Sulforaphane-enriched extracts from glucoraphanin-rich broccoli exert antimicrobial activity against gut pathogens in vitro and innovative cooking methods increase in vivo intestinal delivery of sulforaphane.

PURPOSE: Studies on broccoli (Brassica oleracea var. italica) indicate beneficial effects against a range of chronic diseases, commonly attributed to their bioactive phytochemicals. Sulforaphane, the bioactive form of glucoraphanin, is formed by the action of the indigenous enzyme myrosinase. This study explored the role that digestion and cooking practices play in bioactivity and bioavailability, especially the rarely considered dose delivered to the colon. METHODS: The antimicrobial activity of sulforaphane extracts from raw, cooked broccoli and cooked broccoli plus mustard seeds (as a source myrosinase) was assessed. The persistence of broccoli phytochemicals in the upper gastrointestinal tract was analysed in the ileal fluid of 11 ileostomates fed, in a cross-over design, broccoli soup prepared with and without mustard seeds. RESULTS: The raw broccoli had no antimicrobial activity, except against Bacillus cereus, but cooked broccoli (with and without mustard seeds) showed considerable antimicrobial activity against various tested pathogens. The recovery of sulforaphane in ileal fluids post soup consumption was < 1% but the addition of mustard seeds increased colon-available sulforaphane sixfold. However, when sulforaphane was extracted from the ileal fluid with the highest sulforaphane content and tested against Escherichia coli K12, no inhibitory effects were observed. Analysis of glucosinolates composition in ileal fluids revealed noticeable inter-individual differences, with six "responding" participants showing increases in glucosinolates after broccoli soup consumption. CONCLUSIONS: Sulforaphane-rich broccoli extracts caused potent antimicrobial effects in vitro, and the consumption of sulforaphane-enriched broccoli soup may inhibit bacterial growth in the stomach and upper small intestine, but not in the terminal ileum or the colon.

Impact of Conventional and Integrated Management Systems on the Water-Soluble Vitamin Content in Potatoes, Field Beans, and Cereals.

The reduction of the environmental footprint of crop production without compromising crop yield and their nutritional value is a key goal for improving the sustainability of agriculture. In 2009, the Balruddery Farm Platform was established at The James Hutton Institute as a long-term experimental platform for cross-disciplinary research of crops using two agricultural ecosystems. Crops representative of UK agriculture were grown under conventional and integrated management systems and analyzed for their water-soluble vitamin content. Integrated management, when compared with the conventional system, had only minor effects on water-soluble vitamin content, where significantly higher differences were seen for the conventional management practice on the levels of thiamine in field beans (p < 0.01), Spring barley (p < 0.05), and Winter wheat (p < 0.05), and for nicotinic acid in Spring barley (p < 0.05). However, for all crops, variety and year differences were of greater importance. These results indicate that the integrated management system described in this study does not significantly affect the water-soluble vitamin content of the crops analyzed here.

Novel colon-available triterpenoids identified in raspberry fruits exhibit antigenotoxic activities in vitro.

SCOPE: Ileostomy studies provide a unique insight into digestion of food, allowing identification of physiologically relevant dietary phytochemicals and their metabolites important to gut health. We previously reported the consistent increase of components in ileal fluids of ileostomates after consumption of raspberries with use of nontargeted LC-MSn techniques and data deconvolution software highlighting two major unknown components (m/z 355 and 679). METHODS AND RESULTS: In-depth LC-MSn analyses suggested that the ileal m/z 355 components were p-coumaroyl glucarates. These compounds have not been identified previously and were confirmed in raspberry extracts after partial purification. The major ileal component with m/z 679 was a glycoside with an aglycone of m/z 517 and was present as two peaks in extracts of whole puree, unseeded puree, and isolated seeds. These components were purified using Sephadex LH20 and C18 SPE units and identified as major, novel raspberry triterpenoid glycosides. This triterpenoid-enriched fraction (100 nM) protected against H2 O2 -induced DNA damage in both colon cancer and normal cell lines and altered expression of cytoprotective genes. CONCLUSION: The presence of these novel raspberry triterpenoid components in ileal fluids indicates that they would be colon-available in vivo, so confirmation of their anticancer bioactivities is of key physiological relevance.

Nontargeted LC-MSn Profiling of Compounds in Ileal Fluids That Decrease after Raspberry Intake Identifies Consistent Alterations in Bile Acid Composition.

Ileostomy studies provide a unique insight into the digestion of foods, allowing identification of physiologically relevant dietary phytochemicals and their metabolites that are important to gut health. We previously reported an increase of components, including novel triterpenoids, in ileal fluids of 11 ileostomates following consumption of raspberries using nontargeted LC-MSn techniques in combination with data deconvolution software. The current study focused on components that consistently decreased postsupplementation. After data deconvolution, 32 components were identified that met exclusion parameters of m/z signals and which decreased significantly in ileal fluids from eight of 11 participants post-raspberry supplementation. Two-thirds of these components were identified putatively from their MS properties. Consistent decreases were observed in components that possibly reflected "washing out" of presupplementation intake of common foods/drinks including (poly)phenol metabolites. Metabolites associated with fat metabolism such as hydroxylated fatty acids and cholate-type bile acids were specifically reduced. However, more directed re-examination of the data revealed that although some cholates were consistently reduced, the more polar glyco- and tauro-linked bile acid derivatives increased consistently, by as much as 100-fold over presupplementation levels. The possible reasons for these substantial alterations in bile acid composition in ileal fluids in response to raspberry intake are discussed.

Impacts on the metabolome of down-regulating polyphenol oxidase in potato tubers.

Tubers of potato (Solanum tuberosum L. cv. Estima) genetically modified to reduce polyphenol oxidase (PPO) activity and enzymatic discolouration were assessed for changes in the metabolome using Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography (GC)-MS. Metabolome changes induced over a 48 hour (h) period by tuber wounding (sliced transverse sections) were also assessed using two PPO antisense lines (asPPO) and a wild-type (WT) control. Data were analysed using Principal Components Analysis and Analysis of Variance to assess differences between genotypes and temporal changes post-tuber wounding (by slicing). The levels of 15 metabolites (out of a total of 134 that were detected) differed between the WT and asPPO lines in mature tubers at harvest. A considerably higher number (63) of these metabolites changed significantly over a 48 h period following tuber wounding. For individual metabolites the magnitude of the differences between the WT and asPPO lines at harvest were small compared with the impacts of tuber wounding on metabolite levels. Some of the observed metabolite changes are explicable in terms of pathways known to be affected by wound responses. Whilst some statistically significant interactions (11 metabolites) were observed between line and time after wounding, very few profiles were consistent when comparing the WT with both asPPO lines, and the underlying metabolites appeared to be random in terms of the pathways they occupy. Overall, mechanical damage to tubers has a considerably greater impact on the metabolite profile than any potential unintended effects resulting from the down-regulation of PPO gene expression.

Tracking (Poly)phenol components from raspberries in ileal fluid.

The (poly)phenols in ileal fluid after ingestion of raspberries were analyzed by targeted and nontargeted LC-MS(n) approaches. Targeted approaches identified major anthocyanin and ellagitannin components at varying recoveries and with considerable interindividual variation. Nontargeted LC-MS(n) analysis using an orbitrap mass spectrometer gave exact mass MS data which were sifted using a software program to select peaks that changed significantly after supplementation. This method confirmed the recovery of the targeted components but also identified novel raspberry-specific metabolites. Some components (including ellagitannin and previously unidentified proanthocyanidin derivatives) may have arisen from raspberry seeds that survived intact in ileal samples. Other components include potential breakdown products of anthocyanins, unidentified components, and phenolic metabolites formed either in the gut epithelia or after absorption into the circulatory system and efflux back into the gut lumen. The possible physiological roles of the ileal metabolites in the large bowel are discussed.

Day length dependent restructuring of the leaf transcriptome and metabolome in potato genotypes with contrasting tuberization phenotypes.

Recent advances have defined some of the components of photoperiodic signalling that lead to tuberization in potato including orthologues of FLOWERING LOCUS T (StSP6A) and CYCLING DOF FACTOR (StCDF1). The aim of the current study is to investigate the molecular basis of permissive tuber initiation under long days in Solanum tuberosum Neo-Tuberosum by comparative analysis with an obligate short-day S. tuberosum ssp. Andigena accession. We show that the Neo-Tuberosum accession, but not the Andigena, contains alleles that encode StCDF1 proteins modified in the C-terminal region, likely to evade long day inhibition of StSP6A expression. We also identify an allele of StSP6A from the Neo-Tuberosum accession, absent in the Andigena, which is expressed under long days. Other leaf transcripts and metabolites that show different abundances in tuberizing and non-tuberizing samples were identified adding detail to tuberization-associated processes. Overall, the data presented in this study highlight the subtle interplay between components of the clock-CONSTANS-StSP6A axis which collectively may interact to fine-tune the timing of tuberization.

Physiological, biochemical and molecular responses of the potato (Solanum tuberosum L.) plant to moderately elevated temperature.

Although significant work has been undertaken regarding the response of model and crop plants to heat shock during the acclimatory phase, few studies have examined the steady-state response to the mild heat stress encountered in temperate agriculture. In the present work, we therefore exposed tuberizing potato plants to mildly elevated temperatures (30/20 °C, day/night) for up to 5 weeks and compared tuber yield, physiological and biochemical responses, and leaf and tuber metabolomes and transcriptomes with plants grown under optimal conditions (22/16 °C). Growth at elevated temperature reduced tuber yield despite an increase in net foliar photosynthesis. This was associated with major shifts in leaf and tuber metabolite profiles, a significant decrease in leaf glutathione redox state and decreased starch synthesis in tubers. Furthermore, growth at elevated temperature had a profound impact on leaf and tuber transcript expression with large numbers of transcripts displaying a rhythmic oscillation at the higher growth temperature. RT-PCR revealed perturbation in the expression of circadian clock transcripts including StSP6A, previously identified as a tuberization signal. Our data indicate that potato plants grown at moderately elevated temperatures do not exhibit classic symptoms of abiotic stress but that tuber development responds via a diversity of biochemical and molecular signals.

Pectin engineering to modify product quality in potato.

Although processed potato tuber texture is an important trait that influences consumer preference, a detailed understanding of tuber textural properties at the molecular level is lacking. Previous work has identified tuber pectin methyl esterase (PME) activity as a potential factor impacting on textural properties, and the expression of a gene encoding an isoform of PME (PEST1) was associated with cooked tuber textural properties. In this study, a transgenic approach was undertaken to investigate further the impact of the PEST1 gene. Antisense and over-expressing potato lines were generated. In over-expressing lines, tuber PME activity was enhanced by up to 2.3-fold; whereas in antisense lines, PME activity was decreased by up to 62%. PME isoform analysis indicated that the PEST1 gene encoded one isoform of PME. Analysis of cell walls from tubers from the over-expressing lines indicated that the changes in PME activity resulted in a decrease in pectin methylation. Analysis of processed tuber texture demonstrated that the reduced level of pectin methylation in the over-expressing transgenic lines was associated with a firmer processed texture. Thus, there is a clear link between PME activity, pectin methylation and processed tuber textural properties.

Relationships between volatile and non-volatile metabolites and attributes of processed potato flavour.

Although the flavour of processed potatoes (Solanum tuberosum L.) is important to consumers, the blend of volatile and non-volatile metabolites that impact on flavour attributes is not well-defined. Additionally, it is important to understand how potato flavour changes during storage. In this study, quantitative descriptive analysis of potato samples by a trained taste panel was undertaken, comparing tubers from S. tuberosum group Phureja with those from S. tuberosum group Tuberosum, both at harvest and following storage. The cooked tuber volatile profile was analysed by solid phase micro extraction followed by gas chromatography-mass spectrometry analysis in sub-samples of the tubers that were assessed by taste panels. A range of non-volatile metabolites including the major umami compounds, glycoalkaloids and sugars was also measured in tuber sub-samples. Correlation and principal component analyses revealed differences between the potato cultivars and storage conditions and demonstrated associations of metabolites with the different sensory attributes.

Discerning intra-tuber differences in textural properties in cooked Solanum tuberosum group Tuberosum and group Phureja tubers.

BACKGROUND: The textural properties of potato tubers influence their acceptability and palatability and these properties differ between varieties, groups and progeny. The aim of this study was to compare the textural properties of cooked tubers of Solanum tuberosum group Phureja with those of group Tuberosum. RESULTS: To assess intra-tuber differences, the textural properties of seven cubes from defined positions along the longitudinal axis of tubers of four Tuberosum group cultivars and three Phureja group lines were tested after cooking using an amended wedge fracture method. Tuberosum group tubers gave consistently higher peak force and work done values during fracture than the Phureja group tubers. Moreover, the values for cubes 1-6 from any tuber were not significantly different and only cube 7, from the stem end, gave higher values. Therefore, the use of any of cubes 1-6 is a valid measurement of the tuber as a whole but the central cube 4 may be most conveniently located. The dry matter content of the cubes did not influence the textural properties of the cubes, which suggested that starch swelling is not the main driving force for textural differences. Total pectin methyl esterase (PME) activity was consistently higher in cubes of the Tuberosum group cultivars over the Phureja group lines. CONCLUSION: The method developed is valid and consistent for assessing textural differences within potato germplasm. The relationship between PME activity and enhanced resistance to fracture suggests that PME may modulate pectin cohesiveness, perhaps through increasing Ca(2+)-bridges, to provide greater resistance to fracture.

Flavonoid profiling and transcriptome analysis reveals new gene-metabolite correlations in tubers of Solanum tuberosum L.

Anthocyanin content of potato tubers is a trait that is attracting increasing attention as the potential nutritional benefits of this class of compound become apparent. However, our understanding of potato tuber anthocyanin accumulation is not complete. The aim of this study was to use a potato microarray to investigate gene expression patterns associated with the accumulation of purple tuber anthocyanins. The advanced potato selections, CO97216-3P/PW and CO97227-2P/PW, developed by conventional breeding procedures, produced tubers with incomplete expression of tuber flesh pigmentation. This feature permits sampling pigmented and non-pigmented tissues from the same tubers, in essence, isolating the factors responsible for pigmentation from confounding genetic, environmental, and developmental effects. An examination of the transcriptome, coupled with metabolite data from purple pigmented sectors and from non-pigmented sectors of the same tuber, was undertaken to identify these genes whose expression correlated with elevated or altered polyphenol composition. Combined with a similar study using eight other conventional cultivars and advanced selections with different pigmentation, it was possible to produce a refined list of only 27 genes that were consistently differentially expressed in purple tuber tissues compared with white. Within this list are several new candidate genes that are likely to impact on tuber anthocyanin accumulation, including a gene encoding a novel single domain MYB transcription factor.

A metabolomics study of cultivated potato (Solanum tuberosum) groups Andigena, Phureja, Stenotomum, and tuberosum using gas chromatography-mass spectrometry.

Phytochemical diversity was examined by gas chromatography-mass spectrometry in tubers of genotypes belonging to groups Andigena, Phureja, Stenotomum, and Tuberosum of the potato, Solanum tuberosum. Polar extracts (mainly amino acids, organic acids, sugars, and sugar alcohols) and nonpolar extracts (mainly fatty acids, fatty alcohols, and sterols) were examined. There was a large range in levels of metabolites, including those such as asparagine, fructose, and glucose, that are important to tuber quality, offering considerable scope for selecting germplasm for breeding programmes. There were significant differences in the levels of many metabolites among the groups. The metabolite profiles of genotypes belonging to Phureja and Stenotomum were similar and different from those of Tuberosum and the majority of Andigena genotypes. There was some agreement with the phylogeny of the groups in that Stenotomum is believed to be the ancestor of Phureja and they are both distinct from Tuberosum. Andigena genotypes could be partially distinguished according to geographical origin, Bolivian genotypes being particularly distinct from those from Ecuador. Biosynthetic links between metabolites were explored by performing pairwise correlations of all metabolites. The significance of some expected and unexpected strong correlations between many amino acids (e.g., between isoleucine, lysine, valine, and other amino acids) and between several nonpolar metabolites (e.g., between many fatty acids) is discussed. For polar metabolites, correlation analysis gave essentially similar results irrespective of whether the whole data set, only Andigena genotypes, or only Phureja genotypes were used. In contrast, for the nonpolar metabolites, Andigena only and Phureja only data sets resulted in weaker and stronger correlations, respectively, compared to the whole data set, and may suggest differences in the biochemistry of the two groups, although the interpretation should be viewed with some caution.

Phytochemical diversity in tubers of potato cultivars and landraces using a GC-MS metabolomics approach.

Phytochemical diversity with respect to a range of polar (including amino acids, organic acids, sugars, and sugar alcohols) and nonpolar (including fatty acids, alkanols, and sterols) metabolites was examined within tubers from a total of 29 genetically diverse potato cultivars and Chilean landraces using a metabolomics approach by gas chromatography-mass spectrometry. From principal component analysis of the polar and nonpolar metabolite data there was insufficient variation to differentiate the majority of cultivars and landraces. Analysis of all polar metabolite profiles revealed separation of two cultivars (Glenna and Morag) from the other cultivars and landraces and a separate cluster of one landrace line, largely due to higher levels of sugars. Pentland Javelin was distinct in containing high levels of many amino acids. The two Solanum tuberosum group phureja cultivars (Inca Sun and Mayan Gold) were not particularly similar and were not separated from the S. tuberosum group tuberosum cultivars. Analysis of the nonpolar metabolite data revealed partial separation of two landrace lines and, on the basis of some minor fatty acids, Mayan Gold was distinct. The differences in metabolite profiles are considered in terms of the taxonomy and breeding history of the cultivars and possible influences from other factors such as developmental stage of the tuber. With a view to exploring biosynthetic links between metabolites, a pairwise correlation analysis was performed on all metabolites. The significance of high correlations between many amino acids and between several nonpolar metabolites is discussed.

Metabolomic approach to identifying bioactive compounds in berries: advances toward fruit nutritional enhancement.

Plant polyphenolics continue to be the focus of attention with regard to their putative impact on human health. An increasing and ageing human population means that the focus on nutrition and nutritional enhancement or optimisation of our foodstuffs is paramount. Using the raspberry as a model, we have shown how modern metabolic profiling approaches can be used to identify the changes in the level of beneficial polyphenolics in fruit breeding segregating populations and how the level of these components is determined by genetic and/or environmental control. Interestingly, the vitamin C content appeared to be significantly influenced by environment (growth conditions) whilst the content of the polyphenols such as cyanidin, pelargonidin and quercetin glycosides appeared much more tightly regulated, suggesting a rigorous genetic control. Preliminary metabolic profiling showed that the fruit polyphenolic profiles divided into two gross groups segregating on the basis of relative levels of cyanidin-3-sophoroside and cyanidin-3-rutinoside, compounds implicated as conferring human health benefits.