Performing Department
Food Science & Human Nutrition
Non Technical Summary
Microgreens are an emerging horticultural crop with promise for sustainably diversifying food systems and promoting human, population, environmental, and economic health. They are the edible cotyledons (seed leaves) of many vegetables, herbs, and flowers, and are known to have colorful stems and leaves, a variety of textures and flavors, and acceptability by consumers. Available research indicates the micronutrient and phytochemical contents of microgreens are higher than mature plant counterparts, and that they can be sustainably produced with minimal environmental impacts. They can be grown year-round in most indoor locales thus facilitating adaptations to climate change and population growth, while also addressing seasonal crop availability. Their high nutritional quality and potential ease of meal incorporation suggests they can promote vegetable consumption, micronutrient sufficiency, and increased phytochemical intake for the promotion of health, and to achieve specific health effects as functional foods.To date, no research has explored feasibility of daily microgreen consumption in humans, or their potential impacts on health in humans. One preclinical study has shown promising results. Thus, the overall objective of the proposed study is to determine the feasibility and tolerability of daily microgreen consumption, and to begin to characterize bioavailability and bioactivity of several metabolites associated with microgreen consumption that could positively influence human health. This is essential for preliminary data and hypothesis generation to support large-scale competitive grant proposals aimed at investigating human health outcomes associated with chronic disease development in individuals and populations. The proposed research represents a critical initial step to support a new interdisciplinary research, education, and outreach initiative with several faculty in the Departments of Food Science and Human Nutrition and Horticulture and Landscape Architecture at Colorado State University (CSU). The purpose of this initiative is to systematically advance and disseminate the state of knowledge on microgreens from an agriculture, nutrition and food science, human and population health, and sustainability perspective across food systems. Here, we will characterize the bioactive compounds in select microgreens grown at the CSU Horticulture Center and retention of these compounds post-harvest to better understand their nutritional quality and optimization of nutritional quality for human consumption. We will then conduct a human feeding trial to determine the feasibility and tolerability of daily microgreen consumption, and to gain insight into bioavailability and bioactivity of these compounds.
Animal Health Component
0%
Research Effort Categories
Basic
45%
Applied
45%
Developmental
10%
Goals / Objectives
Microgreens are an emerging horticultural crop with promise for sustainably diversifying food systems and promoting human, population, environmental, and economic health. They are the edible cotyledons (seed leaves) of many vegetables, herbs, and flowers, and are known to have colorful stems and leaves, a variety of textures and flavors, and acceptability by consumers. Available research indicates the micronutrient and phytochemical contents of microgreens are higher than mature plant counterparts, and that they can be sustainably produced with minimal environmental impacts. They can be grown year-round in most indoor locales thus facilitating adaptations to climate change and population growth, while also addressing seasonal crop availability. Their high nutritional quality and potential ease of meal incorporation suggests they can promote vegetable consumption, micronutrient sufficiency, and increased phytochemical intake for the promotion of health, and to achieve specific health effects as functional foods.To date, no research has explored feasibility of daily microgreen consumption in humans, or their potential impacts on health in humans. One preclinical study has shown promising results. Thus, the overall objective of the proposed study is to determine the feasibility and tolerability of daily microgreen consumption, and to begin to characterize bioavailability and bioactivity of several metabolites associated with microgreen consumption that could positively influence human health. This is essential for preliminary data and hypothesis generation to support large-scale competitive grant proposals aimed at investigating human health outcomes associated with chronic disease development in individuals and populations. The proposed research represents a critical initial step to support a new interdisciplinary research, education, and outreach initiative with several faculty in the Departments of Food Science and Human Nutrition and Horticulture and Landscape Architecture at Colorado State University (CSU). The purpose of this initiative is to systematically advance and disseminate the state of knowledge on microgreens from an agriculture, nutrition and food science, human and population health, and sustainability perspective across food systems. Here, we will characterize the bioactive compounds in select microgreens grown at the CSU Horticulture Center and retention of these compounds post-harvest to better understand their nutritional quality and optimization of nutritional quality for human consumption. We will then conduct a human feeding trial to determine the feasibility and tolerability of daily microgreen consumption, and to gain insight into bioavailability and bioactivity of these compounds. We propose the following specific aims:Specific Aim 1: To characterize bioactive compound types and quantities in select microgreens. Primary outcome measures: 1) analysis of metabolites by mass spectrometry (MS) to determine micronutrient and phytochemical types and quantities and their stability during post-harvest fresh storage, and 2) comparison with the compound types and quantities in mature plant counterparts. We hypothesize that bioactive compound types in select microgreens will mirror mature counterparts, but that quantities will be greater in microgreens on a per gram basis.Specific Aim 2: To gain insight into the bioavailability of microgreen phytochemicals and nutrients and their bioactivity in vivo and ex vivo. Primary outcome measures: 1) untargeted and targeted analysis of microgreen bioactive compounds in plasma samples, 2) identification and quantification of gut microbial populations altered by microgreen consumption, and 3) ex vivo stimulation of cytokine secretion in immune cells. Individuals will also be categorized according to usual vegetable intake and diet quality to determine dependence of outcome measures on usual diet. We hypothesize that bioactive compounds present in select microgreens will be bioavailable and bioactive, but impacted by gut microbial populations and usual diet.
Project Methods
Microgreen Production. In coordination with faculty in the Department of Horticulture and Landscape Architecture, microgreens (red beet, arugula, and red garnet amaranth) will be grown at the CSU Horticulture Center using standard greenhouse growing methods for microgreens. These specific microgreens were selected because of their high potential to impact human health, and because there is some pre-existing knowledge on potential bioactive compounds.3, 4, 9-11Metabolomics Analysis of Microgreens. Samples will be harvested and profiled (replicates of 5) for types and quantities of bioactive compound such as micronutrients, carotenoids, flavonoids, glucosinolates, and nitrate using standard laboratory assays and MS platforms and established approaches, data processing and analysis workflow12-17. Changes in types and quantities of bioactive compounds in microgreens (replicates of 2) post-harvest will be assessed daily for 5 days to determine stability of nutritional quality during fresh storage.Clinical Study Overview. A randomized controlled crossover trial will be conducted. Recruited participants will undergo an in-person screening, where blood draw will be performed, resting seated blood pressure will be measured in triplicate, and health history, anthropometrics, and blood lipid profiles and hemoglobin A1C will be assessed to determine eligibility. Qualified subjects (initial n = 28, equal men and women) will be randomly assigned to their treatment order. Treatment periods will be 3 weeks, each separated by a 3-week washout. All procedures will be performed at baseline and 3-week visits after a 12-hour overnight fast and 24 hours after the last bout of moderate-to-vigorous physical activity, and alcohol and medication use. Subjects. Men and postmenopausal women aged 45-75 yrs will be included, as this population is inherently at risk for developing age-related chronic disease. Inclusion of a population with established chronic disease risk factors such as high blood pressure will be evaluated in future studies when more is understood about microgreens as currently proposed. Individuals with HbA1c≤6.4%, blood pressure <140/90 mmHg, total cholesterol <240 mg/dL, LDL cholesterol < 90 mg/dL, triglycerides <350 mg/dL, and a body mass index (BMI) ≥18.5-<30 kg/m2 will be included. Individuals taking blood pressure-, lipid- or glucose-lowering or hormone replacement medications, chronic diseases, >3 days/wk vigorous exercise, weight change >5% in past 3 months, history of smoking in past 12 months, heavy drinkers (>7 drinks/wk for women; >14 drinks/wk for men), or unwillingness to maintain usual diet/physical activity will be excluded.Diet, Physical Activity, and Anthropometrics. 3-day diet records will be completed using ASA2418, 19. Healthy eating index (HEI) will be calculated to determine diet quality according to the 2015-2020 Dietary Guidelines. Physical activity patterns will be assessed using a validated 7-day recall20, 21, and weight, BMI, waist and hip circumferences will be assessed.Biological Sample Collection and Processing. Biological samples will be collected at baseline and 3-week time points. Blood will be centrifuged for separation of serum and plasma, aliquoted, and kept at -80°C until analysis. PBMCs will be isolated using SepMate™-50 ml tubes (Stemcell Technologies) containing a density gradient medium (Lymphoprep™, Stemcell Technologies) per the manufacturers' instructions. Stool samples will be stored at -20°C until analysis.Treatments. In random order, study participants will receive 2 cups (150 g)/day of microgreens (red beet, arugula, or red garnet amaranth) for a 3-week period. This is equivalent to a standard serving of raw leafy greens. Due to their short shelf-life, microgreen distribution will occur immediately after harvest. Frequency of distribution will be determined according to loss of nutritional quality determined in Specific Aim 1, but minimally every 5 days. Participants will be counseled on proper storage, microgreen culinary uses and how to incorporate them into meals, and to consume them fresh. Compliance will be determined using a daily treatment log. Interviews will be performed following study completion to gain insight into issues related to taste fatigue, and perceived barriers and benefits to daily consumption of microgreens. Immune Cell Cytokine Secretion. We will evaluate innate immune responses in isolated immune cells following ex vivo exposure to a pro-inflammatory stimulus. This is important to understanding their bioactivity, impact on immune health, and prevention of chronic inflammation implicated in numerous chronic diseases.22, 23 Peripheral blood mononuclear cells (2 x 106/mL) will be stimulated with 10 µg/mL lipopolysaccharide (LPS) in flat-bottomed 96-well plates, supernatants will be harvested 4 hrs after incubation and kept at -80°C until analysis. Inflammatory cytokines (e.g. IL-6, TNF-α) will be measured in triplicate (Luminex).23, 24Gut Microbiota and Gastrointestinal (GI) Health. The gut microbiota has emerged as a key regulator of human health. Thus, the ability to modulate gut microbes can alter downstream pro-inflammatory pathways, microbial metabolite concentrations, and reduce or exacerbate chronic disease risk.25, 26 In addition, many plant-based beneficial compounds are poorly processed and absorbed by the human digestive system, and bioavailability and bioactivity of these compounds relies heavily on metabolism by the gut microbiota.27 Thus, we will use our previously published methods15, 28, 29 to monitor changes in microbiota communities and correlate them with metabolites in human biofluids. A validated GI health questionnaire will be administered at each time point to assess tolerability of serving-level consumption of microgreens.Metabolomics Analysis of Biological Samples. Untargeted and targeted (e.g. beta-carotene, vitamins/minerals, phenolics, glucosinolates nitrate/nitrite) analysis of plasma samples will be performed using a combination of MS techniques and laboratory assays described above.Sample Size and Statistical Analysis. The sample size required to identify a mean difference of 30% in cytokine secretion from LPS-stimulated PBMCs with α = 0.05 and β = 80% in a crossover design is 28 (assuming 20% attrition). For microbial data, multivariate statistical analyses will be conducted using the adonis function of the vegan package30 in R (a nonparametric method analogous to ANOVA) to determine differences in bacterial communities across treatment groups. P-values will be based on 999 permutations. Bray-Curtis distances generated as output from mothur31 will be used as input for Principle Coordinate Analysis. Specific pairwise tests will be performed to detect treatment differences and a Linear Discriminate Analysis-Effect Size model will be performed to determine which bacterial OTUs. Bacterial diversity indices and relative abundance of phyla will be compared using nonparametric Mann-Whitney/Kruskal-Wallis one-way ANOVA by ranks test using R 3.1.1. Using SAS V9.4, data will be measured on a continuous scale and evaluated for normality prior to performing a linear regression analysis with repeated measures over time. Intervention and time will be included as fixed effects with an interaction term between the variables. Potential confounders (age, sex, and BMI) will be included to adjust for their effects. Post-hoc testing with false discovery correction will be used to determine microbial populations and metabolites that differ at the later sampling point between groups. All outcomes will be evaluated for the total cohort, as well as according to HEI and usual vegetable intake (meeting daily intake recommendation based on dietary guidelines). Statistical significance will be set at P<0.05.