Overexpression of tocopherol biosynthesis genes in guayule (Parthenium argentatum) reduces rubber, resin and argentatins content in stem and leaf tissues.

Natural rubber produced in stems of the guayule plant (Parthenium argentatum) is susceptible to post-harvest degradation from microbial or thermo-oxidative processes, especially once stems are chipped. As a result, the time from harvest to extraction must be minimized to recover high quality rubber, especially in warm summer months. Tocopherols are natural antioxidants produced in plants through the shikimate and methyl-erythtiol-4-phosphate (MEP) pathways. We hypothesized that increased in vivo guayule tocopherol content might protect rubber from post-harvest degradation, and/or allow reduced use of chemical antioxidants during the extraction process. With the objective of enhancing tocopherol content in guayule, we overexpressed four Arabidopsis thaliana tocopherol pathway genes in AZ-2 guayule via Agrobacterium-mediated transformation. Tocopherol content was increased in leaf and stem tissues of most transgenic lines, and some improvement in thermo-oxidative stability was observed. Overexpression of the four tocopherol biosynthesis enzymes, however, altered other isoprenoid pathways resulting in reduced rubber, resin and argentatins content in guayule stems. The latter molecules are mainly synthesized from precursors derived from the mevalonate (MVA) pathway. Our results suggest the existence of crosstalk between the MEP and MVA pathways in guayule and the possibility that carbon metabolism through the MEP pathway impacts rubber biosynthesis.

IgE binding epitope mapping with TL1A tagged peptides.

Linear IgE epitopes play essential roles in persistent allergies, including peanut and tree nut allergies. Using chemically synthesized peptides attached to membranes and microarray experiments is one approach for determining predominant epitopes that has seen success. However, the overall expense of this approach and the inherent challenges in scaling up the production and purification of synthetic peptides precludes the general application of this approach. To overcome this problem, we have constructed a plasmid vector for expressing peptides sandwiched between an N-terminal His-tag and a trimeric protein. The vector was used to make overlapping peptides derived from peanut allergens Ara h 2. All the peptides were successfully expressed and purified. The resulting peptides were applied to identify IgE binding epitopes of Ara h 2 using four sera samples from individuals with known peanut allergies. New and previously defined dominant IgE binding epitopes of Ara h 2 were identified. This system may be readily applied to produce agents for component- and epitope-resolved food allergy diagnosis.

Texture of Hot-Air-Dried Persimmon (Diospyros kaki) Chips: Instrumental, Sensory, and Consumer Input for Product Development.

Persimmon (Diospyros kaki) is an underutilized tree fruit. Previous studies have shown the feasibility of making a hot-air-dried, chip-style product from persimmon. However, the texture of this type of product has not been explored or connected to consumer preference. Thus, for dried samples representing 37 cultivars, this study aimed to (1) predict trained sensory panel texture attributes from instrumental measurements, (2) predict consumer liking from instrumental measurements and sensory texture attributes, and (3) elucidate whether astringency type affects dried product texture. Partial least-squares regression models of fair-to-good quality predicted all measured sensory texture attributes (except Tooth Packing) from instrumental measurements. Modeling also identified that consumer preference is for a moist, smooth texture. Lastly, while astringency type has significant (p < 0.05) effects on several individual texture attributes, astringency type should not be used a priori to screen-in or -out persimmon cultivars for processing into a hot-air-dried product.

Synthesis of descriptive sensory attributes and hedonic rankings of dried persimmon (Diospyros kaki sp.).

This work aimed to characterize the sensory attributes of hot air-dried persimmon (Diospyros kaki) chips, correlate these attributes with consumer hedonic information, and, by doing so, present recommendations for cultivars that are most suitable for hot-air drying. A trained sensory panel evaluated dried persimmon samples (representing 40 cultivars) for flavor, taste/aftertaste, and texture. In addition, in each of two tests conducted in different years, more than 100 consumers provided hedonic evaluations of 21 unique samples in a ranking task with a balanced incomplete block design. A partial least squares regression model correlating the mean hedonic ranking to the trained panel data was developed using the data from the first consumer panel. The predictions from the model were correlated with the second panel to verify the model. It was found that including taste, aftertaste, and texture data (but not specific flavor attribute data) produced a predictive model (Spearman's ρ=0.83). This indicates that flavor is likely secondary to taste and texture in dried persimmon chips. Using the validated predictive model, 6 of the 40 persimmon cultivars tested are recommended for a dried chip product; these cultivars are 'Fuyu', 'Lycopersicon', 'Maekawa Jiro', 'Nishimura Wase', 'Tishihtzu', and 'Yotsumizo'.

Dietary supplementation with purified citrus limonin glucoside does not alter ex vivo functions of circulating T lymphocytes or monocytes in overweight/obese human adults.

Overweight/obesity is associated with chronic inflammation and impairs both innate and adaptive immune responses. Limonoids found in citrus fruits decreased cell proliferation and inflammation in animal studies. We hypothesized that limonin glucoside (LG) supplementation in vivo will decrease the ex vivo proliferation of T cells and the production of inflammatory cytokines by monocytes and T cells. In a double-blind, randomized, cross-over study, 10 overweight/obese human subjects were served purified LG or placebo drinks for 56 days each to determine the effects of LG on immune cell functions. The percentage of CD14+CD36+ cells in whole blood was analyzed by flow cytometry. Peripheral blood mononuclear cells were isolated and activated with CD3 plus CD28 antibodies (T-lymphocyte activation) or lipopolysaccharide (monocyte activation). Interferon γ, tumor necrosis factor α, interleukin (IL) 2, IL-4, and IL-10 were measured in supernatants from activated T cells. Supernatants from activated monocytes were analyzed for the production of tumor necrosis factor α, IL-1ß, and IL-6. Peripheral blood mononuclear cells were prestained with PKH dye and activated with CD3 plus CD28 antibodies to determine the proliferative responses of CD4+ and CD8+ T lymphocytes by flow cytometry. No differences were observed for CD14+CD36+ monocyte populations, T-cell proliferation, or the production of T cell and monocyte cytokines between the 2 treatments. Thus, LG supplementation in vivo did not affect ex vivo functions of T cells and monocytes, whereas it decreased several circulating markers of hepatic inflammation as we previously reported.

Metabolite signature of Candidatus Liberibacter asiaticus infection in two citrus varieties.

Huanglongbing (HLB), also known as Citrus Greening Disease, is caused by the bacterium 'Candidatus Liberibacter asiaticus' (CLas) and is a serious threat to the citrus industry. To understand the effect of CLas infection on the citrus metabolome, juice from healthy (n = 18), HLB-asymptomatic (n = 18), and HLB-symptomatic Hamlin (n = 18), as well as from healthy (n = 18) and HLB-symptomatic (n = 18) Valencia sweet oranges (from southern and eastern Florida) were evaluated using (1)H NMR-based metabolomics. Differences in the concentration of several metabolites including phenylalanine, histidine, limonin, and synephrine between control or asymptomatic fruit and symptomatic fruit were observed regardless of the citrus variety or location. There were no clear differences between the metabolite profiles of Hamlin fruits classified by PCR as asymptomatic and control, suggesting that some of the control fruit may have been infected. Taken together, these data indicate that infection due to CLas presents a strong metabolic response that is observed across different cultivars and regions, suggesting the potential for generation of metabolite-based biomarkers of CLas infection.

Dietary grape powder increases IL-1ß and IL-6 production by lipopolysaccharide-activated monocytes and reduces plasma concentrations of large LDL and large LDL-cholesterol particles in obese humans.

Obese individuals are at an increased risk of developing CVD, hypertension, type 2 diabetes, and bacterial and viral infections when compared with the normal-weight population. In a 9-week randomised, double-blind, cross-over study, twenty-four obese subjects aged between 20 and 60 years and with a BMI between 30 and 45 kg/m2 were fed grape or placebo powder for 3-week intervals to determine the effects of dietary grapes on blood lipid profiles, plasma inflammatory marker concentrations and immune cell function. Blood samples were collected on days 1 and 8 for obtaining baseline information and at weeks 3, 4, 8 and 9. Comprehensive chemistry panels, lipid profile analyses by NMR, measurement of plasma inflammatory marker concentrations, and analyses of cytokine production by activated T lymphocytes and monocytes were performed for each blood draw. Dietary grape powder reduced the plasma concentrations of large LDL-cholesterol and large LDL particles compared with the placebo powder (P< 0·05). The concentrations of interferon-γ, TNF-α, IL-4 and IL-10 were measured in supernatants from peripheral blood mononuclear cells (PBMC) activated with anti-CD3/CD28 antibodies and those of TNF-α, IL-1ß, IL-6 and IL-8 were measured in supernatants from PBMC activated with lipopolysaccharide (LPS). No difference in the production of T-cell cytokines was observed between the two intervention groups. The production of IL-1ß and IL-6 was increased in supernatants from LPS-activated PBMC in the grape powder group compared with the placebo powder group (P< 0·05). These data suggest that dietary grapes may decrease atherogenic lipid fractions in obese individuals and increase the sensitivity of monocytes in a population at a greater risk of developing infections.

Thermal stability of liquid antioxidative extracts from pomegranate peel.

BACKGROUND: Liquid extracts from pomegranate peel have the potential for use as natural antioxidant products. This study investigates the quality changes of liquid extracts before and after thermal treatment during sterilization and storage. Liquid pomegranate peel extracts were prepared, sterilized under ultra-high temperature (UHT) at 121 °C for 10 s and then stored at three temperatures (4, 25 and 37 °C) for up to 180 days. The industrial, color, UV-visible spectrum profile and antioxidant (phenolics) characteristics were measured. RESULTS: Thermal sterilization treatment had no negative effects on the industrial, color, spectral and antioxidant characteristics of the extracts. After 180 days, the extracts stored at 4 °C retained 67% of the initial total soluble phenolic content and 58% of the original scavenging activity. The major antioxidant components in the extracts (stored at 4 °C for 180 days) were gallic acid, punicalagin A, punicalagin B and ellagic acid having concentrations of 19.3, 197.2, 221.1 and 92.4 mg L⁻¹, respectively. CONCLUSION: The results show that liquid pomegranate peel extracts had acceptable thermal stability after sterilization and storage. The recommended storage condition of this product was low temperature.

Fertilisation and pesticides affect mandarin orange nutrient composition.

The effects of the application of foliar fertilisation and pesticide on nutritional quality of mandarin orange juices were evaluated using (1)H NMR metabolomics. Significant differences between the use of fertiliser and pesticides during fruit formation were observed, and included changes in sugar, amino acid and organic acid composition. To determine whether the difference in sugar concentration was enough for the consumer to detect, a sensory experiment was performed in which two orange juice samples were prepared to resemble the sweet/sour taste balance of juice from mandarin oranges in which foliar fertilisation was either applied or not. In a test using non-trained individuals, 68% could correctly identify which juice had a sourer, or less sweet, taste. The implications of this study could impact citrus growers, and ultimately aid in development of fruit with superior sensory quality.

Sensory evaluation of baked chicken wrapped with antimicrobial apple and tomato edible films formulated with cinnamaldehyde and carvacrol.

The addition of plant essential oils to edible films and coatings has been shown to protect against bacterial pathogens and spoilage while also enhancing sensory properties of foods. This study evaluated the effect of adding 0.5 and 0.75% carvacrol (active ingredient of oregano oil) to apple- and tomato-based film-forming solutions and 0.5 and 0.75% cinnamaldehyde (active ingredient of cinnamon oil) to apple-based film-forming solutions on sensory properties of cooked chicken wrapped with these films. Paired preference tests indicated no difference between baked chicken wrapped with tomato and apple films containing 0.5% carvacrol and cinnamaldehyde compared to chicken wrapped with tomato or apple films without the plant antimicrobials. The taste panel indicated a higher preference for carvacrol-containing tomato-coated chicken over the corresponding apple coating. There was also a higher preference for cinnamaldehyde-containing apple films over corresponding carvacrol-containing wrapping. Films containing antibacterial active compounds derived from essential oils can be used to protect raw chicken pieces against bacterial contamination without adversely affecting preferences of wrapped chicken pieces after baking.

Metabolomic analysis of citrus infection by 'Candidatus Liberibacter' reveals insight into pathogenicity.

Huanglongbing (HLB), considered the most serious citrus disease in the world, is associated with the nonculturable bacterium 'Candidatus Liberibacter asiaticus' (Las). Infection of citrus by this pathogen leads to reduced plant vigor and productivity, ultimately resulting in death of the infected tree. It can take up to two years following initial infection before outward symptoms become apparent, making detection difficult. The existing knowledge gap in our understanding of Las and its pathogenesis leading to HLB has stymied development of treatments and methods to mitigate the pathogen's influence. To evaluate the influence of Las on fruit quality in both symptomatic and asymptomatic fruit, and gain further insight into the pathogenesis of the disease, a 1H NMR metabolomics investigation, complemented with physicochemical and analyte-specific analyses, was undertaken. Comparison of the juice obtained from oranges gathered from Las+ (symptomatic and asymptomatic) and Las- (healthy) trees revealed significant differences in the concentrations of sugars, amino and organic acids, limonin glucoside, and limonin. This study demonstrates differing metabolic profiles in the juice of oranges from Las+ and Las- and proposes how Las may be able to evade citrus defense responses.

Ultraviolet-B light treatment increases antioxidant capacity of carrot products.

BACKGROUND: The effect of ultraviolet-B (UV-B) light as a postharvest treatment to enhance the antioxidant content of carrots and fresh-cut carrot products was evaluated. Four levels of UV-B dose ranging from 1.3 to 12 kJ m⁻² were applied to whole, baby and various styles of cut carrots, and the changes in antioxidant capacity, total soluble phenolics and phenylalanine ammonia-lyase (PAL, EC 4.3.1.24) activity were measured after a 3 day incubation period at 15 °C and 45% relative humidity. RESULTS: Both cutting style and dose level were factors in determining carrot responses to UV-B treatment. Antioxidant capacity increased significantly (1.4-6.6-fold). Total soluble phenolic results correlated directly with those of antioxidant capacity (R² = 0.953), indicating that the enhancements achieved were due to an increase in phenolic content. High-performance liquid chromatography analysis revealed that 5-O-caffeoylquinic acid (5-CQA) was the primary phenolic responsible for this increase. Higher PAL activity was also observed in UV-B-treated samples, indicating that the increase in 5-CQA was a biological response to UV-B exposure. CONCLUSION: UV-B treatment has the potential to increase the nutritional value of carrots and offers an exciting opportunity to increase consumer accessibility to dietary choices that are rich in antioxidants.

Effect of UV-B light and different cutting styles on antioxidant enhancement of commercial fresh-cut carrot products.

Wounding stresses resulting from fresh-cut processing are known to enhance the antioxidant capacity (AC) of carrots by increasing the synthesis of phenolic compounds. Ultraviolet-B (UV-B) light exposure further promotes the formation of phenolic compounds. Changes in total soluble phenolics (TSP), 5-O-caffeoylquinic acid (5-CQA), total carotenoids, AC, and phenylalanine ammonia-lyase (PAL) activity of five commercial fresh-cut carrot products (baby carrots, carrot stixx, shredded carrots, crinkle cut coins, and oblong chips) were evaluated after exposure to UV-B dosage at 141.4 mJ/cm(2). Significant increases in TSP, AC and 5-CQA levels were observed for each sample following UV-B exposure. Increases in PAL activity were also observed in all carrot products, except crinkle cut coins. Total carotenoids of the carrot products were unchanged by UV-B exposure. Increases in AC levels corresponded directly with increases in the area/weight ratio (exposure area) of the fresh-cut carrot products.

Limonoid content of sour orange varieties.

BACKGROUND: Modern Citrus cultivars are thought to have arisen from three parents: the pummelo, the mandarin, and citron. Taxological and genetic data support that sweet and sour oranges share a common parentage. However, as their name suggests, the organoleptic properties of the fruit from these two families is distinctly different. Analysis of the limonoid content of sour orange varieties has been limited. RESULTS: Juice samples prepared from a selection of sour orange cultivars were evaluated for their limonoid A-ring lactone, aglycone, and glucoside contents. Limonoate A-ring lactone concentrations ranged from 11.1 to 44. 9 mg L⁻¹, whereas nomilinoate A-ring lactone levels were found not to exceed 1.2 mg L⁻¹. Total limonoid aglycone and total limonoid glucoside concentrations varied from 2.4 to 18.4 mg L⁻¹ and from 149.0 to 612.3 mg L⁻¹, respectively. Limonoid glucoside profiling by liquid chromatography-mass spectrometry suggest that the sour oranges are distinctly different from sweet oranges and other citrus species. CONCLUSION: Limonoid aglycone and A-ring contents across sweet and sour oranges are similar, whereas limonoid glucoside profiles are distinctly different. Juice prepared from Citrus maderaspatana had the highest limonoid concentrations among the samples tested and could potentially be used for the isolation of limonoid A-ring lactones and glucosides.

Elevation, rootstock, and soil depth affect the nutritional quality of mandarin oranges.

The effects of elevation, rootstock, and soil depth on the nutritional quality of mandarin oranges from 11 groves in California were investigated by nuclear magnetic resonance (NMR) spectroscopy by quantifying 29 compounds and applying multivariate statistical data analysis. A comparison of the juice from oranges in groves with deeper soil and trifoliate rootstock versus those with shallow soil and C-35 rootstock revealed differences in the concentrations of 4-aminobutyrate, ethanol, phenylalanine, succinate, and isoleucine. A comparison of fruit from trees grown at higher versus lower elevation revealed that those at higher elevation had higher concentrations of amino acids, succinate, and 4-aminobutyrate and lower concentrations of sugars and limonin glucoside. Such differences indicate that rootstock, soil depth, and differences in elevation influence the fruit nutrient composition. This study highlights how metabolomics coupled with multivariate statistical analysis can illuminate the metabolic characteristics of citrus, thereby aiding in the determination of the grove identity and fruit quality during orange production.

PCR amplification and cloning of tyrosine decarboxylase involved in synephrine biosynthesis in Citrus.

BACKGROUND: The phenolic amine synephrine is a vascoconstrictor and bronchiectatic agent and holds promise as an aid to weight management and obesity reduction. Synephrine is structurally similar to the active ingredients of several commercial cold remedies. Some Citrus contain high concentrations of synephrine. An enzyme involved in synephrine biosynthesis, tyrosine decarboxylase (TYDC), is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that decarboxylates tyrosine to yield CO(2) and tyramine. We used PCR to screen, clone and sequence this gene from various synephrine producing and nonproducing Citrus species and varieties to determine if DNA sequence of this gene correlated with synephrine presence. RESULTS: PCR amplification and comparison of DNA sequence indicates DNA sequence differences that may cause production of truncated proteins to be produced in some nonsynephrine producing Citrus. CONCLUSION: Synephrine production may be genetically determined in part by the gene for TYDC.

Synephrine content of juice from Satsuma mandarins (Citrus unshiu Marcovitch).

Synephrine, the main protoalkaloid in Citrus species, is commonly analyzed as the active component in citrus peel-containing herbal supplements, but the edible parts of mandarins have been largely ignored. The synephrine concentration has been determined in the juices of Citrus unshiu mandarins harvested from 10 different groves located in a major growing region in California. For comparison, the physicochemical properties of the juices, including pH, conductivity, soluble solids content, and titratable acidity, were also measured. The synephrine values among 10 groves ranged from 73.3 to 158.1 mg L (-1). Repeat sampling of fruit from the 10 locations showed that the intragrove variability in synephrine concentrations ranged from 1.0 to 27.7% CV and was grove dependent. Among the physicochemical properties, titratable acidity weakly correlated with synephrine, and for one sample a low maturity index was linked to high synephrine content. The overall mean synephrine concentration of 92.8 mg L (-1) is up to 6-fold higher than values previously determined for orange juices and suggests that mandarin juice could constitute a significant dietary source of synephrine. Furthermore, the results suggest that grove location and maturity affect synephrine content.

Isolation and identification of the first C-17 limonin epimer, epilimonin.

Limonoids are a family of highly oxygenated triterpenoid secondary metabolites found in significant quantities in Citrus and reported to possess multiple health promoting properties. This is the first known report of the isolation and characterization of an epimer of limonin. The epimer, named epilimonin, was isolated by fractional crystallization from a mixture consisting mainly of limonin and epilimonin obtained as byproduct from our efforts to isolate limonin glucoside. Side-by-side comparison of the MS, IR, and (1)H and (13)C NMR data of epilimonin and limonin lead to the assignment of C-17 as the site of epimerization. An earlier study on the bioavailability of limonin glucoside in humans had indicated that limonin glucoside was metabolized to give limonin and a second limonin metabolite. Results from analyzing epilimonin by the same chromatographic conditions used for the bioavailability study suggest that the second limonin metabolite was epilimonin.

Colorimetric method for the estimation of total limonoid aglycones and glucoside contents in citrus juices.

A method for estimating the total limonoid aglycone and glucoside concentrations in Citrus samples in terms of limonin and limonin glucoside equivalents is presented. The method consists of extraction followed by colorimetric quantification. The colorimetric quantification was based on the formation of red to orange colored derivatives resulting from the treatment of limonin, limonin glucoside, or a fruit extract with 4-(dimethylamino)benzaldehyde (DMAB) in the presence of perchloric and acetic acids. Absorbance maxima for the limonin and limonin glucoside derivatives were found to be 470 and 503 nm, respectively. The influence of DMAB concentration, reaction time, and solvent composition on color development and sensitivity were investigated and optimal assay conditions established. With a microplate format under these conditions, the limits of detection and quantification were determined to be 0.25 and 0.50 microg/mL for limonin and 0.50 and 1.0 microg/mL for limonin glucoside.

Evaluation of the antioxidant capacity of limonin, nomilin, and limonin glucoside.

The antioxidant capacity (AOC) of three representative citrus limonoids, limonin, nomilin, and limonin glucoside, was examined by the oxygen radical absorbance capacity (ORAC), Trolox equivalent antioxidant capacity (TEAC), beta-carotene-linoleic acid bleaching, and 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging assays. Pure compounds and proper negative (cinnamic acid) and positive (2,6-di-tert-butyl-4-methylphenol (BHT) and ascorbic acid) controls were used to remove any ambiguity in interpreting results. In all cases, limonin and nomilin gave results equivalent to those of cinnamic acid, indicating that they do not possess any inherent AOC and should not be considered antioxidants. Similar results were observed for limonin glucoside, with the exception of an anomalous result obtained from the beta-carotene-linoleic acid bleaching assay. Limonin glucoside was deemed not to be an antioxidant on the basis of the three unequivocal assays.