UTILIZATION OF NON-THERMAL PROCESSING METHODS ON REDUCTION OF ANTINUTRIENTS AND IMPACT ON NUTRIENTS AND POLYPHENOL CONTENT IN INDUSTRIAL HEMP

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1026499
• Grant No.: 2021-67034-35175
• Cumulative Award Amt.: $164,922.00
• Proposal No.: 2020-10671
• Project Start Date: Jun 15, 2021
• Project End Date: Jun 14, 2024
• Grant Year: 2021
• Program Code: [A7201]- AFRI Post Doctoral Fellowships

Recipient Organization
ALABAMA A&M UNIVERSITY
4900 MERIDIAN STREET
NORMAL,AL 35762

Performing Department
(N/A)

Non Technical Summary
This proposal addresses the 2020 Educational Workforce Development- Postdoctoral Fellowships Grant (Program Code A7201). The major academic program being addressed is Food safety, nutrition, and health. Hemp (Cannabis sativaL.) is an ancient Asian crop that has been grown and cultivated for a long time. The hemp seed contains a myriad of essential nutrients and bioactive compounds. However, the presence of antinutrients in hemp prevents their bioavailability. This research seeks to study the utilization of non-thermal processing methods in reducing the antinutrients and the effect on essential nutrients and polyphenol content in Industrial hemp seeds. This research will be accomplished under four objectives: (i) to optimize the most effective processing method (HPP, ultrasonication, enzyme hydrolysis) for reducing antinutrients in hemp seeds; (ii) to determine the impact of non-thermal processing methods on the nutrition profile and bioactive compounds of hemp seeds; (iii) to determine the functionality and bioactivity of the hemp seed flour post-processing,and (iv) to provide postdoctoral support that will further the PD's training in FAHNS to become an independent investigator.This research is relevant to the goals of AFRI and education workforce development by solving key problems affecting local, national, and global importance. Furthermore, this research will benefit growers of industrial hemp, an underutilized essential crop, also will provide the essential information to improve overall health.

Animal Health Component

30%

Research Effort Categories

Basic

70%

Applied

30%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
501	1730	1150	100%

Knowledge Area
501 - New and Improved Food Processing Technologies;

Subject Of Investigation
1730 - Hemp;

Field Of Science
1150 - Toxicology;

Keywords

bioactive compounds

hemp seeds

nutrients

processing

Goals / Objectives
Themajor goal of this project is to utilize non-thermal processing in reducing antinutrients and impact on essential nutrients and polyphenols in industrial hemp seeds. The objectives include (i) to optimize the most effective processing (HPP, ultrasonication, enzyme hydrolysis) for reducing antinutrients in hemp seeds; (ii) to determine the impact of non-thermal processing methods on the nutrition profile and bioactive compounds of hemp seeds; (iii) to determine the functionality and bioactivity of the hemp seed flour post-processing, and (iv) to provide postdoctoral support that will further the PD's training in FAHNS to become an independent investigator.

Project Methods
The primary goal of this project is to utilize non-thermal processing in reducing antinutrients in industrial hemp seeds. The objectives include (i) to optimize the most effective processing (HPP, ultrasonication, enzyme hydrolysis) for reducing antinutrients in hemp seeds; (ii) to determine the impact of non-thermal processing methods on the nutrition profile and bioactive compounds of hemp seeds; (iii) to determine the functionality and bioactivity of the hemp seed flour post-processing; and(iv) to provide postdoctoral support that will further the PD's training in FAHNS to become an independent investigator. The hemp samples will be procured from the Winfred Thomas Agricultural Research Station, Alabama A&M University. Three different non-thermal processing (HPP, ultrasonication, and enzyme hydrolysis) will be used to optimize the best process for reducing the antinutrients in hemp seeds. The phytic acid, trypsin inhibitors, saponins, and tannin content will be evaluated on pre-processed hemp and the control (unprocessed hemp) using standard procedures. The results collected will provide more data regarding non-thermal processing in reducing antinutrients in hemp seeds. The proximate analysis will be determined based on the official guidelines of AOAC. The hemp seeds flour will be used to analyze the mineral content using the Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES). The hemp flour will be extracted with 80% ethanol (v/c) and analyzed on a Varian Prostar high-performance liquid chromatography (HPLC) system (Varian Inc., Palo Alto, CA, USA). Compounds separation will be performed on a 250 mm × 4.6 mm i.d. Supercosil LC-18 reverse-phase column (Supelco Inc., Bellefonte, PA, USA). Quantitation of identified compounds will be done using the calibration curves of the pure standards at different concentrations. The amino acid composition of non-thermal processed hemp flour will be determined using an Amino Acid analyzer. The fatty acid composition of pre-processed hemp will be determined by extraction with ether and determined using Gas Chromatography (GC MS) with flame ionization detector based on the AOAC 996.06 method. The hemp protein isolate (HPI) will be isolated and freeze-dried to produce HPI powder. The hemp hydrolysate protein (HHP) will be enzymatically hydrolyzed, freeze-dried, and stored at -20° C until needed for further analysis. The protein's functional properties, such as protein solubility, water and oil holding capacity (WHC/OHC), and foam capacity, will be determined. The antioxidant and radical scavenging activities of HPI and HPH will be investigated using the FRAP (ferric-reducing/antioxidant power) assay and DPPH (α,α-Diphenyl-β-picrylhydrazyl) assay. The anti-inflammatory effects of HPI and HPH will be explored using RAW264.7 murine macrophages activated by LPS. The effect of HPI and HPH on the production of the pro-inflammatory cytokines tumor necrosis factor (TNF)- α, interleukin (IL)-1β, IL-6, IL-8, monocyte chemoattractant protein (MCP)-1 and the activity of the metalloproteinase (MMP)-9 will be determined via Elisa and Western blot analysis. The expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2 and regulation of NF-κB signaling pathway will also be determined via Quantitative real-time PCR (qPCR) and Western blot. The level of intracellular ROS will be assessed by measuring the oxidation of the fluorescent probe 2′, 7′ -dichlorofluorescin diacetate. The anti-cancer effects of HPI and HPH will be determined using HDAC Activity/Inhibition Direct Assay kit and the EpiQuik DNMTs Activity/Inhibition Assay Kit (Epigentek, Farmingdale, NY, USA) according to the manufacturer's instructions. The effects of HPI and HPH on the expression of HDAC and DNMT will be determined by Western blot and Q-PCR analysis. Apoptotic population analysis will be done by AnnexinV- Fluorescein isothiocyanate (FITC) apoptosis detection kit (Sigma), strictly following manufacturer instructions. Caspase 3/7 activities will be determined using ApoOne Kit (Promega Corp., Madison, WI, USA). The effects of HPI and HPH on biomarkers relevant to breast cancer, colon cancer, and lung cancer will be determined. Microscopic evaluation to record changes in the morphology of the cells due to the cytotoxicity effect of HPI and HPH will be determined using a phase-contrast (Transmission) microscope. Cells will be imaged on the EVOS® FL Auto system. The morphological changes of the apoptotic cells will be observed using a phase-contrast inverted microscope. Bioactive peptides in hemp have been reported to have beneficial properties on human health. This includes antioxidative, anti-inflammatory, and anti-cancer properties. The bioactivity is conducted to understand how hemp can affect biological change in targeted cells. The results could further be translated into animal models to investigate further their benefits in managing diseases. Statistical analyses of data will be conducted using one-way ANOVA (SPS for Windows, SAS). Tukey's LSD tests will be used to compute individual pairwise comparisons of means (P<0.05).

Progress 06/15/21 to 05/28/24

Outputs
Target Audience:The target audience for this project includes North Alabama hemp growers, U.S. hemp industry stakeholders, the food industry, graduate students, graduate mentees, and the university community. Changes/Problems:Unfortunately, cell culture was not included in the proposed research plan because of budget constraints. Due to these limitations, it was deemed unfeasible to conduct cell culture experiments within the allocated timeframe. What opportunities for training and professional development has the project provided?The USDA-NIFA postdoctoral grant has significantly contributed to my development as an independent investigator. It provided opportunities to enhance my skills in grant writing, project management, and scientific communication through conferences and webinars. How have the results been disseminated to communities of interest?Results from this project were disseminated at several conferences. Association of Research Directors (ARD) biennial conference in Nashville, Tennessee, from April 6th to 9th, 2024, and Atlanta, Georgia, from April 2-5, 2022. IFT Annual Scientific Conference and Food Expo in Chicago, IL, from July 16-19, 2023. Annual STEM Day at Alabama A&M University in April 2023. Virtual Cannabis research conference in August 2022 IFT Annual Scientific Conference and Food Expo, Chicago, IL., from July 11-13, 2022 What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1 experimented with various methods (Cold Plasma [CP], ultrasonication, and enzyme hydrolysis) to reduce antinutrients (tannins, phytic acid, and saponins) in industrial hemp seeds. The results showed that CP was most effective in reducing the antinutrients in industrial hemp seeds. These results demonstrated that utilizing CP technology could help make nutrients more bioavailable by alleviating the problems of antinutrients. The data was presented at the IFT Annual Scientific Conference and the Association of Research Directors (ARD)conferences. Manuscript is in preparation for publication. Objective 2 was conducted to determine the effect of non-thermal processing methods on the composition of industrial hemp seeds.Results showed that ultrasonication led to a higher yield of proteins, fat, and minerals content compared to the other treatments. Furthermore, ultrasonication and enzyme hydrolysis with umamizyme increased the phytochemical contents of industrial hemp seeds. Enzyme hydrolysis with alcalase yielded the most amino acids compared to other treatments. The treated hemp seeds were tested for their techno-functional properties(water absorption and oil capacity, emulsifying capacity and stability, and foam capacity and stability). The results indicated that the enzyme-treated and plasma-treated hemp seeds showed higher water-holding capacity, emulsifying capacity, emulsifying stability, foam capacity, and foam stability than the untreated hemp seeds. These findings suggest that the enzyme hydrolysis and plasma-treated hemp seeds possess unique functional properties, making them an essential ingredient for various food products. This postdoctoral Grant from USDA-NIFA has contributed significantly to my training and development as an independent investigator. This support has enabled me to acquire the skills to initiate and lead my research projects, such as designing experiments, analyzing data, and drawing independent conclusions. Moreover, I received mentorship from experienced investigators, which helped me establish my research program and mentor undergraduate and graduate students. As part of the grant, I attended online and in-person conferences, enhancing my skills in grant writing, project management, and scientific communication. Overall, this postdoctoral fellowship has provided me with the resources and opportunities that have strengthened my research skills, built my professional network, and, ultimately, paved the way to a successful transition to an independent investigator role.

Publications

• Type: Journal Articles Status: Published Year Published: 2024 Citation: Tasie, O., Boateng, J., & Cebert, E. (2024). Screening for Anti-nutrients, Antioxidants, Minerals, and Cannabinoid Content in Hemp (Cannabis sativa L.) Seed Cultivars. EC Nutrition, 19(1), 01-15
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Tasie, O., & Boateng, J. (2024). Effect of Cold Plasma Technology on the Functional Properties of Shelled Hemp Seeds (Cannabis sativa L). Presented at the 22nd Biennial Research Symposium of the Association of Research Directors (ARD) Inc., Nashville, Tennessee, April 6-9th, 2024
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Tasie, O. Boateng, J., and Kassama, L. S. (2023). Utilization of Ultrasonication as a pre-treatment in reducing Antinutrient Factors in Shelled Hemp Seeds (Cannabis sativa L). Presented at the IFT Annual Scientific Conference and Food Expo, July 16 19, Chicago, IL
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Kunisetty, M. S. S., O. Tasie, J. Boateng, L. S. Kassama, A. Jackson-Davis, S. R. Mentreddy, G. Xu, & B. Ghimire. Effect of Cold Plasma on antinutrients and antioxidants in shelled hemp seeds (Cannabis sativa L.). Presented at Science, Technology, Engineering, and Mathematics (STEM) Day, Alabama Agricultural and Mechanical University, Normal AL, April 2023
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Tasie, O. Boateng, J., Cebert, E., & Kuang, X. (2022). Assessment of Hemp (Cannabis sativa L.) cultivars for antioxidant content and antinutrient factors; Virtual Cannabis Research Conference, August 8-10, 2022
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Tasie, O., Cebert, E., & Boateng, J. (2022). Antinutrient factors and antioxidants content of twelve industrial hemp seed (Cannabis sativa L) cultivars. Presented at the IFT Annual Scientific Conference and Food Expo, Chicago, IL. July 11-13, 2022
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Tasie, O. and J. Boateng. (2022). Screening of Hemp (Cannabis sativa L.) for antioxidant content and antinutrient factors. Presented at the Association of Research Directors (ARD) Biennial Symposium 2022.

Progress 06/15/22 to 06/14/23

Outputs
Target Audience:The target audience for this project includes North Alabama hemp growers, U.S. hemp industry stakeholders, the food industry, graduate students, graduate mentees, and the university community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided me with the opportunityto mentorgraduate students. Imoderated a live webinar for the Institute of Food Technologists on Global African, African American, and Black Experiences in Food Science Academia. Additionally, I have collaborated with faculty, staff, and students to organize the Food Science Department Homecoming Alumni Panel at Alabama A&M University. I have also been actively involved in professional societies. I have provided laboratory instruction to both undergraduate and graduate students. How have the results been disseminated to communities of interest?Some results were presented at the Annual Institute of Food Technologists (IFT) conference and at the Annual STEM Day at Alabama A&M University, attended by faculty, scientists, students, and federal agencies. What do you plan to do during the next reporting period to accomplish the goals?Regarding objective 3b, which is to determine the bioactivity of the hemp seed flour, I am currently working on it and aim to complete it by Spring 2024.

Impacts
What was accomplished under these goals? I have completed the first objective of my research project. This objective was to optimize the most effective processing method for reducing antinutrients in hemp seeds. I have also completed the project's second objective, which involved determining the impact of non-thermal processing methods on the nutrition profile and bioactive compounds of hemp seeds. I have collected all the necessary data, and the results are promising. In addition, I have achieved objective 3a, which was to determine the functional properties of the processed hemp seeds. The findings suggest that processed hemp seeds possess unique functional properties, making them an essential ingredient for various food products.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: O. Tasie, J. Boateng, and L. S. Kassama. Utilization of Ultrasonication as a pre-treatment in reducing Antinutrient Factors in Shelled Hemp Seeds (Cannabis sativa L). Presented at the IFT Annual Scientific Conference and Food Expo, July 16-19, Chicago, IL.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Kunisetty, M. S. S., O. Tasie, J. Boateng, L. S. Kassama, A. Jackson-Davis, S. R. Mentreddy, G. Xu, & B. Ghimire. Effect of Cold Plasma on antinutrients and antioxidants in shelled hemp seeds (Cannabis sativa L.). Presented at the Science, Technology, Engineering, and Mathematics (STEM) Day, Alabama Agricultural and Mechanical University, Normal AL, April 2023.

Progress 06/15/21 to 06/14/22

Outputs
Target Audience:The target audience for this project is North Alabama hemp growers, U.S. hemp industry stakeholders, the food industry, graduate students, graduate mentees, and the university community. Changes/Problems:As a result of COVID in 2021, the acquisition of hemp seed cultivars was delayed. Renovation of the FAS instrumentation laboratory from fall 2021 to spring 2022 prevented access to major pieces of analytical equipment, thus some parts of objectives 1 and 2 have not been completed. This will be completed in the summer and fall of 2022. What opportunities for training and professional development has the project provided?I mentored students and had one of my high school mentees graduate this spring of 2022 with a bachelor's degree in food science. Attended webinars and departmental seminars. I participated in a hemp series webinar offered by the Cornell CALS and USDA-ARS and volunteered as a canning specialist to USAID ACDI/VOCA, Farmer-to-Farmer Program Tajikistan, on homemade canning and food preservation virtually to women in Tajikistan. I participated in educational programs at elementary and middle schools in Huntsville Alabama and provided laboratory instruction, and experiential training to undergraduate students. I have also assisted in teaching classes- Food Analysis and Food and Survival of man to undergraduates. Actively engaged in professional societies: Institute of Food Technologists, Committee Member, Membership Qualifications and Evaluation Committee of Phi Tau Sigma Food Science Honor Society, and the American Society for Nutrition How have the results been disseminated to communities of interest?Some of the results were presented at the biennial ARD symposium which was attended by faculty, scientists, students, and federal agencies who were eager to listen and have discussions concerning industrial hemp seed cultivars. What do you plan to do during the next reporting period to accomplish the goals?This summer, enzyme hydrolysis will be completed. Objectives 2 and 3 will be completed by fall.

Impacts
What was accomplished under these goals? Twelve hemp seed cultivars: Altair, Anka,International hemp, CFX-1, CFX-2, Hemp Grandi, Hemp , Rohrer H51, LARA, and Uni seeds Vega were screened for polyphenol, antioxidant activities, and antinutritional factors. The results obtained showed hemp seeds as a good source of polyphenols. Based on the results and availability of hemp seed cultivars, three cultivars have been selected to use in non-thermal processing.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: O. Tasie and J. Boateng. Screening of Hemp (Cannabis sativa L.) for antioxidant content and antinutrient factors. Presented at the Association of Research Directors (ARD) Biennial Symposium 2002. April 2-5, 2022.Atlanta Georgia.