Alcalase-generated proteolysates of stone fish (Actinopyga lecanora) flesh as a new source of antioxidant peptides

Figures & data

Table 1. Peptide content, degree of proteolysis (DP) and antioxidant activity of alcalase digested proteolysates of stone fish flesh during 8 h of proteolysis.

Time (h)	Peptide content (mmol glutathione/g dried sample)	Degree of proteolysis (DP) (%)	FRAP value (mmol/100 mL FeSO4)	DPPH^• scavenging activity		ABTS^• scavenging activity
				(%)	IC50 (mg/mL)		(%)	IC50 (mg/mL)
0	0.0 ^a	0.0 ^a	0.33 ± 0.91^a*	0.0 ± 0.0 ^a	0.0 ^a		0.0 ± 0.0 ^a	0.0 ^a
1	1.36 ± 0.08 ^b	13.45 ± 0.15 ^b	8.83 ± 0.00 ^b	24.26 ± 0.73 ^b	1.54 ^b		40.44 ± 0.25 ^b	1.11 ^b
2	2.39 ± 0.42 ^c	23.73 ± 0.31 ^c	12.26 ± 0.60 ^c	47.5 ± 0.41 ^c	0.98 ^c		59.17 ± 0.14 ^c	0.81 ^c
3	3.60 ± 0.32 ^d	32.61 ± 0.23 ^d	15.97 ± 0.00 ^d	57.45 ± 1.36 ^d	0.80 ^d		71.08 ± 0.23 ^d	0.65 ^d
4	4.85 ± 0.42 ^e	48.20 ± 0.17 ^e	18.54 ± 0.54 ^e	72.12 ± 0.21 ^e	0.69 ^e		78.66 ± 0.11 ^e	0.48 ^e
5	4.92 ± 0.28 ^e	51.21 ± 0.24 ^f	19.11 ± 1.21 ^e	76.11 ± 0.21 ^f	0.55 ^f		91.49 ± 0.13 ^g	0.33 ^f
6	6.45 ± 0.3 ^f	74.68 ± 0.33 ^g	24.18 ± 0.91 ^f	75.37 ± 0.63 ^f	0.57^g		90.56 ± 0.15 ^f	0.35 ^g
7	7.61 ± 0.58 ^g	88.84 ± 0.69 ^h	33.97 ± 0.00 ^g	75.59 ± 0.52 ^f	0.57 ^g		90.29 ± 0.10 ^f	0.35 ^g
8	7.88 ± 0.32 ^g	89.03 ± 0.12 ^h	39.18 ± 0.61 ^h	77.43 ± 0.0 ^g	0.50 ^h		92.73 ± 0.14 ^h	0.33 ^h

* a-h indicated the significant differences at P ≤ 0.05, mean ± SD, n = 3.

Figure 1. Correlation between antioxidant activities ((a) DPPH^•, (b) ABTS^• radical scavenging activity and (c) FRAP reducing power) and degree of proteolysis of stone fish proteolysates during 8 h of digestion with alcalase.

Table 2. Physicochemical properties of stone fish proteolysates in comparison to other marine-based proteolysates.

Physicochemical properties
Source of proteolysate	Emulsifying capacity (EC)	Foaming capacity (FC)	Oil-holding capacity (OHC)	Water-holding capacity (WHC)	Reference
Stone fish	8.5 ± 0.5 mL/g	2.33 ± 0.58 mL/g	2.17 ± 0.29 mL/g	3.33 ± 2.9 mL/g	Present study
Nile tilapia	21.40 ± 0.36 mL/g	124.50%	2.23 ± 0.25 mL/g	1.77 ± 0.06 mL/g	^[Citation11]
Grass carp skin	20–38 mL/g	–	2.4–3.6 mL/g	2.0–4.9 mL/g	^[Citation36]
Blue wing sea robin	20.46–28.06 mL/g	–	4.50–5.52 mL/g	2.44–2.98 mL/g	^[Citation37]
Cobia frame	3–12 mL/g	122.7%	2.4–2.8 mL/g	0.8–1.1 mL/g	^[Citation38]
Fish roe	12–56 mL/g	52–67 mL/g	172–241%	198–315%	^[Citation39]
Shark muscle	25–30 mL/0.5 g	60–140%	0.2–0.5 mL/g	4–6 mL/g	^[Citation13]

Table 3. The yield and DPPH^• radical scavenging activity of stone fish proteolysate ultrafiltration fractions.

MW (KDa)	Yield (%)	DPPH^• scavenging (%)
>10	21.48 ± 1.05^b	52.63 ± 0.24^a
5–10	10.97 ± 0.23^a	68.98 ± 0.4^b
2–5	28.22 ± 0.58^c	72.8 ± 0.33^c
< 2	39.41 ± 0.29^d	73.23 ± 0.18^c

Means ± SD of triplicates determinations. Values with different lower case within each column were significantly different (P ≤ 0.05).

Figure 2. SDS-PAGE patterns of unfractionated stone fish selected proteolysate and its ultrafiltrated fractions. MW: molecular weight, N.F: unfractionated stone fish proteolysate. >10: fraction with MW > 10 KDa, 5–10: fraction with MW between 5 and 10 KDa, 2–5: fraction with MW between 2 and 5 KDa <2: fraction with MW < 2 KDa.

Figure 3. RP-HPLC fractions chromatogram vs. DPPH^• radical scavenging assay. The subscriptions a-n indicated the significant differences at P ≤ 0.05, (mean ± SD, n = 3).

Figure 4. DPPH^• radical scavenging activity of OFFGEL fractions obtained from alcalase proteolysate. The subscriptions a-h indicated the significant differences at P ≤ 0.05, (mean ± SD, n = 3). A: al-2, B: al-3, C: al-19, D: al-20 and E: al-31. F.N: Fraction Number, AA%: antioxidant activity %.

Figure 5. RP-HPLC fractionation chromatogram of final selected fraction (al-20–10).

Figure 6. MS/MS chromatogram, a: peptide 1 (GVSGLHID), score: 76.1, ion intensity: 4.19E+ 05, RT (min): 3.42, precursor m/z: 399.2115, matched m/z: 797.416. b: peptide 2 (PTPSRPQQGPA), score: 58.6, ion intensity: 1.75E+ 06, RT (min): 2.8, precursor m/z: 568.2972, matched m/z: 1135.586. c: peptide 3 (PGRRAA), score: 64.2, ion intensity: 2.59E+ 05, RT (min): 2.35, precursor m/z: 314.1826, matched m/z: 627.369. d: peptide 4 (LAQVAVSNPNN), score: 56.4, ion intensity: 2.03E+ 05, RT (mins): 2.37, precursor m/z: 376.1959, matched m/z: 1126.586.

Table 4. Stone fish-derived peptides characteristics and antioxidant activities.

Peptide no.	Sequence	Residue no.	MW (g/mol)	Net charge	pI	DPPH^• Scavenging activity (% at 5 mg/mL)	IC50 (mg/mL)	ABTS^• Scavenging activity (% at 5 mg/mL)	IC50 (mg/mL)
1	GVSGLHID	8	797	0	5.28	65	4.14 ± 0.21	85	3.28 ± 0.15
2	PTPSRPQQGPA	11	1135	1	10.55	13	19.8 ± 0.28	35	11.23 ± 0.11
3	PGRRAA	6	627	2	12.50	22	6.77 ± 0.34	58	5.72 ± 0.18
4	LAQVAVSNPNN	11	1126	0	6.02	−34	–	−19	–

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