Antioxidant Activity of Collagen Extracts Obtained from the Skin and Gills of Oreochromis sp.

Abstract

Efforts aimed at reduction of fishing waste generated during the evisceration and filleting are scarce. The fishing waste is used in the production of low value-added products, such as flours or silages. It is important to visualize an alternative and profitable use of this waste, as it constitutes a serious environmental problem. This research determined the antioxidant properties of collagenous extracts of skin and galls of Oreochromis sp. The raw materials were characterized by proximal chemical analysis. Three treatments were applied to extract the collagen: salt-soluble collagen, acid-soluble collagen (ASC), and pepsin-hydrolyzed collagen (PHC). The collagenous fractions were hydrolyzed (0.1% pepsin). The recovered collagen yield and antioxidant activity were determined to hydrolyzed collagen (HC) and nonhydrolyzed collagen (NHC). The ASC skin showed the highest extraction yield (3.02%). For galls, only the PHC extraction was feasible (0.16%). Antioxidant analysis of NHC did not reveal radical scavenging activities. HC displayed a 2,2-diphenyl-1-picrylhydrazyl %RSA of 22.58 (ASC skin) and 10.34% (PHC galls), and a 2,2′-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid] %RSA of 30.40% (PHC skin) and 29.43% (PHC galls), respectively. The ASC skin and PHC gall extracts exhibited 94.40% and 81.54% in ferric-reducing antioxidant power, and 43.63 and 38.08 μg ascorbic acid equivalents per milli liter for total antioxidant capacity, respectively. The collagen extracts showed %RSA and chelation of pro-oxidant metal ions. Different mechanism of antioxidant action was identified for the extracts: radical scavengers for HC and metal ion chelators for NHC. In conclusion, red tilapia skin collagen is recommended as an active ingredient of nutraceuticals, pharmaceuticals, or functional foods, for the identified bioactive properties.

Introduction

The world production of catches of fish, crustaceans, mollusks, and other aquatic animals reached 92 million tons for 2016; also, aquaculture production was 76.6 million tons.¹ Of the reported output, 88% is used for human consumption, which represents 6.7% of all proteins consumed.² The remaining production is destined for nonfood purposes, such as in the manufacture of fishmeal, pharmaceutical use, silage and organic fertilizers, and in the preparation of flours/oils. Alternatively, it is discarded without control, which represents high economic and environmental costs. Tilapia production in the Papaloapan River Basin represents 20% of the total national production.³ Of the fishing production in Mexico in 2015 (1,467,203 tons), the catch of tilapia and other cichlids was 709,400 tons.² Physical and chemical characteristics of fishery products depend on the geographical location of growth, the type of species, sexual maturity, seasonal, reproductive, environmental, and diet variations.⁴ The filleting and gutting waste generated from the fish processing industry can represent about a 36% of the initial weight, in the form of blood, guts, and skeleton, without considering the losses by heads, skin, scales, and muscle remnants.

The by-products of the fishing industry can be valorized by recovering their proteins. The collagen (fibrous protein) is an important component of skin, bones, blood vessels, and cartilage.⁵ In the pharmaceutical and food industries, bioactive proteins and peptides find wide applications in functional foods and for conservation purposes. Collagen is usually recovered from by-products of the meat industry (bovine and porcine), but presented the risk of transferring diseased cells to humans (mammalian diseases), such as bovine spongiform encephalopathy and foot/mouth diseases.⁶ Consequently, new and safe alternative sources of collagen extraction are requested. The various physiological and biological functions reported for collagen and its peptides include antioxidant, antimicrobial, antihypertensive, anticoagulant, inhibition of lipid peroxidation, chelation of metal ions,⁷ immunomodulation, regulation of cell proliferation, hypocholesterolemic, and antithrombotic.⁸ In addition, they can also be used in the prevention and/or treatment of chronic degenerative diseases.⁹

The peptides are inactive in the structure (order/composition) of their original protein but can exert bioactive actions in the organism once they are released by various hydrolytic processes. Segura-Campos et al. ¹⁰ described three methods of hydrolysis: fermentative, chemical, and enzymatic. The degree of hydrolysis and the peptides obtained depend on the physicochemical conditions present in the reaction medium.⁵ This study evaluated the antioxidant activity of collagenous extracts obtained from red tilapia (Oreochromis sp.) residues, by in vitro techniques, before and after an enzymatic hydrolysis process.

Materials and Methods

Raw materials and analysis of proximal chemical composition

Red tilapia skin (Oreochromis sp.) of fresh fish from the Temascal Dam of San Miguel Soyaltepec, a municipality belonging to the Papaloapan region, in the state of Oaxaca (18°15′N, 96°24′W), was used. The samples were transferred in thermal containers, under aseptic conditions, to the Food Laboratory of the Higher Technological Institute of Tierra Blanca, Veracruz. Proximal chemical analyses were performed on both materials, by implementing the Association of Official Analytical Chemists¹¹ methods (moisture, fat, ash, and total protein). The skins and gills were washed with distilled water, cut into 1 cm² cubes, and pretreated^12,13 before analyses.

Collagen extraction

Collagen extraction was based on Torres-Arreola et al. ¹⁴ and Chen et al. ¹⁵ with slight modifications. The extraction process was carried out at 5°C. The myofibrillar proteins and proteoglycan were removed with 6 M urea and 0.5 M sodium acetate (pH 6.8), magnetic stirring (24 h), and centrifugation (11,000 g, 45 min). The residue was sequentially extracted with a neutral buffer (salt-soluble collagen [SSC]), acetic acid (acid-soluble collagen [ASC]), and pepsin (pepsin-hydrolyzed collagen [PHC]). The solutions were gradually precipitated with NaCl to a final concentration of 2 M and collected by centrifugation (11,000 g, 45 min). The precipitates were redissolved in 0.5 M acetic acid solution, dialyzed against 0.02 M Na₂HPO₄ (pH 8.6), with a cellulose membrane of 10 kDa (5°C for 12 h), dialyzed against 0.05 M acetic acid (12 h), and against distilled water (24 h). The dialyzed material was lyophilized and the recovered collagen yield (RCY) was determined by the following equation: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{ \rm { RCY } } \left( { \rm { \% } } \right) = \left( { { \frac { { \rm { Weight \ of \ freeze - dried \ collagen } } \left( { \rm { g } } \right) } { { \rm { Total \ weight \ of \ the \ fresh \ sample \ used } } \left( { \rm { g } } \right) } } } \right) \times 100 \tag { 1 } \end{align*} \end{document}

Preparation of the extracts

Solubilization of the collagenous proteins was achieved using 1 N acetic acid (20 mg collagen in 5 mL of 1 N acetic acid solution), with stirring for 24 h. These extracts were named nonhydrolyzed collagen (NHC) and refrigerated until use. The hydrolysates from collagenous extracts were obtained as described by Akagündüz et al. ¹⁶ with some adjustments: the extracts were dissolved in pepsin–HCl solution (0.1% pepsin in 10 mM HCl, pH 2) under magnetic stirring at 37°C for 3 h. Afterward, the enzyme was inactivated by heating at 100°C for 3 min, and the solution was centrifuged (629 g, 10 min). The supernatant was refrigerated until use (hydrolyzed collagen [HC]).

Methods of antioxidant capacity

2,2-Diphenyl-1-picrylhydrazyl antioxidant activity

The 2,2-diphenyl-1-picrylhydrazyl (DPPH^•) radical scavenging activity (RSA) was determined as detailed by Quiñones et al. ¹⁷ with slight modifications. First, a DPPH solution (62 μg/mL) was prepared (6.2 g DPPH dissolved in 100 mL of 99% absolute methanol, vortexed for 5 min). An aliquot (1 mL) of DPPH reagent was combined with 1 mL of extract, incubated in the dark at room temperature (10 min) and the absorbance was measured at 523 nm ( \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${A_{{ \rm{sample}}}}$$ \end{document} ). The blank was prepared under the conditions already described but replacing the extract with absolute methanol ( \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${A_{{ \rm{blank}}}}$$ \end{document} ). The DPPH^• scavenging ability was calculated using Equation (2): \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{ \rm{DPPH \ scavenging \ effect}} \; \left( \% \right) = \left[ { ( {A_{{ \rm{blank}}}} - {A_{{ \rm{sample}}}} ) / {A_{{ \rm{blank}}}}} \right] \times 100. \tag{2} \end{align*} \end{document}

The mean effective concentration (EC₅₀) was calculated from a graph of reducing power percentage against the extract concentration and expressed in milligrams per milliliter (mg/mL). EC₅₀ is defined as the concentration of sample required to reduce the initial concentration of the radical by 50%.

2,2′-Azino-bis[3-ethylbenzothiazoline-6-sulfonic acid] antioxidant activity

The 2,2′-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS^•+) free RSA was established based on Sánchez-Gutiérrez et al. ¹⁸ A 1 mL aliquot of the ABTS solution (7 mM) was mixed with 17.60 μL of the potassium persulfate solution (140 mM) and left to react in the dark at room temperature for 12 h. Once the radical was formed, 1 mL of the extract was mixed with 1 mL of ABTS^•+ solution, incubated in the dark at room temperature (6 min), and the absorbance was measured at 734 nm ( \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${A_{{ \rm{sample}}}}$$ \end{document} ). The blank was prepared under the conditions already described, but instead of sample, 1 mL of bidistilled water was mixed with 1 mL of the ABTS^•+ solution (v/v) ( \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${A_{{ \rm{blank}}}}$$ \end{document} ). The ABTS^•+ RSA (%) of the samples was calculated as follows: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{ \rm{ABTS \ scavenging \ effect}} \left( \% \right) = \left[ { ( {A_{{ \rm{blank}}}} - {A_{{ \rm{sample}}}} ) / {A_{{ \rm{blank}}}}} \right] \times 100. \tag{3} \end{align*} \end{document}

Total antioxidant capacity

The total antioxidant capacity (TAC) was measured using the phosphomolybdenum method reported by Sánchez-Gutiérrez et al. ¹⁸ with slight modifications. Then 100 μL of the sample was combined with 1 mL of the reactive solution (sulfuric acid, sodium phosphate, and ammonium molybdate) and incubated in a thermoblock (95°C, 90 min). Once cooled to room temperature, the absorbance was recorded at 695 nm, against a solution containing all the components of the reaction mixture, but adding bidistilled water. A calibration curve was constructed with ascorbic acid. The TAC values are expressed in micrograms of ascorbic acid equivalents by milliliter of extract (μg AAE/mL), which were calculated using the following calibration curve: A₆₉₅ = 0.0031[ascorbic acid] – 0.0095 (correlation coefficient, r ² = 0.9976), generated from six concentrations of ascorbic acid (0.01–0.10 mg/mL).

Ferric-reducing antioxidant power

The ferric-reducing antioxidant power (FRAP) was measured according to Saikia and Mahanta¹⁹ with modifications. Each sample (75–250 μL) was mixed with 250 μL of 0.20 M phosphate buffer (pH 6.6) and 250 μL of 0.01 g/mL potassium ferrocyanide. The mixture was incubated (50°C for 20 min). Then, 250 μL of 0.11 g/mL trichloroacetic acid was added, the mixture was centrifuged (482 g, 25°C, 10 min), and 500 μL of the resultant supernatant was mixed with 500 μL of deionized water and 100 μL of 0.001 g/mL ferric chloride and the absorbance was recorded at 700 nm ( \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${A_{{ \rm{sample}}}}$$ \end{document} ). The blank was a sample composed of all the components of the reaction mixture but adding bidistilled water instead of extract ( \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${A_{{ \rm{blank}}}}$$ \end{document} ). Ascorbic acid was used as the standard. The FRAP was calculated as shown in Equation (4): \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{ \rm{Chelating \ effect}} \; \left( \% \right) = \left[ {1 - \left( {{A_{{ \rm{sample}}}} / {A_{{ \rm{blank}}}}} \right) } \right] \times 100. \tag{4} \end{align*} \end{document}

Statistical analysis

The RCY and antioxidant activities were performed in triplicate (reported as mean ± standard deviation). The corresponding results were subjected to an analysis of variance at P ≤ .05, and Student's t-test, P ≤ .05, according to data. These analyses were undertaken using IBM SPSS (Armonk, NY, USA) software version 20.

Results

Before extraction of the collagen, proximal chemical composition of the skins and gills of red tilapia (Oreochromis sp.) was analyzed (Table 1). Collagen was extracted from two collected residues (skin and galls of red tilapia) to obtain three sequential fractions (SSC, ASC, and PHC). All fractions were recovered from the skin residue, due to their solubility characteristics and ease of hydrolysis, whereas only one fraction (PHC) was derived from the galls. Table 2 shows the RCY (%) of the fractions obtained from skin and galls evaluated in this study. No DPPH^• and ABTS^•+ %RSA were displayed by the NHC extracts. The DPPH^• and ABTS^•+ antioxidant activities are summarized in Table 3. Table 4 shows the TAC and FRAP data for the different collagenous extracts.

Table 1.

Proximal Chemical Composition of Skin and Gills of Red Tilapia (Oreochromis sp.) Used for Collagen Extraction

Components (%)	Skin	Gills
Moisture^a	73.50 ± 2.60	66.20 ± 5.10
Ethereal extract^a	19.20 ± 4.60	54.90 ± 0.60
Protein	16.14 ± 1.70	31.80 ± 2.80
Ash^a	0.73 ± 0.20	UD

The samples were pretreated with 0.1 M NaOH (0.1 g sample/mL NaOH) under magnetic stirring for 6 h, with a replacement of the solution every 2 h¹²; and degreased with 10% butanol (10% (w/w) for 15 h, with a replacement of the same solution every 5 h.¹³ The results are indicated as mean ± standard deviation (n = 3).

Calculated in dry basis.

UD, undetermined.

Table 2.

Collagen Extraction Yields (Recovered Collagen Yield)

Extraction treatment	Recovered skin yield (%)	Recovered gill yield (%)
SSC	1.46 ± 0.03	N/R
ASC	3.02 ± 0.10	N/R
PHC	2.52 ± 0.20	0.16 ± 0.06

The residue was sequentially extracted with a neutral buffer (0.05 M Tris/1 M NaCl, pH 7.2) (SSC), 0.5 M acetic acid (ASC), and pepsin (10 mg pepsin/g tissue in 0.5 M acetic acid) (PHC). The results are indicated as mean ± standard deviation (n = 3).

ASC, acid-soluble collagen; N/R, not recovered; PHC, pepsin-hydrolyzed collagen; SSC, salt-soluble collagen.

Table 3.

Free Radical Scavenging Activity (%RSA: DPPH^• and ABTS^•+) of the Collagen Extracts from Red Tilapia (1 mg/mL)

Extracts	DPPH^•				ABTS^•+
	Hydrolysate		Not hydrolyzed		Hydrolysate		Not hydrolyzed
	%RSA	EC₅₀ (mg/mL)	%RSA	EC₅₀ (mg/mL)	%RSA	EC₅₀ (mg/mL)	%RSA	EC₅₀ (mg/mL)
SSC skin	21.216 ± 0.620^a	4.862	0	ND	30.381 ± 2.007^a	2.151	0	ND
ASC skin	22.581 ± 0.744^a	5.814	0	ND	24.381 ± 1.438^b	2.201	0	ND
PHC skin	12.283 ± 1.706^b	3.695	0	ND	30.400 ± 0.431^a	7.030	0	ND
PHC guts	10.339 ± 1.153^b	5.340	0	ND	29.429 ± 2.060^a	2.222	0	ND

The extract concentrations used for the reaction were 0.33, 0.50, 0.67, 0.83, and 1.00 mg/mL, using absolute methanol to tare the spectrophotometer. A decrease in the absorbances of the reaction mixtures indicated an inhibition of the radical (DPPH^•, ABTS^•+). The ABTS^•+ solution was diluted with bidistilled water (1:44, v/v) to obtain an absorbance of 0.700 ± 0.01 at 734 nm, before the test with the extracts.

Values with different literals per column indicate significant differences (^a,bTukey, P < .05). The results are indicated as mean ± standard deviation (n = 3).

ABTS^•+, 2,2′-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid]; DPPH^•, 2,2-diphenyl-1-picrylhydrazyl; EC₅₀, effective concentration; RSA, radical scavenging activity; ND, not detected.

Table 4.

Chelating Activity of the Collagen Extracts from Red Tilapia

Extracts	TAC (μg AAE/mL)		FRAP
	Hydrolysate	Not hydrolyzed	Hydrolysate		Not hydrolyzed
	Hydrolysate	Not hydrolyzed	Chelating effect (%)	EC₅₀ (mg/mL)	Chelating effect (%)	EC₅₀ (mg/mL)
SSC skin	23.925 ± 1.834^a	38.763 ± 0.493^a	43.377 ± 0.590^a	1.104^x	92.910 ± 0.636^a	0.583^y
ASC skin	25.860 ± 0.986^a	42.312 ± 1.524^b	43.632 ± 2.335^a	1.083^x	94.403 ± 0.155^a	0.505^y
PHC skin	29.409 ± 1.343^b	41.344 ± 0.493^a,b	30.999 ± 0.308^c	1.190^x	94.522 ± 0.092^a	0.530^y
PHC guts	31.774 ± 1.290^b	29.086 ± 0.372^c	38.078 ± 2.594^b	1.187^x	81.543 ± 1.834^b	0.760^y

The reactive solution contains 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate. The sample concentration varied in 0.010, 0.025, 0.040, 0.055, 0.070, and 0.100 mg/mL. The increase in absorbance of the reaction mixture indicated an increase in the capacity (TAC, FRAP). Values with different literals per column indicate significant differences (^a,b,cTukey, P ≤ .05); ^x,yStudent's t-test, P ≤ .05). The results are indicated as mean ± standard deviation (n = 3).

AAE, ascorbic acid equivalents; FRAP, ferric-reducing antioxidant power; TAC, total antioxidant capacity.

Discussion

Chemical composition of raw materials

The protein (16.14%) and ash (0.73%) contents of red tilapia skin (Table 1) were relatively low compared with the levels stated by Jellouli et al.,⁶ that is, 26.13 and 2.47% (for skin of gray triggerfish, Balistes capriscus). The ash percentage found in this study agrees with that recorded by Zeng et al. ²⁰ of 0.60%, and the protein content (88.50%) of Nile tilapia (O. niloticus) skin gelatin is much higher than that found for red tilapia skin (16.14%). The ethereal extract (19.20%) and moisture (73.50%) contents are similar to those published by Quintero and Zapata,¹³ that is, 18.37 and 69.94% (red tilapia skin), respectively.

Fish gills exhibit complex physiology and structure, comprising spines, cartilages, and bones, so in this discussion, the data obtained for the gills are compared with the literature values pertaining to the most similar parts (spines, cartilages, and bones). In this study, Oreochromis sp. gills presented a moisture content of 62.20%, similar to Di et al.,²¹ who informed contents of 61.83 and 62.37% in spines and skull of skipjack (Katsuwonus pelamis), respectively, but different protein and ether extract contents (14.97% and 6.82% for spines, and 15.55% and 4.39% for skull, respectively).²¹ The variations in the composition could be due to the fish feeding (quantity and type), physiological maturity, period of year of capture, zones and temperature of the water, gender, and reproductive age of the animal,⁴ in addition to the treatment given to the skins and gills after being removed. The knowledge of these characteristics helps the correct handling and treatment of the samples, for the proper development of extraction methods and utilization.

Extraction yields

All fractions were recovered from the skin, due to their solubility characteristics and ease of hydrolysis, whereas only one fraction (PHC) was derived from the galls, implying a relatively lower acid and salt solubility. The presence of cross-links between chains (e.g., aldehyde groups combine with lysine and hydroxylysine in the telopeptide region) increases the internal binding energy, so solubility decreases. Enzymes (pepsin) hydrolyze the bonds, mainly in the telopeptide region, increasing its solubility and improving its extraction yields, in the form of peptides.²² The RCY (Table 2) in the ASC (3.02%) and PHC (2.52%) extracts is higher (P ≤ .05) than in SSC skin and PHC galls (1.46 and 0.16%, respectively). The RCY in the ASC skin of red tilapia was greater than that reported by Quintero and Zapata¹³ of 0.88%. Solari and Córdova²³ acquired 1.00% (ASC) RCY from residues (spines, spines, and scales) of anchoveta (Engraulis ringens). No prior reports were found on the extraction of collagen directly from galls. Some authors claim that enzymes can improve the RCY of skin, scales, fins, and cartilages.^22,24 Besides, the hydrolysates demonstrated enhanced functional activities than the intact protein,²⁵ in addition to its bioavailability.²⁶

Gaurav et al. ²² determined that ASC (with 1500 mM NaCl) has a greater activity for fibril formation than PHC, which is an important property in biomedical and pharmaceutical applications. Peptides from fish have shown biological activities, such as antimicrobial, antihypertensive, and anticarcinogenic, particularly by antioxidant and metal ion chelation abilities.²⁶ An antioxidant can prevent the oxidation of molecules and provide beneficial health effects, associated with the neutralization of reactive oxygen/nitrogen species, free radical scavengers, or the chelation of metal ion pro-oxidants.^27
–29 These mechanisms could represent the working form of the extracts (chain break or preventive) and are considered in the techniques used in this study.

DPPH^• and ABTS^•+ free RSAs

The absence of antioxidant activity means that the extracts did not act as chain-breaking antioxidants,¹⁸ or as donors of hydrogen atoms or electrons to neutralize the radicals. The DPPH^• and ABTS^•+ antioxidant activities (Table 3) reveal differences (P ≤ .05) in the EC₅₀ of the hydrolysates for both analysis tests. The highest DPPH^• %RSA was presented for ASC skin (22.58%). The DPPH^• antioxidant activity for the SSC skin and ASC skin hydrolyzed extracts was significantly different (P ≤ .05) (21.22% and 22.58%, respectively) from that found for the PHC skin and PHC gall extracts (12.28% and 10.34%, respectively). For the HC extracts, a positive dependence between concentration and scavenging of the ABTS^•+ radical was observed. In the same trial (ABTS^•+), the %RSA was higher in treatments of SSC skin (30.38%), PHC skin (30.40%), and PHC galls (29.43%), without significant statistical difference (P ≤ .05). Conversely, Suárez-Jiménez et al. ³⁰ evaluated the %RSA (DPPH^• and ABTS^•+) of collagen extracts extracted from fins and arms of giant squid (Dosidicus gigas), obtaining higher values for the hydrolyzed samples (protease and trypsin) than the intact protein, as already discussed, and consistent with the findings found here for red tilapia residues. Zhuang and Sun³¹ reported OH^• scavenging activities in the range of 40.40–59.20% of Nile tilapia skin gelatin. The phenomenon of %RSA is attributed to the electron- or hydrogen-donating ability of the peptides, which neutralize the radicals, or alter their degree of activity and availability.

The antioxidant activity of hydrolysates is related to lower molecular size, reported under different assays (RSA: DPPH^•, ABTS^•+, OH^•, O₂ ^•; inhibition of linoleic acid peroxidation); besides the low weight, it also influences the increase in the loading and release of hydrophobic groups.³² The increase in the antioxidant activity of collagenous extracts after hydrolysis is because the peptide fractions produced (of smaller size) are electron donors that can react more readily with free radicals and convert them into stable products.³³ Gómez-Guillén et al. ²⁶ mentioned that a peptide fraction with molecular mass <700 Da exhibited a highest %RSA than that of the nonfractionated hydrolysate (20% higher). That investigation concurs with the results presented in this research, whereby smaller molecules impart greater antioxidant activities.

Chelating activity by TAC and FRAP

The chelating activity is the trapping of metal ions forming soluble and stable compounds. An example of the damage of the excessive accumulation of metal ions is iron, which causes tissue damage and leads to pathologies, such as heart failure, diabetes, liver failure, and death. The FRAP and TAC tests are based on the reduction of metal ions, by a single electron transfer. In the FRAP protocol, the ferric complex (Fe³⁺–2,4,6-tripyridyltriazine [TPTZ]) is reduced to its ferrous form (Fe²⁺–TPTZ)³⁴ by oxidation of a reducer (antioxidant agent). Similarly, in the TAC assay, Mo (VI) is reduced to Mo (V) by an antioxidant agent.¹⁸ In the TAC assay (Table 4), it was noticed that for HC, the highest chelating activities were registered in the PHC skin (29.41 μg AAE/mL) and PHC galls (31.77 μg AAE/mL) treatments at 1 mg/mL, without showing statistically significant differences (P ≤ .05). A higher TAC was apparent for the NHC than HC, with ASC skin giving the highest activity (42.31 μg AAE/mL). The NHC exhibited the maximal TAC, could be due to the presence of terminal hydrophobic amino acids or amino acids with an imidazole ring, or both, unlike hydrolyzed samples, where the presence of those amino acids may have been lost or decreased, which decreases the disposition to react and chelate metals. For a complete discussion, it is recommended to study the amino acid sequence present in all the fractions.

In the FRAP assay, the highest chelating activity was presented for the SSC skin (43.38%) and ASC skin (43.63%) HC extracts, without significant difference (P ≤ .05). The difference in properties is attributed to the origin of the sample, the skin being the source with greatest chelating activity. The chelating capacities found in this study were lower than the activity of 56.50% for extracts of HC obtained from jellyfish (Rhopilema esculentum).³⁵ For the HC and NHC, differences were evident in the values of EC₅₀ (FRAP) (P ≤ .05), presenting a higher chelating capacity NHC. Kittiphattanabawon et al. ³⁶ reported that the degree of hydrolysis (%DH) of gelatin from blacktip shark (Carcharhinus limbatus) skin affected the biological capacities; a 40% DH produced peptides with superior antioxidant activity. The authors also evidenced a greater scavenging activity for the gelatin hydrolysates than integral gelatin. Some amino acids have been reported for red tilapia skin gelatin, with the most significant being Gly, Ala, and Pro,²⁰ which are promoters of the antioxidant effects.^8,37 Similar studies, of antioxidant capacity, on red tilapia are scarce, and not of the physiological waste studied in this investigation.

In conclusion, the most effective method for the recovery of collagenous proteins from red tilapia skin with antioxidant activity is an acidic solution. The collagen extracts showed %RSA and chelation of pro-oxidant metal ions. Different mechanism of antioxidant action was identified for the extracts: radical scavengers for HC and metal ion chelators for NHC. Red tilapia skin collagen is recommended as an active ingredient of nutraceuticals, pharmaceuticals, or functional foods for the identified bioactive properties.

Footnotes

Acknowledgment

Author Abel Arce Ortíz thanks National Council for Science and Technology (CONACyT-Mexico) for the scholarship No. 429747. This study is part of the activities of the Thematic Network CONACYT “Network 12.3, To reduce and valorize the losses and the waste of food: Towards sustainable food systems” (Key 294768).

Author Disclosure Statement

No competing financial interests exist.

References

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Antioxidant Activity of Collagen Extracts Obtained from the Skin and Gills of Oreochromis sp.

Abstract

Introduction

Materials and Methods

Raw materials and analysis of proximal chemical composition

Collagen extraction

Preparation of the extracts

Methods of antioxidant capacity

2,2-Diphenyl-1-picrylhydrazyl antioxidant activity

2,2′-Azino-bis[3-ethylbenzothiazoline-6-sulfonic acid] antioxidant activity

Total antioxidant capacity

Ferric-reducing antioxidant power

Statistical analysis

Results

Discussion

Chemical composition of raw materials

Extraction yields

DPPH• and ABTS•+ free RSAs

Chelating activity by TAC and FRAP

Footnotes

Acknowledgment

Author Disclosure Statement

References

DPPH^• and ABTS^•+ free RSAs