Quek, Siew YoungWai, Meng WooDong, Xuan2025-02-182025-02-182024https://hdl.handle.net/2292/71396Hempseed protein isolate (HPI), obtained as a by-product following hempseed oil extraction, has garnered increasing interest due to its sustainability and nutritional quality. The production of HPI typically involves defatting hempseed, extracting protein, and finally dehydrating. Despite its rising popularity, the impact of various processing techniques on HPI remains under-explored, presenting a challenge for the food industry in utilising HPI effectively. This research endeavours to bridge these gaps by investigating the effects of various processing methods on HPI and exploring the bioactive properties influenced by enzymatic hydrolysis. The impact of two prevalent industrial drying methods, spray drying and freeze drying, on the physicochemical properties, functionality, and digestibility of HPI was investigated. Both techniques modified the physicochemical and structural properties compared to undried-HPI. Notably, the protein denaturation temperature increased to approximately 90°C in both freeze-dried and spray-dried samples, compared to 82.5°C in undried HPI. Electrophoresis showed the disappearance of 26 and 17 kDa protein bands post-drying. In vitro simulated gastrointestinal digestion indicated that the digestibility of HPI improved after drying, but there was a 50% reduction in the release of free α-amino groups. Notably, freeze-dried HPI exhibited higher emulsifying stability and oil-holding capacity than the spray-dried sample. Further investigation assessed the impact of processing steps at the proteome level. Specifically, the effects of alkaline and micellisation extraction methods, along with freeze drying and spray drying, on the proteome profile of HPI were analysed. Findings revealed that extraction methods impacted the protein profiles of HPI more than the drying methods. Specifically, micellisation-extracted HPI showed higher albumin, oleosin, and sulphur-containing protein levels than alkaline extracted HPI. The alkali-extracted undried sample (AU) showed more potentially allergenic proteins, including Hsp70 and triosephosphate isomerase, than its micellization-extracted counterpart (MU). Unique potential allergens were identified, including malate dehydrogenase and enolase in AU, and RuBisCo in MU samples. Both drying processes impacted the HPI proteome and reduced RuBisCo in the micellisation extracted HPI. Spray drying demonstrated a more pronounced impact on the proteomic profile of HPI than freeze drying. The bioactive peptides release profiles of HPI samples were further explored through a comprehensive workflow combining in silico screening and prediction with in vitro validation. Using an in silico approach, 13 major HPI proteins were hydrolysed by 20 selected enzymes, predicting 20 potential bioactivities. With papain hydrolysis, dipeptidyl peptidase-IV (DPP4) and angiotensin-converting enzyme (ACE) inhibitory activities emerged as having the highest potential. In vitro experiments confirmed these predictions, with DPP4 and ACE inhibitory activities displaying IC50 values of 0.32–0.42 mg/mL and 6.8–9.17 µg/mL, respectively. A strong correlation (r2 = 0.96) was observed between the in vitro and the in silico predicted data when correlating with protein abundancy. The findings of this study reflect the importance of selecting appropriate methods for optimising HPI processing in the food industry. These insights facilitate improved strategies for HPI production and its application in food products. Additionally, the research showcased a practical, integrative approach for predicting bioactive peptides in food proteins, providing valuable guidance on its processing to create value-added products.https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htmThesisCopyright: The authorAttribution-ShareAlike 4.0 Internationalhttp://creativecommons.org/licenses/by-sa/4.0/