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Characterization of structural and functional properties of fish protein

hydrolysates from surimi processing by-products

Yongle Liu, Xianghong Li , Zhijun Chen, Jian Yu, Faxiang Wang, Jianhui Wang

School of Chemical and Biological Engineering, Changsha University of Science & Technology, 960 South Wanjiali Road, 2nd Section, Changsha 410004, Hunan Province, PR China

a r t i c l e i n f o

Article history:

Received 6 April 2013Received in revised form 30 September 2013

Accepted 18 November 2013

Available online 26 November 2013

Keywords:

Fish protein hydrolysate

Structure

Functional property

Degrees of hydrolysis

a b s t r a c t

Structural and functional properties of fish protein hydrolysates with different degrees of hydrolysis (DH)

from surimi processing by-products, prepared by Protamex and Alcalase, were evaluated. As the DH

increased, the zeta potentials of the hydrolysates increased (p> 0.05). The surface hydrophobicity of

the hydrolysates was significantly affected by DH (p< 0.05). A wide variety of peptides were obtained

after hydrolysis by Protamex and Alcalase. The hydrolysate with DH 10%, prepared by Protamex, con-

tained more large protein molecules than did the others. Hydrolysis by both enzymes increased solubility

to more than 65% over a wide pH range (pH 210). The interfacial activities of hydrolysates decreased

with increasing DH (p< 0.05). The hydrolysate with DH 10%, prepared by Protamex, exhibited the best

interfacial properties among all of the samples. Thermal properties were also affected by the hydrolysis.

The results reveal that structures and functionalities of the hydrolysates were determined both by DH

and enzyme type employed.

2013 Elsevier Ltd. All rights reserved.

1. Introduction

Surimi processing by-products (including fish meat leftover on

bones, head, skin, and viscera, and accounting for about 6070% of

the fish weight), contain approximately 2030% of protein (Torres,

Chen, Rodrigo-Garcia, & Jaczynski, 2007). Most of them are cur-

rently discarded as an industrial solid waste or underutilized as

animal feed or fertilizer (Gehring, Gigliotti, Moritz, Tou, & Jaczyn-

ski, 2011). In China, silver carp (Hypophthalmichthys molitrix) is

the main freshwater species for surimi processing, with an

estimated annual consumption of 3,524,800 metric tons, with pro-

cessing by-products comprising more than 65% or 2,291,120 met-

ric tons of waste (Ministry of Agriculture of the Peoples Republic

of China, 2006). Annual global production is nearly 4.2 mil-

lion metric tons in the AsiaPacific region (Naseri, Rezaei, Moieni,

Hosseni, & Eskandari, 2010). Therefore, utilisation of surimi pro-cessing by-products (such as the recoveries of proteins from the

by-products) for subsequent use in human foods is very important

for the economic viability and increase of add-value of the aquatic

foods industry.

Controlled enzymatic hydrolysis of protein-rich fish wastes is

believed to be a better way to transform wastes into products.

The hydrolysates produced have functional or biological properties

and are appropriate for different applications, compared to those of

native proteins or common food protein ingredients (Gbogouri,

Linder, Fanni, & Parmentier, 2004; Kristinsson & Rasco, 2000;

Suthasinee, Sittiwat, Manop, & Apinya, 2005). Thus, the hydrolysisof surimi processing by-products can reduce the costs of surimi

production. Moreover, the resource waste and environment pollu-

tion associated with disposal could be minimised.

Nowadays, numerous in vitro studies have already focussed on

the bioactivity of fish protein hydrolysates (Khantaphant &

Benjakul, 2008; Klompong, Benjakul, Kantachote, & Shahidi,

2007; Raghavan & Kristinsson, 2009; Theodore, Raghavan, &

Kristinsson, 2008; Thiansilakul, Benjakul, & Shahidi, 2007; Wu,

Chen, & Shiau, 2003), whereas studies on the relationships of

molecular structures to functional properties have been limited.

The latter play a significant role in the application of hydrolysates

as binders, emulsifiers, gelling agents or nutritional supplements

(Sathivel et al., 2004). Generally, the molecular characteristics of

fish protein hydrolysates, such as molecular weight (Adler-Nissen,1986), hydrophobicity (Turgeon, Gauthier, Moll, & Lonil, 1992)

and polar groups of the hydrolysate (Kristinsson & Rasco, 2000) di-

rectly affect the functional properties and uses as food ingredients

(Kristinsson & Rasco, 2000).

To date, little information regarding the structures and func-

tional properties of protein hydrolysates from surimi processing

(with silver carp) by-products is available. A better understanding

of the structural and functional properties of the hydrolysates

would be essential for the control of their properties during pro-

cessing and application. Due to the high production of surimi pro-

cessing by-products every year, the investigation could be

significantly useful to improve the economic value of the aquatic

0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.11.089

Corresponding author. Tel./fax: +86 731 84761181.

E-mail address:[email protected](X. Li).

Food Chemistry 151 (2014) 459465

Contents lists available at ScienceDirect

Food Chemistry

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f o o d c h e m
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foods industry. For the above purpose, the objectives of the present

study were to prepare fish protein hydrolysates with different de-

grees of hydrolysis (DH), using commercial proteinase (Protamex

and Alcalase) and to (i) examine the influences of the hydrolysis

on the structural changes of fish protein by zeta potential, surface

hydrophobicity and high performance size exclusion chromatogra-

phy (SEC-HPLC) tests and (ii) characterise their functionality in

terms of solubility, emulsifying, foaming and thermal properties.

2. Materials and methods

2.1. Materials

Surimi processing (with silver carp (H. molitrix)) by-products,

including fish meat leftover on bones, head, skin, and viscera,

was supplied by Hunan Yiyang Yihua Aquatic Products Co., Ltd.

The company has been certified as exporting aquatic products by

the European Economic Community and American Food and Drug

Administration. Its main products are fresh-water fish surimi, sur-

imi products, and fillets. The supplied by-products were ground

into uniformity with ice and sealed in polyethylene bags and

stored at 40C until used. Protamex (120,000 U/g) and Alcalase(200,000 U/g) were obtained from Novozymes China Inc. (Suzhou,

Jiangsu). All other reagents and chemicals were of analytical grade.

2.2. Preparation of protein hydrolysates

The ground by-products were defatted with isopropanol

(1:5, g:ml) for 1 h at 30 C with continuous stirring. The superna-

tants were recovered using a Buchner funnel and then air-dried

at room temperature.

The defatted materials were suspended in distilled water

(3%, w/v) and homogenised at a speed of 10,000 rpm for 1 min

using a T10 homogenizer (IKA, Germany). The homogenates were

pre-incubated at each optimal temperature for 30 min prior to

enzymatic hydrolysis. The homogenates were hydrolysed by Prota-mex and Alcalase to the same DH (1030%) in bioreactors under

optimal enzyme conditions (pH 7.0 and 50 C for Protamex; pH

8.5 and 60 C for Alcalase). The hydrolysis reactions were started

by the addition of Protamex and Alcalase at a level of 2400 and

3000 (U/g, enzyme/substrate), respectively, and the DH of the

hydrolysates was determined, using the pH-stat method (Adler-

Nissen, 1986). The pH values of the mixtures were maintained con-

stant during hydrolysis, using 1 M NaOH. Once the desired DH was

reached, the pH of the sample solution was adjusted to 7.0 and

then the solution was heated at 90 C for 10 min to inactivate the

proteases. The hydrolysates were centrifuged at a speed of

10,000 rpm at 4 C for 15 min to separate insoluble and soluble

fractions. Finally, the supernatants were dialyzed at 4 C for 24 h,

freeze-dried, and then stored at 4

C. In the present study, theDHs of the hydrolysates were as follows: Protamex DH

10 0.28%, Protamex DH 20 0.35%, Protamex DH 30 0.50%,

Alcalase DH 10 0.19%, Alcalase DH 20 0.31% and Alcalase DH

30 0.46%. Each difference of the DH prepared by Protamex and

Alcalase was not significant (p> 0.05). Therefore, DH 10%, 20%

and 30% were used for the experiments.

2.3. Determination of structures

2.3.1. Zeta potential measurements

Zeta potentials of hydrolysates with different DHs were deter-

mined, using a Zetasizer 2000 (Malvern Instruments, Southbor-

ough, UK). The samples were diluted by a factor of 105 with

distilled water and then injected into the apparatus. The averagesof five measurements were reported as zeta potentials.

2.3.2. Surface hydrophobicity measurements

Surface hydrophobicities of hydrolysates with different DHs

were determined, using the fluorescence probe, 1-anilino-8-naph-

thalene-sulfonate (ANS), as described by Kato and Nakai (1980).

40 ll of 8 mM ANS were added to the samples with a concentra-tion ranging from 0.005 to 1 mg/ml. The relative fluorescence

intensities (RFI) of the samples were measured, using a 650-60

spectrometer (Hicathi, Tokyo, Japan) at 365 and 484 nm as theexcitation and emission wavelengths, respectively. The initial slope

of the RFI against hydrolysate concentration (mg/ml) was calcu-

lated by linear regression analysis and reported as an index of sur-

face hydrophobicity of hydrolysate.

2.3.3. Determination of molecular weight distributions

The molecular weight distributions of hydrolysates with dif-

ferent DHs were estimated by high performance size-exclusion

chromatography (SEC-HPLC). Various samples were first solubi-

lised using 0.1 M Na2SO4 in 0.1 M sodium phosphate buffer (pH

6.7). The suspensions were centrifuged at a speed of

10,000 rpm for 15 min and the supernatants were filtered

through cellulose acetate membranes with pore size of 0.45 lm(Merck, Germany) to remove any insoluble particles. A Shimadzu

liquid chromatography system (Shimadzu Corporation, Kyoto,

Japan) equipped with a TSKgel 2000 SWXL column (30 mm

i.d. 7.8 mm, Tosoh, Tokyo, Japan) and a Shimadzu ultraviolet

detector were used. The hydrolysates were applied to the column

and eluted at a flow rate of 1 ml/min and monitored at 220 nm

at 25C. A molecular weight calibration curve was prepared from

average retention times of following standards: bovine serum

albumin (Mw: 67,000 Da), peroxidase (Mw: 40,200 Da), ribonu-

clease A (Mw: 13,700 Da), glycine tetramer (Mw: 246 Da) and

p-aminobenzoic acid (Mw: 137.14 Da) (Sigma Co., St. Louis, MO,

USA).

2.4. Determination of functional properties

2.4.1. Solubility

The hydrolysates with different DHs (100 mg) were dispersed in

10 ml of distilled water and pHs of the solutions were adjusted to

2.0, 4.0, 7.0 and 10.0 with 1 M HCl and 1 M NaOH. Each solution

was magnetically stirred for 1 h at 25C. The solutions were centri-

fuged at a speed of 3000 rpm for 10 min, and the soluble fractions

were collected. Then the protein contents in the supernatants were

determined according to the method ofLowry, Rosebrough, Farr,

and Randall (1951):

Solubility % protein content in supernatant

total protein content in sample 100%: 1

2.4.2. Emulsifying propertiesEmulsifying properties of the hydrolysates with different DHs,

including emulsifying activity index (EAI) and emulsion stability

index (ESI), were determined according to the method of Pearce

and Kinsella (1978) with slight modifications. 30 ml portions of

2 mg/ml of each hydrolysate solution were homogenised in a mix-

er at high speed and 10 ml of soybean oil was added and the pH va-

lue of each sample was adjusted to 2.0, 4.0, 7.0 and 10.0. The

mixtures were homogenised using a homogenizer (IKA, Germany)

at a speed of 10,000 rpm for 1 min. 50 ll of the emulsion waspipetted from the bottom of the mixture at 0 and 10 min after

homogenisation and diluted to 5 ml with 0.1% (w/v) dodecyl sul-

fate sodium salt (SDS). The absorbance of the diluted solution

was measured at 500 nm, using a UV2600 spectrophotometer

(UNICO Instruments, Shanghai, China). The absorbances (A0 andA10) were used to calculate the EAI and ESI:

460 Y. Liu et al./ Food Chemistry 151 (2014) 459465


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EAI m2=g

2 2:303 100 A

c 0:25 10; 000 2

whereA = absorbance at 500 nm; c= protein concentration (g/ml).

ESI min A0 10

A0 A103

2.4.3. Foaming propertiesFoaming properties of the hydrolysates with different DHs,

including foaming capacity and foam stability, were determined

according to the method ofSathe and Salunkhe (1981)with slight

modifications. 200 ml of 10 mg/ml hydrolysate solutions in 250 ml

cylinder were adjusted to pH 2.0, 4.0, 7.0 and 10.0 and homoge-

nised using homogenizer (IKA, Germany) at a speed of

10,000 rpm for 1 min to incorporate the air at 25 C. The total vol-

ume after whipping was read immediately and used to calculate

the foaming capacity, based on the following equation (Sathe &

Salunkhe, 1981):

Foaming capacity % A B

B 100% 4

whereA is the volume after whipping (ml) and B is the volume be-fore whipping (ml).The total volume of the whipped sample was re-

corded after standing for 10 min at 25 C, and used to calculate the

foam stability:

Foam stability % A B

B 100% 5

whereA is the volume after standing (ml) andB is the volume be-

fore whipping (ml).

2.4.4. Differential scanning calorimetry measurements

The net heat energy (enthalpy, DH) and the onset (Tonset) and

maximum (Tmax) temperatures for endothermic transitions of the

hydrolysates with different DHs, as a function of temperature were

determined using DSC (differential scanning calorimeter, InfinitySeries F5010, Instrument Specialists, Inc., Spring Grove, IL, USA).

2.5 mg of each sample were hermetically sealed in an aluminium

pan and scanned from 30 to 150C at a heating rate of

10C/min. Temperature calibrations were performed prior to mea-

surements according to the manufacturer and an empty pan was

used as reference. The averages of six measurements were used

to report the results.

2.5. Statistical analysis

All determinations were carried out at least three times. Data

were analysed by analysis of standard deviations and variances

using DPS V7.05 software (Data Processing System, Zhejiang Uni-

versity, China). The software has been developed to execute arange of standard numerical analyses and operations used in

experimental design, statistics and data mining (Tang & Zhang,

2013).

3. Results and discussion

3.1. Structures of the hydrolysates with different DHs prepared by

Protamex and Alcalase

3.1.1. Zeta potential observation

Zeta potentials of the hydrolysates with different DHs prepared

by Protamex and Alcalase are shown inTable 1. The zeta potentials

of the samples hydrolysed by both enzymes increased with

increase of DH. Generally, a high absolute value of zeta potentialgenerates a repulsive electrostatic force between the molecules,

which is a key property of an aggregation resistant suspension.

Therefore, it can be reasonably inferred that an increase of electro-

static repulsive forces between the hydrolysate molecules would

favour an increase of their solubility and other solubility-related

functional properties.

Besides, although the specificities of Protamex and Alcalase for

peptide bonds adjacent to certain amino acid residues were quite

different (Khantaphant & Benjakul, 2008), the difference of pro-

duced zeta potential after the hydrolysis of surimi processing by-

products by the two enzymes was not significant (p> 0.05). Thus,

both Protamex and Alcalase could be efficient enzyme choices for

preparing hydrolysates from surimi processing by-products.

3.1.2. Surface hydrophobicity

Surface hydrophobicity of the hydrolysates with different DHs

prepared by Protamex and Alcalase was measured (Table 2).

Changes in surface hydrophobicity mainly influence the interfacial

properties of the hydrolysates. The results showed that the surface

hydrophobicity of the hydrolysates was significantly affected by

DH (p< 0.05). Calderon de la Barca, Ruiz-Salazar, and Jara-Marini

(2000) reported that proteolysis, due to shortening of peptide

chains, is accompanied by gain or loss of hydrophobicity, which

mainly depends on the nature of the hydrolysed protein and

molecular weight of the formed peptides. In the present work,

enzymatic hydrolysis by Protamex was accompanied by a decrease

of surface hydrophobicity. The possible reason is that peptides re-

leased from protein in surimi processing by-products gradually

adopted a conformation with hydrophilic groups exposed outward,

while surface hydrophobicity of hydrolysates prepared by Alcalase

increased along with the increasing DH.Liu, Kong, Xiong, and Xia

(2010)found that enzymatic hydrolysis of porcine plasma protein

by Alcalase was coupled with a decrease of surface hydrophobicity.

Probably the difference of protein nature could explain the oppo-

site tendency.

3.1.3. Molecular weight distribution

The calibration curve of five standard substances on a TSKgelG2000SWXL column is shown in Fig. 1and this was obtained to

Table 1

Zeta (f) potential of the hydrolysates with different DHs prepared by Protamex and

Alcalase.

Sample f Potential (mV)

Protamex DH 10% 22.5 (1.1)b

Protamex DH 20% 26.9 (0.8)ab

Protamex DH 30% 29.8 (3.1)a

Alcalase DH 10% 24.0 (1.3)b

Alcalase DH 20% 26.1 (2.4)abAlcalase DH 30% 29.2 (0.9)a

Data show mean values (SD) for five replicates.

The different letters in columns indicate significant difference at p < 0.05.

Table 2

Surface hydrophobicity of the hydrolysates with different DHs prepared by Protamex

and Alcalase.

Sample Surface hydrophobicity

Protamex DH 10% 565.5 (4.8)a

Protamex DH 20% 265.4 (5.2)c

Protamex DH 30% 225.1 (3.8)d

Alcalase DH 10% 229.8 (3.1)d

Alcalase DH 20% 262.1 (2.4)c

Alcalase DH 30% 393.1 (3.5)b

Data show mean values (SD) for three replicates.


Y. Liu et al./ Food Chemistry 151 (2014) 459465 461
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interpret the results. The elution patterns (chromatograms not

shown), corresponding to the hydrolysates prepared by Protamex

and Alcalase with different DHs, displayed 67 major elution peaksat 220 nm. Comparing with standard molecular weights and con-

sidering the exclusion limit of the column, the major elution peaks

corresponded to Mws of >150,000, 2468, 1360, 799, 502, 387, 226

and 138 Da, respectively. Relative proportions (%) of each peak are

presented in Table 3. The hydrolysate with DH 10%, treated by

Protamex, had the highest proportion of larger molecules

(Mw > 150,000 Da), which was about 8.3%. Those of others ranged

from 3.4% to 6.9%. Although the proportion of the larger molecules

was lower, it is speculated that they were crucial to the function-

ality (such as the interfacial and gelling properties). The hydroly-

sate with DH 10%, treated by Protamex also contained peptides

with Mw of 2468 Da, and the relative proportion reached 20.6%.

Besides, the molecular weights of peaks of all the other hydroly-

sates were all lower than 1400 Da. These results suggested that

hydrolysis by Protamex and Alcalase yielded a wide variety of pep-

tides. Some studies have demonstrated that molecular weights of

hydrolysates were closely related to the solubility (Dong et al.,

2008; Gbogouri et al., 2004). Lee, Shimizu, Kaminogawa, and

Yamauchi (1987)supported the conclusion that there is an opti-

mum molecular size for peptides to be good emulsifiers. Slizyte

et al. (2009) further found that the hydrolysates, which had the

highest amount of peptides in the molecular weight range

80,0001000 Da, displayed best emulsifying properties. Therefore,

the peptides prepared by Protamex and Alcalase can also be con-

sidered as potential functional agents in food.

The present results also show that molecular weights of the

hydrolysates with the same DH, produced by Alcalase, were gener-

ally lower than those by Protamex, which could be associated with

the higher activities of the former (Klompong et al., 2007).

3.2. Functional properties of the hydrolysates with different DHs

prepared by Protamex and Alcalase

3.2.1. Solubility

The solubilities of the hydrolysates with different DHs, prepared

by Protamex and Alcalase in the pH range of 210, are shown in

Fig. 2. The solubilities increased from about 10% for raw material

(data not shown) to more than 65% over a wide pH range. The in-crease of solubility was positively correlated with DH. And the dif-

ference of solubility for the hydrolysates prepared by Protamex at

different DHs was significant (p< 0.5). Characterisation of molecu-

lar structures testified that the zeta potential, molecular size and

surface hydrophobicity of protein hydrolysates were affected by

the enzymatic hydrolysis and DH. The results of solubility indi-

cated that the degradation of large protein molecules to smaller

peptides was critical for marked increase of solubility. Gbogouri

et al. (2004)andDong et al. (2008)also reported that the solubility

of the hydrolysates increases with the protein fraction with lower

molecular mass, and the smaller peptides from proteins are ex-

pected to have proportionally more polar residues, with the ability

1.0E+02

1.0E+03

1.0E+04

1.0E+05

6 8 10 12 14

Elution timemin

Mw(Da)

Fig. 1. Calibration curve of standard substances on TSKgel G2000SWXL column at

220 nm. Five standard substances were bovine serum albumin (Mw: 67,000 Da),

peroxidase (Mw: 40,200 Da), ribonuclease A (Mw: 13,700 Da), glycine tetramer

(Mw: 246 Da) and p-aminobenzoic acid (Mw: 137.14 Da), respectively.

Table 3

Relative proportion (%) of each molecular weight in the hydrolysates with different DHs prepared by Protamex and Alcalase.

Mw (Da)

>150,000 2468 1360 799 502 387 226 138

Protamex DH 10% 8.3 20.6 29.5 17.0 5.9 12.8 3.5

Protamex DH 20% 4.9 43.4 22.1 17.4 8.2 2.5

Protamex DH 30% 4.7 40.6 27.3 11.6 10.9 3.1

Alcalase DH 10% 6.9 40.8 22.8 5.8 5.1 11.6 6.1

Alcalase DH 20% 4.7 41.7 18.8 2.5 9.6 13.7 7.4

Alcalase DH 30% 3.4 45.5 23.0 4.6 3.2 9.2 9.8

: Not detected.

(a)

60

70

80

90

100

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0

pH

Solubility(%)

DH 10% DH 20% DH 30%

(b)

60

70

80

90

100

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0

pH

Solubility(%)

DH 10% DH 20% DH 30%

Fig. 2. Solubility of the hydrolysates with different DHs prepared by Protamex and

Alcalase as influenced by pHs. (a) Samples prepared by Protamex; (b) samples

prepared by Alcalase. Bars represent standard deviations from triplicate

determinations.



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to form hydrogen bonds with water. The hydrolysates prepared by

Alcalase at lower DH showed higher solubility. This lends further

support to the finding that molecular weights of that hydrolysate

were generally lower.

Some previous studies showed that the surface hydrophobicity

of peptides was another crucial influence on the solubility of pro-

tein hydrolysate (Gbogouri et al., 2004; Klompong et al., 2007).

However, in the present study the effects of surface hydrophobicityon the solubility were less important than were the smaller size

and charge group of the peptides produced during the hydrolysis

process. For example, the surface hydrophobicity of the samples

hydrolysed by Alcalase increased with increase of DH, while the

solubility also increased.

The solubilities of protein hydrolysates prepared by both

enzymes were relatively lower at about pH 4.0. The results of

SEC-HPLC evidenced that some protein and/or peptides with high

molecular weight (Mw) remained after hydrolysis. Such molecules

could precipitate at this pH, which was close to the isoelectric

point (pI) of fish proteins.

Due to the high solubility of the hydrolysates over a wide pH

range, it was presumed that the hydrolysates with different DHs,

prepared by Protamex and Alcalase, were good sources of protein

and appropriate for many functional applications.

3.2.2. Emulsifying properties

EAI (m2/g) and ESI (min) of the hydrolysates with different DHs,

prepared by Protamex and Alcalase as influenced by pHs are shown

in Table 4. The hydrolysate with DH 10%, treated by Protamex,

exhibited the best emulsifying properties among all the samples

(p< 0.05).Mutilangi, Panyam, and Kilara (1996)found that higher

solubility accompanied the higher EAI of hydrolysates. In the pres-

ent study, the hydrolysate with DH 10% prepared by Protamex

showed lower solubility than did other samples. Thus, a higher

proportion of larger molecular weight peptides and higher surface

hydrophobicity, which were verified by the above characterisation

of molecular structures, played more important roles in the emul-

sifying properties once the solubility of the sample reached a cer-tain value. The results also showed that EAI and ESI of

hydrolysates, prepared by both enzymes, decreased with increas-

ing DH, and the difference of the emulsifying properties for the

hydrolysates prepared by Protamex was significant (p< 0.05) with

increasing DH. Both EAI and ESI of the hydrolysates with DH 20%

and 30%, prepared by Alcalase, were better than those of samples

with the same DH prepared by Protamex (p< 0.05). Thus, the size

and molecular weight of the hydrolysates played the most signifi-

cant roles in the emulsifying properties of the present hydroly-

sates. Lee et al. (1987)also reported that there was an optimum

molecular size for peptides to be good emulsifiers. Moreover, the

solubility, surface hydrophobicity and amino acid composition of

the hydrolysates prepared by different enzymes may also be vital

factors in governing the emulsifying properties.

When considering the effect of pH on EAI and ESI, the worst EAI

and ESI were found at pH 4.0. It is probable that the pH value was

close to the isoelectric point (pI) of fish proteins; therefore, some

large molecules of the hydrolysates precipitated or the net charges

of the large molecules were reduced, which led to the decrease of

emulsifying properties. Similar results were also found in the study

ofKlompong et al. (2007).

3.2.3. Foaming properties

Foaming capacity (%) and foam stability (%) of the hydrolysates

with different DHs, prepared by Protamex and Alcalase as influ-

enced by pHs, are shown in Table 5. The hydrolysate with DH

10% prepared by Protamex also exhibited the best foaming proper-

ties among all the samples (p< 0.05). As DH increased, the hydrol-

ysates prepared by both enzymes displayed a lower foaming

capacity and foam stability (p< 0.05). The results were in agree-

ment withKlompong et al. (2007)and Van der Ven, Gruppen, De

Bont, and Voragen (2002). These authors also stressed that high

molecular weight peptides are generally positively related to foam

stability of protein hydrolysates, and surface hydrophobicity of un-

folded proteins has also been shown to positively correlate with

foaming characteristics.

The foaming properties of the hydrolysates were also affected

by pH value. For foaming capacity and foam stability, the lowest

value was found at pH 4.0 for all the samples, which coincided with

the precipitation of the large protein molecules at their isoelectric

pH.Klompong et al. (2007)found that foaming capacity of protein

hydrolysate of yellow stripe trevally, prepared by Alcalase and Fla-

vourzyme, decreased at very acidic or alkaline pH due to the repul-

sion of peptides (via ionic repulsion). But, in the present study,

higher foaming properties were found at pH 2.0, while the solubil-

ities of the samples were lower at pH 2.0 than those at pH 7.0 and

10.0 (Fig. 2). Thus, it seems that the effects of composition and net

charge of peptides, in hydrolysates produced in the present study,

on the foaming properties outweighed that of solubility.

The hydrolysates with DH 20% and 30% prepared by Alcalase,

exhibited foam properties superior to those of samples with thesame DH prepared by Protamex (p< 0.05). Possibly, the differences

of the surface hydrophobicity, size and charge of peptides could ex-

plain the difference in the foam properties.

3.2.4. Thermal properties

Table 6compares the onset (Tonset) and maximum (Tmax) tem-

peratures for endothermic transitions, as well as the net heat en-

ergy (enthalpy, DH) required for the reaction to occur.

Hydrolysates prepared by Protamex exhibited an endotherm withTmax shifted to lower temperatures. A shift to lower transition

temperature signified destabilization of protein structure and

therefore led to lower energy required to denature the proteins.

Interestingly, the Tmax values of hydrolysates prepared by

Protamex were higher than those prepared by Alcalase, which

Table 4

Emulsifying activity index (EAI, m2/g) and emulsion stability index (ESI, min) of the hydrolysates with different DHs prepared by Protamex and Alcalase as influenced by pHs.

EAI (m2/g) ESI (min)

pH pH

2.0 4.0 7.0 10.0 2.0 4.0 7.0 10.0

Protamex DH 10% 67.7 (4.4)a 49.7 (3.2)a 54.9 (2.5)a 89.7 (2.9)a 24.0 (2.1)a 15.6 (1.9)a 25.3 (2.2)a 19.7 (1.6)a

Protamex DH 20% 39.0 (2.2)cd 31.4 (3.3)bc 39.2 (4.5)b 61.4 (2.1)c 14.1 (1.1)b 12.7 (2.0)ab 18.7 (1.0)bc 13.6 (1.7)bc

Protamex DH 30% 32.4 (4.3)d 28.1 (3.4)bc 33.9 (3.7)b 58.3 (4.0)c 13.4 (2.4)b 10.5 (1.4)b 17.7 (1.7)c 12.4 (2.0)c

Alcalase DH 10% 57.6 (4.2)ab 34.7 (3.6)b 36.2 (3.7)b 75.6 (3.9)b 20.8 (2.3)a 13.6 (1.4)ab 23.2 (1.7)ab 17.2 (2.0)ab

Alcalase DH 20% 47.7 (4.6)bc 31.7 (3.1)bc 34.9 (2.7)b 71.6 (2.8)b 15.0 (1.4)b 11.8 (2.3)ab 19.6 (1.5)bc 14.7 (1.9)bc

Alcalase DH 30% 37.9 (2.1)cd 23.3 (3.1)c 30.2 (2.0)b 61.0 (2.6)c 14.2 (1.2)b 11.5 (1.3)ab 18.2 (2.5)bc 14.0 (1.1)bc

Data show mean values (SD) for three replicates.The different letters in columns indicate significant difference at p < 0.05.

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6/7

suggested that the former showed higher thermal stability than

the latter.

The enthalpy change of the hydrolysates, prepared by both en-

zymes, decreased with the increase of DH. The net DHindicates

cumulative effects of endothermic events, such as the breakdown

of hydrogen bonds and exothermic phenomena (Murray, Arntfield,

& Ismond, 1985). And the DH represents the extent of ordered

structure of a protein (Arntfield & Murray, 1981). Thus the data

suggested that the extent of the ordered structure of the samples

gradually decreased with increasing DH, which was in agreement

with the results of SEC-HPLC.

4. Conclusion

Structural and functional properties of fish protein hydrolysates

from surimi processing by-products, prepared by Protamex and

Alcalase with different DHs, were evaluated. The results reveal that

structures and functional properties of the hydrolysates were

determined by the DH and by the enzyme type employed. The

structurefunctionality relationships revealed by this study sug-gest that it might be possible to select specific forms of enzymatic

hydrolysis to meet different application needs.

Acknowledgements

This research project was funded by Natural Science Foundation

of China (Project No. 31101214 and 31201427), Science Founda-

tion of Hunan Province, PR China (Project No. 13JJ4054), and the

Ministry of Science and Technology Support Program, PR China

(Project No. 2012BAD31B08).

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Table 5

Foaming capacity (%) and foam stability (%) of the hydrolysates with different DHs prepared by Protamex and Alcalase as influenced by pHs.

Foaming capacity (%) Foam stability (%)

pH pH

2.0 4.0 7.0 10.0 2.0 4.0 7.0 10.0

Protamex DH 10% 58.1 (1.4)a 40.3 (3.3)a 46.2 (2.5)a 50.5 (2.9)a 97.5 (1.6)a 80.6 (2.4)a 88.6 (1.7)a 90.0 (2.9)a

Protamex DH 20% 40.2 (3.4)bc 30.1 (3.5)bc 34.7 (2.7)b 36.2 (2.0)bc 81.0 (3.2)cd 74.5 (1.3)bc 79.9 (2.7)b 81.2 (1.0)b

Protamex DH 30% 29.4 (3.2)d 22.1 (3.3)c 27.5 (2.5)c 30.2 (2.1)c 72.1 (2.4)e 70.7 (1.4)c 72.5 (1.7)c 73.3 (2.0)cAlcalase DH 10% 48.2 (3.0)b 32.0 (2.3)ab 36.1 (1.6)b 40.6 (4.1)b 87.5 (3.0)b 78.6 (1.4)ab 80.6 (2.9)b 85.0 (1.0)ab

Alcalase DH 20% 46.5 (5.0)b 32.8 (3.2)ab 34.0 (2.0)b 36.0 (2.8)bc 87.0 (2.0)bc 77.5 (1.8)ab 75.9 (2.1)bc 83.3 (2.0)b

Alcalase DH 30% 36.6 (3.2)cd 28.3 (3.3)bc 32.1 (2.5)bc 33.0 (3.0)bc 78.1 (1.0)de 76.7 (2.3)ab 71.5 (1.5)c 81.3 (1.9)b

Data show mean values (SD) for three replicates.


Table 6

The onset (Tonset) and maximum (Tmax) temperatures for endothermic transitions and

a net heat energy (enthalpy,DH) required for these transitions for the hydrolysates

with different DHs prepared by Protamex and Alcalase.

Sample Tonset(C) Tmax (C) DH(J/g)

Protamex DH 10% 76.7 (2.2)a 105.2 (5.1)a 148.5 (2.7)a

Protamex DH 20% 73.7 (3.5)

ab

102.4 (4.6)

a

127.6 (4.2)

b

Protamex DH 30% 68.2 (4.3)ab 100.9 (3.8)a 102.5 (3.6)d

Alcalase DH 10% 56.8 (4.4)c 95.8 (3.1)a 119.0 (3.3)c

Alcalase DH 20% 64.4 (2.8)bc 94.0 (5.7)a 113.4 (1.2)c

Alcalase DH 30% 56.3 (5.6)c 96.1 (2.6)a 110.6 (2.8)cd

The Tonset, Tmax, and DH were determined from differential scanning calorimetry

(DSC) thermograms (figures not shown).

Data show mean values (SD) for six replicates.


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