Progress 05/01/21 to 04/30/24
Outputs
(N/A)
Impacts
What was accomplished under these goals?
This study investigated the impact of anthocyanin (ACN)-rich functional foods on the mouse gut microbiome's response over time and examined how ACN structural diversity influenced microbiome composition. We hypothesized that the microbiome would respond rapidly to ACN-rich food intake, with ongoing consumption essential for lasting changes in composition, and that distinct ACN profiles of food powders would influence microbiome diversity and specific taxa. We conducted a multifactorial experiment with C57BL/6J male mice, testing eight diets across exposure durations of 1, 3, or 7 days and measuring responses at baseline, 0, 1, 3, or 7 days post diet exposure. Mice were housed individually and fed a Western diet supplemented with 2 g/kg of anthocyanins (ACN) from freeze-dried powders or extracts of bilberry (BB), tart cherry (TC), chokeberry (CB), elderberry (EB), black currant (BC), and black raspberry (BRB), 2 g/kg of cocoa polyphenols (CP) or no supplement (CON). Fecal microbiome samples were collected at baseline and after diet cessation at specified response intervals for 16S rRNA sequencing analysis. Alpha diversity significantly changed in mice fed the BB diet (evenness) and EB, BC, BB, or CP diets (richness), regardless of exposure duration or response time. Interestingly, for BRB, TC, and EB diets, day 0 alpha diversity spiked above baseline, but this response was not maintained through days 1-7. Similarly, unweighted unifrac beta diversity showed significant, yet transient, shifts in the fecal microbiome for BRB, BC, EB, or TC diets, with changes fading by day 1 (BC) or day 3 (EB, BRB, TC). This observation suggests a possible structure-activity relationship, where foods rich in cyanidin-3-O-glucoside or cyanidin-3-O-rutinoside altered the microbiome profile, unlike those high in cyanidin-3-galactoside (CB and BB). This study confirmed that ACN-induced microbiome changes do not persist after consumption of the ACN-rich food ceases. Furthermore, despite equal anthocyanin concentrations in the ACN-supplemented diets, the varied ACN profiles prompted distinct microbiome responses, suggesting a potential structure-activity relationship.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2021
Citation:
Presentations:
Benninghoff, A. (Presenter & Author), Rodriguez, D. (Author Only), Hintze, K. J. (Author Only), American Council on Medicinally Active Plants 10th Annual Conference, "Dynamics of the gut microbiome in response to dietary intervention with black raspberries in the context of a standard or Western type diet in a mouse model of inflammation-associated colorectal cancer," Virtual. (June 24, 2021 - June 25, 2021)
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Progress 05/01/22 to 04/30/23
Outputs
Target Audience:global scientific community Changes/Problems:As noted above, some samples required resequencing, which was successful. One challenge is the extraordinarily expansive dataset and complicated analyses of a three-factor experiment. Data analyses is predicted to take longer than anticipated. Yet, the final dataset and analyses should be very informative. What opportunities for training and professional development has the project provided?One graduate student, Mohammed Almatani, is overseeing this work as part of his dissertation research. He has participated in several professional development workshops (research presentations, data management, etc.) and other trainings offered at USU. He has also mentored three undergraduate students who worked in the laboratory to assist in the sample processing. Additionally, in the past year, three undergraduate students worked as laboratory research assistants on this project, where they learned about experiment design, animal husbandry (working with available sentinel mice), study management, animal necropsy (working with available sentinel mice), data analysis and presentation preparation. Students also learned bench life science skills through the preparation of fecal samples for high-throughput sequencing. All students interacted closely with the PI through weekly meetings or one-on-one appointments, and these students worked very closely with other research personnel, including other graduate students. How have the results been disseminated to communities of interest?Not yet applicable. What do you plan to do during the next reporting period to accomplish the goals?The remainder of the project period will include analyses of the resulting microbiome profiles and preparation of a publication detailing the findings.
Impacts
What was accomplished under these goals?
As of this report, we have completed the animal study with the diverse anthocyanin-rich foods (black raspberries, bilberries, black currant, chokeberries, tart cherry, and elderberries) with cocoa polyphenol as a non-anthocyanin comparison group. Per the experiment design, more than 700 fecal samples were collected; these were processed according to our standard operating protocol and then subject to 16s rRNA sequencing. The resulting data sets were processed using Qiime2 and mapped to the Silva database to determine the taxa present in these samples. Initial review of the sequencing results indicated that about 10% of samples required reprocessing and/or resequencing to obtain the necessary sequencing depth. This resequencing has been completed, and the final data set processed via Qiime2 with mapping to Silva. Analyses of the resulting microbiome profiles using Microbiome Analyst is ongoing.
Publications
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Progress 05/01/21 to 04/30/22
Outputs
Target Audience:global scientific community Changes/Problems:Based on the anthocyanin profile of the fruit powders, six anthocyanin-rich powders were selected for this animal study including bilberry, tart cherry, chokeberry, elderberry, black currant and black raspberry. Anthocyanin content of red raspberry and strawberry fruit powders was too low to use in this experiment without chemical extraction, a method not applied to any of the other fruit powders. Thus, one of these fruits was replaced by tart cherry, which did have a substantially high anthocyanin concentration by our testing. For the last experimental group, we selected a food item that would have a vastly different polyphenol profile than the others, cocoa polyphenols, with the expectation of a strong contrast in microbiome response. What opportunities for training and professional development has the project provided?One graduate student, Mohammed Almatani, is overseeing this work as part of his dissertation research. She has participated in several professional development workshops (research presentations, data management, etc.) and other trainings offered at USU. He attended the American Society of Nutrition conference in June 2022, which was held as a virtual conference. Additionally, in the past year, three undergraduate students worked as laboratory research assistants on this project, where they learned about experiment design, animal husbandry (working with available sentinel mice), study management, animal necropsy (working with available sentinel mice), data analysis and presentation preparation. Students also learned bench life science skills through the preparation of fecal samples for high-throughput sequencing. All students interacted closely with the PI through weekly meetings or one-on-one appointments, and these students worked very closely with other research personnel, including other graduate students. How have the results been disseminated to communities of interest?Not yet applicable. What do you plan to do during the next reporting period to accomplish the goals?The animal study is planned for summer 2022, with microbiome analyses following during the fall 2022 term. Data analysis and reporting will conclude in spring 2023.
Impacts
What was accomplished under these goals?
As of this report, we have performed polyphenol analyses via HPLC of eight different fruit powders reportedly high anthocyanin content, including black raspberries, bilberries, black currant, red raspberries, strawberries, chokeberries, and tart cherry. The anthocyanin content varied greatly, from 0.037% (w/w) for strawberry to 18.1% for a standardized bilberry powder from Artemis-International. Profiling also identified the major anthocyanin chemical components, which were compared to the USDA polyphenol database. Subsequently, the food powders were subject to composition analyses for determination of carbohydrate, protein, fat and fiber content. With these data, the experimental diets were formulated in partnership with a nutrition scientists at Envigo. As of this report, diets were in preparation, with the animal study to be performed during the summer 2022. During this time, students have also worked on the microbiome analysis methodology to optimize for high throughput analyses. Students have also learned analytical approaches for microbiome datasets using practice datasets.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2021
Citation:
Presentations
Benninghoff, A. (Presenter & Author), Rodriguez, D. (Author Only), Hintze, K. J. (Author Only), American Council on Medicinally Active Plants 10th Annual Conference, "Dynamics of the gut microbiome in response to dietary intervention with black raspberries in the context of a standard or Western type diet in a mouse model of inflammation-associated colorectal cancer," Virtual. (June 24, 2021 - June 25, 2021)
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