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

Location: Methods and Application of Food Composition Laboratory

2022 Annual Report

Objectives
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)

Approach
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
Evaluation of literature precedent for climate-induced nutritional variation in food, develop strategy for incorporation of this variability into the FoodData Central. This literature search focused on variability observed in plant biology that should have an impact on the food system, specifically, the impact of surface temperatures on the essential fatty acids linoleic (LA) and a-linolenic (ALA). Meteorological metadata will be used to determine the fatty acid variability of these oils as a function of origin and climate. Evaluation of physical methods for starch and dietary fiber analysis. Better methods are needed to assess starch and dietary fiber in foods, and these methods will rely on polymer science techniques. Several techniques were identified that can be developed within Methods and Application of Food Composition Laboratory (MAFCL), including size-exclusion chromatography with triple detection. Using this technique polysaccharides with mass ranges up to several million Daltons can be analyzed for molecular weight and branching. High throughput method for triacylglycerol (TAG) and fat-soluble vitamin (FSV). A method has been developed and applied to numerous samples, from beans, lentils, and chickpeas to Dairy Grand Challenge milk samples using a unique chromatography separation with dual parallel mass spectrometry detection. Two mass spectrometers, used simultaneously in parallel, provide different and complementary data using electrospray ionization (ESI) with high-resolution, accurate-mass (HRAM) mass spectrometry (MS) and atmospheric pressure photoionization (APPI) MS. The method has been applied to more than 207 of 360 Dairy Grand Challenge milk samples (58%). Calibration of triacyclglycerols in milk. A two-tier set of calibration standards was developed and employed for quantification of triacylglycerols (TAGs), as well as low levels of fat-soluble vitamins (FSVs), in Dairy Grand Challenge milk extracts and pulse extracts. Two calibration sets were made that contained different regioisomers (fatty acids arranged in different orders) of short-chain TAGs like those in milk and infant formula, and also polyunsaturated normal TAGs like those in seed oils. The 130-minute higher resolution chromatographic separation has been shortened to 75 minutes to making it more useful. The shorter 75-minute run has been combined with silver-ion and multi-cycle chromatography to provide the single most effective TAG separation technique reported to date. Data were collected from ten pulse varieties using the newly developed three-dimensional liquid chromatography technique. Development of Botanical Supplements Database. With the support of the Office of Dietary Supplements (ODS) at the National Institute of Health (NIH) and in collaboration with Middle Tennessee State University, MAFCL is building a botanical supplements database for the identification of more than 60,000 secondary metabolites (phytochemical components) found in botanical supplements and plants and for the authentication of botanicals. This database will make it possible to evaluate the impact of secondary metabolites on human health. Building web-based platforms for FlavonQ and GLS-finder. The 6,000 dietary flavonoids and 200 glucosinolates are families of secondary metabolites of considerable interest for their health beneficial effects. Two programs, FlavonQ and GLS-finder, have been developed for rapid analysis of complex mass spectral data. These programs are being developed for on-line access by researchers in the nutrition field. Development of a one-class method of authentication of botanical supplements. With the support of ODS at NIH, MAFCL is developing an authentication method based only on a model for reference standard profiles, no profiles for adulterants is needed. The one-class model essentially examines the difference between the reference standards and any unknown sample at each component of the profile. Analysis of dry beans from the Pulse Crop Health Initiative (PCHI). 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 a third year and will provide data critical to the selection of varieties best suited for locations and climates. Evaluate methods for the analysis of dietary fiber using an automated fiber analyzer. Dietary fiber, classified as soluble dietary fibers (SDF) and insoluble dietary fibers (IDF), were analyzed using an Automated Ankom Fiber Analyzer and two commonly used precipitation methods. Results were compared to analysis of carbohydrate linkages performed by collaborators at the University of California, Davis. Impact of different Light-Emitting Diode Illumination on bioactive compounds in mustard greens. The impact of different light-emitting diode (LED) illumination on bioactive compounds in mustard was investigated in collaboration with the Food Quality Laboratory, USDA-ARS. “Ruby Streaks” a popular microgreen species with rich phenolic and glucosinolate content was grown under three LED light treatments and compared to the white (2700k) control. Phytochemical profiles of anthocyanins, non-anthocyanin phenolics, and glucosinolates (GLs) were analyzed. The red (660 nm) light and magenta (450 nm+ 650 nm) light promoted the accumulation of both total and individual anthocyanin/aliphatic GLs, while blue (450 nm) light was found to be the dominant factor in the accumulation of non-anthocyanin phenolics. The results suggested the significant and complex effect of lighting conditions on phytochemical accumulation in microgreens, and thus the potential of modulating nutritional profiles by varying wavelength of the light source. Identification of phenolic compounds in tomato seeds. A collaborative project with the University of Maryland identified phenolic compounds, free radical scavenging activities, along with the anti-proliferation effects against human colorectal cancer cell line (HCT-116 in tomatoes). Soluble free, soluble conjugated, and insoluble bound phenolic fractions, including indole-3-acetic acid derivatives, flavonoids and feruloyl tyramine were identified. The insoluble bound phenolic fraction was observed to have stronger free radical scavenging activities as well as a stronger inhibitory effect against HCT-116 cells compared with the soluble free and the soluble conjugated fractions. The results may provide a foundation for future application of tomato seeds as nutraceuticals in dietary supplements and functional foods. Steroidal alkaloids in anthracnose resistant and susceptible lines of tomatoes. A collaborative project with Genetic Improvement for Fruits and Vegetables Laboratory, USDA-ARS targeted analysis of steroidal alkaloids in anthracnose resistant and susceptible lines of tomatoes. Anthracnose is a widespread plant disease caused by various species of the hemi biotrophic fungal pathogen Colletotrichum. Steroidal glycoalkaloids (SGAs), nitrogenous secondary metabolites, accumulated in anthracnose resistance tomatoes. Alpha-tomatine, as well as several derivatives, were shown to accumulate at higher levels, in comparison to fruit of the susceptible tomato cultivars. Expression analysis suggests an important role for GAME31 enzymes, which catalyzes the synthesis of precursors of these metabolites, in anthracnose resistance. These findings support a role for SGAs in defense against Colletotrichum in tomato. Chemical differences between wild and cultivated American ginseng (Panax Quinquefolius L.). Examined the chemical differences between wild and cultivated American ginseng (Panax Quinquefolius L.) using untargeted ultra-high performance liquid chromatography coupled to a high-resolution mass spectrometer (UHPLC-HRMS) based metabolomics. The wild roots have greater market value and contain a higher level of active pharmacological compounds (i.e., ginsenosides). Chemometric methods were used to analyze the variance induced by the experimental factors such as wild vs. cultivated ginseng and age effect. The data matrix was further deconvoluted into the ginsenoside relevant and non-ginsenoside relevant matrices. Biomarkers will be identified to allow for a better understanding of the biochemical and biological significance occurring between American ginseng grown via traditional cultivation methods and American ginseng found in its natural habitat. Systematic review of the cruciferous vegetables and cancer prevention. Working with scientists from Diet, Genomics and Immunology Laboratory and Food and Nutrition Research of MAFCL, USDA-ARS, and National Agricultural Library on systematic review of the cruciferous vegetables and cancer prevention.

Accomplishments
1. A multi-dimensional liquid chromatography-mass spectrometry method for Infant formula lipidomics. Infant formula and milk contain highly complex and difficult to analyze fats (triacylglycerols) containing short-chain fatty acids as well as medium/long-chain fatty acids. ARS researchers developed a new method based on multi-dimensional chromatography that separated fats from infant formula more effectively than previously possible. The newly developed technology allows identification and quantification of numerous triacylglycerols, including isomers, while still allowing quantification of fat-soluble vitamins. The multi-cycle chromatography approach provides a new tool and opens new avenues for investigation using multi-dimensional chromatography.

2. Profiling of polyphenols and glucosinolates in kale and broccoli microgreens in growth chambers and windowsill by UHPLC-HRMS. Brassica vegetables contain many health-enhancing components, such as high levels of polyphenols and glucosinolates (GSLs). Levels of these components are dependent on age (enhanced in newly sprouted plants, microgreens) and environmental factors (abiotic and biotic stresses). This study compared the polyphenol and GSL composition of kale and broccoli microgreens growing under chamber and windowsill environments. Ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) and pattern recognition methods were used to identify discriminative components. The study revealed clear variations in the secondary metabolites between plants and growing conditions demonstrating that environmental factors have a significant impact on the plant secondary metabolites composition affecting their health-enhancing potential.

3. A metabolite-ratio rule-based method for differentiation of the four cinnamon species. Accurate identification of cinnamon species is difficult due to the variability of chemical composition with time and location. A method for automated metabolite profiling and differentiation of four major cinnamon species was developed using ultra-high performance liquid chromatography - high-resolution mass spectrometry (UHPLC- HRMS). The method compares the ratio of key cinnamon metabolites to critical ratios established using a stepwise classification strategy. Over 95% classification accuracy was achieved, far exceeding other metabolomic-based modeling methods. This research offers great value for the authentication of cinnamon samples and evaluation of their potential health benefits.

4. Secondary metabolite differences between pastas from single varietal common bean (Phaseolus vulgaris L.) and Durum wheat (Triticum durum). Gluten-free bean pastas utilizing bean composite flour (bean flour, tapioca starch, and xanthan gum) from three bean varieties and the controlled durum wheat pasta were analyzed by ultrahigh performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS). Secondary metabolite profiles of the pastas were compared between each variety of bean and wheat to determine the impact of bean genotype, cooking status (raw vs cooked), and processing conditions (extrusion and drying temperature) using factorial multivariate analysis of variance (MANOVA). Processing and cooking do not contribute significantly to compositional disparities. These data are important for consumers looking for wheat flour alternatives.

5. Variation of phytochemicals in leaves of seven accessions of Hibiscus sabdariffa grown under field, green roof, and high tunnel conditions. Urban farming modes are of considerable interest for the development of healthy produce. The impact genetics and environment were examined for five accessions of H. sabdariffa from the United States National Plant Germplasm System (USNPGS) grown in three agricultural production systems: field; green roof; and high tunnel. Significant differences in composition were observed between cultivars and production systems. Phenolic compounds were higher in roof samples and, total amino acids and sugars were higher in field samples. This study showed that H. sabdariffa leaves provide a good source of bioactive phenolics, free amino acids, and sugars and can be a nutritious addition to meals. These results are relevant to researchers interested in urban farming and modes to increase global food production.

6. Rapid assessment of curcuminoids and adulterants in turmeric. There is an increased commercial demand for turmeric because it contains health-promoting bioactive curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin). The demand for turmeric has resulted in adulteration with azo dyes and other chemicals, some of which have toxic effects on human health. A rapid method was developed for the 3 curcuminoids and 3 common adulterants that can detect adulterants as low as 0.002% in turmeric with 97.3-100.3% accuracy. These results are relevant to researchers in the food and dietary supplement industry for identifying adulterants in turmeric.

7. Non-Targeted and Targeted Analytical Approaches in Fingerprinting and Metabolomics of Food and Agricultural Research: A Review. Published an invited review for the Journal of Agricultural and Food Chemistry on mass spectrometry (MS)-based techniques extensively applied in food and agricultural fields. The purpose of this review is to address the advances and application of MS-based analytical strategies in non-targeted and targeted analysis. We summarize the applications of flow injection MS fingerprinting, chromatography tandem MS metabolomics, direct analysis using ambient mass spectrometry, as well as advances in MS data deconvolution software packages and databases for the metabolomic studies. Different non-targeted and targeted approaches are employed in marker compounds identification, material adulteration detection, and specific class of secondary metabolite analysis. In the newly emerging applications, the in-house developed computer tools for fast deconvolution of MS data in targeted secondary metabolites analysis are highlighted.

Review Publications
Liu, Z., Shi, J., Wan, J., Pham, Q., Zhang, Z., Sun, J., Yu, L., Luo, Y., Wang, T.T.Y., Chen, P. 2021. Profiling of polyphenols and glucosinolates in kale and broccoli microgreens grown under chamber and windowsill conditions by ultrahigh-performance liquid chromatography high-resolution mass spectrometry. ACS Food Science and Technology. 2(1):101-113. https://doi.org/10.1021/acsfoodscitech.1c00355.
Geng, P., Hooper, S., Sun, J., Chen, P., Cichy, K.A., Harnly, J.M. 2022. Contrast study on secondary metabolite profile between pastas made from three single varietal common bean (Phaseolus vulgaris L.) and durum wheat (Triticum durum). ACS Food Science and Technology. 2(5):895–904. https://doi.org/10.1021/acsfoodscitech.2c00050.
Durrazo, A., Sorkin, B.C., Lucarini, M., Gusev, P.A., Kuszak, A.J., Crawford, C., Boyd, C., Deuster, P.A., Saldanha, L., Gurley, B.J., Pehrsson, P.R., Harnly, J.M., Turrini, A., Andrews, K.W., Lindsey, A. 2022. Analytical challenges and metrological approaches to ensuring dietary supplement quality: International perspectives. Frontiers in Pharmacology. 12:1-23. https://doi.org/10.3389/fphar.2021.714434.
Byrdwell, W.C., Hari, K.K., Goldschmidt, R.J., Jakubec, P., Novakova, L. 2022. Three-dimensional liquid chromatography with parallel second dimensions and quadruple parallel mass spectrometry for infant formula. Journal of Chromatography. 1661:462682. https://doi.org/10.1016/j.chroma.2021.462682.
Fukagawa, N.K., Mckillop, K.A., Pehrsson, P.R., Moshfegh, A.J., Harnly, J.M., Finley, J.W. 2021. USDA’s FoodData Central: What is it? and why is it needed today? American Journal of Clinical Nutrition. https://doi.org/10.1093/ajcn/nqab397.
Raghavendhar, K.R., Fakir, T.S., Byrdwell, W.C., Luthria, D.L. 2021. Rapid and sensitive analytical assessment of curcuminoids and common turmeric adulterants using liquid chromatography and tandem mass spectrometry. ACS Food Science and Technology. 1(11):2174-2181. https://doi.org/10.1021/acsfoodscitech.1c00356.
Luthria, D.L., Tareq, F.S., Kotha, R.R., Marupaka, R., Harnly, J.M., Arlotta, C.G., Richardson, M.L. 2021. Variation of phytochemicals in leaves of seven accessions of Hibiscus Sabdariffa grown under field Row, green roof and high tunnel conditions. ACS Food Science and Technology. 1(9):1702-1710. https://doi.org/10.1021/acsfoodscitech.1c00204.
Coates, L.C., Storms, D.H., Finley, J.W., Fukagawa, N.K., Lemay, D.G., Kalscheur, K., Kable, M.E. 2022. A low starch and high fiber diet intervention impacts the microbial community of raw bovine milk. Current Developments in Nutrition. 6(6). Article nzac086. https://doi.org/10.1093/cdn/nzac086.
Harnly, J.M., Picklo, M., Bukowski, M.R., Kalscheur, K., Magnuson, A.D., Fukagawa, N.K., Finley, J.W. 2021. Deriving information from complex data sets: Impact of forage on fatty acids in cow milk. Journal of Food Composition and Analysis. 107:104179. https://doi.org/10.1016/j.jfca.2021.104179.