Jurnal Ilmu dan Teknologi Hasil Ternak, March 2024 Vol. 19 No.

1
ISSN : 1978 – 0303 DOI : 10.21776/ub.jitek.2023.018.03.

Enhancement of Antibacterial Activity from Chicken Head Protein

Hydrolysate Using Dual-Enzyme Hydrolysis
Pramudya Andiana1), Moch. Geerhan Miraja Syahdan1), Arif Hendra Utama1), Kasri2), 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: manabfpt@ub.ac.id

Submitted 22 November 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

 Jurnal Ilmu dan Teknologi Hasil Ternak, March 2024
ISSN : 1978 – 0303
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
*Corresponding author:
Abdul Manab
Email: manabfpt@ub.ac.id
Animal Product Technology Department, Faculty
of Animal Science, Universitas Brawijaya, Jl.
Veteran, Malang, East Java, 65145, Indonesia
Vol. 19 No. 1
DOI : 10.21776/ub.jitek.2023.018.03.
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
How to cite:
Andiana, P., Syahdan, M. G. M., Utama, A. H.,
Kasri, 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), -
 Jurnal Ilmu dan Teknologi Hasil Ternak, March 2024
ISSN : 1978 – 0303
Cryptozona bistrialis showed antibacterial
activity against Pseudomonas aeruginosa
and 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,
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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
 Jurnal Ilmu dan Teknologi Hasil Ternak, March 2024
ISSN : 1978 – 0303
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).
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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.
 Jurnal Ilmu dan Teknologi Hasil Ternak, March 2024
ISSN : 1978 – 0303
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,
Table 1. Chemical composition of chicken head
Chemical composition
Protein
Collagen
Moisture
Fat
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.
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
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
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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).
Mean (%) ± SD
16.82 ± 0.2121
4.13 ± 0.0424
77.06 ± 0.0778
8.50 ± 0.0849
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).
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,
 Jurnal Ilmu dan Teknologi Hasil Ternak, March 2024
ISSN : 1978 – 0303
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 histidine residues. The
cationic nature of antimicrobial peptides
has an important role in the adsorption
process onto the surface of bacterial cells
Table 2. Antibacterial activity of chicken head protein hydrolysate against L. casei, E. coli, and S. typhimurium
Inhibition Zone (mm)
Treatment
L. casei
A0 1.72 ± 0.4685a
A1 5.92 ± 0.5438b
b b
A2 4.97 ± 0.8438
A3 2.68 ± 0.3883a
*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
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(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.
E. coli S. typhimurium
1.48 ± 0.2515a 1.03 ± 0.0975a
4.47 ± 0.4698b 1.01 ± 0.1475a
3.65 ± 0.6892 3.62 ± 0.8628b
1.19 ± 0.1851a 1.02 ± 0.1352a
against microbes vary and may be distinct
for different bacterial species. The initial
stage in bacterial destruction involves the
peptide attaching to bacterial 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
 Jurnal Ilmu dan Teknologi Hasil Ternak, March 2024
ISSN : 1978 – 0303
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.
Table 3. Antibacterial activity of chicken head protein hydrolysate against S. aureus, P. aeruginosa, and B. subtilis
Inhibition Zone (mm)
Treatment
S. aureus
A0 1.21 ± 0.3959
A1 1.45 ± 0.2725
A2 1.00 ± 0.1837
A3 0.93 ± 0.3074
*Different superscript letters in the same column show a significant difference (P<0.05)
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
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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).
P. aeruginosa B. subtilis
1.64 ± 0.6077a -
1.83 ± 0.3633ab -
2.28 ± 0.3328b -
2.46 ± 0.3975b -
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
 Jurnal Ilmu dan Teknologi Hasil Ternak, March 2024
ISSN : 1978 – 0303
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.
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