京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18327号 / 農博第2052号 / 新制||農||1022(附属図書館) / 学位論文||H26||N4834(農学部図書室) / 31185 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 河田 照雄, 教授 金本 龍平, 教授 入江 一浩 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

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Our study focuses on the application of volatile metabolomics and chemometrics in plant pathology. Specifically, volatile metabolites or volatile organic compounds (VOCs) from the American chestnut tree (Castanea dentata) and its pathogenic fungus Cryphonectria parasitica have been investigated. The American chestnut was once a dominant tree species in the eastern forests of the United States. However, it was nearly devastated by the fungal pathogen C. parasitica. The loss of this tree species has significantly impacted the ecosystem. Therefore, preservation and restoration of American chestnut are crucial. Chapter one provides an overview of mass spectrometry based volatile metabolomics and their implementation in the investigation of plant pathology. The study of volatile metabolites profiles from virulent and hypovirulent strains of C. parasitica are presented in chapter two. The microbial volatile organic compounds (MVOCs) profiles were analyzed via nondestructive sampling method, headspace solid phase microextraction (HS-SPME), combined with gas chromatography (GC)-mass spectrometry (MS). The results indicate that the MVOCs profiles emitted from these two strains are significantly different. In general, compared with its hypovirulent strains, high emissions of sesquiterpenes were observed in the virulent strains. Furthermore, the study explored MVOCs differences associated with hypovirulence processes. The study found that both hypovirulence and aging can alter the virulent strains' MVOCs, and the process can be observed via their volatile metabolites. Chapter three describes the effects of aging, cultivation medium, and pH on fungal volatile metabolite profiles, all of which can change the strength of MVOCs emission and their composition. An acidic environment favors fungal bioactivity and therefore enhanced MVOCs emission. However, due to the inherently low MVOCs production from hypovirulent strains, the pH effect was less apparent in the hypovirulent isolates. The strength of MVOCs emission was highly correlated to the fungal expansion in virulent strains for the first 14 days. The overall emission from hypovirulent strains was relatively steady during the 28-day observation. Finally, the cultivation media are critical to the fungal MVOCs production. Among the tested media, cornmeal was least favorable for MVOCs production for both strains. Finally, Chapter Four presents a study of the total constitutive phenolic content estimation and volatile organic compounds identification from four species of chestnut tree leaf tissues. Folin Ciocalteu reagent assay with UV/Vis spectrophotometry was applied to estimate the total phenolic content in leaf tissues of American chestnut (Castanea dentata), Chinese chestnut (Castanea mollissima), and their backcross breeding generations (B3F2 and B3F3). The results from leaf tissue extraction in methanol/water (95:5 v/v), pH 2, and analyzed under the UV/Vis at 765 nm show that the variations among these tree species are significant (ANOVA, p < 0.05). The kinetics of phenolic compound solid-liquid extraction was elaborated using Peleg, second order, and power law models. Moreover, the analysis of VOCs collected from these species indicated that the distinction of American and Chinese chestnut could be archived via their VOCs, while the hybrids’ leaf VOCs are different from their parents’.

Occupational health surveys reported that first responders such as firefighters (FF) have some of the highest levels of cardiovascular disease in the nation from poor eating habits and lack of exercise. Three studies were established with goals to identify oxidative stress (OS) biomarkers and improve cardiovascular health for FF including: 1) a 28-day-carbohydrate restricted diet (CRD), 2) a heat-house search and clear protocol in personal protection equipment (PPE) plus curcumin supplementation, and 3) a treadmill exercise protocol in PPE with ketone salt supplementation. During those studies, stored blood plasma subsamples were evaluated for targeted antioxidants or untargeted metabolite concentration fluctuations using 1H NMR. Results from the 28-day-CRD tracked 40 metabolites consistently pre- and post-diet using 1H NMR platform. Of these metabolites’ acetone, β-hydroxybutyrate, leucine, and valine significantly upregulated while isoleucine downregulated. The plasma from the curcumin supplementation study contained 34 metabolites that were consistently identified. Lactate significantly upregulated immediate after exercise but returned to pre-exercise levels at 30 min post exercise while all the other metabolites were similar. From the ketone salt study 38 metabolites that were consistently identified from the pre- and post-exercise samples. Mean concentrations of acetone and β-hydroxybutyrate were significantly upregulated as were leucine and valine pre- and post-exercise while isoleucine downregulated. Lactate increased with ketone salt ingestion post-exercise and up to 30 min post-exercise but returned to normal at 24 h post-exercise compared to pre-exercise levels. Six other metabolites significantly differed in concentrations when compared across sampling times with no discernable impacts to OS or other notable trends. Multivariate analyses using principal components analysis (PCA), partial least squares-discriminant analysis (PLS-DA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) models were not supported using cross-validation for Q2 coefficients and permutations values at p ≤ 0.05. From these results no metabolites were shown to support transient OS suppression.

<p>  </p> <p>Shikonin is 1,4-naphthoquinones produced exclusively in Boraginaceae species. The compound and its derivatives are predominantly made in roots where they function in mediating plant-plant (allelopathic) and plant-microbe interactions. Moreover, this compound has been a target for drug development due to its strong anti-cancer properties. Our genome assembly and analysis of <em>Lithospermum erythrorhizon</em> uncovered metabolic innovation events that contributed to the evolution of the shikonin biosynthesis. This metabolic innovation also reveals the evolutionary link between shikonin biosynthesis and ubiquinone biosynthesis, one of the central metabolism functions in aerobic cellular respiration. To explore additional links between these two pathways, we used a transcriptome-based network analysis which uncovered a shikonin gene network model that predicts strong associations between primary metabolic pathway genes and known shikonin biosynthesis genes, as well as links with uncharacterized genes. <em>L. erythrorhizon</em> geranyldiphosphate (GPP) synthase (<em>LeGPPS</em>) is one of the candidates predicted by the network analysis, of which encodes a cytoplasmic enzyme shown in vitro to produce GPP. Knocking down of <em>LeGPPS</em> in <em>L. erythrorhizon </em>hairy roots (<em>LeGPPSi </em>lines) results in reduced shikonin content. This result provides functional evidence that cytoplasmic LeGPPS supplies GPP precursor to the shikonin biosynthesis. <em>LeGPPSi </em>lines also increased ubiquinone content, further supporting our hypothesis on the metabolic and evolutionary connection between shikonin and ubiquinone biosynthesis. Further RNA-seq analysis of the <em>LeGPPSi</em> line showed that downregulating <em>LeGPPS</em> significantly reduces the expression of benzenoid/phenylpropanoid genes, indicating the presence of factors that coordinately regulate the pathways providing the 4-hydroxybenzoic acid and GPP precursors to the shikonin pathway. In addition to <em>LeGPPS</em>, we also found<em> ubiquinone biosynthesis protein COQ4-like </em>gene (<em>LeCOQ4-L</em>) which provided another evolutionary link between shikonin and ubiquinone biosynthesis. The enzymatic activity of canonical COQ4 is unknown. In yeast, the protein is essential for ubiquinone biosynthesis and its metabolon formation. With the existing connections between shikonin and ubiquinone biosynthesis, if LeCOQ4 functions in the same manner as yeast COQ4, it is possible that <em>LeCOQ4-L </em>has an analogous function in shikonin biosynthesis as a structural protein for stabilizing biosynthesis metabolon. This leads us to the characterization of<em> COQ4</em> ortholog in Arabidopsis (<em>AtCOQ4</em>) to gain insight into its functional mechanism. Characterization of <em>atcoq4 </em>T-DNA mutant line showed that reduced <em>AtCOQ4</em> expression resulted in reduced ubiquinone. Further subcellular localization study revealed that AtCOQ4 and <em>LeCOQ4-L</em> localize in mitochondria without conventional transit peptide. We also performed pull-down assay to identify AtCOQ4 interactors which might be the missing enzymes that cannot be identified based on homology. 80 potential AtCOQ4 interactors were found including proteins like AtCHLM, GRIM-19, and AtSSLs. However, further study is needed to verify the protein interactions captured by pull-down assay. Taken all together, our study sheds light on the metabolic innovations that give rise to shikonin biosynthesis from ubiquinone biosynthesis and provide insight into the dynamics of the metabolic networks.</p>

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Ideally, a nutritional biomarker serves as an objective measure of the intake of a particular food or nutrient, may provide a reflection of health and disease processes, and can aid in the development of personalized nutritional recommendations. However, few food biomarkers have been validated and most have yet to be critically appraised in the literature. With the increased use of metabolomics in population-based studies, it is important to identify the sources of variability in nutritional biomarkers that may be attributed to intrinsic physiologic characteristics and extrinsic factors so that exposure-outcome associations can be examined more accurately. Additionally, circulating metabolites are associated with obesity-related changes in gut microbiome but there has been limited integration of metabolomics with microbiome in childhood obesity, and even less is known in non-white populations. This dissertation presents a series of studies that provide direct support for utility of nutritional biomarkers in population-based studies. The first study, presented in Chapter 2, contributes to the growing literature on food-based biomarkers by generating a comprehensive list of metabolites associated with a comprehensive list of all individual foods and food groups, and rated the evidence based on interstudy repeatability and study design. Chapter 3 identifies sources of variability in serum metabolite concentrations in White Europeans and South Asian pregnant women, thereby guiding appropriate statistical modeling when utilizing metabolomics in nutritional epidemiological studies. Chapter 4 provides results from a multi-omics integration analysis of serum metabolites and amplicon sequence variants of 16S ribosomal RNA genes to identify biomarkers that discriminate children with and without obesity. Collectively, the results showed that a specific food/food group may give rise to many metabolites, however in several cases, a single metabolite can be a good indicator of food intake. Dietary factors explained the highest proportion of variability in exogenous food-based biomarkers relative to non-dietary factors, whereas the contribution of non-dietary factors was either similar or lower for metabolites that can either be produced endogenously, biotransformed by gut microbiota, and/or derived from more than one food source. Most of the circulating metabolites differed by ethnicity (South Asian and White Europeans). Biomarkers with good evidence can be considered direct surrogates for food intake, however, they can be influenced by several non-dietary factors, which require appropriate consideration during the statistical analyses of the data. Finally, the results showed notable differences in serum metabolome and specific gut bacterial species, and between specific metabolites and bacterial species related to childhood obesity. Obesity related metabolic pathways such as glutamate and carnitine metabolism may provide insight into the metabolic processes related to early onset of obesity in childhood. / Dissertation / Doctor of Philosophy (Medical Science)

Plant-based proteins have gained popularity because of their high nutritional value and more sustainable alternative to animal-based proteins. Soybean and chickpea are two widely consumed plant-based proteins, whereas tempeh is a popular plant-based fermented whole food product that is rich in protein. With the increase in the development of plant-based food products, there is little research into how plant proteins affect gut microbiota characteristics and metabolites. Therefore, there is a need to understand the underlying mechanisms surrounding the consumption of these foods. The purpose of this study was to investigate the health benefits of soybean, chickpea, and their tempeh counterparts’ consumption as whole foods on the gut microbiota and metabolites. Our results showed that soybean tempeh significantly increased the abundance of beneficial probiotic bacteria such as Roseburia and Ruminiclostridium 5 in the gut microbiota of mice. Additionally, soybean tempeh and soybean significantly increased Muribaculaeceae abundance, known to increase SCFA production in the colon. Lachnospiraceae NKA136 was significantly increased in soybean tempeh, soybean, and chickpea groups which may allow these foods to be used as a way of probiotic restoration. Our results showed that all dietary supplementation groups had significantly altered metabolic profiles compared to the control group. The soybean tempeh group had higher levels of peroxide (vitamin B6), myoinositol, and tetrahydrobiopterin while the chickpea tempeh group had higher levels of metabolites such as 3 hydroxyanthranilic acid. The soybean group had higher levels of metabolites such as 3-hydroxytryptophan (Oxitriptan) whereas the chickpea protein group had higher levels of metabolites such as 3-hydroxyanthranilic acid and oxitriptan. In conclusion, our study suggests that different plant-based foods can have distinct effects on gut microbiota and metabolic profiles in mice. These findings may have implications for human health and warrant further investigation into the effects of plant protein consumption on human metabolism.

The objective of this study was to investigate the diet-induced metabolic changes that affect the brain tissue of juvenile pigs with NAFLD. This study explored the liver- brain axis and metabolic markers in the frontal cortex (FC) affected by liver damage. 18 male (M) and 20 female (F) Iberian pigs were randomly allocated to 1 of 3 high-fructose high-fat liquid diets (lard, olive oil, and coconut oil) and fed for 10 consecutive weeks. “Healthy” Iberian pigs were fed a eucaloric diet to establish a physiological baseline. Protein precipitation extraction using ultra-performance liquid chromatography-tandem quadrupole mass spectrometry was performed for primary metabolic and bile acid assays on FC samples. Univariate data were analyzed by one-way ANOVA, which included diet as the fixed effect and both replicate and pen nested in the diet as random effects. A more pronounced separation was observed in the PLS-DA between the COC and LAR/OLI diets. LC, C14:0 SM, and kynurenine, all metabolites linked to brain health, showed elevated levels in COC-fed pigs and reduced levels in animals fed OLI. Each of the three diets demonstrated heightened ratios of secondary bile acids to primary bile acids, with OLI-fed pigs showing increased TDCA:CA and TLCA:CA ratios associated with neurodegeneration (MahmoudianDehkordi et al., 2019). In conclusion, results may suggest that LAR could represent a more favorable dietary intervention for promoting brain health in pediatric NAFLD but further research is required.

Date palm (Phoenix dactylifera L.) is a fruit-bearing tree with numerous potential sustainable applications. Since ancient times, it has been considered a stable, secure, and sustainable food. This work provides comprehensive metabolic profiling of both parts, flesh and seed, of four P. dactylifera cultivars; Ajwa, Anbara, Sukkari, and Degelt Nour, which originated from two countries, Saudi Arabia, Tunisia. The analysis performed using mass spectrometry-untargeted metabolomics approaches, included a combination of LC-MS and GC-MS coupled to multivariate statistical analysis to reveal the differences in metabolite compositions among date varieties. The LC-MS seed results showed several classes of metabolites that belong to the flavonoids, phenolic acids, and amino acids derivatives, including citric acid, malic acid, lactic acid, hydroxyadipic acid, caffeic acid, which were at high concentrations in AJS followed by DNS and ARS. The LC-MS flesh analysis displayed that DNF had a high level of Isoquercitrin (flavonoid compound) and sinapic acid, and AJF was high concentrations level in hydroxyadipic acid and ascorbic acid. GC-MS concluded that seed samples of four date varieties are richer in metabolites classes than the flesh samples. The metabolites contributed to the seed metabolite compositions included several classes of amino acids, hydrocinnamic acids (caffeic, ferulic and sinapic acids), antioxidant phenolics, and long-chain fatty acids. The PCA and its loading analysis demonstrated the discriminating metabolites that were responsible for date varieties segregation, as HCA displayed the metabolic patterns and groups of metabolites that drive the clustering of the date samples, two groups of dates clustered together (AR and AJ) and (SR and DN). These clusterings are based on the similarities and differences observed in the metabolite compositions of the studied date samples that could be explained by differences in various metabolic responses and the environmental conditions, genotypes and geographical regions. This extensive date palm information would increase the potential of date fruits and seeds as low-cost sources of natural diet that may provide nutritious and bioactive components in the food and pharmaceutical fields to produce value-added products.