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Can Tho University Journal of Science Vol. 13, No. 2 (2021): 52-60
52
DOI: 10.22144/ctu.jen.2021.030
Investigation of the antibacterial activity against Cutibacterium acnes of lysozyme
purified from “Co Co” duck egg whites
Tran Khoa Nguyen and Vo Van Song Toan*
Biotechnology Research and Development Institute, Can Tho University, Viet Nam
*Correspondence: Vo Van Song Toan (email: vvstoan@ctu.edu.vn)
Article info.
ABSTRACT
Received 01 Mar 2021
Revised 22 May 2021
Accepted 19 Jul 2021
Lysozyme has been applied in various fields such as food technology,
medicine, and diagnostics because it can resist many types of bacteria. In
this research, lysozyme from duck egg whites was studied to evaluate the
antibacterial activity against Cutibacterium acnes (C. acnes) often caus-
ing acne on human skin. Lysozyme was purified from duck egg whites by
ion-exchange chromatography and gel-filtration chromatography. After
that, this enzyme was used to investigate the resistance to C. acnes at dif-
ferent pH, temperature, concentration, and storage conditions. The re-
sults presented that lysozyme exhibited the best resistance to C. acnes at
pH 6.0 and 6.5 on trypticase - yeast extract - heart extract - glycerol agar
(TYEG) medium, at 30°C and 35°C. Additionally, these conditions had
the least effect on lysozyme antibacterial activity. The minimal inhibitory
concentration (MIC80) and minimum bactericidal concentration (MBC) of
lysozyme to C. acnes were 0.55 mg/mL and 1.11 mg/mL, respectively.
Lysozyme could keep up the best antimicrobial activity when stored at -
20oC and -10oC; After 30 days, it still kept nearly 80% of its activity.
These findings will offer a basis for larger-scale production of lysozyme
powder for further research and commercial purposes, especially skin-
care products.
Keywords
Antibacterial activity, Cuti-
bacterium acnes, lysozyme,
storage, “Co Co” duck
1. INTRODUCTION
Lysozyme, also known as N-acetylmuramide glu-
canhydrolase, is an antibacterial enzyme that can
hydrolyze the β (1,4)-glycosidic bonds between N-
acetylmuramic acid (NAM) and N-
acetylglucosamine (NAG) of the peptidoglycan
layer in the bacterial cell walls (especially Gram-
positive bacteria) thereby destroying the cell wall
of bacteria (Cotterill, 1954). Lysozyme is consid-
ered as one of the enzymes having diverse applica-
tions in the food industry, medicine, and diagnos-
tics. Although lysozyme can be found in different
sources such as milk, saliva, tears, urine, stomach
mucus, or egg whites (Jollès et al., 1990; Maroni &
Cuccuri, 2001), lysozyme-powder in Viet Nam has
been mainly imported from abroad at very high
prices.
“Co Co” duck is an egg-laying breed imported
from Zhejiang, China through the quota. It is the
second-largest duck-head breed in Viet Nam after
Tau duck. “Co Co” duck has an early laying age,
about 90 - 120 days, the average egg yield per year
reaches 260 - 300 eggs, higher than both grass
duck and Khaki Campbell duck (Nguyen Duc
Trong et al., 2006). “Co Co” duck eggs are rich
sources of proteins as well as many essential amino
acids, fats, and minerals necessary for the body.
The protein content of a fresh duck egg is 11.8%,
Can Tho University Journal of Science Vol. 13, No. 2 (2021): 52-60
53
of which lysozyme accounts for 3.5% of the 11
proteins in egg whites (Kovacs-Nolan et al., 2005).
Many previous publications have figured out that
the antibacterial ability of lysozyme was markedly
affected by temperature and pH conditions of the
agar medium or buffer solution. Lysozyme is a
heat-resistant enzyme, with a denaturing tempera-
ture up to 81.5oC (Campbell et al., 2003). Majid
(2015) also performed research on lysozyme ex-
tracted from cow's milk and indicated that lyso-
zyme at 37oC gave the best anti-Micrococcus lute-
us activity. Lysozyme is acid-soluble and thermally
stable in a neutral medium because it can persist at
100°C for 30 minutes and loses its activity at pH
values higher than 7.0. Besides, many studies have
been conducted to determine the minimal inhibito-
ry concentration (MIC) and minimum bactericidal
concentration (MBC) values of lysozyme extracted
from different sources for the bacteria that cause
foodborne-diseases such as Escherichia coli and
Clostridium botulinum or acne and skin inflamma-
tion such as Staphylococcus aureus, Staphylococ-
cus epidermis. However, the information on MIC
and MBC values of lysozyme for C. acnes have not
been published yet.
Therefore, the study was conducted to take ad-
vantage of cheap and plentiful duck eggs in Viet
Nam to obtain lysozyme high values, serving in
research and human needs in many areas of life.
2. MATERIALS AND METHODS
2.1. Materials
“Co Co” duck eggs were purchased in Hau Giang
province, Viet Nam. SP-Streamline and Sephadex
G-50 resins were bought from Pharmacia Fine
Chemicals (Sweden). Bovine Serum Albumin and
Coomassie Brilliant Blue G-250 were obtained
from Merck (Germany). The C. acnes ATCC
11827 strain was purchased from Microbiologics
company (USA). Micrococcus lysodeikticus ATCC
4698 and commercial lysozyme were obtained
from Sigma-Aldrich (USA). Other reagents were of
analytical grade from local sources.
2.2. Preparation of crude protein extract and
lysozyme from duck egg whites
The procedure was implemented based on the re-
search by Vo Thi Truc Ngan (2018) with small
modifications. Duck egg whites was diluted with
0.1 M sodium phosphate buffer at pH 7.0 (with the
ratio of 1 egg whites:2 buffer solution) for crude
sample extraction. The filtrate was centrifuged at
5,000 rpm for 10 minutes at 4°C to remove resi-
dues. The supernatant was continuously treated
with Streamline-SP and Sephadex G-50 resins for
lysozyme purification. The absorbance measure-
ment at 280 nm (A280) was conducted with Hitachi
U-1500 spectrophotometer (Japan) and 100-QS
quartz cuvette (10 mm) (Hellma Analytics, Germa-
ny) to identify target protein fractions.
The lysozyme solution obtained from the purifica-
tion process was immobilized with maltodextrin in
the form of physical adsorption and drying. The
lysozyme powder was store at -20oC and dissolved
in buffer solution before using in the experiments.
2.3. Investigation of the impact of the external
medium pH on the antibacterial effect of
duck egg-white lysozyme against C. acnes
2.3.1. Impact of the TYEG medium pH on the
antibacterial effect of duck egg-white
lysozyme against C. acnes using the agar
diffusion method
Bacterial colonies were suspended in 0.9% saline
solution and then the bacterial suspension was ad-
justed to a density equipvalent to 106 cells/mL. For
C. acnes, with an absorbance of 0.132 at 600 nm,
the bacterial suspension is equipvalent to 108
cells/mL (Crane et al., 2013), 106 cells/mL in den-
sity can be obtained with 100 times diluting. The
pH values of the TYEG medium (6.0, 6.5, 7.0 and
7.5) were prepared using NaH2PO4 and Na2HPO4.
A volume of 30 µL of the bacterial suspension was
inoculated onto the agar plates, then a 6-mm-in-
diameter glass punch was used to make wells on
the agar surfaces. Next, 20 µL of lysozyme solu-
tion was pipetted into the wells above (negative
control was lysozyme inactivated at 100oC, posi-
tive control was commercial lysozyme). All the
agar plates were then incubated at 35°C and meas-
ured the diameter of inhibition zones after 14
hours.
2.3.2. Impact of liquid medium pH on the change
of antibacterial activity of duck egg-white
lysozyme
Micrococcus lysodeikticus powder was dissolved in
0.06 M potassium phosphate solution at different
pH values and the bacteria suspension was then
measured spectroscopically at 450 nm to adjust the
absorbance values in the range of 0.6 - 0.7. The
buffer solutions at investigated pH values (6.0, 6.5,
7.0 and 7.5) were prepared using KH2PO4 and
K2HPO4 at different ratios. 100 µl of lysozyme
Can Tho University Journal of Science Vol. 13, No. 2 (2021): 52-60
54
sample was added to a cuvette containing 2.5 mL
of the bacterial suspension of pH to be tested. A 2-
minutes-decrease in an absorbance value at 450 nm
using 10 mm glass cuvette (Hellma Analytics,
Germany) was recorded to calculate the lysozyme
activity presenting in the lysis reaction.
2.4. Investigation of the impact of the external
medium temperature on the antibacterial
effect of duck egg-white lysozyme against
C. acnes
2.4.1. Impact of the TYEG medium temperature on
the antibacterial effect of duck egg-white
lysozyme against C. acnes
The procedure was conducted similarly to the pre-
vious experiments that followed Kirby’s method
(1956) on TYEG medium with the pH was chosen
from the previous experiment. All the plates were
incubated at 25oC, 30oC, 35oC, and 40oC. The di-
ameter values of inhibition zones were recorded
after 14 hours.
2.4.2. Impact of liquid medium temperature on the
change of antibacterial activity of duck egg-
white lysozyme
The experimental design was implemented based
on the research by Miyazaki (1998) with small
modifications. The suspension of Micrococcus
lysodeikticus was prepared as described in 2.3.2
section. pH value of the potassium phosphate solu-
tion used to dissolve the bacteria powder was cho-
sen from the result of the previous experiment.
A volume of 100 µl of lysozyme sample was added
to a cuvette containing 2.5 mL of the bacterial sus-
pension. Record the absorbance value at 0 min.
Then incubate the mixture of bacteria and enzyme
solutions at the temperatures to be examined (25oC,
30oC, 35oC, and 40oC). After 90 minutes of incuba-
tion, re-measure the absorbance value and record
the results to calculate the percentage of remaining
enzyme activity compared to the initial one.
2.5. Determination of the MIC and MBC values
of the duck egg-white lysozyme for C. acnes
Broth microdilution and colonies forming units
counting assay (Somasegaran & Hoben, 1994)
were carried out for MIC and MBC determination.
A volume of 100 µL of bacterial suspension (106
cells/mL) was added to 900 µL of trypticase - yeast
extract - heart extract - glycerol broth medium. 50
µL of this bacterial solution was respectively trans-
ferred into wells of a 96-well microplate, which
was then mixed with 50 µL of lysozyme in increas-
ing concentration as well as control samples. The
plate was incubated at 35oC for 14 hours. Bacterial
counting was carried out by serially diluting the
samples in the 96-well microplate with 0.9% saline
solution to give a bacterial suspension within the
countable range on agar plates. The inhibition level
of lysozyme to C. acnes growth was calculated
from the bacterial density in the sample wells and
in the negative control after the incubation period.
2.6. Impact of storage conditions on the
antibacterial activity of duck egg-white
lysozyme
The experiment was a two-factor completely ran-
domized design, including temperature (-20oC, -10
oC, 4oC, and 25oC) and time (1, 15, 30, and 60
days). Lysozyme powder samples after being im-
mobilized were stored at the storage temperatures
above and determined the antibacterial activity
after each timeline required to be examined. The
results of this experiment were also evaluated
based on the diameter of C. acnes inhibition zones
(Kirby et al., 1956) and the lytic action of lyso-
zyme in potassium phosphate buffer solution
(Shugar, 1952). The remaining lysozyme activity
percentage was calculated based on its lytic activity
at the time to be tested versus initially.
2.7. Statistical analysis
The data were entered, stored, and processed using
Microsoft Excel 2016 software. Statistical analysis,
mean value, and standard deviation were calculated
using Minitab 16. The mean values were compared
with the Tukey test and the experiments were per-
formed with three replicates.
3. RESULTS AND DISCUSSION
3.1. Impact of the external medium pH on the
antibacterial effect of duck egg-white
lysozyme against C. acnes
As can be seen in Table 1, duck egg-white lyso-
zyme (at 3,000 U/mL) inhibited the growth of C.
acnes in pH agar from 6.0 to 7.5. At pH 6.0, the
largest inhibition zone was obtained, reaching
12.50 ± 0.50 mm, but the difference was not statis-
tically significant at 5% level compared to the in-
hibition zone at pH 6.5. Meanwhile, the smallest
one was obtained from the medium of pH 7.5,
reaching 3.67 ± 0.58 mm. This result was also in
line with the positive control when using commer-
cial lysozyme with the same activity (3,000 U/mL),
the antibacterial zone diameter reached the highest
value at pH 6.0 and lowest at pH 7.5. These results
Can Tho University Journal of Science Vol. 13, No. 2 (2021): 52-60
55
are in line with some early works. Hjelmeland et al.
(1983) studied lysozyme from the skin mucus of
rainbow trout (Oncorhynchus mykiss) and showed
that pH 6.0 of the medium had the least effect on
lysozyme activity, where its antibacterial activity
was strongest. Besides, Sonomi et al. (2001) also
concluded that lysozyme extracted from the kid-
neys of Japanese flounder (Paralichthys oliva-
ceuscho) kept the best antibacterial activity at pH
ranging from 5.0 to 6.5.
In this experiment, when observing the growth of
C. acnes on TYEG agar, the bacteria grew quickly
on the agar surface after only 12 hours. Besides,
Achermann et al. (2014) also demonstrated that C.
acnes grows best in media with pH between 6.0
and 7.0, better than an acidic or more alkaline me-
dium. Therefore, although pH 6.0 and 6.5 were the
most suitable pH for the C. acnes growth, lyso-
zyme in this experiment still illustrated very clear
resistance to C. acnes at these pH values.
Table 1. Antibacterial ability of duck egg-white lysozyme at different external pH medium
pH
Diameter of C. acnes inhibition zone
(mm)
Antibacterial activity in phosphate buffer
(U/mL)
6.0
12.50 ± 0.50a
9,227 ± 377.54a
6.5
10.70 ± 0.58ab
9,127 ± 100.66a
7.0
10.30 ± 1.15b
8,107 ± 930.02ab
7.5
3.67 ± 0.58c
7,500 ± 200.33b
P-value
0.00
0.01
CV (%)
11.11
6.06
*Note: The data was an average of 3 replicates. Means in the same column that do not share a letter are significantly
different.
The positive control was commercial lysozyme (3,000 U/mL) giving inhibition zone diameters at pH 6.0: 6.33 ± 0.58a
mm; pH 6.5: 5.67 ± 1.00a mm; pH 7.0: 5.00 ± 0.58a mm; pH 7.5: 1.00 ± 0.00b mm.
The influence of the pH values of agar plates and
buffer solution on lysozyme activity was relatively
similar. Specifically, at the buffer of pH 6.0, lyso-
zyme showed the highest lysis activity on the sub-
strate, reaching 9,227 ± 377.54 U/mL, not statisti-
cally different compared with the buffer of pH 6.5
and 7.0 (Table 1). The smallest value was also ob-
tained from the buffer medium with pH 7.5.
According to Chang and Charles (1971), the de-
creasing activity of lysozyme in phosphate buffers
when the medium pH increased from 6.0 to 8.0 was
mainly due to the sharp increase in ionic strength.
The ionic strength required for suitable lysis action
is abided by a principle that at the lower pH the
optimum lies at the high ionic strength, while at a
higher pH value the optimum is converted to rela-
tively low ionic strength (Davies et al., 1969). In
this experiment of the study, the ionic strength of
buffer solution at pH 6.0 was I1 = 0.124 M and that
of buffer solution at pH 7.5 was I2 = 0.268 M > I1.
From these values, it can be explained why lyso-
zyme put in the pH 6.0 of phosphate buffer exhib-
ited higher antibacterial activity than at pH 7.5.
From the analyzed results, it can be seen that the
pH of the external medium influences the expres-
sion of lysozyme antibacterial activity. When per-
forming this experiment, the conductors took into
account the possibility that the environmental pH
will simultaneously affect the growth of bacteria
and lysozyme activity. This is consistent with the
practical conditions if lysozyme is applied in skin-
care products. Because everyone's skin has a dif-
ferent pH, acne and skin cleansing solutions can
change the pH of facial skin in a long enough peri-
od of time (Gfatter et al., 1997; Lambers et al.,
2006; Prakash et al., 2017), the results of this study
can help propose recommendations to the consum-
er who will suit this lysozyme product and which
skincare solution is suitable to be used together.
3.2. Impact of the external medium
temperature on the antibacterial effect of
duck egg-white lysozyme against C. acnes
As evidence from the second column in Table 2, it
can be noted that the inhibition zone reached the
largest value at 30oC (10.67 ± 0.58 mm) and was
not statistically different at 5% level compared
with at 35oC (10.00 ± 0.00 mm). A similar propen-
sity was also observed from the reactions in the
potassium buffer solution (the third column in Ta-
ble 2) in which lysozyme was lost only 36% and
39% of its activity at 30oC and 35oC, respectively,
while lost until 80% of activity at the other two
temperatures. Kawahara and Kusuda (1988) also
showed that lysozyme from the slime of Japanese
eel (Anguilla japonica) at pH 6.0 also acted best at
30oC of the external medium. Similarly, Miyazaki
Can Tho University Journal of Science Vol. 13, No. 2 (2021): 52-60
56
(1998) performed a study on plasma-derived lyso-
zyme of pink dot salmon (Salverinus leucomaenis)
and reported that lysozyme exhibited optimum
activity at medium treated with 30oC.
When incubated at 40°C, no growth of C. acnes
was observed resulting in an inability to assess
enzyme activity. However, the measurement of
lysozyme activity in potassium buffer solution il-
lustrated that such high temperature made lyso-
zyme lose up to 80% of its activity within 90
minutes.
In 1978, Frasco et al. used infrared spectroscopy to
study the mechanism of lysozyme heat denatura-
tion. As the temperature increases, water molecules
are released from the outer polar amino acid-base.
Water moves to the peptide-peptide bonds within
the structure, encouraging the transformation of
peptide-peptide bonds into water-peptide bonds,
causing proteins to begin to swell and reduce the
degree of coils, from there affecting the expression
of lysozyme activity. However, many previous
publications have shown that lysozyme can even
show a relatively strong activity at higher than
40°C at suitable external pH conditions. In 2013,
Venkataramani et al. presented that egg-white ly-
sozyme has a temperature range of 25°C to 95°C in
an acidic solvent. Cunningham and Lineweaver
(1965) found that lysozyme was 50 times more
thermally stable in phosphate buffer (pH 6.2) than
in egg whites (pH 9.0) at 62.5oC.
Table 2. Antibacterial ability of duck egg-white lysozyme at different external pH medium
Temperature (oC)
Diameter of inhibition zone (mm)
Decrease in lysozyme activity after 90 minutes
of reaction (%)
25
9.00 ± 1.00b
81.36 ± 10.54b
30
10.67 ± 0.58a
36.21 ± 3.64a
35
10.00 ± 0.00ab
39.11 ± 4.38a
40
0.00 ± 0.00c
80.95 ± 10.52b
P-value
0.00
0.00
CV (%)
7.78
13.41
*Note: The data was an average of 3 replicates. Means that do not share a letter are significantly different.
The positive control was commercial lysozyme (3,000 U/mL) giving inhibition zone diameters at 25oC: 8.00 ± 0.58b mm;
30oC: 10.00 ± 0.58a mm; 35oC: 7.00 ± 0.58c mm; 40oC: 0.00 ± 0.00d mm.
3.3. MIC and MBC values of duck egg-white
lysozyme for C. acnes
MIC80 value is defined as the lowest concentration
of a reagent in which bacterial colonies still devel-
op but are inhibited by 80% of their density (Ku-
mar & Pandey, 2013). Table 3 illustrated that duck
egg-white lysozyme at the concentration of 0.55
mg/mL (corresponding to 5,000 U/mL) inhibited
more than 80% of the C. acnes colonies compared
with results from negative control (inactivated ly-
sozyme gave 0% in inhibitory ability), so lysozyme
concentration of 0.55 mg/mL was considered to be
the MIC80 for C. acnes. Because lysozyme from
the concentration of 0.66 mg/mL onwards could
inhibit more than 90% of the bacteria, so the MIC90
value of the duck eggs lysozyme for C. acnes was
0.66 mg/mL. MBC value is defined as the lowest
reagent concentration that can kill bacterial growth
by up to 99.9% (Levison, 2004). Therefore, the
MBC value of lysozyme was chosen for C. acnes
was 1.11 mg/mL. In addition, according to Levison
(2004), if the MBC/MIC ratio is bigger than 4.00,
the reagent used has bacteriostatic effects, while if
this ratio is smaller than 4.00, the reagent is con-
sidered to be effective bactericidal. The MBC/MIC
ratio of the duck egg-white lysozyme in this study
was 2.00 < 4.00, so it can be concluded that this
lysozyme was effective bactericidal against C. ac-
nes.
Until now, there have been not many studies relat-
ed to MIC and MBC of lysozyme from egg whites.
Xiu et al. (2008) used lysozyme from marine mi-
croorganisms to treat Staphylococcus aureus and
Staphylococcus epidermis and found MIC values
of 0.5 mg/mL to 1.0 mg/mL; MBC values were
also reported to be 1.0 mg/mL to 4.0 mg/mL. Anti-
biotics have long been also used to treat acne, such
as tetracycline, minocycline, and azithromycin
(Farrah & Tan, 2016). Cha et al. (2020) studied the
MIC of some antibiotics and compounds common-
ly used to treat C. acnes, the results demonstrated
that the minimum concentration of erythromycin
and salicylic acid to inhibit the C. acnes growth
was both about 1.25 mg/mL, which was equivalent
to MBC values of duck egg lysozyme (1.11
mg/mL) and ampicillin concluded from the present
study. In addition, many studies have also shown
that long-term use of these antibiotics often cause
Can Tho University Journal of Science Vol. 13, No. 2 (2021): 52-60
57
side effects such as mood disturbances, dry skin,
dermatitis, burning (Eichenfield, 2015).
Side effects of antibiotics and the emergence of
multidrug-resistant bacteria have become a threat
to the health of consumers, so the main concern
today is finding compounds or proteins derived
from nature that possess strong antibacterial, anti-
inflammatory activity and cause fewer side effects
(Neamsuvan et al., 2015), such as natural com-
pounds from herbal plants, tea or grapefruit essen-
tial oils, and even some brown algae extract. Lyso-
zyme is also a protein that can be obtained from
abundant, easy-to-find, and inexpensive ingredients
like egg whites. Therefore, lysozyme is believed to
be a potential protein that can replace antibiotics in
the treatment of skin problems.
Table 3. Percentage of inhibited C. acnes density by the concentration of duck egg-white lysozyme
Lysozyme concentration (mg/mL)
Lysozyme activity (U/mL)
Percentage of inhibition (%)
MIC/MBC
0.11
1,000
4.00 ± 2.35c
0.22
2,000
11.85 ± 5.59c
0.44
4,000
36.30 ± 6.68b
0.55
5,000
86.37 ± 1.05a
MIC80
0.66
6,000
93.78 ± 0.44a
MIC90
0.88
8,000
94.52 ± 0.26a
0.99
9,000
99.08 ± 0.25a
1.11
10,000
99.99 ± 0.00a
MBC
1.66
15,000
100.00 ± 0.00a
LysozymeTM - 0.1 mg/mL
14,500
100.00 ± 0.00a
Ampicillin – 1.0 mg/mL
100.00 ± 0.00a
Inactivated lysozyme
0.00 ± 0.00c
P-value
0.00
CV (%)
7.42
3.4. Impact of storage conditions on the
antibacterial effect of duck egg-white
lysozyme against C. acnes
Observing in Table 4, from 15 days to after 30 days
of storage, the inhibition zone values obtained at all
temperatures were almost not statistically different
at 5%, but they were all significantly smaller than
the first day. In addition, at 4oC and 25oC, the re-
duction in inhibition zone diameters was more than
the first two temperatures, which decreased 3.5
times and 4.1 times after 30 days. This is consistent
with Wasserfall’s (1977) conclusion, more de-
crease in the activity of lysozyme was observed
when increasing storage temperature and the more
stable lysozyme activity was when stored at low
temperature. After 60 days of storage, the values in
all treatments were 0.00 mm, however, lysozyme
has still partly retained its initial activity, which is
proved in Table 5.
Table 4. The C. acnes inhibition zone of duck egg-white lysozyme at different storage temperatures
and times
Storage time (days)
Storage temperature (oC)
-20
-10
4
25
1
23.00 ± 0.00a
21.00 ± 0.00a
22.00 ± 0.58a
22.00 ± 0.58a
15
8.67 ± 1.53b
7.00 ± 1.00bc
6.00 ± 3.61bc
5.33 ± 0.58c
30
7.67 ± 0.58bc
7.00 ± 1.00bc
6.33 ± 0.58bc
5.33 ± 0.58c
60
0.00 ± 0.00d
0.00 ± 0.00d
0.00 ± 0.00d
0.00 ± 0.00d
P-value
0.19
CV (%)
12.23
*Note: The antibacterial zone diameter figure is an average of 3 replicates. Means that do not share a letter are signifi-
cantly different.
The results in Table 5 reflected the causes of data
of Table 4. Accordingly, lysozyme after the first 30
days of storage retained 70% to 79% of its activity
when stored at -20°C to 4oC; whilst 25oC only
maintained more than 30% of lysozyme activity.
Can Tho University Journal of Science Vol. 13, No. 2 (2021): 52-60
58
After 30 to 60 days, at -20°C, lysozyme retained up
to 63% of the activity, while at the remaining tem-
peratures, lysozyme activity kept only 15% to 35%.
Uddin et al. (2017) also reported that lysozyme had
almost unchanged activity in the first 30 days when
stored under cold conditions (from ≤ 0oC - 4oC)
and low humidity (from 12% - 25%), the activity
of lysozyme would decrease slightly when stored at
room temperature and higher humidity (about
65%).
There have been many studies demonstrating that
lysozyme activity during storage also depends on
how the enzyme is immobilized, the material hold-
ing the enzyme, or the substance dissolving en-
zymes. In 1972, Uchida et al. used lysozyme in a
mirin wine sample and found that more than 95%
of the lysozyme activity was remained after one
year, meanwhile, a combination of mirin with glu-
tentannin and activated carbon reduced lysozyme
activity by 15%. Zi-xuan et al. (2012) reported that
free lysozyme almost lost 100% of its activity after
one month, whereas, under the same storage condi-
tions, the decrease in the activity of immobilized
lysozyme was 2 to 3 times lower. In 2017, Uddin et
al. immobilized lysozyme in aerogel and then
found that lysozyme activity almost unchanged
after 30 days. Lysozyme in the present study when
immobilized with 5% maltodextrin also presented a
good protective effect, maintaining more than 60%
of the activity after 2 months.
Types of containers used to store lysozyme solu-
tions also influence its activity. Goldblum et al.
(1981) reported that when breast milk was con-
tained in flasks made of pyrex and polypropylene,
lysozyme concentrations decreased by 40% after
24 hours of refrigeration. Kravchenko et al. (1967)
found that the lysozyme adhered to the glassware
and causing a poor ability to maintain enzyme ac-
tivity. Preservation of lysozyme samples in a glass
capillary tube for 9 days at -4°C also reduced lyso-
zyme activity by about 20% to 50% (Copeland et
al., 1982). Lysozyme in this experiment was pre-
served in plastic Eppendorf tubes, compared with
the data in Table 5 and the references above, it can
be assumed that the plastic material helped stabi-
lize the activity. Lysozyme properties were better
and longer when it was stored after two months at
minus temperature, retaining up to 65% of the ac-
tivity.
Table 5. The activity maintenance level of the egg-white lysozyme stored at different temperatures and
times
(Unit: %)
Storage time (days)
Storage temperature (oC)
-20
-10
4
25
1
100.00 ± 0.00a
100.00 ± 0.00a
100.00 ± 0.00a
100.00 ± 0.00a
15
97.67 ± 3.94a
81.33 ± 14.01ab
100.00 ± 0.00a
38.67 ± 0.68cde
30
78.67 ± 37.09ab
70.67 ± 9.47abc
78.67 ± 15.40ab
30.67 ± 7.53de
60
63.00 ± 2.94bcd
34.67 ± 0.14de
15.33 ± 0.41e
21.67 ± 2.39e
P-value
0.00
CV (%)
16.00
*Note: The antibacterial zone diameter figure is an average of 3 replicates. Means that do not share a letter are signifi-
cantly different.
4. CONCLUSIONS
Lysozyme from “Co Co” duck eggs was most ef-
fectively resistant to C. acnes at pH of 6.0 and 6.5,
between 30oC and 35oC. At pH 7.5 and 40oC, lyso-
zyme exhibited weak antibacterial ability. Lyso-
zyme stored at -20°C or -10°C retained the most
effective and longest lysozyme activity, after 60
days still retained up to 63% of the activity. The
authors strongly recommended that before preserv-
ing, lysozyme can be investigated with some kinds
of carriers for the immobilization, compounds that
can have synergistic effects with lysozyme, or ma-
terials holding lysozyme.
The MIC80, MIC90, and MBC values of the duck
egg-white lysozyme for C. acnes were 0.55
mg/mL, 0.66 mg/mL, and 1.11 mg/mL, respective-
ly. This study expressed that the effects of duck
egg-white lysozyme were in line with or better than
many antibiotics used for skin treatments nowa-
days. Therefore, in the future, lysozyme is hoped to
be studied more and produced on an industrial
scale, creating a base for acne treatment products
with many good properties, efficient and safe for
consumers.
Can Tho University Journal of Science Vol. 13, No. 2 (2021): 52-60
59
ACKNOWLEDGMENTS
The authors gratefully acknowledge Prof. Cao
Ngoc Diep and Dr. Tran Thi Giang, Biotechnology
Research and Development Institute (BiRDI) of
Can Tho University, for their valuable and con-
structive suggestions during the planning and de-
velopment of this research work. We also im-
mensely appreciate Ms. Le Thi Truc Mai, Ms. Phan
Thi Hong Tiep, and Mr. Nguyen Minh Thuan,
BiRDI of Can Tho University, for their comments
on an earlier version of the manuscript.
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