Antioxidant and α-Amylase Inhibitory Properties Generated from Chicken Head Proteins by Dual-Enzyme Hydrolysis

Today’s lifestyles associated with unhealthy diets, lack of physical activity, use of pesticides, food additives, and air pollution can result in oxidative stress in the body. Oxidative stress results from the inability of the biological system to balance the production and accumulation of reactive oxygen species (ROS) with its detoxification. The result of oxidative stress is chronic diseases, such as cancer, diabetes and stroke (Sharifi-Rad et al., 2020). Free radicals and oxidants that are produced in excess will produce oxidative stress in the body which results in damage to cell membranes, DNA, proteins, lipoproteins, and lipids. Free radicals can trigger lipid peroxidation which can damage cell membranes and lipoproteins and generate malondialdehyde which is mutagenic and cytotoxic (Pham-Huy et al., 2008). Natural antioxidants derived from phytochemical compounds such as flavonoids and polyphenols can be used to reduce the harmful effects of oxidative stress (Pizzino et al., 2017). Apart from that, active peptides derived from meat muscle and by-products are also known to have the potential to be used as antioxidants, due to their ability to scavenge free radicals and chelate metals (Liu et al., 2016).

Antioxidant peptides can come from vegetable proteins such as legumes, or from animal proteins from muscle tissue, milk, egg, and by-products. Bioactive peptides derived from poultry by-products, such as chicken skin (Onuh et al., 2014), chicken cartilage (Yang et al., 2019), chicken blood (Hamzeh et al., 2019) and chicken feathers (Alahyaribeik et al., 2021) showed the ability and potential as antioxidant. Bioactive peptides have good electron donor ability and hydrophobic character, so they can act as antioxidants by donating electrons to free radicals and converting them into stable compounds (Najafian and Babji, 2014). The use of poultry by-products as a source of bioactive peptides is based on the high protein content, especially collagen which can be a good precursor of bioactive peptides (Lafarga and Hayes, 2014). Chicken heads are also one of the poultry by-products that can be used to produce bioactive peptides because the protein content is high, namely around 10.58% (Du et al., 2013). The conversion of chicken heads into bioactive peptides is also expected to change its status to a high-value added product.

Bioactive peptides are protein fragments that generally have 2-20 amino acid residues. Some of the biological activities possessed by bioactive peptides include antioxidant, antimicrobial, antidiabetic and antihypertensive (Zaky et al., 2022). Bioactive peptides can only be biologically active in their simple form and are inactive in their parent protein (Gruppi et al., 2022). The generation of bioactive peptides from livestock by-products can be done through chemical and enzymatic hydrolysis. However, enzymatic hydrolysis is preferred because of the specific action of the enzyme and reduces the risk of amino acid damage (Andiana et al., 2023). Each protease has a different cleavage site during the protein hydrolysis process. Papain tends to cleave on residues Ala, Val, Leu, Ile, Phe, Trp, and Tyr which are hydrophobic amino acids (Zhao et al., 2022), while bromelain on alanine, glycine, and leucine (Colletti et al., 2021). Hydrolysis by combining more than one enzyme simultaneously or separately can be more effective in producing peptides that have certain biological activities compared to single enzyme treatment (Wickramasinghe et al., 2022). However, scarce information is available regarding the use of combined enzymes, especially bromelain and papain, to produce peptides with antioxidant and α-amylase inhibitory activity. Therefore, this study aims to evaluate the effect of using different combined concentrations of bromelain and papain on the hydrolysis of chicken head protein in terms of the physicochemical characteristics and bioactivity of the hydrolysate.

The material used in this research was chicken head. The part of the chicken head used was the entire head to the neck, except the beak. Chicken head protein was extracted using the pH-shifting method as described by Darine et al. (2010) with slight modifications. Papain was provided by Hunan Insen Biotech Co., Ltd. (Hunan Province, China). Bromelain was purchased from Shaanxi Rainwood Biotech Co., Ltd. (Shaanxi, China). All other chemicals used in this work were analytical grade.

The research method used was a laboratory experiment with a completely randomized design with 4 treatments and 5 replications. The treatment was as follows: CHP (without hydrolysis); CHP 1 (hydrolysis using bromelain 0.25% and papain 0.75%); CHP2 (hydrolysis using 0.5% bromelain and 0.5% papain); and CHP3 (hydrolysis using bromelain 0.75% and papain 0.25%). The hydrolysis process was carried out for 3 hours for each enzyme, so the total duration of hydrolysis was 6 hours and was carried out at optimum conditions for both enzymes (pH 7 and 50 oC).

Chicken head protein was extracted using the alkaline solubilization method and concentrated by acid precipitation as described by Darine et al. (2010) with slight modifications. Fresh ground chicken heads are dried for 6 hours using an oven at 40℃, after drying they are then ground into flour. Chicken head flours were made into a suspension with a concentration of 10% (w/v). The suspension was homogenized using magnetic stirrer for 5 min, then the pH was adjusted to 12 by adding 10 M NaOH. After the pH reached 12, the suspension was stirred continuously for 1 hour. The suspension was then centrifuged at 4,000 rpm for 15 min and the supernatant was obtained. The chicken head protein in the supernatant obtained was then precipitated by lowering the pH of the supernatant to 4 by adding 1 M HCl, then continued with centrifugation at 5,000 rpm for 15 min. The proteins concentrate obtained in paste form were dried with a microwave dryer on low mode (± 39 0C) for 5 min.

The hydrolysis process was carried out separately for both enzymes (bromelain and papain). Chicken head protein was first hydrolyzed using bromelain for 3 hours, then continued with hydrolysis using papain for 3 hours. The hydrolysis process was carried out at pH 7 and 50 0C for both enzymes. The total percentage of enzyme used for both enzymes is 1% (w/w) of the weight of the protein concentrate used. In the first stage of hydrolysis, the protein concentrate was homogenized with distilled water at a concentration of 2% (w/v). The pH was adjusted to 7 by adding 2 M NaOH. The pre-incubation process was carried out at a temperature of 50 oC for 20 min followed by the addition of bromelain (0.25%; 0.5%; and 0.75%). The incubation process was carried out for 3 hours, then the enzymatic reaction was stopped by heating the mixture at 85 oC for 20 min. The same steps were carried out for papain hydrolysis. The percentage of papain added was 0.75%; 0.5% and 0.25%, so the total percentage of the two enzymes was 1%. After the hydrolysis process using papain was carried out, centrifugation was carried out at 4,000 rpm for 15 min, the supernatant obtained was then stored at -20 oC (Yuan et al., 2020).

The pH was measured using pH meter (Hanna Hi98107). The soluble protein concentration was determined spectrophotometrically at 280 nm using a Nano Drop/ND-1000 UV/Vis (Thermo Fisher Scientific). The A280 protein program was used to quantify the soluble protein concentration. Peptide concentration and degree of hydrolysis were determined by the method described by Zhan et al. (2021). A series of tryptone casein concentrations of 0, 0.04, 0.08, 0.12, and 0.16 mg/ml were prepared to make a tryptone casein standard curve. The standard curve was used to determine the peptide concentration based on the linear regression equation y = ax + b. The degree of hydrolysis (DH) was calculated using the following equation:

DH (%)= A/Bx 100

Where A (mg/ml) is the peptide concentration in the sample after hydrolysis and B (mg/ml) is the protein concentration in the sample before hydrolysis.

Assay of DPPH scavenging activity was determined according to a method reported by Hu et al. (2018). Antioxidant activity against DPPH radicals was expressed in IC50 values obtained by plotting DPPH radical scavenging activity (%) against the concentration of hydrolysate (mg/ml). The reducing power of hydrolysates was evaluated according to the procedure described by Latorres. et al (2018). The method described by Najafian and Babji (2015) and Sellal et al. (2019) was used to evaluate the ability of samples to chelate iron and copper, respectively. Alpha amylase inhibitory activity assay was performed by following the previous description (Fadimu et al., 2022).

The data obtained were presented in the form of mean ± standard deviation and analyzed using one-way analysis of variance (ANOVA) based on significance levels of 1% and 5%. Duncan’s multiple range test was performed to compare means. Statistical analysis was performed using SPSS Statistics 26.

The difference in the percentage of the combination of bromelain and papain enzymes in the hydrolysis of chicken head protein showed a significant difference (P<0.01) on the pH value of the hydrolysate. The data in Table 1 showed the decrease on pH in chicken head protein before hydrolysis (CHP) and after hydrolysis using bromelain and papain (CHP1, CHP2, and CHP3). The highest value was found in CHP, while the lowest value was found in CHP3, namely protein hydrolysate using 0.75% bromelain and 0.25% papain. A decrease in pH after hydrolysis was also reported to occur in undersized crawfish minced meat hydrolysate (Bonilla et al., 2022) and whey protein hydrolysate (Kleekayai et al., 2022). The decrease in pH after hydrolysis may be caused by the release of carboxyl groups from protein chains during the hydrolysis process by proteases (Banjongsinsiri et al., 2016). The decrease in pH in the hydrolysate can also be caused by the specific cleavage site of an enzyme. It is known that papain has specific cleavage sites at Arg, Lys, and Phe residues (Hou et al., 2017). Arginine and lysine are known to contain base groups in the side chains that are attached to the core of the two amino acid molecules. These two amino acids have pKa values of 12-13.7 for arginine and ~10.5 for lysine (Li et al., 2013). This reason may also explain why CHP1 which used a higher papain concentration (0.75%) had a higher pH value compared with CHP2 and CHP3.

Different percentages of the combination of bromelain and papain enzymes gave a significant difference (P<0.01) on the average soluble protein concentration. The data in Table 1 showed that the average soluble protein concentration is in the range of 1.47-6.02 mg/ml. The highest value was at CHP1 and the lowest was at CHP. Susanto et al. (2018) reported that chicken feet protein hydrolysate using the papain enzyme had a dissolved protein content of 0.76-1.38 mg/ml determined by the Bradford method. This showed that chicken head protein hydrolysate using a combination of bromelain and papain has a higher soluble protein concentration compared to previous study. The enzymatic hydrolysis process can increase protein solubility by increasing protein-water interactions (Noman et al., 2022). Protease can reduce the molecular mass of proteins and release ionisable groups, resulting in increased protein solubility (Saengsuk et al., 2021). This can be seen in the data that chicken head protein that has been hydrolysed had a higher concentration of soluble protein compared to before hydrolysis. Good protein solubility is related to the efficiency of adding protein to a food ingredient. There is an indirect correlation between the level of protein solubility and the degree of hydrolysis (Nongonierma and FitzGerald, 2011).

The concentration of TCA-soluble peptide can be used as an indicator to measure the level of protein degradation. Analysis of variance showed that different concentrations of the enzyme combination bromelain and papain had a significant effect (P<0.01) on the peptide concentration of chicken head protein hydrolysate. The peptide concentrations in the samples can be seen in Table 1. A peptide concentration of 0.1550 mg/ml was found in CHP which is a chicken head protein that was not hydrolysed. When the chicken head protein was hydrolysed, the peptide concentration increased in CHP1, CHP2, and CHP3 with combined enzyme hydrolysis treatment compared to CHP. Similar results were also reported by Saengsuk et al. (2021) that the hydrolysis of restructuring pork steak using bromelain had a higher peptide concentration when compared to before hydrolysis. Enzymatic hydrolysis allows for an increase in peptide concentration in the hydrolysate sample, due to the simplification of the protein form into peptides (Ibrahim and Ghani, 2020).

The difference in concentration of the combination of bromelain and papain enzymes gave a significant difference (P<0.01) on the degree of hydrolysis. The degree of hydrolysis in chicken head protein hydrolysate is in the range of 10.54-55.99% as shown in Table 1. The lowest degree of hydrolysis was in CHP, while the highest was in CHP1. These findings were similar to that reported by Lee et al. (2012) who found that duck skin gelatin hydrolysate using a combination of collagenase+papain and collagenase+α-chymotyrpsin had a degree of hydrolysis of 51% and 55.21%, respectively. The degree of hydrolysis can be defined as the percentage of the number of peptide bonds cleaved to the total number of peptide bonds (Bao et al., 2017). The success of a hydrolysis process can also be assessed from the degree of hydrolysis, because antioxidant activity can be influenced by the degree of hydrolysis and amino acid compositions (Puspawati et al., 2021). A high degree of hydrolysis will also increase protein solubility (Taheri et al., 2013) as can be seen that CHP1 which had the highest degree of hydrolysis also had a highest concentration of soluble protein.

Analysis of variance showed that different concentrations of the enzyme combination bromelain and papain had a significant difference (P<0.01) on the IC50 of DPPH. The IC50 value of chicken head hydrolysate using bromelain and papain was in the range of 4.62-15.63 mg/ml. The lowest value was in CHP1, while the highest was in CHP as can be seen in Table 2. The IC50 value is the sample concentration required to scavenge 50% of DPPH free radicals. A lower IC50 value indicates better antioxidant activity in the sample (Fadimu et al., 2022). In this study, it was seen that CHP1 using 0.25% bromelain and 0.75% papain had the strongest antioxidant activity when compared with other samples. Yang et al. (2019) reported that chicken

Table 1: Physicochemical analysis of chicken head protein hydrolysate.

Data expressed as mean ± SD. Mean values with different letters in the same column were significantly different (P<0.01) according to Duncan’s multiple range test.

Table 2: Bioactivity assay of chicken head protein hydrolysate.

Data expressed as mean ± SD. Mean values with different letters in the same column were significantly different (P<0.01) according to Duncan’s multiple range test.

cartilage hydrolysate, fractions >10 kDa, and <10 kDa using trypsin for 3 hours had IC50 values of 9.80, 18.91, and 7.35 mg/ml, while Alahyaribeik et al. (2021) reported that the <3 and <10 kDa fractions from chicken feather hydrolysate using fermented by Bacillus licheniformis had IC50 values of 5.03 and 6.64 mg/ml, respectively. This showed that chicken head protein hydrolysate had antioxidant activity that was equivalent or even better than chicken cartilage and feather hydrolysate.

The antioxidant capability found in protein hydrolysate samples may originate from peptides that engage in hydrogen donation, thereby interrupting free radical reactions. These peptides react with free radicals to generate more stable products and prevent further oxidative reactions (Najafian and Babji, 2015). Generally bioactive peptides have less than 50 amino acid residues. Bioactive peptide sequences are usually composed of proline, arginine, and lysine which have hydrophobic groups (Akbarian et al., 2022). The antioxidant activity of peptides is influenced by composition, structure and hydrophobicity. The antioxidant properties of a peptide are also influenced by the treatment given to the protein, such as the type of protease enzyme used, the degree of hydrolysis, and the structure of the peptide (Padaga and Aulanniam, 2017). Papain is known to have cleavage sites on residues Ala, Val, Leu, Ile, Phe, Trp, and Tyr which are hydrophobic amino acids (Zhao et al., 2022), this may be the reason why CHP1 which used the highest concentration of papain had higher antioxidant activity than other hydrolysates. The degree of hydrolysis, peptide concentration, and soluble protein content in the samples may also influence its antioxidant activity.

The difference in concentration of the combination of bromelain and papain enzymes had a significant difference (P<0.01) on the reducing power. The reducing power of chicken head protein hydrolysate using a combination of bromelain and papain was in the range of 0.1554-0.2610 as can be seen in Table 2. The highest value is in CHP2 and the lowest is in CHP, namely 0.2610 and 0.1554, respectively, showing a significant (P<0.01) difference. CHP was a chicken head protein that was not hydrolysed and had the lowest capability among the other samples, this can be caused by the large size of the peptide in CHP. Peptides that have a smaller size have a better ability to donate electrons and interact with free radicals (Onuh et al., 2014). Reducing power can be indicated from the increase in absorbance at 700 nm in the sample. Acidic amino acids such as aspartic acid and glutamic acid can strengthen the reducing power. Grass turtle protein hydrolysate was reported to have a reducing power of 0.88 at a concentration of 15 mg/ml and DH of 19.52% (Islam et al., 2021). That value was higher than the reducing power of chicken head protein hydrolysates. Reducing power assay is often used to evaluate the ability of antioxidants to provide an electron to free radicals. Samples with higher reducing power have better ability to donate electrons (Jemil et al., 2014). Bioactive peptides in protein hydrolysate will donate an electron and perform a reaction with potassium ferricyanide (Fe3+) to produce potassium ferrocyanide (Fe2+), followed by reacting with ferric chloride to generate a ferric-ferrous complex (Chalamaiah et al., 2015).

The difference in concentration of the combination of bromelain and papain enzymes had a significant difference (P<0.01) on iron and chopper chelating ability. As can be seen in Table 2, the iron chelating ability of chicken head protein hydrolysate was in the range of 42.15-81.07%, while the copper chelating ability was 7.13-35.97%. CHP1 showed the strongest iron and copper chelating ability compared to other samples, namely 81.07% and 35.97%, respectively. CHP showed the lowest iron and copper chelating activity when compared with all treatments and showed a significant (P<0.01) difference. This may be caused by the lowest degree of CHP hydrolysis compared to other treatments, because the degree of hydrolysis indicates the amount of simple peptides produced. Small peptides have a simple spatial structure and more exposed metal ion binding sites, resulting in higher ability of metal chelation (Zhang et al., 2021). Thus, the smaller the amount of simple peptides in the protein hydrolysate, the lower the ability to chelate metals. Abeyrathne et al. (2016) reported negative results on the copper chelating ability of ovomucin hydrolysate, while Islam et al. (2021) reported that grass turtle protein hydrolysate had an iron chelating ability of 63.25% at a DH of 11.96% and a concentration of 20 mg/ml and a copper chelating ability of 66.90% at a DH of 11.96% and a concentration of 6 mg/ml. The ability to chelate metals by protein hydrolysates can be influenced by the type of substrate, type of enzyme, and enzyme concentration (Onuh et al., 2014).

Ferrous ions are catalysts that play a role in the lipid oxidation process. Hydroxyl radicals can be formed from hydrogen peroxide through the Fenton reaction and metal-catalyzed Haber-Weiss reactions. Therefore, ferrous ion chelation can reduce the formation of hydroxyl radicals (Famuwagun et al., 2021). Amino acids and peptides that contain sulphur, such as cysteine, methionine, and glutathione have the ability to chelate and remove metals from the body (Flora and Pachauri, 2010). The ability to chelate iron ions is also associated with the presence of the -NH group of the imidazole ring on the histidine amino acid residue, especially when the histidine is located at the C-terminal (Canabady-Rochelle et al., 2015).

Analysis of variance showed that different concentrations of the enzyme combination bromelain and papain had a significant effect (P<0.01) on the α-amylase inhibitory activity. CHP showed the lowest α-amylase inhibitory activity, namely 4.87%, while the highest activity of 32.65% was in CHP1 as can be seen in Table 2. The inhibition value was relatively higher when compared with collagen hydrolysate as reported by Gaspardi et al. (2022). The inhibitory activity of the α-amylase enzyme by bioactive peptides may originate from the amino acid residues Tyr, Trp, Phe, Lys, and cationic residues that have a tendency to bind α-amylase (Islam et al., 2021). Protein hydrolysate can inhibit α-amylase through disrupting enzyme-substrate interactions at the active site of the enzyme. Peptides with certain amino acid sequences have the ability to block the active site of α-amylase and delay the hydrolysis of carbohydrates into glucose (Fadimu et al., 2022).

Differences in the percentage combination of bromelain and papain enzymes could affect the physicochemical characteristics and bioactivity of chicken head protein hydrolysate. The highest soluble protein concentration, peptide concentration and degree of hydrolysis were in CHP1 with values 6.02 mg/ml, 0.8230 mg/ml, and 55.99%. Meanwhile, the pH value for all samples was in the range of 5.98-7.00. The hydrolysis process of chicken head protein using a combination of papain and bromelain can increase the bioactivity of the hydrolysate. This was shown in the non-hydrolyzed (CHP) sample which had the lowest bioactivity compared to other samples. This research briefly showed that CHP1 using 0.25% bromelain and 0.75% papain had the best antioxidant activity when assessed from its DPPH inhibitory activity with an IC50 value of 4.62 mg/ml, iron and copper chelating ability of 81.07 and 35.97%, respectively. CHP1 also showed the highest an α-amylase inhibitory activity, namely 32.67%. However, CHP2 using 0.5% bromelain and 0.5% papain showed the best reducing power among the others. Thus, chicken head protein hydrolysate using a combination of bromelain and papain has potential as an antioxidant and α-amylase inhibitor.

The author would like to thank the Animal Products Technology Laboratory, Faculty of Animal Husbandry, Universitas Brawijaya for providing research facility.

The novelty in this research is the use of a combination of bromelain and papain enzymes with different concentrations to produce chicken head protein hydrolysate which has antioxidant and amylase inhibitory activity.

PA and MGMS performed laboratory analysis, analyzed data, and wrote the manuscript. KUAA and AM designed the research, provided guidance, and revised the manuscript. All authors have read and approved the final manuscript.

The authors have declared no conflict of interest.