Chicken Combs and Wattles as Sources of Bioactive Peptides: Optimization of Hydrolysis, Identification by LC-ESI-MS2 and Bioactivity Assessment

Abstract

The production of bioactive peptides from organic by-waste materials is in line with current trends devoted to guaranteeing environmental protection and a circular economy. The objectives of this study were i) to optimize the conditions for obtaining bioactive hydrolysates from chicken combs and wattles using Alcalase, ii) to identify the resulting peptides using LC-ESI-MS² and iii) to evaluate their chelating and antioxidant activities. The hydrolysate obtained using a ratio of enzyme to substrate of 5% (w/w) and 240 min of hydrolysis showed excellent Fe²⁺ chelating and antioxidant capacities, reducing Fe³⁺ and inhibiting 2, 2'-Azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. The mapping of ion distribution showed that a high degree of hydrolysis led to the production of peptides with m/z ≤ 400, suggesting low mass peptides or peptides with multiple charge precursor ions. The peptides derived from the proteins of cartilage like Collagen alpha-2(I), Collagen alpha-1(I), Collagen alpha-1(III) and elastin contributed to generation of bioactive compounds. Hydrolysates from chicken waste materials could be regarded as candidates to be used as ingredients to design processed foods with functional properties.

Keywords: LC-ESI-MS2; bioactive compounds; chicken by-products; free radicals; protein hydrolysate.

Figure 1

(A) Response surface for the dependent variable “degree of hydrolysis”. (B) Desirability graph of the degree of hydrolysis. DH (%), degree of hydrolysis; E:S (%), enzyme to substrate ratio; Time (minutes), hydrolysis time.

Figure 2

(A): lectrophoretic profile (SDS-PAGE) and (B): electrophoretic profile (SDS-PAGE- Tricine). C + W: mixture of combs and wattles; <DH: protein hydrolysate of the chicken comb and wattle mixture with the lowest degree of hydrolysis; >DH, protein hydrolysate of the chicken comb and wattle mixture with the highest degree of hydrolysis.

Figure 3

(A) Reducing capacity of Fe³⁺ and chelation to the Fe²⁺ metal of the chicken comb and wattle mixture (1:1 w/w) and protein hydrolysates; (B) ability of the chicken comb and wattle mixture (1:1 w/w) and protein hydrolysates to eliminate ABTS^● and DPPH^● radicals. C + W, chicken comb and wattle mixture; <DH, protein hydrolysate of the chicken comb and wattle mixture with the lowest degree of hydrolysis; >DH, protein hydrolysate of the chicken comb and wattle mixture with the highest degree of hydrolysis. Different uppercase letters indicate significant differences (p ≤0.05) between the samples regarding the chelating capacity analysis. Different lowercase letters indicate significant differences (p ≤ 0.05) between the samples regarding the Fe³⁺ reducing activity.

Figure 4

Comparison of derived peptide profiles from the different degrees of hydrolysis. 3D LC-MS showing distribution of ion peak (m/z × retention time × intensity) followed by characteristics of the identified peptides in the homology search according to molecular weight (MW) in high degree (A) and low degree (B) hydrolysis processes. Identified peptides number distribution according to MW range in each hydrolysate (C).

Figure 5

Representation of the potential antioxidant peptide based on amino acid composition. MS/MS spectrum of the peptide GADGAP (+15.99)GKDGLR (m/z 565.27+2) with a possible ion chelating effect through iron binding on the carboxyl oxygen of Asp, guanidine nitrogen of Arg and amino group of Lys. Post-translational modifications: hydroxylation on proline (+ 15.99 Da).

Figure 6

Identified post-translational modifications (PTM) in peptides from protein hydrolysis. (A) Assembly result for the identified peptide sequences from precursor protein Collagen alpha-2(I) chain in >DH. Gray lines indicate coverage regions from peptide spectrum matches and the tags indicate identified hydroxylation (blue), dihydroxy (yellow) and deamidation (purple). (B) MS/MS spectrum of the peptide GPP(+15.99)GPSGPPGKDGRN (m/z 703.34⁺²) showing hydroxylation in Proline¹⁰⁵⁵ with b (blue) and y (red) ions matched.