Biocatalysis and Agricultural Biotechnology 35 (2021) 102100
Available online 13 July 2021
1878-8181/© 2021 Elsevier Ltd. All rights reserved.
In-vitro angiotensin converting enzyme (ACE), antioxidant activity and some functional properties of silver catfish (Pangasius sp.) protein hydrolysate by ultrafiltration
Mannur Ismail Shaik, Siti Nor Ashimah Adilah Mohd Noor, Norizah Mhd Sarbon
*Faculty of Fisheries and Food Science, Universiti Malaysia Terengganu, 21030, Kuala Nerus, Terengganu, Malaysia
A R T I C L E I N F O Keywords:
Protein hydrolysate Bioactive peptide Silver catfish
Angiotensin converting enzyme (ACE) Antioxidant
Molecular size
A B S T R A C T
The aim of this study was to investigate the bioactivity and functional properties of silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH) at different molecular size (3, 5 and 10 kDa) via ultrafiltration membrane.
Bioactivity measures included angiotensin-converting enzyme (ACE) inhibitory activity and antioxidant activity (DPPH radical-scavenging activity, reducing power and Fe2+ chelating effects). The functional properties examined included emulsifying, foaming and solubility. There were significant differences (p <0.05) in ACE inhibitory activity among the SCMH fractions, with the <3 kDa SCMH fraction showing the highest ACE inhibitory activity (87.68%). There were significant differences (p <0.05) in DPPH radical-scavenging activities, reducing power and Fe2+chelating effects among the different SCMH fractions. Fraction <3 kDa showed the highest DPPH radical-scavenging activities and reducing power, while fraction <10 kDa showed the highest chelating effect among all fractions (p <0.05). In addition, there were no significant difference (p >0.05) among all SCMH fractions in the emulsifying activity index. However, there were significant differences in terms of emulsifying stability index and foaming capacity properties (p <0.05). Moreover, there was a significant dif- ference (p >0.05) among the SCMH fractions in terms of solubility. In conclusion, variations in the molecular size of the SCMH fractions showed a variety of potential applications for silver catfish protein hydrolysates in functional foods, as it possessed high bioactivity and good functional properties.
1. Introduction
Silver catfish (Pangasius sp.) is the second most popular freshwater fish in Malaysia according to the Malaysian Department of Fisheries (2009). This species of fish, from the Pangasidae family, lives in fresh- water in the Mekong basin. A good environment gives a good impact in growing fast. Pangasius is now cultured in Laos, Thailand, Nepal, Cambodia, India, Pakistan, Bangladesh, Vietnam, Myanmar, and Indonesia. It can be cultured in several ways, such as in fishponds, concrete tanks, fish cages or fish pens (Fernando (2016)). Several studies have successfully investigated the potential applications of silver catfish.
The fatty acid composition of silver catfish (Pangasius sp.) has been successfully studied by (Hashim et al., 2015). Furthermore, the potential of acid soluble collagen (ASC) and pepsin soluble collagen (PSC) to be extracted from skin of silver catfish (Pangasius sp.) has been successfully demonstrated by Hukmi and Sarbon (2018).
Angiotensin I-converting enzyme (ACE) leads to an increase in blood
pressure by producing the vasoconstrictor peptide angiotensin II and by degrading the vasodilator peptide bradykinin (Sarbon et al., 2019).
Hence, ACE inhibitors are used as therapeutic agents against hyperten- sion. Recently, the physiological functions such as antihypertensive and antioxidative activities of the bioactive peptide produced from fish by-products have received growing attention; they may be suitable for applications in healthcare and pharmaceuticals (Rasli and Sarbon, 2018). Several studies on ACE inhibitors from fish protein have exam- ined the viscera of Rohu (Labeo rohita) (Chalamaiah et al., 2016), waste (Ishak and Sarbon, 2017), and the fish skin gelatin of shortfin scad (Decapterus macrosoma) (Rasli and Sarbon, 2018).
Antioxidants are well known for their role in protecting human health against reactive oxygen species (ROS), which may damage membrane lipids, protein molecules and DNA. For the past decade, re- searchers have paid greater attention to the production of antioxidant peptides from fish sources. For instants, hydrolysate of eel has been found to contain peptides exhibiting antioxidant activity through in vitro
* Corresponding author.
E-mail address: [email protected] (N. Mhd Sarbon).
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Biocatalysis and Agricultural Biotechnology
journal homepage: www.elsevier.com/locate/bab
https://doi.org/10.1016/j.bcab.2021.102100
Received 10 April 2021; Received in revised form 9 July 2021; Accepted 11 July 2021
studies (Halim et al., 2018). The ability of antioxidant peptides depends on the amino acid compositions, presence of free amino acids in hy- drolysate and peptide size. Several studies have been done to determine the antioxidant properties using the fish protein hydrolysate such as Cobia skin gelatin hydrolysate (Razali et al., 2015), Indian mackerel (Rastrelliger kanagurta) (Sheriff et al., 2014) and shortfin scad (Kang et al., 2018; Nasir and Sarbon, 2019).
The functional properties of the protein can be modified and repaired for use as food and industrial products and are more valuable if the enzyme technology for protein modification is developed (Onuh et al., 2014). The protein hydrolysates and by-products from the seafood have been shown to have different functional properties that provides a va- riety of applications in the food and pharmaceutical industries (Chi et al., 2014). Fish protein hydrolysate also offers better functional properties such as emulsifying activity and stability; foaming capacity;
solubility; water and oil holding capacities; which are important as food ingredient properties (Ishak and Sarbon, 2018a).
The biological and functional properties of a hydrolysate depend on the average molecular weight (AMW) due to a correlation between the AMW and functional properties of the hydrolysate (Chi et al., 2014).
Molecular size also influences bioactive properties. Protein fractions derived from the fish source with short amino acid sequences and low molecular size are more effective as bioactive peptides compared to large sequences and high molecular size fractions (Ishak and Sarbon, 2018b). The relative distribution of the peptides with different molec- ular weight and residual amino acid sequences affects the ACE inhibi- tory activity of the hydrolysate (Elavarasan and Shamasunder, 2014).
The low molecular size peptides have been reported to have greater ACE inhibitory activity compared to the larger molecular weight peptides.
Peptides with low molecular weight (<1000Da) are well associated with greater health benefits (Li-Chan, 2015). In addition, Dong et al. (2008) reported that mackerel (Scomber austriasicus) protein hydrolysate shows the strongest antioxidant properties due to it containing free amino acids, small peptides, and a low molecular weight of about 1400 Da. This shows that the lower molecular weight of fish protein hydrolysate makes it the strongest antioxidant. Molecular weight also has a strong effect on the functional properties. Smaller molecular weight hydrolysate frac- tions were observed to contain higher water binding capacity and emulsifying activity index (Razali et al., 2015). However, those fractions with the highest molecular weight also had the highest fat binding ca- pacity and emulsifying stability index. Therefore, the aim of this study is to determine the bioactivity and functional properties of silver catfish (Pangasius sp.) muscle protein hydrolysate as affected by changes in molecular weight.
2. Material and methods 2.1. Materials
Silver catfish (Pangasius sp.), were purchased from a local supplier.
The alcalase, Hippuryl-l- histidyl-L-leucine (HHL), hippuric acid (HA), captopril and angiotensin converting enzyme (ACE) were purchased from the Sigma–Aldrich (M) Sdn. Bhd. Selangor, Malaysia. All other reagents and chemicals used in this study were of analytical grade.
2.2. Sample preparation
The silver catfish were beheaded, gutted, filleted and cleaned. The fish filled was minced and stored at − 40 ᵒC for further use. Before the experiment was conducted, the silver catfish mince was thawed in a chiller overnight.
2.3. Preparation of the silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH)
The silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH)
samples were prepared according to the method of Razali et al. (2015).
About 44 g of homogenized fish mince was added with 44 ml of distilled water. Each mixture was then heated at 85 ◦C and stirred for 20 min to deactivate the endogenous enzymes in the sample. Hydrolysis was initiated by adding 2.26% alcalase. The pH of the mixture was adjusted to the desired value (7.89) using 1N NaOH and 1N HCl. In order to deactivate the enzyme activity during the hydrolysis process, the mixture was again heated at 85 ◦C for 20 min before being centrifuged (GYROZEN, KOREA) at 4035×g for about 20 min. The supernatant of hydrolysate was then freeze-dried after being filtered. After the super- natant was collected, fractionation then took place for different molec- ular weights (3, 5 and 10 kDa) with the help of ultrafiltration membrane (Vivaspin 20, Sartorius Stedim Biotech, Goettingen, Germany). The protein hydrolysate was allowed to filter through a series of filtration membranes, starting with the 10 kDa molecular size cutoff (MWCO) membrane. The resultant supernatants were filtered with 5 kDa followed by 3 kDa MWCO membranes, followed by freeze-drying.
2.4. Angiotensin converting enzyme (ACE) activities of fractionated silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH)
The ACE inhibitory activity assay of fractionated (<3, <5, and <10 kDa) silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH) depended on the liberation of hippuric acid (HA) from hippuryl-L-his- tidyl-L-leucine (HHL) catalyzed by the angiotensin-converting enzyme (ACE). The ACE inhibitory activity assay was accomplished with some modification as reported by Azemi et al. (2017). The reaction mixture was made up of 10 μl of SCMH solution (all prepared with 50 mM borate buffer, containing 300 mM NaCI, pH 8.3), 50 μl of 2.17 mM HHL, and 10 μl of 2 mU of ACE. The SCMH solution and HHL were added together and incubated at 37 ◦C for 10 min in 2 ml polyethylene micro-centrifuge tubes. ACE inhibitory activity was also measured using a similar method, with incubation at 37 ◦C for 10 min before the two solutions (HHL and SCMH solution) were mixed together and incubated at 37 ◦C for 30 min with continuous agitation. After 30 min, the reaction was stopped with the 85 μl of 1M HCl and the mixture was then vortexed.
The blank (HHL and buffer) and the positive control (HHL and enzyme) were also prepared in a similar manner. Captopril was used as a standard ACE inhibitor. The ACE activity was calculated as follows:
a) ACE inhibition activity (%) =(Ac – As / Ac – Ab) x 100 where.
Ab =the absorbance of the blank Ac =the absorbance of the control
As =the absorbance of the mixture containing sample 2.5. DPPH radical-scavenging activity of fractionated silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH)
According to Azemi et al. (2017), a synthetic free radical compound 1, 1- diphenyl-2-picrylhydrazyl (DPPH) can be used to analyze the ability of fractionated (<3, <5, and <10 kDa) protein hydrolysates to scavenge the free radicals. Approximately 500 μl of ethanol (99.5%) and 125 μl (0.02%, w/v) of DPPH were mixed with 500 μl of each protein hydrolysate fraction (<3, <5 and <10 kDa). The mixture was shaken using vortex mixture and incubated in the dark place for 60 min. A UV–Visible spectrophotometer (SPECTROQUANT PHARO 300, INDIA) was used to measure absorbance at 517 nm in triplicate for each frac- tion. DPPH radical-scavenging activities were calculated as follows:
DPPH scavenging activity(%) =Acontrol − Asample
Acontrol
x 100 where.
Acontrol is absorbance of the control reaction; Asample is absorbance of
protein hydrolysate.
2.6. Reducing power of fractionated silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH)
The ability of fractionated (<3, <5, and <10 kDa) silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH) to reduce iron (III) was determined as described by Azemi et al. (2017). Each of the 500 μl SCMH samples (<3, <5 and <10 kDa) were mixed with 1.25 ml of 1%
potassium ferricyanide and 1.25 ml of 0.2M phosphate buffer (pH, 6.6).
Each mixture was then mixed with 1.25 ml of 10% (w/v) trichloroacetic acid after incubation at 50 ◦C for 30 min. Each mixture was then centrifuged (GYROZEN, KOREA) at 10,000 rpm for 10 min after about 1.25 ml of supernatant was mixed with 250 μl of 0.1% (w/v) ferric chloride and 1.25 ml of distilled water. The absorbance for each resulting solution was then measured at 700 nm using a UV–Visible spectrophotometer after 10 min of reaction with synthetic antioxidant BHT as a reference. The absorbance increased with increased reducing power. The reducing power of each SCMH fraction was measured in triplicate.
2.7. Chelating effects of the ferrous ion of fractionated silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH)
The chelating effects of ferrous ions for each silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH) fraction (<3, <5 and <10 kDa) were determined according to Razali et al. (2015). Briefly, 500 μl of each SCMH fraction at 10 mg/ml concentration was mixed with 0.05 ml of FeCl2 and 1.6 ml of distilled water. After 15 min, the reactions were initiated with the addition of 0.1 ml of ferrozine (5 mM). Then, each mixture was vigorously shaken before being left at room temperature for 10 min for stabilization. The absorbance of the Fe2+ferrozine complex was then measured at 562 nm with the help of UV–Visible spectropho- tometer. The chelating effect of Fe2+was calculated as follows:
Chelating effect(%) =Acontrol − Asample
Acontrol
x 100 where.
Acontrol is absorbance of the control reaction; Asample is absorbance of the tested SCMH fraction. Butylated hydroxytoluene (BHT) was pre- pared in the same manner except that distilled water was used instead of a trial sample. BHT also was used as a positive control. The measure- ments were conducted in triplicate.
2.8. Emulsifying properties of fractionated silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH)
The emulsion stability index (ESI) and the emulsion activity index (EAI) of silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH) were determined according to the methods described by Razali et al.
(2015) with slight modification. Each sample was homogenized and incubated at 55 ◦C in a water bath for 30 min in order to determine the emulsifying stability index (ESI). Then, the samples were centrifuged at 2000 rpm for 5 min, after which the two layered emulsions were measured. Similarly, each assay was measured in triplicate for each molecular fraction as follows:
ESI(%) = Height of emulsified layer after heating Height of the total content before heatingx 100
To determine the emulsifying activity index (EAI), approximately 5 ml of each SCMH solution (<3, <5 and <10 kDa) at 10 mg/ml con- centration was mixed with 5 ml palm oil and homogenized at 18,000 rpm for 1 min. Each emulsion was then centrifuged at 1180 rpm for 5 min to determine the emulsifying activity index EAI of the sample. The height of the emulsifier layer and the height of the total content were
measured as follows:
EAI(%) =Height of emulsified layer Height of the total contentx 100
2.9. Foaming capacity of fractionated silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH)
The foaming capacity index for each silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH) fraction was measured following Jamil et al. (2016). Approximately 250 mg of each SCMH fraction (<3,
<5 and <10 kDa) was mixed with 12.5 ml of distilled water. The so- lution was blended in a 50 ml cylinder at 16,000 rpm (Ultraturrax T25) to consolidate air for 1 min. The total volume was measured at 0 min and 30 min after whipping, respectively, for fractions <3, <5 and <10 kDa.
Similar procedure repeated for triplicate determinations of each mo- lecular fraction. The foaming capacity index was expressed as a foam development from time at 0 min and calculated as follows:
Foam capacity index (%) =[(A-B)/B)] X 100 where.
A is volume after whipping (ml), and B is volume before whipping (ml).
2.10. Solubility properties of fractionated silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH)
Protein solubility of fractionated (<3, <5, and <10 kDa) silver cat- fish (Pangasius sp.) muscle protein hydrolysate (SCMH) was determined as described by Jamil et al. (2016) with slight modification. Approxi- mately 200 mg of each molecular weight of SCMH were mixed with 20 ml of distilled water. The mixture was stirred for 30 min at room tem- perature and centrifuged at 7500×g for 15 min at 4 ◦C. The Kjeldahl method was used to regulate protein content in the supernatant and protein solubility, which was calculated as follows:
Protein solubility(%) =Protein content in supernatant Total protein content in samplex 100
2.11. Data analysis
The data analysis was carried out in triplicate. The one-way analysis of variance (ANOVA) was calculated and all data were stated as mean and ±standard deviation. The significant differences (p <0.05) between the different molecular weights of the fish muscle protein hydrolysate were further analyzed using Fisher’s LSD test.
3. Results and discussion
3.1. Bioactivity properties of fractionated silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH)
3.1.1. Angiotensin converting enzyme (ACE) activity
Fig. 1 shows the ACE inhibitory activity of silver catfish muscle hy- drolysate (SCMH) at different molecular size (<10, <5 and <3 kDa) and captopril as a standard at 10 mg/ml concentration. Based on Fig. 1, the ACE inhibitory activity showed an increased with the decreased of the SCMH molecular size fraction. A lower molecular size was associated with higher ACE inhibitory activity. Fraction <3 kDa of SCMH was depicted as having the highest ACE inhibitory activity (87.68%), fol- lowed by <5 kDa (54.58%) and <10 kDa (29.14%). Significant differ- ences (p < 0.05) in ACE inhibitory activity were observed between different molecular size fractions. Captopril showed the highest ACE inhibitory activity (90.44%); however, this was not significantly different (p >0.05) from fraction <3 kDa.
Similarly, the highest ACE inhibitory activity was found in fraction
<3 kDa fraction (77.40%), compared to fraction <5 kDa (62.18%) and fraction <10 kDa (32.53%) of shortfin scad muscle protein hydrolysate, as studied by Nasir and Sarbon (2019). Specifically, the lowest fraction (<3 kDa) showed the highest ACE inhibitory activity. According to Azemi et al. (2017), the ACE inhibitory activities of the peptides are strongly influenced by the molecular size of the peptides. Furthermore, the hydrophobic amino acid present in the hydrolysate also plays a vital role in the inhibition of ACE activity. ACE inhibitory activity was significantly influenced by the hydrophobicity of the C-terminal amino acids and three-dimensional chemical properties, revealing that the higher volume and greater hydrophobicity of the amino acids leads to the highest ACE inhibitory activity (Sarbon et al., 2019). Therefore, the hydrophobic amino acids at the C-terminal containing dipeptides, such as tryptophan, phenylalanine and tyrosine, will have highest ACE inhibitory activity. Based on the results obtained from the ACE inhibi- tory activity of SCMH at different molecular size, the level of hydro- phobic amino acids present in the SCMH might increase with the decrease in molecular size during the hydrolysate fractionation. There- fore, the lowest molecular size fraction shows the highest inhibition of ACE activity.
These findings are in agreement with a study conducted by Azemi et al. (2017), who reported that the <3 kDa fraction of eel protein hy- drolysate (EPH) showed the greater ACE inhibitory activity (71.90%), followed by the <5 kDa (49.05%) and <10 kDa (17.52%) fractions. The lower molecular size hydrolysate fraction (<2 kDa) exhibits the higher ACE inhibitory activity in chicken skin gelatin hydrolysates (Sarbon et al., 2019). Hence, the results obtained indicate that higher ACE in- hibition activity was recorded at lower molecular size (<3 kDa).
3.1.2. Antioxidant activity of fractionated silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH)
3.1.2.1. DPPH radical-scavenging activity. Table 1 shows the DPPH radical-scavenging activities of SCMH with different molecular size (<10, <5 and <3 kDa) fractions and positive control at a concentration of 10 mg/ml. DPPH radical-scavenging activities were increased with the decrease in molecular size molecular size of the fraction. The <3 kDa fraction showed the highest antioxidant activity, followed by <5 kDa and <10 kDa fractions. There were significant differences (p <0.05) in DPPH radical scavenging activity among all molecular size fractions and also the BHT as a positive control.
According to the results, it may be stated that antioxidant activity was augmented by the effects of low molecular size peptide(s). A recent
study conducted by Nasir and Sarbon (2019) revealed that the shortfin scad muscle protein hydrolysate fraction with low molecular size (<3 kDa) exhibited the highest DPPH scavenging activity when compared to fractions <5 kDa and <10 kDa. In addition, antioxidant activity can be enhanced by the presence of hydrophobic amino acid. Amino acids which contain hydrophobic side chain residues could promote the electron transfer from the peptides to the DPPH radical, thus stabilizing the N radical (Azemi et al., 2017).
Similarly, eel protein hydrolysate with 3 kDa molecular size fraction exhibited the highest DPPH radical scavenging activity (73.17%) when compared to crude (70.11%), 10 kDa (70.38%) and 5 kDa (68.80%) fractions (Halim et al., 2018). This finding was supported by a study by Razali et al. (2015), who reported that the 3 kDa fraction had highest level of DPPH radical inhibitory activity compared to other three cobia skin gelatin hydrolysate fractions. Similarly, Qin et al. (2011) found that a higher percentage of DPPH radical-scavenging activity was exhibited due to the lower molecular size of the purple sea urchin gonad hydro- lysate. However, the radical scavenging activity of Golden apple snail hydrolysate at 3 kDa fraction (79.10%) was higher compared to 5 kDa and 10 kDa fractions and positive control BHT. The fraction 3 kDa exhibited higher DPPH radical scavenging activity compared to other fractions, possibly due to its amino acid sequence and low molecular size (Saallah et al., 2020).
3.1.2.2. Reducing power. Fig. 2 shows the reducing power of SCMH with different molecular size fractions (<10, <5 and <3 kDa) and BHT as a positive control at a concentration of 10 mg/ml. Reducing power was increased with decreasing molecular size molecular size fraction. The Fig. 1. ACE inhibition activity of fractionated silver catfish muscle protein hydrolysate at different molecular weights (<10, <5 and <3 kDa) and Captopril as a standard at a concentration of 10 mg/ml *Values with different superscripts (a-c) are significantly different (p <0.05).
Table 1
Antioxidant activity of fractionated silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH).
Sample Metal chelating (%) DPPH radical scavenging activity (%)
<10 kDa 43.32 ±7.40a 17.63 ±2.22d
<5 kDa 17.77 ±2.79b 48.75 ±3.15b
<3 kDa 6.03 ±1.78c 55.12 ±1.54a BHT 49.48 ±0.672a 33.24 ±0.28c
Antioxidant activity of fractionated silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH) at different molecular weights (<3 kDa, <5 kDa, <10 kDa).
BHT used as a positive control. The values are reported as mean ±SD of trip- licate determination (n =3).Value with different superscript letter (a-d) indicates significant different (p <0.05).
reducing power for the <3 kDa fraction of SCMH was higher in absor- bance compared to the <5 kDa and <10 kDa fractions, which were 2.50%, 2.45% and 2.29%, respectively. However, there was no signifi- cant difference between the <3 kDa and <5 kDa fractions (p >0.05).
Based on these result, shorter peptide chains had higher electron reduction potential, allowing for the highest reducing power (Liu et al., 2010). The SCMH with lower molecular size might have more electron donating substances collected during hydrolysis, as compared to SCMH with a higher molecular size. The reducing capacity of the compound may serve as a significant indicator of potential antioxidant activity. An electron-donating reducing agent is capable to donate an electron to a free radical.
Moreover, Razali et al. (2015) also reported that, the reducing power for the 3 kDa fraction was higher in absorbance compared to fraction 5 kDa and 10 kDa at 0.5–20 mg/ml concentrations. However, all hydro- lysate fractions showed lower reducing power compared to BHT at all concentrations. The results also indicated that the reducing power of SCMH was significantly affected (p <0.05) by molecular weight. The lower molecular size showed higher reducing power activity.
3.1.2.3. Chelating effects of ferrous ion. Table 1 shows the SCMH scav- enging of ferrous ions at different molecular size fractions (<10, <5 and
<3 kDa), with BHT as a positive control at a concentration of 10 mg/ml.
The chelating effects of the fraction increased with the increase in mo- lecular size of the fraction. Fraction <10 kDa of SCMH showed the highest percentage of ferrous chelating activity compared to other mo- lecular size at 43.32%, and was not significantly different (p >0.05) from BHT. However, there were significant differences (p <0.05) in chelating among all molecular size sample fractions.
Moreover, all fractions exhibited the capability to reduce Fe+3 into Fe+2 and ferrous-ferrozine complex. Transition metal ions, like Cu2+and Fe2+, can catalyze the reactive oxygen species generation which accel- erates the lipid oxidation. Moreover, Fe2+can catalyze the Haber-Weiss reaction and also induce the superoxide anions to form more hazardous hydroxyl radicals, especially lipid oxidation (Xie et al., 2008). Therefore, the chelation of transition metal ions by antioxidative peptides could retard the oxidation reaction. The fractionation of SCMH leads to the collection of hydrolysates, which contains chelating agents. However, the chelating agents present in the hydrolysate were reduced when the SCMH was fractionated at low molecular size (<3 kDa).
These findings were supported by He et al. (2012), who reported that the low molecular size fraction (<3 kDa) exhibited weaker Fe2+ chelating activity, while the high molecular size (5–10 kDa) fractions
showed stronger chelating capacity. This finding also agreed with those of Razali et al. (2015), who found that the chicken skin gelatin hydro- lysate (CSGH) exhibited the highest percentage of ferrous chelating activity at 5 kDa and was significantly different (p <0.05) from BHT. In addition, a study reported by Azemi et al. (2017) also supported that the fraction 5 kDa (75.44%) of eel protein hydrolysate showed the highest chelating activity, followed by 10 kDa and 3 kDa. Moreover, Fe2+at different concentrations (5.0, 10 and 20 mg/ml) of chicken peptides exhibited chelating activity levels of 98, 99, and 99%, respectively (Sarbon et al., 2018).
3.2. Functional properties of fractionated silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH)
3.2.1. Emulsifying activity index (EAI)
There were no significant differences (p >0.05) in EAI among all molecular size fraction (Table 2). Proteins or peptides must be migrating rapidly into the water or oil interface in order to exhibit better emulsi- fying activity where they quickly unfold and rearrange the interface.
Peptide with a low molecular size can migrate rapidly to the interface.
According to a study by Chi et al. (2014), higher average molecular size fractions displayed higher EAI by the Spanish mackerel hydrolysate. The molecular weight of the fraction strongly affects the emulsifying activity index of the fish protein hydrolysate. A different sample fraction indi- cated a different EAI value.
Fig. 2. Reducing power of SCMH with different molecular weights (<10, <5 and <3 kDa) with BHT as a positive control at a concentration of 10 mg/ml *Values with different superscripts (a-c) are significantly different (p <0.05).
Table 2
Functional properties of fractionated silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH).
Sample Foaming capacity index (%)
Emulsion activity
index (EAI) (%) Emulsion stability index (ESI) (%)
Solubility (%)
<10
kDa 41.22 ±2.85a 44.92 ±1.26a 47.15 ±1.15a 2.12 ± 0.53ab
<5 kDa 3.85 ±0.00c 45.78 ±3.56a 44.13 ±1.13b 1.59 ± 0.00b
<3 kDa 30.39 ±0.65b 44.86 ±2.39a 41.56 ±1.12c 2.65 ± 0.53a Functional properties of fractionated silver catfish (Pangasius sp.) muscle protein hydrolysate (SCMH) at different molecular weights (<3 kDa, <5 kDa, <10 kDa).
The values are reported as mean ±SD of triplicate determination (n =3). Value with different superscript letter (a-c) indicates significant different (p <0.05).
3.2.2. Emulsifying stability index (ESI)
Table 2 showed that ESI was increased with the increasing of mo- lecular weight. The fraction <10 kDa of SCMH shows the highest per- centage of emulsifying stability index (47.16%). There was a significant difference (p <0.05) between all the molecular size fractions which was 41.56% and 44.14% for <3 kDa and <5 kDa, respectively.
The fraction <3 kDa of SCMH shows the lowest ESI value. This finding was supported by Razali et al. (2015) who reported that less emulsion stability results in smaller molecular size fractions due to un- balanced hydrophilic or hydrophobic groups. The hydrophilic or hy- drophobic balances of the smaller peptides are insufficient to stabilize the emulsion, even peptides with lower molecular weights can easily migrate into the interface which impacts on emulsifying stability (Deng et al., 2011). Therefore, the smaller molecular size fractions result in less emulsion stability due to unbalanced hydrophilic or hydrophobic groups. This study is agreement with studies carried out by Chi et al.
(2014) on the Spanish mackerel skin collagen hydrolysate and Razali et al. (2015) on cobia skin gelatin hydrolysate (CSGH), which stated that the high molecular size fractions showed the highest emulsifying sta- bility indexes (ESI).
3.2.3. Foaming capacity
Table 2 shows the foaming capacity index of silver catfish muscle protein hydrolysate at different molecular size (<10, <5 and <3 kDa).
Sample fraction of <10 kDa shows the highest foaming capacity (41.22%) followed by <3 kDa and <5 kDa which was 30.39% and 3.85%, respectively. There was a significant difference (p <0.05) in the foaming capacity of SCMH between all molecular size fraction.
A higher molecular size leads to higher foaming capacity. The higher molecular size polypeptides resulting from the hydrolysate fractions allow the formation of a stable film around the gas bubbles, which might be the primary cause of higher foaming properties (Chi et al., 2014). This finding was in contrast with the study done by Baharuddin et al. (2016), who stated that the excellent capability of proteins in rapidly migrating into air-water interface result in a superior foaming property, as the proteins will unfold and rearrange at the interface. A similar finding was reached in a study on eel protein hydrolysate by Halim and Sarbon (2020), who found that a lower molecular size will increase migration, thus increasing foaming properties.
In contrast, a study by Razali et al. (2015) has claimed that the quick adsorption capability of a protein in the water or air interface was required by foam formation, thereby lowering surface tension. They found that the fraction 5 kDa of CSGH had the highest index at 11%.
Foam stability mainly depends on the protein-protein interactions extant within the matrix of the film’s surrounding air bubbles, as well as protein structure and flexibility (Tsumura et al., 2005). Low molecular size proteins or peptides may rapidly form a foam, but such foams are not stable.
3.2.4. Solubility properties
Table 2 shows the protein solubility of silver catfish muscle protein hydrolysates with various molecular size (<10, <5 and <3 kDa). There were no significant differences (p >0.05) in solubility of <3 kDa and
<10 kDa fractions. However, there was a significant difference (p <
0.05) between fraction <3 kDa and <5 kDa. Fraction <3 kDa showed the highest protein solubility, followed by <10 kDa and <5 kDa. The lower molecular size fractions exhibit the highest solubility. According to Gbogouri et al. (2004), the smaller peptides of myofibrillar proteins have the ability to form hydrogen bonds with water and enhance solu- bility, as they are expected to have proportionally more polar residues.
According to Li et al. (2013), peptides with the lower molecular size, which are more hydrophilic and more solvated in aqueous solutions, can be produced by enzymatic hydrolysis.
This result can be supported by Li et al. (2013) who reported that peptides with low molecular weight (1–20 kDa) showed greater solu- bility. In addition, solubility also has the correlation to the degree of
hydrolysis. Baharuddin et al. (2016) found that the higher the degree of hydrolysis (DH), the higher the solubility. However, the presence of hydrophilic, polar amino acids such as asparagine, serine, threonine and glutamine, could help the solubility efficiency of the hydrolysate (Baharuddin et al., 2016). Chi et al. (2014) also found that the reduction of average molecular size significantly enhanced the solubility of Spanish mackerel hydrolysate fractions.
4. Conclusion
In conclusion, ACE inhibitory activity increased with decreasing molecular size. Fraction <3 kDa showed the highest ACE inhibitory activity, followed by <5 kDa and <10 kDa. Meanwhile, fraction <3 kDa exhibited the highest DPPH radical-scavenging activity and reducing power. However, fraction <10 kDa showed the highest value for the chelating effects of ferrous ion. The molecular size of the fractions was strongly affected the functional properties of the fractions. However, there was no significant differences of emulsion activity index and sol- ubility among fractions were recorded. While fraction <10 kDa showed the highest values for emulsion stability index (ESI) and foaming properties. Therefore, the findings from this study show that different molecular size for peptides may contribute to different bioactivity and functional properties, and thus potential uses in many applications in numerous industries.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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