populations and germplasm lines. Chemical mutagenesis played an essential role in soybean breeding to generate novel and desired seed composition traits.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19779号 / 農博第2175号 / 新制||農||1041(附属図書館) / 学位論文||H28||N4995(農学部図書室) / 32815 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 阪井 康能, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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<p>U okviru doktorske disertacije ispitan je nutritivni kvalitet mleka magarice balkanske rase tokom laktacije. Prosečna suva materija mleka magarice balkanske rase iznosi 9,26%. Sadržaj proteina tokom laktacije kreće se od 1,40% do 1,92%. Prosečan sadržaj mlečne masti je 0,61%, a sadržaj laktoze iznosi 6,50%. Sadržaj analiziranih minerala: Ca, Na, K, Mg, P i Zn se povećava tokom laktacije i maksimalna vrednost utvrđena je 170. dana. Primenom kapilarne elektroforeze definisan je profil proteina mleka magarice balkanske rase. Identifikovane su sledeće proteinske frakcije: &alpha;s1-kazein, &alpha;s2-kazein, &beta;-kazein (A, F), &alpha;-laktalbumin (A, C), &beta;-laktoglobulin, lizozim, laktoferin, serum albumin i imunoglobulin čiji sadržaj opada tokom perioda laktacije. Sadržaj &alpha;-laktalbumina se kreće od 3090 mg/l do 1990 mg/l, a lizozima varira od 1040 mg/l do 2970 mg/l. Navedene frakcije proteina su najzastupljenije u mleku magarice balkanske rase. Laktoferin i imunoglobulin su frakcije sa najmanjim udelom u mleku magarice balkanske rase. Kori&scaron;ćenjem gasne hromatografije/masene spektrometrije utvrđen je sastav masnih kiselina mleka. Udeo esencijalne linolne kiseline (C18:2 n6) kreće se u opsegu od 7,08%, do 9,69%, a udeo &alpha;-linoleinske kiseline (C18:3 n3) varira od 5,85% do 7,83%.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Sastav mleka magarice balkanske rase kompariran je sa nutritivnim karakteristikama humanog mleka tokom 40. i 90. dana laktacije. Utvrđene su značajne razlike u sadržaju proteina mleka, mlečne masti i minerala. Odnos kazeina i proteina surutke kreće se od 0,68 do 0,75 u mleku magarice, dok u humanom mleku varira od 0,59 do 0,70. Udeo -linoleinske kiseline (C18:3 n3) je oko 2,5 puta veći u mleku magarice u odnosu na humano mleko.<br />Generalno se može zaključiti da mleko magarice balkanske rase ima specifične nutritivne karakteristike koje variraju u zavisnosti od sastava hrane za životinje i analiziranog perioda laktacije.</p> / <p>Nutritional quality of Balkan donkey milk during lactation was investigated within this thesis. The mean content of dry matter, fat and lactose in the Balkan donkey milk was 9.26%, 0.61% and 6.50%, respectively. Protein content during lactation period ranged from 1.40% to 1.92%. Content of the analyzed minerals: Ca, Na, K, Mg, Zn and P increased during the lactation period and reached their maximum value at 170th day. The protein profile of Balkans donkey milk was defined by application of capillary electrophoresis when the following protein fractions: &alpha;s1-kazein, &alpha;s2-kazein, &beta;-kazein (A, F), &alpha;-laktalbumin (A, C), &beta;-laktoglobulin, lysozyme, lactoferrin, serum albumins and immunoglobulins, whose content decreases during lactation period,were identified. &alpha;-lactalbumin contents ranged from 3090 mg/l to 1990 mg/ and lysozyme varies between 1040 mg/l to 2970 mg/l. These two protein fractions were the most abundant in the Balkan donkey milk, while lactoferrin and immunoglobulin were at least represented. The fatty acid composition of Balkan donkey milk was determined using gas chromatography/mass spectrometry. The share of the essential linoleic (C18: 2 n6) and &alpha;&nbsp;- linolenic (C18: 3 n3) acid rangred from 7.08 % to 9.69% and from 5.85 % to 7.83 %.</p><p>Nutritional quality of Balkan donkey milk has been compared with the nutritional quality of human milk during the 40th and 90th day of lactation. Significant differences in the protein content of milk, fat and minerals were found. The ratio of casein and whey protein ranged from 0.68 to 0.75 in the Balkan donkey milk, while in human milk this value varies from 0.59 to 0.70. The share of &alpha;-linolenic acid (C18:3 n3) is around 2.5 times higher in donkey than in human milk.<br />The main conclusion is that Balkan donkey milk has specific and unique nutritional quality which depend on the feed composition and on the analyzed period of lactation.</p>

Red blood cells (RBC) have been shown to mediate plaque development seen in coronary artery disease (CAD). This study determined whether differences in RBC fatty acid (FA) composition were related to CAD risk. FAs were extracted from RBCs of 38 individuals who have undergone cardiac catheterization, 9 of whom had obstructive CAD, and analyzed via gas chromatography. Ferric reducing ability of plasma (FRAP) assay was used to determine oxidative stress. Food frequency questionnaires were used to correlate RBC omega-3 FA to daily intake of omega-3 FA. No correlation was found between RBC content and intake of omega-3 FA. FRAP values and RBC FA composition did not differ between the 2 groups with exception of the saturated FA, palmitic acid (p=0.018). These results suggest that RBC FA composition may differ between individuals with or at risk for CAD. Additional research is needed to validate this biomarker as a predictor of CAD.