Performing Department
(N/A)
Non Technical Summary
The prevalence of cardiometabolic disease risk factors is reaching an epidemic with the highest risk among individuals with nutrient insecurity. To combat this, traditional plant breeding has begun to shift from a calorie-centric approach to improving nutrients in crops. Potatoes are an ideal crop to begin this shift due to their high consumption, affordability, accessibility, and high nutritional value. Furthermore, potatoes' gene pool provides a unique opportunity to use their novel traits in developing cultivars.The long-term goal of this research is to improve access to a healthy and sustainable source of nutrients for different socioeconomic groups while boosting economic viability for growers and stakeholders. I hypothesize that potato carbohydrates and associated phytochemicals (PCAP) provide a high-quality source of nutrients that improve gut health by supplying prebiotics to the gut microbiota. The field of potatoes' matrices in the context of gut health is understudied. We have two goals; 1) to develop a standard profile of PCAP, and 2) to determine the effect of PCAP on gut health, using a multi-omics approach.This rigorous training will provide me with a diverse skillset, setting me apart as a dynamic researcher, with a unique vision of fusion of food-omics, metagenomics, and preclinical research to better understand how crop variation and their dietary component and gut microbiome interact.With its focus on solving current challenges in human nutrition and health in the United States, this project is well aligned with the goals of AFRI NIFA Fellowship Grant Program.
Animal Health Component
40%
Research Effort Categories
Basic
30%
Applied
40%
Developmental
30%
Goals / Objectives
One of the emerging challenges of our century isnutrient insecurity, which is correlated withobesity and associated disorders such as cardiometabolicdiseases (CMDs). To combat this, research in food and agriculture is evolving fromcaloric-centric toward nutrient-centricapproaches.My goalis to understand the relationship between the complexmatrix of potatoes' carbohydrates and associated phytochemicals (PCAP)andgut health. This will guide breeders and public health providers in developing and providing nutritious crop varieties. With my unique research experience across agri-food sectors, I can establish strategic links between multi-omics approaches and breeding functional foods while addressing consumer demands.This proposal is based on three key tenets:Foods are not just isolated compounds undergoing separate digestion. Quite the opposite. Carbohydrates can sequester phytochemicals and modulate their bioavailability and absorption. In turn, phytochemicals can modulate carbohydrate metabolism, making them less digestible and decreasing the glycemic response. New omics approaches have reached the capability of allowing science to be free from a single-dimensional view of food science. This project will break this paradigm by exploring the important matrix interaction, unlike previous studies on in vitro or isolated metabolites.A diverse potato gene pool provides a unique chance to explore various matrices of phytochemicals and carbohydrates. These authentic crops' distinct chemical compositions create a unique avenue for precise food recommendations. This will open the door for utilizing different potato varieties to facilitate crucial targeted health interactions. Potatoes' bioactive (resistant carbohydrates, phenolics, etc.) have mainly been studied as isolated chemicals. Limited preclinical or clinical studies investigated whole potato matrices but are limited to one variety and overlook the variation in this crop. This study will introduce an innovative perspective by employing cutting-edge omics techniques. It not only endeavors to construct a standardized PCAP profile across diverse potato cultivars but also pushes the boundaries by delving into a profound comprehension of the metagenomic shifts.We hypothesize that potatoes' carbohydrates and associated phytochemicals (PCAP) are nutrient-rich resources that hold the potential to augment health. Moreover, this complex matrix, encompassing prebiotic elements is capable of mitigating risk factors associated with CMDs.Goal 1: Evaluate the composition of the PCAP in selected potato genotypes.Goal 1 is defined to address the question, "what is there?". Previous research has described distinctions in phytochemicals, minerals, and carbohydrates among various potato varieties. However, the impact of these studies is limited due to the lack of standardized methods, hindering direct comparison across laboratories and crops and a tendency to focus on a limited set of phytochemical classes, leading to the oversight of the broader spectrum of essential bioactive constituents within this crop. I hypothesize that the comprehensive phytochemical composition of potatoes will vary among phenotypically divergent potato varieties. To address this, the proposed study will leverage the metabolomics tools developed by the Periodic Table of Food Initiative (PTFI) toolset. Specifically, the PTFI has developed standardized analytical methods to enable the comprehensive characterization of food in a way that ensures the results are accessible and comparable worldwide. Furthermore, analysis will be performed on raw potatoes and as potatoes prepared using a range of common cooking methods. Finally, the total dietary fiber (TDF) will be characterized using an established method in Dr. Thompson's laboratory.Goal 1 - Objective 1: Characterize phytochemicals profile in selected potato varieties. High-throughput omics platforms will be utilized to profile the phytochemicals in potato tubers. We will use the established Periodic Table of Food Initiative (PTFI) methods for small molecule characterization (LC-MS) and elemental mineral analysis (ICP-MS).Goal 1 - Objective 2: Characterize the dietary fiber content in potato variety, including soluble and non-soluble polymers. Acknowledging that many aspects of carbohydrates in potatoes need to be elucidated, this proposal will focus on TDF, soluble and insoluble dietary fiber (SDF and IDF), and non-digestible oligosaccharides (NDO).Goal 2: Determine whether a whole potato diet (i.e., PCAP) will differentially alter gut health when compared to a refined carbohydrate source. Goal 2 is defined to address the question, "What do they do?". Evidence from limited studies indicates a positive impact of potatoes' metabolites, such as phenolics, on alleviating CMD risk factors and improving gut health. Furthermore, resistant carbohydrates (RCs) in potatoes exhibit prebiotic qualities due to their lower digestibility in the upper gastrointestinal tract. These RCs can engage with gut bacteria and their fermentation metabolites like short-chain fatty acids, which are known for their health-enhancing benefits. Knowledge gaps: 1) The primary focus in these studies is mainly on isolated metabolites from potatoes rather than considering the context of the whole food matrix. 2) These studies overlook the genotypic variation in potatoes, neglecting the variation of nutrient matrices. 3) Importantly, to our knowledge, there hasn't been a comprehensive metabolomics-metagenomics study that assesses the impact of different potato varieties' whole food matrices' health outcomes. To address these gaps, we will utilize a metagenomics approach to study the health outcome of 3 distinct potato varieties (microwaved with skin). We hypothesize that PCAP from distinct varieties of intricate matrices of nutrients in whole potatoes with prebiotic potential can enhance gut health and potentially alleviate CMD risk factors.Goal 2- Objective 1: Evaluate the impact of PCAP from different varieties on microbial community composition and function in the cecum.We will use metagenomics techniques to study cecum microorganisms as it contains the densest and most diverse microbiota of the GI. Metagenomics will provide a taxonomic composition of the microbiome as well as their functional contribution.Goal 2 - Objective2: Investigate how PCAP ingredients (per goal 1) alter gut barrier integrity.This objective is designed to provide information regarding the interaction of the host with the microbiome and the potential impacts of gut microbiota changes on systemic inflammation and possible downstream disease development.It is well-established that diet has an impact on intestinal morphology, including tight junction (TJ) integrity. The TJ proteins play a critical role as barriers by selectively filtering the passage of noxious luminal microbes and harmful substances. A healthy gut with maintained barrier function reduces the likelihood of exposure to microbial products, such as lipopolysaccharide (LPS). To evaluate the effect of PCAP on the intestinal barrier integrity, the following assays will be conducted:TJ mRNA expression: The mRNA expression of important apical junctional complex components (claudin, occluding, JAM-A, and E-cadherin) will be measured. These proteins play a crucial role in maintaining paracellular permeability.LBP/sCD14 Measurement:A leaky gut facilitates the translocation of LPS and other noxious stimuli, as they are continuously produced in the gut by resident bacteria. LPS binding protein (LBP) is a soluble acute phase protein that binds and presents LPS to its co-receptor CD14. Serum LBP and soluble CD14 (sCD14) are established markers of circulating LPS and are independently associated with cardiometabolic risk.
Project Methods
Goal 1 - Objective 1: Samples will be analyzed using the established PTFI methods for small molecule characterization (LC-MS) and elemental mineral analysis (ICP-MS). Briefly, freeze-dried potato tuber samples will be extracted in 80% methanol containing the PTFI internal standard reagent, and lipids and proteins will be removed using solid-phase extraction. Extracted and purified samples will be analyzed using standardized reverse phase and HILIC separation coupled with high-resolution mass spectrometry (both positive and negative mode ionization). Data will be analyzed using standardized data preprocessing methods established by the PTFI. This pipeline will include compound annotation based on searching data against a library generated by the analysis of authentic standards (1500+). This process will result in high-confidence annotation of a wide range of important phytochemicals, including phenolics, organic acids, amino acids, flavonoids, and simple sugars. In parallel, another aliquot of the freeze-dried tubers will be digested for ICP-MS analysis, where 26 elements are profiled.Goal 1 - Objective 2: we will use the standard method AOAC 2022.01, which has been set up in Dr. Thompson's laboratory to measure TDF, SDF, ISD, and NDO. Briefly, samples undergo digestion (pancreatic α-amylase and amyloglucosidase) resulting in hydrolyzing non-resistant starch to glucose. The enzyme reaction will be terminated by pH adjustment to 8.2. The IDF and SDF are collected via a vacuum filtration process. The SDF, which will permeate the first filtration, will undergo treatment with heat and 95% ethanol, resulting in precipitation followed by vacuum filtration. The IDF and SDF are dried and quantified using gravimetric method while accounting for protein and ash content corrections. The remaining NDO retrieved from the precipitation step is subjected to analysis via High-Performance Liquid Chromatography (HPLC).The TDF, IDF, and SDF will be presented as mean ± standard error of the mean (SE). Significance will be considered with a P-value of < 0.05. One-way ANOVA with Tukey's post hoc test will be used for variables measured between the variety. Student t-tests with multiple comparison testing using the Benjamini, Krieger, and Yekutieli two-stage step-up method will be used to compare these fibers in samples.Goal 2- To evaluate the effect of whole potato food matrix (PCAP) from different varieties on gut health, we will use a model of diet-induced obesity in mice, C57Bl6/J. Female mice will not be used for this initial experiment to reduce experimental confounders, but it is recognized that sex differences need to be explored in follow-up experiments. Utilizing methods established in Dr. Thompson's laboratory, a purified SUMO32 formulation will be the base for all diets. The control diet will be designed to contain 32% fat which is physiologically relevant to human high-fat diet (HFD). Three varieties (PM, YG, and RN) will be microwaved with skin, lyophilized, and added to the experimental diets The experimental diets (HFD-PM, HFD-YG, and HFD-RN) will contain SUMO32 with 32% fat, while carbohydrate and protein will be substituted with potato powder from 3 varieties. All diets will have consistent nutrient density per calorie across, with matched protein and carbohydrates. The potato powder and formula will be sent to ENVIGO (USA, Madison WI) to make the animal food. To account for cage effects, we will set up multiple cages for each study group/mice and treat the cage as a variable in the final statistical analyses. After 10 weeks of diet, mice will be euthanized, and tissues harvested. Preliminary data obtained from the Thompson and Weir labs have been used for power calculations to determine the appropriate sample size for experimental groups. With the statistical power set to 80% and alpha set to 0.01, we calculated a sample size of 10 animals per dietary group to detect inter-group differences of 25% (effect size = 1.1).Goal 2- Objective 1: Using the established protocols in Drs. Thompson and Weir's labs, briefly, cecum content will be harvested and snap-frozen in liquid nitrogen. The DNA will be extracted using the QIAamp PowerFecal DNA Kit (Qiagen, Germantown, MD, USA) following the manufacturer's protocol. Libraries will be prepared and sequenced on an Illumina NovaSeq system at the Genomics Core at the University of Colorado Denver, Anschutz Campus. Briefly, genomic DNA will be used to produce 200-bp fragments using Agencourt AMPure XP beads (Beckman Coulter). Ovation Ultralow System V2 (Tecan) will be used to prepare Illumina libraries following the manufacturer's instructions. Libraries are quality-checked for size and concentration with electrophoresis using a high-sensitivity D1000 kit on a 4200 TapeStation (Agilent). Sequencing generates a total of 30 million reads per sample.Goal 2- Objective 2:TJ mRNA expression: The mRNA expression of important apical junctional complex components (claudin, occluding, JAM-A, and E-cadherin) will be measured. These proteins play a crucial role in maintaining paracellular permeability. Total RNA will be extracted from the colon and complementary DNA (cDNA) will be generated using RT-PCR reagents.LBP/sCD14 Measurement:A leaky gut facilitates the translocation of LPS and other noxious stimuli, as they are continuously produced in the gut by resident bacteria. LPS binding protein (LBP) is a soluble acute phase protein that binds and presents LPS to its co-receptor CD14. Serum LBP and soluble CD14 (sCD14) are established markers of circulating LPS and are independently associated with cardiometabolic risk. The levels of each will be measured using an immunoassay ELISA kit from Cell Sciences (Canton, MA), at the baseline and at the harvest.All samples were normalized with 18s rRNA housekeeping gene levels with subsequent calculation of fold-change in mRNA expression (β-actin). The LPS will be standardized by sCD15 measurement (LBP/sCD14). Data will be presented as mean ± SEM and will be evaluated by one-way ANOVA or Kruskal-Wallis test depending on the distribution of the data. Tukey pairwise post-hoc testing will be used to determine whether there is a difference between the mean of all possible pairs.