Digestomics of Japanese abalone in real food matrix

Abstract

Objective: Haliotis discus (Japanese abalone), mollusks among various shellfish, is a highly nutritive food resource in the world, but also among the eight allergic food groups accounting for approximately 90% of all immunoglobulin E food allergies worldwide. The general objective of our research is to comprehensively investigate stability and structures of pepsin-resistant allergens, of their larger fragments, and of short digestion resistant peptides (SDRPs) released by pepsin digestion of whole raw and extract of shellfish, under standardized and physiologically relevant gastric conditions. Materials and Methods: Extract of raw whole shellfish (eRSS) and whole raw shellfish (wRSS), were pepsin digested according to standardized static digestion protocol. Controls were treated in a same manner without adding pepsin. Supernatant of samples and its counterpart controls were precipitated with TCA/acetone. Obtained proteins were assessed by 2D SDS PAGE and 1D SDS- PAGE, under reducing and non-reducing conditions. 1D SDS-PAGE of RSS were analyzed by ncLC-MS/MS (Orbitrap LTQ) shot-gun proteomics. Relative quantification was performed by LFQ algorithm within Peaks 8.5 software package Bioinformatics Solutions Inc. (BSI), Waterloo, Canada. Results and Conclusion: 1D SDS-PAGE analysis of eRSS and wRSS, and its controls showed a range of proteins in varied concentrations between 10-250 kDa. In extracted and whole raw shellfish, approximately 22 prominent protein bands were observed including the distinct bands corresponding with the molecular weights of recognized shellfish allergen, tropomyosin (37- 39kDa). Fewer high molecular weight proteins were observed followed by protein smearing, specifically around the low molecular weight protein bands. The smearing could possibly be due to the breakdown products and the glycation. There were slight differences between the protein profiles under reducing and non-reducing conditions as well. Nevertheless, there was the retention of a band in the 37kDa molecular weight marker in all 4 samples, likely consistent with heat stable tropomyosin (TM). Mass spectrometry showed allergens that are characterized (Hal d 1 and Hal di 1), with 90% of sequence homology with main tropomyosin allergens from seafood. Scientific impact and relevance: The results will highlight effects of food matrix on shellfish allergens digestibility proving its relevancy in molecular allergology. Moreover, an insight will be obtained on the differences in digestibility of allergenic versus non-allergenic tropomyosins in the real food matrix.