Progress 05/01/24 to 04/30/25
Outputs
Target Audience:The target audience was successfully engaged through various channels. Posters werepresented at an international conference, while oral presentations were delivered at a departmental seminar andconference. Additionally, research findings were published in peer-reviewed journals. Changes/Problems:One of the significant challenges encountered was the need to repeat experiments multiple times due to difficulties in maintaining microbial strains free from contamination. Ensuring pure cultures was a considerable challenge, as distinguishing them from plate cultures was not always possible. Additionally, the optimization of techniques and protocols for probiotic characterization was another critical hurdle. This process was extensive, requiring significant time and effort to refine methodologies and ensure accurate results. What opportunities for training and professional development has the project provided?- Graduate student was trained in microbial DNA isolation and molecular biology techniques. - Postdoctoral researchers were trained in experimental design, data interpretation, presentation at the conference levels and departmental seminars, scientific proposal writing, and report writing. How have the results been disseminated to communities of interest?- Project outcomes were published in the peer-reviewed journals. - Posters were presented at the international conference. - Oral presentations were delivered at the conference and departmental seminar level. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Microbial endophytic populations were isolated from the muscadine cultivars. These isolates were further subjected to molecular and functional characterization.The nine distinct bacterial species were selected for characterization and subsequently deposited in the NCBI database with corresponding accession numbers. Phylogenetic analysis of these isolates revealed that the dominant phylum was Bacillota, which included the strains Staphylococcus aureus, Staphylococcus warneri, Bacillus tropicus, Paenibacillus cineris, Calidifontibacillus erzurumensis, and Bacillus aerius. The remaining three strains belonged to the phyla Actinomycetota (Curtobacterium oryzae and Curtobacterium citreum) and Pseudomonadota (Tatumella ptyseos). A phylogenetic tree based on partial 16S rDNA sequences was constructed to examine the relationships among these bacterial isolates. This analysis indicated that Curtobacterium oryzae AM-38 and Curtobacterium albidum AM-42 exhibited 100% homology, while Tatumella ptyseos AM-36 showed 99% phylogenetic similarity to AM-38 and AM-42. Additionally, Staphylococcus aureus AM-39 and Staphylococcus warneri AM-41 demonstrated 100% homology with each other and exhibited 91% phylogenetic similarity to Bacillus tropicus AM-40. Moreover, Calidifontibacillus erzurumensis AM-46 and Bacillus aerius AM-48 showed 92% homology with each other. The morphological and microscopic characteristics of the nine bacterial isolates revealed considerable variation in colony appearance, cell shape, and size. On LB agar plates, colony colors ranged from pale to yellow. Differential Interference Contrast imaging and membrane-stained images further illustrated distinct cellular morphologies. While AM-39 and AM-41 displayed a cocci shape, the remaining isolates exhibited rod-shaped structures with variations in rod length and thickness. Notably, AM-40 exhibited the largest colony size and widest rod shape, suggesting physiological differences among the strains. The bacterial isolates were tested for growth in LB and MRS media at three different temperatures (25°C, 30°C, and 37°C). All strains were capable of growth across all tested conditions. However, AM-36 and AM-38 exhibited slower growth rates and a prolonged lag phase compared to the other strains. This slower growth pattern was consistent across both media types and all tested temperatures. In contrast, AM-40 demonstrated the fastest growth, particularly at 25°C and 30°C. The ability of probiotic bacteria to survive in gastrointestinal conditions is a critical factor in their effectiveness.Ourin vitrostudies demonstrated that all bacterial isolates survived at 37°C, in intestinal juice and bile salts conditions, indicating their adaptability to intestinal environments.Notably, isolates from the families Bacillaceae and Paenibacillaceae exhibited exceptional resilience, tolerating pH levels below 3 and toleranceto gastric juice. These results suggest that these strains possess intrinsic mechanisms to withstand acidic stress, making them strong candidates for probiotic applications. Among all tested strains, only Paenibacillus cineris AM-44 was found to be safe for probiotic applications, as evidenced by its negative results in DNase and hemolysis tests. This finding aligns with previous studies indicating that Paenibacillus species exhibit probiotic potential and beneficial effects on intestinal microbiota in poultry. Our study also analyzed yeast communities and their potential for survival under gastrointestinal conditions. The yeast isolates exhibited more than 50% survival at pH 4 and 3, similar to bacterial isolates. However, they could not tolerate gastric juice conditions and low pH, suggesting they may not be suitable candidates for probiotic applications. A correlation analysis conducted between muscadine phytochemicals (e.g., phenolics) and endophytic microbial diversity revealed no direct relationship. However, our studies showed that extracts from high-phytochemical cultivars did not inhibit bacterial growth at ≤2 mg/mL, suggesting compatibility for synbiotic formulations (prebiotic and probiotic combinations). Overall, our findings contribute to understanding the endophytic diversity of muscadine grapes, their functional characterization, and their potential use as pro/pre-biotics to improve gut health and related disorders. In addition, the faculties, scientists, and students were provided training on microbial techniques.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Messeha, S.S., Agarwal, M., Gendy, S.G., Mehboob, S.B. and Soliman, K.F., 2024. The Anti-Obesogenic Effects of
Muscadine Grapes through Ciliary Neurotrophic Factor Receptor (Cntfr) and Histamine Receptor H1 (Hrh1) Genes in 3T3-
L1 Differentiated Mouse Cells. Nutrients, 16(12).
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Agarwal, M. and Sheikh, M.B., 2025. Isolation and Functional Characterization of Endophytic Bacteria from Muscadine
Grape Berries: A Microbial Treasure Trove. Cells, 14(5), p.369.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Sheikh MB, Rahman MA, Ahmed IM, Agarwal M, Tushar Dhanani T. 2025 Transcriptome Profiling Reveal Molecular
Evidence of Muscadine Grape cv. Noble During Berry Development.Plant and Animal Genome Conference, San Diego,
CA, USA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
M. Agarwal*, A. Kaplan, and M. B. Sheikh. Muscadine Grape Bioactives: A Natural Source of Antimicrobial Power. ARD
Research Symposium, 2024, USA.
|
Progress 05/01/23 to 04/30/24
Outputs
Target Audience:This research is aimed at developing knowledge, technology, and nutraceutical products for the benefit of people suffering from gut health and other digestion-related ailments, who stand to benefit greatly from the information generated and nutraceutical products developed to treat gut health related ailments. The project aims to assist grape growers, customers, and the business by increasing sales, market value, and income, in addition to persons with digestive health concerns. This project aims to construct cutting-edge nutraceutical and product development programs in both public and private sectors, with an emphasis on employing muscadine grape to generate novel therapeutic goods and agropharmaceuticals for prevention and treatment. Faculty and students from disciplines spanning biology, microbiology chemistry, and community groups received training/instructionsin cellular biology, microbiology, and participatory courses aimed at enhancing their proficiencies and understanding. To effectively communicate project outcomes, materials such as posters, presentations, and research articles were generated in collaboration with extension specialists to facilitate the dissemination of findings. Changes/Problems:- Wediscovered that endophytic bacterial diversity was extremely low among the numerous muscadines studied.To reduce the chance of handling error, we collected other muscadine cultivars and repeated the procedure. -Several isolated microbial strains showed significantly delayed growth under laboratory conditions, thus we had to cultivate and collect measurements for up to 40-50 hours concurrently with 2-3 hour intervals, which was a lengthy and difficult operation. Dr. Agarwal, leading this project, had to spend many nights to achieve these measurements. - To record the growth pattern of individual microbial species, several medium components and temperatures were modified and tested. What opportunities for training and professional development has the project provided?1. One undergraduate student was trained in berry sample collection and processing, phytochemical extraction andquantification,biochemical techniques. The project participants were also provided training in experimental design and a wide range of biochemical and analytical techniques, data collection and analysis, and report writing. 2. Two post-doctoral research associates were trained in berry developmental stage assessment, biochemical evaluation, sample processing, andPCR technique,bioinformatics, related molecular and cellular techniques, cell culture, isolation and characterization of bioactive molecules,instrumental analysis, and nutraceutical product development and testing. The researchers were also trained in experimentaldesign, troubleshooting, data collection, and processing, scientific proposal writing, project execution, and management, reportwriting and manuscript preparation. How have the results been disseminated to communities of interest?This project is aimed at determining the usefulness of muscadine grape to serve as pre- and pro-biotic components for preventing/remedying gut ailments as well as benefit the growers, consumers, and individuals suffering from gut ailments. Further, the outcome of this research is also intended to support the grape growers and sustain the muscadine grape industry. Accordingly, the research results were shared with the concerned groups and stakeholders through various outreach activities and other means. Some of these activities and method of communications are identified below. a. Conference presentations b. Workshops c. Social Media pages etc. d. Florida Legislature Appreciation Day e. Professional meetings What do you plan to do during the next reporting period to accomplish the goals? Complete the endophytes characterization studies including hemolytic and DNase assays Comprehend the endophytes diversity population analysis among the different muscadine cultivars along with their phytochemicals content and composition Determine the adherence and permeability properties of endophytic isolates to gut cells Determine the effect of bacterial consortia on intestinal permeability in mice
Impacts
What was accomplished under these goals?
Previously, we optimized the methodto isolate and identify endophytes from various muscadine grape genotypes to assess their potential to improve gastrointestinal health through prebiotic and probiotic properties. Bacterial endophytes isolated from seven distinct muscadine cultivars were found to belong to just one or two unique bacterial species. To avoid the possibility of mistreatment, we included an additional seven different muscadine cultivars in our study. A similar procedure was used to separate microorganisms, which were then cultured on plates specifically developed for bacterial and yeast/fungal strains.Several rounds of serial dilutions were performed to obtain a completely uncontaminated culture of the microbial isolates. Multiple colonies with distinct visual characteristics were selected and examined under a microscope to distinguish between bacteria, yeast, and fungi. The microbial isolates were subjected to DNA extraction, and their quality and purity were evaluated. The concentration of the isolated DNA varied from 20 ng/µl to 50 ng/µl for the bacterial and fungal samples, and the DNA purity ratio was determined to be optimal. We employed primers designed to specifically target the coding areas of 16SrRNA and 18SrRNA in order to determine the bacterial and fungal species, respectively. The PCR amplification was conducted, followed by purification and sequencing of the resultant DNA. After processing the sequences, we have successfully identified 13 bacterial isolates from selected muscadine cultivars. Out of these, nine isolates were determined to belong to different species. Furthermore, we have observed that gram-positive bacteria were dominant over gram-negative bacteria. The bacterial species identified were classified into the following families: Enterobacteriaceae, Staphylococcaceae, Microbacteriaceae, Bacillaceae, and Paenibacillaceae. Fungi, specifically yeast, were determined to be the dominant population. Furthermore, we have effectively identified more than 25 fungal isolates, out of which 10 were determined to be different species belonging to the families Saccharomycodaceae, Saccharomycetaceae, Debaryomycetaceae, Pichiaceae, and Trichomonascaceae. Some of these microorganisms are classified as endophytes in grapes and other plants, and their potential as probiotics has been emphasized. Furthermore, the isolates were subjected to characterization, which encompassed the analysis of their media composition and culture conditions, growth temperature, colony morphology, colony color, and cell shape. In addition, suitable microbial stocks were generated and utilized to sustain the microbial isolates. Currently, we are in the process of evaluating their susceptibility to different carbon sources and identifying their tolerance to acidic pH. In addition, we employed a culture-independent method called metagenomic analysis to identify the whole population of endophytes that are unable to be grown in laboratory conditions. To accomplish this, the raw sequences obtained from Illumina sequencing were extracted and fed into Quantitative Insight into Microbial Ecology as paired-end reads using the manifest format. Trimmomatic version 0.39 and Cutadapt version 2.10 (M. Martin, 2011) were employed to remove low-quality sequences (with an average score of 20) and adapters from the raw reads, which had a read length of 100 bp. The deblur algorithm version 1.1.0 (-deblur denoise-16S) was used to denoise the quality-filtered connected reads. The taxonomic classification was conducted using the 16S rRNA sequences obtained from the Greengenes v.13_8 database. In addition, the file was fitted using the naive Bayes method with qiime's feature-classifier. The classifier file that resulted from the process was utilized for taxonomy classification. The obtained taxonomy files were subsequently utilized to construct the OTUs table and Biom file. The BIOM data were evaluated using the Microbiome Analyst online application. The metagenomics study revealed that the bacterial families with the highest number of reads were Enterobacteriaceae, followed by Actinomycetaceae, Bacillaceae, Cellulomonadaceae, Streptomycetaceae, Paenibacillaceae, Lactobacillaceae, Planococcaceae, Caulobacteraceae, and Rickettsiaceae. Our findings from the culture-dependent technique support this observation, as the isolated bacteria were found to belong to the same families. We are currently examining the findings of the fungal community analysis. Furthermore, we conducted a comparison to see whether cultivars with a greater number of microorganisms also exhibited a higher number of phytochemicals, in order to establish the correlation between the endophytic community and phytochemicals. We conducted a comparison of the levels of catechin, epicatechin, epicatechin gallate, resveratrol, viniferin, and pterostilbene, as well as the quantity and diversity of microorganisms collected using culture-dependent procedures. This comparative investigation revealed that the concentrations of total stilbenes and total catechins differed among muscadine cultivars. Nevertheless, all muscadine cultivars contained bacterial and yeast species, exhibiting some commonalities in the specific microbial species found. In addition, our future research will also assess the correlation between the quantity and diversity of microorganisms analyzed by the culture-independent method and the phytochemical composition present in each cultivar. In addition, we conducted a study on the utilization of the muscadine grape as a prebiotic along with the combination of microbial isolates. To do so, we prepared the grape extract from fully ripe berries and examined its effect on the growth of microbial isolates. Our objective was to determine whether the grape extract had any inhibitory effects on the growth of these isolates. Our research revealed that bacterial isolates could grow upto concentrations of 1-2 mg/ml. Whereas, yeast strains were able to grow up to 4 mg/ml or more concentration of grape extract. This demonstrates that grape extract does not impede the growth of the isolates in an in vitro system at specific concentrations. To assess the effectiveness of muscadine grape berry extract on gut health, we have successfully developed a cell culture system using cell lines, A549, LNCaP, Caco-2, and 3T3L1. The IC50 doses (lowest and highest) of several muscadine grape extracts were determined to be 298 and 410 µg/mL for A549 cells, 310 and 456 µg/mL for LNCaP cells, and 301 and 465 ng/mL for Caco-2 cells, respectively. Further, the human intestinal epithelial cell line, T84 is being procured and cultivated in a 1:1 blend of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 media, enriched with heat-inactivated fetal bovine serum (FBS) as well as antibiotics and glutamine. The T84 cells will be placed in culture tubes that have been coated with extracellular matrix proteins such as collagen or fibronectin, which will facilitate the attachment and proliferation of the cells. The cells will be cultured in a controlled environment with a 5% concentration of CO2 at a temperature of 37°C to facilitate their growth and multiplication. The extent of genetic variation in the endophytes population among the muscadine grape genotypes will be also assessed both individually and in combination. Students and faculty are being provided hands-on experiential learning in various aspects of research and bioinformatics to enhance their skills and sustain their careers in science and research.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Muscadine Grape Bio-Prospecting: A Path to Adiposity Control.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Exploring Muscadine Grape Phytochemicals to Support Healthy Aging.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Muscadine Grape: A Dietary Source of Stilbenes with Potential Anti-Inflammatory Properties
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Muscadine Grape Endophytes Diversity: Exploring Their Potential as Probiotics
|
Progress 05/01/22 to 04/30/23
Outputs
Target Audience:This research is focused on developing knowledge, technology and nutraceutic products for the benefit the individuals suffering from gut health and other digestion related ailments who stand to greatly benefit from the information generated and nutraceutic products developed to remedy the gut health associated ailments. Besides the individuals with gut health ailments, the other interested groups and audience will be the grape growers, consumers, and the industry by and large who stand to greatly benefit from the project outcome through increased sales, market value and income. Besides, this research will also help establish cutting edge research and development programs in nutraceutics, and product development at the public and private sectors with focus on using muscadine grape for developing novel therapeutic products and agro-pharmaceuticals to prevent/remedy various ailments. The current research activities will help establish and strengthen nutragenomics research capability at FAMU for enhancing or altering phytochemical constituent content and composiiton of selected fruit and vegetable crops for medicinal use to meet the consumer/stakeholder/clientele needs and demands as well as help provide training ground for current and future students in cutting edge nutraceutic technologies. The project accomplishments will be communicated to the consumers, grape growers, industry, academic community, USDA, collaborators and other 1890 and 1862 land-grant Universities, and outreach personnel using vrious educational and outreach activities. The project results will be communicated to the stakeholders through workshops, presentations and demonstrations to train farmers, grape growers on varietal selection, and cultural practices for superior quality grape production to safeguard product health value and marketing. The researach data will be communicated to the stakeholders through publications in peer reviewed scientific journals and platform presentations. The summary of interesting findings will be also published as popular articles in leading newspapers and pamphlets. The literature and educational materials will be made available to potential beneficiaries, consumers, growers, and industry at CVSF/FAMU website as documents. Besides, the project will also provide hands on training to students in molecular and cellular biology, proteomics, and transcriptomics. The interested faculty and students from biology, chemistry and pharmacy, and community groups will be trained in cell biology involving nutraceutical screening and interactive courses focused on enhancing their skills and knowledge. Suitable propagation materials like posters, brochures and publications will be made with the help of extension specialist and used in dissemination of project outcome. Changes/Problems:Initially we had some difficulty in isolating and identifying the endophytes of muscadine grape. However, we were able to optimize the culturing method by modifying the culture media and growth conditins. This issue has slowed the progress requiring a detailed evaluation and modification of protocol to be able to culture the cells. What opportunities for training and professional development has the project provided?One of the pbjectives of this project is to provide handson training to undergraduate students in cell biology, nutraceutics, biochemistry, proteomics and metabolomics to build their expertise in cutting edge technologies for meeting the workforce needs of the 21st Century. In this regard the following training activities were conducted to increase students experise and competency in latest biotechnologies. a). Twe undergraduate students were trained in grape berry collection based on their maturity stage. They were also exposed to phytochemicals extraction from collected samples and their quantification for use in evalautaing thier bioactivity using cell culture technique, protein and RNA isolation and characterization techniques and other related biochemical techniques. The project participants were also provided training in experimental design and a wide range of biochemical and analytical techniques, data collection and analysis, and report writing. b). Two graduate students were provided training in experimental design, berry maturity determination, phytochemicals isolation, purification and quantification, RNA and protein extraction and other related molecular and cellular techniques, data collection and processing to obtain training in cutting edge gene and protein techniques as well as data collection, processing and analysis tools for use in their thesis research. c). Two post-doctoral research associates were trained in berry developmental stage assessment, biochemical evaluation, sample processing, RNA and protein extraction and quantification, electrophoretic techniques, Primer design, PCR technique, bioinformatics, related molecular and cellular techniques, cell culture, isolation and characterization of bioactive molecules, instrumental analysis, and nutraceutic product development and testing. The researchers were also trained in experimental design, troubleshooting, data collection and processing, scientific proposal writing, project execution and management, report writing and manuscript preparation. How have the results been disseminated to communities of interest?This project is aimed at determining the usefulness of muscadine grape to serve as pre- and pro-biotic components for preventing/remedying gut ailments as well as benefit the growers, consumers, individuals suffering from gut ailments. Further, the outcome of this research is also intended to support the grape growers and sustain the muscadine grape industry. Accordingly, the research results were shared with the concerned groups and stakeholders through various outreach activities and other means. Some of these activities and method of communications are identified below. a. Conference presentations b. Workshops c. Social Media pages etc. d. Florida Legislature Appreciation Day e. Guest lectures f. Professional meetings What do you plan to do during the next reporting period to accomplish the goals?This research was primarily designed to assess the prebiotic and probiotic effects of muscadine grape and know the identity of phytochemical components involved to remedy the gastrointestinal problems. The muscadine grape berry found to contain various phytochemical constituents with known pre- and pro-biotic effects which have been successfully discovered and characterized. To further our knowledge in this matter we have successfully isolated and characterized the microbial community and quantified their prevalence among various muscadine grape genotypes we have investigated. In addition, we have also isolated and processed microbial samples and created cDNA libraries. Further, we have developed and refined methodology to quantify the stilbenes and catechins in muscadine berry to build on- campus capacity. We are now intent to determine the identity of microbial phylum, family, and species found in grape berry among diverse muscadine genotypes employing next-generation sequencing followed by the bioinformatic analysis. The phytochemical profiles thus obtained will be connected to the endophytic microbial population of the berry. To further ensure the accuracy of DNA extractions and determine the seasonal effects on microbial population, we will collect and analyze grape berry samples from the current season. We have also established Caco-2 cell cultures to further examine the effect of muscadine grape berry extracts at the molecular and cellular levels. Grape berry extracts will be prepared from selected muscadine grape cultivars, phytochemicals content and composition determined. The berry extracts will be added to the Caco-2 cell cultures and incubated for 24 to 48 hrs. The performance of Caco-2 cells post incubation with berry extract will be monitored at the gene and protein levels to determine the effect of muscadine berry extract at the molecular and cellular levels. For this purpose, RNA will be isolated from Caco-2 cell cultures treated with and without berry extract, purity confirmed, and the transcriptome sequence obtained to ascertain the effect of berry extract treatment on cell performance. The derived information will be compared with microbiome data to correlate the relationship between berry phytochemicals content and composition, and its endophytic population. Lectures and presentation will be made to students on nutraceuticals and functional foods to enhance their knowledge and expertise in food, agriculture, and nutritional sciences. Enhanced core knowledge and skills, critical thinking, and ability of students' to become self-directed learners and become future researchers will be the expected benefits from the practical experience they will receive while working in the project and comes across a wide range of subjects and disciplines.
Impacts
What was accomplished under these goals?
As muscadine grape phytochemicals have been shown to exhibit a variety of health benefits, including anticancer, anti-inflammatory, anti-hypertension, and antioxidant properties, they are considered to be beneficial to human health. However, little is known about the prebiotic and probiotic potential of phytochemicals and endophytes in muscadine grapes for improving gastrointestinal health. Earlier, we collected grape berries from various muscadine cultivars for isolating and identifying the endophytic microbial community. We also optimized the protocol for the isolation of purified microbial isolates and extraction of microbial DNA to identify the microbial community present within the grape. As objective of this project is also to establish institutional capacity, we prepared the library in-house for microbial identification. As a first step, we analyzed the microbial richness and diversity of the selected muscadine cultivars using a low-cost technique called "Automated Ribosomal Intergenic Spacer Analysis" (ARISA). The whole microbial DNA extract was used as a template for PCR amplification with primers between 16S and 23S genes amplifying the bacterial community, and primers from ITS regions amplifying the fungal community. The amplified products were verified on the 1% agarose gel following electrophoresis and further purified and quantified using nanodrop spectrophotometer. ARISA was performed on these samples in accordance with the manufacturer's protocol. We found that muscadine cvs. African Queen and Dixieland exhibited lower number of bands as compared to cvs. Noble, Carlos, Sweet Jenny, and Florida Fry. It should be noted that the presence of a fewer bands in the selected cultivars may be also attributable to erroneous extraction of microbial DNA. Previously we had encountered some difficulty in this regard due to plant host DNA obscuring the microbial DNA. Further, we have prepared the library for Miseq sequencing to identify the microbial community. We amplified the bacterial samples using 16s primer and ITS primers for fungal samples. Individual PCR reactions were utilized to amplify a 5' Nextra adaptor sequence designed for each target. All amplicons were pooled and prepared for sequencing. The sequence data obtained from these samples will be analyzed to ascertain the phylum, genus, and species levels of the microbial community. Furthermore, to establish the relationship between the endophytic community and phytochemicals, we utilized the same batch of muscadine berries harvested from several muscadine cultivars for phytochemicals extraction and analysis. Catechins have been demonstrated to improve intestinal barrier function, decrease inflammation, and modulate gut microbiota, thus contributing to preserving gut health. Also, Catechins have been demonstrated to improve gut bacterial composition by increasing Bifidobacteria and Lactobacilli and decreasing Clostridia and Enterococci. In addition to their anti-inflammatory and gut-healing properties, stilbenes are also widely used as gut health promoting agents. Inflammatory bowel disease (IBD) and colorectal cancer are two of the diseases of the digestive tract that have been linked to persistent inflammation. Resveratrol and vineferin serve as powerful antioxidants that may prevent oxidative damage to cells in the digestive tract. Hence, we determined total stilbenes (Resveratrol, Viniferin, and Pterostillbene) and Catechins (including catechin, epicatechin, and epicatechin gallate) content and composition in berry samples to determine their role in gut health promotion using HPLC. Ten grams of whole berries were crushed using liquid nitrogen and a portion of the powdered sample then extracted with methanol, vortexed, homogenized, and sonicated. After centrifuging the extract, the supernatant was collected, filtered and analyzed by HPLC using a Prodigy column, PDA detector and a gradient mobile phase of water and acetonitrile. The resulting profiles were used to quantify various metabolites using corresponding standards. Chromatograms were monitored at wavelengths of 280 and 320 nm, and the data was then evaluated. The results showed that total catechin content of cv. Supreme was 3646.48±341.90 µg/g FW which was the highest amount found among the cultivars studied. Other cultivars, Carlos, African Queen, Florida Fry, Sweet Jenny, and Dixieland contained 1359.32±24.16, 1263.46±135.62, 823.20±29.99, 431.05±15.38, 344.76±10.70, µg/g FW respectively. This observation is in line with our findings which showed that the cv. Dixieland lacked significant amount of microbial diversity. It is important to highlight that although we saw less microbial diversity in the cv. African Queen than in other cultivars, the amount of catechins was not significantly lower. In the case of stilbenes, the highest concentration detected was 127.51+0.86 µg/g FW among the various muscadine cultivars tested. In the cv. Dixieland, no stilbenes were detected, while the cvs. Carlos, Sweet Jenny, Florida Fry, and African Queen showed stilbenes content ranging from 11.03+0.10 to 14.57+0.20 g/g FW. This agrees with our findings about microbial diversity, which indicated lower amounts of stilbenes and catechins in cv. Dixieland as well as in the microbial diversity revealed by ARISA. Additionally, a more precise understanding of next-generation sequencing will reveal whether catechin and stilbene also affects the type of microbial species present in the muscadine cultivars.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Muscadine grape as a potential prebiotic and probiotic source to promote gut health
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Identification and characterization of molecular and cellular components to augument sugar metabolism for increasing
sugar content of muscadine grape
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Efficacy of muscadine grape phytochemicals to modulate prostate cancer progression
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Synergistic action of stilbenes has potent cytotoxic activity resveratrol alone
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Exploiting grape phytochemicals to promote healthy ageing
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Identifying and characterizing parthenocarpic gene in muscadine grape
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Towards developing non-leathry muscadine table grape:identifying and characterizing gene associated with leathery skin
in muscadine grape
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Development of value added functional foods from muscadine grapes
|
Progress 05/01/21 to 04/30/22
Outputs
Target Audience:The focus of this research is to develop knowledge, technology and nutraceutic products for the benefit of gut health victims suffering from leaky gut and other age-related gut ailments who stand to greatly benefit from the information generated and nutraceutic products developed to remedy the gut associated ailments. Besides the gut health victims, the other interested groups and audience will be the grape growers, consumers, and the industry by and large who stand to greatly benefit from the project outcome through increased sales, market value and income. This project will also help establish cutting edge nutraceutics research and development programs at the public and private sectors with focus on using muscadine grape for developing novel nutraceutic products and agro-pharmaceuticals to prevent/remedy various ailments. The proposed research activities will help establish and strengthen nutrigenomics research capability at FAMU for enhancing or altering phytochemical constituent content of fruit and vegetable crops for medicinal use to meet the consumer/stakeholder/clientele needs and demands as well as help provide training ground for current and future students in cutting edge technologies. The project accomplishments will be communicated to the consumers, grape growers, industry, academic community, USDA, collaborators and other 1890 and 1862 land-grant Universities, and outreach personnel using diverse educational and outreach activities. The research outcome will be communicated to the stakeholders through field demonstrations and workshops to train farmers, grape growers on varietal selection, application of appropriate cultural practices for high quality grape production to safeguard health value and marketing strategies. The results obtained in the experimental studies will be published in peer reviewed scientific journals. The summary of interesting findings will be also published as popular articles in leading newspapers. The literature and educational materials will be made available to potential beneficiaries, consumers, growers, and industry at CVSF/FAMU website as documents, provide hands on training to students in molecular and cellular biology, proteomics, and transcriptomics. The interested faculty and students from biology, chemistry and pharmacy, and community groups will be trained in cell biology involving nutraceutical screening and interactive courses focused on enhancing their skills and knowledge. Suitable propagation materials like posters, brochures and publications will be made with the help of extension specialist and used in dissemination of project outcome. Changes/Problems:The Research Associate who was hired to conduct the project research left the university for a faculty position requiring finding another qualified individual. Because of the required expertise to conduct this specialized research and lack of availability of suitable candidates it was difficult to find and recruit a qualified individual who is experienced in this area and capable of tackling the experiments proposed in the project. Fortunately, a person with appropriate expertise was identified and recruited into the project. This individual is currently onboard and conducting the required experiments. In addition, the pandemic and prescribed guidelines also limited options for in person training of undergraduate and graduate students in the lab. Hence, it was not possible to provide in person training in various sample collection and processing protocols, analytical techniques, instrumental analysis and data collection and analysis protocols using laboratory facilities. Further, social distancing, density limitation and access to the lab was limited which curtailed training activities. However, we were able to provide appropriate training in various laboratory procedures and experimental protocols, literature review, experimental design, data collection and processing protocols, etc., virtually to enhance student knowledge and skill sets. The project personnel also successfully collaborated with the scientists from the FAMU College of Pharmacy and worked on optimizing the cell culture techniques to execute the prescribed experiments in a timely manner as well as provide student experiential learning to accomplish the project tasks for meeting project objectives. Presently we are following the proposed experimental workplan and intend to complete the project tasks in a timely manner. We believe with the new collaborative arrangements and support from other researchers we will be able to execute the project experiments to successfully complete the project assignments to meet the objects. What opportunities for training and professional development has the project provided?Because of the pandemic, limited options were available for in person training of students. However, as the guidelines were relaxed towards the later part of the year limited access was allowed for in person training of students. Some of these activities are described: 1. One undergraduate student was trained in berry sample collection and processing, phytochemicals extraction and quantification, adipose cell culturing methods, protein and RNA isolation and characterization techniques and other related biochemical techniques. The project participants were also provided training in experimental design and a wide range of biochemical and analytical techniques, data collection and analysis, and report writing. 2. Two graduate students were provided training in experimental design, berry maturity determination, phytochemicals isolation, purification and quantification, RNA and protein extraction and other related molecular and cellular techniques, data collection and processing to obtain training in cutting edge gene and protein techniques as well as data collection, processing and analysis tools for use in their thesis research. 3. Two post-doctoral research associates were trained in berry developmental stage assessment, phytochemicals extraction, biochemical evaluation, sample processing, RNA and protein extraction and quantification, electrophoretic techniques, Primer design, PCR technique, bioinformatics, other related molecular and cellular techniques, cell culture, isolation and characterization of bioactive molecules, instrumental analysis, and nutraceutic product development and testing. The researchers were also trained in experimental design, troubleshooting, data collection and processing, scientific proposal writing, project execution and management, report writing and manuscript preparation. How have the results been disseminated to communities of interest?The main goal of this research is to determine the usefulness of muscadine grape phytochemicals as pro- and pre-biotic components to promote gut health, for controlling age related ailments including leaky gut syndrome. Thus, the beneficiaries of this research arethe growers, students, consumers, individuals with gut related ailments and the grape industry. Accordingly, the research results were shared with the concerned groups through various outreach activities, lectures, discussions and using other diverseelectronic and print media. Some of these means and media are identified below. 1. Journal articles, 2. Conference presentations, 3. Florida Grape Grower Association meeting, 4. Workshops, 5. Florida Legislature Appreciation Day, 6. Professional meetings, 7. Social Media pages, and 8. Guest lectures. These results will be also shared with the community groups and other interested groups through various outreach activities including annual Grape Harvest Festival and Extension outreach activity days, and other community events. What do you plan to do during the next reporting period to accomplish the goals?This project is intended to evaluate the efficacy of muscadine grape extracts to help control gut ailments by promoting pre- and pro-biotic activity of the gut microbiota. In this regard we have successfully isolated and characterized the phytochemical constituents of muscadine grape berry with potential pre- and pro-biotic activities. Further, we have also isolated, identified and characterized the predominant endophytic community of the muscadine berry. These research activities enabled us to learn about the phytochemical components and the identity of resident microbial community of muscadine grape berry. The follow up studies will include evaluating the effects of muscadine grape berry extracts on physiological functions and performance of gut microbiome. Fully ripe muscadine grape berries will be collected and aqueous and methanolic extracts prepared and used for bioactivity evaluation. For this purpose, selected gut microorganisms known to be crucial for gut health will be planted in Brucella agar plates and cultured with and without muscadine grape berry extract. The minimal inhibitory concentration and effective dose of berry extract resulting in change of appearance of microbial growth as compared to Control will be recorded to establish optimal microbial strain concentration for each test. In addition, the microbial components will be evaluated for pre- and pro-biotic properties and safety by determining their hemolytic activity, DNase activity and antioxidant activity. The aqueous and methanolic extracts of muscadine berries will be tested for prebiotic potential using gut microbial isolates identified above. The parameters including Tolerance to Acids and Bile Salts, Resistance to Phenol, Auto Aggregation, Cell Surface Hydrophobicity, Survival in Simulated Gastric and Pancreatic Digestion, Quantification of biofilm formation on polystyrene, and Exopolysaccharide production that indicate a healthy gut microbiome will be studied. The muscadine grape genotypes exhibiting highest synbiotic activity will be selected and their berry extracted with various aqueous and non-aqueous solvents and phytochemicals quantified with reference to their phenolics content and composition, and other specific chemical markers. In addition, stilbenes content and composition of these berry extracts will be determined by HPLC to learn about the identity of phytochemicalconstituents. Subsequently, additional muscadine grape genotypes differing in phytochemicals content and composition will be screened to determine the extent of genetic variation in bioactivity level among muscadine genotypes and increase the portfolio of genotypes to obtain a comprehensive idea on their bioactivity level. We will also apply a variety of post-harvest stilbene induction techniques to increase the resveratrol content of berry for enhancing the bioactivity level of non-performing muscadine genotypes. We will further explore the observed pro-gut health effects at the molecular and cellular levels by monitoring muscadine berry extract induced changes at the gene and protein levels in Caco-2 cells. For determining the transcriptomics profile, RNA will be isolated from the muscadine berry extract treated and control cells and sent for RNA sequencing to determine the treatment effect on gene expression. For determining the effect of muscadine berry extract treatment on expression/suppression of related pathway genes, protein will be isolated from treated cells and sent for protein sequencing. These studies will help identify the genes and pathways responding to the treatment of Caco-2 cells with muscadine grape berry extract. These genomics and proteomics studies will help better understand the global genome and proteome expression changes in Cao-2 cells treated with muscadine berry extract. Student training on nutraceuticals and functional foods development will be continued for enhancing their career opportunities in food, agriculture, and nutritional sciences. This will include providing hands-on experiential learning and training in diverse subject matter areas and disciplines to build student's basic skills, enhance critical thinking and proficiency, and mold them into independent researchers. This will increase student expertise and confidence, and support their careers in agriculture, food, and biomedical sciences.
Impacts
What was accomplished under these goals?
The incidence of gut related disorders is on the rise, and due to complex nature of the disease and organ, no effective treatment is available to address the malady. It has been shown that dysbiosis and oxidative stress in the gut contributes to the progression of intestinal inflammatory bowel disease (IBD) and leaky gut syndrome. The quantity and quality of microbiota in the gut is determined by food intake, health status of the host and environment. Research has established that enteric bacterium plays a major role in maintaining homeostasis. Application of phytochemicals to modulate gut microbiome and improve gut integrity may be a potential solution to remedy this disorder. Muscadine berry phytochemicals have also shown to exhibit numerous health benefits including anticancer, anti-inflammatory, anti-hypertension, and antioxidant activities. However, little is known about the pre- and pro-biotic potential of muscadine grape phytochemicals and endophytes in enhancing gut health. In this regard, we have hypothesized that muscadine grape could be a superior source of phytochemicals to modulate gut microbiome and reduce age induced 'leaky gut'. Accordingly, this research is being conducted to determine the efficacy of muscadine grape for augmenting gut microbiome and reduce age related 'leaky gut. This would provide a potential opportunity to establish the rejuvenative effect of muscadine grape, identity of bioactive components and its potential as a nutraceutical supplement for promoting gut health. Towards this end we have investigated the phytochemicals content and composition of muscadine grape genotypes to determine their effects on gut microbiome and gut health. The results showed that muscadine grape contained a range of polyphenolics, flavonoids, anthocyanins, stilbenes, etc., which differed widely in their content and composition. Further, we have also isolated and characterized several bacterial and fungal communities residing in the muscadine grape berry. Research is also being conducted to determine the prebiotic potential of these phytochemicals in modulating the quantity, quality, and functioning of isolated gut microbiome using in vitro cell culture system. Fully ripe berries were collected from the selected muscadine grape genotypes from the FAMU vineyard at the Center for Viticulture and Small Fruit Research. The phytochemicals were extracted from berry tissue and used for the quantification of various polyphenolics including stilbenes using various biochemical assays and HPLC. Further, the predominant endophytic microbial population from muscadine grape berry tissue were isolated and evaluated for their probiotic impact and safety. Whole berry was surface sterilized by washing with 70% ethanol and rinsing in sterile distilled water. The skin was peeled aseptically, and seeds removed with pincers. The deseeded muscadine berry tissue was homogenized and used for isolating its endophytic microbial community. The microbial isolates were purified, their individual stocks prepared and the microbial community present in muscadine berry characterized following metagenomic approach. The amount and quality of DNA was determined in the preparations. The Primers were designed from 16S rDNA and 18SrDNA region to amplify the bacterial and fungal communities from the genomic DNA extracts and samples prepared for the sequencing study. Following these protocols, we have successfully measured the phenolics, flavonoids and stilbenes content of muscadine grape berries and found them to contain a widerange of polyphenolic components including flavonoids, stilbenes, anthocyanins, etc. Further, these studies also showed that muscadine berry contains four different stilbenes including t-piceid, t-resveratrol, viniferin and t-pterostilbene where the t-piceid and t-resveratrol formed the predominant components. We were also able to isolate and purify several microbial isolates belonging to the bacterial, yeast, and filamentous fungus as well as maintain their stocks. The entire endophytic microbial community was successfully isolated and confirmed by PCR amplification employing the primers from the 16S rRNA and 18S rRNA gene coding regions. The PCR amplification studies revealed that fungal communities were more prevalent in the selected muscadine grape genotypes than the bacterial population. These polyphenolic components and microbes are being studied to determine their role/function in the gut environment, safety, pre- and pro-biotic effects using human gut cell culture. These studies would reveal the impact of muscadine grape endophytes and phytochemicals on gut microbial community and help assess their pro-biotic effects as well determine the role of individual microbial species and polyphenolics on gut health.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Rahman MA, Balasubramani SP and Sheikh MB. 2021. Molecular characterization and phylogenetic analysis of MADSbox gene AGL11 associated with stenospermocarpic seedlessness in muscadine grapes. Genes 12(2): 232. doi: 10.3390/genes12020232
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Darwish AG, Das PR, Ismail A, Gajjar P, Balasubramani SP, Sheikh MB, Tsolova V, Sherif SM and El-Sharkawy I. 2021. Untargeted Metabolomics and Antioxidant Capacities of Muscadine Grape Genotypes during Berry Development. Antioxidants 2021, 10, 914. https://doi.org/10.3390/ antiox10060914.
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