BIOACTIVE AND ALLERGENIC PROPERTIES OF EDIBLE CRICKET (GRYLLODES SIGILLATUS) PEPTIDES
Creators: name: Hall, Felicia G
Publisher: name: Purdue University Graduate School
Year: 2020
Subjects: Food sciences not elsewhere classified
Medical biochemistry - proteins and peptides (incl. medical proteomics)

description: Cardiovascular diseases (CVD) and their risk factors remain the leading cause of morbidity and mortality in North America. Food-derived bioactive peptides (BAP) have been shown to play a role in regulating physiological pathways of CVD risk factors including hypertension, diabetes, and chronic inflammation. Common sources of BAP include dairy and plant proteins. In addition to being an alternative protein source, it is now accepted that edible insect proteins can also confer health benefits beyond nutrition. However, as with any novel protein source, allergenicity remains a major concern surrounding edible insect consumption. This dissertation aimed to: 1) Evaluate the bioactive potential of peptides from an edible cricket species and; 2) determine the effects of BAP production methods on immunoreactivity. First, peptide-rich extracts were generated from farmed food-grade crickets via enzymatic hydrolysis techniques with the commercial protease Alcalase™. To measure the <i>in vitro</i> bioavailability, cricket peptides were also subject to simulated gastrointestinal digestion (SGD). Peptides and their digests were tested for relevant bioactivities and active groups were further fractionated by chromatographic methods to identify the major peptides responsible for the bioactivity. When tested for <i>in vitro</i> antihypertensive and anti-glycemic properties, cricket peptides were found to inhibit the activities of angiotensin converting enzyme, dipeptidyl peptidase-4, α-amylase, and α-glucosidase. The anti-inflammatory potential was expounded using RAW-264.7 macrophages and human umbilical vein endothelial cells (HUVEC). Cricket peptides (after SGD) effectively lowered NF-κB, MCP-1, and IL-6 production in cells without affecting their viability. Proteomic analyses identified 18 sequences from the enriched cationic peptide fraction that showed the highest activity. Three novel peptides were identified via molecular docking, as potent ACE-inhibitors and binding was similar to that of the commercial drug captopril. Key binding characteristics included interaction with hydrophobic amino acids (Phe, Pro, Leu) near the C-terminal position and coordination with Zn (II) ions near the ACE active site. Immunological reactivity was measured by IgE-binding from shrimp-allergenic patient sera to antigens present within cricket peptides. Our studies demonstrate that immunoreactivity was impacted by enzymatic hydrolysis, depending on the conditions and heating source used. Tropomyosin (a major shrimp allergen) was extracted from both untreated crickets and protein hydrolysates, and verified as the major reactive protein. Tropomyosin reactivity decreased (under both partial and extensive hydrolysis) or retained (under conditions which prevented epitope cleavage). However, using microwave-assisted enzymatic hydrolysis was effective at decreasing tropomyosin reactivity in all immunoassays tested (IgG and IgE). Proteomic and immunoinformatic analyses revealed prominent allergen binding regions of cricket tropomyosin available for cleavage during enzymatic hydrolysis. Conserved antigen regions showed greater homology with other crustacean species, but not with other well studied allergenic insect proteins (i.e., cockroach). Lastly, LC-MS/MS and FT-Raman spectrometry suggests that reactivity was affected due to distinct epitope cleavage within the protein instead of decreased antigen extractability/solubility. The findings of this dissertation support that edible cricket proteins are a potential source of bioactive peptides for functional food or nutraceutical development. Additionally, using protein extraction methods such as microwave-assisted hydrolysis seems a promising tool for minimizing the immunoreactivity of the allergen present in this edible cricket species.