The world\'s hunger for novel food ingredients drives the development of safe, sustainable, and nutritious novel food products. For foods containing novel proteins, potential allergenicity of the proteins is a key safety consideration. One such product is a fungal biomass obtained from the fermentation of Rhizomucor pusillus. The annotated whole genome sequence of this strain was subjected to sequence homology searches against the AllergenOnline database (sliding 80-amino acid windows and full sequence searches). In a stepwise manner, proteins were designated as potentially allergenic and were further compared to proteins from commonly consumed foods and from humans. From the sliding 80-mer searches, 356 proteins met the conservative >35% Codex Alimentarius threshold, 72 of which shared ≥50% identity over the full sequence. Although matches were identified between R. pusillus proteins and proteins from allergenic food sources, the matches were limited to minor allergens from these sources, and they shared a greater degree of sequence homology with those from commonly consumed foods and human proteins. Based on the in silico analysis and a literature review for the source organism, the risk of allergenic cross-reactivity of R. pusillus is low.

In this study, we present a systematic proteomic overview of macadamia nut using a label-free shotgun proteomic approach. We identified 947 proteins in 723 clusters and gene ontology analysis revealed proteins across 46 functional categories including carbohydrate metabolism (10%), protein metabolic processes (5%), amino acid metabolism (4%), transport (4%), stress response (3%), lipid metabolism (3%), protein folding (3%) and defense response (1.4%). The defense response proteins accounted for 24% of the total peptide abundance. The vicilin-like macadamia antimicrobial peptides 2-3 (MiAMP2) was the most abundant protein, followed by glyceraldehyde-3-phosphate dehydrogenase 3, 11S legumin-like protein, 2-phospho-D-glycerate hydrolase and heat shock 70 kDa protein among others. The cascading of amino acid and carbohydrate metabolic pathways in macadamia nut were constructed against reference maps from KEGG and proposed for the first time. Results were also indicative of useful protein candidates with possible allergenic potential and cross-reactivity in macadamia nut. The in-silico analysis revealed homology and linear epitope similarities to known allergens such as conglutin β allergen from lupin, Jug r2 vicilin allergens from walnut, Ara h3 11S globulin from peanut, small rubber particle protein Hev b3, hevein, enolase 2, HSP 70kDa Cla h4, Der f28 allergen, and methylglyoxalases. Label-free shotgun proteomics reveal valuable insights into the genetic and biological makeup of macadamia nut proteome and provide guidance on protein candidates with allergenic potential for further immunological investigation. Data are available via ProteomeXchange with identifier PXD015364.