Biofunctionality of Enzymatically Derived Peptides from Codfish (Gadus morhua) Frame: Bulk in vitro Properties, Quantitative Proteomics, and Bioinformatic Prediction

Abstract

Protein hydrolysates show great promise as bioactive food and feed ingredient and for valorization of side-streams from e.g. the fish processing industry. This study characterizes bulk emulsifying, foaming, and in vitro antioxidative properties of hydrolysates derived from cod frame by application of Alcalase and Neutrase, individually and sequentially as well as the influence of heat-treatment prior to hydrolysis. We present a novel approach that utilizes proteomics data for calculation of weighted mean peptide properties (length, molecular weight, and charge) and peptide-level abundance estimation. Using subsequent bioinformatic prediction of biofunctional properties to describe observed bulk properties, we are able to provide an in-depth hydrolysate characterization not previously seen. All hydrolysates displayed comparable or higher emulsifying activity and stability than sodium caseinate. Heat-treatment significantly increased stability but showed a negative effect on the activity and degree of hydrolysis. Combining peptide abundance with predicted emulsifying activity, we were able to identify several peptides that are likely linked to the observed differences in bulk emulsifying properties. In general, decreased hydrolysis resulted in significantly higher chelating activity, while the opposite was observed for radical scavenging activity. The study highlights the prospects of applying proteomics and bioinformatics for hydrolysate characterization and in food protein science.

Targeted hydrolysis of native potato protein: A novel route for obtaining hydrolysates with improved interfacial properties

“…Degree of hydrolysis (DH) was determined based on α-amino nitrogen content as previously described (Jafarpour, Gregersen, et al, 2020) with minor modifications, adjusting for the α-amino nitrogen content of the untreated substrate. Briefly, free α-amino content of PPHs was determined using the PFAN-25 free amino nitrogen assay kit (PractiChrom, USA) measured using A 530 on a Picoexplorer (USHIO INC, USA), according to manufacturer guidelines, using glycine as reference for standard curve generation.…”

“…Emulsifying activity index (EAI) and emulsion stability index (ESI) were determined using the method described by (Pearce & Kinsella, 1978) with slight modifications, previously described (Jafarpour, Gregersen, et al, 2020). Briefly, 15 mL PPH in distilled water (2 mg/mL) was mixed with 5 mL rapeseed oil by an ultraturax homogenizer (IKA, Germany) at speed of 9,500 rpm for 60 s without pH adjustment.…”

“…Foaming capacity (FC) and foaming stability (FS) were determined according to the method of (Elavarasan, Naveen Kumar, & Shamasundar, 2014) and calculated as previously described (Jafarpour, Gregersen, et al, 2020). Accordingly, 0.1 % (w/v) PPH in distilled water was homogenized at 9,500 rpm for 120 s using an Ultraturrax (IKA, Germany), and poured into a 200 mL graduated cylinder.…”

“…Although the specific mechanisms and prerequisites for potent peptide emulsifiers still remains only superficially characterized, our understanding of the underlying molecular properties continue to expand (Ricardo, Pradilla, Cruz, & Alvarez, 2021). Recent work has investigated the influence of factors such as interfacial peptide structure (Dexter, 2010; Du et al, 2020; García-Moreno et al, 2021; Lacou, Léonil, & Gagnaire, 2016), physico-chemical properties such as length and charge (García-Moreno, Gregersen, et al, 2020; Lacou et al, 2016; Liang et al, 2020; Yesiltas et al, 2021), amino acid composition (Enser, Bloomberg, Brock, & Clark, 1990; Saito, Ogasawara, Chikuni, & Shimizu, 1995; Siebert, 2001), and specific sequence patterns (Jafarpour, Gregersen, et al, 2020; Mondal et al, 2017; Nakai et al, 2004; Wychowaniec et al, 2020). Although various factors do appear to influence emulsification, the potential appears to indeed depend significantly on the propensity of a given peptide to adopt a more well-defined amphiphilic structure at the interface (Dexter & Middelberg, 2008; Enser et al, 1990; Saito et al, 1995).…”

“…This property, in turn, is governed by these underlying factors. Although not appearing to be governed by the exact same molecular mechanisms, the stabilization of the air-water interface in foams have been suggested to also depend on peptide amphiphilicity (Enser et al, 1990; Jafarpour, Gregersen, et al, 2020).…”