Enhancement of Antibacterial Activity from Chicken Head Protein Hydrolysate Using Dual-Enzyme Hydrolysis
Pramudya Andiana1), Moch. Geerhan Miraja Syahdan1), Arif Hendra Utama1), Kasri2),Lilik Eka Radiati1), Khothibul Umam Al Awwaly1), Abdul Manab1)
1) Animal Product Technology Department, Faculty of Animal Science, Universitas Brawijaya, Jl. Veteran, Malang, East Java, 65145, Indonesia
2) Animal Nutrition and Feed Department, Faculty of Animal Science, Universitas Brawijaya, Jl. Veteran, Malang, East Java, 65145, Indonesia
*Corresponding Email: [email protected] Submitted 13 December 2023; Accepted 12 February 2024
ABSTRACT
The chicken head is one of the by-products with a high protein content. Therefore, chicken heads can be used as raw materials to produce protein hydrolysates containing bioactive peptides that have biological activities, such as antibacterial, anti-inflammatory, and antioxidant activities. This research aimed to evaluate the use of the combined ratio of papain and bromelain enzymes to produce chicken head protein hydrolysate that has antibacterial activity. The research method used in this study was a laboratory experiment using a completely randomized design (CRD) with four treatments and five replications. Statistical significance was done using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT). The inhibition zones of chicken head protein hydrolysate using a combination of papain enzymes against Lactobacillus casei, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella typhimurium were 1.72-2.68, 1.19-4.47, 0.93-1.45, 1.64-2.46, and 1.01-3.62 mm, respectively. The result showed that the highest antibacterial activities against Lactobacillus casei, Escherichia coli, and Staphylococcus aureus were in A1 (hydrolysis using papain 75% and bromelain 25%), the highest antibacterial activities against Pseudomonas aeruginosa was in A3 (hydrolysis using papain 25% and bromelain 75%), and the highest antibacterial activity against Salmonella typhimurium was in A2 (hydrolysis using papain 50% and bromelain 50%). However, all the hydrolysate didn’t exhibit antibacterial activity against Bacillus subtilis. Chicken head protein hydrolysate had the potential to be an antibacterial agent against pathogenic bacteria.
Key words: By-product; chicken head; antibacterial activity; bioactive peptide
INTRODUCTION
Chicken heads are one of the secondary products from the poultry processing industry. Parts of a chicken’s head include the neck, tongue, eyes and brain (Akimova et al., 2023). Chicken heads have often been used as feed for livestock and processed into meals for humans.
Another use is as an ingredient for producing gelatin, because of the collagen content in it. Chicken heads have a proportion of approximately 2.0% per live weight and also have a high protein content (Gál et al., 2020). Al Awwaly et al. (2020) reported a protein content in chicken heads of 12.29%, while Akimova et al. (2023) reported a higher protein content in ground mass of chicken head and feet, namely 17.5%. Therefore, chicken heads have the potential to be used as raw material for producing protein hydrolysates which contain bioactive peptides.
In recent years, researchers have paid attention to the utilization of poultry by- products as raw materials for protein hydrolysates which have health benefits and biological activity. Among them are the antioxidant activity from chicken feet hydrolysate (Susanto et al., 2018), the α- amylase inhibitory activity from chicken intestines (Vimalraj et al., 2022), and the antibacterial activity from chicken blood plasma (Tian et al., 2022). Protein hydrolysate can be defined as a mixture of peptides and amino acids obtained from protein hydrolysis activities. In this process the peptide bonds in proteins are cleaved, so that the proteins are converted into peptides and amino acids. Protein hydrolysis makes the protein size to peptide, so it can also modify the functional characteristic and
enhance their quality (Ulagesan et al., 2018).
The methods commonly used in the hydrolysis process are enzymatic, chemical, and microbial methods (Hou et al., 2017).
The enzyme group used in the protein hydrolysis process is the hydrolase enzyme group which works to catalyze the hydrolysis process (Sutrisno, 2017). The advantage of enzymatic hydrolysis compared to chemical methods is its specific action.
Protease enzymes can come from animal, vegetable, and microbial sources.
Some examples of protease enzymes are papain, bromelain, pepsin, trypsin, and alcalase (Cruz-Casas et al., 2021). Papain selectively hydrolyzes peptide bonds that include basic amino acids, with a preference for arginine, lysine, and amino acid residues following phenylalanine (Gomez et al., 2019), while the bromelain enzyme is an endopeptidase enzyme which cleaves peptide bonds from non-terminal amino acids. The bromelain enzyme tends to cleave peptide bonds in the amino acid residues alanine, glycine, and leucine (Colletti et al., 2021).
Each protease used to hydrolyze proteins has a unique cleavage site.
Therefore, protein hydrolysis by combining more than one enzyme is expected to be more effective than single enzyme treatment in producing peptides that have certain bioactivities (Wickramasinghe et al., 2022).
Porcine liver hydrolysate produced using bromelain exhibited the best antibacterial activity against B. thermosphacta and L.
monocytogenes compared to alcalase (Borrajo et al., 2022), while papain-digested protein hydrolysate from snail Cryptozona bistrialis showed antibacterial activity against Pseudomonas aeruginosa and
*Corresponding author:
Lilik Eka Radiati
Email: [email protected]
Animal Product Technology Department, Faculty of Animal Science, Universitas Brawijaya, Jl. Veteran, Malang, East Java, 65145, Indonesia
How to cite:
Andiana, P., Syahdan, M. G. M., Utama, A. H., Kasri., Radiati, L. E., Al Awwaly, K. U., & Manab, A. (2024). Enhancement of Antibacterial Activity from Chicken Head Protein Hydrolysate Using Dual-Enzyme Hydrolysis. Jurnal Ilmu dan Teknologi Hasil Ternak, 19 (1), 15-24
Staphylococcus aureus (Ulagesan et al., 2018). The antibacterial activity of a peptide may originate from its destructive action on the physical structure of the microbial membrane or by targeting intracellular bodies (Sultana et al., 2021). These two studies showed that the enzymes, both papain and bromelain, have the potential to be used to produce protein hydrolysates that have antibacterial activity.
However, scarce information is available on the use of the combination of papain and bromelain enzymes to produce protein hydrolysates that have antibacterial activity. The novelty of this research is the use of a combination of papain and bromelain enzymes to produce chicken head protein hydrolysate containing antibacterial peptides.
This research aims to examine different ratios in the use of a combination of papain and bromelain enzymes to produce chicken head protein hydrolysate which has antibacterial activity.
MATERIALS AND METHODS The research was carried out from September to November 2023 at the Animal Product Technology Laboratory, Faculty of Animal Science, Universitas Brawijaya and Meat Science and Technology Laboratory, Faculty of Animal Science, Universitas Gadjah Mada.
Research Materials and Equipment The materials used in this study were chicken head, aquadest, NaOH (Makmur Sejati), HCl (Merck), bromelain (Shaanxi Rainwood Biotech), papain (Hunan Insen Biotech), buffered peptone water (OXOID), nutrient agar (Merck), EMB agar (Himedia), SS agar (Himedia), MRS agar (TM Media).
Bacterial cultures of Salmonella typhimurium, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were obtained from Biotechnology Laboratory, Faculty of Agricultural Technology, Universitas Brawijaya, while Lactobacillus casei and Bacillus subtilis were obtained from Animal Product Technology Laboratory, Faculty of Animal Science, Universitas Brawijaya.
The equipment used in this research were a centrifuge (Corona 80-2), oven, waterbath shaker (Jisico), incubator, pH meter (Hanna), micropipette (Dragonlab), magnetic stirrer, and FoodScan™ NIR Spectrophotometer (FOSS).
Research Method
The research method used in this study was a laboratory experiment using a completely randomized design (CRD) with four treatments and five replications. The treatment in this study was the different ratios of the combination of papain and bromelain enzymes in the hydrolysis process of chicken head protein. The research design can be seen as follows:
A0 : Without enzyme addition
A1 : Hydrolysis using 75% papain + 25% bromelain A2 : Hydrolysis using 50% papain + 50% bromelain A3 : Hydrolysis using 25% papain + 75% bromelain Procedure for Making Chicken Head
Protein Concentrate
Chicken head proteins were extracted using the pH-shifting method as described by Al Awwaly et al. (2020) with slight modification. Fresh chicken heads were ground and dried using an oven at 40 0C for 6 hours. Dried chicken heads were then made into powder. Chicken head powder
was mixed and homogenized with deionized water (10% w/v). The pH of the homogenized sample was then adjusted to 12 using 10 M NaOH and stirred using a magnetic stirrer for 1 hour. The mixture was then centrifuged at 4.000 rpm for 15 min.
The supernatant was separated and adjusted pH to 4 using 1 M HCl, then centrifuged at 5.000 rpm for 15 min to recover chicken
head protein in pelleted material. The pellets were stored at -20 0C overnight and then dried using a microwave dryer on low mode (± 39 0C) for 5 min.
Procedure for Making Chicken Head Protein Hydrolysate
Chicken head protein was hydrolyzed using bromelain and papain separately. The hydrolysis process was done at optimum conditions for bromelain and papain (55 0C and pH 7). The ratio of both enzymes to chicken head protein concentrate was 1:100 (w/w).
The amount of each enzyme was adjusted to the percentage in each treatment as mentioned in the experimental design.
The percentage of enzyme in each treatment was multiplied by the total enzyme used in the experiment (e.g., if the enzyme used was 20 mg, then the papain and bromelain enzymes used in A1 were 15 and 5 mg, respectively). In the bromelain hydrolysis step, the chicken head protein concentrate was mixed with deionized water at a ratio of 2% (w/v) and adjusted pH to 7 using 2 M NaOH, then pre-incubated at 55 0C for 20 min.
Bromelain enzyme according to the treatment was added to the mixture and incubated for 3 hours at optimum conditions. The incubation process for the bromelain was stopped by heating the mixture at 85 0C for 10 min. After that, the mixture was pre-incubated for papain by readjusting pH to 7 using 2 M and carried out at 55 0C for 20 min. Papain enzyme according to the treatment was added to the mixture and incubated for 3 hours at optimum conditions. The incubation process for the bromelain was stopped by heating the mixture at 85 0C for 10 min. The mixture was then cooled at room temperature and
centrifuged at 4.000 rpm for 15 min. The supernatant was collected and stored at -20
0C (Yuan et al., 2021).
Chemical Compositions Analysis Procedure
Chemical compositions (protein, collagen, moisture, and fat content) of chicken head were determined using FoodScan™ NIR Spectrophotometer (FOSS) according to AOAC (2015).
Antibacterial Activity Test Procedure The Kirby-Bauer disc diffusion method was used to evaluate the antibacterial activity of chicken head protein hydrolysate against L. casei, E. coli, S.
aureus, S. typhimurium, P. aeruginosa, and B. subtilis. The procedure as described by Sukarno et al. (2023) with slight modification. L. casei, S. aureus, and B.
subtilis were chosen to observe the antibacterial activity of the hydrolysate against gram-positive bacteria, while S.
typhimurium, E. coli, and P. aeruginosa were used to represent the antibacterial activity of the hydrolysate against gram- negative bacteria. One ml of each culture was added to a petri dish separately, then poured 20 ml medium agar (SS agar for S.
typhimurium, EMB agar for E. coli, MRS agar L. casei, and NA for S. aureus, P.
aeruginosa, and B. subtilis) into each petri dish. After it solidifies, place the paper disc that has been soaked with the hydrolysate sample on the medium.
Agar was then incubated at 37 0C for 24 hours. After the incubating process, the inhibitory zone was measured using a caliper. The diameter of the paper disc used was 6 mm. The results were presented in mm and calculated using the following equation:
Inhibition zone diameter (mm)
= (B−A)−(C−A) 2
Where A (mm) is the diameter of paper disc; B (mm) is the diameter of vertical clear zone; and C (mm) is the diameter of horizontal clear zone.
Statistical Analysis
The data were analyzed using a one- way analysis of variance. Duncan’s multiple range test was used to determine a significant difference (P<0.05) or (P<0.01) among treatments. All the results were presented as the mean ± SD. Analysis of data was done using the Microsoft Excel Program.
RESULTS AND DISCUSSION
Chemical Composition of Chicken Head Table 1 showed that chicken heads had moisture as a major component with contents of 77.06% followed by protein, fat, and collagen with contents of 16.82, 8.50,
and 4.13%, respectively. Akimova et al.
(2023) reported the higher protein content in ground mass of chicken head and feet, namely 17.5%. The moisture content was reported to be similar, while the fat content was lower than in this study, namely 3.05%.
The collagen content in chicken heads was 4.13%. The most abundant protein fractions in poultry by-products are collagen and elastin (Akimova et al., 2023). The collagen content and homogeneous combination of skin and bone in chicken heads make it a good raw material for producing halal gelatin (Aidat et al., 2023).
Collagen from livestock also has the potential to produce antibacterial peptides (Lima et al. 2015).
Table 1. Chemical composition of chicken head
Chemical composition Mean (%) ± SD
Protein 16.82 ± 0.2121
Collagen 4.13 ± 0.0424
Moisture 77.06 ± 0.0778
Fat 8.50 ± 0.0849
Antibacterial Activity against L. casei L. casei is a bacteria that can be found in vegetables and fruits. This bacteria is also generally used as a fermentation starter and as a probiotic. These bacteria can adhere to
the intestinal mucosa and form colonies (Jung et al., 2021). The results for antibacterial activity against L. casei can be seen in Table 2.
Table 2. Antibacterial activity of chicken head protein hydrolysate against L. casei, E. coli, and S. typhimurium
*Different superscript letters in the same column show a highly significant difference (P<0.01)
Antibacterial Activity against S.
typhimurium
The results showed that different combinations of papain and bromelain in chicken head protein hydrolysis gave a significant difference (P<0.05) in antibacterial activity against S.
typhimurium. The highest and the lowest antibacterial activity was in A2 and A1, respectively. The inhibition zone formed is in the range 1.01-3.62 mm as can be seen in Table 2. Even though all samples showed an inhibition zone below 5 mm, the chicken
head protein hydrolysate sample still showed potential as an antibacterial agent.
The area of the inhibition zone in S.
typhimurium was almost the same as the desalted duck egg white hydrolysate that was hydrolyzed for 6 hours using the enzyme pepsin, namely 2.3-3.0 mm, reported by Thammasena and Liu (2020).
The mechanisms by which peptides act against microbes vary and may be distinct for different bacterial species. The initial stage in bacterial destruction involves the peptide attaching to bacterial
Treatment Inhibition Zone (mm)
L. casei E. coli S. typhimurium
A0 1.72 ± 0.4685a 1.48 ± 0.2515a 1.03 ± 0.0975a
A1 5.92 ± 0.5438b 4.47 ± 0.4698b 1.01 ± 0.1475a
A2 4.97 ± 0.8438b 3.65 ± 0.6892b 3.62 ± 0.8628b
A3 2.68 ± 0.3883a 1.19 ± 0.1851a 1.02 ± 0.1352a
membranes, exerting electrostatic forces.
Peptides can impact intracellular structures and processes, such as cell wall synthesis, DNA, RNA, and protein synthesis.
Additionally, the plasma membrane is affected, facilitating the entry of peptides into the bacterial cytoplasm (Roy et al., 2020).
Antibacterial Activity against S. aureus The difference in the treatment ratio of the combination of papain and bromelain enzymes gave no significant difference (P>0.05) in antibacterial activity against S.
aureus among all the hydrolysates. The results presented in Table 3 showed that the inhibition zone of all hydrolysates against S.
aureus was in the range 0.93-1.45 mm. It showed that the antibacterial activity against S. aureus in all samples was weak.
A study by Sukarno et al., (2023) showed that egg white protein hydrolysate at the concentration of 20 mg/mL from local duck, chicken breed, local chicken, and muscovy duck did not have antibacterial activity against S. aureus. Antibacterial activity could be detected at a concentration of 40 mg/mL.
The antimicrobial activity of a peptide is largely determined by its charge, molecular weight, secondary structure and amino acid sequence (Thammasena and Liu, 2020).
Table 3. Antibacterial activity of chicken head protein hydrolysate against S. aureus, P. aeruginosa, and B. subtilis
*Different superscript letters in the same column show a significant difference (P<0.05)
Different combination ratios of papain and bromelain enzymes gave a highly significant difference (P<0.01) on antibacterial activity from chicken head protein hydrolysate against L. casei. A1 exhibited the highest antibacterial activity against L. casei, while the lowest activity was shown by A0. Differences in antibacterial activity in each sample can be caused by the size of the peptides produced.
Simpler peptides may be able to contact target sites on the bacterial surface more easily (Abd Rashid et al., 2022). The antibacterial activity of a peptide can come from interactions between the peptide and the membrane formed, resulting in membrane damage. Peptide-membrane interactions result in pore formation, cell lysis, and transfer of peptides into the cytoplasm, these are the things that result in membrane damage in microorganisms (Moghaddam et al., 2015).
Antibacterial Activity against E. coli The results showed that different combinations of papain and bromelain in
chicken head protein hydrolysis gave a highly significant difference (P<0.01) in antibacterial activity against E. coli. The results of the antibacterial activity of all hydrolysates against E. coli can be seen in Table 2. The highest and the lowest antibacterial activity were showed by A1 and A3, respectively. All the hydrolysates exhibited weak antibacterial activity against E. coli. The diameter of the inhibition zone
≤ 5 mm can be categorized as weak antibacterial activity (Malinggas et al., 2015). The strength of the antibacterial activity of protein hydrolysate is due to the specific peptides contained in it. The specific amino acid sequence of the peptide in the protein hydrolysate contributes to its antibacterial properties (Raharjo et al., 2021).
E. coli is facultative anaerobic and gram-negative bacteria (Li et al., 2021).
Peptides that have cationic amino acids in their sequences will have higher antibacterial activity (Ulagesan et al., 2018).
Antimicrobial peptides also typically have a small excess of lysine, arginine, and
Treatment Inhibition Zone (mm)
S. aureus P. aeruginosa B. subtilis
A0 1.21 ± 0.3959 1.64 ± 0.6077a -
A1 1.45 ± 0.2725 1.83 ± 0.3633ab -
A2 1.00 ± 0.1837 2.28 ± 0.3328b -
A3 0.93 ± 0.3074 2.46 ± 0.3975b -
histidine residues. The cationic nature of antimicrobial peptides has an important role in the adsorption process onto the surface of bacterial cells (Santos et al., 2018). In chicken head protein hydrolysate, peptides containing lysine and arginine residues may originate from hydrolysis carried out by papain, because this enzyme cleaves these two residues. Hakim et al. (2023) reported that bioactive peptides from chicken feet showed antibacterial activity against E. coli through in vivo experiments. Increasing the concentration of bioactive peptides in feed showed a reduction in the total of E. coli colonies in the intestine of chickens.
Bioactive peptides can destroy bacterial cell membranes, thereby inhibiting cell wall synthesis. Weak cell walls in bacteria will trigger lysis which results in the death of the bacteria.
Antibacterial Activity against P.
aeruginosa
P. aeruginosa is a gram-negative bacteria. This bacterial species is a pathogenic bacteria that causes infections of the respiratory tract and urinary system (Qin et al., 2021). The difference in the treatment ratio of the combination of papain and bromelain enzymes gave a significant difference (P<0.05) in antibacterial activity against P. aeruginosa among all the hydrolysates. The inhibition zone of all samples was 1.64-2.46 mm as can be seen in Table 3. A3 exhibited the highest antibacterial activity, while the lowest antibacterial activity was shown by A0.
Increasing the concentration of the bromelain enzyme used in chicken head protein hydrolysate from A0 to A3 showed a positive correlation with increasing the zone of inhibition in P. aeruginosa. The protein hydrolysis process using a suitable protease enzyme can produce antimicrobial peptides. Antimicrobial peptides designed for targeting gram-negative bacteria such as P. aeruginosa need to attach to the lipopolysaccharide present on the outer membranes. Subsequently, they must traverse the cytoplasmic membrane to
initiate the breakdown of the membrane structure.
These peptides generally possess a combination of positive charges and hydrophobic properties (Tian et al., 2022).
Hydrophobic peptides in chicken head protein hydrolysate can be obtained from hydrolysis carried out by bromelain.
Bromelain has cleavage sites on alanine, glycine, and leucine residues which are hydrophobic amino acids (Colletti et al., 2021). This may explain why increasing bromelain concentrations from A0 to A3 can increase antibacterial activity against P.
aeruginosa.
Antibacterial Activity against B. Subtilis All the hydrolysates didn’t exhibit an antibacterial activity against B. subtilis. The possible reason why chicken head protein hydrolysate can’t inhibit B. subtilis is the existence of a bacterial resistance mechanism. Similar to other bacteria, B.
subtilis has diverse resistance mechanisms, such as generating proteases and forming biofilms, that can diminish susceptibility to antimicrobial peptides (Henriques et al., 2020).
CONCLUSION
Chicken head protein hydrolysate using a combination of papain and bromelain enzymes showed antibacterial activity against all bacteria tested, except B.
subtilis. The inhibition zones of chicken head protein hydrolysate using a combination of papain enzymes against Lactobacillus casei, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella typhimurium were 1.72-2.68, 1.19-4.47, 0.93-1.45, 1.64- 2.46, and 1.01-3.62 mm, respectively. A1 exhibited the highest antibacterial activity against L. casei, E. coli, and S. aureus, while A2 exhibited the highest activity against S.
typhimurium.
At the same time, A3 possessed the strongest antibacterial activity against P.
aeruginosa. This shows that chicken head
protein hydrolysate produced using a combination of papain and bromelain has potential as an antibacterial agent.
CONFLICT OF INTEREST
The authors declare no potential conflict of interest in this research.
ACKNOWLEDGEMENT
The authors would like to express their sincere gratitude to Prof.Dr.Ir. Lilik Eka Radiati, MS., IPU for generously funding and providing facilities for the research and publication of this scientific journal. Support and encouragement by professor have been invaluable throughout this endeavor.
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