Very long chain polyunsaturated fatty acids (VLCPUFAs) such as arachidonic acid (ARA, 20:4ù6), eicosapentaenoic acid (EPA, 20:5ù3) and docosahexaenoic acid (DHA, 22:6ù3) have been shown to have many health benefits, some of which include lowering blood pressure, providing protection against cardiovascular diseases and improving brain and eye functions. Entomopathogenic fungi, a group of fungal pathogens able to infect insects, were previously reported to produce substantial amounts of VLCPUFAs, however the genes involved in the biosynthesis of these fatty acids have yet to be identified. This research started with fatty acid analysis of five entomopathagenic fungi, of which Conidiobolus obscurus and Conidiobolus thromboides were found to produce high levels of VLCPUFAs such as ARA and EPA. Thus, these two fungal species were selected as potential gene sources for the enzymes involved in the biosynthesis of VLCPUFAs. Using degenerate reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE) methods; we cloned two full-length putative ∆6 desaturase cDNAs (CoD6 and CtD6) from the two fungi.<p> Functional expression of CoD6 in Saccharomyces cerevisiae showed it codes for a functional Ä6 desaturase, which can introduce a Ä6 double bond into linoleic acid and á-linolenic acid, respectively. However, expression of CtD6 in S. cerevisiae showed it does not have any Ä6 desaturase activity. Using degenerate RT-PCR and RACE, we also cloned two full-length ∆6 elongase cDNAs (CoE6 and CtE6) from the C. obscurus and C. thromboides species. Functional expression of these genes in S. cerevisiae showed CoE6 and CtE6 code for functional ∆6 elongase. Substrate specificity analysis indicated that both preferentially elongate 18-carbon Ä6 desaturated fatty acids, such as ã-linolenic acid and stearidonic acid. In addition, CtE6 can also elongate 20-carbon VLCPUFAs, such as ARA and EPA. The entire eicosatetraenoic acid (ETA, 20:4ù3) biosynthetic pathway was reconstituted in yeast using four genes, CoD6 (a ∆6 desaturase) and CoE6 (a ∆6 elongase) from Conidiobolus obscurus, CpDes12 (a Ä12 desaturase) and CpDesX (a ù3 desaturase) from Claviceps purpurea. Yeast transformant expressing the four genes produced several new fatty acids. Among them, eicosatetraenoic acid (ETA) accounts for approximately 0.1% of the total fatty acids. Although the level of ETA in the transformant is low, this represents the first report describing the reconstitution of the entire ETA pathway in yeast without exogenous supplementation of any fatty acids.

Scientific evidence shows that dietary intake of the omega-3 polyunsaturated fatty acids is beneficial to human health. Fish oil is a rich source of omega-3 fatty acids. However, fish oil with high levels of omega-3 PUFA is very susceptible to oxidative deterioration during storage. The objective of this study was to investigate the effect of xanthan gum (XG)-locust bean gum (LBG) mixtures on the physicochemical properties of whey protein isolate (WPI) stabilized oil-in-water (O/W) emulsions containing 20% v/v menhaden oil. The O/W emulsions containing XG/LBG mixtures were compared to emulsions with either XG or LBG alone. The emulsions were prepared using a sonicator by first mixing menhaden oil into the WPI solution and then either XG, LBG or XG/LBG mixtures were added. WPI solution (2 wt%) and gum solutions (0.0,0.05, 0.1, 0.15, 0.2 and 0.5 wt%) were prepared separately by dissolving measured quantities of WPI in distilled water. XG and LBG gums were blended in a synergistic ratios of 50:50 for the mixture. The emulsions were evaluated for apparent viscosity, microstructure, creaming stability and oxidative stability. Addition of 0.15, 0.2 and 0.5 wt% XG/LBG mixtures greatly decreased the creaming of the emulsion. The emulsion with 0.15, 0.2 and 0.5 wt% XG/LBG mixtures showed no visible serum separation during 15 d of storage. The apparent viscosity of the emulsions containing XG/LBG mixtures was significantly higher (p < 0.05) than the emulsions containing either XG or LBG alone. The viscosity was sharply enhanced at higher concentrations of XG/LBG mixtures. Microstructure images showed depletion flocculation for LBG (0.05-0.5 wt%), XG (0.05- 0.2 wt%) and XG/LBG mixtures (0.05 and 0.1 wt%) emulsions. Flocculation was decreased with the increased biopolymer concentration in the emulsion. The decrease in flocculation was much pronounced for the emulsion containing XG/LBG mixtures. The rate of lipid oxidation for 8 week storage was significantly (p < 0.05) lower in emulsions containing XG/LBG mixtures than in emulsions containing either of the biopolymer alone. The results suggested that the addition of XG/LBG mixtures greatly enhanced the creaming and oxidative stability of the WPI-stabilized menhaden O/W emulsion as compared to either XG or LBG alone.

Very long chain polyunsaturated fatty acids (VLCPUFAs) such as arachidonic acid (ARA, 20:4ù6), eicosapentaenoic acid (EPA, 20:5ù3) and docosahexaenoic acid (DHA, 22:6ù3) have been shown to have many health benefits, some of which include lowering blood pressure, providing protection against cardiovascular diseases and improving brain and eye functions. Entomopathogenic fungi, a group of fungal pathogens able to infect insects, were previously reported to produce substantial amounts of VLCPUFAs, however the genes involved in the biosynthesis of these fatty acids have yet to be identified. This research started with fatty acid analysis of five entomopathagenic fungi, of which Conidiobolus obscurus and Conidiobolus thromboides were found to produce high levels of VLCPUFAs such as ARA and EPA. Thus, these two fungal species were selected as potential gene sources for the enzymes involved in the biosynthesis of VLCPUFAs. Using degenerate reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE) methods; we cloned two full-length putative ∆6 desaturase cDNAs (CoD6 and CtD6) from the two fungi.<p> Functional expression of CoD6 in Saccharomyces cerevisiae showed it codes for a functional Ä6 desaturase, which can introduce a Ä6 double bond into linoleic acid and á-linolenic acid, respectively. However, expression of CtD6 in S. cerevisiae showed it does not have any Ä6 desaturase activity. Using degenerate RT-PCR and RACE, we also cloned two full-length ∆6 elongase cDNAs (CoE6 and CtE6) from the C. obscurus and C. thromboides species. Functional expression of these genes in S. cerevisiae showed CoE6 and CtE6 code for functional ∆6 elongase. Substrate specificity analysis indicated that both preferentially elongate 18-carbon Ä6 desaturated fatty acids, such as ã-linolenic acid and stearidonic acid. In addition, CtE6 can also elongate 20-carbon VLCPUFAs, such as ARA and EPA. The entire eicosatetraenoic acid (ETA, 20:4ù3) biosynthetic pathway was reconstituted in yeast using four genes, CoD6 (a ∆6 desaturase) and CoE6 (a ∆6 elongase) from Conidiobolus obscurus, CpDes12 (a Ä12 desaturase) and CpDesX (a ù3 desaturase) from Claviceps purpurea. Yeast transformant expressing the four genes produced several new fatty acids. Among them, eicosatetraenoic acid (ETA) accounts for approximately 0.1% of the total fatty acids. Although the level of ETA in the transformant is low, this represents the first report describing the reconstitution of the entire ETA pathway in yeast without exogenous supplementation of any fatty acids.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成23年3月25日 村松崇氏の博士論文として提出された

A study was performed to investigate the effect of diet modifications on skin and hair coat condition in canines. The study included 24 normal adult dogs fed a baseline diet (Ol'Roy[trademark]), during an acclimation period of 12 wk (Phase I). Nine female Beagles and 15 male Hound mix-breed dogs were used. For the next 12 wk (Phase II) the dogs were divided into three groups and fed one of three specially formulated diets. They contained similar ingredients and had similar nutrient profiles except for the following differences: Diet A contained lower but adequate amounts of dietary zinc and linoleic acid than diet B. Diet C was similar to B with respect to zinc and linoleic acid but contained more α-linolenic acid. An evaluation panel conducted skin and hair coat condition scoring on wk 0, 4, 7, and 12 (Phase I) and wk 14, 16, 19, and 24 (Phase II). The panel evaluated the dogs for glossiness, softness, scale, greasiness, and overall condition. Transepidermal water loss (TEWL) and skin hydration (HYDR) assessments were determined on wk 3, 7, and 11 (Phase I) and wk 11, 12, 15, 19, and 23 (Phase II) using a Tewameter[trademark] and Corneometer[trademark] respectively. Blood samples were collected on d 0, 5, 8, 16, 28, 56, and 84. Profiles of plasma phospholipid fatty acids were determined at each collection period. Serum zinc concentrations were analyzed on wk 12, 14, and 24. The hypothesis was that a diet containing increased LA, ALA, and zinc concentrations (diet C) would show improvements of skin and hair coat condition in dogs compared to the other diets. All three test diets caused significant improvements compared to Ol'Roy[trademark]. Diet B caused more improvement than diet A in both subjective and objective assessments of skin and hair coat. Based on mean values diet B is better to be fed to dogs that need to improve skin hydration and diet C should be fed to dogs that need to decrease TEWL. Diet C not only led to improvements in skin and hair coat condition, but also provided additional benefit by producing less pro-inflammatory conditions in the skin.

Long-chain polyunsaturated fatty acids (LCPUFA) are essential for proper neural and retinal development in many mammalian species. One objective of this research was to investigate the effects of dietary &#945;-linolenic acid (ALA) and LCPUFA on the fatty acid composition of canine plasma phospholipids (PL) and milk during the gestation and lactation periods. The fatty acid composition of plasma PL and the retinal development of puppies reared on the same experimental diets as their mothers were also investigated. Enriching the canine gestation/lactation diet with ALA (6.8% DM) does not result in enrichment of docosahexaenoic acid (DHA) in the milk. From this data it can be inferred that peroxisomal elongation and desaturation of LCPUFA does not occur in canine mammary tissue. Dose responses of linoleic acid (LA), ALA and DHA were observed in the plasma of adult dogs during gestation and lactation and in puppies during both the suckling and post-weaning periods. Plasma PL fatty acid data from puppies indicate that canine neonates are capable of synthesizing LCPUFA from ALA, but that plasma enrichment of the newly-synthesized DHA does not compare with that obtained from preformed DHA in the diet. Visual function was assessed via electroretinography (ERG) in 12-wk old canines. One-way ANOVA revealed significantly better visual performance in dogs fed the highest amounts of n-3 LCPUFA. Puppies in this group demonstrated the greatest rod response as measured by the amplitude and implicit time of the a-wave. Neonates reared on the lowest dietary levels of both ALA and n-3 LCPUFA exhibited the poorest visual function. A novel parameter devised in this study was the threshold intensity, which was the initial intensity at which the a-wave was detectable. Again, puppies consuming the greatest concentrations of n-3 LCPUFA responded significantly sooner, i.e. exhibited greater rod sensitivity, than other diet groups. The findings of this research underscore the importance of preformed n-3 LCPUFA in the diet, rather than ALA, as a means of enriching neural tissues in DHA during the developmental period. Moreover, dietary DHA appears to be related to improved visual performance in developing canines.

Adipose tissue is well-recognized as an endocrine organ which secretes a variety of bioactive molecules, including angiotensin II and its precursor angiotensinogen (Agt). There is mounting evidence linking the adipose renin-angiotensin system (RAS) and diet to obesity and obesity-related disorders. However, research addressing dietary regulation and function of adipose RAS is limited, and the specific mechanisms by which PUFAs modulate the endocrine function of adipose tissue remain largely unclear. There are several potential mechanisms that may mediate PUFA effects on Agt, including toll-like receptor signalling, prostaglandins or PPAR-gamma. Thus, we propose to investigate whether PUFAs differentially modulate Agt expression and secretion and to examine possible mechanisms by which PUFA alter Agt expression using the 3T3-L1 cell line.Differentiated 3T3-L1 adipocytes were treated with arachidonic acid (AA), eicosapentaenoic acid (EPA), AA + EPA, or vehicle (C) for 48 hours. Results showed a significant increase in intracellular Agt protein following treatment with PUFAs. Agt secretion, however, was only increased by AA. Interestingly, there is a dose-dependent decrease in Agt protein levels by EPA suggesting that a minimum concentration of n-3 PUFAs is required to elicit an Agt response. Agt mRNA levels were measured by RT-PCR and results showed a significant increase in Agt mRNA in response to treatment with AA but not EPA. These findings suggest that Agt regulation by PUFAs is complex and occurs both post-transcriptionally and post-translationally.Changes in mRNA stability may account for the observed effects of PUFAs. Adipocytes were treated with the transcriptional inhibitor actinomycin D (Act D) and Agt mRNA expression was measured over time. Total RNA was also measured at each time point to ensure that Act D treatment was effectively decreasing transcription. Agt mRNA expression was not significantly altered by treatment with EPA while treatment with AA increased Agt mRNA levels. These results suggest that Agt mRNA stability is differentially increased by n-6 but not n-3 PUFAs. Although there are clear effects of AA on Agt secretion and mRNA stability, the signaling pathways mediating this response remain to be determined, and additional studies are necessary to further dissect the underlying mechanisms of this regulation.

Oil-in-water emulsions have wide range of applications in food industry because of their structure-forming properties, and as delivery systems of polyunsaturated fatty acids into foods. The thermodynamic instability of oil and water, and high susceptibility of unsaturated fatty acids to oxidation lead to physical and oxidative stability in oil-in-water emulsions. These instability processes are generally controlled by incorporating proteins and polysaccharides into oil-in-water emulsions. The objective of this study was to evaluate xanthan/enzymatically modified guar (XG/EMG) polysaccharides on the physical and oxidative stability of 2 wt% whey protein stabilized oil-in-water emulsions containing 20% v/v menhaden fish oil. Enzymatic modified guar gum was obtained by hydrolyzing native guar gum using α-galactosidase enzyme. Emulsions were prepared for guar gum (GG), xanthan gum (XG), xanthan/guar (XG/GG), and xanthan/enzymemodified guar (XG/EMG) gum mixtures using 0, 0.05, 0.1, 0.15, 0.2, and 0.3% gum concentrations. Emulsions were then evaluated for creaming, viscosity, particle size, and microstructure to evaluate the physical stability, and peroxide value, TBARS value and GC-MS solid phase micro extraction (SPME) experiments were performed to evaluate the oxidative stability. Emulsions containing XG/EMG gum mixtures exhibited better creaming stability and higher viscosity of all the emulsion types. However, XG/EMG gum concentrations did not affect the droplet size of the emulsions. The microstructures revealed decreased flocculation in emulsions with XG/EMG mixtures. The primary and secondary lipid oxidation measurements indicated that emulsions containing XG/EMG gum mixtures were more effective in preventing the lipid oxidation of all the emulsion types. From the results, it is evident that XG/EMG gum mixtures can be used as emulsifiers in oil-in-water emulsions to increase both physical and oxidative stability.

The biological activity of α-linolenic acid (ALA) is poorly understood and primarily associated with its conversion to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). This study used the Δ6 desaturase knockout (D6KO) mouse, which lacks Δ6 desaturase and therefore cannot convert ALA, to evaluate the independent effects of ALA on preventing non-alcoholic fatty liver disease (NAFLD). First, the capacity of very long chain fatty acids to rescue the D6KO lipid profile was established. Next, to evaluate the independent effects of ALA, D6KO or wild-type mice were fed diets containing lard, canola, flaxseed, or fish oil. Following treatment, liver phospholipid fatty acid composition was evaluated and livers were scored for steatosis and inflammation. Glucose tolerance was also evaluated. D6KO mice fed ALA-rich diets had lower liver lipid accumulation, lower hepatic inflammation (8 weeks) and improved glucose tolerance (20 weeks) relative to lard-fed D6KO mice. Overall, this thesis supports an independent biological role for ALA. / D.W.L. Ma is funded by the Canola Council of Canada, Natural Sciences and Engineering Research Council of Canada and the Canada Foundation for Innovation Leaders Opportunity Fund with matching from the Ontario Research Fund; Jessica Monteiro is funded Ontario Graduate Scholarship.

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