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ARS Home » Northeast Area » Beltsville, Maryland (BHNRC) » Beltsville Human Nutrition Research Center » Methods and Application of Food Composition Laboratory » Research » Research Project #436105

Research Project: Advanced Technology for Rapid Comprehensive Analysis of the Chemical Components

Location: Methods and Application of Food Composition Laboratory

2021 Annual Report

Objective 1. Utilize comprehensive, non-targeted methods for classifying foods and for identifying candidate compounds that can then be quantified by specific targeted methods to determine the variance of nutritionally important food components in the western diet. (NP 107, Problem Statement 2A). Objective 2. Apply comprehensive non-targeted methods to identify, and apply specific targeted methods to quantify, nutritionally important compounds in food crops that may be impacted by genetics, environment, management, and processing (GxExMxP). (NP 107, Problem Statement 1A and NP 216, Component 5) Objective 3. In collaboration with other laboratories, utilize metabolite fingerprinting, metabolomics, and lipidomics to: A) characterize the impact of genetics, environment (including geographical location) and management on the nutritional characteristics of dry beans and soybeans; and B) evaluate the impact of bovine diet and environment on the nutritional composition (with emphasis on lipids) of milk and dairy products. (NP 107, Problem Statement 1A and 2A and NP 216, Component 5) Objective 4. Demonstrate that comprehensive non-targeted analysis of individual samples from selected national studies prior to compositing is a critical compliment to targeted data in the new USDA Food Composition Database. (NP 107, Problem Statement 2A)

New analytical technology will be adapted to the analysis of foods to help nutritionist, health practitioners, and the public to understand the link between agricultural systems, nutrition, and health. The food supply is changing rapidly with new genotypes from the farm and new processed foods in the marketplace. Every food consists of thousands of compounds, each with the potential to impact human health. Each must be identified, quantified, and added to a database. For a database to keep pace with the new foods, high throughput must be combined with even more detailed, comprehensive analyses. Rapid screening methods, based on metabolite fingerprinting, will allow classification of foods and determination of the relative variance associated with food production factors. Selected samples from each class will be subjected to metabolomic and lipidomic analysis. These methods will produce libraries of compounds that will allow metabolite fingerprinting methods to be used for rapid identification and quantification and will fill out nutrient databases. The combination of fingerprinting, metabolomic, and lipidomic methods will be used to analyze commodities and processed foods and to evaluate the nutritional qualities of crops and food products as a function of genetics, environment, management, and processing. This data is critical to establishing relationships between agricultural systems, nutrition, and Health. Ultimately, this data will be combined into a single database available to researchers and the public.

Progress Report
Developed three-dimesional-liquid chromatography method for analysis of fat-soluble vitamins (FSVs) and triacylglycerols (TAGs) in NIST Standard Reference Material (SRM) 1849 (Adult/Infant Formula). A new three-dimensional liquid chromatography method (LC3) with quadruple parallel mass spectrometry (MS4), was developed and applied to NIST standard reference material (SRM) adult/infant formula, #1849a. Showed that the combination of an extraction internal standard and an analytical internal standard allowed accurate quantification of FSVs in SRM 1849a. Showed that lipidomics software (LipidSearch) can be used instead of manual integration to quantify TAGs using high-resolution, accurate-mass (HRAM) MS. This proved that the method worked well and is ready for use on milk samples. Initial results for National Institute of Standards and Technology (NIST) SRM 1849a were included in a published manuscript. Analyzed ten types of beans using Fuzzy Chromatography with dual parallel mass spectrometry detection (2x MS). Extracted and analyzed bean samples and then applied “fuzzy chromatography” (a shortened 10-minute analysis) with dual parallel mass spectrometry. A new set of calibration standards was developed that allows quantification of fat-soluble vitamins (FSVs) and triacylglycerol (TAG) regioisomers at different concentrations. High-resolution, accurate-mass ESI-MS data have been acquired for all samples, and lipidomic data analysis has started, using LipidSearch and Compound Discoverer software. Analyzed bovine milk extracts using Fuzzy Chromatography with dual parallel mass spectrometry (2x MS). Applied “fuzzy chromatography” (a shortened 10-minute analysis) with dual parallel mass spectrometry to bovine milk samples as part of the Dairy Agriculture for People and the Planet (DAPP) Grand Challenge. A new set of calibration standards was used that allowed quantification of fat-soluble vitamins (FSVs) as well as triacylglycerol (TAG) regioisomers and short-chain TAGs at different concentrations. Profiling of Polyphenols and Glucosinolates in Kale and Broccoli Microgreens Growing under Chamber and Windowsill Conditions by ultra-highperformance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). The purpose of this study was to determine and compare the polyphenol and glucosinolates (GLS) composition of kale and broccoli microgreens growing under chamber and windowsill environment. The samples were analyzed using ultra-high-performance liquid chromatography, high-resolution mass spectrometry, and principal component analysis. Flavonol derivatives (21 in kale and 7 in broccoli) and hydroxycinnamic acid derivatives (14 in kale and 22 in broccoli) were identified. Additionally, GSLs, including 7 in kale, and 9 in broccoli, were also detected. The results demonstrated clear variations in secondary metabolism between the two growing conditions. The results suggested that the environmental factor may have a significant impact on the plant secondary metabolites composition. Building a mass spectrometry (MS) database for food and botanical materials based on metabolomic methods. More than 60,000 secondary metabolites have been reported for plants, and these numbers can be considered to apply to botanical supplements as well. Supplements may have a significant short- and/or long-term impact on human Health as a result of their interaction with the human metabolome or microbiome. It is not possible to evaluate the impact of secondary metabolites on human Health unless they can be identified in foods and botanicals and their presence quantified in the diet. Thus, nutritional epidemiology for secondary metabolites and the determination of synergism between compounds require a botanical composition database. The lab is in the process of building a web-based platform to make the database searchable. Building web-based plat forms for FlavonQ and GLS finder. Dietary flavonoids and glucosinolates are two kinds of compounds mostly researched in the nutritional area for their health beneficial effects. There are more than 6,000 known flavonoids and 200 glucosinolates reported. Both the profiles and levels of flavonoids and glucosinolates have direct impacts on their related health benefits. However, profiling flavonoids and glucosinolates pose a great challenge due to the structure diversity, the lack of commercially available standards, and the complexity of matrices. Two programs, FlavonQ and GLS-finder, have been developed successfully for fast profiling flavonoids and glucosinolates in food and botanicals. The lab is currently working on online platforms to allow researchers worldwide to use FlavonQ and GLS finder to process the UHPLC-HRMS data that uses an intuitive graphical interface that does not require installation or extensive expertise. Variation of Phytochemicals in Leaves of Seven Accessions of Hibiscus sabdariffa Grown under Field Row, Green Roof, and High Tunnel Conditions. Five accessions of H. sabdariffa from the United States National Plant Germplasm System (USNPGS), one commercial variety, and one variety were developed at the University of the District of Columbia (UDC). All five accessions were cultivated in three agricultural production systems: field rows; green roof; and high tunnel. Data from NIR spectral fingerprinting of the seven accessions were initially investigated by multivariate analysis to identify similarities and differences. Analysis of dry beans from the Pulsed Crop Health Initiative (PCHI). Methods and Application of Food Composition Laboratory (MAFCL) was one of 5 collaborators awarded funding under the USDA PCHI. Dry beans of multiple varieties were grown at multiple sites in North Dakota and Washington state. More than 2000 samples were collected over the first two years and have been analyzed by near-infrared (NIR) spectrometry and high-resolution mass spectrometry (HRMS). The study is ongoing for the third year and will provide data critical to selecting varieties best suited for locations and climates. Correlation of soil conditions with nutrient values of corn. This study considers previously published soil and corn composition data and presents an integrated consideration of the soil quality, the plant quality, yield, and correlation of the soil quality and yield. Initial results showed that soil character below 15 cm was the same for all treatments, and character above 15 cm was impacted by fertilization. Corn composition across 2 years was constant. Plant yield (1000 kernel mass) for dry manure and urea treatment exceeded that of the control. The correlation of yield with soil components was significant for total soluble carbon and nitrogen, soluble organic carbon, phosphorous, potentially mineralizable nitrogen, and beta-glucosidase. Chemical analysis and classification of black pepper (Piper nigrum L.) based on country of origin using mass spectrometric methods and chemometrics. Gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and thermal desorption direct analysis in real-time mass spectrometry (DART-MS) methods were used to analyze black pepper (Piper nigrum L.) samples. Partial least squares-discriminant analysis (PLS-DA) was used to classify black pepper samples based on their origins. Mass spectral data representations were 94.1±0.6%, 87.7±0.6%, and 97.0±0.3%, respectively for the 3 methods. The determination of geographical origins can play an important role in ensuring the food quality and authenticity, maintaining the prestige of the producers, and protect consumers Chemical composition of tomato seed flours, and their radical scavenging, anti-inflammatory and gut microbiota modulating properties. The chemical composition and total phenolic content of tomato seed flours were examined using two different cultivars. Eight compounds were identified in the tomato seed flour, including malic acid, 2-hydroxyadipic acid, salicylic acid, naringin, N-acetyl-tryptophan, quercetin-di-O-hexoside, kaempferol-di-O-hexoside, and azelaic acid. The total phenolic contents of tomato seed flour were 1.97–2.00 mg gallic acid equivalents/g. The mRNA expression of the pro-inflammatory markers, interleukin-1 beta (IL-1_), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-), were dose-dependently suppressed by tomato seed flour extracts. The extracts altered five of the eight bacterial phyla and genera evaluated. These data provide scientific support for using tomato seed flour as value-added food ingredients for consumers, farmers, and industries.

1. Impact of elevated CO2 levels on the nutritional composition of wheat. Increasing levels of CO2 in the atmosphere are affecting the nutritional composition of plants, most notably proteins and minerals. This study examined the metabolite profiles for nutrients and other small molecules of two genetic lines of wheat grown under ambient and free-air CO2 enrichment (FACE) conditions. Pattern recognition analysis of the plant compositions showed distinct differences between all 4 test groups (high and low responders at high and ambient CO2) and identified 50 compounds (amino acids, sugars, phenolic acids, flavonoids, and lipids) that varied with the CO2 level, with variation in the sugars, lipids, and amino acids having the greatest impact. These data demonstrated that genetics and environment (elevated CO2) impacted the whole wheat biochemistry, not just proteins, and minerals.

2. Consumption of kale and Daikon radish impact human urinary profiles. Brassica vegetables are rich in healthy beneficial plant secondary metabolites, including phenolics and glucosinolates, and are hypothesized to significantly affect human metabolism. These compounds are broken down into smaller molecules by the digestion process and can be detected in urine. Healthy human volunteers fed a single kale and daikon radish meal showed changes in numerous urinary compounds including acyl carnitines, uric acid, hippuric acid, phenylacetylglutamine, sulphates using a non-targeted analytical method. Using a method targeted on compounds in specific metabolic targeted method, 18 metabolites were found in urine for the first time. Several metabolites could be used as marker compounds for kale and radish consumption. The combination of non-targeted and targeted analyses can provide insight into human metabolism, and urine is an excellent non-invasive indicator of food consumption for human dietary intervention studies.

3. Identification of marker compounds for browning of fresh-cut lettuce. Enzymatic browning negatively impacts product quality and shelf-life of packaged fresh-cut lettuce. This study identified specific compounds that predict lettuce browning and can be used to identify accessions suited for commercial production and industrial breeding programs. Mass spectrometry and pattern recognition programs were used to identify seven compounds that increased or decreased with storage time. Two additional phenolic compounds were identified as potential marker compounds, whose presence immediately after cutting was negatively correlated with browning development. This study identified compounds that can predict browning-resistant accessions and assist in the development of edible and attractive foods.

4. Identification and classification of steroidal saponins in onion-type foods. Tens of thousands of secondary metabolites found in plant foods constitute the black matter of nutrition. Steroidal saponins are large, complex molecules consisting of more than 30 carbons ubiquitous in the plant kingdom but are difficult to detect. A strategy based on mass spectrometry was developed for the identification of saponins in onions. More than 152 saponins previously reported in the literature were categorized based on their structure and a method developed for systematic identification. The method successfully identified a total of 108 steroidal saponins in scallions, giant green onions, petite sweet Vidalia onions, and leeks; seventeen were reported for the first time. Researchers can now routinely identify saponins in foods for the first time and determine their role in human nutrition.

5. Impact of salinity on nutrients in foods. Accumulation of nutrients and bioactive compounds in green vegetables is significantly altered by preharvest abiotic stress imposed by extreme conditions of temperature, humidity, drought, salinity, mineral deficiencies, CO2, and UV radiation. Spinach (Spinacia oleracea L.) is consumed globally due to its diverse nutritional value. It contains considerable amounts of fiber, vitamins A, C, and K, carotenoids, folic acid, minerals, and phytochemicals. Two spinach genotypes were grown in a greenhouse under ten salinity treatments (five sodium and two potassium concentrations). Simple analytical screening methods developed in the laboratory showed a significant difference between control and high salinity plants. The detailed analysis identified significant differences in the concentration of polyphenols, phenolic acids, and soluble sugars. These results are critical to understanding the differences in metabolism for crops grown using recycled and saline waters as sustainable alternatives to freshwater to accommodate the increased demand for global food production.

Review Publications
Tareq, F.S., Kotha, R.R., Ferreira, J.F., Sandhu, D., Luthria, D.L. 2021. Influence of moderate-to-high salinity on the phytochemical profile of two salinity-tolerant spinach genotypes. ACS Food Science and Technology. 1 205–214.
Li, Y., Bahadur, R., Ahuja, J.K., Pehrsson, P.R., Harnly, J.M. 2021. Macro-and micronutrients in raw plant foods: the similarities and implication for dietary diversity. Nature Food.
Weisberg, A., Kotha, R., Kramer, C., Chang, J., Luthria, D.L., Clarke, C.R. 2020. Whole-genome sequencing of five Streptomyces isolates identifies a new pathogenic species group that causes common scab disease of potato. Molecular Plant-Microbe Interactions. 34(1):39-48.
Sun, J., Charron, C.S., Zhihao, L., Novotny Dura, J., Harrington, P.B., Ross, S.A., Seifried, H.E., Chen, P. 2020. Study on human urinary metabolic profiles after consumption of kale and daikon radish using high-resolution mass spectrometry-based non-targeted and targeted metabolomic approach. Journal of Agricultural and Food Chemistry. 68:14307-14318.