The major goal of this proposal is to increase the knowledge and applications of hemp products, and for this particular project, of hempseed meal after protein extraction at pH 10 (HM-PI), protein isolate extracted at pH 10 (HPI-10), and protein isolate extracted at pH 12 (HPI-12) in food manufacturing. This will make industrial hempseed an alternative source of food and food ingredients which will have a long-term positive impact on US agriculture and population, most importantly with the passage of Farm Bill 2018. HM-PI and HPI-12 are novel food ingredients whose physicochemical property and functionality, as well as safety and food applications, are unknown. Our preliminary data show unique characteristics of these ingredients warranting further study. This proposal will then lead to value addition to co-products after isolation of industrial HPI at pH 10 and the production of industrial hempseed oil. This proposal demonstrates innovative approaches in the fundamental understanding on how HM-PI, HPI-10, and HPI-12 can serve as novel food ingredients by 1) determining the nutritional quality and safety profile (allergenicity, impact on metabolome, and blood chemistry) of HM-PI, HPI-10, and HPI-12; 2) evaluating the impact of enzymatic treatment on the physicochemical and functional properties of HM-PI, HPI-10, and HPI-12; and 3) evaluating antioxidant effect of HM-PI, HPI-10, and HPI-12 in foods.OBJECTIVE 1. Assess the nutritional quality and safety of HM-PI, HPI-10, and HPI-12The nutrional quality of HM-PI, HPI-10, and HPI-12 will be assessed using both in vitro and in vivo methods as detailed below. Another important parameter determining potential application of protein-based ingredients is allergenicity. . The reactivity of HM-PI, HPI-10, and HPI-12 to BIG 8 allergens plus sesame (now the 9th major allergen as promulgated by Food Allergy, Safety, Treatment, Education, and Research Act). will be studied in this project. We hypothesize that HM-PI, HPI-10, and HPI-12 as novel food protein ingredients from hempseed with nutritional quality that are safe for consumption. The goal is to determine the nutritional quality and safety profile (allergenicity, blood chemistry, effect on the metabolome) of HM-PI, HPI-10, and HPI-12. HM-PI refers to the meal after extraction at pH-10, HPI-10 is the protein isolate from defatted meal, and HPI-12 is the protein isolate extracted from defatted meal. We expect to show the high nutritional quality of HM-PI, HPI-10, and HPI-12 for their potential use as food ingredients. In addition, the allergenicity test and feeding study will establish the safety of these novel ingredients from industrial hempseed.OBJECTIVE 2. Evaluate physicochemical properties and functionality of HM-PI, HPI-10, and HPI-12 as affected by enzymatic modificationProtein-based ingredients serve both nutritional and non-nutritional purposes in foods. The non-nutritional purpose of protein in foods is mostly associated with the ability of proteins to interact with water as well as to provide and stabilize the structure in individual foods known as their functional properties. This objective will evaluate these functional properties as well as the effect of enzymatic modification on the physicochemical and functional proprerties of HM-PI, HPI-10, and HPI-12. We hypothesize that HM-PI, HPI-10, and HPI-12 can be used as novel food ingredients with unique physicochemical and functional properties with adequate nutritional quality. The goal is to characterize HM-PI, HPI-10, and HPI-12 and determine the effect of enzymatic modifications on their physicochemical and functional properties. We expect to determine the functionality of HPI-12 which has not been reported previously. Also, the effect of enzymatic modifications on the physicochemical and functional properties of HM-PI, HPI-10, and HPI-12 will be established in comparison to SPI, most commonly used plant-based protein ingredients in food.OBJECTIVE 3. Evaluate the antioxidant effect of HM-PI, HPI-10, HPI-12 and their enzymatic derivatives in food systems.Proteins are known to contain amino acid sequences encrypted in their native structure that can be released upon hydrolysis. These hydrolysates contain bioactive peptides due to low molecular weight that can easily escape enzymatic degradation in the gastrointestinal tract, and therefore can be absorbed and circulate in the bloodstream. One of these biological activities include antioxidant property. There are multiple studies showing in vitro antioxidant property of hempseed protein hydrolysates. Our previous study also showed that pepsin-pancreatin hydrolysis of HPI-10 resulted in better in vitro antioxidant activity. This increased antioxidant activity of the hydrolysates agree with previous reports, but the application of these enzymatic derivatives of HM-PI, HPI-10, and HPI-12 as food ingredients with antioxidant activity is currently unknown. Hence, we hypothesize that enzymatic derivatives of HM-PI, HPI-10, and HPI-12 can be used as functional food ingredients with antioxidant activity in food systems. The objective is to determine the antioxidant potential of HM-PI, HPI-10, HPI-12, and their enzymatic derivatives in food emulsion systems. Hempseed oil-in-water emulsion, ground turkey meatball, and fish cake will be used as model systems to evaluate antioxidant activity. We expect to establish the application of HM-PI, HPI-10, and HPI-12 enzymatic derivatives as antioxidants in food emulsion systems. This will be the first investigation on how these industrial hempseed components can serve as antioxidant ingredients in both hempseed oil-in-water emulsion and meat emulsion.The expected results of this project will improve understanding of the physicochemical properties, functionality, and nutritional quality of hemp-based novel food ingredients leading to industrial hemp value addition and US agriculture enhancement.