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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