High-pressure effects on the activity of cathepsins B and D of mackerel and horse

mackerel muscle

Liliana Fidalgo1, Jorge A. Saraiva1, Santiago P. Aubourg2, Manuel Vázquez3, J. Antonio

Torres4

1 Research Unit of Organic Chemistry, Natural and Agro-food Products (QOPNA),

Chemistry Department, Aveiro University, Campus Universitário de Santiago, 3810-

193 Aveiro, Portugal.2 Department of Food Technology, Instituto de Investigaciones Marinas (CSIC), Vigo,

Spain. 3 Department of Analytical Chemistry, Facultad de Ciencias Veterinarias,

Universidad de Santiago de Compostela, Lugo, Spain.4 Food Processing Engineering Group, Department of Food Science and Technology,

Oregon State University, Corvallis, OR, USA.

Abstract:

This work sought to determine high pressure processing effects on the activity of

cathepsins B and D in the muscle of mackerel (Scomber scombrus) and horse mackerel

(Trachurus trachurus). In mackerel, the cathepsin B activity decrease reached 40%

while in horse mackerel, low and intermediate pressures (150 and 300 MPa) caused an

activity increase (30%) but a decrease of up to 60% at 450 MPa. In both species,

cathepsin D activity increased after a 300 MPa treatment (up to 2-fold for mackerel and

60% for horse mackerel) while decreasing after a 450 MPa treatment. The activity

increase is probably due to pressure treatment damage of lysosome releasing enzymes

into the fish muscle. Based on the high-pressure processing effects on the activity of

cathepsins B and D, 450 MPa may be used to reduce the proteolytic activity of

cathepsin B prior to chilled or frozen storage of these fish species.

Keywords: High hydrostatic pressure, Scomber scombrus, Trachurus trachurus,

cathepsins B and D

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INTRODUCTION

Small pelagic fish species, such as Atlantic mackerel (Scomber scombrus) and horse

mackerel (Trachurus trachurus), are important fishery resources in many coastal areas

in the South European countries (MBARKI et al. 2009). Atlantic mackerel is a fatty fish

(fat content higher than 10%), while horse mackerel (Trachurus trachurus) is a

medium-fatty fish (fat content between 5 to 10%). Both species are abundant in the

Northeast Atlantic (AUBOURG et al. 2005; BECHET et al. 2005). Horse mackerel has

been attracting great attention because of its moderate price and large quantities

captured by most West-European countries (Holland, Ireland, Spain, France, Germany

and Portugal) (FAO 2003).

For the consumer, freshness is the most important quality aspect in raw fish, this

defining the quality of fish as food. Loss of freshness is caused by a combination of

physical, biochemical, and microbiological reactions. Enzymatic degradations cause the

postmortem softening of fish muscle and facilitate the proliferation of bacterial flora

(CHÉRET et al. 2006; HERNÁNDEZ-ANDRÉS et al. 2008). Endogenous protease activity is

responsible for the proteolysis of fish myofibrillar proteins (CHÉRET et al. 2007).

Cathepsins are one of the characterized proteolytic systems known to hydrolyze

myofibrillar proteins during postmortem storage of fish muscle (JIANG 2000) and it is

found widely distributed in muscles and organs. After death and during muscle storage,

cathepsins B and D may be released from the lysosomal matrix into both the cytoplasm

and the intracellular spaces as a consequence of lysosomes breakdown (BECHET et al.

2005). Cathepsin B enzyme is a cysteine protease that hydrolyzes a wide range of

proteins and has an important role in the hydrolysis of tissue proteins (AOKI et al.

2002), showing an optimal pH for activity around 6.0 (NIELSEN et al. 2006). Cathepsin

D, an aspartic acidic protease, is considered the most important enzyme in the

postmortem degradation of muscle because of its optimum pH and the absence of a

specific inhibitor in the muscle (CHÉRET et al. 2005). Cathepsin D can be found in

several isoenzyme forms with an activity pH range in muscle tissue between pH 3.0 and

6.0 (ASHIE et al. 1997).

High-pressure processing (HPP) is now a commercially well-established new food

preservation technology and an effective alternative to thermal treatments or the use of

chemical preservatives (CHÉRET et al. 2006; RAMIREZ et al. 2009). HPP applications in

food processing are of great interest because of the ability to inactivate food borne

microorganisms and endogenous enzymes (CASTRO et al. 2008; CASTRO et al. 2011)

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while preserving the nutritional and sensory attributes of foods (WIMALARATNE et al.

2008). However, HPP can affect also proteins, particularly myofibrillar proteins,

resulting in structural modifications and texture changes in muscle foods

(ANGSUPANICH et al. 1999). In addition, HPP can disrupt lysosomal membranes causing

the release into the fish muscle of enzymes, such as cathepsins, leading to possible

alterations of myofibrillar proteins mediated by these enzymes (OHMORI et al. 1992).

HPP treatment effects on fish muscle depend on several factors causing activation or

inactivation of muscle enzymes. For instance, studies have shown that HPP treatment

causes an activity increase of cathepsin B at 500 MPa, while cathepsin D activity would

increase after a pressure treatment up to 300 MPa and decrease at higher pressures

(CHÉRET et al. 2005). However in cold-smoked salmon, cathepsin B activity was

reduced by treatments at pressures up to 300 MPa (LAKSHMANAN et al. 2005).

During processing and storage of fish, quality declines as a result of several factors.

Freezing and frozen storage of fish have been largely used to retain their sensory and

nutritional properties before consumption (ERICKSON 1997) but there are several

problems caused/promoted by fish endogenous enzymes occurring during frozen

storage of fish (AUBOURG 1999; LUND et al. 2001; HULTMANN et al. 2004; AUBOURG et

al. 2005; CHAIJAN et al. 2005) resulting in an off-taste and a dry and tough texture

(SIMEONIDOU et al. 1997). The successful applications of HPP in other food systems

suggest this technology as a potential treatment to minimize changes that occur during

the frozen storage of fish. However, the effect of HPP treatment on fish muscle is still

poorly studied, particularly enzymes with proteolytic activity. Thus, the aim of this

work was to determine the effect of HPP treatments on the activity of cathepsins B and

D in mackerel and horse mackerel muscles, and to evaluate whether HPP could be used

to improve fish quality by reducing their proteolytic activity in Atlantic mackerel and

horse mackerel before freezing and subsequent frozen storage.

MATERIALS AND METHODS

Preparation of samples

Atlantic mackerel (Scomber scombrus) and Atlantic horse mackerel (Treachusrus

trachurus) caught near the Bask coast in Northern Spain (Ondarroa harbor, Bizkaia,

Spain) were transported under refrigeration to the AZTI Tecnalia (Derio, Spain) pilot

plant for HPP treatment. Whole mackerel (28-33 cm and 230-280 g range) and whole

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horse mackerel individuals (25-30 cm and 200-250 g range) were placed in flexible

polyethylene bags (three individuals per bag) and vacuum sealed at 400 mbar.

Treatments

Whole fish were treated by high pressure in a 55-L high pressure unit (WAVE

6000/55HT; NC Hyperbaric, Burgos, Spain) at 150, 300 and 450 MPa with 0.0, 2.5 and

5 min holding time (the 0.0 min holding time samples were carried out to study the

effect of just the pressure come-up and depressurizing time). Non-pressure treated

samples (controls) were also studied. The pressurizing medium was water applied at 3

MPa.s-1, yielding come up times of 50, 100 and 150 s for treatments at 150, 300 and 450

MPa, respectively while decompression time took less than 3 s. Pressurizing water was

cooled down to maintain room temperature (20 ºC) conditions during HPP treatment.

Enzymatic activity

Preparation of enzyme extract

The preparation of enzymatic extract was performed by the methodology used by

LAKSHMANAN et al. (2005). Samples (10 g) were homogenized with 50 mL ice cold

distilled water for 2 min and the homogenate allowed standing in ice for 30 min with

occasional stirring. After 30 min, the homogenate was centrifuged at 14,600g at 4 ºC for

20 min. The supernatant (used as cathepsins extract) was filtered through a filter

(Whatman nº1) and stored at -20 ºC prior to enzymatic activity quantifications.

Cathepsins activity

The activity of cathepsin B was assayed by the methodology described by

LAKSHMANAN et al. (2005). Enzyme extract (0.1 mL) and 0.1 mL of substrate solution

(0.0625 mM of Z-Arg-Arg-7-amido-4-methylcoumarin hydrochloride in 100 mM Bis-

Tris buffer, with 20 mM EDTA and 4 mM dithiothreitol, pH 6.5) were incubated at 37

ºC during 5 min. The reaction was stopped by the addition of 1 mL 3% SDS (w/v) in 50

mM Bis-Tris (pH 7.0) and the fluorescence of 7-amino-4-methylcoumarin (AMC)

liberated was measured (excitation: 360 nm, emission: 460 nm). Cathepsin B activity

was expressed as FU.min-1.g-1. Three replicates were performed for each treatment.

Cathepsin D activity assay was based on the procedure described by BUCKOW et al.

(2010), with small modifications. Enzyme extract (0.2 mL) was mixed with 0.6 mL of

substrate solution, 2% denatured hemoglobin (w/v) in 200 mM citrate buffer (pH 3.7),

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and incubated at 37 ºC during 3 h. The reaction was stopped by the addition of 0.6 mL

10% trichloroacetic acid (w/v). After vigorous stirring, the precipitate was removed by

centrifugation (13,000 rpm, 15 min) and the soluble peptides measured at 280 nm.

Cathepsin D activity was expressed as µg tyrosine.min-1.g-1. Three replicates were

performed for each treatment.

Statistical analysis

Analysis of variance (ANOVA) followed by Fisher’s Least Significant Difference

(LSD) Test was used to identify differences between treatments. Differences between

treatments were considered significant when p < 0.05. Results are reported as mean

values ± standard deviation.

RESULTS AND DISCUSSION

Cathepsin B

The activity of cathepsin B in non-processed samples (controls) was 218839 ± 13147

and 13302 ± 360 FU.min-1.g-1 for mackerel and horse mackerel, respectively (Table 1),

i.e., about 16-fold higher for mackerel than for horse mackerel.

Error: Reference source not found and 2 show the effect of the HPP treatments on

cathepsin B activity on mackerel and horse mackerel, respectively. In mackerel,

significant differences were observed in HPP treated samples compared with the

control, being the activity significantly (p < 0.05) lower for the processed samples with

the exception of samples treated at 150 MPa/2.5 min and 300 MPa/5 min, for which no

statistical difference was observed. Generally, a gradual decrease in the activity of

cathepsin B was observed when the pressure level was increased, up to around 40%

both at 300 and 450 MPa (

Error: Reference source not foundA). Activity increased for the 150 MPa for 2.5 min and

at 300 MPa for 2.5 and 5 min treatments. Globally, a higher effect of pressure level as

compared to pressure holding time was observed, except for 300 MPa, for which the

activity increased significantly with pressure holding time.

In horse mackerel, low and intermediate pressures (150 and 300 MPa for 0 min) caused

an enzyme activity increase of up to 30% (

Error: Reference source not found), with a decrease being observed for 2.5 and 5 min

reaching an activity value lower than the control for 150 MPa/5 min and equal for the

300 MPa for 2.5 and 5 min treatments (

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Error: Reference source not foundB). For 450 MPa, the activity of the HPP samples was

lower than the control, regardless of the holding time.

Comparing the results for the two species, the main conclusions could be expressed as

follows. The intermediate pressure levels (150 and 300 MPa) showed a lower effect on

activity of cathepsin B in horse mackerel as compared to mackerel. Pressure holding

time increased the activity in mackerel and decreased it in horse mackerel. Pressure-

treated mackerel showed an activity lower and up to equal to that of the control, while

for horse mackerel intermediate pressure levels caused an increase in activity. For both

species, the higher pressure level studied (450 MPa) caused a significant decrease in

cathepsin B and D activity.

In general, the results observed for the effect of pressure on cathepsin B are in

agreement with those reported by other authors. For instance, CHÉRET et al. (2005)

observed that when sea bass muscle is pressurized at 300 MPa for 5 min, the activity of

cathepsin B slightly increased, being this explained by the possible release of the

enzyme from the lysosomes. ASHIE et al. (1996) applied HPP treatments (300 MPa for

30 min) on sheephead and bluefish cathepsin C, having observed that the activity

decreases up to 80% and 91%, respectively.

Cathepsin D

The activity of cathepsin D in horse mackerel is higher (about 50%) than in mackerel

while the reverse was observed for cathepsin B (Table 1). In mackerel, all treatments

caused a statistically significant (p < 0.05) increase in the activity of cathepsin D for

mackerel, with the exception of 150 MPa/2.5 min and 450 MPa/5 min for which

treatments no effects were observed

Error: Reference source not found. The activity increase was higher for 300 MPa, reaching more than 2-fold for 5 min, compared to the control. Pressure holding time showed small effects on activity when a 150 MPa treatment was applied; however, a 300 MPa treatment led to a significant increase, while a 450 MPa previous treatment provoked an activity decrease. For horse mackerel, similar results than for mackerel were obtained (

Error: Reference source not found). In general, activity increased with pressure except for

150 MPa/5 min, 300 MPa/0 min, and 450 MPa/0 and 2.5 min treatments. Similar results

to those obtained in this work for cathepsin D of mackerel and horse mackerel were

obtained for sardine (HERNÁNDEZ-ANDRÉS et al. 2008) and sea bass (CHÉRET et al.

2005) muscle. For instance, CHÉRET et al. (2005) observed that pressure treatments at

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300 MPa increased the activity of cathepsin D, while for higher pressure levels the

activity decreased to values similar to those obtained when a 100 MPa pressure was

applied. These results can be hypothetically explained by the liberation of the enzyme

from the lysosomes, which appears to be the dominant effect at lower pressures and

shorter holding times (JUNG et al. 2000), while at higher pressures enzyme inactivation

would prevail. Results obtained point out the occurrence of two effects of pressure

treatments on the activity of cathepsin D. At lower pressures (150 MPa and 300 MPa),

activity increases, possibly because of enzyme release from the lysosomes into the fish

muscle while at higher pressure (450 MPa) the activity decreases, possibly due to a

higher on cathepsin D pressure inactivation effect, over its release from lysosomes.

CONCLUSION

In general, lower pressures and shorter pressure holding times caused cathepsin B and D

activity increases, while higher pressures/longer pressure holding times had the opposite

effect. The former effect can be attributed to enzyme release from lysosomes while the

latter may reflect pressure inactivation. HPP reduced cathepsin B activity in mackerel

and horse mackerel, in all cases for the former and at 450 MPa for the latter. For

cathepsin D, all pressure treatments studied resulted in an equal or higher enzyme

activity as compared to the control. In conclusion, pressure treatments studied in this

work (150, 300 and 450 MPa, with holding times of 0, 2.5 and 5 min) can reduce the

endogenous cathepsin B activity of mackerel and horse mackerel and thus opening the

possibility for a better quality retention during subsequent chilled or frozen storage

preservation; however such activity inhibition was not observed for cathepsin D.

Acknowledgements

To the Xunta de Galicia (Spain) for financing the Project 10TAL402001PR (2010-

2012) and Research Unit 62/94 QOPNA (project PEst-C/QUI/UI0062/2011).

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Tables

Table 1 – Cathepsin B and D activities in untreated mackerel and horse mackerel

Fish speciesCathepsin B activity

(FU.min-1.g-1)

Cathepsin D activity

(µg tyrosine.min-1.g-1)

Mackerel 218839 ± 13147 2.044 ± 0.138

Horse mackerel 13303 ± 361 3.31 ± 0.263

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Figures

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Non processed

B

Non processed

2.5

Statistical analysis# 150 MPa 300 MPa 450 MPa

0 min bx* by* ay*

2.5 min ax ax* ay*

5 min abx* ax ay*

(#) Different letters denote significant differences (p < 0.05) between pressure levels (x-y) or between pressure holding times (a-b), and the symbol * denotes significant differences compared with non-processed samples (0.1 MPa).

Fig. 1 – Pressure level and holding time effects on cathepsin B activity in mackerel

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0 min ax* ax* ay*

2.5 min ax* by bz*

5 min by* bx bz*

(#) Different letters denote significant differences (p < 0.05) between pressure levels (x-z) or between pressure holding times (a-b), and the symbol * denotes significant differences compared with non-processed samples (0.1 MPa).

Fig. 2 – Pressure level and holding time effects on cathepsin B activity in horse

mackerel

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acke

rel

(µg

tyro

sine m

in-1

.g-1

)

Pressure (MPa)

0 min 2.5 min 5 min

Non processed

A B

Non processed

2.5

Statistical analysis# 150 MPa 300 MPa 450 MPa

0 min az* cy* ax*

2.5 min bz bx* by*

5 min ay* ax* cz(#) Different letters denote significant differences (p < 0.05) between pressure levels (x-z) or between pressure holding times (a-b), and the symbol * denotes significant differences compared with non-processed samples (0.1 MPa).

Fig. 3 – Pressure level and holding time effects on cathepsin D activity in mackerel

14

344

345

346

347

2728

2

3

4

5

6

7

0 2,5 5

Cat

heps

in D

act

ivity

in h

orse

mac

kere

l(µ

g ty

rosin

e min

-1.g

-1)

Pressure holding time (min)

0.1 MPa 150 MPa300 MPa 450 MPa

2

3

4

5

6

7

0 150 300 450

Cat

heps

in D

act

ivity

in h

orse

mac

kere

l (µ

g ty

rosin

e min

-1.g

-1)

Pressure (MPa)

0 min 2.5 min 5 min

Non processed

A B

Non processed

2.5

Statistical analysis: 150 MPa 300 MPa 450 MPa

0 min ax* bxy By

2.5 min aby* ax* By

5 min by ax* ax*(#): Different letters denote significant differences (p < 0.05) between pressure levels (x-y) or between pressure holding times (a-b), and the symbol * denotes significant differences compared with non-processed samples (0.1 MPa).

Fig. 4 – Pressure level and holding time effects on cathepsin D activity in horse

mackerel

15

348

349

350

351

2930