Progress 10/01/20 to 09/30/21
Outputs
Target Audience:The target audience of this project will be food and nutritional scientists, plant biochemists, plant geneticists, corn breeders, farmers in the US, and developing countries, consumers, and the general public. The health beneficial effects of protein-derived peptides and phytochemicals are emerging topics having great potential to improve our understanding of food functions and nutrition. The combination of food science, metabolomics, and quantitative genetics will have significant impacts on the research field of food and nutritional science, biochemistry, and genetics. The project will investigate novel traits of maize grains, which can be the potential selection target of maize germplasms with health beneficial biological activities above and beyond the nutritional value. The project will also provide biochemical and genetic markers that have the potential to be used for marker-assisted breeding. Once the maize varieties with high health beneficial effects were generated, they will improve commercial values and public perception of corn-derived food products. They will also improve nutritional values and food security, especially in developing countries. Changes/Problems:We had planned to analyze a smaller set of maize inbreds due to the limited capacity of the regular GI digestion workflow to process a large number of samples. We changed the plan to analyze the entire set of inbreds in the genetic diversity panel since it is now feasible by the semi-high-throughput GI-digestion system developed in this reporting period. The larger dataset will improve the detection power and reliability of the correlation and quantitative genetics analysis, which is the key to identifying the genetic and chemical factors associated with the bioactivities. What opportunities for training and professional development has the project provided?One Ph.D. student (Rajnee Hassan, Center for Biological Chemistry, UNL) and one master student (Prabhashis Bose, Food Science & Technology Masters' Program, UNL) have joined to work on this project. They have been trained to conduct metabolite profiling and small-scale GI digestion. One postdoc (Michael Meier, Department of Agronomy and Horticulture, UNL) will participate in this project to conduct a quantitative genetic analysis from the beginning of 2022. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We will finalize metabolite profiling of the kernel samples from 2019 kernel materials and conduct a preliminary quantitative genetic analysis to identify the genetic components associated with kernel metabolite composition. The same genetic materials will be processed by the semi-high-throughput GI digestion, and the anti-inflammatory and antioxidant activities and the metabolite contents in the digested kernels will be determined. The candidate bioactive compounds will be identified by the correlation analysis between bioactivities and individual metabolite levels in genetically diverse maize genotypes. Additionally, preliminary quantitative genetic analysis will identify the associated loci or possibly candidate genes that are potentially playing a key role in accumulating bioactivities and metabolite contents at the intestine. The kernel samples from the 2021 trials will also be processed in the next reporting period.
Impacts
What was accomplished under these goals?
The overarching goal of the research team is to improve the health beneficial values of maize by enhancing the accumulation of health beneficial bioactive compounds through genetic improvement. In the proposed project, the genetic diversity of the composition of chemical compounds before and after gastrointestinal digestion and anti-inflammatory effects of corn-derived peptides and chemicals will be tested to identify the protein-derived peptides and phytochemicals responsible for the anti-inflammatory activity of corn digests in the gastrointestinal tract. The genes associated with the compositional variations of these health-beneficial peptides and chemicals will be identified by using the GWAS approach.? Impacts Experimental systems and materials to analyze the genetic diversity of the health-beneficial bioactivities of maize kernels have been established in this reporting period. The newly developed semi-high-throughput digestion system simulating human gastrointestinal (GI) digestion enables three times faster sample processing for the 300 genotypes in the maize population panel. The newly established cell culture systems are suitable for confirming the bioactivities in the cells closely related to normal human intestinal cells. Additionally, two graduate researchers and one postdoc were trained to conduct these experiments, and the kernel samples from the 2019 and 2021 field trials are ready for analysis. These accomplishments prepare us to perform chemical profiling and bioactivity assays for the GI-digested kernels of all 300 genotypes. Genetic diversity information is essential to identify genes and compounds related to the antioxidant and anti-inflammatory activities in GI-digested maize kernels by quantitative genetics approaches and regression analysis. Identification of bioactive compounds will elucidate the biochemical basis underlying the health-beneficial activities. The scientific evidence will improve the consumers' perception of corn-derived diets and their consumption. The genes responsible for the bioactivities can be genetic markers for marker-assisted breeding of maize cultivars with enhanced health benefits. This can improve the health outcomes of consumers and commercial values of dietary corn. Potential health and commercial impacts can be enormous since health benefits are the major concern of consumers today, and nearly 60 million US individuals suffer from chronic metabolic disorders. Major activities/experiments Kernels of 282 inbred maize lines from the Goodman-Buckler genetic association panel were analyzed to determine the genetic diversity in metabolite contents and anti-inflammatory activities. The kernels from the 2019 field trial were processed in the last reporting period. The metabolite contents in these samples were analyzed by gas chromatography-mass spectrometry (GC-MS) to gain the profiles of primary metabolites, including sugars, amino acids, polyamines, and polyols. The bioactivity assays require a time-consuming and labor-intensive digestion process that prevents the analysis of the kernels from a large genetic diversity panel. We have developed a semi-high throughput workflow for simulated gastrointestinal (GI) digestion. Another limitation in our regular assay system is based on the immortal intestinal cells lines (Caco2 cells); Caco2 cells often do not reflect the actual physiology of normal intestinal cells. Thus, to mitigate any potential issues associated with the Caco2 cells, we established two alternative ways; a) HT-29 cells to test the biological activity either individually or in combination with Caco2 cells in a co-culture system and b) human induced pluripotent stem cell (iPSC)-derived intestinal cells. Data collected The GC-MS chromatography data of the kernels of 278 inbred lines from the 2019 trial has been collected. We are currently analyzing to identify and quantify the metabolites in these kernels using the chromatography data. Due to the COVID-19 limitations, we did not conduct the field trial in 2020. We continued the field experiment in 2021. The kernels from the 2021 field trial have been sorted and ready for the metabolite and bioactivity assays. The newly developed simulated GIdigestion and the modified cell culture experiment workflow now enable us to process at least 96 samples within a week. Additionally, the samples collected during this experiment can be used to test multiple cellular biomarkers that may be critical for inflammation and other immunological responses. Discussion The metabolite contents data will be used to identify genetic components which influence the accumulation of individual metabolites in kernels by the quantitative genetic analysis of the relationship between the metabolite contents and genetic variation. Comparison of the data from kernels grown in 2019 and 2021 enables us to evaluate the effects of environmental factors on metabolite levels. This evaluation is essential to identify the metabolites whose levels are genetically controlled rather than affected by environmental factors. Key outcomes and other accomplishments We established the experimental procedures and materials of two-year trials in this reporting period. Newly employed semi-high-throughput GI digestion workflow allows processing three times more samples than the regular system. This system makes the bioactivity assays of all inbreds in the genetic diversity panel possible. The newly established cell line systems will be a valuable system to confirm the bioactivities of specific metabolites, which will be identified in the rest of the project period. The samples from both the 2019 and 2021 trials are ready for processing. We also hired two graduate students and one postdoc to work on this project and trained them to conduct the experiments.
Publications
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Progress 12/29/19 to 09/30/20
Outputs
Target Audience:The target audience in this reporting period were food and nutritional scientists, plant biochemists, and plant geneticists. The health beneficial effects of protein-derivedpeptides and phytochemicals are emerging topics having great potential to improve our understanding of food functions and nutrition. The results shown in this reporting periodwill have significant impacts on the researchfield of food and nutritional science, biochemistry, and genetics. The project investigated novel traits of maize grains, which can be the potential selection target of maize germplasms with health beneficial biological activities above and beyond the nutritional value. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One high school student, Rohan Tatineni, at the Lincoln Southwest Highschool was trained by a PhD student, Nathan Abshire, in the Obata laboratory for GC-MS chromatogram analysis and analyzed the GC-MS data of the raw kernels as a part of Young Nebraska Scientist High School Research program. One undergraduate student with a major in food science and technology, Emily Jundt was trained by a PhD student, Emerson Nolasco, in the Majumder laboratory, on conducting simulated gastrointestinal digestion using an high-end automated titrator (Titrando). A postdoctoral fellow, Dr. Catherine Paul was trained by Dr. Kaustav Majumder to conduct the cell culture experiments, anti-inflammatory, and antioxidant assays. One undergraduate student with a major in Agricultural and Environmental Science Communications, Jenifer Velazquez-Perfecto was trained by a PhD student, Semra Palai Delen, in the Yang laboratory, on conducting plant phenotyping and field crossing. A PhD student, Zhikai Yang was trained by Dr. Jinliang Yang to develop statistical models to analyze genomics, metabolomics and phenomics datasets. He has been awarded the Summer Institute Scholarship to attend the 2020 Online Summer Institute in Statistical Genetics (SISG). How have the results been disseminated to communities of interest?The results are still preliminary. Once the results are confirmed, these will be published in a scientific journal such as Food Chemistry. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Antioxidant and anti-inflammatory effects of maize kernels in human intestinal cells were elucidated in this reporting period. These results indicate the potential of corn diet to ameliorate adverse effects of chronic inflammation in the gastrointestinal tract. This knowledge may enhance the consumption of corn derived foods and improve the wellness of the individuals suffering from chronic inflammatory disorders. This can also improve the commercial values of food corn. Additionally, the results in this reporting period also indicated the possibility that the anti-inflammatory activity of corn depends on the compositions of proteins and chemical compounds in the kernels. Since these characteristics are largely genetically controlled, the genes responsible for the anti-inflammatory activity can be identified by analyzing the variation of anti-inflammatory activities in various maize lines in relation to their genomic sequence. The identification of the genes will lead to the generation of maize hybrids to produce kernels with high anti-inflammatory activity and enhance the health beneficial effects of the corn derived diets. Objective 1. Identification of bioactive peptides and phytochemicals in maize grains exhibiting anti-inflammatory activity in gastrointestinal tract Major activities/experiments To confirm that maize kernels exhibit antioxidant and anti-inflammatory activities and these varies between genotypes, the kernels of B73, a classical maize inbred line, and the opaque2 mutant under B73 genetic background (B73-Op), which has an altered composition of seed storage proteins with higher lysine and tryptophan contents, were cooked and processed by simulated gastrointestinal (GI) digestion. The biological effects of these GI digests were evaluated using intestinal epithelial cell culture, Caco2. For the anti-inflammatory activity assay, the Caco2 cells were first pre-incubated with the GI digests for two hours and then inflammation was induced by treatment with Interleukin (IL)-1β and incubated for 24 hours. The presence of pro-inflammatory cytokine IL-8 was measured to evaluate the significance of inflammatory reaction. The digests were also tested for the antioxidant activity by inducing oxidative stress by TNF-α treatment to the cells pre-treated with digests and measured the accumulation of superoxide by dihydroethidium staining. Additionally, metabolite profiling of raw kernels was conducted by GC-MS to characterize the difference in chemical composition between kernels of B73 and B73-Op.The metabolite contents in the digested kernels were also analyzed by GC-MS and the results are being analyzed. Data collected The rate of digestion was significantly higher for the boiled B73-Op samples compared to the B73_WT samples only during the gastric phase with no significant difference was observed during intestinal digestion. At the end of GI digestion, significantly higher amounts of peptides (molecular mass < 300Da) were produced from kernels of B73_Op compared to the wild type. The anti-inflammatory activity assay indicated that the B73-WT digest, but not the B73_Op digest significantly reduces the accumulation of pro-inflammatory cytokine IL-8 after IL-1β induction. The B73 kernel digest reduced the accumulation of IL-8 to the half of the control cells which was not treated by the kernel digests with the concentration of 500 µg/mL. This is an attainable concentration that can be obtained by dietary intake of the corn. Kernels of both B73 and B73-Op varieties exhibit significant antioxidant effects with the concentration as low as 10 µg/mL. The raw B73_Op mutant kernels contain higher amount of most of amino acids and sugars including aspartate, glutamate, asparagine, lysine, tyrosine, and fructose although the contents of carboxylic acids were comparable in these genotypes. Discussion The results clearly indicated that the GI digested B73 kernel has both antioxidant and anti-inflammatory activity in intestinal cells while the anti-inflammatory activity was not observed with B73_Op kernels. As the B73_Op has different compositions and structure of storage proteins compared to the B73, the results indicate the effects of storage proteins on the bioactivities in maize kernels. Additionally, remarkable differences in metabolite profiles of raw kernels were observed between these genotypes, suggesting the contribution of phytochemicals in the bioactivities. It should also be noted that the B73_Op showed greater protein digestibility in simulated GI digestion. This is most likely due to the specificity and accessibility of the GI proteolytic enzymes (i.e., pepsin) to the storage protein with altered composition. Additionally, the alteration of the chemical composition by GI digestion occurred differentially between the two lines. It is well-known that gastric and intestinal digestion could modulate the accessibility and absorption of bioactive compounds from the digested foods in the human GI tract. The difference in peptide production and chemical alteration in different maize varieties after GI digestion in our results suggests that digestion can modulate the accessibility and availability of the compounds with health-beneficial biological activity in maize kernels. Key outcomes and accomplishments The results indicate that corn derived diet has anti-inflammatory and antioxidant activities in the GI epithelial cells. This is a novel information which can facilitate dietary consumption of corn and potentially improve health outcomes of the individuals with chronic inflammatory disorder. The anti-inflammatory activity varies between genotypes most likely due to the difference of storage protein composition and/or phytochemical contents. These are the prerequisite of the further genetic approach to identify the genes responsible for the bioactivities from genetic variation and eventually the generation of maize hybrids to produce kernels with higher health beneficial activities. Objective 2. Identification of genes regulating the accumulation of functional compounds Major activities/experiments Kernels of more than 200 maize inbred lines from the Goodman and Buckler diversity panel were obtained from the field trial in the summer 2018 and 2019. Metabolites were extracted from all samples from the 2019 trial and ready for metabolomics analysis. The GC-MS analysis is currently ongoing. The data will be used to identify the genes responsible for the accumulation of specific metabolites in kernels.
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