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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

2023 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
FY2023 Obstacles. Methods and Application of Food Composition Laboratory (MAFCL) suffered a number of setbacks in 2023. In the fall of 2022, the building transformer failed as did two back-up generators and the building elevator. Staff moved freezers with critical samples to B307C. While the move to B307C had been planned, the timing was very premature. Temporary accommodations were made for most equipment and for the 6 mass spectrometers critical to the lab’s research. Lack of a liquid nitrogen supply for the mass spectrometers was another factor. Failure of many pieces of equipment (including several mass spectrometers) also occurred. As a result, lab research was delayed a minimum of 6 months. Climate-induced nutritional variation in oil composition of oils. In support of our efforts to determine the impact of climate change on the essential fatty acid content of vegetable oils in the food supply, we are procuring canola oil composition data from the Canadian Grains Commission. When used in conjunction with publicly available data, we will generate a complementary model to the one we developed for soybean oil composition under variable growing temperatures. Evaluation of physical methods for resistant starch and dietary fiber analysis. Complex carbohydrates like starch and dietary fiber are polymeric materials and require a different array of instrumentation for analysis than traditional small-molecule nutrients such as vitamins. In support of our projects, we have identified techniques and instrumentation we need to successfully perform carbohydrate analysis. Where it made economic sense and could serve multiple users, as is the case for size exclusion chromatography and the quadrapole- time-of-flight mass spectrometer, we invested in the instrumentation. In other cases, such as nuclear magnetic resonance spectrometry, X-ray diffraction, and terahertz Raman spectroscopy, we negotiated access to the necessary instrumentation. Working in conjunction with scientists at FDA in College Park, Maryland, and Rowan University in Glassboro, New Jersey, we have developed a suite of analyses. A member of MAFCL will Chair the Dietary Fiber committee for Cereals and Grains. We are evaluating and updating the AACC methods. New methodology for the evaluation of plant phytochemicals. In collaboration with the Food Quality Lab, USDA-ARS, a rapid and simple method for optimizing controlled environment agricultural (CEA) conditions was developed to shorten the assessment cycle, save energy and increase the profits of CEA industries. This method specifies a minimum sample size of two cotyledons and uses UHPLC-HRMS for sample analysis. Image-based data normalization coupled with chemometrics-based strategies including principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were applied for the post-acquisition data analysis. This method successfully distinguished between Brassica microgreens grown under different CEA settings in a shortened cycle. This method will be beneficial for evaluation of different CEA growing practices. Food and botanical secondary metabolites database. In collaboration with Middle Tennessee State University (MTSU), a prototype web-based database has been developed. It was successfully moved from MTSU’s server to Amazon Web Services (AWS). The graphic user interface (GUI) has been improved and bugs are still being fixed. More plant and compound information has been added to the database. Molecular relative response factors for flavonoids are being added to the database for quantification. Secondary metabolites and crop management on a USDA Long-Term Agroecosystem Research (LTAR) site. Collaborating with Northern Great Plains Research Laboratory (NGPRL), one of the LTAR sites to evaluate the secondary metabolites and their relevance in agroecosystems at the soil-food-health interface, especially for the different management strategies that will influence concentrations of secondary metabolites in different parts of the crops, e.g. roots, leaves, and grain. Assisted in the non-targeted metabolomic data analysis of the first batch of wheat and corn samples from different management approaches. Dietary Biomarkers Development Consortium (DBDC). Participating in the collaborative project with Harvard, Fred Hutchinson Cancer Center, and University of California, Davis to identify food biomarkers. MAFCL will perform non-targeted food composition analysis on the feeding materials. Developed a Standard Operating Procedure (SOP) for sampling and analysis. MAFCL will receive food samples from 3 clinical sites. The first batch of food samples from UC Davis have been freeze-dried. Pig fecal metabolome. In collaboration with the Diet, Genome, and Immunology Laboratory (DGIL), changes in the pig fecal metabolome will be determined after long-term feeding of mixed fruits and vegetables, and a dextran sulfate sodium (DSS) challenge. The sample extraction has been completed and the non-targeted metabolomic study has been initiated. The data analysis is ongoing. Analysis of cranberry supplements. In collaboration with the Office of Dietary Supplements (NIH), cranberry supplements have been collected for analysis. MAFCL will analyze proanthocyanidins using a thiolysis-based ultra high-performance liquid chromatography-diode array detection-mass spectrometry method. DAPP milk study. As part of the milk grand challenge, samples were analyzed and reported using classical quantification from workstation software programs (WSPs), using proprietary lipidomics software, and now open-source metabolomics and lipidomics software platforms. All milk data have been analyzed by XCMS Online, and are now starting through MetaboAnalyst online, and MS-DIAL standalone. Approximately 60% of milk samples from the bovine dietary invention trial have been extracted and analyzed by fast LC-MS. Pulse Crop Health Initiative bean study. In collaboration with North Dakota State University, more than 400 samples were analyzed by near Infrared (NIR) spectrometry and flow injection mass spectrometry. Both methods could discriminate between samples from the 3 market classes (pinto, navy, and black) and from the 4 growing locations. Bean genotype was less impacting. Loadings from principal component analysis were used to identify ions (and compounds) that were critical for discrimination. These compounds are being used for genome wide association studies. Different thermal treatments impact the chemical compositions of tomato seeds. The total antioxidant content and the antioxidant properties of soluble-free, soluble-conjugated, and insoluble-bound phenolics are regarded as indicative of nutritional quality. Total phenolic content (TPC) and free radical scavenging activities were investigated in tomato seeds. The phenolic composition of tomato seeds was significantly altered by thermal treatment. These results indicated that thermal treatments may alter the bioactivity of tomato seeds and steaming can be an effective way to enhance their health-benefits. Identification of phenolics in tomato seeds and their activities. The soluble free, soluble conjugated, and insoluble bound phenolic compounds in tomato seeds are considered to be health promoting. Phenolics were extracted and analyzed using ultra-high-performance liquid chromatography–high-resolution mass spectrometry. Total phenolic content (TPC) and free radical scavenging activity and antiproliferative effects against the human colorectal cancer cell line (HCT-116) were examined. The results suggest a future application of tomato seeds as nutraceuticals in dietary supplements and functional foods.

1. Impact of climate change on lipids in soyeans. The nutritional value of the food we eat is not well understood. Conventional modeling approaches correlate yield with nutrition under the assumption that high yields translate to good nutrition. Scientists at the USDA-Beltsville Human Nutrition Research Center, Beltsville, Maryland, and the Pasture Systems and Watershed Management Unit, University Park, Pennsylvania, are challenging this assumption. Most Americans get the essential fatty acid a-linolenic acid (ALA) from the soybean and canola oil incorporated into the foods we eat. As temperatures during the growing season increase, the production of this essential fatty acid in the plants decreases. These researchers built a model demonstrating the reduction in ALA we can expect for soybean oil across the food system as temperatures increase, which will result in dietary insufficiency for a wide-ranging population of Americans.

2. Comparison of phytochemicals in wild and cultivated American ginseng. American ginseng (Panax quinquefolius L.) is a “cash crop” in many states and the roots are used as dietary health supplements and additives to food, beverages, and cosmetics and for the treatment of many human ailments. Limited research on the wild populations of American ginseng is available due to the scarcity. In collaboration with Middle Tennessee State University the chemical profiles of wild American ginseng were compared with its cultivated counterpart using an ultrahigh-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS)-based metabolomics approach. Both ginsenoside and non-ginsenoside profiles of wild and cultivated ginseng were well-discriminated by untargeted analysis. The results suggested that characteristic components can be used as chemical markers to differentiate between cultivated and wild America ginseng. This strategy is beneficial for the quality evaluation of America ginseng and quality control by manufacturers.

3. Steroidal glycoalkaloids contribute to anthracnose resistance in Solanum lycopersicum. Anthracnose is a widespread plant disease, caused by the fungal pathogen Colletotrichum, that affects a wide range of crops including tomato fruit. In collaboration with the Genetic Improvement for Fruits & Vegetables Laboratory, Beltsville, Maryland, the biochemistry and genetic control of anthracnose resistance in different lines of tomatoes was investigated. It was determined that a class of secondary metabolites known as steroidal glycoalkaloids (SGAs) provide anthracnose resistance and confirmed a role for corresponding genes (GAME genes) that regulate SGA biosynthesis. This work provides a foundation for new genetic tools for the management of anthracnose in tomatoes and other crops and is valuable for scientists investigating fundamental aspects of plant disease resistance.

4. Effects of different light-emitting diode illumination on bioactive compounds in mustard. Light-emitting diodes (LEDs) have been increasingly used to promote the content of bioactive compounds in commercially grown plants, particularly in controlled environment agriculture (CEA) settings. In collaboration with the Food Quality Laboratory of USDA-ARS, the effect of LED light wavelengths on the phytochemical profiles of the major secondary metabolites (i.e., anthocyanins, non-anthocyanin phenolics, and glucosinolates) of mustard “Ruby Streak” microgreens was investigated. Red and magenta light promoted the accumulation of both total and individual anthocyanin/aliphatic glucosinolates, while blue light was found to be the dominant factor in the accumulation of non-anthocyanin phenolics. The results suggest a significant and complex impact of lighting on phytochemical accumulation in microgreens and the potential of modulating plant nutritional profiles by varying wavelengths of the light source.

5. Chemical composition of cinnamon, clove, and honeysuckle. The possibility of dietary approach to enhance resistance to COVID is appealing. In collaboration with the University of Maryland, the chemical composition of commonly used botanical-based spices was evaluated for their capacity to suppressing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binding to angiotensin-converting enzyme 2 (ACE2), inhibiting ACE2, and scavenging free radicals. New compounds were discovered with the potential of reducing the risk of SARS-CoV-2 infection and COVID-19 development, as well as scavenging free radicals using in vitro models. The dietary approach has an interesting potential.

6. Extractability of bioactive curcuminoids from turmeric. Curcuminoids in turmeric have been purported to have a series of health promoting qualities. A major problem with curcuminoids is its low solubility in aqueous media, poor bioavailability, and pharmacokinetic profiles. The extractability of three curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin) from a series of solvents was investigated using high-performance liquid chromatography. The results showed that the extractability varied significantly with lipid and alcohol content. This study showed that the bioavailability and the bioefficacy of phytochemicals from various foods or dietary supplements significantly change with the mode of consumption. This provides consumers with a better knowledge of how to prepare the supplements for efficient consumption.

7. Variations in fiber content associated with the commonly applied methodologies. Analysis of fiber is currently non-specific and highly ambiguous. Analysis of total dietary fiber (TDF), insoluble dietary fiber (IDF), and soluble dietary fiber (SDF) was investigated in two foods using an automated fiber analyzer to implement two commonly used gravimetric methods (AOAC 2017.16 and AOAC 991.43). The fiber results were compared to the total monosaccharide, glycosidic linkage, and free saccharide levels in collaboration with the University of California, Davis. Results showed that for oats, both AOAC methods gave similar results while, for raw potato starch, each AOAC method gave markedly different results attributable to resistant starch. This information will be of significant interest to health care and nutrition professionals to investigate the role of dietary fiber as it relates to health.

8. Improved and simplified fatty acid analysis. Analysis of the fatty acid composition of foods has been of significant nutritional interest. Improvements to the gas chromatography (GC) separation with flame ionization detection (FID) and MS confirmation for fatty acid methyl ester (FAME) analysis were implemented and reported. Statistical treatment of the GC-FID FAME data showed the same grouping pattern by principal component analysis (PCA) of the triacylglycerol composition by LC-MS but using a simpler analysis. The modified method will simplify and speed up fatty acid analysis.

9. Lipidomics of pulses. Methods developed for infant formula and bovine milk analysis were successfully applied to the analysis of lipids in pulses. These analyses are important due to the gap in knowledge on pulse lipids. We successfully applied both lipidomic and classical identification and quantification approaches to pulses and applied them to dairy feeding trial milk samples. We demonstrated both a long, detailed method for analysis and a fast, high-throughput method for analysis. Both the detailed and the fast approaches allow quantification of triacylglycerol regioisomers and fat-soluble vitamins. We proved that the method we are using for milk samples also worked well for pulse lipids.

Review Publications
Fabian, M.L., Zhang, C., Sun, J., Price, N.P., Chen, P., Clarke, C.R., Jones, R.W., Stommel, J.R. 2023. Steroidal glycoalkaloids contribute to anthracnose resistance in solanum lycopersicum. Journal of Experimental Botany. 74(12):3700-3713.
Liu, Z., Teng, Z., Pearlstein, D.J., Chen, P., Yu, L., Zhou, B., Luo, Y., Sun, J. 2022. Effects of different light-emitting diode illumination on bioactive compounds in mustard “Ruby Streak” microgreens by ultra-high performance liquid chromatography high-resolution mass spectrometry. ACS Food Science and Technology. 2(9):1483–1494.
Li, Y., Liu, Z., Tamia, G., He, X., Sun, J., Chen, P., Lee, S., Wang, T.T., Gao, B., Xie, Z., Yu, L. 2022. Identification of soluble free, soluble conjugated and insoluble bound phenolics in tomato seeds and their radical scavenging and antiproliferative activities. Journal of Agricultural and Food Chemistry. 70(29):9039-9047.
Zhihao, L., Mengliang, Z., Chen, P., Harnly, J.M., Sun, J. 2022. Mass spectrometry based non-targeted and targeted analytical approaches in fingerprinting and metabolomics of food and agricultral research. Journal of Agriculture and Food Chemistry. 70(36):11138–11153.
Li, Y., Liu, Z., Zeng, M., El Kadiri, A., Huang, J., Kim, A., He, X., Sun, J., Chen, P., Wang, T.T., Zhang, Y., Gao, B., Xie, Z., Yu, L. 2022. Chemical compositions of clove (Syzygium aromaticum (L.) Merr. & L.) extracts and their potentials in suppressing SARS-CoV-2 spike protein-ACE2 binding, inhibiting ACE2, and scavenging free radicals. Journal of Agricultural and Food Chemistry. 70(45):14403-14413.
Li, Y., Wu, K., Liu, Z., He, X., Sun, J., Zeng, M., Lee, S., Wang, T.T., Gao, B., Xie, Z., Wei, C., Yu, L. 2022. Effect of thermal treatments on soluble-free, soluble-conjugated, and insoluble-bound phenolic components and free radical scavenging properties of tomato seeds. ACS Food Science and Technology. 2(10):1631-1638.
Gao, B., Zhu, L., Liu, Z., Li, Y., He, X., Wu, X., Pehrsson, P.R., Sun, J., Xie, Z., Slavin, M., Yu, L. 2023. Chemical composition of honeysuckle (Lonicerae japonicae) extracts and their potential in inhibiting the SARS-CoV-2 spike protein and ACE2 binding, suppressing ACE2, and scavenging radicals. Journal of Agricultural and Food Chemistry. 71(15):6133-6143.
Bacalzo, N., Couture, G., Ye, C., Castillo, J., Phillips, K., Fukagawa, N.K., Lebrilla, C. 2022. Quantitative bottom-up glycomic analysis of polysaccharides in food matrices using liquid chromatography – tandem mass spectrometry. Analytical Chemistry. 95:1008.
Huang, Y., Bruna, P., Fukagawa, N.K., Barile, D. 2022. Comprehensive oligosaccharide profiling of commercial almond milk, soy milk, and soy flour. Food Chemistry. 409:135267.
Bacalzo,.P., Couture, G., Ye, C., Luthria, D.L., Phillips, K.M., Fukagawa, N.K., Lebrilla, C.B., Tareq, F.S., Harnly, J.M., Pehrsson, P.R., McKillop, K.A. 2022. Multi-glycomic characterization of fiber from AOAC methods defines the carbohydrate structures. Journal of Agricultural and Food Chemistry. 70(45):14559-14570.