 *Corresponding author.

Email: norizah@umt.edu.my

eISSN: 2550-2166 / © 2020 The Authors. Published by Rynnye Lyan Resources

  Food Research 4 (4) : 1363 - 1370 (August 2020)
  Journal homepage: http://www.myfoodresearch.com

F
U

L
L

 P
A

P
E

R

Effect of different molecular weight on the antioxidant activity and

physicochemical properties of golden apple snail (Ampullariidae) protein

hydrolysates

Saallah, S., Ishak, N.H. and *Sarbon, N.M.

Faculty of Fisheries and Food Science, Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu,

Malaysia

Article history:

Received: 20 October 2019

Received in revised form: 11

January 2019

Accepted: 23 January 2020

Available Online: 17

February 2020

Keywords:

Golden apple snail,

Protein hydrolysate,

Molecular weight,

Antioxidant activity

DOI:
https://doi.org/10.26656/fr.2017.4(4).348

Abstract

Golden apple snail (GAS) is reported to have a medicine effect for treat broken bones,

back pain and stool bloody. This study was aimed to investigate the fractionation of

protein hydrolysate from the golden apple snail (GAS) using an ultrafiltration technique

with molecular weight cut-offs of 3, 5, and 10 kDa, followed by the determination of its

antioxidant activity and functional properties. In terms of antioxidant activity, fraction of 3

kDa showed the highest reducing power and DPPH radical scavenging activity.

Additionally, fraction of 5 kDa had the highest chelating effect on ferrous ion (85.6%).

The results also revealed that fraction of 3 kDa showed good emulsion activity index and

water binding capacity, while 5 kDa showed good foaming capacity and solubility. These

results suggested that GASH showed potential for application in the food industry as its

antioxidant, with strong functional properties able to be obtained by varying its molecular

weights.

1. Introduction

An antioxidant is a molecule or compound which is

able to inhibit the oxidation of a given substrate

(Chalamaiah et al., 2012). Butylated hydroxyanisole

(BHA) and butylated hydroxytoluene (BHT) are

synthetic antioxidants used to protect food products from

lipid oxidation (Hamid et al., 2015). However, these

synthetic antioxidants may be hazardous to the

consumer, which has led to increased demand for natural

antioxidant (Halim et al., 2016). There have been many

studies on hydrolysates with antioxidant properties, such

as those in eel (Halim and Sarbon, 2017) and scallops

and abalone muscle (Zhou et al., 2012). Therefore, there

was a great interest in finding new and safe natural

antioxidant compounds such as fish proteins hydrolyzed

peptides (Jemil et al., 2014).

Protein hydrolysates are breakdown products from

protein compounds to smaller peptides which contain 2-

20 amino acids by enzymatic conversion (Hamid et al.,

2015). However, bioactive peptides only function when

released from native protein (Ranamukhaarachchi et al.,

2013). Generally, bioactive peptides can be obtained

from fish proteins via enzymatic hydrolysis (Ishak and

Sarbon, 2017). The proteolytic enzymes most commonly

used to hydrolyze fish proteins for the production of fish

protein hydrolysates are usually sourced from plants

(papain, bromelain), animals (pepsin, trypsin, α-

chymotrypsin), and microbes (Alcalase®, protamex,

flavourzyme, neutrase) (Putra et al., 2018). Due to its

thermostability, enzyme Alcalase® is a highly efficient

bacterial protease used for hydrolyzing muscle proteins

for preparing protein hydrolysate (Hamid et al., 2015).

Ultrafiltration (UF) is a membrane technology used

in diverse fields such as food, pharmaceutical, and

biotechnological industries for the concentration,

purification and fractionation of various products (Foh et

al., 2010). The antioxidant activities of hydrolyzed

protein were influenced by its molecular weight. This

has been supported by work from Razali et al. (2015),

who found that the low molecular weight of cobia skin

gelatin hydrolysate (<3 kDa) is thought to have stronger

DPPH radical scavenging activity.

In addition, the peptides and free amino acids

produced via the enzymatic hydrolysis of native proteins

will determine the changes in functional properties that

may occur (Halim et al., 2016). The functional quality of

the hydrolysates produced commonly measured by

properties such as solubility, emulsifying properties,

foaming properties, and water holding capacity. The

modification and improvement of functional

characteristics of controlled enzymatic hydrolysis of

protein result in the production of a series of smaller

 Saallah et al. / Food Research 4 (4) (2020) 1363 - 1370

eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources

polypeptides for different applications (Jamil et al.,

2016).

Ampulariidae, commonly known as the apple snail,

is a large family of freshwater snails. It typically

consumes 7 to 24 rice seedlings per day of the young

stem and leaves of paddies, causing extreme damage to

growing paddies (Salleh et al., 2012). The Golden apple

snail is a major rice pest in Asia. It has been reported that

these snails were initially introduced in Keningau,

Sabah, Malaysia. However, the invasion of snail was

spread to Peninsular Malaysia resulted in extensive

damage to the young rice plants especially in Perlis and

Kedah (Salleh et al., 2012). In a previous study,

optimization of the production of golden apple snail

hydrolysate using Alcalase® was successfully conducted

by Hamid et al. (2015). The hydrolysate produced

showed high in antioxidant activity, chelating effects and

hydroxyl scavenging activity. Therefore, this study aims

to investigate the antioxidant activity and functional

properties of fractionated protein hydrolysate from

golden apple snails at three different molecular weight

fractions (3, 5, 10 kDa).

2. Materials and methods

2.1 Materials

Golden apple snails (Ampullariidae) (GAS) were

obtained from Pekan, Pahang, Malaysia and were

brought to the laboratory alive. The flesh was separated

manually and thoroughly washed before mincing using

Waring Blender (model HGB2WTS3). Then, the minced

GAS was stored at -80oC for further analysis. The

enzyme Alcalase® 2.4L FG used was purchased from

Novo Industry (Denmark). All chemicals used in this

study were of analytical grade.

2.2 Preparation and fractionation of golden apple snail

(Ampullariidae) hydrolysate (GASH) by ultrafiltration

Hydrolysate of Golden apple snail (GASH) was

prepared following the method as described by Hamid et

al. (2015) and Razali et al. (2015) with some

modification. Approximately 22±1 g of minced GAS

flesh was mixed with 66 g distilled water. The GAS

solution mixture was heated at 80oC (15 mins) to

inactivate endogenous enzyme that presents in the

golden apple snail flesh. About 20 g of diluted

Alcalase® enzyme at an enzyme-substrate concentration

of 2% (prepared by diluting the required enzyme mass to

a final weight of 20 g with distilled water) was added

into the solution mixture. The GAS solution mixture was

heated again in a water bath shaker at 45oC and adjusting

the pH using 4 N NaOH. The pH of the solution was

kept constant at pH 6.7 while continuously stirring for

159 mins. In order to inactivate the Alcalase® enzyme

activity, the hydrolysis was terminated by heating the

solution at 90oC for 15 mins. The resultant solution was

centrifuged (Cryozen 1580R, Korea) at 5000 rpm (4oC)

for 20 mins. The supernatant obtained was then collected

and fractionated to 3, 5 and 10 kDa using vivaspin

ultrafiltration membrane (Vivaspin 20, Sartorius Stedim

Biotech, Goettingen, Germany). Firstly, the GASH

solution was allowed to pass through 10 kDa molecular

weight cut-off (MWCO) membrane. Then, the filtrate

obtained was consecutively filtered using 5 kDa and 3

kDa MWCO membranes. The obtained GASH fractions

(<3, <5 and <10 kDa) were then freeze-dried and kept at

-80oC for further analysis.

2.3 Determination of antioxidative activities

2.3.1 Reducing power

Reducing power of GASH was measured according

to the procedure described by Hamid et al. (2015).

Hydrolysate at 1.0 mg/mL for 1.25 mL (diluted with

distilled water) of various molecular weights (3, 5, and

10 kDa) was mixed with phosphate buffer (1.25 mL)

(0.2M, pH 6.6) and potassium ferricyanide (1.25 mL)

(1%, w/v). The solution mixtures were then subjected to

incubation at 50oC (30 mins) before 1.25 mL of

trichloroacetic acid (10%, w/v) were added. The mixture

was then subjected to centrifugation (Cryozen 1580R,

GRYOZEN Co. Ltm, the Yuseong, Daejon, Korea) at

10,000 rpm (10 mins). Then, the supernatant of solution

(1.25 mL) was collected and were mixed with distilled

water (1.25 mL) and 0.1% (w/v) Ferric Chloride (2.50

mL). Following incubation for 20 mins at room

temperature, the solution absorbance was measured at

700 nm. BHT as synthetic antioxidant was used as

positive control. A higher absorbance value was

understood as indicating higher reducing power ability of

the hydrolysate. Each test sample was examined in

triplicate for each molecular weight.

2.3.2 DPPH radical scavenging activities

The DPPH radical-scavenging assay was used to

investigate the ability of compounds to act as free radical

scavengers or hydrogen donors per the methods used by

Razali et al. (2015). Briefly, a volume of 500 μL of each

hydrolysate with different molecular weight (3, 5, and 10

kDa) was mixed with ethanol (500 µL) and DPPH in

99.5% ethanol (125 µL) at 0.02%, (w/v). The solution

mixture was vortexed before subjected to incubation in a

dark place at room temperature. The absorbance of the

supernatant was measured via UV-Visible

spectrophotometer at 517 nm (Spectroquant® Pharo 300,

Darmstadt, European Union) after 60 mins incubation.

The DPPH-radical scavenging activity was calculated as

follows:

F
U

L
L

 P
A

P
E

R

1364

 Saallah et al. / Food Research 4 (4) (2020) 1363 - 1370

eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources

DPPH radical scavenging activity (%) = [(A0 – A1)/ A0]

x 100

Where A0 = The absorbance of the control, and A1 = The

absorbance of the mixture containing sample.

The synthetic antioxidant reagent Butylated

Hydroxytoluene (BHT) was used as a positive control.

Lower absorbance of the reaction mixture indicates

higher DPPH radical scavenging activity. The tests were

conducted in triplicate.

2.3.3 Metal chelating activity

Metal chelating activity on ferrous ion of different

molecular weight (3, 5, and 10 kDa) of GASH fraction

was determined following Hamid et al. (2015) with

slight modification. Aliquot solution of GAS hydrolysate

(500 µL) at a concentration of 1 mg/mL was mixed with

FeCl2 (0.05 mL). The addition of 5mM ferrozine (0.1

mL) was initiated the reaction to occur. Then after

shaken vigorously, the mixture was left to stand at room

temperature for 10 min. The resulting solution was

measured at an absorbance of 562 nm using a

spectrophotometer (Spetrouant Pharo 300, Europe). The

chelating antioxidant activity was calculated as follows:

Metal chelating effect (%) = [(A0 – A1)/ A0] X 100

Where A0 = The absorbance of the control, and A1 = The

absorbance of the mixture containing sample

The positive control (Butylated hydroxytoluene,

BHT) was prepared in the same manner, except that

distilled water was used instead of a trial sample. The

tests were conducted in triplicate.

2.4 Determination of functional properties

2.4.1 Emulsifying properties

 The emulsifying properties were measured

according to the procedure of Razali et al. (2015). The

emulsions were prepared by homogenizing 45mL GASH

solution with pure vegetable oil (Vsawit) (15 mL) using

a mechanical homogenizer (Ultraturrax T18) at 16 000

rpm (1 min). After homogenization, samples were taken

(500 µL) at 0 min and 10 mins from the bottom of the

container before diluted with 10 mL SDS solution

(0.1%). Diluted emulsion formation at 0 min and 10

mins was then measured at 500 nm. The emulsifying

activity index (EAI) and emulsifying stability index

(ESI) of each emulsion were calculated as follows:

Where ∆A =A0 - A10, and ∆t= 10 mins

The test was conducted in triplicate.

2.4.2 Foaming properties

Foaming capacity was measured using the method

described by Shahidi et al. (1995). Each of GASH

fraction (1 g) was added with distilled water (50 mL).

The solution mixture was then homogenized (16,000

rpm) for 1 min by using Ultraturrax T18 homogenizer.

The height of solution was measured at 0, 0.5, 1, 5, 10,

20, and 30 min after whipping, respectively and the

foaming capacity index (%) was calculated as follows:

Measurements were conducted in triplicate.

2.4.3 Solubility

Protein solubility was measured according to the

method described by Castro and Sato (2014). About 100

mg of GASH was dispersed in 10 mL of distilled water

(pH 6.5±0.3) at room temperature. Then, the mixture was

centrifuged (Cryozen 1580R, GRYOZEN Co. Ltm, the

Yuseong, Daejon, Korea) at 17,000 x g for 10 mins. The

total protein content of the GASH was determined using

Kjedahl method (AOAC, 2002), while the protein

content in the supernatant was determined using the

Biuret method. Protein solubility was calculated using

the following expression:

2.4.4 Water binding capacity

Water binding capacity was determined using the

method described by Razali et al. (2015). Each GASH

fraction (0.5 g) was dissolved in distilled water (10 mL)

in centrifuged tube followed by vortexing for 30 s. After

that, the solution mixture was allowed to stand at room

temperature for 30 mins before being centrifuged at 4840

rpm for 25 mins (Cryozen 1580R, GRYOZEN Co. Ltm,

the Yuseong, Daejon, Korea). The mixture was filtered

by using Whatman No. 1 and the volume recovered was

measured. The water binding capacity of GASH was

calculated as follows:

Measurements were conducted in triplicate.

2.6 Statistical analysis

All the tests were done in triplicate and the data were

averaged. All data obtained was subjected to one-way

analysis of variance (ANOVA) and stated as mean ±

standard deviation. MINITAB Statistical Software 14.0

and Microsoft Excel were used for all analyses and the

F
U

L
L

 P
A

P
E

R

1365

 Saallah et al. / Food Research 4 (4) (2020) 1363 - 1370

eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources

Fisher LSD test was used to evaluate significant

differences (p<0.05) between the means for each sample.

3. Results and discussion

3.1 Yield of Golden apple snail hydrolysate (GASH)

Table 1 shows the yield obtained for the Golden

apple snail hydrolysate (GASH) and its fractions (3, 5,

and 10 kDa). The yield of unfractionated GASH was

12.62%, which was higher than that of a study conducted

by Hamid et al. (2015), (9.72%). While, the fractionate

GASH yielding 1.45%, 3.67% and 3.34%, respectively

for fraction 3, 5, and 10 kDa. The lowest yield at low

molecular weight was because the peptides formed

during protein hydrolysis reaction were themselves

degraded into smaller peptides. The same trends were

reported by Razali et al. (2015) for cobia skin gelatin

hydrolysate. However, this finding contrasts with a study

conducted on rapeseed hydrolysate which reported the

yield increased with decreasing molecular weight

fractions (He et al., 2012). The differences in yield

obtained depended on several factors such as pre-

treatment, extraction time, temperature, pH, and peptide

chains during the hydrolysis process.

3.2 Antioxidative properties

3.2.1 Reducing power

The reducing power of the GASH fractions was

measured by Potassium ferricyanide. Higher reducing

power of the sample was indicated by higher absorbance

obtained. The reducing power of different Golden apple

snail hydrolysate (GASH) fraction (3, 5, and 10 kDa)

and BHT that acts as a positive control at a concentration

of 1 mg/mL was shown in Table 2. All GASH fractions

(3, 5, and 10 kDa) were significantly different (p<0.05)

as compared to the positive control, BHT. Based on the

results obtained, 3 kDa shows the highest (p<0.05)

reducing power compared to 5 kDa and 10 kDa.

However, BHT demonstrated the highest reducing power

for all tested samples at 1.75 nm. The higher reducing

power obtained by 3 kDa was due to lower molecular

weight fractions having higher hydrophobic amino acid

levels compared to the high molecular weight fraction

that more easily to donate an electron and hydrogen atom

in order to stabilize the Fe2+. A similar finding was also

obtained by Saidi et al. (2014). The findings show that a

low molecular weight of hydrolysate (1-4 kDa) has better

reducing power than those of higher molecular weight

(>4 kDa). The highest reducing power increase with

decreasing peptide length or molecular weight indicates

that higher electron reduction potential resulted from

lower peptide chains (Razali et al., 2015). Compounding

with reducing power indicates an electron donor and can

reduce the oxidized intermediates of lipids peroxidation

process that makes the compound develop into primary

and secondary antioxidants (Jayanthi and Lalitha, 2011).

3.2.2 DPPH radical scavenging activity

Table 2 shows the DPPH radical scavenging activity

of different Golden apple snail hydrolysate (GASH)

fractions (3, 5, and 10 kDa) with BHT as the positive

control. There were significant differences in DPPH

radical scavenging activity for all GASH fractions (3, 5,

and 10 kDa) as compared to the positive control (BHT)

(p<0.05). The radical scavenging activity of 3 kDa

fraction (79.10%) was higher compared to BHT, 5 kDa

and 10 kDa. Fraction 3 kDa showed higher DPPH radical

scavenging activity compared to other fractions, perhaps

due to its low molecular weight of peptides. Low

molecular peptides have more exposure to the residue

side chain and are responsible for facilitating the reaction

between peptides and free radicals, thus increasing

DPPH radical scavenging activity (Zhou et al., 2012).

These findings were in agreement with a study

conducted by Razali et al. (2015), in which scavenging

activity of GASH was able to stabilize the DPPH radical

by acting as a proton donating substance.

F
U

L
L

 P
A

P
E

R

Hydrolysate Yield (%)

Unfractionated GASH 12.62±0.74

3kDa 1.45±1.25b

5kDa 3.67±0.92a

10kDa 3.34±1.82a

Table 1. The yield of Golden apple snail hydrolysate and its

fractions at different molecular weights (3, 5, 10 kDa).

All results expressed as the mean (± SD) of three trials.

Different superscript letters indicate significant differences

between means (p<0.05).

Hydrolysate
Antioxidant activity

Reducing power (nm) DPPH radical scavenging (%) Metal chelating (%)

BHT 1.75±0.03a 66.35±1.43b 90.16±0.07ab

3kDa 0.90±0.03b 79.10±0.30a 80.86±0.87bc

5kDa 0.82±0.02bc 18.37±0.06cd 85.60±0.71a

10kDa 0.72±0.02d 20.42±0.69c 72.84±3.49cd

All results expressed as the mean (± SD) of three trials. Different superscript letters indicate significant differences between

means (p<0.05).

Table 2. Antioxidant activity of Golden apple snail hydrolysate and its fractions at different molecular weight (3, 5, 10 kDa).

1366

 Saallah et al. / Food Research 4 (4) (2020) 1363 - 1370

eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources

3.2.3 Metal chelating activity

The metal chelating activity on fractionated Golden

apple snail hydrolysate (3, 5, and 10 kDa) at 1mg/mL

was shown in Table 2. These findings indicate that metal

chelating activity was not affected significantly (p>0.05)

by 3 and 5 kDa from BHT. GASH exhibited the highest

metal chelating activity (85.60%) at 5 kDa, yet the result

still lower than BHT value (90.16%) that acts as the

positive control. Higher metal chelating activity was

probably due to the increasing carboxylic group (COO-)

and amino group in branches of the acidic and basic

amino acids with low molecular weight, thus enhancing

the binding of Fe2+. These results are in agreement with

research conducted by Taheri et al. (2014), with herring

brine, as 10-1 kDa molecular weight of herring brine has

higher chelating activity compared to molecular fraction

>50 kDa. Taheri et al. (2014) also stated that low

molecular fraction 10-1 kDa is the best metal chelator.

Iron is one of the common initiators of lipid

peroxidation, resulting in the acceleration of lipid

decomposition (Liu et al., 2012). Ferrozine can

quantitatively form complexes with Fe2+. In the presence

of chelating agents, this formation is disrupted, resulting

in a decrease in the red color of the complex (Zhu et al.,

2006). Metal chelating capacity was significant since it

reduced the concentration of the catalyzing transition

metal in lipid peroxidation. Thus, the chelating agent can

decrease the redox potential and therefore stabilize the

oxidized form of the metal ion.

3.3 Functional properties

3.3.1 Emulsifying properties

 Figure 1 (a) shows the emulsion activity index

(EAI) in different Golden apple snail hydrolysate

(GASH) fraction. Results indicated that EAI value was

affected significantly (p<0.05) with the different fraction

of GASH. Emulsion activity index (EAI) decreased as

fractional molecular weight increased and the hydrolysis

process appeared to improve GASH EAI for 3 kDa to 10

kDa fractions. A study reported by Razali et al. (2015)

also indicated that increasing EAI values led to

decreasing molecular weight fractions. The higher

surface hydrophobicity was obtained by 3 kDa fraction.

The protein structure and function alteration of the

hydrolysate via partial hydrolysis may facilitate diffusion

at the oil/water interface which resulted from higher

solubility and smaller molecular size (Wu et al., 1998).

In addition, partial hydrolysis could lead to the

unfolding of protein molecules. Nonpolar amino acid

groups buried inside protein molecules could be

exposed on the surface of protein molecules after

unfolding (Miroljub et al., 2006). Thus, the enhanced

interactions between proteins and lipids would be

obtained by hydrophobic groups exposed (Razali et al.,

2015).

In addition, the emulsion stability indexes (ESI) for

all three GASH fractions are shown in Figure 1b. It was

observed that 3 kDa was significantly affected (p<0.05)

with 5 and 10 kDa fractions. In contrast with EAI value,

the ESI for all samples decreased (106.87%, 30.35%,

23.71%) as the molecular weight of the hydrolysate

fraction decreased (10, 5, 3 kDa). Similar results were

obtained by Razali et al. (2015) on cobia skin gelatin

hydrolysate (CSGH). The higher ESI for large molecular

peptides compared to low molecular peptides is due to

small peptides having short chain of amino acid that lead

to the lack of unfolding at the interface and have low

efficiency in reducing the interfacial tension compared to

the large molecular peptide (Gbogouri et al., 2004). In

order to exhibit better emulsifying activity, proteins and/

or peptides must migrate rapidly to the water/oil

interface where they quickly unfold and rearrange the

interface (Kotlar et al., 2013). The hydrophobic/

hydrophilic balance of smaller peptides is insufficient for

the stabilization of emulsions, although peptides with

lower molecular weights can migrate rapidly to the

interface (Deng et al., 2011). Hence, due to a reduction

in the hydrophobic/hydrophilic balance, smaller

hydrolysate fractions result in decreasing emulsion

stability.

F
U

L
L

 P
A

P
E

R

(a)

(b)

Figure 1. The (a) Emulsifying activity index (EAI) (b)

Emulsifying stability index (ESI) at different molecular

fraction (3, 5, and 10 kDa) of Golden apple snail hydrolysate

(GASH) at concentration of 1 mg/mL. Results presented as

mean ± SD. The different letters indicate there is significant

difference (p<0.05) between samples.

1367

 Saallah et al. / Food Research 4 (4) (2020) 1363 - 1370

eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources

3.3.2 Foaming capacity

Figure 2 (a) shows the foaming capacity for all

GASH fractions. The results showed that the foaming

capacity of all molecular weights (3, 5, and 10 kDa)

decreased over time. However, there was no significant

difference in foaming capacity between these molecular

weights in terms of time (p>0.05). Among all molecular

weights (3, 5, and 10 kDa), fraction 5 kDa had the

highest value compared to 3 kDa and 10 kDa, at 50.0,

5.66 and 40.0%, respectively. The decrease in peptide

molecular weight leads to the decreased in foaming

capacity because small peptides do not have the ability to

stabilize the air cells of the foam. Furthermore, smaller

peptides allow quick absorption of water thus lowering

the surface tension. Similar results were obtained by

Barac et al. (2011). In smaller molecular peptide

fractions, protein hydrolysate is unable to form a strong

bond at air/water interface thus experienced more

gravitational pull than unmodified isolates. However,

research conducted by Razali et al. (2015) on cobia skin

gelatin hydrolysate showed different results from the

findings in GASH fraction, as 5 kDa obtained the highest

foaming capacity, followed by 10 kDa and 3 kDa. This

may be due to the different protein-protein interaction of

the fraction in the surface of water and air, thus affecting

the foaming capacity of the protein hydrolysate at the

different molecular weight. Cohesive interactions

between molecules at air-liquid interfaces lead to the

formation of surface tension. Thus, substances with

amphiphilic properties can adsorb at air-liquid interfaces,

decreasing the surface tension and resulting in foams

with increased kinetic stability (Patino et al., 2008).

3.3.3 Solubility

Figure 2 (b) shows the solubility index of different

Golden apple snail hydrolysate fraction (3, 5, and 10

kDa). There were significant differences (p<0.05) in

solubility index for all fractions and 5 kDa had the

highest solubility index compared to 3 kDa and 10 kDa.

This might be due to the presence of more hydrophilic

amino acid exposed to the water, leading to the

formation of hydrogen bonds with water and increasing

its solubility index. According to Wu et al. (1998), when

amino acids are exposed, they will interact with water

molecules through hydrogen bonds and electrostatic

interaction and thus solubilize the hydrolysate. These

findings were slightly different from a previous study

conducted by Wang et al. (2006) on wheat gluten, in

which the lowest molecular weight 20 kDa shows the

highest solubility index compared to the other fractions,

i.e. 50 kDa and 100 kDa in various range of pH. Smaller

peptide has shorter peptide length and have fewer

hydrophobic binding sites but more hydrophilic group

that exposed to the water surrounding thus increasing its

solubility (Wu et al., 1998). In addition, the exposure of

more charged and polar groups of low molecular weight

to the surrounding water increased the solubility of low

molecular peptide fraction (Jemil et al., 2014). Thus,

molecular fractions were shown to have an effect on the

solubility index of protein hydrolysate in water. GASH

with high solubility over a wide pH range can, therefore,

be widely applied in formulated food systems.

3.3.4 Water binding capacity

The water binding capacity (WBC) of fractionate

Golden apple snail hydrolysate (GASH) at the different

molecular weight (3, 5, and 10 kDa) were shown in

Figure 2 (c). This finding indicates that fraction (3 kDa)

had higher water binding capacity compared to fraction 5

kDa and 10 kDa which were 5.47, 3.07 and 2.53 mL/g,

respectively. However, there was no significant

difference (p<0.05) on WBC between fraction 5 kDa and

F
U

L
L

 P
A

P
E

R

(a)

(b)

(c)

Figure 2. The (a) foaming capacity (b) solubility (c) water

binding capacity (WBC) at different molecular fraction (3, 5,

and 10 kDa) of Golden apple snail hydrolysate (GASH) at

concentration of 1 mg/mL. Results presented as mean ± SD.

The different letter indicates a significant difference (p<0.05)

between samples.

1368

 Saallah et al. / Food Research 4 (4) (2020) 1363 - 1370

eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources

10 kDa. The greater water binding capacity of 3 kDa

may be due to enzymatic hydrolysis, resulting in fewer

peptide fragments, as smaller peptides have higher

hydrophilic functional groups. Furthermore, low

molecular weight peptide fragments are more effective in

holding water than larger peptide sizes (Cumby et al.,

2007). These results are similar to previous findings on

cobia skin gelatin hydrolysate as reported by Razali et al.

(2015). They show that the 3 kDa and 5 kDa peptide

fractions had the highest water binding capacity

compared to 10 kDa. Therefore, GASH may act as a

good water binder for food products.

4. Conclusion

In conclusion, protein hydrolysate from golden apple

snail (GAS) with different molecular weight (3, 5, and

10 kDa) affects the yields, antioxidant activity and also

the functional properties of hydrolysate produced. The

results indicate that golden apple snail hydrolysates

(GASH) with different molecular weights have potent

antioxidant activity and good functional properties. The

functional properties of GASH were significantly

affected by the size of molecular weight. In other words,

fraction of <3 kDa may have good antioxidant activity

but not necessarily it will be good in functional

properties. Thus, antioxidant and functional properties

are significantly affected by the molecular weight of

hydrolysate.

References

Association of official analytical chemists (AOAC).

(2002). Official Methods of Analysis. 16th ed.

Washington, D.C., USA: Association of Official

Analytical Chemists.

Barac, M., Cabrilo, S., Pesic, M., Stanojevic, S.,

Pavlicevic, M., Macej, O. and  Ristic, N. (2011).

Functional properties of Pea (Pisum sativum, L.)

protein isolates modified with Chymosin.

International Journal of Molecular Science, 12(12),

8372–8387. https://doi.org/10.3390/ijms12128372

Castro, R.J.S. and Sato, H.H. (2014). Comparison and

synergistic effects of intact proteins and their

hydrolysates on the functional properties and

antioxidant activities in a simultaneous process of

enzymatic hydrolysis. Food Bioproduction Process,

92(1), 80–88. https://doi.org/10.1016/

j.fbp.2013.07.004

Chalamaiah, M., Dinesh, B., Hemalatha, R. and

Jyothirmayi, T. (2012). Fish protein hydrolysates:

Proximate composition, amino acid composition,

antioxidant activities and applications: A review.

Food Chemistry, 135(4), 3020–3038. https://

doi.org/10.1016/j.foodchem.2012.06.100

Cumby, N., Zhong, Y., Naczk, M. and Shahidi, F.

(2007). Antioxidant activity and water holding

capacity of canola protein hydrolysates. Food

Chemistry, 109(1), 144-148. https://doi.org/10.1016/

j.foodchem.2007.12.039

Deng, Q., Wang, L., Wei, F., Xie, B., Huang, F., Huang,

W., Shi, J., Huang, Q., Tian, B. and  Xue, S. (2011).

Functional properties of protein isolates, globulin

and albumin extracted from Ginkgo biloba seeds.

Food Chemistry, 124(4), 1458-1465. https://

doi.org/10.1016/j.foodchem.2010.07.108

Foh, M.B.K., Qixing, J., Amadou, I. and Xia, W.S.,

(2010). Influence of ultrafiltration on antioxidant

activity of tilapia (Oreochromis niloticus) protein

hydrolysate. Advance Journal of Food

Science Technology, 2(5), 227–235.

Gbogouri, G.A., Linder, M., Fanni, J. and  Parmentier,

M. (2004). Influence of hydrolysis degree on the

functional properties of salmon byproducts

hydrolysates. Food Chemist Toxicology, 69(8), 615-

622. https://doi.org/10.1111/j.1365-

2621.2004.tb09909.x

Halim, N.R.A., Yusof, H.M. and Sarbon, N.M. (2016).

Functional and bioactive properties of fish protein

hydolysates and peptides: A comprehensive review.

Trends in Food Science Technology, 51, 24-33.

https://doi.org/10.1016/j.tifs.2016.02.007

Halim, N.R.A. and Sarbon, N.M. (2017). A response

surface approach on hydrolysis condition of eel

(Monopterus Sp.) protein hydrolysate with

antioxidant activity.  International Food Research

Journal, 24(3), 1081-1093.

Hamid, S.A., Halim, N.R.A. and Sarbon, N.M. (2015).

Optimization of enzymatic hydrolysis conditions of

Golden apple snail (Pomacea canaliculata) protein

by Alcalase®. International Food Research Journal,

22(4), 1615–1622.

He, R., Girgih, A.T., Malomo, S.A., Ju, X. and Aluko,

RE. (2012). Antioxidant activities of enzymatic

rapeseed protein hydrolysates and the membrane

ultrafiltration fractions. Journal of Functional Food,

5(1), 219–227. https://doi.org/10.1016/

j.jff.2012.10.008

Ishak, N.H. and Sarbon, N.M. (2017). Optimization of

the enzymatic hydrolysis conditions of waste from

shortfin scad (Decapterus Macrosoma) for the

production of angiotensin I-converting enzyme

(ACE) inhibitory peptide using response surface

methodology. International Food Research Journal,

24(4), 1735-1743.

Jamil, N.H., Halim, N.R.H. and Sarbon, NM. (2016).

F
U

L
L

 P
A

P
E

R

1369

https://doi.org/10.3390/ijms12128372

https://doi.org/10.1016/j.fbp.2013.07.004

https://doi.org/10.1016/j.fbp.2013.07.004

https://doi.org/10.1016/j.foodchem.2012.06.100

https://doi.org/10.1016/j.foodchem.2012.06.100

https://doi.org/10.1016/j.foodchem.2007.12.039

https://doi.org/10.1016/j.foodchem.2007.12.039

https://doi.org/10.1016/j.foodchem.2010.07.108

https://doi.org/10.1111/j.1365-2621.2004.tb09909.x

https://doi.org/10.1111/j.1365-2621.2004.tb09909.x

https://doi.org/10.1016/j.tifs.2016.02.007

https://doi.org/10.1016/j.jff.2012.10.008

https://doi.org/10.1016/j.jff.2012.10.008

 Saallah et al. / Food Research 4 (4) (2020) 1363 - 1370

eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources

Optimization of enzymatic hydrolysis condition and

functional properties of eel (Monopterus sp.) protein

using response surface methodology (RSM).

International Food Research Journal, 23(1), 1-9.

Jayanthi, P. and Lalitha, P. (2011). Determination of the

invitro reducing power of the aqueous extract of

Eichhornia crassipes (Mart.) Solms. Journal of

Pharmaceutical Research, 4, 4003–4005.

Jemil, I., Jridi, M., Nasri, R., Ktari, N., Salem, RBSB.,

Mehiri, M., Hajji, M. and  Nasri, M. (2014).

Functional, antioxidant and antibacterial properties

of protein hydrolysates prepared from fish meat

fermented by Bacillus subtilis A26. Process

Biochemistry, 49(6), 963–972. https://

doi.org/10.1016/j.procbio.2014.03.004

Kotlar, C.E., Ponce, A.G. and Roura, S.I. (2013).

Improvement of functional and antimicrobial

properties of brewery byproduct hydrolysed

enzymatically. LWT - Food Science Technology, 50

(2), 378-385. https://doi.org/10.1016/

j.lwt.2012.09.005

Liu, S., Sun, J., Yu, L., Zhang, C., Bi, J., Zhu, F., Qu, M.

and Yang, Q. (2012). Antioxidant activity and

phenolic compounds of Holotrichia parallela

Motschulsky extracts. Food Chemistry, 134(4), 1885

–1891. https://doi.org/10.1016/

j.foodchem.2012.03.091

Miroljub, B.B., Snezana, T.J., Sladjana, P.S. and

Mirjana, B.P. (2006). Effect of limited hydrolysis on

traditional soy protein concentrate. Sensor, 6(9),

1087–1101. https://doi.org/10.3390/s6091087

Patino, J.M.R., Sanchez, C.C. and Nino, M.R.R. (2008).

Implications of interfacial characteristics of food

foaming agents in foam formulations. Adv. Colloid

Interface Science, 140(2), 95-113. https://

doi.org/10.1016/j.cis.2007.12.007

Putra, S.N.K.M., Ishak, N.H. and Sarbon, N.M. (2018).

Preparation and characterization of physicochemical

properties of golden apple snail (Pomacea

canaliculata) protein hydrolysate as effected by

different proteases. Biocatalysis Agriculture

Technology, 13, 123- 128. https://doi.org/10.1016/

j.bcab.2017.12.002

Ranamukhaarachchi, S., Meissner, L. and Moresoli, C.

(2013). Production of antioxidant soy protein

hydrolysates by sequential ultrafiltration and

nanofiltration. Journal of Membrane Science, 429,

81–87. https://doi.org/10.1016/j.memsci.2012.10.040

Razali, A.N., Amin, A.M. and Sarbon, N.M. (2015).

Antioxidant activity and functional properties of

fractionated cobia skin gelatin hydrolysate at

different molecular weight. International Food

Research Journal, 22(2), 651–660.

Saidi, S., Deratani, A., Belleville, M.P. and Ben Amar,

R. (2014). Antioxidant properties of peptide

fractions from tuna dark muscle protein by-product

hydrolysate produced by membrane fractionation

process. Food Research International, 65(Part C),

329–336. https://doi.org/10.1016/

j.foodres.2014.09.023

Salleh, N.H.M., Arbain, D., Daud, M.Z.M., Pilus, N. and

Nawi, R. (2012). Distribution and Management of

Pomacea canaliculata in the Northern Region of

Malaysia: Mini Review. APCBEE Procedia, 2, 129–

134. https://doi.org/10.1016/j.apcbee.2012.06.024

Shahidi, F., Han, X.Q. and Synowiecki, J. (1995).

Production and characteristics of protein

hydrolysates from capelin (Mallotus villosus). Food

Chemistry, 53(3), 285–293. https://

doi.org/10.1016/0308-8146(95)93934-J

 Taheri, A., Farvin, K.H.S., Jacobsen, C. and Baron, CP.

(2014). Antioxidant activities and functional

properties of protein and peptide fractions isolated

from salted herring brine. Food Chemistry, 142, 318

–326. https://doi.org/10.1016/

j.foodchem.2013.06.113

Wang, J., Zhao, M., Yang, X. and Jiang, Y. (2006).

Improvement on functional properties of wheat

gluten by enzymatic hydrolysis and ultrafiltration.

Journal of Cereal Science, 44(1), 93–100. https://

doi.org/10.1016/j.jcs.2006.04.002

Wu, W.U., Hettiarachchy, N.S. and Qi, M. (1998).

Hydrophobicity, solubility, and emulsifying

properties of soy protein peptides prepared by papain

modification and ultrafiltration. Journal of American

Oil Chemists' Society, 75(7), 845–850. https://

doi.org/10.1007/s11746-998-0235-0

Zhou, D.Y., Tang, Y., Zhu, B.W., Qin, L., Li, D.M.,

Yang, J.F., Lei, K. and Murata, Y. (2012).

Antioxidant activity of hydrolysates obtained from

scallop (Patinopecten yessoensis) and abalone

(Haliotis discus hannai Ino) muscle. Food

Chemistry, 132(2), 815–822. https://doi.org/10.1016/

j.foodchem.2011.11.041

 Zhu, K., Zhou, H. and Qian, H. (2006). Antioxidant and

free radical-scavenging activities of wheat germ

protein hydrolysates (WGPH) prepared with

Alcalase®. Process Biochemistry, 41(6), 1296–1302.

https://doi.org/10.1016/j.procbio.2005.12.029

F
U

L
L

 P
A

P
E

R

1370

https://doi.org/10.1016/j.procbio.2014.03.004

https://doi.org/10.1016/j.procbio.2014.03.004

https://doi.org/10.1016/j.lwt.2012.09.005

https://doi.org/10.1016/j.lwt.2012.09.005

https://doi.org/10.1016/j.foodchem.2012.03.091

https://doi.org/10.1016/j.foodchem.2012.03.091

https://doi.org/10.3390/s6091087

https://doi.org/10.1016/j.cis.2007.12.007

https://doi.org/10.1016/j.bcab.2017.12.002

https://doi.org/10.1016/j.bcab.2017.12.002

https://doi.org/10.1016/j.memsci.2012.10.040

https://doi.org/10.1016/j.foodres.2014.09.023

https://doi.org/10.1016/j.foodres.2014.09.023

https://doi.org/10.1016/j.apcbee.2012.06.024

https://doi.org/10.1016/0308-8146(95)93934-J

https://doi.org/10.1016/0308-8146(95)93934-J

https://doi.org/10.1016/j.foodchem.2013.06.113

https://doi.org/10.1016/j.foodchem.2013.06.113

https://doi.org/10.1016/j.jcs.2006.04.002

https://doi.org/10.1016/j.jcs.2006.04.002

https://doi.org/10.1007/s11746-998-0235-0

https://doi.org/10.1007/s11746-998-0235-0

https://doi.org/10.1016/j.foodchem.2011.11.041

https://doi.org/10.1016/j.foodchem.2011.11.041

https://doi.org/10.1016/j.procbio.2005.12.029


Skip to content
PENUNTUT ILMU
Dr. Abdullaah Jalil
40 Hadith (13)

Academic Advisory, Supervision and Review

Academic Courses

ASWJ

Awards

Books (40)

Chapter in Books

Conferences & Workshops – Participant/ Moderator

Conferences – Presenter/ Author/ Co-Author

Consultation

Courses & Workshops – Trainer/ Member

Educational Background

Fiqh Muamalat

Hak Cipta

Halal

Home

Islamic Banking and Finance

Journals

Kuliah & Kursus Agama – Pengajar/ Pembentang

Laws and Regulations

Mirath & Fara’id

Mutun Ilmiyyah

News Paper / Magazines

PCVE (Deradicalization and Rehabilitation)

Penyelidik IMARAH

Policy / Industry

Q & A

References & Links

Research

Risalah Penuntut Ilmu (RPI)

Selected Fatwa

Services/ Modules Offered

Shariah Compliance and Governance

Takaful

Talaqqi

Waqf

Zakat

Zikrullah and Supplications

About

Blog
Bengkel Nvivo
December 3, 2016
Penuntut Ilmu
Thanks for signing up and participating in this workshop.

Location:
Reezqi Academy
7A, Tingkat 1,
Jalan Ceongsam 10/11
Seksyen 10, 40100,
Shah Alam, Selangor.

Next to the Azira Restoran & Butik Indah.

Pay parking on weekdays between 9am to 1pm.
Get your coupun parking from the Restoran Azira.

Weekends is FREE parking.

If you using WAZE, type REEZQI ACADEMY or REEZQI CINEMATOGRAPHY.

The More You Give
The More You Get.
Share this:
Twitter

Facebook
Like this:
Related
Uncategorized
Published by Penuntut Ilmu
Seniour Lecturer, USIM
View all posts by Penuntut Ilmu
Post navigation
‹ PreviousG2.TMC1.FM2.Sports Toto
Next ›Bengkel Panel Penyelidik Fatwa Negeri Sembilan
Leave a Reply
Cancel reply
Blog at WordPress.com.
Comment

Follow

Following

PENUNTUT ILMU

Join 37 other followers

												Sign me up

Already have a WordPress.com account?
Log in now.
PENUNTUT ILMU

Customize

Follow

Following

Sign up

Log in

Copy shortlink

Report this content

View post in Reader

Manage subscriptions

Collapse this bar