Food Chemistry 135 (2012) 583–589

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Food Chemistry

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Characterisation and partial purification of proteolytic enzymes from sardineby-products to obtain concentrated hydrolysates

Ana Bertha Castro-Ceseña, M. del Pilar Sánchez-Saavedra ⇑, Facundo J. Márquez-RochaCentro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Carretera Ensenada a Tijuana 3918, Zona Playitas, CP 22860 Ensenada, Baja California, Mexico

a r t i c l e i n f o

Article history:Received 11 December 2011Received in revised form 12 April 2012Accepted 1 May 2012Available online 11 May 2012

Keywords:Sardine by-productEnzyme concentrateProtein hydrolysatesEnzyme recovery and purification

0308-8146/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2012.05.024

⇑ Corresponding author. Tel.: +52 646 175 0500; faE-mail address: psanchez@cicese.mx (M. del Pilar

a b s t r a c t

A procedure to recover proteases and lipases from the by-products of Monterey sardine (Sardinops sagaxcaerulea) has been developed, comprising 2 steps: a centrifugation at low temperature to eliminate morethan 90% of the initial fat content, and an acetone precipitation step. After this treatment, enzymaticactivity increased by 33.8% for lipase, 15.5% for trypsin, 14.8% for chymotrypsin, 93.4% for aminopepti-dase, and 19.7% for pepsin. The extents of hydrolysis of fish by-product proteins by endogenous enzymeby-product extract, viscera concentrate extract, and commercial Alcalase� were 62%, 85%, and 28%,respectively. The two extract preparations from sardine by-product (viscera and by-product concentrateextracts) produced 3-fold greater hydrolysis than with the commercial enzyme. The recovery of enzymeconcentrates from sardine waste has both ecological and economical advantages for the fish industry.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Baja California has one of the highest fishery production capac-ities in Mexico; sardine is the chief specie that is captured locallyand nationally (36,622 and 175,012 tons, respectively, in 2006)(SIAP, 2007).

During the canning of sardines, 40% of the body is removed-spe-cifically, the head, tail, and viscera-collectively termed the by-products. Globally, the annual discard of fisheries is estimated tobe twenty million tons per year (Rustad, 2003). Moreover, thismaterial is discarded many times without undergoing furthertreatment, causing environmental pollution and ecological prob-lems. Thus, ecologically and economically, the recovery and useof high-value by-products from these materials are importantstrategies.

The value of fish by-products, specifically as a source of enzymeconcentrates, can be increased by using such materials in differentdisciplines, such as medicine and the cosmetics, food, wastewatertreatment, and pharmaceutical industries (Alarcón-López, 1997;Burkert, Maugeri, & Rodrigues, 2004), and in the production of fishprotein hydrolysates (Aspmo, Horn, & Eijsink, 2005; Gilmartin &Jervis, 2002; Kristinsson & Rasco, 2000).

Several laboratory preparations of fish digestive enzymes havebeen characterised and purified, from which trypsin and chymotryp-sin have been the most extensively studied (Castillo-Yañez,Pacheco-Aguilar, García-Carreño, & Navarrete-Del Toro, 2004,2005; Nayak, Wiswanathan, Ammu, & Mathew, 2003;

ll rights reserved.

x: +52 646 175 0534.Sánchez-Saavedra).

Olivas-Burrola, Ezquerra-Brauer, Rouzaud-Sánchez, & Pacheco-Aguilar, 2001). Nevertheless, other enzymes, such as aminopepti-dase and lipase, should be examined, because fish viscera constitutean abundant source of these enzymes.

Conversely, enzymatic proteolysis by other sources has beenstudied, e.g., Atlantic salmon (Salmo salar) (Kristinsson & Rasco,2000) and Atlantic cod (Gadus morhua) viscera (Aspmo et al.,2005). On the other hand, proteolytic enzymes from sources otherthan fish viscera, such as alcalase 2.4L, flavorzyme 1000L, corolasePN-L, corolase 7089, endogenous digestive proteases (Kristinsson& Rasco, 2000), neutrase 0.8L, Protamex, papain, bromelain andactidin (Aspmo et al., 2005), validase TSP, and validase FP 60(Gilmartin & Jervis, 2002) have been used for enzymatichydrolysis.

Hydrolysates with different molecular weights have been ob-tained, using preparations of different enzymes of complementaryactivities (Gilmartin & Jervis, 2002). Notably, endogenous enzymeactivity alone is sufficient to obtain a solubilisation yield up to75% (in terms of solubilised dry matter) for enzymatic hydrolysisby Atlantic cod viscera (Aspmo et al., 2005). García-Sánchez andMárquez-Rocha (personal communication) have studied the recov-ery of soluble protein from sardine by-products and preliminaryproteases concentration conditions at low and high alkaline pH(2.5 and 11.5, respectively).

The present study describes a process for the recovery of prote-ases and lipases from cannery sardine by-products and their partialactivity characterisation, besides the preparation of endogenousprotein hydrolysates. The enzyme concentrates may be useful forthe production of endogenous protein hydrolysates to increasethe commercial value of these materials.

584 A.B. Castro-Ceseña et al. / Food Chemistry 135 (2012) 583–589

2. Materials and methods

2.1. Reagents

Hydrochloric acid (HCl), sodium hydroxide (NaOH), Tris–HCl,sodium chloride (NaCl), trypsin, chymotrypsin, azocasein, trichlo-roacetic acid (TCA), calcium chloride (CaCl2), dimethyl sulfoxide(DMSO), acetone, Na-benzoyl-DL-arginine-p-nitroanilide (BAPNA),succinyl-(Ala)-2-Pro-Phe-p-nitroanilide (SAPNA), Ala-p-nitroani-lide, haemoglobin, hippuryl-L-phe, hippuryl-L-arg, p-nitrophenylacetate, potassium phosphate, Triton X-100, 1-propanol, trinitro-benzenesulfonic acid (TNBS), bovine albumin serum (BSA), chlor-operoxidase, and ribonuclease were purchased from Sigma. Thesoluble protein determination kit was obtained from Bio-RadLaboratories.

2.2. Samples

Monterey sardine (Sardinops sagax caerulia) by-product wasprovided by the Productos Marinos ABC cannery (Ensenada, BC,México). Samples were delivered to us frozen, inside polyethylenebags. The by-product was placed between layers of crushed ice andtransported to the CICESE Marine Bioprocess Engineering Labora-tory at Ensenada, and the samples were kept at �20 �C prior to use.

2.3. Isolation and characterisation of proteolytic enzymes

2.3.1. GeneralThe complete procedure for the preparation of enzymatic con-

centrates is presented in Fig. 1a.

2.3.2. Preparation of the sardine viscera crude extractThe frozen viscera were thawed and minced in a RIVAL� blender

to homogeneity. A convenient quantity of the mix was weighedand blended with distiled water, 1:1. The mix was homogenisedon a PRO250� homogeniser for 5 min in an ice bath. The pH wasadjusted to 11.5 with 0.2 N NaOH and constant agitation at 4 �C.Next, the material was centrifuged at 15,000g for 45 min at 4 �C.The lipid phase was discarded, and the supernatant was collected;this layer was considered the viscera crude extract.

2.3.3. Preparation of total by-product (heads, tails, and viscera) crudeextract

Two kilograms of by-product were thawed at 4 �C, eviscerated,and weighed separately (heads and tails, viscera, and blood and

Fig. 1. (a) Experimental strategy for the preparation of enzymatic concentrates. (b) En

other body fluids) to determine the proportion of each component.The material was minced (together) in a RIVAL� blender, to homo-geneity. A sample of the blended by-product was weighed. Then,distiled water was added (1:2, by-product:water). The materialwas homogenised in a PRO250� homogeniser for 5 min in an icebath. The pH was adjusted to 11.5 with 0.2 N NaOH with constantagitation at 4 �C. The solution was centrifuged at 15,557g for50 min at 4 �C. The lipid phase was discarded, and the supernatantwas collected; this layer was considered the by-product crudeextract.

2.3.4. Partial enzymatic characterisation2.3.4.1. Quantification of proteolytic activity. Total proteolytic activ-ity was assayed according to González-Agráz (2000) with somemodifications: 0.5 ml of 1% azocasein, 0.5 ml of Tris–HCl, pH 7.5,and 0.02 ml of the sardine protein extract were mixed. Reactionswere performed at 30 �C. The reaction was initiated by addingthe sample and was stopped 10 min later with 0.5 ml of 10% tri-chloroacetic acid (TCA), incubating at 0 �C for 10 min. The reactionmixture was centrifuged in Eppendorf tubes for 5 min at 10,000g.The supernatant was separated from the undigested substrate,and the absorbance at Abs366nm of the released dye was measured.The blank was prepared by adding TCA before the substrate. Prote-ase activity was expressed as the number of protease units per mgof protein and was calculated using Eq. (1):

Uðmg proteinÞ ¼

ðDAbs=minÞðfinal reaction volÞðmg proteinÞ ð1Þ

2.3.4.2. Trypsin. Trypsin activity was evaluated according to Castil-lo-Yañez et al. (2005), using BAPNA as substrate with slight modi-fications: 0.005 ml of the sardine protein extract was mixed with0.1 ml of 0.5 M Tris–HCl, pH 7.5, and 0.1 ml of 0.2 M CaCl2;0.04 ml of 0.02 M BAPNA in dimethyl sulfoxide (DMSO) was alsoadded to the mixture. The final reaction volume was adjusted to1 ml with distiled water. Assays were performed at 30 �C. Oneactivity unit was defined as the production of 1 lmol min�1 of freep-nitroanilide released, which was measured by monitoring thechange in absorbance at Abs410nm every 5 s for 1 min. BAPNAhydrolysis units per mg of protein was calculated using Eq. (2):

Uðmg proteinÞ ¼

ðDAbs=minÞð1000Þðfinal reaction volÞð8800Þðmg proteinÞ ð2Þ

where 8800 is the molar extinction coefficient of p-nitroanilide inM�1 cm�1.

zymatic hydrolysis process for the production of sardine by-product hydrolysates.

A.B. Castro-Ceseña et al. / Food Chemistry 135 (2012) 583–589 585

2.3.4.3. Chymotrypsin. Chymotrypsin activity was measuredaccording to Castillo-Yañez et al. (2005), with some modifications:0.003 ml of the sample was mixed with 0.1 ml of 0.5 M Tris–HCl,pH 7.5, and 0.1 ml of 0.2 M CaCl2; 0.08 ml of 0.001 M SAPNA inDMSO was also added. The final reaction volume was adjusted to1 ml with distiled water. Reactions were performed at 30 �C. Oneactivity unit was defined as the production of 1 lmol min�1 of freep-nitroanilide that was released. Chymotrypsin activity was mea-sured by monitoring the change in absorbance at Abs410nm every5 s for 1 min. SAPNA hydrolysis units per mg of protein were calcu-lated using Eq. (2).

2.3.4.4. Aminopeptidase. Aminopeptidase activity was assayedaccording to Donovan, Horn, and Gawlicka (2004), with the follow-ing modifications: 0.006 ml of the sardine protein extract wasmixed with 0.1 ml of 0.5 M Tris–HCl, pH 7.5; 0.1 ml of 0.2 M CaCl2,and 0.05 ml of 0.02 M ala-p-nitroanilide in 0.5 M Tris–HCl, pH 7.5at 30 �C. The final reaction volume was adjusted to 1 ml with dist-iled water. One activity unit was defined as the production of1 lmol min�1 of free p-nitroanilide released, measured by moni-toring the change in absorbance at Abs410nm every 5 s for 1 min.Ala-p-nitroanilide hydrolysis units per mg of protein were calcu-lated using Eq. (2).

2.3.4.5. Pepsin. Pepsin activity was evaluated as by Donovan et al.(2004), using haemoglobin as substrate with slight modifications:0.5 ml of 0.5% haemoglobin in 0.06 M HCl, pH 2.0, was incubatedwith 0.015 ml of sample in a microcentrifuge tube at 30 �C. Thereaction was initiated with the addition of sample and stopped30 min later with 0.5 ml of 10% TCA, and incubated at 0 �C for10 min. The reaction mixture was centrifuged in Eppendorf tubesfor 10 min at 4500g. The supernatant was separated from the undi-gested substrate, and the absorbance at Abs280nm was measured. Ablank was prepared by adding TCA before the substrate. One activ-ity unit was defined as the production of 1 lmol/min of free tyro-sine released and was calculated using Eq. (3):

Uðmg proteinÞ ¼

ðDAbs=minÞð1000Þðfinal reaction volÞð1449:6Þðmg proteinÞ ð3Þ

where 1449.6 is the molar extinction coefficient of tyrosine inM�1 cm�1.

2.3.4.6. Carboxypeptidase A. Carboxypeptidase A activity was mea-sured according to González-Agráz (2000), with some modifica-tions: 0.89 ml of distiled water was mixed with 0.1 ml of 0.001 Mhippuryl-L-phe in 0.025 M Tris–HCl, pH 7.5, at 30 �C, 0.1 M NaCl,and 0.01 ml of protein solution in 1.0 M NaCl. Reactions were per-formed at 30 �C. One activity unit was defined as the production of1 lmol/min of hippuric acid, which was measured by monitoringthe change in absorbance at Abs254nm every 5 s for 1 min. Hippu-ryl-L-phe hydrolysis units per mg of protein were calculated byEq. (4):

Uðmg proteinÞ ¼

ðDAbs=minÞð1000Þðfinal reaction volÞð360Þðmg proteinÞ ð4Þ

where 360 is the molar extinction coefficient of hippuric acid inM�1 cm�1.

2.3.4.7. Carboxypeptidase B. Carboxypeptidase B activity was evalu-ated as by González-Agráz (2000), with the following modifica-tions: 0.75 ml of distiled water was mixed with 0.25 ml of0.001 M hippuryl-L-arg in 0.025 M Tris–HCl, pH 7.5, 30 �C (0.5 MNaCl) and 0.007 ml of protein solution. Reactions were carriedout at 30 �C. One activity unit was defined as the production of1 lmol min�1 of hippuric acid, which was measured by monitoring

the change in Abs254nm every 5 s for 1 min. Hippuryl-L-arg hydroly-sis units per mg of protein were calculated using Eq. (4).

2.3.4.8. Lipase. Lipase activity was determined according to Yang,Koga, Nakano, and Yamane (2002) with slight modifications:0.48 ml of 0.5 mM p-nitrophenyl acetate in 0.5% Triton X-100 in25 mM potassium phosphate, pH 7.0, 37 �C, was mixed with0.02 ml of protein solution. The reaction was initiated with theaddition of protein solution and stopped 60 min later with 0.5 mlof 1-propanol. Abs405nm was measured immediately. Lipase activitywas expressed as the production of 1 lmol min�1 of p-nitrophenolat 405 nm, under assay conditions, using Eq. (5):

Uðmg proteinÞ ¼

ðDAbs=minÞð1000Þðfinal reaction volÞð17;800Þðmg proteinÞ ð5Þ

where 17,800 is the molar extinction coefficient of p-nitro-phenyl in M�1 cm�1.

2.4. Production of sardine by-product hydrolysates

2.4.1. Substrate preparationSubstrate was prepared as described above.

2.4.2. Enzymatic hydrolysisSardine by-product enzymatic hydrolysis was performed using

Alcalase�, viscera enzymatic concentrate, and total by-productconcentrate, the two latter were obtained as described above, forwhich the sardine crude extracts, concentrated with acetone, werefreeze-dried in a Labconco� freeze dryer, using 30 ml samples inEppendorf tubes, for 96 h.

Enzymes were added, based on the protein concentration in thesubstrate at a 25% (w/w) enzyme/substrate ratio. For example: foreach 100 mg of substrate protein, 25 mg of enzyme were added.Reactions were performed in triplicate, using 2 ml Eppendorfmicrocentrifuge tubes at 30 �C for 312 min with occasional agita-tion. The reaction volume was 1 ml. The hydrolysis was ended byheating the tubes to 95 �C for 15 min. The hydrolysates were thencentrifuged at 15,000g for 15 min at 4 �C, and the supernatant wassaved (Aspmo et al., 2005; Estrada Corona, 2002; Gilmartin &Jervis, 2002; Kristinsson & Rasco, 2000). Aliquots were stored formolecular characterisation. Supernatant and aliquots were keptat �20 �C prior to use. (Fig. 1b) shows the procedure for the enzy-matic hydrolysis process for the production of sardine by-producthydrolysates.

2.4.3. Evaluation of the degree of hydrolysis (DH)%DH was calculated by the trinitrobenzene-sulphonic acid

(TNBS) technique of Adler-Niessen (1979) and Estrada Corona(2002) as follows: 0.064 ml of 1:100 diluted sample was pouredinto 7 ml amber vials and mixed with 1 ml of 0.2 M sodium phos-phate buffer (pH 8.2) and 0.5 ml of 0.05% TNBS (fresh). Reactionswere performed in a water bath at 50 �C for 60 min. Vials with dist-iled water instead of sample were used as blanks. Reactions werestopped with 1 ml of 0.1 N HCl. The tubes were allowed to standat room temperature for 15 min, after which the absorbance wasmeasured at A420nm. DH was calculated by the following Eq. (6):

DH ¼ a� NH2 tgroups� a� NH2 t0 groupsa� NH2 maxgroups� a� NH2 t0 groups

� �ð100Þ ð6Þ

where a-NH2t groups = a-amino groups at a given time, a-NH2t0

groups = a-amino groups at the beginning of the hydrolysis, anda-NHmax groups = a-amino groups from total acidic hydrolysis;a-amino groups were determined, based on a L-leucine standardreference curve.

Table 1Proximate analysis of the by-product1 before and after applyingour purification procedure.

Analysis Raw material (%) Crude extracta (%)

Crude protein 36.3 36.0Lipids 29.0 2.80Ashes 11.9 22.7

1 Head, tails and viscera.a Obtained after protein concentration with acetone of the raw

material.

586 A.B. Castro-Ceseña et al. / Food Chemistry 135 (2012) 583–589

Total acidic hydrolysis was measured as described by EstradaCorona (2002): 0.5 ml of crude extract and 4.5 ml of 6 N HCl weremixed in a 15 ml Pyrex tube. Oxygen was displaced using nitrogengas, and the tubes were immediately closed. The reactions wereperformed in triplicate at 100 �C for 24 h. The reaction was stoppedwith 4.5 ml 6 N NaOH and the mixture filtered to eliminate ashes.

2.4.4. Molecular exclusion chromatography/molecular weightdistribution

The molecular weight distribution of protein hydrolysates wasobtained by molecular exclusion chromatography-HPLC using aZORBAX Bio series GF-250 (Agilent) column. The mobile phasewas 0.018 M sodium phosphate, pH 7.0, 0.13 M NaCl; the flow ratewas 1 ml min�1, and the absorbance was monitored at 210, 214,220, 230, and 254 nm; the run time was 15 min. The values at254 nm were used to measure protein hydrolysis. A proteincalibration curve was generated as by Aspmo et al. (2005),with the following modifications: BSA (66,430 Da), chloroperoxi-dase (42,000 Da), trypsin type IX (23,800 Da), ribonucleasa A(13,700 Da), and Ala-p-nitroanilide (245.7 Da) were used as proteinstandards. The plot of the retention time versus the log molecularmass of the peptides fitted well, nearly linearly, and the linearequation from this plot was used to calculate the molecular weightdistribution of the protein hydrolysates.

2.5. Other analytical methods

2.5.1. Protein evaluationProtein concentration was measured by the Bradford method

(Rosenberg, 1996). Bovine serum albumin (BSA), 0.2 mg ml�1,was used as the standard.

2.5.2. Proximate analysis of viscera crude extractTo determine the amounts of soluble protein that were

recovered and the lipids that were eliminated, we performed aproximate analysis [crude protein by Kjeldahl method by NMX-F-608-NORMEX-2002 (official Mexican regulation), lipids basedon ethereal extract by NOM-086-SSA1–1994 (official Mexicanregulation), and ash by gravimetric method] of the viscera crudeextract of the raw material and crude extract.

2.6. Statistical analysis

We performed the student’s t-test to analyse the differences inaverage values of the 3 experimental replications for enzymaticcharacterisation, DH, and molecular weight distribution. The sig-nificance level was determined at the 99% probability level(a = 0.01). Stats v1.1 and Microsoft EXCEL were used to processthe data and analyse the results.

3. Results

3.1. Preparation of the enzyme concentrates

The purification procedure for the preparation of concentratesconsisted on analysing two sardine by-product groups: a) completesardine by-product (heads, tails and viscera, and b) sardine visceraconcentrate, both contained some amount of body fluids, but nomore than 5%. The soluble protein was concentrated in each groupby acetone precipitation and then the enzyme activity was charac-terised by using specific substrates (Fig. 1a).

Proximate analyses indicated no considerable loss of crude pro-tein, since raw material contained 36.3%, and crude extract 36.0%,which means no significant differences between them. However,lipid content in raw material was 29% while crude extract

contained 2.8%. These results confirmed that acetone concentratedprotein, but eliminated lipids (Table 1).

3.2. Characterisation of proteolytic and lipase activities on by-productand viscera extracts

Protease and lipase activities were performed on crude extractfrom raw material, crude acetone extract and freeze-dried mate-rial, for both by-product and viscera samples. Table 2 shows thatprotease and lipase activities were present in by-product and vis-cera crude extracts, as well as in the acetone precipitates. Acetoneprecipitation notably increased protease and lipase activities in by-product and viscera extracts. The next step was to quantify certainproteolytic activities, such as: trypsin, chymotrypsin, aminopepti-dase, pepsin, and carboxypeptidases A and B, which are the mainproteolytic enzymes found in the digestive system. Trypsin, chy-motrypsin, aminopeptidase, and pepsin were found in completeby-product and viscera crude extracts, whereas carboxypeptidaseA was found only in viscera crude extract. Carboxypeptidase Bwas not detected with the procedure used (Table 3). Acetone pre-cipitation allowed 3-fold concentrations of trypsin and chymotryp-sin, and 48-fold concentration of aminopeptidases for visceraacetone concentrate. Purification folds for trypsin and chymotryp-sin were 0.46 and 2.19, respectively for complete by-product. Tryp-sin and chymotrypsin showed the highest activities for bothcomplete by-product and viscera (Table 3). These results mightindicate that sardine cannery by-product may be rich in digestiveenzymes; therefore, their concentration in one step with acetonecould be advantageous.

3.3. Sardine by-product hydrolysates

3.3.1. Enzymatic hydrolysisTable 4 shows the degree of hydrolysis (DH) of total sardine by-

product (heads, tails, and viscera), using three enzymatic prepara-tions: 1) sardine viscera concentrate, 2) total sardine by-product,and 3) Alcalase�. A significantly higher (P < 0.01) DH was achievedusing viscera concentrate (85%), 23% higher than that obtainedwith by-product concentrate (62%), possibly because the total pro-teolytic activity in the viscera concentrate was significantly higher(P < 0.01) than that of by-product. The lowest DH (28%) was ob-served with Alcalase�, which was three times lower than that ofviscera concentrate.

3.3.2. Molecular weight distribution of the sardine hydrolysatesThe molecular weight profile of total sardine by-product

hydrolysates differed significantly (P < 0.01) between enzymaticpreparations (viscera concentrate, complete by-product concen-trate, and Alcalase�) (Fig. 2). The highest concentration of solublepeptides (product), based on the signal at 254 nm, for sardinehydrolysates was achieved using viscera concentrate, followed bysardine total by-product concentrate. The lowest concentration ofproduct was obtained with Alcalase�. The hydrolysis of sardineproteins could be achieved by endogenous proteases, such as those

Table 2Recovery of proteases and lipases from sardine by-product1 and viscera.

Fraction Protein,mg g�1

by-prod

Proteolyticactivity,U mg�1 prot

Proteolyticactivity, U g�1

by-prod

Lipaseactivity,U mg�1 prot

Lipaseactivity,U g�1 by-prod

Proteo-lyticactivityyield (%)

Proteasepurif. fold

Lipaseactivityyield (%)

Lipasespurif. fold

Proteinyield (%)

Complete by-productCrude extract 15.5 ± 0.96a 0.15 ± 0.017c 1.66 ± 0.28a 0.01 ± 0.0005c 1.02 ± 0.1a 100 1 100 1 100Acetone 1.14 ± 0.09b 0.68 ± 0.06a 0.54 ± 0.07b 0.17 ± 0.006a 0.224 ± 0.006b 32.5 4.53 22.1 15.5 7.34Freeze-dried 0.04 ± 0.004c 0.30 ± 0.026b 0.02 ± 0.002c 0.03 ± 0.003b 0.015 ± 0.001c 1.20 2 1.47 2.82 0.26

VisceraCrude extract 6.15 ± 0.49ª 2.86 ± 0.35b 13.1 ± 1.40 a 0.025 ± 0.001c 1.36 ± 0.18ª 100 1 100 1 100Acetone 0.59 ± 0.07b 6.77 ± 0.31ª 2.95 ± 0.37b 0.441 ± 0.032ª 0.459 ± 0.02b 22.5 2.37 33.8 17.6 9.59Freeze-dried 0.01 ± 0.001c 7.34 ± 0.87ª 0.07 ± 0.006c 0.174 ± 0.02b 0.019 ± 0.002c 0.53 2.57 1.40 6.96 0.16

⁄Means with different superscript in a column are significantly different (P < 0.01: a > b > c).By-prod = by-product.Prot = protein.

1 Head, tails and viscera.

Table 3Summary of the partial characterisation of proteolytic activities found in by-product1 and viscera.

Enzyme Crude extract Acetone concentrate

Activity,U mg�1

prot

Activity,U g�1 by-prod

Activityyield (%)

Purificationfold

Activity,U mg�1

prot

Activity,U g�1 by-prod

Activity yield, % Purificationfold

Complete by-productTrypsin 0.77 ± 0.06a 9.89 ± 0.96a 100 1 0.36 ± 0.02b 0.33 ± 0.03b 3.34 0.46Chymotrypsin 0.81 ± 0.03b 10.5 ± 0.25a 100 1 1.77 ± 0.04a 1.64 ± 0.22b 15.7 2.19Aminopeptidase 0.005 ± 0.0005 0.63 ± 0.05 100 1 N.D. N.D. – –Pepsin 0.02 ± 0.0013b 0.4 ± 0.037a 100 1 0.05 ± 0.002a 0.08 ± 0.01b 20 2.8Carboxypeptidase A N.D. N.D. – – N.D. N.D. – –Carboxypeptidase B N.D. N.D. – – N.D. N.D. – –

VisceraTrypsin 2.42 ± 0.22b 13.1 ± 1.74a 100 1 7.92 ± 0.93a 2.03 ± 0.17b 15.5 3.27Chymotrypsin 21.57 ± 1.55b 116 ± 3.50a 1 00 1 67.0 ± 4.52a 17.3 ± 1.51b 14.8 3.11Aminopeptidase 0.12 ± 0.01b 3.17 ± 0.37a 100 1 5.77 ± 0.59a 2.96 ± 0.20a 93.4 48.08Pepsin 0.09 ± 0.006b 0.76 ± 0.10a 100 1 0.18 ± 0.009a 0.15 ± 0.01b 19.7 2.09Carboxypeptidase A 0.46 ± 0.04 1.02 ± 0.12 100 1 N.D. N.D. – –Carboxypeptidase B N.D. N.D. – – N.D. N.D. – –

⁄Means with different superscript in a row are significantly different (P < 0.01: a > b).By-prod = by-product.Prot = protein.N.D. = no enzymatic activity was detected.

1 Head, tails and viscera.

Table 4Degrees of hydrolysis of sardine by-product1 using Alcalase� (a commercial enzymepreparation), sardine viscera, or sardine by-product concentrate. An enzyme:substrate ratio of 25% was used at 30 �C for 312 min*.

Enzyme preparation DH (%)

Alcalase� 28 ± 0.37c

By-product concentrate 62 ± 3.96b

Viscera concentrate 85 ± 1.02a

1 Head, tails and viscera.* Means with different superscript are significantly different (P < 0.01: a > b > c).

A.B. Castro-Ceseña et al. / Food Chemistry 135 (2012) 583–589 587

found in by-product and viscera, but lipid elimination and enzymeconcentration allowed a much better controlled hydrolysis ofendogenous proteins, as shown in this paper.

4. Discussion

Two environmental factors are important for the separation oflipids and the recovery of proteins, in general, and enzymes in par-ticular: temperature and pH. Low temperature helped the solublepart of proteins to separate from the lipid part at low (2.5) and highpH (11). However, high pH stabilised enzyme activity better than

low pH (data not shown). Lipid separation should be performedas soon as possible after filleting, because lipids accelerate proteinoxidation, producing a significant detriment to protein quality(Kanner & Rosenthal, 1992). As well, lipid oxidation inhibits prote-ase activity (Matsushita, Kobayashi, & Nitta, 1970). The procedureused in this paper produced 99% of soluble protein recovery and90% of lipid separation.

In the present study, the enzyme concentrates showed a wideenzymatic activity spectrum (Table 3), in both by-product and vis-cera extracts. Carboxypeptidase A was found in viscera extract butnot in by-product extract, it is possible that carboxypeptidase A,including carboxypeptidase B, were present in both extracts butquantities were sufficiently low to be out of the detection rangeof the methodology used in the present work. Among enzymaticactivities, chymotrypsin and trypsin seemed to play a major rolein the digestive tract. In this paper, chymotrypsin activity washigher than that reported for Monterrey sardine by Castillo-Yañezet al., (2005). They used ammonium sulphate to precipitate protein.However, precipitation with ammonium sulphate was less effectivethan acetone precipitation, based on our results (data not shown).

The purification procedure can be defined by the final applica-tion of the enzyme extracts. Specific size and composition of thepeptides can be driven by an enzymatic cocktail, which might be

Fig. 2. Molecular exclusion chromatography–HPLC of supernatants obtained after312 min of hydrolysis: (a) substrate; (b–d) hydrolysates obtained from enzymatichydrolysis using Alcalase�, viscera, and total by-product concentrates, respectively(n = 3). The top of the Figure shows a molecular weight axis, derived from theexperiments, with standard compounds, which gives a rough estimation of the sizeof the mixture compound.

588 A.B. Castro-Ceseña et al. / Food Chemistry 135 (2012) 583–589

obtained by a specific purification procedure or addition of com-mercial enzymes. Viscera and complete by-product extracts wereconcentrated with acetone after lipid separation, and then each ex-tract was freeze-dried. Notably, there were no differences in prote-olytic activity after freeze-drying for viscera concentrates, at asignificance level of 0.01, whereas proteolytic activity diminishedafter freeze-drying for complete by-product concentrates (Table 2).These results indicated that target proteins in this case, proteolyticenzymes, were lost during purification and freeze-drying treat-ment, whereas the sardine viscera had such high concentrationsthat resulted in no significant differences in proteolytic activity be-tween the steps of acetone concentration and freeze-drying.

The acetone by-product and viscera concentrates were testedfor (DH) using the crude extract of by-product as the substrate,and the results were compared with the commercial enzyme Alca-lase. The highest DH was reached using viscera concentrate (85%),and the lowest DH (28%) was observed with Alcalase�, which was 3times lower than the DH obtained using viscera concentrate. A 65%DH was reached with the by-product concentrate. Therefore, thetwo extracts from sardine showed higher proteolytic activity thandid the commercial Alcalase�, under the reaction conditions usedin the present work. These results are comparable with those ofKristinsson & Rasco (2000) the citations suggested by the copyed-itor are in the appropriate place, and correct if necessary. who re-ported a DH that was 2.8 times lower in the production of Atlanticsalmon (Salmo salar) hydrolysates, using Alcalase 2.4L, when com-pared to an endogenous extract prepared with Atlantic salmonpyloric ceca. They performed enzymatic hydrolysis for 180 min,using an enzyme:substrate ratio of 7.5% at 40 �C.

Determining the size distribution of peptides can be useful inpredicting their antigenicity and revealing differences betweenthe mixtures of enzymes that were used (Guadix, Guadix, Páez-Dueñas, González-Tello, & Camacho, 2000). The molecular weightdistribution chromatogram (Fig. 2) showed that the majority ofprotein hydrolysates eluted in the interval between 4000 and15000 Da, revealing a correlation between the mass distributionof soluble peptides and DH for each enzymatic preparation

(Table 4). For example, the highest concentration of product wasobtained with the viscera concentrate; consequently, the visceraconcentrate produced the highest DH (Table 4). Conversely, theseresults indicated that the molecular weight distribution of the sol-uble peptides from hydrolysis was a function of the enzyme andwas substrate-specific, yielding different peptides. Guadix et al.(2000) and Kristinsson and Rasco (2000) have reported differencesin peptide profiles, depending on DH.

5. Conclusion

The present paper set up a procedure to prepare two proteinconcentrates with high proteolytic activity, called viscera and by-product concentrates. Both concentrates produced hydrolysis ofthe endogenous proteins of the by-product crude extract. Proteasesand lipase activities were considerably high in viscera concentrate.Among the enzymes, chymotrypsin seemed to be the predominantenzymatic activity in by-product and viscera extracts.

Sardine viscera concentrate yielded the highest DH (85%), fol-lowed by total by-product concentrate (62%) and Alcalase� (28%).The molecular weight distribution profile generated was between4000 and 15000 Da.

In this report, we propose a process that increases the value ofsardine by-products. Based on our characterisation, sardine hydrol-ysates may be used to treat organic loads in wastewater to reduceits viscosity. Fish protein hydrolysates might have applications as anitrogen source in diet formulations and ingredients in microbialculture media, as well as in the production of new therapeuticagents, since some sardine hydrolysates contain molecules thatare related to biologically active proteins that mediate the develop-ment of diseases.

Acknowledgements

This work was funded by CONACYT-SAGARPA, ProjectNo. 185/A-1. We thank Productos Marinos A.B.C., especially RagnarGutierrez Abarca, for providing sardine by-product. Special thanksgo to M.Sc. Eduardo Morales and M.Sc. Miriam Poumian for theirtechnical support.

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