Antibacterial Activity and RP-HPLC Characteristic of Lysozyme

from Local Chicken Egg White after Modification Treatments

Syahrizal Nasution

, Didah Nur Faridah

, Eni Kusumaningtyas

, Zakiah Wulandari

and Harsi Dewantari Kusumaningrum

Study Program of Food Science, Graduate School, IPB University, Bogor 16680, Indonesia

Department of Food Science and Technology, Faculty of Agricultural Technology, IPB University, Bogor 16680, Indonesia

Indonesian Research Center for Veterinary Sciences, Bogor 16114, Indonesia

Department of Animal Production and Technology, Faculty of Animal Science, IPB University, Bogor 16680, Indonesia

Keywords: Antibacterial, Food Safety, Lysozyme, MIC, RP-HPLC.

Abstract: Lysozyme is a globular protein and is a hydrolase enzyme. Lysozyme isolated from chicken egg whites can

damage the membrane of bacteria hence it can be used as an antibacterial. The purpose of the study was to

modify, to measure antibacterial activity, and to characterize the lysozyme from local chicken egg whites.

The isolate of lysozyme was modified by heat modification treatments at 60°C, 75°C, and 90°C in pH seven

buffer solution. The antibacterial activity of lysozyme was measured by the micro-dilution method. The

isolate and the modified lysozyme were characterized by reversed phase-high performance liquid

chromatography (RP-HPLC). The results showed that heat modification treatments decreased the minimum

inhibitory concentration of lysozymes to Gram-positive bacteria up to 2-fold from 6 mg/mL to 3 mg/mL. Heat

modification also improved antibacterial spectrum of lysozyme against Gram-negative bacteria. The RP-

HPLC chromatograms of modified lysozyme showed the peak of lysozyme at 43.59 ± 0.09 minutes retained

time had decreased. It was suggested that any concentration of lysozyme was retained on the different retained

times doe to the modification of lysozyme. The characteristic of RP-HPLC had explained the reason for

increasing the antibacterial activities of lysozyme after modification treatments.

1 INTRODUCTION

Lysozyme is a hydrolase enzyme in the form of

antibacterial protein that can be isolated from the

chicken egg white (Gyawali and Ibrahim 2014). The

chicken egg white has excellent potential as a source

of lysozyme because it contains about 2500-3000

µg/mL of lysozyme and can be obtained from some

of the industrial waste (Lesnierowski and Kijowski

2007). Lysozyme can be used directly, combined

with antimicrobials on food, also added to edible

packaging so that it can improve food safety

(Kijowski et al. 2002; Herath et al. 2015).

The food industry had been using lysozyme

because it has a stable primary structure at heat and

low pH treatments. The mechanism of antibacterial

activity of lysozyme occurs through peptidoglycan

destruction on bacterial cell wall membrane by active

site of the lysozyme, which breaks the glycosidic

bond between N-acetylglucosamine and N-

acetylmuramic acid (Susanto et al. 2013).

The antibacterial activity of isolate lysozyme in

native form is still limited to Gram-positive bacteria,

whereas Gram-negative bacteria could be food

contaminants (Lesnierowski et al. 2001). Therefore,

it is necessary to modify lysozyme to improve its

antibacterial activity against Gram-negative bacteria

that are more resistant because the bacterial cell wall

is coated by lipopolysaccharide (Cegielska-

Radziejewska et al. 2008; Susanto et al. 2013). One

of simple methods that used to modify lysozyme was

heat treatments (Nasution et al. 2018). Besides, after

heat modification treatments, the characterization of

lysozyme by RP-HPLC used to explained the

changed of antibacterial activity of modified

lysozyme that have never been reported.

2 MATERIALS AND METHODS

2.1 Lysozyme

Respectively, a local and commercial isolate of

lysozyme were obtained from Sentul chicken eggs at

Nasution, S., Faridah, D., Kusumaningtyas, E., Wulandari, Z. and Kusumaningrum, H.

Antibacterial Activity and RP-HPLC Characteristic of Lysozyme from Local Chicken Egg White after Modiﬁcation Treatments.

DOI: 10.5220/0009979300002833

In Proceedings of the 2nd SEAFAST International Seminar (2nd SIS 2019) - Facing Future Challenges: Sustainable Food Safety, Quality and Nutrition, pages 84-88

ISBN: 978-989-758-466-4

the Research Institute for Animal Production

(Balitnak, Ciawi-Bogor) and commercial chicken

eggs wholesaler (Kurnia Jaya, Dramaga-Bogor).

Both of lysozyme was isolated with Amberlite

FPC3500 resin. The standard lysozyme (L-6876,

from chicken egg white, 47,000 unit/mg solid) was

obtained from Sigma-Aldrich. The lysozyme sample

was prepared in 10 mL of 10 mM potassium

phosphate buffer pH seven.

2.2 Modification of Lysozyme

The lysozyme was modified by heat treatment

(Carrillo et al. 2018). As much as 6 mg/mL of each

isolate lysozyme in 10 mL of 10 mM potassium

phosphate buffer pH seven was heated in a water bath

in each heat temperature of treatments (60°C, 75°C,

and 90°C) for 20 minutes. The solution was placed in

the refrigerator (4°C) after having heated.

2.3 Measurement of Antibacterial

Activity

Antibacterial activity of lysozyme was measured

based on the micro-dilution method concerning on

Clinical and Laboratory Standards Institute (CLSI)

(Balouiri et al. 2016). As much as 50 μL MHB was

put into each 96-well microplate. Then, 50 μL from 6

mg/mL of each isolate lysozyme or modified as initial

lysozyme was added. Afterward, the initial lysozyme

was diluted four times to the Mueller Hinton Broth

(MHB) until it reached 0.38 mg/mL of lysozyme

dilution concentration. Before having incubated, it

was added 50 μL tested bacteria culture having

prepared to each well. The bacteria stock was made

in sideways MHA media and incubated at 37°C as

long as 24 hours in MHB when it will be utilized.

Control as much as 150 μL used was put in separated

well as MHB media, tested bacteria, and standard

lysozyme solution. Then, a 96-well microplate in

covered condition was incubated (37°C) for 24 hours

before the MIC of lysozyme being evaluated.

The MIC lysozyme was considered as a

concentration that triggers the viewless growth of

bacteria in well after growing as long as 24 hours (not

turbid and no precipitate). Determining MIC, it was

confirmed with a microplate reader at 655 nm. The

minimum bactericidal concentration (MBC) was

obtained by scratching the MIC result to the MHA in

the dish. The not turbid mixture in the well was taken

and scratched to MHA in the dish, then incubated

(37°C) for 24 hours. MBC of lysozyme was

considered as a concentration having no growth of

bacteria in media of each scratch.

2.4 The RP-HPLC Characterization

The lysozyme was characterized by RP-HPLC

(Carrillo et al. 2014 and Kusumaningtyas et al. 2015).

The sample of the isolate or the modified of lysozyme

was injected as much as ten μL utilizing Colom C-18,

possessing reverse-phase high-performance liquid

chromatography (RP-HPLC) system in 215 nm

wavelength for 50 minutes (1 mL/minutes speed at

29.3°C). Before being injected, the sample was

refined with nylon membrane 0.45 μm. Dissolver A

contains 0.37% trifluoroacetic acid in double distilled

water, while dissolver B consists of 0.27 %

trifluoroacetic acid in acetonitrile grade HPLC. The

HPLC system was balanced with 95% solution A (5

minutes), followed by gradient 5-45% of solution A

(5 minutes). The concentration of isolate lysozyme

was calculated by comparing the peak of local isolate

lysozyme to the peak of standard lysozyme.

2.5 Data Analysis

The data was portrayed as the average result ±

standard deviation and also analyzed with Microsoft

Excel, XLSTAT, and SPSS. The result of the analysis

was used to compare the significance of the data.

3 RESULTS AND DISCUSIONS

3.1 Antibacterial Activity of Lysozyme

The research on improving the antibacterial activity

of lysozyme by modification of heat on 60°C, 75°C,

and 90°C through measurements of MIC value of

lysozyme from the local chicken have never been

published ultimately. The MIC value was the

minimum inhibitory concentration of lysozyme

against bacteria or a concentration that does not show

bacterial growth in the well (not cloudy and no

deposits) after 24 hours of incubation. The results

showed that the MIC values of isolate lysozyme were

the same as standard lysozyme in all four bacteria. All

of modified lysozyme from local chicken could

inhibit all bacteria from Gram-positive and Gram-

negative but have slightly different in MIC values.

The MIC values of isolate and modified lysozyme

from local chicken were 3 mg/mL and 6 mg/mL,

which were the concentrations of 50% and 100% of

the solution of lysozyme tested (Table 1).

Antibacterial Activity and RP-HPLC Characteristic of Lysozyme from Local Chicken Egg White after Modiﬁcation Treatments

Table 1: The minimum inhibitory concentration of lysozyme towards Gram-positive and Gram-negative bacteria.

Lysozyme types and treatments

modifications

The minimum inhibitory concentration of lysozyme (mg/mL)

Gram-positive bacteria Gram-negative bacteria

Staphylococcus

aureus ATCC

25923

Bacillus

cereus ATCC

10876

Escherichia

coli ATCC

25922

Salmonella

Typhimurium

ATCC 14028

Standard lysozyme (7) 6

˃ 6

Before modification treatments

Isolate lysozyme (native) (7) 6

˃ 6

After heat modification treatments

Heat-modified lysozyme 60°C (7) 6

Heat-modified lysozyme 75°C (7) 3

Heat-modified lysozyme 90°C (7) 3

Numbers followed by the same letter were not significantly different (p>0.05) on each bacteria

The improvement of antibacterial activity of

lysozyme from local chicken egg white by heat

modification at pH seven towards Gram-positive and

Gram-negative bacteria can be seen in Table 1. The

heat-modified lysozyme at 75°C and 90°C, when

compared to standard and isolate lysozyme, can

decrease MIC of lysozyme from local chicken egg

white against S. aureus ATCC 25923, and lysozyme

modified by 90°C also decreased MIC of lysozyme

against B. cereus ATCC 10876, whereas at all heat-

modified temperatures, also decreased MIC of

lysozyme against E. coli ATCC 25922 and S.

Typhimurium ATCC 14028.

The MIC values of lysozyme in Gram-negative

bacteria were still higher than in Gram-positive

bacteria. This condition showed that the antibacterial

activity of isolate and modified lysozyme was higher

in Gram-positive bacteria than Gram-negative

bacteria, which were more resistant due to the

presence of lipopolysaccharide as protection

(Lesnierowski et al. 2001). The results f the

improvement of antibacterial activity were in

accordance with other research (Carrillo et al. 2014),

which showed an increasing in the antibacterial

spectrum.

The difference of MIC values at each heat

modification at 60°C, 75°C, and 90°C for the two

Gram-positive bacteria tested was suggested to be

due to changes different in the lysozyme sample. The

improvement of antibacterial activity by heat

modification occurs because it was triggered by

changes in the conformation of lysozyme molecules

from the monomers form to dimers or polymers

(Lesnierowski et al. 2001; 2004; Carrillo et al. 2014;

Vilcacundo et al. 2018). The research conducted by

Cegielska-Radziejewska et al. (2008) reported that

dimer forms in heat-modified lysozyme make the

lysozyme easily attach to lipopolysaccharide in the

bacterial membrane of Gram-negative cell due to

changes in the composition and hence increased the

hydrophobicity of the outside part of lysozyme

molecule. The lysozyme attaches membrane will

disrupt the electrochemical process and the stability

of the lipid bilayer so that it can form holes in the

bacterial membrane. Heat modification can increase

the antibacterial activity against Gram-negative

bacteria through opening the folds of the lysozyme

and expanding the lysozyme bonds in the membrane.

This condition was explained by Ibrahim et al.

1996 that the globular structure of lysozyme has been

dominanted by hydrophobic amino acids inside the

molecule and hydrophilic amino acids outside the

molecule. Heat modification causes the denaturation

process of the globular structure of lysozyme doe to

the two cysteine (Cys) as lysozyme bridges were cut

off so that the hydrophobicity of lysozyme increases.

Breaking disulfide (Cys64-Cys80 bond) and (Cys76-

Cys94 bond) by heat modification causes the

tryptophan 62, 63, and 108 (hydrophobic) in the

globular structure of lysozyme to be exposed and will

come to outside and then contact with bacterial

membranes. The denaturation process causes some or

all of the globular structure of lysozyme to change the

tertiary and secondary lysozyme structures into

primary structures. Heat modification will cause the

hydrophobicity of the outside part of the lysozyme

molecule, and the attach ability of lysozyme to the

bacterial membrane increased. This condition was the

reason for the improvement of the antibacterial

activity of lysozyme against Gram-negative bacteria

and still maintains against Gram-positive bacteria.

2nd SIS 2019 - SEAFAST International Seminar

3.2 RP-HPLC Characteristic of

Lysozyme

The characterization of isolate and modified

lysozyme by RP-HPLC method aims to see the

effect of heat modification treatments on the

lysozyme structure from local chicken egg white

based on the tertiary structure of lysozyme as

globular protein and its relation to the antibacterial

activity of isolate and modified of lysozyme. The

results showed that the lysozyme was detected on

43.59 ± 0.09 minutes retained time close to

researched by Carrillo et al. (2016) that detected the

lysozyme on 38 minutes retained time.

Table 2: The concentration of lysozyme.

Lysozyme types

Concentration

(mg/g)

Before heat modification treatments

Isolated lysozyme 41.07 ± 4.44

After heat modification treatments

Heat-modified lysozyme 60°C 38.87 ± 4.20

Heat-modified lysozyme 75°C 8.53 ± 0.92

Heat-modified lysozyme 90°C 0.99 ± 0.11

Numbers followed by the same letter were not significantly

different (p>0.05) among the isolate and modified

lysozyme.

The modification decreased the concentration of

lysozyme as globular proteins detected in lysozyme

from local chicken egg white, which was calculated

based on comparison with standard lysozyme

(Table 2). The reduction of isolated lysozyme

concentration detected at 215 nm wavelength

indicates that isolated lysozyme from local chicken

egg white was not as same as the concentration of

standard lysozyme. The increasing of heat

temperature makes lowering the concentration of

lysozyme. The highest concentration of isolate

lysozyme was found in lysozyme without heat

modification treatments compare to heat-modified

lysozymes.

The reduction of concentration was suggested

because by the structure of lysozyme from local

chicken egg white underwent reactions in running

process through the RP-HPLC column such as

denaturation due to the modifications that caused

changes the lysozyme so that only some

concentration of lysozyme was detected at 43.59 ±

0.09 minutes retained time as same with the

standard lysozyme. Some concentration of

lysozyme from local chicken egg white was thought

to undergo dimerization at an oven temperature of

29.3°C during running RP-HPLC because dimer

conformation can also be formed between 20°C to

30°C as the reversible character of lysozyme at

(Cegielska-Radziejewska et al. 2008).

Based on Table 2, although the detected

lysozyme concentration was reduced, it is evident

in the lysozyme sample that there were other

proteins of modified lysozyme, which were the

reason for the increasing of antibacterial activity of

lysozyme after modification. That protein might be

detected at other retention times. Research

Cosentino et al. (2015) showed that the

concentration of lysozyme in the type of milk

detected by the HPLC method still had the same

concentration after pasteurization (63° C) for 30

minutes. This condition showed that although the

modification of lysozyme heat in this study caused

the lysozyme concentration to decrease, the

concentration of lysozyme contained in the food

system can survive after heat treatment.

Figure 1: The profile of RP-HPLC chromatograms of lysozyme.

Antibacterial Activity and RP-HPLC Characteristic of Lysozyme from Local Chicken Egg White after Modiﬁcation Treatments

The heat modification to the lysozyme showed

that decreasing the peak of lysozyme on (Figure 2).

The increasing of heat temperature makes the peak of

lysozyme to be lower. This condition showed that the

structure of lysozyme was changed that changing the

composition of amino acid of lysozyme from some of

the concentrations of lysozymes. So in the highest

heat temperature of modification treatments made the

peak of lysozyme was the lowest among others.

Besides, column C-18 in RP-HPLC will attach the

modified lysozyme based on the hydrophobicity of

modified lysozymes. This condition showed that the

heat modification treatments can decrease the

concentration of lysozyme from the local chicken egg

white and change the hydrophobicity of modified

lysozymes.

4 CONCLUSIONS

The lysozyme from local chicken egg white was

modified by heat modification treatments. The heat

modification treatments improved antibacterial

activity of lysozyme by decreasing the MIC value.

Otherwise, the isolate and the modified lysozyme

were characterized by RP-HPLC, and the RP-HPLC

chromatograms explaining the reason for the

increasing of antibacterial activity of lysozyme after

modification treatments.

ACKNOWLEDGEMENTS

This work was funded by Direktorat Riset and

Pengabdian Masyarakat, Ditjen Penguatan Riset,

dan Pengembangan, Kementerian Riset, Teknologi

and Pendidikan Tinggi (142/SP2H/LT/DRPM/

2018). Authors thank the Indonesian Research

Institute for Animal Production (Balitnak Ciawi-

Bogor) that providing Sentul chicken eggs.

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2nd SIS 2019 - SEAFAST International Seminar