Marine and Fishery sciences 38 (2): xxx-xxx (2025)

https://doi.org/10.47193/mas.3822025010106

ABSTRACT. The valorization of shery byproducts is essential to reduce waste and create

high-value products. Waste from Argentine hake (Merluccius hubbsi) could enhance its functional

and antioxidant properties through hydrolysis, releasing peptides with bioactive properties. Protein

hydrolysates of Argentine hake were produced through autolysis (Aut) and enzymatic hydrolysis

using Alcalase 2.4L at concentrations of 0.24% and 2% (v/v) (Alc-0.24 and Alc-2), respectively,

over 150 min. Alkaline peptidase activity, degree of hydrolysis, and antioxidant activity were assessed

using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic

acid) radical ABTS•+ scavenging assays. All hydrolysates retained alkaline peptidase activity through-

out the process. Alcalase-treated hydrolysates exhibited signicantly higher peptidase activity and

hydrolysis degree compared to autolysis. At 60 min, Alc-0.24 reached peptidase activity levels similar

to Alc-2, and by 30 min, both had comparable degrees of hydrolysis. ABTS•+ scavenging activity

increased over time for Alc-0.24, with both Alcalase 2.4L concentrations outperforming autolysis.

No signicant differences were found between Alc-0.24 and Alc-2. Although all hydrolysates showed

DPPH scavenging activity, no signicant differences were detected between treatments or reaction

times. These ndings highlight the potential for producing value-added protein hydrolysates from

Argentine hake waste.

Key words: Waste management, sh protein hydrolysate, peptidase activity, hydrolysis degree, an-

tioxidant property.

De residuo a valor: hidrolizados proteicos de subproductos del procesamiento de la merluza

argentina (Merluccius hubbsi) utilizando enzimas endógenas y Alcalasa 2.4L

RESUMEN. La valorización de los subproductos pesqueros es fundamental para reducir los residuos

y crear productos de alto valor. Los residuos de la merluza argentina (Merluccius hubbsi) podrían

potenciar sus propiedades funcionales y antioxidantes a través de la hidrólisis, liberando péptidos

con propiedades bioactivas. Los hidrolizados proteicos de merluza argentina tienen un gran potencial

como ingredientes funcionales debido a sus propiedades bioactivas, pero optimizar los procesos

de hidrólisis es esencial para mejorar el rendimiento y las características biofuncionales, como la

actividad antioxidante. Se obtuvieron hidrolizados proteicos de merluza argentina mediante autólisis

ORIGINAL RESEARCH

From waste to value: protein hydrolysates from byproducts of the

Argentine hake (Merluccius hubbsi) processing using endogenous enzymes

and Alcalase 2.4L

clara liebana1, nair de los Ángeles Pereira1, analia V. FernÁndez-giMenez1 and Maria Florencia Fangio2, 3, *

1Instituto de Investigaciones Marinas y Costeras (IIMyC-CONICET), Facultad de Ciencias Exactas y Naturales, Universidad Nacional de

Mar del Plata (UNMdP), Consejo Nacional de Investigaciones Cientícas y Técnicas (CONICET), CC 1260, Mar del Plata, Argentina.

2Departamento de Química y Bioquímica, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata (UNMdP),

Funes 3350, B7602AYL - Mar del Plata, Argentina. 3Instituto de Investigaciones Físicas de Mar del Plata (IFIMAR-CONICET), Facultad de

Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata (UNMdP), Consejo Nacional de Investigaciones Cientícas y Técnicas

(CONICET), Funes 3350, B7602AYL - Mar del Plata, Argentina. ORCID Clara Liebana https://orcid.org/0000-0002-7062-8675,

Nair de los Ángeles Pereira https://orcid.org/0000-0002-7341-5333, Analia V. Fernández-Gimenez https://orcid.org/0000-0001-9232-4560,

Maria Florencia Fangio https://orcid.org/0000-0001-5860-6513

Marine and

Fishery Sciences

MAFIS

*Correspondence:

mfangio@mdp.edu.ar

Received: 12 November 2024

Accepted: 23 December 2024

ISSN 2683-7595 (print)

ISSN 2683-7951 (online)

https://ojs.inidep.edu.ar

Journal of the Instituto Nacional de

Investigación y Desarrollo Pesquero

(INIDEP)

This work is licensed under a Creative

Commons Attribution-

NonCommercial-ShareAlike 4.0

International License

Marine and Fishery sciences 38 (2): xxx-xxx (2025)

INTRODUCTION

Protein hydrolysates are produced through the

enzymatic cleavage of peptide bonds in food pro-

teins, resulting in peptides of varying molecular

weights. These hydrolysates have broad applica-

tions in food formulations, functional foods, animal

nutrition, pharmaceuticals, and cosmetics (Gao et

al. 2021). They may contain bioactive peptides

with diverse benecial properties, such as anti-

oxidant, antimicrobial, antithrombotic, immuno-

modulatory, and functional effects (Nirmal et al.

2022). In particular, antioxidant peptides derived

from protein sources have garnered signicant at-

tention due to their health-promoting benets and

lower toxicity compared to synthetic pharmaceu-

ticals (Wang et al. 2021). Antioxidant activity in

protein hydrolysates obtained from various sh

species has been widely reported (Gao et al. 2021;

Moya-Moreira et al. 2023; Shekoohi et al. 2024).

These properties depend on the type of peptides re-

leased during enzymatic hydrolysis, which can be

carried out using endogenous enzymes (autolysis)

or commercial enzymes, such as Alcalase 2.4L

(Gao et al. 2021).

The vast marine coastline of Argentina is homes

to a thriving shing industry, with Merluccius

hubbsi being the most widely caught species in

the country. In 2023, 297,354 t of Argentine hake

were captured, making it one of the nation’s most

valuable shery resources (MAGyP 2024). Byprod-

ucts of this processing represent approximately 50-

60% of the total sh weight (Karoud et al. 2019;

Ananey-Obiri et al. 2019). If not properly managed,

these materials are often discarded, leading to in-

efciencies and potential environmental pollution

due to the accumulation of organic waste (Karoud

et al. 2019; Ananey-Obiri et al. 2019). The sub-

stantial volume of byproducts generated during

processing highlights the need for more effective

strategies to increase the value of this resource

(Góngora et al. 2012). Previous efforts have aimed

at revalorizing M. hubbsi shery byproducts by

producing chemical and biological silages (Góngo-

ra et al. 2012; Fernández-Herrero et al. 2015) and

extracting digestive enzymes for characterization

or biotechnological applications (Lamas et al. 2015;

Friedman et al. 2022, 2024). However, to the best

of our knowledge, only Martone et al. (2005) have

produced a protein hydrolysate from these residues,

specically seeking its use as a culture medium.

Various hake species have been treated enzy-

matically to produce protein hydrolysates, either

by autolysis or by using commercial enzymes

(Samaranayaka et al. 2007; Pacheco-Aguilar et al.

2008; Karoud et al. 2019). To enhance the value of

sh protein byproduct, this study aimed to produce

a protein hydrolysate from M. hubbsi processing

waste using both endogenous enzymes and the

commercial enzyme Alcalase 2.4L (Novozymes).

(Aut) y hidrólisis enzimática utilizando Alcalasa 2.4 L a concentraciones de 0,24% y 2% (v/v) (Alc-0.24 y Alc-2), respectivamente,

durante 150 min. Se evaluó la actividad de peptidasas alcalinas, el grado de hidrólisis y la actividad antioxidante utilizando ensayos de

inhibición del radical 2,2-difenil-1-picrilhidrazilo (DPPH) y 2,2’-azino-bis (ácido 3-etilbenzotiazolina-6-sulfónico) ABTS•+. Todos los

hidrolizados mantuvieron actividad de peptidasas alcalinas a lo largo del proceso. Los hidrolizados tratados con Alcalasa 2.4L mos-

traron una actividad de peptidasas e un grado de hidrólisis signicativamente mayores en comparación con Aut. A los 60 min, Alc-0.24

alcanzó niveles de actividad de peptidasas similares a los de Alc-2, y a los 30 min, ambos presentaron grados de hidrólisis comparables.

La actividad de captura de ABTS•+ aumentó con el tiempo para Alc-0.24, siendo ambas concentraciones de Alcalasa 2.4 L superiores

a la autólisis. No se encontraron diferencias signicativas entre Alc-0.24 y Alc-2. Aunque todos los hidrolizados mostraron actividad

de captura de DPPH, no se detectaron diferencias signicativas entre tratamientos o tiempos de reacción. Estos hallazgos destacan el

potencial para producir hidrolizados proteicos de valor agregado a partir de residuos de merluza argentina.

Palabras clave: Gestión de residuos, hidrolizado de proteínas de pescado, actividad de peptidasa, grado de hidrólisis, propiedad

antioxidante.

Liebana et aL.: Protein hydroLysates from argentine hake byProducts 3

The enzymatic treatments were then evaluated and

compared on the basis of their degree of hydrolysis

and antioxidant activity.

MATERIALS AND METHODS

The processing waste of M. hubbsi used in this

study was supplied by Grupo Polo Sur (Mar del

Plata, Argentina). This waste included heads, skele-

tons and viscera. Materials were uniformly minced

and subsequently stored at -20 °C until needed.

Preparation of hake protein hydrolysate

Hake protein hydrolysate was prepared accord-

ing to the method of Pereira et al. (2022) with slight

modications. To produce the hake protein hydro-

lysate by autolysis (Aut), the mixture was digested

in a water bath maintained at 45 ± 2 °C for 150 min

with stirring, followed by an increase in temper-

ature to 80 °C for 20 min to deactivate the en-

zymes. Subsequently, the mixture was centrifuged

at 10,000 g at 4 °C for 15 min (KIMADI 3H12RI,

China), resulting in the protein hydrolysate being

present in the supernatant. For the hydrolysate pro-

duction using 0.24% and 2% (w/v) Alcalase 2.4L

(designated as Alc-0.24 and Alc-2, respectively),

the mixture was preheated to 80 °C for 20 min

to inactivate endogenous enzymes before adding

the commercial enzyme. Finally, the Aut, Alc-0.24,

and Alc-2 were lyophilized (RIFICOR L-A-B3, Ar-

gentina) and stored at -20 °C until further use. All

protein hydrolysates were made in triplicate.

Alkaline peptidase activity

The activity of alkaline peptidases was assessed

using the method described by García-Carreño

(1992). A 0.5% (w/v) azocasein solution (Sigma

A2765) dissolved in a 50 mM Tris-HCl buffer at

pH 7.5 was used as substrate. For the assay, 5 μl

aliquots of the protein hydrolysates (prior to the en-

zyme deactivation) were incubated for 30 min with

a mixture containing 250 μl of substrate and 250 μl

of the same buffer. The reaction was terminated by

adding 250 μl of 20% (w/v) trichloroacetic acid

(TCA). In control treatments, TCA was added be-

fore the substrate. Absorbance readings were tak-

en at a wavelength of 366 nm using a microplate

spectrophotometer (Epoch BioTek, Gen5 Soft-

ware, USA). Total alkaline peptidase activity was

calculated as activity units, representing the change

in absorbance per minute measured in the crude

extract, expressed per milliliter of crude extract

(U ml-1).

Degree of hydrolysis

The degree of hydrolysis (DH) was assessed

using the methodology described by Baek and

Cadwallader (1996). Aliquots from the three rep-

licates were collected at 15, 30, 60, 90, 120, and

150-min intervals. Enzyme activity was then halted

by heating the samples to 80 °C for 20 min. After

this, 500 μl of the sample was mixed with 1,000 μl

of 0.3 M trichloroacetic acid and incubated at room

temperature for 20 min before centrifugation at

2,000 g for 5 min. The resulting supernatant (25 μl)

was then combined with 225 μl of distilled water,

125 μl of 0.5 N NaOH and 0.25 ml of 1 N Fo-

lin-Ciocalteu reagent (Sigma F 9252). The mix-

ture was stirred and incubated at 30 °C for 30 min,

followed by centrifugation at 2,000 g for 10 min.

The absorbance of the supernatant was measured

at 578 nm. A blank was prepared by adding 1,000

μl of 0.3 M TCA to unhydrolyzed shrimp protein

(zero time), representing the baseline concentration

of TCA-soluble peptides as tyrosine. The maxi-

mum hydrolysis level, indicating the highest con-

centration of TCA-soluble peptides, was obtained

by hydrolyzing 0.1 g of shrimp substrate with 4 ml

of 6 N HCl at 110 °C for 24 h. The DH was calcu-

lated using the following equation:

DH = Abs t - Abs t0 × 100

Abs max

Marine and Fishery sciences 38 (2): xxx-xxx (2025)

where Abs t is the absorbance at a specic time

point, Abs t0 is the initial absorbance, and Abs max

is the absorbance at the nal hydrolysis time.

Antioxidant activity

The antioxidant activity was evaluated to deter-

mine the hydrolysis reaction time that maximizes

antioxidant capacity, with evaluations performed

at 0, 30, 60, and 90 min of the reaction. The

2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic

acid) radical (ABTS•+) scavenging activity was

also measured according to the method of Re et

al. (1999). The ABTS•+ was prepared by mixing

7 mM ABTS•+ with 2.45 mM potassium persulfate,

allowing it to stand in the dark at room temper-

ature for 16 h. The ABTS•+ solution was diluted

with 5 mM sodium phosphate buffer (pH 7.4) to

achieve an absorbance of 0.70 ± 0.02 at 734 nm. A

20 μl aliquot of hydrolysate at various concentra-

tions (0.5-10 mg ml-1) was combined with 2 ml of

the ABTS•+ solution and incubated in the dark for

6 min. Absorbance was measured at 734 nm using

a spectrophotometer (SPECTROstar Nano BMG

LABTECH, Germany). The control solution was

prepared similarly, replacing the sample with dis-

tilled water. The ABTS•+ scavenging activity (sa)

was calculated using the following formula:

ABTS•+ sa (%) = 1 - Abs sample × 100

Abs control

where Abs control represents the absorbance of the

control and Abs sample represents the absorbance

of sample. The IC50, dened as the concentration

of the sample required to scavenge 50% of DPPH,

was subsequently calculated for each protein hy-

drolysate from the plot of sample concentration

versus percentage inhibition.

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) rad-

ical scavenging activity was determined following

the method of Shimada et al. (1992). A 1 ml aliquot

of each SPH hydrolysate (0.25-10 mg ml-1) was

combined with 1 ml of DPPH solution (0.1 mM

DPPH in 95% ethanol). The mixture was kept in

the dark at room temperature for 30 min. Absorb-

ance was measured at 517 nm using a spectropho-

tometer (SPECTROstar Nano BMG LABTECH,

Germany). A control sample was prepared using

distilled water in place of the hydrolysate solution.

The DPPH radical scavenging activity (sa) was

calculated as the percentage inhibition of DPPH

according to the following equation:

DPPH sa (%) = 1 - Abs sample × 100

Abs control

where Abs sample and Abs control refer to the ab-

sorbance of sample and control, respectively. The

IC50 value for this assay was also determined as

previously described.

Statistical analysis

All experiments were conducted in triplicate.

Results were reported as mean ± SE. Analysis of

variance (ANOVA) was carried out followed by

Tukey’s test for post-hoc pairwise comparisons of

mean values, using R statistical software (version

4.3.1) (R Core Team 2022). A signicance criterion

of p < 0.05 was applied.

RESULTS

Enzymatic activity of alkaline peptidases

The enzymatic activity in Aut (with an initial

activity of 0.53 ± 0.041 U ml-1) decreased signif-

icantly after 90 min, reaching a value of 0.23 ±

0.024 U ml-1, and remained stable for the rest of the

150 min (Figure 1). The enzymatic activity in the

hydrolysates treated with Alcalase 2.4L was sig-

nicantly higher at all evaluated times compared

to Aut. Although Alc-0.24 initially showed enzy-

Liebana et aL.: Protein hydroLysates from argentine hake byProducts 5

matic activity comparable to Aut, it experienced a

sharp increase after 15 min (25.46 ± 2.87 U ml-1)

and remained stable throughout the reaction. At

15 min, the activity was similar to Alc-2 (26.61

± 1.62 U ml-1), and this similarity persisted after

60 min (30.70 ± 3.32 and 33.36 ± 1.79 U ml-1 for

0.24% and 2%, respectively), continuing until the

end of the reaction.

Degree of hydrolysis

The DH of Aut began to increase after 120 min,

reaching 14.09 ± 0.451%, remaining stable until

the end of the 150-min study (Figure 2). In contrast,

protein hydrolysates obtained with Alcalase 2.4L

exhibited signicantly higher degrees of hydrolysis

at all time points, indicating a marked difference

in enzymatic activity between M. hubbsi endoge-

nous enzymes and Alcalase 2.4L. Notably, Alc-

0.24 reached a DH comparable to Alc-2 starting at

30 min, maintaining this level for the remainder of

the reaction. At 120 min, both Alcalase-treated hy-

drolysates reached a plateau that persisted until the

end of the reaction, with values of 64.39 ± 2.56%

for Alc-0.24 and 72.84 ± 1.79% for Alc-2.

Antioxidant capacity

ABTS•+ radical scavenging activity

Both enzymatic treatment and reaction time had

a signicant impact on the ABTS•+ radical scav-

enging activity (Table 1). Regarding differences

between time points within the same enzymatic

treatment, a signicant increase in the antioxidant

capacity of the M. hubbsi hydrolysate treated with

0.24% Alcalase 2.4L was observed at 30 and

60 min. Additionally, when comparing different

enzymatic treatments, hydrolysates treated with

Alcalase 2.4L at 0.24% and 2% exhibited a greater

inhibitory effect on the ABTS•+ radical compared

Figure 1. Alkaline peptidase activity (U ml-1) of protein hydrolysates from Merluccius hubbsi over the hydrolytic reaction time.

Different uppercase letters indicate signicant differences (p < 0.05) between treatments at the same time point, while

different lowercase letters indicate signicant differences (p < 0.05) between time points within the same treatment. Aut:

M. hubbsi hydrolysate obtained through autolysis; Alc-0.24: M. hubbsi hydrolysate obtained using 0.24% commercial

Alcalase 2.4L enzyme; Alc-2: M. hubbsi hydrolysate obtained using 2% commercial Alcalase 2.4L enzyme.

Aa AabAab AabAbAbAb

Cbc Bbc Bbc Bc

Bb Bb

Bb Bb Bb

-1

015306090 120 150

Alkaline peptidase activity (U ml )

-1

Time (min)

Alc-2AutAlc 0.24-

Marine and Fishery sciences 38 (2): xxx-xxx (2025)

Table 1. Antioxidant activity of Merluccius hubbsi hydrolysates obtained through autolysis (Aut) or with 0.24% and 2% v/v Al-

calase 2.4L (Alc-0.24 and Alc-2, respectively), determined by DPPH and ABTS•+ inhibition assays.

Time (min) Aut Alc-0.24 Alc-2

ABTS•+ radical inhibition assay (IC50)

0 1.60 ± 0.11Aa 1.45 ± 0.179ABa 0.98 ± 0.210B

30 1.34 ± 0.13Aab 1.47 ± 0.301Aa 0.86 ± 0.113B

60 1.47 ± 0.116Aab 1.09 ± 0.140ABb 0.83 ± 0.158B

90 1.02 ± 0.026Ab 0.90 ± 0.125Ab 0.83 ± 0.012A

DPPH radical inhibition assay (IC50)

0 3.52 ± 0.238Aa 3.89 ± 0.261Aa 3.54 ± 0.291Aa

30 3.64 ± 0.050Aa 3.96 ± 0.141Aa 3.58 ± 0.221Aa

60 3.50 ± 0.046Aa 3.79 ± 0.369Aa 3.69 ± 0.148Aa

90 3.22 ± 0.533Aa 3.27 ± 0.623Aa 3.29 ± 0.310Aa

Values are expressed as IC50 (mg ml-1) (mean ± SE). Lowercase letters within the same column indicate signicant differences

(p < 0.05) between time points for each treatment. Uppercase letters within the same row indicate signicant differences among

treatments at the same hydrolytic reaction time (p < 0.05).

Figure 2. Degree of hydrolysis (%) of protein hydrolysates from Merluccius hubbsi over the hydrolytic reaction time. Different

uppercase letters indicate signicant differences (p < 0.05) between treatments at the same time point, while different

lowercase letters indicate signicant differences (p < 0.05) between time points within the same treatment. Aut: M. hubbsi

hydrolysate obtained through autolysis; Alc-0.24: M. hubbsi hydrolysate obtained using 0.24% commercial Alcalase® 2.4L

enzyme; Alc-2: M. hubbsi hydrolysate obtained using 2% commercial Alcalase® 2.4L enzyme.

Aa Aa Aa Aa

Ab Ab

Cb Bb Bb

Bbc

Bc Bc Bc

015306090 120 150

Degree of hydrolysis (%)

Time (min)

Alc-2AutAlc 0.24-

Liebana et aL.: Protein hydroLysates from argentine hake byProducts 7

to the hydrolysate obtained by autolysis at all eval-

uated time points. Furthermore, no signicant dif-

ferences were found between hydrolysates treated

with different concentrations of Alcalase 2.4L.

DPPH scavenging assay

Results showed no signicant differences (p <

0.05) either across various reaction times within

the same enzymatic treatment or among different

enzymatic treatments at any given time point. This

suggested that neither reaction time nor the type

of enzymatic treatment had a signicant effect on

antioxidant activity (Table 1).

DISCUSSION

In this study, wastes from Argentine hake (M.

hubbsi) were hydrolyzed using endogenous en-

zymes or Alcalase 2.4L at concentrations of

0.24% and 2% (v/v) for 150 min. Based on the

results of peptidase activity of different protein

hydrolysates (Aut, Alc-0.24 and Alc-2), it can be

concluded that enzymes remained active in all

cases, suggesting that the hydrolytic reaction was

sustained over time. The high stability of endog-

enous enzymes from M. hubbsi is consistent with

the ndings of Friedman et al. (2022), who evalu-

ated intestinal peptidase activity in this species and

observed that these enzymes were highly stable

within a pH range of 7 to 11.5 and temperatures

between 10 and 50 °C over a period of 150 min.

In the same way, Samaranayaka et al. (2007), Ma-

zorra-Manzano (2008, 2012), and Cheung et al.

(2012) reported peptidase activity in endogenous

enzymes of the Pacic whiting (M. productus).

Overall, enzymatic activity was higher in protein

hydrolysates produced with Alcalase 2.4L than

those obtained through autolysis. Additionally, the

enzymatic activity of the hydrolysate produced

with Alcalase 2.4L 0.24% v/v reached a compara-

ble level to that of Alcalase 2.4L 2% hydrolysate

after 60 min of reaction.

The DH is a key indicator of the progress of

protein hydrolysis and the extent of product deg-

radation. Generally, a higher DH is linked to im-

proved bioactive properties due to the release of

smaller bioactive peptides, while a lower degree is

associated with better sensory qualities as shorter

peptides often contribute to increased bitterness

(Idowu and Benjakul 2019; Nirmal et al. 2022). In

our study, according to results of enzymatic activ-

ity, DH was signicantly lower in protein hydro-

lysates obtained by autolysis compared to those

treated with Alcalase 2.4L.

The hydrolysate obtained through autolysis

achieved a peak of around 14% after 120 min of

reaction, which is greater than the percentage re-

ported by Mazorra-Manzano et al. (2012) for pro-

tein hydrolysates from M. productus. On the other

hand, Alc-0.24 and Alc-2 showed values of approx-

imately 64% and 72% of DH. These values were

higher than those reported by Pires et al. (2013,

2024), who found a degree of hydrolysis ranging

from 10.7% to 36.4% for hake hydrolysates treated

with Alcalase 2.4L. However, other studies have

reported closer values of DH, ranging from 42.5%

to 78.26%, in hydrolysates of other sh species

treated with Alcalase 2.4L (Piotrowicz and Mel-

lado 2015; Gómez et al. 2020). In agreement with

results of this study, Ovissipour et al. (2013) report-

ed a signicantly lower DH in protein hydrolysates

of the anchovy sprat (Clupeonella engrauliformis)

produced by autolysis (17.4%) compared to those

treated with Alcalase 2.4L (55.8%). Furthermore,

the low enzymatic activity and DH observed in the

Aut treatment could be associated with the initial

quality of byproducts, which may have impacted

the activity of endogenous enzymes, as indicated

in studies in rainbow trout byproducts and other

marine resources (Nikoo et al. 2021, 2022).

The signicantly higher alkaline peptidase ac-

tivity and DH observed in the protein hydrolysate

of hake prepared with Alcalase 2.4L, compared

to the autolysate (p < 0.05), can be attributed to

the broad specicity for peptide bond hydrolysis

of Alcalase 2.4L (Sbroggio et al. 2016) and its

Marine and Fishery sciences 38 (2): xxx-xxx (2025)

high efciency in achieving elevated degrees of

hydrolysis in a short time (Ovissipour et al. 2009).

Similarly, Cheung et al. (2012) found that although

endogenous enzymes in M. productus exhibited

activity, commercial enzymes achieved a greater

degree of hydrolysis in their hydrolysates.

Consistent with the enzymatic activity ndings,

the protein hydrolysate produced with Alcalase

2.4L 0.24% reached a DH similar to that of the

Alcalase 2.4L 2% hydrolysate starting at 30 min,

maintaining this level throughout the remainder

of the reaction, with both hydrolysates reaching a

plateau at 120 min.

Antioxidants are stable compounds that neutral-

ize reactive oxygen species, helping to prevent dis-

eases through various mechanisms (Ananey-Obiri

et al. 2019). Protein hydrolysates or peptides are

commonly evaluated for their antioxidant capacity

using methods such as DPPH and ABTS•+ radical

inhibition, which indicates their ability to scav-

enge radicals through hydrogen donation or elec-

tron transfer (Sila and Bougatef 2016; Idowu and

Benjakul, 2019).

The ABTS•+ radical inhibition assay revealed that

only Alc-0.24 exhibited an increase in antioxidant

activity at 30 and 60 min, while the ABTS•+ inhi-

bition remained stable over time for both Aut and

Alc-2. Regarding the comparison between different

enzymatic treatments, although Aut showed a sig-

nicant inhibitory effect on the ABTS•+ radical, Alc-

0.24 and Alc-2 demonstrated a superior inhibitory

effect on this radical at all evaluated time points.

These results align with the ndings of Cheung et

al. (2012), who reported improved ABTS•+ radical

inhibition with the addition of exogenous peptidas-

es. Additionally, no signicant differences were ob-

served between the hydrolysates treated with the two

concentrations of Alcalase 2.4L, suggesting that a

lower concentration of Alcalase 2.4L can produce

a protein hydrolysate with the same ABTS•+ radical

inhibitory activity as a higher concentration.

Results of the ABTS•+ assay indicate a strong

correlation between the DH and ABTS•+ radical

scavenging activity in the Argentine hake. These

ndings are in line with those of Raghavan et al.

(2008) and Phanturat et al. (2010), who observed

that an increase in the DH enhances the ABTS•+

free radical scavenging activity in hydrolysates

from other sh species. On the other hand, it is gen-

erally reported that a higher DH releases a greater

number of low molecular weight peptides, which

exhibit higher DPPH scavenging activity (Cha-

lamaiah et al. 2015; Henriques et al. 2021). For

example, Karoud et al. (2019) found an increase

in DPPH scavenging activity as the DH increased

in protein hydrolysates from M. merluccius heads.

However, in this study, no signicant differences

were observed among hydrolysates produced with

different enzymatic treatments, despite their var-

ying DH levels. This lack of correlation between

DPPH scavenging activity and DH has also been

reported in other studies. Klompong et al. (2007),

for instance, found that hydrolysates with a low DH

exhibited higher antioxidant power in the yellow

stripe trevally (Selaroides leptolepis). Similarly,

Pires et al. (2024) observed that smaller peptides

had lower DPPH scavenging activity in protein

hydrolysates from Atlantic salmon (Salmo salar)

and Cape hake (M. capensis) byproducts.

In this study, although hydrolysates did not show

improvements in DPPH inhibitory activity during

the hydrolysis process, they maintained a high ac-

tivity level from the beginning. This initial scav-

enging ability, sustained over time, may be attrib-

uted to the natural presence of various antioxidant

compounds in the hake byproducts (Ognjanović et

al. 2008; Karoud et al. 2020). On the other hand,

while the DH, the enzyme used, and the protein

substrate play signicant roles in the antioxidant

capacity of protein hydrolysates (Klompong et al.

2007), the net hydrophobicity of peptides and free

amino acids also inuences this activity (Asaduz-

zaman et al. 2020). It is important to note that the

DPPH assay is conducted in a lipophilic medium,

whereas the ABTS•+ assay is performed in an aque-

ous medium. This difference inuences the radi-

cal scavenging capabilities: hydrophobic peptides

demonstrate greater efcacy in the DPPH scav-

Liebana et aL.: Protein hydroLysates from argentine hake byProducts 9

enging assay, whereas less hydrophobic peptides

perform better in the ABTS•+ scavenging assay

(Latorres et al. 2018; Singh et al. 2024). Therefore,

it is possible that the hydrophobic peptides released

in this study did not demonstrate a greater antiox-

idant capacity than natural compounds present in

the hake byproducts.

This study offers valuable insights into the hy-

drolysis process and the antioxidant activity of

hake hydrolysates. The complexity of the relation-

ship between hydrolysis and antioxidant properties

is highlighted by the absence of sig nicant differ-

ences in DPPH radical scavenging activity among

treatments, despite notable varia tions in DH and

peptidase activity. Future research should focus

on analyzing the molecular weight distribution and

amino acid composition of these protein hydro-

lysates to provide a deeper understanding of the

results and inform their potential applications in

feed or food-grade products.

Results showed that protein hydrolysates of

Argentine hake obtained with Alcalase 2.4L ex-

hibited higher alkaline peptidase activity, degree

of hydrolysis, and ABTS•+ scavenging activity

compared to autolysis. Notably, Alc-0.24 pro-

duced a hydrolysate with similar characteristics

to Alc-2 after 120 min of hydrolysis. This nd-

ing is particularly interesting since it implies that

less commercial enzyme may be needed to make

protein hydrolysates with comparable characteris-

tics, which could minimize the cost associated to

the enzymatic hydrolysis of these residues. It also

highlights the potential of using Argentine hake

waste to produce value-added protein hydrolysates,

offering a more efcient and sustainable approach

to the use of shery byproducts.

ACKNOWLEDGEMENTS

This study is part of Clara Liebana’s postgrad-

uate PhD Thesis (National University of Mar del

Plata, Argentina) supported by CONICET fel-

lowship. We are grateful to Juan Lancia from the

Invertebrate Laboratory (IIMyC/CONICET-UN-

MDP) for his invaluable assistance with the ly-

ophilization of the samples. Financial support

from the Universidad Nacional de Mar del Plata

(EXA 1066/2022 and EXA 1075/2022), CON-

ICET (PIP K7 11220200101093CO and PUE

22920200100016CO-IFIMAR), and FONCyT

AGENCIA (PICT-2020-SERIEA-01851) is grate-

fully acknowledged.

Conict of interest

The authors declare that there is no conict of

interest and that the research meets the required

ethical guidelines.

Author contributions

Clara Liebana: data curation; formal analysis;

writing-original draft; project administration. Nair

de los Ángeles Pereira: funding acquisition; project

administration; supervision; writing-review and

editing. Analia V. Fernández Gimenez: funding

acquisition; project administration; supervision;

writing-review and editing. Maria Florencia Fan-

gio: funding acquisition; project administration;

supervision, writing-review and editing.

REFERENCES

ananey-obiri d, Matthews lg, tahergorabi r.

2019. Proteins from sh processing by-products.

In: galanakis cM, editor. Proteins: sustainable

source, processing and applications. Academic

Press. p. 163-191. DOI: https://doi.org/10.1016/

B978-0-12-816695-6.00006-4

asaduzzaMan akM, hasan i, rahMan Mh,

tareq arM. 2020. Antioxidant and antipro-

liferative activity of phytoconstituents identi-

ed from Sargassum binderi seaweed extracts

Marine and Fishery sciences 38 (2): xxx-xxx (2025)

cultivated in Bangladesh. Int J Biosci. 16 (3):

481-494.

baek hh, cadwallader kr. 1996. Volatile com-

pounds in avor concentrates produced from

craysh-processing byproducts with and with-

out protease treatment. J Agric Food Chem. 44

(10): 3262-3267. DOI: http://doi.org/10.1021/

jf960023q

chalaMaiah M, JyothirMayi t, diwan PV,

dinesh kuMar b. 2015. Antioxidant activity

and functional properties of enzymatic protein

hydrolysates from common carp (Cyprinus car-

pio) roe (egg). J Food Sci Technol. 52: 5817-

5825. DOI: https://doi.org/10.1007/s13197-015-

1714-6

cheung iwy, cheung lky, tan ny, li-chan

ecy. 2012. The role of molecular size in an-

tioxidant activity of peptide fractions from Pa-

cic hake (Merluccius productus) hydrolysates.

Food Chem. 134 (3): 1297-1306. DOI: https://

doi.org/10.1016/j.foodchem.2012.02.215

FernÁndez herrero a, Vittone M, saloMone a.

2015. Biological silage of Merluccius hubbsi.

Amino acid composition, degree of hydrolysis,

and peptide size. Issues Biol Sci Pharm Res. 3

(6): 57-62.

FriedMan is, behrens la, Pereira ndla, con-

treras eM, FernÁndez-giMenez aV. 2022.

Digestive proteinases from the marine sh

processing wastes of the South-West Atlantic

Ocean: their partial characterization and com-

parison. J Fish Biol. 100 (1): 150-160. DOI:

https://doi.org/10.1111/jfb.14929

FriedMan is, contreras eM, FernÁndez-giMe-

nez aV. 2024. Catalytic stability of aspartic

proteinases recovered from viscera of Merluc-

cius hubbsi, Percophis brasiliensis, Urophicis

brasiliensis, and Cynoscion guatucupa. Waste

Biomass Valor. DOI: https://doi.org/10.1007/

s12649-024-02717-8

gao r, yu q, shen y, chu q, chen g, Fen s,

yang M, yuan l, MccleMents dJ, sun q.

2021. Production, bioactive properties, and

potential applications of sh protein hydro-

lysates: developments and challenges. Trends

Food Sci Technol. 110: 687-699. DOI: https://

doi.org/10.1016/j.tifs.2021.02.031

garcía-carreño Fl. 1992. The digestive pro-

teases of langostilla (Pleuroncodes planipes,

decapoda): their partial characterization, and

the effect of feed on their composition. Comp

Biochem Physiol B Comp Biochem. 103:

575-578. DOI: https://doi.org/10.1016/0305-

0491(92)90373-Y

góMez lJ, góMez na, zaPata Je, lóPez-garcía

g, cilla a, alegría a. 2020. Optimization

of the red tilapia (Oreochromis spp.) viscera

hydrolysis for obtaining iron-binding peptides

and evaluation of in vitro iron bioavailability.

Foods. 9: 883. DOI: https://doi.org/10.3390/

foods9070883

góngora hg, ledesMa P, ValVo Vrl, ruiz

ae, breccia Jy. 2012. Screening of lactic acid

bacteria for fermentation of minced wastes of

Argentinean hake (Merluccius hubbsi). Food

Bioprod Process. 90 (4): 767-772.

henriques a, VÁzquez Ja, Valcarcel J, Mendes

r, bandarra nM, Pires c. 2021. Characteriza-

tion of protein hydrolysates from sh discards

and by-products from the North-West Spain

shing eet as potential sources of bioactive

peptides. Mar Drugs. 19: 338. DOI: https://doi.

org/10.3390/md19060338

idowu at, benJakul s. 2019. Bitterness of sh

protein hydrolysate and its debittering pros-

pects. J Food Biochem. 43 (9): e12978. DOI:

https://doi.org/10.1111/jfbc.12978

karoud w, ghlissi z, krichen F, kallel r, bou-

gateF h, zarai z, boudawara t, sahnoun z,

sila a, bougateF a. 2020. Oil from hake (Mer-

luccius merluccius): characterization, antioxi-

dant activity, wound healing and anti-inamma-

tory effects. J Tissue Viability. 29 (2): 138-147.

DOI: https://doi.org/10.1016/j.jtv.2020.01.002

karoud w, sila a, krichen F, Martinez-alVa-

rez o, bougateF a. 2019. Characterization,

surface properties and biological activities of

protein hydrolysates obtained from hake (Mer-

Liebana et aL.: Protein hydroLysates from argentine hake byProducts 11

luccius merluccius) heads. Waste Biomass Val-

or. 10: 287-297. DOI: https://doi.org/10.1007/

s12649-017-0069-9

kloMPong V, benJakul s, kantachote d, shahi-

di F. 2007. Antioxidative activity and function-

al properties of protein hydrolysate of yellow

stripe trevally (Selaroides leptolepis) as inu-

enced by the DH and enzyme type. Food Chem.

102: 1317-1327. DOI: https://doi.org/10.1016/j.

foodchem.2006.07.016

laMas dl, yeannes Mi, Massa ae 2015. Partial

purication of proteolytic enzymes and char-

acterization of trypsin from Merluccius hubbsi

by-products. Internat J Food Nutrit. Sci. 4 (5):

2-11.

latorres JM, rios dg, saggioMo g, wasiele-

sky w, Prentice-hernandez c. 2018. Func-

tional and antioxidant properties of protein

hydrolysates obtained from white shrimp (Li-

topenaeus vannamei). J Food Sci Technol. 55:

721-729. DOI: https://doi.org/10.1007/s13197-

017-2983-z

[MagyP] secretaría de agricultura, ganade-

ría y Pesca. 2024. Desembarques de capturas

marítimas totales. [accessed 2024 Sep 17].

https://www.magyp.gob.ar/sitio/areas/pesca_

maritima/desembarques/lectura.php?imp=1&

tabla=especie_mes_2018.

Martone cb, borla oP, sÁnchez JJ. 2005. Fish-

ery by-product as a nutrient source for bacteria

and archaea growth media. Bioresour Technol.

96 (3): 383-387.

Mazorra-Manzano Ma, Pacheco-aguilar r,

raMirez-suarez Jc, garcía-sÁnchez g. 2008.

Pacic whiting (Merluccius productus) un-

derutilization in the Gulf of California: muscle

autolytic activity characterization. Food Chem.

107 (1): 106-111. DOI: https://doi.org/10.1016/j.

foodchem.2007.07.056

Mazorra-Manzano Ma, Pacheco-aguilar r,

raMírez-suÁrez Jc, garcia-sanchez g, lu-

go-sÁnchez Me. 2012. Endogenous proteases

in Pacic whiting (Merluccius productus) mus-

cle as a processing aid in functional sh protein

hydrolysate production. Food Bioproc Tech-

nol. 5: 130-137. DOI: https://doi.org/10.1007/

s11947-010-0374-9

Moya-Moreira tF, gonçalVes oh, leiMann FV,

ribeiro rP. 2023. Fish protein hydrolysates: bi-

oactive properties, encapsulation and new tech-

nologies for enhancing peptides bioavailability.

Curr Pharm Des. 29 (11): 824-836. DOI: https://

doi.org/10.2174/1381612829666230110141811

nirMal nP, santiVarangkna c, raJPut Ms, ben-

Jakul s, Maqsood s. 2022. Valorization of sh

byproducts: sources to end-product applications

of bioactive protein hydrolysate. Compr Rev

Food Sci Food Saf. 21 (2): 1803-1842. DOI:

https://doi.org/10.1111/1541-4337.12917

nikoo M, beJakul M, benJakul s, ahMadi

gablighi h. 2022. Protein hydrolysates de-

rived from aquaculture and marine byproducts

through autolytic hydrolysis. Compr Rev Food

Sci Food Saf. 21 (6): 4872-4899.

nikoo M, regenstein JM, noori F, gheshlaghi

sP. 2021. Autolysis of rainbow trout (Oncorhy-

nchus mykiss) by-products: enzymatic activities,

lipid and protein oxidation, and antioxidant ac-

tivity of protein hydrolysates. LWT Food Sci

Techol. 140: 110702.

OgnjanOvić Bi, ĐOrĐević nZ, Perendija Br,

desPOtOvić sg, Žikić rv, Štajn aŠ, saičić Zs.

2008. Concentration of antioxidant compounds

and lipid peroxidation in the liver and white

muscle of hake (Merluccius merluccius L.) in

the Adriatic Sea. Arch Biol Sci. 60 (4): 601-607.

DOI: https://doi.org/10.2298/ABS0804601O

oVissiPour M, rasco b, shiroodi sg, Modan-

low M, gholaMi s, neMati M. 2013. Anti-

oxidant activity of protein hydrolysates from

whole anchovy sprat (Clupeonella engrauli-

formis) prepared using endogenous enzymes

and commercial proteases. J Sci Food Agric. 93

(7): 1718-1726. DOI: https://doi.org/10.1002/

jsfa.5957

oVissiPour M, saFari r, MotaMedzadegan

a, shabanPour b. 2009. Chemical and bio-

chemical hydrolysis of Persian sturgeon (Ac-

Marine and Fishery sciences 38 (2): xxx-xxx (2025)

ipenser persicus) visceral protein. Food Bio-

process Technol. 5: 460-465. DOI: https://doi.

org/10.1007/s11947-009-0284-x

Pacheco-aguilar r, Mazorra-Manzano Ma,

raMírez-suÁrez Jc. 2008. Functional prop-

erties of sh protein hydrolysates from Pacic

whiting (Merluccius productus) muscle pro-

duced by a commercial protease. Food Chem.

109 (4): 782-789. DOI: https://doi.org/10.1016/j.

foodchem.2008.01.047

Pereira ndla, Fangio MF, rodriguez ye,

bonadero Mc, harÁn ns, FernÁndez-giMe-

nez aV. 2022. Characterization of liquid protein

hydrolysates shrimp industry waste: analysis

of antioxidant and microbiological activity,

and shelf life of nal product. J Food Process

Preserv. 46 (8): e15526. DOI: http://dx.doi.

org/10.1111/jfpp.15526

Phanturat P, benJakul s, Visessanguan w,

roytrakul s. 2010. Use of pyloric caeca

extract from bigeye snapper (Priacanthus

macracanthus) for the production of gelatin

hydrolysate with antioxidative activity. LWT

Food Sci Technol. 43: 86-97. DOI: https://doi.

org/10.1016/j.lwt.2009.06.010

Piotrowicz ibb, Mellado MMs. 2015. Antiox-

idant hydrolysates production from Argentine

anchovy (Engraulis anchoita) with different

enzymes. Int Food Res J. 22 (3): 999-1007.

Pires c, cleMente t, batista i. 2013. Functional

and antioxidative properties of protein hydro-

lysates from Cape hake by-products prepared by

three different methodologies. J Sci Food Agric.

93 (4): 771-780. DOI: https://doi.org/10.1002/

jsfa.5796

Pires c, leitão M, saPatinha M, gonçalVes a,

oliVeira h, nunes Ml, teixeira b, Mendes

r, caMacho c, Machado M, et al. 2024. Pro-

tein hydrolysates from salmon heads and Cape

hake by-products: comparing enzymatic meth-

od with subcritical water extraction on bioac-

tivity properties. Foods. 13 (15): 2123. DOI:

https://doi.org/10.3390/foods13152418

r core teaM. 2022. R: A language and environ-

ment for statistical computing (Version 4.3.1).

R Foundation for Statistical Computing. https://

www.r-project.org.

raghaVan s, kristinsson hg, leeuwenburgh

c. 2008. Radical scavenging and reducing abil-

ity of tilapia (Oreochromis niloticus) protein

hydrolysates. J Agric Food Chem. 56: 10359-

10367. DOI: https://doi.org/10.1021/jf8017194

saMaranayaka ag, ho tc, li-chan ec. 2007.

Correlation of Kudoa spore counts with prote-

olytic activity and texture of sh mince from

Pacic hake (Merluccius productus). J Aquat

Food Prod Technol. 15 (4): 75-93. DOI: https://

doi.org/10.5555/20073066467

re r, Pellegrini n, Proteggente a, Pannala

a, yang M, rice-eVans c. 1999. Free radi-

cal. Free Radic Biol Med. 26: 1231-1237. DOI:

https://doi.org/10.1016/s0891-5849(98)00315-3

sbroggio MF, Montilha Ms, Figueiredo Vrgd,

georgetti sr, kurozawa le. 2016. Inuence

of the degree of hydrolysis and type of enzyme

on antioxidant activity of okara protein hydro-

lysates. Food Sci Technol. 36 (2): 375-381. DOI:

https://doi.org/10.1590/1678-457X.000216

shekoohi n, carson bP, Fitzgerald rJ. 2024.

Antioxidative, glucose management, and mus-

cle protein synthesis properties of sh protein

hydrolysates and peptides. J Agric Food Chem.

72: 21301-21317. DOI: https://doi.org/10.1021/

acs.jafc.4c02920

shiMada k, FuJikawa k, yahara k, nakaMura

t. 1992. Antioxidative properties of xanthan on

the autoxidation of soybean oil in cyclodextrin

emulsion. J Agric Food Chem. 40 (6): 945-948.

DOI: https://doi.org/10.1021/jf00018a005

sila a, bougateF a. 2016. Antioxidant peptides

from marine by-products: isolation, identica-

tion and application in food systems. A review.

J Funct Foods. 21: 10-26. DOI: https://doi.

org/10.1016/j.jff.2015.11.007

singh a, kadaM d, gautaM ar, rengasaMy

kr, aluko re, benJakul s. 2024. Angioten-

sin-I-converting enzyme and renin inhibitions

by antioxidant shrimp shell protein hydrolysate

Liebana et aL.: Protein hydroLysates from argentine hake byProducts 13

and ultraltration peptide fractions. Food Bio-

sci. 60: 104524. DOI: https://doi.org/10.1016/j.

fbio.2024.104524

wang z, liu x, xie h, liu z, rakariyathaM k,

yu c, zhou d. 2021. Antioxidant activity and

functional properties of Alcalase-hydrolyzed

scallop protein hydrolysate and its role in the

inhibition of cytotoxicity in vitro. Food Chem.

344: 128566. DOI: https://doi.org/10.1016/j.

foodchem.2020.128566