Progress 05/01/23 to 04/30/24
Outputs
Target Audience:The target Audience of this project include scientists in food and ingredient manufactures, nutraceutical or pharmaceutical industry, health professional, researchers and students in the area of food science and nutrition, peanut producers and processors. Changes/Problems:The only change made to the project is that two MS students are involved in the project instead of one Ph.D. student and one MS student. What opportunities for training and professional development has the project provided?Training activities: During this reporting period, two MS graduate students, Sukanya Poddar and Seyi Adebayo were trained. The training for Sukanya Poddar mainly include data analysis, thesis and publication writing, presentation and communication skill. The training for Seyi Adebayo includes PPC from peanut flour, enzymatic hydrolysis of peanut protein of PPC to produce PPH, experimental design for simulated digestion of PPC and PPH, methods for monitoring PPH digestion including total free amino acid quantification, ACE inhibitory activity tests and SDS-PAGE. Professional development: Sukanya Poddar been trained to present her research result in different professional conferences, including 2023 SERMACS Meeting in Durham, North Carolina, 105th Annual Conference of North Carolina Association of Family and Consumer Sciences. Participated in graduate student research completion in North Carolina A&T State University and the 1890 ARD 21st Biennial Research Symposium. She is accepted by both NC A&T State University and University of Arkansas for Ph.D. study in Food Science. Her research results are recently published in the International Journal of Molecular Science. Seyi Adebayo just completed his first semester in NC A&T State University, but the research results he generated are sufficient for a poster presentation. I am still teaching him data analysis, writing abstract, and literature review for conference presentation. How have the results been disseminated to communities of interest?The research results were disseminated in 105th Annual Conference of North Carolina Association of Family and Consumer Sciences, 2023 SERMACS Meeting in Durham, North Carolina, 1890 ARD Research Symposium, 2024 North Carolina Small Farms Field Day hosted by the Cooperative Extension, NC A&T State University, and published in International Journal of Molecular Science as shown in the Products section. What do you plan to do during the next reporting period to accomplish the goals?I plan to complete Objective 2 by the end August 2024, and start Objective 4 from September 2024. Because Object 3 has not begun, and part of Objective 4 depends on the fecal samples from the activities of Objective 3, I do not anticipate completing the project by April 30, 2025. Therefore, I plan to apply a one-year No Cost Extension. In addition, we proposed to hire a Ph.D. student and a Master student to conduct research activities of this project at NC A&T. However, the Ph.D. student was not available, thus two MS students are involved in this project. Due to the difference of assistantship for PhD and MS students, I will make a budget revision to move part of the research assistantship fund to the tuition account to support the graduate student Seyi Adebayo who is working hard for the project.
Impacts
What was accomplished under these goals?
Major activities completed during the reporting period 05/01/2023 - 04/30/2024: We accomplished the optimization of Alcalase hydrolysis process of PPC using the ACE inhibitory activity of PPH as measurement. The renin inhibitory activities of PPH samples produced at 3, 4 and 5% of Alcalase for 6-10 hours, which exhibited higher ACE inhibitory activities, were determined. The bioavailability of PPH was evaluated by testing the resistance of PPH to pepsin and pancreatin digestion followed by measuring ACE-inhibitory activity of digested samples, and SDS-PAGE. The IC50 values of ACE inhibition and renin inhibition of crude PPH and different fractions of PPH were determined. Data obtained were analyzed using non-linear regression and post-ANOVA Tukey tests at a significance level of 5%. A manuscript was published in a peer-reviewed journal, and 1.2 kg of PPH was produced and sent the Subcontractor of the project for in vivo study using rat model. Specific objectives met: During this reporting period, we completed Task 3 of Objective 1, and Task 1 and 2 of Objective 2. The task 3 of Objective 2 and tasks of Objective 3 are undergoing. Significant results achieved, including major findings, developments, or conclusions: The data generated in this reporting period indicate that the peanut protein hydrolysate (PPH) of extensively hydrolyzed PPC had significant renin inhibitory activities. The optimal condition for renin inhibitory PPH was Alcalase concentration 4-5% and hydrolysis time 6-8hr at the optimal pH of Alcalase (pH8). A remarkable higher renin inhibitory activity was observed in the fractions smaller than 5kDa (F3). Statistical analysis shown that the renin inhibitory activities of PPH samples produced by 6 and 8 hour-hydrolysis of PPC were not significantly different, but PPH samples from 10 hour-hydrolysis of PPC displayed significantly lower renin inhibitory activities. Therefore, the optimal hydrolysis condition should be Alcalase concentration of 4-5% and hydrolysis time 6-8 hours. Simulated gastric digestion (SGD) of PPH for 1hr significantly reduced the ACE inhibitory activity of PPH, but 2hr-SGD resulted significantly increased ACE-inhibiting activity compared to the undigested PPH. Similarly, 1hr-simulated intestinal digestion (SID) of PPH significantly reduced the ACE-inhibiting activity of PPH, but 2hr SID significantly increased the ACE-inhibitory activity of PPH at pancreatin concentration 0.2-2.0 mg/mL. The IC50 of PPH after 2hr-SGD was between 2.30-3.05 mg/mL which decreased with pepsin concentration, and the IC50 of PPH after 2hr-SID was 3.4-1.5 mg/mL. The increase of ACE-inhibition by the longer SGD and SID might be resulted from the formation of more potent shorter peptides. The SDS-PAGE of pepsin and pancreatin digested samples showed that all protein/peptide bands with molecular weight larger than 2 kDa disappeared after 2hr digestion. Thus, the ACE inhibitory peptides in the PPH may not be all resistant to the gastric digestion and small intestine digestion, but new and smaller peptides formed due to gastrointestinal digestion of PPH might have stronger antihypertensive potential. Key outcomes or other accomplishments realized: The results from the research activities of this reporting period revealed that the hydrolysis of PPC with Alcalase under optimal condition could result in crude PPH with moderate capacity to inhibit the activities of ACE and renin, two critical enzymes in blood pressure regulation, which is an important advantage over the protein hydrolysates/peptides derived from animal proteins because most of animal protein derived peptides/hydrolysates, do not have renin inhibitory activity. Simulated gastric and intestinal digestions significantly enhanced the ACE-inhibitory activity of PPH, thus the its antihypertensive potential. Therefore, PPH derived from peanut flour could be a potential product for food therapy of hypertension. The results also show the great opportunity for value-added utilization of peanut flour.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Poddar, S., & Yu, J. (2024). Angiotensin-Converting Enzyme and Renin-Inhibitory Activities of Protein Hydrolysates Produced by Alcalase Hydrolysis of Peanut Protein. International Journal of Molecular Sciences, 25(13), 7463.
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2023
Citation:
Yu, J. (2023). Peanut flour and use in foods as a protein supplement. In Nutritional Aspects of Dietary Protein (Victor R. Preedy, Ed)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
Poddar, S., & Jianmei Yu. Effects of Protease Concentration and Hydrolysis Time on Antihypertensive properties of Alcalase Hydrolyzed Peanut Protein Concentrate. 2024 ARD Biennial Research Symposiums, April 4-10, 2024, Nashville, TN
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
Poddar, S., & Jianmei Yu. Renin-Inhibitory Activity of Alcalase Hydrolyzed Peanut Protein Concentrate. 2024 NCAFCS Conference, March 1, 2024, Raleigh, NC.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Poddar, S., & Jianmei Yu, J. ACE-inhibitory activity of extensively hydrolyzed peanut protein concentrate. Abstract submitted to 2023 Southeastern Regional Meeting of American Society of Chemistry (SERMACS), October 25-28, 2023, Durham, NC.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Poddar, S., & Jianmei Yu, J. Effects of protease concentration and hydrolysis time on the ACE-inhibitory activity of Alcalase hydrolyzed peanut protein concentrate. 2023 APRES Annual Meeting. July 10-14, 2023, Savannah, GA.
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Progress 05/01/22 to 04/30/23
Outputs
Target Audience:The target Audience of this project include scientists in food and ingredient manufactures, nutraceutical or pharmaceutical industry, health professional, researchers, and students in the area of food science and nutrition, peanut producers and processors. Changes/Problems:Problems: The renin inhibitory activity of PPH was tested using the Renin Inhibitor screen kit but the result were fluctuated too much because the wavelength of our fluorescence detector was not exactly the same as that required in the procedure. Thus, a pair of new filter was purchased, but was not received until late May, 2023. Change: It was planned to use the PPH fraction smaller than 5kDa for the rat study. However, we found that the yield of this fraction is very small and it is difficult to produce sufficient amount for rat study. Thus we will use crude PPH at higher dosages for the animal study. The current IACUC protocol will be modified to reflect the changes and the modified IACUC protocol will be submitted for approval. What opportunities for training and professional development has the project provided?Training activities: Master student Sukanya Poddarhas been trained to conduct protein extraction from peanut flour, enzymatic hydrolysis of peanut protein, total soluble protein and free amino acid quantification, ACE and renin inhibitory activity tests, as well as in vitro allergenicity test (IgE-binding test). Professional development: She has been trained to do data analysis and poster making, as well as how to make presentations in professional conference. Sukanya was given opportunities to present research results in 104th Annual Conference of North Carolina Association of Family and Consumer Sciences and 55th Annual Meeting of American Peanut Research and Education Society. How have the results been disseminated to communities of interest?The research results were disseminated in 104th Annual Conference of North Carolina Association of Family and Consumer Sciences and 55th Annual Meeting of American Peanut Research and Education Society. The posters were put on the poster wall outside of the lab for all faculty, staffs and student to view after the conferences. What do you plan to do during the next reporting period to accomplish the goals?For the project period starting May 1, 2023 through April 1, 2024, we plan to produce sufficient amount of PPH for animal study using SHR model by the end of August 31, 2023. The PPH will be sent to the subcontract PI at UNC-Chapel Hill to conduct animal study (Objective 3). Meanwhile, the PI and co-PI at NC A&T state University will continue to study renin inhibitory activity the effects of PPH on growth of different species of gut bacteria.
Impacts
What was accomplished under these goals?
Major activities completed: During the reporting period of May 1, 2022 to April 30, 2023, materials required for the research activities were purchased, one master student was recruited and she started the research activity in September 2022, sufficient amount (1.2 kg) of peanut protein concentrate (PPC) was produced from peanut flour with average PPC yield of about 20%. The PPC hydrolysis conditions including Alcalase concentration and hydrolysis time were optimize for producing peanut protein hydrolysate (PPH) with high ACE-inhibitory activity. The PPH samples hydrolyzed for 6, 8 and 10 hours at different Alcalase concentrations were fractionated using ultrafiltration (UF) centrifugal tubes into three fractions with molecular weight range >1 kDa, 5-10kDa and <5kDa, respectively, and the ACE-inhibitory activity of each fraction was tested. The in vitro allergenicity of crude PPH was tested and compared to unhydrolyzed PPC using a completion (inhibition) ELISA using pooled serum of 7 patients who are allergic to peanuts. Specific objectives met: Task 1 and Task 2 of Objective 1 are completed during this reporting period. One new student is recruited and he will start in 2023 Fall. Hopefully, the project progress will be accelerated after the student join the team. Significant results achieved, including major findings, developments, or conclusions (both positive and negative): The protein concentration decreased while the free AA concentration of PPH increased significantly as a result of Alcalase hydrolysis. At same enzyme concentration the free AA increasedlinearly with treatment time (P<0.0001, R2=0.886-0.974) and the percentage of ACE inhibition increased with hydrolysis time in sigmoid pattern, while the protein concentration decreased near linearly (R2=0.811-0.887). At same treatment time, the protein concentration did not change significantly with Alcalase concentration, while free AA and ACE-inhibition% increased significantly (P<0.05). For crude PPH, samples produced at 6hr and 5% Alcalase demonstrated the highest ACE inhibition with IC50 of 5.6 mg/mL, followed by the samples hydrolyzed for 8 or 10 hrs. At concentration of 8 mg/mL, all PPH sample showed more than 50% ACE inhibition. At concentration 10 mg/mL, all PPH sample exhibited more than 60% ACE inhibition which was not affected by protease concentration and hydrolysis time. For fractionated samples, the fraction 3 (F3) (<5kDa) had significantly higher ACE inhibitory activity than other fraction (P<0.05) at same concentration regardless of hydrolysis time and enzyme concentration. Among all fractions, the F3 of PPH produced by hydrolysis of PPC for 6 hours exhibited the highest ACE inhibition with IC50 < 1 mg/mL and reached 61% inhibition at a protein concentration of 1.5mg/ml. However, the yield of F3 is low. The IgE-binding inhibition test results demonstrate significantly reduced allergenicity of PPH compared to unhydrolyzed PPC. The PPH exhibited obviously higher IgE-binding inhibition at concentration of 1 µg and higher. At the same Alcalase concentration, the percentage of IgE-binding inhibition of PPH increased with hydrolysis time. However, there was no further increase after 5hr of hydrolysis, indicating that the maximum reduction of allergenicity could be achieved by 5-hr hydrolysis of PPC under the experimental condition used. Key outcomes or other accomplishments realized: The results from this reporting period shows that the extensive hydrolysis of PPC by Alcalase could result in PPH with moderate ACE-inhibitory activity. Instead of using purified food peptides or the small molecular fraction of PPH which are too expensive to produce, the crude PPH produced under the optimized hydrolysis condition of this study has potential to serve as affordable dietary supplement or therapeutic food to regulate blood pressure for people with hypertension. This will add value to peanut flour which is the protein source for PPH production. Additional benefit of the hydrolysis of PPC is significantly reduced allergenicity which may lowered the incidence of severe peanut allergy due to accidental exposure.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Poddar, S., & Jianmei Yu, J. Free amino acid concentration and ACE-inhibitory activity of Alcalase hydrolyzed peanut protein concentrate. 2023 NCAFCS 104th Annual Conference, April 3-5, 2023, Asheville, NC.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Poddar, S., & Jianmei Yu, J. Effects of PROTEASE CONCENTRATION AND HYDROLYSIS TIME on the ACE-inhibitory activity of alcalase hydrolyzed peanut protein concentrate. 2023 APRES Annual Meeting. July 10-14, 2023, Savannah, GA.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Poddar, S., & Jianmei Yu, J. ACE-inhibitory activity of extensively hydrolyzed peanut protein concentrate. Abstract submitted to 2023 Southeastern Regional Meeting of American Society of Chemistry (SERMACS), October 25-28, 2023, Durham, NC
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