Antihypertensive

Antihypertensive, Antioxidant and Functional Properties of Peptide
from Dried Squid Head
Pattraporn Sukkhown1, Tantawan Pirak1*, Kamolwan Jangchud1,
Yaowapa Lorjaroenphon2 & Witoon Prinyawiwatkul3
1 Department of Product Development, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand.

2 Department of Food Science and Technology, Faculty of Agro-Industry,
Kasetsart University, Bangkok 10900, Thailand.

3 School of Nutrition and Food Sciences, Louisiana State University Agricultural Center, Baton Rouge, LA 70803-4200, USA
* Corresponding author. Tel: +662 562 5004
E-mail address: [email protected], [email protected], [email protected]
The research aimed to produce functional peptides from dried squid head, the by-product from the squid snack industry, using commercial enzymes (Alcalase® and Flavouzyme®). A CRD design was conducted with reaction times of 0, 60, 120, 180, 240, 300, 450, 600 and 750 min. The optimal hydrolysis time for Alcalase® and Flavouzyme® was 750 min. The resulting supernatant had the highest protein content (mg/mL), total soluble solid (oBrix), degree of hydrolysis (%), ash content (%) and antioxidant, antimicrobial properties. SDS-PAGE revealed that short chain peptides with molecular weights lower than 10 kDa were obtained. Hydrolysis with Flavouzyme® had the highest antimicrobial activity as shown by it producing the largest clear zones (10.00 mm for S. aureus and 9.50 mm for E. coli). Hydrolysis with Alcalase® produced a low molecular weight peptide which had the highest inhibitory activity (56.98%) of angiotensin I converting enzyme (ACE) for 600 min.
Keywords—Dried squid head, By-product, Functional Peptide, Ingredient, Antioxidant peptide
Introduction
The fisheries industry in Thailand has been expanding up in recent years and generates a lot of waste from manufacturing. In 2010, almost 90 million of food was described as waste from the food manufacturing industry (Ravindran and Jaiswal, 2016). Processing in the seafood industry utilizes only 20-50% as edible portions and the remains (50-80%) are by-products, averaging 20 million globally (Pal and Suresh, 2016). The utilization of seafood by-product is of interest because it can create high-value products and income for the country. Pal and Suresh (2016) reported that seafood was source of protein as a functional component including collagen and gelatin, protein and peptide, oil and lipids, chitin, vitamins, minerals, enzyme, pigment and flavor for human health. Enzymatic hydrolysis is one of the alternative methods that can be used to produce high value-functional peptides from seafood by-products (Sukkhown et al., 2017). The appropriate conditions for the production of protein peptide are very important because the antioxidant activity of protein peptides mainly depends on the amino acid sequence of the generated peptide (Shavandi et al., 2017). For antioxidant peptides, Slizyte et al. (2016) found that the highest AEC inhibition and the antioxidant peptide with a small molecular weight (>2,500 Da) was produced using the enzymatic hydrolysis of defatted salmon backbones with Trysin enzyme at 0.1% (w/w of raw material mixture) at 50oC in a water bath for 120 min followed by enzyme inactivation using microwave heating for 5 min at a temperature > 90oC. Moreover, by-products from shrimp shell were used to produce bioactive peptide using Alcpalase, Trypsin, Chymotrypsin and pepsin. Enzymes were added in the ratio of 1:100 (w/w) for the enzyme to substrate ratio at 50oC in a water bath for 1 h. They found that shrimp shell by-products hydrolyzed with trypsin, as well as protein extracted with Trypsin followed by Alcalase hydrolysis, exhibited the highest ACE inhibitory activity. The peptide research studies implied that the use of food- protein-derived bioactive peptide as a functional ingredient in food was a suitable area to develop for food innovation (Ejike et al., 2017). Hence, the current research aimed to study the appropriate conditions for the production of functional ingredients from dried squid head (one of the by-products from the snack industry) using enzymatic hydrolysis and its application in a snack product.

Materials and methods
Dried squid head, a by-product from the squid snack industry, peanut and wasabi were supplied by a local company in Bangkok, Thailand and stored at -18oC until use. Flavourzyme® 1000L and Alcalase®2.4L FG enzyme were supplied by Brenntag Ingredient (Thailand) Public Co Ltd, Bangkok, Thailand and stored at 4oC prior to use.

Method
Preparation of functional ingredients from dried squid head using enzymatic hydrolysis for studying of appropriate conditions
Dried squid head and water at a ratio of 1:2, respectively were selected from preliminary study. The aqueous squid-water mixture was homogeneously mixed using a Waring blender at 11,000 rpm for 1 min and heated under high pressure and autoclaved at 121oC. After that, the sample was cooled down to room temperature and kept in a refrigerator until the next step.

The samples were hydrolyzed using the commercial protease Alcalase® and the Flavouzyme® enzyme. The experiments were conducted at pH 8 and 55oC with Alcalase®, pH 6.5 and 55oC with Flavouzyme®, and the ratio of enzyme to protein in squid was fixed at 3% (w/w). In this experiment, a CRD design was used with the reaction time varying (0, 60, 120, 180, 240, 300, 450, 600 and 750 min) as shown in the figure. The supernatant was collected at various reaction times. Then, each sample was heated at 90oC for 15 min to inactivate the enzyme (Muzaifa et al., 2012). The peptide was separated using centrifugation at 8,000×g for 10 min according to the method of Fang et al. (2012) and then vacuum filtered. The sample was freeze dried at -57oC for 48 h. The powder was stored at -18oC until further analysis as follows.

Determination of chemical characteristics of dried squid head peptide
The protein content of the obtained peptide solution was determined according to the method of AOAC (2000). The salt content in samples was determined using the method of Hjalmarsson et al. (2007). Total soluble solids (oBrix) were determined by the refractive index measured using a refractometer (Carl Zeiss IMT Corp. Brighton, Michigan, USA) based on the method of Hjalmarsson et al. (2007). The pH values of all samples were measured using a pH meter (Sartorius, Docu-pH-Meter, USA) in a 50 mL sample (Hjalmarsson et al., 2007).

The degree of peptide (DH; %) was defined as the percentage of number of peptide bonds or free amino groups cleared from protein compared with the total number of peptide bonds in substrate. The DH was calculated from spectrophotometric readings of the serine standard and the test sample. The OPA method was investigated according to the method of Nielsen et al. (2001).
Peptide solubility was determined using the modified method of Zhang et al. (2013). A sample of 1 g (m1) squid peptide powder was placed in a 50 mL beaker then 10 mL of deionized water (50 ± 1?C) was added, with constantly stirring for 60 s and then maintained for 60 s, using a stopwatch. Then, 1 mL was transferred into an aluminum can five times and then dried in an oven at 105 ± 2?C for 4 h. The samples were removed from the oven, cooled in a desiccator and weighted (m2). The drying and weighing processes were repeated until constant weight were obtained. The solubility was calculated as
Peptide solubility (%) = 10m2/m1× 100%
m1 = weight of sample before drying in an oven
m2 = weight of sample after removal from the oven and cooling in a desiccator.

Determination of physical characteristic of dried squid head peptide
A chromaticity instrument (CM-3500d Minolta Co., Japan) was used to measure the surface color of the samples. A white standard board was used for calibration. The color values were expressed as L* (whiteness/darkness), a* (redness/greenness) and b* (yellowness/blueness). An average value of five replications was reported (CIE., 1986).

The percent yield (% yield) of each hydrolyzed sample was calculated using the modified method of Kim et al. (2014). The yield of the dried squid head peptide was determined by calculating the difference in weight after and before producing peptide with the following equation:
% yield = weight of dried squid head peptide powder (g) / weight of raw sample (g) × 100
Determination of antioxidant property of dried squid head peptide powder
ABTS assay of dried squid head peptide
The ABTS radical (2,20-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) scavenging capacity of dried squid head peptide was determined according to the method of Gimenez et al. (2009). The stock solution of ABTS radical consisted of 7 mM ABTS in potassium persulfate 2.45 mM, kept in the dark at room temperature for 16 h. An aliquot of stock solution was diluted with deionized water in order to prepare the working solution of ABTS radical with absorbance at 734 nm of 0.70±0.02. Each treatment of dried squid head peptide was dissolved in deionized water. A 60 µL aliquot of sample (dissolved peptide) or deionized water (the control) was mixed with 2,940 µL of ABTS radical working solution and the reduction of absorbance at 734 nm was measured after incubation at 37oC for 10 min in the dark. The sample was replaced by BHT (200 ppm) for comparative purposes. A standard curve of gallic acid was determined, which related the concentration of gallic acid to the amount of absorbance reduction caused by gallic acid. Antioxidant capacity was expressed as milligrams of gallic acid equivalent antioxidant capacity (VCEAC) per gram of sample sample. All determinations were performed at least in triplicate.

FRAP assay of dried squid head peptide
The FRAP assay of dried squid head peptide was determined using the modified method of Gimenez et al. (2009). The ferric reducing ability of plasma (FRAP) method was used to measure the ferric ion reducing capacity of the squid peptides. This is based on the increase in absorbance at 595 nm due to the formation of the complex tripiridiltriazine (TPTZ)–Fe(II) in the presence of reducing agents at 37oC. The squid peptides were dissolved in deionized water. Absorbance was read at 30 min using a UV- spectrophotometer. A standard curve of FeSO4.7H2O was determined, which relates the concentration of FeSO4 . 7H2O (uM) to the absorbance at 595 nm. Results were expressed as mmol FeSO4 . 7H2O equivalents/g sample. At least three replicates were carried out for each sample.

DPPH scavenging activity (%) of dried squid head peptide:
The antioxidant activity was measured by the scavenging effect on DPPH free radicals according to the method of Fang et al. (2012). Briefly, a volume of 1.5 mL squid peptide (1.5 mg/mL) in 95% ethanol was added to 1.5 mL of 0.1 mmol/L DPPH in 95% ethanol. The mixture was shaken and left for 30 min at room temperature and the absorbance of resulting solution was measured at 517 nm using a spectrophotometer. When DPPH encountered a proton-donating substance such as an antioxidant, the radical would be scavenged and the absorbance reduced. A lower absorbance indicated higher DPPH scavenging activity. The scavenging effect can be expressed as shown in the following equation:
DPPH scavenging activity (%) =
(blank absorbance?sample absorbance)/blank absorbance × 100
where the DPPH blank is the value of 1.5 mL of ethanol mixed with 1.5 mL of ethanol containing 0.1 mmol/L DPPH.

Metal chelating assay (%) of dried squid head peptide
The Fe2+ chelating activity of sole and squid peptides at 25 mg/ml assay concentration was measured using the method of Gimenez et al. (2009). Briefly, a test sample of 800 µL was mixed with 10 µL of 2 mM FeCl2 and 20 µL of 5mM ferrozine; the mixture was vortexed and kept at room temperature for 10 min prior to measuring the absorbance (Abs.) at 562 nm. The chelating ability (%) was calculated as follows:
Chelating ability (%) = 1 – (Abs.sample / Abs. control) × 100
The control consisted of a mixture composed of 800 µL of water, 10 µL of 2 mM FeCl2 and 20 µL of 5 mM ferrozine. All determinations were performed at least in triplicate.

Determination of antimicrobial property of dried squid head peptide
The antimicrobial assay was tested using the disc diffusion method of Sruthy et al., (2012). The crude peptide was loaded on a 6 mm disc (Whatman No. 1) using a micropipette with a sterile tip, with each disc containing 20 mg of crude peptide. The samples were loaded onto discs such that each disc was impregnated with 30 µL of each sample. These discs were carefully placed on Petri dishes and seeded with the microbial strains in nutrient agar medium. The plates were incubated overnight at 37?C for S. aureus and E.coli. The plates were then observed for a zone of inhibition (measured in mm).

Determination of angiotensin-converting enzyme (ACE) inhibitory property of dried squid head peptide
The ACE inhibitory activity assay was performed based on the method described by Asoodeh et al., (2012) using FAPGG as the substrate with only slight protocol modifications. In each test sample, the assay mixture was composed of the following components: 22 µL of ACE (50 mU/mL), 50 µL of peptide or peptide (1 mg/mL) and 100 µL of FAPGG (0.5 mM) and 150 µL of ACE buffer (50 mM of Tris-HCl pH 7.5, 0.3 M NaCl and 1 mM ZnCl2). The control sample contained 22 µL of ACE (50 mU/mL), 100 µL of FAPGG and 200 µL of ACE buffer. The reaction was monitored at 340 nm for 60 min. The ACE inhibition was measured using a comparison of the absorbance changes of the test and the control, according to the equation:
ACE inhibition (%) = 1 – (?Ainhibitor / ?Acontrol) × 100
Determination of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-PAGE of crude extract and partially purified glycyl endopeptidase from both cultivars was performed according to Laemmli (1970). Protein solutions were mixed in a 1:1 (v/v) ratio with the sample buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol). The mixture was boiled for 10 min. The samples (25 mg/mL) were loaded onto gel made of 4% stacking and 18% separating gels. They were subjected to an electrophoresis set at a constant current of 250 V per gel using a Mini Protean Tetra Cell unit (Bio-Rad Laboratories, Richmond, CA). After electrophoresis, the gel was stained overnight with staining solution (0.02% (w/v) Coomassie Brilliant Blue R-250) in 50% (v/v) methanol, and 7.5% (v/v) acetic acid. Protein patterns were then visualized after destaining with 30% methanol and 10% acetic acid until a clear background was obtained.

Statistical analysis
All experimental data were expressed as the mean±standard deviation. The analysis of differences between variable were divided for significance by Duncan’s New Multiple Rang Test (DMRT) using SPSS software, 12.0 (Version 12, SPSS Inc., Thailand). The statistically significant was considered at p?0.05.

Results and Discussion
Determination of chemical characteristics of dried squid head peptide
The dried squid head was hydrolyzed using Alcalase® and with Flavouzyme®, a commercial protease enzyme. A CRD design was conducted with reaction times of 0, 60, 120, 180, 240, 300, 450, 600 and 750 min. The optimal conditions for Alcalase® and Flavouzyme® were determined. The chemical properties of the dried squid head peptide are shown in the Figure 1-3.

The protein content (mg/mL and %), ash content (%) and salt content (%) all increased with hydrolysis time (Figure 1). The protein content in the peptide solution increased drastically from 0 to 300 min and then continually increased from 300 to 750 min (Figure 1a, Figure 1b). The dried squid head hydrolyzed with Alcalase® had the highest protein content (47.58 mg/mL, 13.24%) at the end of hydrolysis (750 min). Solubility of small molecules, such as salts or ash increased with increasing hydrolysis time from 0 to 750 min (Figure 1c, Figure 1d). The reaction could destroy protein molecules and break them up into several subunits; hence, ash and salt were then released (Nissen, 1976). The salt content of the Alcalase® and Flavouzyme® treatments increased from 0.42 to 0.64% and from 0.44 to 0.69%, respectively, when the hydrolysis time increased to 750 min (Figure 1d). This result implied the liberation of salt from the squid head during hydrolysis resulting in an increase in the protein concentration, ash and salt contents with increasing hydrolysis time.
The pH, total soluble solids (°Brix), degree of hydrolysis (DH, %) and peptide solubility of the peptide solution are shown versus the hydrolysis time in Figure 2. The pH of the peptide with Alcalase®and Flavouzyme® treatments increased slowly from 0 to 750 min (Figure 2a). Peptides were released that may have increased the ability to expose more hydrophilic groups in the aqueous system. With deeper hydrolysis, the released peptides may change their conformations to expose hydrophobic amino acid residues, so the pH increased (Xia et al., 2012). There was an increase in total soluble solids (TSS) for the Alcalase® and Flavouzyme® treatments during the 0-750 min of hydrolysis from 15 to 18.5oBrix and from 14.8 to 17.2 oBrix, respectively (Figure 2b). This result might have occurred due to the degradation of large molecular weight proteins into free amino acids and short chain peptides (Dong et al., 2014). These amino acid and peptides were easily dissolved in water, resulting in the increase in the TSS with increasing hydrolysis time. There were significant differences (p?0.05) in the DH (%) of samples with different hydrolysis times (Figure 2c). The DH increased from 0 to 71.87% (Alcalase® treatment) and from 0 to 66.39% (Flavouzyme® treatment) with the increasing hydrolysis time to 750 min. The selected enzyme, Alcalase®, has a specificity fit to meat protein and can produce smaller-size peptides and free amino acids (Xia et al., 2012). The peptide solubility of the dried squid head hydrolysis solution gradually increased during the hydrolysis period (Figure 2d). Proteins or peptides with high solubility could provide homogeneous dispersibility of the molecules in colloidal systems and enhance the interfacial properties (Thiansilakul et al., 2007). The peptide solubilities of samples with Alcalase® and Flavouzyme® treatments were not significantly different. The samples of both enzymes had more than 90% peptide solubility. This result suggested that the peptide powders had high water solubility and this property is required in many functional applications such as emulsions, foams, thickeners and flavoring agents (Thiansilakul et al., 2007).
The pH of the dried squid head peptide powder with Alcalase® and Flavouzyme® increased slowly from 0 to 750 min (Figure 3). The samples with Alcalase® had a higher protein content (84.17%) than those with Flavouzyme® (81.07%) at the completion of hydrolysis (750 min). This result implied that the obtained protein peptide was composed of short chain peptides with hydrophilic amino acids. The enhanced solubility of the peptides was due to their smaller molecular size compared with the intact protein and the newly exposed ionizable amino and carboxyl groups of the amino acids, that increased peptide hydrophilicity (Kristinsson and Rasco, 2000).

Determination of physical characteristic of dried squid head peptide
The percent yield of the dried squid head peptide powder with Alcalase® and Flavouzyme® increased slowly from 0 to 750 min (Figure 3).

The samples with Alcalase® had a higher percent yield (48.66%) than those with Flavouzyme® (39.95%) at completion of hydrolysis (750 min). The yield increased with hydrolysis time perhaps because the enhanced solubility of the peptides was due to their smaller molecular size compared with the intact protein, and the newly exposed ionizable amino and carboxyl groups of the amino acids that increase the peptide hydrophilicity (Kristinsson and Rasco, 2000). The amount of protein in solution then increased and resulted in an increasing yield as hydrolysis proceeded.

The color of the protein peptide solution changed significantly (p?0.05) during hydrolysis (Table 1). The solution became darker after 60 min of hydrolysis. The color change occurred continually until the end of hydrolysis (750 min). The lightness (L*), redness (a*) and yellowness (b*) continually decreased after hydrolysis for 60 min. The sample with Alcalase® had lower lightness (L* of 13.61) than that of Flavouzyme® (19.01%) at the completion of hydrolysis (750 min) due to the higher protein content of samples with Alcalase®. This phenomenon suggested that increased hydrolysis time affected the protein content and TSS as shown in Figure 1 and Figure 2. The increases in those parameters implied increased amounts of peptide in the solution. Hence, a dark brown-clear solution resulted. The enzymatic hydrolysis reaction is assumed to have contributed to reduction in the luminosity and the resultant darker appearance of the peptide (Kotlar et al., 2013). Samples of dried squid head peptide powder at 0, 300, 450, 600 and 750 min are shown in Figure 4. The results indicated that dark color increased with the increase of hydrolysis time.

Determination of antioxidant property of dried squid head peptide
The results indicated that DPPH scavenging, FRAP scavenging, ABTS scavenging and metal chelating activity increased with increasing hydrolysis time (Figure 5).The hydrolysis time was important in maintaining controlled and limited hydrolysis to achieve the desired functional properties.

The antioxidant activity of dried squid head hydrolysis was also assessed using DPPH radical scavenging ability (Figure 5a). The DPPH radical ability of the 2 enzyme was significantly different (p?0.05). The Flavouzyme® samples had the highest DPPH radical capacity (75.6%), while it was less in those with Alcalase® (69.3%). This result indicated that the antioxidant peptide had activity in the hydrolysis of Flavouzyme® rather than of Alcalase®. In the FRAP assay, the dried squid head peptide with Flavouzyme® had the highest ferric iron reducing ability (53.43 mmol Fe2SO2.7H2O/g) than the corresponding peptide with Flavouzyme® (60.20 mmol Fe2SO2.7H2O/g) (Figure 5b). The FRAP assay of the 2 enzyme was significantly different (p?0.05). The peptide from hydrolysis of Alcalase® had higher ability to quench ABTS radicals (102.50 mg/g) than Flavouzyme® (98.02 mg/g) (Figure 5c). The ABTS assay results revealed that the type of enzyme significantly affected the antioxidant ability of protein (p?0.05). Hydrolysis with Alcalase® could help protect the structure of the peptide and also the functional properties. The ability to react with the ABTS radical still remained because of the proper chain length and structure of the peptide. The mechanism of the oxidation reaction was terminated with the aid of their hydrophobicity, so they were able to scavenge radicals (Zou et al., 2016). The metal chelating ability of dried squid head peptide is shown in Figure 5d. The chelating effect of Alcalase® at 56.8 mg/mL and and Flavouzyme® at 60.3 was high (but not significantly different). Therefore, the metal chelating assay revealed that dried squid head peptide may act as a chelator of metal ions and is likely prevent lipid oxidation via metal chelating ability.
Determination of antimicrobial properties of dried squid head peptide
The antimicrobial properties of the dried squid head peptide were investigated. The results showed the antimicrobial peptides of dried squid head peptide were small-sized,with an amphipathic structure and a cationic character so that these gene-encoded peptides could rapidly diffuse to the point of infection after microbial infection and act rapidly to neutralize a broad range of microbes and shared several common properties as shown in the Table 2.

The results indicated the antimicrobial properties of dried squid head hydrolysis increased with increasing hydrolysis time. The hydrolyzed sample with Flavouzyme® had a higher antimicrobial action (clear zone of S. aureus at 10.00 mm and E. coli at 9.50 mm) than that of Alcalase® (clear zone of S. aureus at 7.50 mm and E. coli at 6.50 mm) in the completion of hydrolysis (750 min). The highest clear zone of dried squid head peptide was using Flavouzyme® after 750 min.

The hydrolyzed peptide using Flavouzyme® after 750 min of reaction time displayed impressive activity against both Gram-positive and Gram-negative bacteria. The antimicrobial peptides killed cells by disrupting membrane integrity via interaction with negatively charged cell membrane components including proteins, DNA and RNA synthesis, or by interacting with certain intracellular targets. Therefore, rapidly death of antimicrobial peptides was not only due from membrane disruption but also from the inhibition of these functional proteins. Normally, there are four types of peptides according to the base on their secondary structures (?-sheet, ?-helix, extended and loop) but the most common structures are ?-sheet and ?-helix; hence, it is relatively easy to modify the structure and immobilize antimicrobial peptides on the surface (Bahar and Ren, 2013).

Determination of angiotensin-converting enzyme (ACE) inhibitors property of dried squid head peptide
The angiotensin-converting enzyme (ACE) inhibitors (%) of the dried squid head peptide with Alcalase® and Flavouzyme® increased with increasing hydrolysis time from 0 to 600 min and then the ACE inhibitors (%) decreased at 750 min (Table 3). The hydrolyzed samples with Alcalase® had higher levels of angiotensin-converting enzyme (ACE) inhibitors (56.98%) than Flavouzyme® (15.47%) after 600 min of hydrolysis. The results recorded for the ACE inhibitors of the dried squid head peptide can be explained by the ACE inhibition pattern of the ACE inhibitory peptides probably being related to the structure of the inhibitory peptides. The study of the structure-activity relationships demonstrated that the presence of a positive charge on the guanidine or e-amino group of the C-terminal Arg and Lys side-chains, respectively, has a key role in the binding to ACE (Asoodeh et al., 2013).

The results from SDS-PAGE revealed short chain peptides with molecular weights lower than 10 kDa (Figure 6). The band pattern of dried squid head peptide that was hydrolyzed using Alcalase® was less intense than for Flavouzyme®, probably due to the smallest peptides passing through the electrophoresis gel. The dried squid head peptides using Flavouzyme® had various molecular weights due to the long band pattern. The results indicated that dried squid head peptide could effectively cleave the peptide bond yielding useful functional peptides with prominent antioxidant and antimicrobial properties.
Conclusion
The optimal conditions for Alcalase® and Flavouzyme® were determined. The appropriate incubation time was 750 min. The resulting supernatant possessed the highest protein content (mg/mL), total soluble solid (oBrix), degree of hydrolysis (DH, %), ash content (%), antioxidant and antimicrobial properties. The results from SDS-PAGE revealed the presence of short chain peptides with molecular weights lower than 10 kDa and these peptides effectively inhibited oxidation reactions. As a result, the highest antioxidant inhibitory effect was achieved. The clear zone obtained from disc diffusion testing directly depended on the molecular weight of the peptide. The lowest molecular weight peptide after 750 min of incubation had the highest antimicrobial activity as shown by the largest clear zone. A comparison between samples from Alcalase® hydrolysis and those from Flavouzyme® hydrolysis indicated that peptides from Flavouzyme® hydrolysis had significant antioxidant activity (DPPH scavenging at 61.20%, FRAP scavenging at 60.20 mmol Fe2SO4.7H2O/g, metal chelating activity at 60.30% and ABTS scavenging at 98.02 µg/mL) and antimicrobial properties (clear zone of S. aureus at 10.00 mm and E. coli at 9.50 mm). Hence, this flavored-functional protein peptide from dried squid heads was suited for using in foods, especially as an antioxidant.

Acknowledgments
The authors gratefully acknowledge financial support from the Research and Researchers for Industries-RRI, T Thai Snack Food Co. Ltd. for financial support and supplying ingredients, the Department of Product Development, Faculty of Agro-Industry, Kasetsart University, Thailand for providing access and use of instruments in this project.

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