J Food Sci Technol

DOI 10.1007/s13197-011-0512-z

ORIGINAL ARTICLE

Antioxidative activity of protein hydrolysate produced

by alcalase hydrolysis from shrimp waste (Penaeus
monodon and Penaeus indicus)
Satya Sadhan Dey & Krushna Chandra Dora

Revised: 25 July 2011 / Accepted: 16 August 2011

# Association of Food Scientists & Technologists (India) 2011

Abstract Protein hydrolysates prepared by hydrolysis of consumer. The alternative natural compounds with efficient
shrimp waste (Penaeus monodon and Penaeus indicus) for antioxidative activity have been paid increasing attention.
90 min. using Alcalase enzyme following pH-stat method. Byproducts from shrimp waste have attracted considerable
Antioxidative activities of SWPH were assessed determining attention due to their different food application as inferred
FRAP, ABTS and DPPH radical scavenging activities, which from different research works in the recent past. Addition-
increased linearly with increasing concentration of protein ally, shrimp waste comprising head and shell is enriched in
hydrolysate upto 5 mg/ml maintaining good correlation. amino acids, peptides, proteins and other useful nutrients.
SWPH showed high stability over wide ranges of pH (2–11) Penaeid shrimps have become the economically important
and temperature (up to 100 °C for 150 min), in which the species in India and are widely cultured in ponds. Black
activity of more than 80% was retained. Protein hydrolysate tiger shrimp (Penaeus monodon) and white shrimp
solution with a concentration of 5 mg/ml significantly (Penaeus indicus) are commonly cultured and exported
lowered TBA values of Croaker fish fillet and maintained with a catch volume over 1000 t per year. Waste after
yellowishness of skin colour compared to untreated control processing of shrimp may account for up to 35–40% of the
sample during 10 days of refrigerated storage at 4 °C. original weight of raw material (Shahidi et al. 1992). Those
SWPH also restricted the increase of PV and FFA values in by-products consist of 71.4% head and 28.6% shell
Croaker fish fillet within acceptable limit. (Meyers 1986) and may pose a disposal problem due to
the ease of spoilage causing environmental pollution
Keywords Shrimp waste . Protein hydrolysate . (Quaglia and Orban 1987; Gildberg 1993). Effective
Antioxidative activity utilization of this waste would be a promising approach to
reduce such a problem as well as earn revenue for its
application in food industry. Protein hydrolysates from
Introduction different fish species such as mackerel (Wu et al. 2003),
herring (Sathivel et al. 2003), tuna cooking juice (Jao and
Food quality is directly affected by oxidation, and is Ko 2002) and black soy bean (Shih et al. 2002) have been
commonly associated with changes of flavour and texture. found to show antioxidative activity against peroxidation of
Therefore, prevention of lipid oxidation is a great concern lipids or fatty acids. Different initiative for recovery of
in the food industry. The use of synthetic antioxidants is an protein and amino acids (Mandeville et al. 1992), colourants
old practice and their safety could be questioned by the (Chen and Meyers 1982), flavourants (Pan 1990) and
chitin, chitosan (Coward-kelly et al. 2006) from shrimp
S. S. Dey : K. C. Dora (*) head and shell has been undertaken of which enzymatic
Department of Fish Processing Technology, hydrolysis approach was effective (Ruttanapornvareesakul
Faculty of Fishery Sciences, et al. 2006). Very few research has been undertaken
West Bengal University of Animal and Fishery Sciences 5,
regarding antioxidative effect of protein hydrolysate or
Budherhat Road P.O: Panchasayar,
Kolkata 700 094, West Bengal, India peptides from fish and shellfish, as well as their by-
e-mail: kc_dora@yahoo.co.in products (He et al. 2006). However, little information
 J Food Sci Technol

regarding antioxidants of a protein concentrate from shrimp 0.05% (w/w) based on protein concentration and the
waste and its application has been reported. Therefore, the reaction conducted at pH 8.5 and temperature 60 °C
objective of this investigation was to characterise and using pH stat method as described by Adler-Nissen
determine antioxidative activities of shrimp waste protein (1986) for 90 min. The reaction was terminated by heating
hydrolysate and study the use of it as a natural antioxidant at 90 °C for 15 min in a water bath with occational
in comparison to traditional chemical antioxidant used in stirring. Samples were cooled and then centrifuged at
food industry. 16000 g for 15 min at 4 °C using REMI centrifuge. The
supernatants were collected, concentrated and freeze dried
using a freeze drier (Vertis Freeze Mobile, 6ES, USA).
Materials and methods Protein hydrolysate powder prepared by Alcalase was kept
in polyethylene bag under vaccum at room temperature in
Industrial shrimp waste from a processing plant at Kolkata, dessicator until use.
India mainly comprising of cephalothorax, shell and tail of
Penaeus monodon and Penaeus indicus were collected. The Proximate analysis and determination of physical properties
waste was washed under running water, freeze dried,
ground, homogenized, vacum packed and kept in refriger- Moisture, protein, fat, ash and salt contents of raw waste
ated storage at 4 °C and used within a month. Prior to the and protein hydrolysate powder were determined according
hydrolysis process, a portion of this waste was thawed for to the method of AOAC (1999). Chitin was determined
analysis of proximate composition and physical character- according to the method of Spinelli and Mahnken (1978).
istics of raw material. Colour was measured by Hunter lab and reported in CIE
system. L*, a* and b* parameters indicate lightness,
Chemicals redness/greenness and yellowness/blueness, respectively.
Water activity was measured using water activity analyzer
Alcalase 2.4 L was obtained from Novo Nordisk (Bagsvaerd, (Thermoconstanter, Novasina, Swizerland). pH was deter-
Denmark). Ethanol and methanol were obtained from Merck mined by pH meter CG 842 (Schott, Germany) as described
(Darmstadt, Germany). 2,2-azino-bis(3-ethylbenzothiazoline- by Benjakul et al. (1997).
6-sulfonic acid) diammonium salt (ABTS), 6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and Estimation of DPPH and ABTS radical scavenging activity,
2,2-diphenyl-1-picryl hydrazyl (DPPH) were purchased FRAP (Ferric reducing antioxidant power)
from Sigma Chemical Co. (St. Louis, MO, USA). 2,4,6-
Tripyridyls-triazine (TPTZ), ferric chloride hexahydrate The DPPH and ABTS radical scavenging activity was
and potassium persulfate were procured from Fluka determined using the method of Yen and Wu (1999) and
Chemical Co. (Buchs, Swizerland). Arnao et al. (2001) respectively with some modification.
Sample (1.5 ml) was added with 1.5 ml of 0.15 mM 2,2-
Enzymatic hydrolysis diphenyl-1-picryl hydrazyl (DPPH) in 95% ethanol and
working solution mixing stock solution of 7.4 mM ABTS
Shrimp waste protein hydrolysate prepared according to and 2.6 mM potassium per sulphate respectively. The
method of Holanda and Netto (2002) with slight modifica- mixture in both the cases was mixed vigorously and allowed
tion. Freeze-dried raw waste thawed and suspended (1:1, to stand at room temperature in the dark for 30 min. The
w/v) in distilled water in Sorvall bottles and the mixture absorbance of the resulting solution was measured at 517 nm
was heated at a temperature of 90 °C for 30 min to using a UV-1601 spectrophotometer (Shimadzu, Kyoto,
inactivate the endogenous hydrolyzing enzyme. The Japan). The blank was prepared in the same manner, except
mixture was then homogenized and pH was adjusted that distilled water was used instead of the sample. A
to 8.5 with 1N NaOH at 60 °C for enzymatic hydrolysis standard curve was prepared using Trolox in the range of 10–
using Alcalase. The Sorvall bottles were then preheated 60 lM. The activity was expressed as μmol Trolox
in a water bath to optimum temperature of 5 °C. equivalents (TE)/g shrimp protein hydrolysate. A standard
Reactions were done in duplicates in 1 L polyethylene curve of Trolox ranging from 50 to 600 μM was prepared.
jacketed glass vessel in a thermostatically controlled
water bath with an automatic temperature compensator FRAP (Ferric reducing antioxidant power)
(ATC) probe, a pH electrode, a mixer shaft for addition
of alkali to maintain the desired pH. The hydrolytic FRAP was assayed according to Benzie and Strain (1996).
reaction was started by addition of enzymes at levels of Stock solutions included 300 mM acetate buffer (pH 3.6),
J Food Sci Technol

10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution in iced water after the designated time. The residual antiox-
40 mM HCl, and 20 mM FeCl3. 6H2O solution. A working idative activities were measured by DPPH, ABTS, and
solution was prepared freshly by mixing 25 ml of acetate FRAP assays.
buffer, 2.5 ml of TPTZ solution and 2.5 ml of FeCl3. 6H2O
solution. The mixed solution was incubated at 37 °C for Application of antioxidative activity
30 min and was referred to as FRAP solution. A sample
(150 μl) was mixed with 2850 μl of FRAP solution and Samples of whole Croaker fish (Johnius gangeticus) with
kept for 30 min in dark. The ferrous tripyridyltriazine skin on were individually dipped into the following
complex (coloured product) was measured by reading the solutions as different treatments for 5 min: control contain-
absorbance at 593 nm. The standard curve was prepared ing only distilled water, 0.1%, 0.2%, and 0.5% (w/v) crude
using Trolox ranging from 50 to 600 μM. The activity was antioxidant solution, or 0.05% (w/v) sodium erythorbate
expressed as μmol Trolox equivalents (TE)/g shrimp (C6H7NaO6) solution. For each treatment, three whole fish
protein hydrolysate. were used as replicates. After treatment, whole fish samples
were put into individual Fisher brand polyethylene bags
Effect of concentration of protein hydrolysate and stored in the refrigerator at 4 °C for 10 days. One half
on antioxidative activity fillet of a whole fish on the skin side was used to measure
color while the other half on the cut exposed flesh side was
Protein hydrolysate with 15% DH, which exhibited the used to determine TBA, PV and FFA values.
highest antioxidative activity, compared with those with
DHs of 5 and 25% (Klompong et al. 2007), were dissolved Color measurement
in distilled water to obtain the various concentration of 0.5,
1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 mg ⁄mL. The skin color of Croaker fish was measured as described
Antioxidative activity of protein hydrolysates at different earlier before and during refrigerated storage. Tristimulus
concentrations was measured by monitoring the DPPH, L*, a*, b* values were obtained using a white color
FRAP and ABTS assays. standard. The measuring head of chroma meter CR 300 had
an 8 mm diameter measuring area and used diffuse
Effect of pH on antioxidative activity illumination. Each fillet was measured in three locations
average was taken. Averages and standard deviations of L*,
Protein hydrolysate with 15% DH at a concentration of a*, b* values were calculated.
3 mg ⁄mL were prepared using distilled water as a medium.
The 5 mL of sample solutions were adjusted to pHs TBA analysis
2,3,4,5,6,7,8,9,10 and 11 with 1 or 6 m HCl and 1 or 6 m
NaOH and incubated at room temperature for 30 min. The 2-Thiobarbituric acid value (TBA) was determined for
pHs of sample solutions were then adjusted to 7.0 with 1 m Croaker fish (Johnius gangeticus) fillets during the refriger-
phosphate buffer. The final volume of all solutions was ated storage as described by Yu and Sinnhuber (1975). The
brought up to 20 mL using the distilled water. Residual fillets were ground, mixed, and subjected to analysis. TBA
antioxidative activity of protein hydrolysates was deter- number was expressed as mg malonaldehyde/kg sample.
mined by DPPH, FRAP and ABTS assays.
Peroxide value (PV) and Free fatty acid (FFA)
Effect of temperatures on antioxidative activity
The PVof the lipid was determined from lipid extract using
Protein hydrolysate with 15% DH were dissolved in iodometric method as described by Jacobs (1958). The FFA
distilled water to obtain the concentration of 3 mg ⁄ mL at content in sample was determined by the method recom-
pH 7.0. The sample solutions were incubated at 30, 40, 50, mended by Takagi et al. (1984).
60, 70, 80, 90 and 100 °C placed in a temperature
controlled water bath (Memmert, Schwabach, Germany) Statistical analysis
for 30 min. The sample without incubation (25 °C) was
used as the control. The residual antioxidative activities Data were taken triplicate and subjected to Analysis of
were determined by DPPH, ABTS and FRAP assays. The Variance (ANOVA) and the differences between means were
effect of heating times at 100 °C on antioxidative activity evaluated by Duncan’s Multiple Range Test (Steel and Torrie
was also investigated by heating for 30, 60, 90, 120 and 1980). SPSS statistic program (Version 10.0) (SPSS, 1.2)
150 min. The treated samples were immediately cooled in was used for data analysis.
 J Food Sci Technol

Results and discussion reported to possess antioxidative properties (Je et al. 2005).
Additionally, peptides also exhibited their antioxidative
Proximate compositions of raw shrimp waste and protein activity via radical scavenging activity, as well as reducing
hydrolysate are shown in Table 1. SWPH consisted of power (Benjakul et al. 2005). The effect of antioxidants on
8.05% moisture, 42.24% protein, 16.11% ash and 5.84% DPPH radical scavenging was thought to be due to their
fat. It was found that protein and carbohydrate were the hydrogen donating ability (Siddhuraju and Becker 2007).
major components in it. SWPH is produced by hydrolyzing ABTS assay is an excellent tool for determining the
with enzyme and then the supernatant collected was freeze antioxidant activity of hydrogen-donating antioxidants
dried where the extract was concentrated, water was mostly (scavengers of aqueous phase radicals) and of chain
removed and the protein became concentrated, as indicated breaking antioxidants (scavenger of lipid peroxyl radicals)
by the high protein content in the finished product. The (Leong and Shui 2002). Therefore, the water soluble
high content of carbohydrate (27.65%) most likely resulted protein hydrolysate was able to scavenge free radicals,
from the addition of sugar to improve the flavour and taste. thereby preventing lipid oxidation via a chain breaking
SWPH contained 3.58% salt. This might contribute to high reaction. The ferric reducing antioxidant power, generally
ash content found in the sample. Chitin constituted in the measures the antioxidant effect of any substance in the
sample at very low content (0.29%). Since chitin was not reaction medium as its reducing ability. Effect of different
soluble in water, it could be removed during the filtration factors on the antioxidative effect has been studied in the
process of the extract before evaporation. SWPH was dark following section.
brown in colour as evidenced by low L*-value (9.42). The
brown colour of SWPH might be developed via the reaction Effect of the concentration of SWPH on antioxidative
between sugar and amino acid known as the Maillard activity
reaction, which takes place in thermally processed food
(Carabasa-Giribet and Ibarz-Ribas 2000). SWPH had a* The antioxidant property of protein hydrolysate determined
value of 8.64 and b*-value of 12.34. Water activity (Aw) of by DPPH, ABTS radical scavenging activities and FRAP
SWPH was 0.78. Normally, the browning rate is usually assays, measured as Trolox equivalent antioxidant capacity
maximum in intermediate moisture foods in which Aw is (TEAC) generally increased (p<0.05) with the concentra-
about 0.7 (Fennema 1996). SWPH had neutral pH (7.21). tion up to 5 mg/ml as depicted in Fig. 1 (I), (II) and (III).
Alcalase is endopeptidase capable of hydrolysing pro- Despite of same DH (35%) the antioxidant activities varied
teins with broad specificity for peptide bonds and prefer for with the concentration of protein in the hydrolysate
a large uncharged residue. Hydrolysed short chain peptides solution. These results were in agreement with Suetsuna
containing water soluble hydrophilic peptides have been (2000) who reported that prawn protein hydrolysate showed
varying antioxidative activity with different concentration
applied. The results suggested that antioxidative com-
Table 1 Chemical composition and some physical properties of pounds in the fraction tested were capable of radical
SWPH prepared by alkaline hydrolysis scavenging with reducing power to a greater extent when
higher concentrations were used. This result was in
Properties Shrimp waste protein hydrolysate
accordance with Jao and Ko (2002), who reported that the
Moisture 8.0±0.07 DPPH radical scavenging activity of protein hydrolysates
Protein 42.2±0.49 from tuna cooking juice, increased when the concentration
Lipid 5.9±0.85 increased from 17% to 75%. The greater reducing power
Ash 16.1±0.02 indicated that hydrolysates could donate electron to free
Carbohydrate 27.6±0.64 radical, leading to the prevention or retardation of propa-
Salt 3.6±0.10 gation. In the propagation step, the free radical reacts with
Chitin 0.2±0.01 oxygen to form peroxy radical, which reacts with more
Aw 0.7±0.00 unsaturated lipids to form hydroperoxide. However, the
pH 7.2±0.05 continuous increase in metal chelating activity (Fig. 1 (III))
Color was found with increasing concentrations up to 5 mg/mL.
L* 9.4±0.22 Metal ion is an effective prooxidant, which can accelerate
a* 8.6±0.25
the initiation process (Gordon 2001). As a consequence, the
b* 12.3±0.34
ability of hydrolysate in chelating those metal ions could
lead to the prevention of lipid oxidation. From the result, it
Means ± SD from triplicate determinations can be recommended that SWPH could function as both
J Food Sci Technol

0.6

ABTS radical scavenging actiovity

0.6 0.7
DPPH radical scavenging activity

0.5 0.5 0.6

FRAP (µ mol /TE ml)

(µ mol / TE ml )
(µ mol / TE ml)

0.4 0.5
0.4
0.4
0.3 0.3
0.3
0.2 0.2
0.2
0.1 0.1 0.1

0 0 0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
CONCENTRATION (mg / ml) CONCENTRATION (mg /ml) CONCENTRATION (mg/ml)
(I) (II) (III)

DPPH FRAP ABTS DPPH FRAP ABTS

100 DPPH FRAP ABTS
110 100

RELATIVE ACTIVITY (%)

RELATIVE ACTIVITY (%)

105 RELATIVE ACTIVITY (%) 95

96
100 90
92
95 85
88
90 80

85 84 75

80 80 70
1 2 3 4 5 6 7 8 9 10 11 20 30 40 50 60 70 80 90 100 0 30 60 90 120 150
pH TEMPERATURE (ºC) TIME (minutes)

(IV) (V) (VI)

Fig. 1 Antioxidative activities [DPPH (I), ABTS (II) radical scavenging depicting pH and thermal stability of shrimp waste protein hydrolysate
activity and FRAP (III)] and variation of relative activity (combination of prepared by alcalase hydrolysis at different concentration. (n=3, Mean
three antioxidative activities) with pH (IV), temperature (V) and time (VI) data represented in the graph)

primary and secondary antioxidant via scavenging the free mostly affected antioxidative activities of the protein
radical and chelating the metal ions. hydrolysates. Binsan et al. (2008) who reported that ABTS
radical-scavenging activity of Mungoong was activated at
Effect of pH on antioxidative activity of protein hydrolysate alkaline pH.

The DPPH, ABTS radical scavenging activity and FRAP of Effect of temperature and time on antioxidative activity
hydrolysate were stable over a wide pH range (Fig. 1, IV). of protein hydrolysate
However, the DPPH radical scavenging activity and FRAP
tended to decrease in high alkalinity i.e. above eight. Thermal stability of hydrolysate as monitored by DPPH,
Peptides with the short chain peptides and amino acids in ABTS radical scavenging activities and FRAP is shown in
protein hydrolysate are not much affected by charge Fig. 1 (V) as relative activity (%). An increase in FRAP
modification governed by pH changes. Basically, protein was noticeable when subjected to higher temperature,
hydrolysate is soluble over a wide pH range, showing low especially upto 60 °C with increasing heating time (45–
influence by pH, whereas native proteins with tertiary and 60 min), where activities of more than 80% remained which
quarternary structure are affected considerably by pH got reduced with further increase in temperature and
(Gbogouri et al. 2004). Nevertheless, metal chelating heating time. Some losses in ABTS radical scavenging
activity of hydrolysate decreased in high alkaline and activity was also found after the water soluble fraction was
acidic pH ranges (p<0.05). Conversely, the increase in incubated at 30 °C for 30 min. Thereafter, no further
ABTS radical scavenging activity was noticeable. At changes in activity were observed with increasing temper-
alkaline pH, antioxidative compounds exhibiting ABTS ature up to 100 °C. The result indicated that antioxidant
radical scavenging activity might be activated, while activity of peptides with low molecular weight were most
those with DPPH radical scavenging activity and FRAP likely stable at very low temperature and time. In general,
lost their activity to some extent. Thus, alkaline pHs proteins were vulnerable to heat treatment, leading to the
 J Food Sci Technol

aggregation of protein and exposure of hydrophobic Table 3 Correlation coefficient (r) between average values of auto
oxidation parameters of Croaker fish fillet varied with different
domain (Sikorski and Naczk 1981). Low MW fish protein
antioxidant treatment and refrigerated storage days
hydrolysates were influenced by heat treatment minutely,
whereas proteins with second, tertiary and quarternary Between treatment Between storage days
structure were affected greatly by heat (Mutilangi et al.
PV and FFA 0.75a 0.87a
1996). The antioxidant activity slightly decreased after
PV and TBA 0.68 0.76a
being heated for 30 min but the activity remained after
PV and Color 0.89a 0.99b
heating for up to 120 min, in which 80% activity was still
FFA and TBA 0.64 0.71
retained. Accumulated energy or enthalpy might be
sufficient for antioxidative compounds to undergo denatur- FFA and Color 0.99b −0.92b
ation and loss of their activities (Pokorny and Schmidt TBA and Color 0.52 −0.81
2001). This result reconfirmed thermal stability of protein a
Correlation is significant at the 0.05 level (2-tailed)
hydrolysate is a important character of a good antioxi- b
Correlation is significant at the 0.01 level (2-tailed)
dant. Binsan et al. (2008) reported that antioxidative
activities of Mungoong slightly decreased with prolonged
heating time. storage, it could be visually observed that the yellowish
Table 2 shows that all the three parameters are well color at the skin side of control sample, without antioxi-
correlated in respect of increase in concentration of SWPH dant, got faded, while the fillet treated with antioxidants
and the correlation values are (ABTS and DPPH: r=0.978 maintained the yellowish skin color. At the end of storage,
ABTS and FRAP: r=0.989, FRAP and DPPH: r=0.952) the b* value of the control was lower (p≤0.05) compared to
significant at 1% level. Significant correlation between the antioxidant treatments and decreased from 18.8 to 8.8
parameters was also noticed in respect temperature and during 10 days of refrigerated storage, while no significant
heating time though for pH parameters were negatively difference was found in respect of L* (lightness) and a*
correlated except DPPH and FRAP (r=0.757) which was (redness) among treatments or storage time . Protein
significant at 5% level. Antioxidant capacity of Mungoong hydrolysate at a higher concentration (5% w/v) equally
(an extracted paste from shrimp waste), determined by prevented color degradation in Croaker fish fillet though
DPPH and ABTS radical-scavenging assays and FRAP the effectiveness was lower (p≤0.05) at later stage of
showed similar co relationship as described by Binsan et al. storage compared to sodium erythorbate (0.05% w/v).
(2008) (Table 3). Hsieh and Kinsella (1989) illustrated that lipid hydro-
peroxides interacted with pigments and other macromole-
Antioxidative effect of SWPH during refrigerated storage cules in fish causing discoloration and off-flavor.
of Croaker fish fillet Antioxidant extracted from shrimp shell waste in the form
of protein hydrolysate could decrease the carotenoid
Changes in colour degradation by inhibiting autoxidation and/or lipoxygenase
activity.
Results in Fig. 2 (I) indicated that during refrigerated
storage, b* values (yellowness) for the control decreased Changes in TBA value
more rapidly than samples dipped in protein hydrolysate
solution and sodium erythorbate. After 4 days refrigerated Thio barbiuteric acid (TBA) values of all samples tended to
increase throughout the storage time specially 0 to 6 days of
refrigerated storage after which no significant increment
Table 2 Correlation coefficient (r) between average values of was noticed in Fig. 2 (III). The increase in TBA indicated
antioxidant activities of shrimp waste protein hydrolysate varied with
concentration, temperature, time and pH formation of secondary lipid oxidation products (Kolakowska
2002). TBA value has been used to measure the concentra-
pH Temperature Time Concentration tion of relatively polar secondary reaction products, espe-
of SWPH
cially aldehydes (Nawar 1996). The highest TBA values of
ABTS and DPPH activity −0.51 0.71a 0.75 0.97b Croaker muscle was found in control and 4% SWPH
a
DPPH and FRAP activity 0.758 0.93 0.91a 0.95b solution sample throughout the storage period (p<0.05).
ABTS and FRAP activity −0.11 0.72a 0.85a 0.98b
The marked increase in TBA at the end of storage was found
in control (pre-soaked with distilled water) which most likely
a
Correlation is significant at the 0.05 level (2-tailed) due to higher rate of lipid oxidation in the sample.
b
Correlation is significant at the 0.01 level (2-tailed) Furthermore, the released free iron and other prooxidants
J Food Sci Technol

20 36
PV C PV A0

Meq of oxygen / kg fat

b* (lightness) values

PV A1 PV A2
32 PV A3
16

28
C A0
12 A1 A2
A3
24

8 20
0 2 4 6 8 10 0 2 4 6 8 10
STORAGE DAYS STORAGE DAYS
(I) (II)
4 16
TBA (mg MA/kg sample)

C A0 A1 FFA C FFA A0
3.5 14 FFA A1 FFA A2
A2 A3

% of Oleic acid
FFA A3
3 12
2.5 10
2 8
1.5 6
1 4
0.5 2
0 2 4 6 8 10 0 2 4 6 8 10
STORAGE DAYS STORAGE DAYS
(III) (IV)

Fig. 2 Physico chemical changes in terms of b* (lightness) value for graph). C = Control (no additive), A0 = with 0.2% of 5 mg/ml
skin side of Croaker fillet (I), per oxide (PV) value (II), thio barbituric concentration SWPH solution, A 2 = with 1.0% of 5 mg/ml
acid (TBA) content (III), free fatty acid (FFA) content (IV) of Croaker concentration SWPH solution, A 1 = with 0.5% of 5 mg/ml
fish fillet with four different antioxidant treatments during 10 days of concentration SWPH solution, A3 = with 0.05% w/v sodium
refrigerated storage at 4 °C. (n=3, Mean data represented in the erythroborate (C6H7NaO6) solution

from the muscle during storage time increased the oxidation increased (p<0.05) with storage days for all the samples.
of lipid in control fillet. The presence of sodium erythrobo- However, no significant differences in PV values between
rate and 5% SWPH solution could prevent the oxidation antioxidant treatments were observed at the end of storage
initiated by metal ions probably by several mechanisms period. Lajolina et al. (1983) opined that a PV value upto
including free radical-scavenging action, metal ion-chelating 30 Meq of O2/Kg of fat should be acceptable for fishery
property and/or reducing activity. Results suggested that both products suggesting fillets were consumable even after
color degradation and TBA values were significantly 10 days of refrigerated storage. Reddy and Srikar (1991)
different with effects of treatments and soaking in protein observed PV values for products prepared from Pink perch
hydrolysate solution effectively arrested oxidation of fat in and Croaker as 11.35 and 10.23 Meq of O2/Kg of fat
Croaker muscle. respectively during 98 days of frozen storage. Free fatty
acids are the first product of lipid hydrolysis due to the
Changes in PV and FFA action of lipolytic enzyme. The release of non-heme iron
with absence of antioxidants in croaker muscle during
The lipid oxidation as measured by the primary lipid extended refrigerated storage might enhance the oxidation
oxidation products, per oxide value occurred in Croaker process in the muscle (Chaijan et al. 2005). Changes in FFA
fillet for control rapidly increased continuously during first values of Croaker fillet pre-soaked with sodium erythrobo-
10 days of refrigerated storage, probably due to the absence rate and SWPH occurred increased (p<0.05) with storage
of antioxidants added [Fig (II)]. However PV value was days though it was compoaratively lower than that of
comparatively lower (p≤0.05) in the presence of sodium control sample (p≤0.05). Significant differences (p<0.05)
erythroborate and SWPH solution (5%). The sample treated in FFA values were also observed between treatment, where
with sodium erythroborate solution showed the lowest PV SWPH showed almost similar effectivity though both were
upto 10 days of storage when compared to treatment of lower than that of control [Fig (IV)]. Reddy and Srikar
SWPH solution and distilled water, respectively. PV values (1991) observed FFA value in increasing trend through out
 J Food Sci Technol

the storage period of fish finger prepared from Pink perch Jao CL, Ko WC (2002) 1,1-Diphenyl-2-picrylhydrazyl (DPPH)
radical scavenging by protein hydrolysates from tuna cooking
and Croaker.
juice. Fish Sci 68:430–435
Je JY, Park PJ, Kim SK (2005) Antioxidant activity of peptide isolated
from Alaska pollack (Theragra chalcogramma) frame protein
References hydrolysate. Food Res Int 38:45–50
Klompong V, Benjakul S, Kantachote D, Shahidi F (2007) Antiox-
idative activity and functional properties of protein hydrolysate
Adler-Nissen J (1986) Enzymatic hydrolysis of food proteins. Elsevier of yellow stripe trevally (Selaroides leptolepis) as influenced by
Applied Science, London, p 427 the degree of hydrolysis and enzyme type. Food Chem
AOAC (1999) Official method of analytical chemists, 15th edn. The 102:1317–1327
Association of Official Analytical Chemists, Inc, Arlington Kolakowska A (2002) Lipid oxidation in food systems. In: Sikorski
Arnao MB, Cano A, Acosta M (2001) The hydrophilic and ZE, Kolakowska A (eds) Chemical and functional properties of
lipophilic contribution to total antioxidant activity. Food Chem food lipids. CRC, New York, pp 133–160
73:239–244 Lajolina P, Laine J, Linko P (1983) Quality changes in minced fish
Benjakul S, Seymour TS, Morrissey MT, An H (1997) Protein during cold and frozen storage. In: Zerthan et al (eds) Thermal
hydrolysates from Pacific whiting solid wastes. J Agric Food processing and quality of foods. Elsvier Applied Science
Chem 45:3423–3430 Publishers Ltd.
Benjakul S, Visessanguan W, Thongkaew C, Tanaka M (2005) Effect Leong LP, Shui G (2002) An investigation of antioxidant capacity of
of frozenstorage on chemical and gel-forming properties of fish fruits in Singapore markets. Food Chem 76:69–75
commonly used for surimi production in Thailand. Food Hydro- Mandeville S, Yaylayan V, Simpson BK (1992) Proximate analysis,
colloids 19(2):197–207. isolation and identification of amino acids and sugars from raw
Benzie IFF, Strain JJ (1996) The ferric reducing ability of plasma and cooked commercial shrimp waste. Food Biotechnol 6:51–
(FRAP) as a measure of antioxidant power the FRAP assay. Anal 64
Biochem 239:70–76 Meyers SP (1986) Utilization of shrimp processing waste. Infofish
Binsan W, Benjakul S, Visessanguan W, Roytrakul S, Tanaka M, Marketing Digest 4:18–19
Kishimura H (2008) Antioxidative activity of Mungoong, an Mutilangi WAM, Panyam D, Kilara A (1996) Functional properties of
extract paste, from the cephalothorax of white shrimp hydrolysates from proteolysis of heat-denatured whey protein
(Litopenaeus vannamei). Food Chem 106:185–193 isolate. J Food Sci 61(270–274):303
Carabasa-Giribet M, Ibarz-Ribas A (2000) Kinetics of colour Nawar WW (1996) Lipids. In: Fennema OR (ed) Food Chemistry, 3rd
development in aqueous glucose systems at high temperatures. edn. Marcel Dekker, New York, pp 225–319
J Food Eng 44:181–189 Pan BS (1990) Recovery of shrimp waste for flavorant. In: Voigt
Chaijan M, Benjakul S, Visessanguan W, Faustman C (2005) Changes MN, Botta JR (eds) Advances in fisheries technology and
of pigments and colour in sardine (Sardinella gibbosa) and biotechnology for increased profitability. Technomic Pub,
mackerel (Rastrelliger kanagurta) muscle during iced storage. Basel, pp 437–447
Food Chem 93:607–617 Pokorny J, Schmidt S (2001) Natural antioxidant functionality during
Chen HM, Meyers SP (1982) Extraction of astaxanthin pigment food processing. In: Pokorny J, Yanishlieva N, Gordon M (eds)
from crawfish waste using a soy oil process. J Food Sci Antioxidant in food practical applications. Woodhead, Abington,
47:892–896 pp 331–351
Coward-Kelly G, Agbogbo FK, Holtzapple MT (2006) Lime Quaglia GB, Orban E (1987) Enzymic solubilisation of proteins of
treatment of shrimp head waste for the generation of highly sardine (Sardina pilchardus) by commercial proteases. J Sci Food
digestible animal feed. Carbohydr Polym 97:1515–1520 Agric 38:263–269
Fennema OR (1996). Amino acids, peptides, and proteins. In: Food Reddy GVS, Srikar LN (1991) Preprocessing ice storage effects on
chemistry. Marcel Dekker, New York, pp 321–420 functional properties of fish mince protein. J Food Sci 56:965–
Gbogouri GA, Linder M, Fanni J, Parmentier M (2004) Influence of 968
hydrolysis degree on the functional properties of salmon Ruttanapornvareesakul Y, Somjit K, Otsuka A, Hara K, Osatomi K,
byproduct hydrolysates. J Food Sci 69:615–622 Osako K, Kongpun O, Nozaki Y (2006) Cryoprotective effects of
Gildberg A (1993) Enzymatic processing of marine raw materials. shrimp head protein hydrolysate on gel forming ability and
Process Biochem 28:1–15 protein denaturation of lizardfish surimi during frozen storage.
Gordon M (2001) Antioxidants and food stability. In: Pokorny J, Fish Sci 72(2):421–428
Yanishlieva N, Gordon M (eds) Antioxidant in food. CRC, New Sathivel S, Bechtel P, Babbitt J, Smiley S, Crapo C, Reppon K
York, pp 7–21 (2003) Biochemical and functional properties of herring
He H, Chen X, Sun C, Zhang Y, Gao P (2006) Preparation and (Clupea harengus) byproducts hydrolysates. J Food Sci
functional evaluation of oligopeptide-enriched hydrolysate from 68:2196–2200
shrimp (Acetes chinensis) treated with crude protease from Shahidi F, Synowiecki J, Naczk M (1992) Utilization of shellfish
Bacillus sp. SM98011. Bioresour Technol 97:385–390 processing discards. In: Graham BE (ed) Seafood science
Holanda HD, Netto FM (2002) Optimization of the conditions for the andtechnology. Fishing News Books, Canada, pp 300–304
enzyme hydrolysis of shrimp residue, using response surface Shih MC, Yang KT, Kuo SJ (2002) Quality and antioxidative activity
methodology (RSM). In book of abstracts, 2002 IFT Annual of black soybean tofu as affected by Bean Cultivar. J Food Sci
Meeting, Anaheim, Calif., USA. p 194 67:480–484
Hsieh RJ, Kinsella JE (1989) Oxidation of polyunsaturated fatty acids: Siddhuraju P, Becker K (2007) The antioxidant and free radical
mechanisms, products, and inhibition with emphasis on fish. In: scavenging activities of processed cowpea (Vigna unguiculata
Kinsella JE (ed) Advances in food and nutrition research, vol 33. (L.) walp.) seed extracts. Food Chem 101:10–19
Academic, San Diego, pp 233–341 Sikorski ZE, Naczk M (1981) Modification of technological
Jacobs MB (1958) The chemical analysis of foods and food products. properties of fishprotein concentrate. Crit Rev Food Sci Nutr
Krieger, New York, pp 393–394 14:201–230.
J Food Sci Technol

Spinelli J, Mahnken C (1978) Carotenoid deposition in pen reared Wu HC, Chen HM, Shiau CY (2003) Free amino acids and
salmonids fed diets containing oil extracts of red crab peptides as related to antioxidant properties in protein hydro-
(Pleuronnocodes planipes). Aquaculture 13:213–216 lysates of mackerel (Scomber austriasicus). Food Res Int
Steel RGD, Torrie JH (1980) Principle and procedure of statistics. 36:949–957
MacGraw-Hill, New York Yen G, Wu J (1999) Antioxidant and radical scavenging properties of
Suetsuna K (2000) Antioxidant peptides from the protease digest of extract from Ganoderma tsugae. Food Chem 65:375–379
prawn (Penaeus Japonicus) muscle. Mar Biotechnol 2:5–10 Yu TC, Sinnhuber RO (1975) An improved 2-thiobarbituric acid
Takagi T, Hayashi K, Itabashi Y (1984) Toxic effect of free unsaturated (TBA) procedure for the measurement of autoxidation in fish oil.
fatty acid in mouse assay diarluric shell fish toxin by intra- J Am Oil Chem Soc 44:256–258, Ms received 8/4/97; revised 11/
peritoinal injection. Bull Jap Soc Sci Fish 50(8):1413–1418 10/97; accepted 11/17/97