Location: Citrus and Other Subtropical Products Research
Project Number: 6034-30600-001-000-D
Project Type: In-House Appropriated
Start Date: Jul 3, 2025
End Date: Jul 2, 2030
Objective:
Objective 1: Develop a new process to produce pectin with improved gelling and water holding capacity to develop pectin nano/micro-carriers.
Objective 2: Identification of novel flavonoid formulations from HLB tolerant citrus clones for targeted health conditions.
Approach:
Pectin will be extracted from citrus peel using high-pressure processing followed by a commercial extraction method. The pectin will be used to encapsulate gluten-degrading enzymes. We hypothesize that encapsulation of gluten-degrading enzymes with pectin hydrogel will protect the enzymes from acid inactivation in the stomach and deliver the enzymes to the intestine to degrade the human digestion-resistant gluten peptides, thereby alleviating gluten-related disorders in gluten-intolerant people. Three food-grade gluten-degrading enzymes will be encapsulated, individually or in different combinations. Enzyme-loaded pectin mini-hydrogel beads will be prepared in 9-12 formulations and tested in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) to assess the melting rate of the hydrogel and the release of the enzyme under the conditions of gastrointestinal tract. The optimal formulation of the hydrogel will be determined based on the enzyme encapsulation efficiency, enzyme release rate and activity in SIF after challenge in SGF. The enzyme activities and the effects on the gut microbiota of the enzyme-loaded pectin mini-gels will be further evaluated using a complete human gastrointestinal tract simulation system and a mouse model and compared with the non-encapsulated enzyme control and the hydrogel carrier control. Enzyme activity will be quantified using SDS-PAGE, a commercial lateral flow test kit and an ELISA assay kit. Gut microbiota will be analyzed using 16S rRNA sequencing and the reads will be taxonomically classified based on the Greengenes reference database. Microbial communities will be compared between the treatments and the controls. Statistical analysis will be performed using the R and corrected for false discovery.
The fruit of 21 different citrus clones will be harvested and the peel will be removed and solvent extracted. The extracts will be analyzed for the composition and concentration of various flavonoids. The composition and concentration will be compared among the 21 different citrus clones and their parentage using data from the literature. Four citrus clones with unique flavonoid composition and concentration will be harvested and used to produce extracts for in vitro testing of different doses on a macrophage and human trophoblast cell lines. Cells will be treated with and without extracts ± lipopolysaccharide and/or fatty acids to simulate inflammation or high fat or western diet environment. Inflammatory cytokine panel will be measured to evaluate the anti-inflammatory effect of the citrus flavonoids. The 2 -3 flavonoid clones that showed the significant effect in inflammation from in vitro studies will be considered as candidates for vivo testing. Thus, to test the citrus clones flavonoids ability to reduce inflammation and insulin resistance and improve cognitive function, mice will be exposed to control and western diet and orally gavaged with citrus clone variety extracts. Mice will be euthanized to collect samples for inflammation, gut microbiota, and metabolite measurements. Mice will be subjected to behavioral tests to determine cognition, memory, and sociability.