Progress 06/01/24 to 05/31/25
Outputs
Target Audience:During this reporting period, the audience reached included graduate and undergraduate students, peer researchers and scientists, and professionals from the food industry and nutrition; Extension educators and public members interested in diet and health promotion. Changes/Problems:We have not encountered any problems in this project. Our initial delay was related to student recruitment, but we have made significant progress towards both project aims. Moreover, due to the substantial quantity of prebiotic nanoparticles needed for the study, an extension is warranted for an additional year. What opportunities for training and professional development has the project provided?This project has provided valuable opportunities for professional development and training, particularly for two undergraduate students and one graduate student recruited over the past two years. Students received hands-on training in various aspects of scientific research, including the measurement of total phenolics and anthocyanins, cell culture techniques, gene expression analysis, and protein quantification. Additionally, they were trained in statistical analysis, academic abstract writing, and professional oral and poster presentation skills. Moreover, other lab students were engaged indirectly in this project and assisted with various aspects of the experimentation and gained diverse sets of skills. As a result, data generated from this research was presented at the Texas Tech Transformative Undergraduate Experiences Symposium in Fall 2024, where two undergraduate students provided oral presentations. Furthermore, an undergraduate researcher will present the project titled "Novel Prebiotic Chitosan Nanoparticles for Enhanced Adsorption of Anthocyanins in Tart Cherry Juice" at the Texas Tech Undergraduate Research Conference (URC) in April 2025. Due to its innovative approach to leveraging prebiotic nanotechnology to improve human health and its potential applications in the food industry, this project has been selected as a finalist for the 2025 TTU URC Commercialization Poster Competition. The same project will also be presented by undergrad at the American Society of Nutrition (ASN) Annual Meeting in May 2025. How have the results been disseminated to communities of interest?The findings from this project have been disseminated to various communities of interest through multiple academic and professional platforms. Nationally, in the second year of the project, two conference abstracts from this project have been accepted for publication, ensuring broad dissemination to the scientific community. One oral and two poster presentations are scheduled to be presented at the American Society for Nutrition (ASN) Annual Meeting 2025, providing opportunities for students, scientists, and professionals to engage with the research findings firsthand. Regionally, project data have been shared through oral and poster presentations at various academic meetings, including the TTU Transformative Undergraduate Experiences Symposium in the fall of 2024 and the Undergraduate Research Conference in the spring of 2025. Notably, the prebiotic nanotechnology project has been selected for presentation at the TTU URC Commercialization Poster Competition in May 2025, where it will be reviewed by experts. This evaluation will allow for a rapid assessment of the market potential of this technology, expanding its impact beyond academia. What do you plan to do during the next reporting period to accomplish the goals?We plan to request a first one-year no-cost extension to finalize the characterization of Alg-Tnano hydrogel beads (ongoing). As part of this extension, we will also conduct the mouse model study to assess the bioavailability of ACNs protected by Alg-Tnano beads compared to the free form of ACNs in tart cherry juice. Given the substantial number of prebiotic nanoparticles required for the study, we are actively preparing and collecting nanoparticles to generate Tnano, followed by the production of Alg-Tnano beads. Tart Cherry encapsulated beads will be incorporated into a high-fat, high-sucrose diet to investigate their potential anti-obesity and anti-inflammatory effects in a diet-induced obese mouse model. During the next reporting period, we will complete the animal study and conduct serum, tissue, and data analyses, including inflammatory cytokine profiling in the bloodstream, adipose tissue, and liver, microbiota composition analysis, gut integrity assessment, and other key metabolic markers. Ultimately, we aim to prepare and publish two peer-reviewed research papers based on our findings. One is in preparation and the other one will be prepared after we complete the animal study.
Impacts
What was accomplished under these goals?
In the second year of this project, we aimed to develop novel prebiotic nanoparticles to enhance the bioavailability of anthocyanins in tart cherry juice (TCJ) while improving gut health in an obese mouse model. Significant progress was made toward Aim 1: Developing nano-encapsulated TC anthocyanin (Tnano) technologies and formulations using prebiotic compounds as follows: Novel prebiotic chitosan nanoparticles were developed to adsorb anthocyanins from tart cherry juice (Tnano). Void nanoparticles (Vnano) were synthesized via ionic gelation of prebiotic polysaccharides, chitosan (C) and carboxymethyl chitosan (CMC). The concentration of C and CMC have been adjusted to optimize the mean particle size of Vnano to 100-120nm. After purification, Vnano was incorporated into TCJ to produce Tnano. Incorporating TCJ increased the particle size of Tnano to approximately 180nm, with ACNs smoothing the rough surface of Vnano. Both Vnano and Tnano displayed PDI values near 0.2 and zeta potentials of 25-30mV. The ACNs adsorption process was optimized by adjusting specific ratios (w/w), temperature, and reaction time, followed by solid-liquid separation and ACNs quantification. Optimized conditions for ACNs adsorption were achieved by combining TCJ with Vnano at a 1:300 (w/w) ratio and incubating for 16 hours at 37°C with agitation, resulting in over 80% ACNs adsorption efficiency. To investigate selective binding, Vnano was incubated with various anthocyanin monomers, including cyanidin-3-glucoside (C3G), cyanidin-3-O-rutinoside (C3R), and cyanidin chloride (CC). We found that nanoparticles exhibited the highest binding affinity for CC (93.8%), followed by C3G (66.8%) and C3R (63.6%). The release profiles of ACNs from TCJ and Tnano were evaluated in vitro via the dialysis method. Tnano-adsorbed ACNs demonstrated a sustained release profile in acidic conditions, with a 50% reduction in cumulative release over 24 hours compared to free ACNs in TCJ. Investigate the anti-inflammatory and anti-adipogenic effects of Tnano in 3T3-L1 cells.The MTT assay was used to assess the effects of TCJ and Tnano on the viability of 3T3-L1 pre-adipocytes. TCJ and Tnano reduced 3T3 cell viability from 92% to 43% and 99% to 52% at 2 to 128 µg/mL of ACNs, respectively, after 72 hours. Anti-inflammatory effects of these treatments were evaluated by treating lipopolysaccharide (LPS)-stimulated pre-adipocytes with 4 µg ACNs/mL TCJ and Tnano. Tnano demonstrated superior anti-inflammatory effects in LPS-stimulated pre-adipocytes, and reduced IL6 and MCP1 levels by 39% (p<0.0001) and 20% (p<0.0001), compared to TCJ (11.5% and 4.5%), respectively. Similarly, in differentiated adipocytes, compared to LPS group, Tnano reduced IL-6 and MCP-1 levels by 56.8% (p=0.05) and 20%, respectively, while TCJ achieved reductions of 12.6% and 4.5%. Inflammatory gene expression, including Il6, Mcp1, and Tlr-4, was also reduced by Tnano compared to the LPS-stimulated group (p=0.0004, 0.027 and 0.004). Anti-adipogenic effects were analyzed in differentiated cells treated with 1 µg ACNs/mL TCJ and Tnano. Using oil Red O staining, we observed that lipid content in adipocytes differentiated with TCJ and Tnano was reduced by 14.2% and 35% (p=0.0003), respectively, compared to control cells. Additionally, to conduct the animal study proposed in Aim 2: Evaluating the bioavailability and gut microbiota-modulating effects of optimized prebiotic Tnano in diet-induced obese B6 mice, alginate core-shell beads were designed to protect the nanostructure of Tnano from degradation during diet preparation and in the stomach. To optimize the loading capacity of Tnano in alginate hydrogel beads, Tnano was mixed with varying concentrations of calcium chloride (CaCl?) solution and extruded dropwise through a syringe pump into an alginate solution. Capsules were formed as Ca²? ions from the droplets interacted with alginate, initiating gelation. After the gelation process, the resulting wet Alg-Tnano beads, prepared under different conditions, were spherical, monodisperse, and measured approximately 2-3 mm in diameter. These beads consisted of an aqueous core surrounded by a calcium-alginate membrane. The beads were further cured in a 2% CaCl? solution for 1 hour and subsequently washed multiple times with distilled water. Following vacuum drying, Alg-Tnano hydrogel beads of approximately 2 mm in size were obtained, achieving a Tnano loading efficiency of up to 95%. Light microscopy was used to assess the physical appearance of the wet Alg-Tnano beads, revealing that bead size and alginate shell thickness increased with CaCl? concentration (1-3%). Dry beads were analyzed using Fourier-transform infrared spectroscopy and scanning electron microscopy, confirming that the nanostructure of Tnano was preserved post-drying. Further characterization of Alg-Tnano beads, including ACNs release patterns in simulated gastrointestinal digestion in vitro, bioavailability in vivo, and their potential metabolic benefits in a diet-induced obese mouse model, will be conducted during the 2025-2026 period.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2025
Citation:
Zu Y, Adegbile T, Walker T, Scoggin S, Moustaid-Moussa N. Tart Cherry Anthocyanins-Adsorbed Nanoparticles Reduces Inflammation and Adipogenesis in 3T3L1 Adipocytes.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2025
Citation:
Walker T, Zu Y, Scoggin S, Moustaid-Moussa N. Novel Prebiotic Chitosan Nanoparticles for Enhanced Adsorption of Anthocyanins in Tart Cherry Juice.
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Progress 06/01/23 to 05/31/24
Outputs
Target Audience:The audience reached during this reporting period included students (graduate and undergraduate students), postdocs, peer researchers, scientists, and professionals from nutrition science, food industry, and food engineering. The research outcomes of this project will be shared with the target audience in two posters that will be presented during the Texas Tech Undergraduate Research Conference in April 2024 and the American Society for Nutrition Annual Meeting(Nutrition 2024 in June 2024). Changes/Problems:No changes are expected. What opportunities for training and professional development has the project provided?The project has provided an opportunity for the professional development and training of one undergraduate student recruited during the past year. The student received hands-on training in various aspects of scientific research, including the measurement of total phenolics and anthocyanins, statistical analysis, academic abstract writing, and professional oral and poster presentation skills. As a result, the student will showcase the research outcomes at the Texas Tech Undergraduate Research Conference 2024, demonstrating the practical application of their acquired skills. The student, who is in the nutrition program, gained significant experience in transdisciplinary training through our collaboration with an engineering team in this project. How have the results been disseminated to communities of interest?A conference abstract for a poster presentation of this project has been accepted for publication at the annual meeting (Nutrition24) of the American Society for Nutrition, ensuring dissemination to a broad scientific audience. Additionally, the undergraduate student has submitted and will be presenting a poster at the Texas Tech Undergraduate Research Conference 2024 that includes various fields from STEM, social sciences, and humanities providing a broader dissemination of project information and outcomes. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we will involve additional undergraduate and graduate students in this project. And we will continue to explore the additional physical and chemical properties of the TCJ-loaded microparticle delivery system. The TCJ-loaded microparticles will be freeze-dried to assess their in vivo bioavailability when incorporated into the diet.
Impacts
What was accomplished under these goals?
During the initial year of this study, we developed several protocols to quantify total phenolics and anthocyanins and characterize the anthocyanin profile in tart cherry juice (TCJ). Total phenolics were assessed using the Folin-Ciocalteu assay, while anthocyanins were measured via the pH differential method and further characterized using HPLC-MS. Secondly, a range of nano/microparticle carrier systems were evaluated for encapsulating anthocyanin-rich TCJ. 1) Liposomes, prepared using soy-phosphatidylcholine via extrusion, were employed for encapsulation using both passive and pH-gradient loading methods. While TCJ-loaded liposomes exhibited particle sizes ranging from 100-300nm, the encapsulation efficiency was found to be below the desired threshold (<30%) in both methods. 2) Whey protein isolates and β-lactoglobulin (β-Lg) were examined as potential encapsulation materials for protein-biopolymer-based nanoparticles. In pursuit of a prebiotic carrier system, methods involving ethanol or chemical cross-linking agents such as ethyl carbodiimide hydrochloride were avoided, opting instead for the use of natural polymers. Through coacervation, tart cherry juice (TCJ) was successfully encapsulated within nanoparticles composed of β-lactoglobulin and pectin (pec) in a specified ratio. The resulting TCJ-loaded β-Lg/pec nano-complex exhibited a particle size of approximately 300 nm with a polydispersity index below 0.3. 3) Chitosan, pectin, and alginate were investigated as potential materials for encapsulating polysaccharide-based nanoparticles. Among them, alginate particles demonstrated promising spherical morphology when electrospraying under various conditions, with an average size ranging from 300 to 500 μm, making it suitable for encapsulating tart cherry juice (TCJ). Significantly, in a pioneering effort to enhance TCJ retention time and bioavailability, we have synthesized electrospray alginate hydrogel microcapsules to encapsulate TCJ-loaded β-Lg/pec nano complexes, proposing a novel gut delivery system.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Development and Characterization of Tart Cherry Juice Encapsulated Prebiotic Particles
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Tart Cherry Juice Encapsulated Prebiotic Particles: Comparison of Characteristics
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