The evaluation of environmental and genotypic effects on fruit physical and chemical characteristics enables assessment of the feasibility of selecting diploid and/or triploid cultivars for either specific or more diverse locations. Isolation and characterization of genes encoding enzymes in the carotenoid biosynthetic pathway provides fundamental genetic information which can facilitate breeding of watermelon cultivars having desirable flesh colors and enhanced beneficial carotenoids. For the environmental studies, the effects of deficit irrigation on lycopene content, total soluble solids, firmness, and yield of diploid and triploid watermelon were evaluated in different locations and growing seasons. Irrigation regimes were 1.0 evapotranspiration (ET), 0.75 ET, and 0.5 ET. To investigate if there is a consistent response in cultivars across diverse locations, studies were conducted in three distinct Texas regions. Deficit irrigation reduced total marketable yield, and increased the yield of small fruits. Location and irrigation regimes had major influences on yield. Soluble solids content increased with deficit irrigation at 0.5 ET in triploids, but not in diploids. Flesh firmness also increased in triploids compared to diploids. Lycopene content increased with maturity at all irrigation regimes and cultivars. This work confirms that deficit irrigation directly reduces yield, but does not reduce lycopene and fruit quality of the triploids used in this study. From the genetic studies, a total of eight genes encoding enzymes in the carotenoid biosynthetic pathway were isolated and characterized. Two members of the phytoene synthase (PSY) gene family were identified; PSY-A was expressed in all type of tissues, but PSY-B transcript was detected only in ovary, leaf, and root tissues. Gene expression of carotenoid isomerase (CRTISO) was not detected in salmon yellow. A color inheritance study of watermelon flesh indicated that a single gene might determine color difference between canary yellow and red without an inhibitory effect. A cleaved amplified polymorphic sequence (CAPS) marker developed from the SNP marker tagging two different lycopene β-cyclase (LCYB) alleles cosegregated perfectly with color phenotypes. It was concluded that color determination may be due to a reduced activity of LCYB enzyme in red, whereby a phenylalanine is conserved among canary yellow and valine is conserved among red watermelon.

Plants contain a host of secondary metabolites that may be of dietary use to man. A comprehensive approach to plant-based nutrition would include investigating all aspects of a nutrient, from creation through storage and consumption. Here, experiments address each of these facets for a group of important antioxidant and pigment compounds, the carotenoids. The carotenoid biosynthetic pathway regulatory mechanisms leading to lycopene accumulation are well defined in the model fruit, tomato. Those leading to accumulation of other carotenoids and flesh colors, however, are poorly understood. The variety of flesh colors available in watermelon fruit (red, orange, salmon yellow, and canary yellow) makes it an ideal candidate for investigating the regulation of the full pathway. Carotenoid accumulation was measured in ten watermelon varieties, representing the four flesh colors and three ploidy levels, throughout fruit maturation. It was found that the putative regulatory mechanisms controlling lycopene accumulation in red-fleshed fruit may be applied in a generalized fashion to each flesh color in respect to the major carotenoid accumulated at maturity. Additionally, triploid varieties were generally found to have higher accumulation levels than diploids, and tetraploids were intermediate to both. In addition to total carotenoid content, many factors are important in determining perceived benefit. Several of these factors involve components of the food matrix, cellular and subcellular species-specific characteristics of the food which act as barriers to nutrient release. Cell size, cell wall, and chromoplast (the carotenoid storage organelle) characteristics were observed in nine fruits and vegetables using light and transmission electron microscopy. Watermelon, tomato, and melon have the largest cells. Sweet potato, butternut squash, carrot, and mango have the most fibrous cell walls; mango and papaya additionally had the thickest walls. Chromoplast globular, tubular, crystalline, and membranous substructures were described for each food. These food matrix factors may be related to differences in carotenoid bioaccessibility between food sources. An in vitro digestion experiment was used to determine carotenoid bioaccessibility for each of these foods. Per serving, grapefruit yielded the most lycopene while carrot gave the most ?-carotene, ?-carotene, lutein, and phytoene, and mango proved a good source of violaxanthin.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

In oviparous animals, maternal traits such as the investment of resources in eggs and oviposition site selection are often important determinants of offspring phenotypic quality, and may have an adaptive role in tailoring offspring phenotypes to local environmental conditions. This thesis examines the adaptive significance of two specific maternal traits in the green turtle (Chelonia mydas); namely the deposition of fat-soluble antioxidants in egg yolk, and the selection of nest sites via natal homing. (Please view 'front matter' file for full abstract)

A diet rich in fruit and vegetables is associated with better bone health although the reason behind this remains unclear. Mounting evidence suggests that it may be particular fruit and vegetables or a variety of fruit and vegetables that are important and it has been suggested that bioactive compounds, considered to be non-essential nutrients, may play a role. The aims of this project were to study the roles of three classes of dietary compounds on bone health: flavonoids (represented by hesperetin), anthocyanidins and carotenoids. Methods The influence of six major anthocyanidins, six major carotenoids and the citrus flavanone hesperetin were examined by three methodological approaches: (i) a cross-sectional study using outcomes of bone mineral density (BMD) and markers of bone turnover, (ii) a randomised controlled trial with bone turnover markers as the outcome measures and (iii) in vitro examinations in osteoblasts and osteoclasts. The epidemiological part of this thesis was carried out in the Aberdeen Prospective Osteoporosis Screening Study (APOSS) cohort. Women were recuited to the study between 1990 and 1994 and returned for a follow-up visit between 1997 and 1999 (n=3214, mean age 54.8 y at follow-up). Measurements of BMD at the spine and hip were taken at both visits; and urinary markers of bone resorption total deoxypyridinolines (DPD) and total pyridinolines (PYD), and a serum bone formation marker N-terminal propeptides of type 1 procollagen (P1NP), were analysed at the follow-up visit. Diet was recorded by food frequency questionnaire and dietary anthocyanidins and carotenoids were estimated using a database of food compositions developed for this purpose. Analysis was carried out to determine if anthocyanidin or carotenoid intakes were associated with BMD, change in BMD (between the 2 visits), or markers of bone resorption or formation, within the APOSS population. The effect of the carotenoid lycopene on bone turnover was assessed in a 3-month randomised controlled trial in 214 apparently healthy men and women. Participants were randomised into 3 groups: high dietary lycopene (minimum 10 mg/d); low tomato diet with lycopene capsule vi (10 mg/d) or a low tomato diet as the control. Marker of bone resorption plasma carboxyterminal collagen crosslinks (CTX) and marker of bone formation serum P1NP were analysed at baseline (after washout) and after 12 weeks of intervention. In order to study if the aglycone or glycoside compounds directly affect bone metabolism, the effects of a series of anthocyanidins on osteoblast differentiation were analysed in vitro. The effects of hesperetin on osteoblast differentiation and mineralisation and on osteoclast formation and function in vitro were also assessed. Results Regarding the epidemiological part of the project, associations between both dietary anthocyanidins and carotenoids and markers of bone health were observed in the APOSS population. Higher total dietary anthocyanidin intake was found to be associated with higher spine BMD and lower concentrations of bone resorption markers. In addition higher total dietary anthocyanidin intake was associated with less BMD loss at the spine in the period between baseline and follow-up, which was illustrated by a 13.2% difference in annual percent bone loss between the highest and lowest quartiles of anthocyanidin consumption. Individual anthocyanidins were also found to be associated with different markers of bone turnover. Total dietary carotenoid intake was found to be associated with BMD at the spine and lower concentrations of bone resorption markers. Analysis of the individual carotenoids showed that lycopene was associated with higher BMD at the hip; β-carotene was associated with less BMD loss at the spine; and β-carotene, lycopene, β-cryptoxanthin and lutein/zeaxanthin were found to be associated with lower concentrations of bone resorption markers. Each of these findings remained significant after adjusting for confounding factors. In the 3-month randomised controlled trial, lycopene supplementation did not alter bone turnover markers CTX or P1NP. These results are in contrast to those of a previous, smaller randomised controlled trial in postmenopausal women where a decrease in a marker of bone resorption (N-telopeptide of collagen cross-links (NTX)) was observed. Therefore these results suggest that the potential beneficial effect of lycopene may be specific to a population at risk of bone loss. Alternatively, lycopene may have a cumulative protective effect over the lifetime but short-term effects may only be observed in groups with high bone turnover, where there is greater potential to see measureable effects. The results of the in vitro investigations of this project showed that neither anthocyanidins nor hesperetin had an affect on osteoblasts or osteoclasts at physiologically relevant concentrations. Almost all of the anthocyanidin compounds tested had no effect on osteoblast differentiation, and none at physiological concentrations. Similarly, hesperetin had no effect on osteoblast differentiation or mineralisation although it did have an effect on both osteoclast formation and function, but only at concentrations which were not considered to be physiologically relevant. These results add weight to the suggestion that the metabolites of dietary compounds may be responsible for the action on bone metabolism rather than the dietary compounds directly or that a combination of compounds, as found in foods, may be required. Conclusions Taken together, these results support the evidence that a diet rich in fruit and vegetablederived bioactive compounds is beneficial to bone health. Future work could include: observational studies to examine the association of lifetime consumption and long-term risk of fracture; larger dietary intervention trials; and in vitro studies to examine the effects of the compound metabolites and elucidate their mechanism of action.

Corn gluten meal (CGM) is a high protein (60% crude protein), highly digestible feed ingredient widely used in diets for salmonids, however its use has been related to reduction in muscle pigmentation possibly due to pigment interaction. Therefore, laboratory scale and in vivo trials were conducted to reduce pigment level in CGM and to assess its effect on fish muscle pigmentation, respectively. In the first chapter, a bench-scale study was carried out to investigate factors that affect bleaching of carotenoids in CGM, using white soy flake flour (WSFF) as a lipoxygenase (LOX) source. Plackett-Burman and Box-Behnken designs were used to screen and optimize factors, respectively. Furthermore, a 12-week growth trial was conducted in order to assess the effects of dietary regular and pigment bleached CGM on growth and muscle pigmentation of rainbow. In the second chapter, a 24-week growth trial was carried out in order to assess the effects of increasing levels of CGM on growth and muscle pigment deposition in rainbow trout. In the third chapter, a bench-scale (10 g) corn wet milling procedure was used to assess the bleaching of carotenoids from CGM during steeping. Studies from this thesis confirmed the negative effects of CGM on fillet pigmentation and highlighted the need for evaluation of muscle quality traits such as colour in response to inclusion of new feed ingredients. Furthermore, this thesis gives insight on how to reduce pigments from corn gluten meal using cost-effective and practical bleaching approaches.

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In processing lobster, approximately 70% of the body weight is discarded as waste. The solid waste consists of shell, viscera and a small amount of meat. Lobster waste is an excellent source of protein (26.6%) crude fat (2.7%) and pigment (98 $ mu$g/g) on a dry weight basis which could be processed into a stable complex known as carotenoprotein. / Procedures for extraction of carotenoprotein from lobster waste have been developed, along with investigation of its distinctive physicochemical properties. The main factors were: demineralization agents (EDTA, HCl or heat) and precipitation agents ((NH$ sb4$)$ sb2$SO$ sb4$ or HCl). Lobster carotenoprotein recovered under different extraction procedures showed significant differences in terms of quantity and quality of the product recovered. However the procedures did not appear to affect the physical properties and amino acid profiles of carotenoprotein. / Optimal recovery involved treatment with EDTA as demineralization agent and precipitation with (NH$ sb4$)$ sb2$SO$ sb4$. Proximate and amino acid analyses indicated the potential of the recovered lyophilized protein as feed ingredient for cultured salmonids.

While carotenoids are essential for visual function, their potential role in diabetic retinopathy is not known. By providing carrot powder, this study examined carotenoid metabolism and visual function in Type 1 diabetes. Wistar rats (n=30) were assigned to diet either with or without carrot enrichment (15%, w/w) for 12 weeks. Type 1 diabetes was induced with streptozotocin at 3 weeks. Retinal function and anatomical integrity were assessed along with retinoid and carotenoid levels in the serum, liver, and retina. Loss of ERG oscillatory potentials, with normal histology indicated early stage retinopathy. Healthy animals fed carrot diet showed highest b-wave amplitudes; reflecting higher phototransduction. Diabetic animals fed carrot diet had the lowest b-wave amplitudes, reduced retinoids liver reserves, and highest α- and β-carotene, suggesting disturbance of conversion during diabetes. Consequently carrot powder at concentrations used by this study cannot be recommended for diabetic retinopathy.

Carotenoids are essential for visual function, however their potential role in Computer Vision Syndrome (CVS) is not known. By providing carrot powder, this study examined carotenoid metabolism and visual function in CVS. CVS participants were recruited into a double-blind, placebo-controlled, repeated measures trial (n=19, ages 20-65) and were randomized to 2 supplementation groups; control (15g cream of wheat powder) or carrot enriched (15g carrot powder, 33% of vitamin A RDA for adults) in an isocaloric pudding and yogurt for 4 weeks. Retinal function, self-perceived vision status, and plasma carotenoids/retinoids were assessed, along with plasma lipids and oxidative stress markers. Photopic b-waves marginally improved following supplementation reflecting higher phototransduction, possibly due to increased plasma carotenoid/retinoid levels. LDL cholesterol and oxidative stress markers showed trending reductions illustrating a protective role of the carrot. Carrot powder, at a minimal supplementation dose, can be recommended for CVS.