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Journal of Functional Foods 38 (2017) 273–279
Contents lists available at ScienceDirect
Journal of Functional Foods
journal homepage: www.elsevier.com/locate/jff
The antibacterial activity and antibacterial mechanism of a
polysaccharide from Cordyceps cicadae
Yu Zhang a,1, Yu-Ting Wu a,1, Wei Zheng a, Xiao-Xuan Han a, Yao-Huang Jiang a, Pei-Lin Hu a,
Zhen-Xing Tang b, Lu-E Shi a,⇑
a
b
College of Life and Environmental Sciences, Hangzhou Normal University, 310016 Hangzhou, Zhejiang, China
Hangzhou Tianlong Group Co. Ltd, 310021 Hangzhou, Zhejiang, China
a r t i c l e
i n f o
Article history:
Received 19 January 2017
Received in revised form 5 September 2017
Accepted 14 September 2017
Keywords:
Cordyceps cicadae
Polysaccharide
Antibacterial activity
Antibacterial mechanism
a b s t r a c t
In this study, the antibacterial characteristics and mechanism of Cordyceps cicadae polysaccharide were
determined. A water-soluble polysaccharide from Cordyceps cicadae was separated, and antibacterial
activity against common pathogens was investigated by Oxford cup method. To investigate its antibacterial mechanism, the change of bacterial (Escherichia coli as the indicator bacteria) growth curve, electric
conductivity, alkaline phosphatase (AKP) and b-galactosidase activity were measured. And the mycoprotein, membrane protein were also studied. The results showed that Cordyceps cicadae polysaccharide had
strong antibacterial activity against Escherichia coli, Staphyloccocus aureus, Bacillus subtilis, Salmonella
paratyphi and Pseudomonas aeruginosa. The minimum inhibitory concentration (MIC) to Escherichia coli
was 0.10 mg/mL. In addition, the electric conductivity, AKP and b-galactosidase activity in microbial
broth increased. The growth curve, mycoprotein, membrane protein changed. Our results revealed that
Cordyceps cicadae polysaccharide exerted its bactericidal activity by damaging bacterial cell wall and cell
membranes, increasing the cell permeability which resulted in the structural lesions and release of cell
components, thus led to cell death.
Ó 2017 Published by Elsevier Ltd.
1. Introduction
Cordyceps cicadae is one kind of medicinal fungus beetle, a complex of coremium and stroma formed by cicadae that is infected by
Paecilomyces cicadae (Zeng et al., 2014). As the valuable traditional
medicine, the same to Cordyceps sinensis, Cordyceps cicadae belongs
to ergot fungus Branch, Cordyceps (Shao et al., 2003). Cordyceps
cicadae has similar fermentation broth to Paecilomyces cicadae.
Many studies indicate that Cordyceps cicadae contains many active
ingredients such as nucleosides, cephalosporins, ergosterol and its
peroxide, cordyceps acid, hyaluronic acid, macromolecular
polysaccharide and more kinds of essential amino acids, trace elements, etc. Chen and Lu et al. studied the nutritional and bioactive
components of solid-state fermented substrate of Cordyceps cicadae. The results indicated that the contents of protein, total sugar
and fat acid, were 16.17%, 38.87% and 1.43%, respectively. The
unsaturated fat acid content was 1.18% (Chen, 1991; Lu, Jiang,
Mu, Hou, & Wang, 2006). Modern pharmacological studies indicate
⇑ Corresponding author.
1
E-mail address: shilue@126.com (Lu-E Shi).
These authours contributed equally to this paper.
http://dx.doi.org/10.1016/j.jff.2017.09.047
1756-4646/Ó 2017 Published by Elsevier Ltd.
that Cordyceps cicadae contains many active ingredients that perform many functions such as resist inflammation and relieve pain,
suppress immunity, modulate the immune system, anti-fatigue,
improve renal function, anti-bacterial effect, etc. (Xu, Mo, Yu, &
Mao, 2010). Chai isolated and purified the insecticidal active substance from fruiting body of Paecilomyces cicadae (Miquel) Samson
that could kill the larvae of Plutella xylostella (Lepidoptera: Plutellidae) for the first time (Chai et al., 2007). Cordycepin is one kind
of antibacterial substances, which is firstly isolated from the fungus. Li et al. found the content of cordycepin was 2.77 mg/g in
the mycelium of Cordyceps cicadae, which was far higher than that
of cordyceps sinensis (Li et al., 2014). In addition, due to lower content of heavy metals such as As, Hg and Pb in Cordyceps cicadae
than that in Cordyceps sinensis, low toxicity and easy cultured,
Cordyceps cicadae has been showing the potential in ediblemedicinal applications. Polysaccharide, one of bioactive
compounds from Cordyceps cicadae, is the natural high molecular
compound composed of many monosaccharides. The CMP- 40,
CMP- 60 and CMP- 80 of Cordyceps minlitaris polysaccharide have
been obtained by fractionated precipitation. The results of antioxidation demonstrated that Cordyceps minlitaris polysaccharide had
the ability to remove the oxygen free radicals and hydroxide free
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Y. Zhang et al. / Journal of Functional Foods 38 (2017) 273–279
radicals, and showed the bacteriostasic activity to Pseudomonas
aeruginosa, Escherichia coli, Staphylocouus aureus and Micrococcus
tetragenus. As one of the main available constituents in many Chinese herbs including Cordyceps sinensis, polysaccharides play an
important role in anti-bacterial activity (Duarte, Ferreira, Martins,
Viveiros, & Amaral, 2007). In recent years, with the rapid development and wide applications of antibiotics, drug resistance of bacterium and fungus are posing a threat to clinical treatment. So it
appeals to screen of new antibiotics in order to meet the needs
of clinical practice. In our previous work, the antibacterial activity
of organic acids from Enterococcus faecium KQ 2.6 isolated from
peacock feces, were determined (Zheng et al., 2015). Organic acids,
as the growth promoter, show antibacterial effects on bacteria
through different mechanisms. Researchers have reported the five
possible antibacterial mechanisms of organic acids, such as energy
competition, permeabilizing through outer membrane, increase of
cellular osmolarity, inhibition of macromolecule synthesis and
induction of antimicrobial peptide in host cells (Zhang et al., 2011).
Nowadays, studies on Cordyceps cicadae polysaccharide have
been mainly focus on the exosomatic antibacterial effect (Weng,
2010; Xu et al., 2010). Only antimicrobial activities of exopolysaccharides (EPS) and intracellular polysaccharides (IPS) produced by Cordyceps cicadae were tested under different factors.
IPS showed the strongest bacteriostatic ability which was observed
against Pseudomonas aeruginosa (Sharma, Gautam, & Atri, 2015).
However, the specific antibacterial studies of Cordyceps cicadae
have not been fully studied, particularly for the study of antibacterial mechanism. Cordyceps cicadae is one of Chinese edible herbal
resources, including many bioactive substances. It shows great
potential in food relative fields. Therefore, in our present study,
Cordyceps cicadae was collected from Zhejiang province, China,
stronger antibacterial activity of which was observed. In addition,
by analyzing the destruction effects towards cell wall and the
membrane, antibacterial mechanism of the polysaccharide from
Cordyceps cicadae were investigated in detail. It is the first report
on the study of antibacterial mechanism of the polysaccharide
from Cordyceps cicadae. The results would provide scientific basis
for further research and new antimicrobial product development
from Cordyceps cicadae polysaccharide.
2. Materials and methods
2.1. Materials
Escherichia coli, Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes,
Enterococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumonia and Salmonella paratyphi were obtained from fermentation
engineering lab of Hangzhou Normal University. The Oxford cups
were purchased from Changji Apparatus and Equipment Cooperation Ltd (Dongtai, China). All chemicals were purchased from Sangon (Shanghai, China).
2.2. Extraction and determination of Cordyceps cicadae polysaccharide
1.0 g Cordyceps cicadae powder was dissolved into 10 mL water,
and heated in the microwave for 1.5 min. 3-times volume 95%
alcohol was added into the above solution. The mixture was kept
at 4 °C for 12 h. After centrifugation, the supernatant was discarded. 9.0 mL water was added to dissolve the dried precipitation.
Then 2.4-times volume Sevage solution were used to extract crude
polysaccharide of Cordyceps cicadae. The supernatant and 3-times
volume 95% alcohol were incubated at 4 °C for 4 h. After centrifuged at 4000g for 15 min, the purified polysaccharide was
isolated and polysaccharide content was determined with
phenol-vitriolic colorimetry (Kiho, Nagai, Miyamoto, Watanabe, &
Ukai, 1990). The linear relation of polysaccharide content and the
absorption at 590 nm was determined. Glucose solution was used
as the standard (Xing et al., 2013).
2.3. Bacteriostatic spectrum analysis
Antimicrobial activity of Cordyceps cicadae polysaccharide
against pathogenic bacteria including Escherichia coli, Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Enterococcus faecalis,
Pseudomonas aeruginosa, Klebsiella pneumonia and Salmonella
paratyphi, was investigated. The antimicrobial assay was performed using the agar well diffusion method (Zheng et al., 2015).
250 lL of indicator bacteria cultured in 10 mL liquid medium
was poured into a plate with solid medium containing 1.5% (w/v)
agar. Oxford cups (8 mm in diameter) were filled with 200 lL
Cordyceps cicadae polysaccharide solution (final concentration of
15.23 mg/mL). The same amount of sterile water was taken as
the control. The plates were kept for 2 h at 4 °C before being incubated at 37 °C for 24 h. After incubation, the antimicrobial activity
was detected by observing the clear zones around the wells containing the cell free supernatant. The clear zones were regarded
as inhibitory zones and recorded in mm (Cheikhyoussef et al.,
2009).
2.4. Analysis of growth curve of E coli
5.0 mL Cordyceps cicadae polysaccharide solution and 2.0 mL
Ecoli bacterium at the phase of logarithm (approximately 108–
109 CFU/mL), were added into 100 mL culture solution. The same
amount of sterile water was taken as the control. The mixture
was incubated at 37 °C at the speed of 200 rpm. Absorption at
600 nm was measured. The bacterial growth curve was made in
order to analysis effect of Cordyceps cicadae polysaccharide on E
coli growth.
2.5. Determination of the MIC
Cordyceps cicadae polysaccharide was diluted by twofold dilution method (Kang & Siragusa, 2001). The MIC against E coli was
measured using turbidimetric method (Kyhseandersen et al.,
1994). 100 lL overnight cultured of E coli solution and 2.0 mL
Cordyceps cicadae polysaccharide solution with different concentrations, were added in test tubes, respectively. The mixture of
100 lL bacterium solution and 2.0 mL sterile water was used as
the control. Absorptions at 600 nm were measured. Each bacteriostatic rate was calculated as the following formula (Weng, 2010).
Bacteriostatic rateð%Þ ¼ ð1 AX =A0 Þ 100
2.6. Determination of cell wall permeability
5.0 mL Cordyceps cicadae polysaccharide solution and 2.0 mL E
coli bacterium at the phase of logarithm (approximately
108–109 CFU/mL) were added into 100 mL bacterial culture solution. The same amount of sterile water was taken as the control.
The mixture was incubated at 37 °C, 200 rpm for 4 h. After the mixture was centrifuged at 8000g for 10 min, the supernatant was collected and AKP content was measured. AKP chromogenic substrate
was dissolved in buffer. p-nitrophenol solution was treated as the
standard working solution, and was also diluted in buffer to reach
final concentrations of 0.50 mM. Different amounts of the standard
and 50 lL samples were injected into 96-well plate. After 50 lL
chromogenic substrate solutions were added, the mixture was
incubated at 37 °C for 10 min. Finally, 100 lL termination agents
Y. Zhang et al. / Journal of Functional Foods 38 (2017) 273–279
were added to stop the reaction. Absorptions at 405 nm were measured. AKP concentration was calculated according to the standard
curve. Amount of AKP for every lM pAnitrophenol producing per
minute was defined as a unit of AKP activity(Ishibe, Rosier, &
Puzas, 1991).
2.7. Specific conductance analysis
The collected E coli cells were washed three times with PBS
(10 mM, pH 7.4) and fit to the approximate concentration of 108
CFU/mL (Hoehn et al., 2008). 5.0 mL Cordyceps cicadae polysaccharide solution and 5.0 mL E coli bacterium were adequately mixed.
The same amount of sterile water was taken as the control. The
mixture was incubated at 37 °C with the speed of 200 rpm. Specific
conductance was measured per 10 min.
2.8. Determination of inner membrane permeability
Inner membrane permeability was determined through measuring the release of cytoplasmic b-galactosidase activity from
the bacteria (Han et al., 2013). Aliquots (10 mL) of E coli cells
(approximately 108–109 CFU/mL) were centrifuged at 4500g for
15 min. The precipitation was washed three times with sterile saline, and re-suspended in 100 mL of M9 lactose induction media
(0.050% MgSO4, 0.50% lactose, 0.0010% CaCl2, 1.28% Na2HPO4,
0.30% NaH2PO4, 0.050% NaCl, and 0.10% NH4Cl). E coli culture solution was centrifuged at 4500g for 15 min after incubation at 37 °C
for 10 h. The collected E coli cells were washed three times with
sterile saline and re-suspended in fresh b-galactosidase buffer
(0.80% NaCl, 0.020% KCl, 0.29% Na2HPO4, 0.024% NaH2PO4, 0.125%
MgSO4 and 0.39% b-mercaptoethanol) to obtain an around absorbance 0.20 at 630 nm. Aliquots of (0.50 mL) O-nitrophenyl-b-D-g
alactopyranoside (ONPG, 1.0 mg/mL) and 0.50 mL of Cordyceps
cicadae polysaccharide solution were added to of E coli culture
solution (4.0 mL). The mixture was shaken thoroughly, and then
incubated at 37 °C. Every 30 min, the absorbance of the mixture
at 415 nm was measured. The variation of E. coli inner membrane
permeability (OD) is calculated with the following equation.
Where ODt is the value of the sample treated with 0.50 mL Cordyceps cicadae polysaccharide solution every 30 min, OD0 is the control treated with 0.50 mL of sterile water at 415 nm every 30 min,
0.01 per change of OD is defined as a unit of enzyme activity,
OD = ODtOD0 (Han et al., 2013).
2.9. Whole-cell proteins analysis
E coli cells treated with Cordyceps cicadae polysaccharide after
incubation at 37 °C for 4 h, were centrifuged at 8000g for 10 min.
After that, the supernatant was discarded, E. coli cells were concentrated 100 times after mixing well by sterile water. 5.0 lL mycoproteins combined with 20 lL of sample buffer (pH 6.8; 1.0 M
Tris-HCl, 50% glycerol, 10% SDS, 10% b-mercaptoethanol, and
0.10% bromophenol blue) (Frank et al., 2003), was heated to
100 °C for 10 min, then cooled to room temperature (25 °C). The
proteins were electrophoresed on 5.0% stacking gel at 80 V for
30 min, then 12% resolving gel at 110 V for 1 h. The gel was dyed
with Coomassie Brilliant Blue R250, decolorized with decolorizing
agent (10% glacial acetic, 10% acid alcohol and 80% distilled water).
After 24 h, the protein bands were visualized on the gels (Blakesley
& Boezi, 1977; Chai et al., 2007; Cheikhyoussef et al., 2009).
2.10. Measurement of protein concentration in the medium
Protein content in the medium was determined by BCA Protein
Quantitation Kit (Li et al., 2014). BCA working solution and BCA
protein standard solution (0.50 mg/mL) were prepared. BCA work-
275
ing solution was added to each tube with protein standard solution, separately. After mixing sufficiently, each mixture was
stored in 60 °C for 30 min. Then, absorption at 562 nm was measured after the samples were cool to room temperature. 10 lL
the sample and 90 lL PBS were added into centrifuge tube, the
absorption at 562 nm was measured according to the method
described above. E coli cells treated with Cordyceps cicadae
polysaccharide were centrifuged at 8000g for 10 min after incubation at 37 °C for 4 h. The supernatant was diluted with phosphate
buffer. Protein content of the sample was calculated by the protein
standard curve.
2.11. Membrane proteins analysis
SDS-Page of E coli membrane protein was performed according
to a previous method with slight modifications (Han et al., 2013).
2.0 mL Cordyceps cicadae polysaccharide solution with different
concentrations, were added to 18 mL bacterial culture solutions
(approximately 108-109 CFU/mL). The polysaccharide with final
concentrations of 0, 4, 2 and 1 MIC were reached. The treated
bacterial suspensions were incubated at 37 °C for 4 h, and then
centrifuged at 4500g for 15 min. The collected E coli cells were
washed three times with PBS (10 mM, pH 7.4) and re-suspended
in 4.0 mL of PBS containing 8.0% Triton X-114. After incubation at
4 °C for 3 h, the bacterial suspensions were centrifuged at
10,000g for 30 min. Then, the supernatant was incubated at 37 °C
for 2 h, and centrifuged at 3000g for 15 min for phase separation.
The separated organic phase was blended with anhydrous alcohol
(36 mL), and incubated on an ice bath for 10 h, followed by centrifugation at 10,000g for 30 min at 4 °C. The collected membrane
proteins were combined with sample buffer (pH 6.8; 1.0 M TrisHCl, 50% glycerol, 10% SDS, 10% b-mercaptoethanol, and 0.10% bromophenol blue), heated to 100 °C for 10 min, cooled to room temperature (25 °C), and loaded onto a 5.0% stacking and 12% resolving
gel, then subjected to SDS-Page in the same protocol with wholecell protein analysis.
2.12. Statistical analysis
Each experiment was performed in triplicates and mean value
with standard deviation was calculated. SPSS 15.0 software was
used for all statistical analysis. The data were analyzed by analysis
of variance. Graphs were produced using Microsoft Excel 2010.
Regression analysis was used to determine the significant difference at 5% confidence intervals. The significant differences were
compared using t test and statistical significance was at the level
of P < 0.05.
3. Results and discussion
3.1. Antibacterial spectrum of Cordyceps cicadae polysaccharide
Water extraction-alcohol precipitation is a classic method for
polysaccharide extraction. According to the linear relationship
between polysaccharide concentration and OD490, the standard
equation is Y = 0.0122X 0.0259 (R2 = 0.9990). The linear range
is 10–60 mg/mL. The content of Cordyceps cicadae polysaccharide
is 29 mg/mL according to the standard equation. As shown in
Fig. 1, inhibition zone of the control sterile water was not detected
in negative group. There were inhibition zones around the Oxford
cup when Cordyceps cicadae polysaccharide solution was infiltrated
into the medium. Table 1 showed that Cordyceps cicadae polysaccharide had strong antibacterial activity against Escherichia coli,
Staphyloccocus aureus, Bacillus subtilis, Salmonella paratyphi and
Pseudomonas aeruginosa. By comparing the inhibition zone
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Y. Zhang et al. / Journal of Functional Foods 38 (2017) 273–279
Fig. 1. Antimicrobial spectrum of Cordyceps cicadae polysaccharide. (A) sterile water (negtive control); (B) Cordyceps cicadae polysaccharide; (C) chloramphenicol (positive
control, 0.10 mg/mL).
Table 1
Antimicrobial spectrum of Cordyceps cicadae polysaccharide.
2.5
Medium
Temperature
(°C)
Anti-bacterial
activitya
G+
Bacillus subtilis
Staphylococcus aureus
Bacillus cereus
Streptococcus pyogenes
Staphylococcus epidermidis
Enterococcus faecalis
LB
LB
LB
LB
LB
MRS
37
37
37
37
37
37
+++
+++
–
–
–
–
G﹣
E. coli
Pseudomonas aeruginosa
Salmonella paratyphi
Klebsiella pneumoniae
LB
LB
LB
LB
37
37
37
37
++
++
++
–
a
No inhibition zone; +: Inhibition zone diameter less than 15 mm; ++: Inhibition
zone diameter between 15 and 20 mm; +++: Inhibition zone diameter more than
20 mm.
diameter, Cordyceps cicadae polysaccharide showed better antibacterial effect to gram-positive bacteria than gram-negative ones,
which showed different antibacterial effect than previous studies
(Sharma et al., 2015). Anti-bacterial activity polysaccharides from
different fungus have been studied (Kita, Isono, Misaki, Endo, &
Yamasaki, 1974). The antibacterial activity is not completely same,
that is likely related to the sources and concentration of Cordyceps
cicadae polysaccharide (Ren, He, Cheng, & Chang, 2014). Weng
(2010) reported that the concentration of polysaccharide extracted
from Cordyceps cicadae was 10.25 mg/g. It showed better antibacterial activity against Escherichia coli and Staphylococcus aureus
than Bacillus subtilis.
3.2. Effect of Cordyceps cicadae polysaccharide on the growth curve of
E. coli
As shown in Fig. 2, bacterial growth in the control group followed the model sAshaped growth curve. E. coli began to be at logarithmic phase after 4 h, and then stationary phase after 10 h.
Bacterium grow slower when Cordyceps cicadae polysaccharide
2
1.5
OD600
Indicator bacteria
1
Control group
Experimental group
0.5
0
0
2
4
6
8
10
12
14
16
18
20
22
24
Time (h)
Fig. 2. Changes of anti-bacterial curve of E coli treated with Cordyceps cicadae
polysaccharide.
(final concentration of 15.23 mg/mL) was added into the medium.
Absorptions at 600 nm were almost lower than the control in each
growth phase. Cordyceps cicadae polysaccharide showed an antibacterial activity during 3 to 10 h. The strongest anti-bacterial
activity happened at 4 h. E coli cells treated by Cordyceps cicadae
polysaccharide, grow slower than the control. The inhibition effect
gradually decreased after 4 h, and reached to the minimum until
24 h. The reason for this was that Cordyceps cicadae polysaccharide
could inhibit the growth of E coli, but to some extent it could serve
nutrition to the growth of E coli. When the inhibition dominated in
the interaction, the growth of E coli was inhibited. However, the
inhibition effect decreased relatively when Cordyceps cicadae
polysaccharide was as its necessary nutrition to promote the
growth of E coli (Tolstoguzov, 2004). However, the specific relationships among time, inhibition effects and polysaccharide concentration needed to further investigate.
3.3. MIC of Cordyceps cicadae polysaccharide against E coli
Cordyceps cicadae polysaccharide was diluted to 0.80, 0.40, 0.20,
0.10, 0.05 mg/mL, and the anti-bacterial rates of Cordyceps cicadae
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Y. Zhang et al. / Journal of Functional Foods 38 (2017) 273–279
polysaccharide against E coli were 33.0%, 16.13%, 6.93%, 1.47% and
0%, respectively. Anti-bacterial rate decreased with the decrease of
Cordyceps cicadae polysaccharide concentration. The lowest antibacterial concentration (MIC) of Cordyceps cicadae polysaccharide
against E coli was 0.10 mg/mL. In Sharma’s study, the MIC of both
EPS and IPS fractions ranged from 60–100 mg/mL (Sharma et al.,
2015). In addition, based on the MIC results, the higher polysaccharide concentration it was, the better inhibition effect it was. Therefore, optimization of the extracting technique of Cordyceps cicadae
polysaccharide needed to further investigate to improve the antibacterial activity.
trate into the culture out of E coli cells to inhibit its growth.
Otherwise, we found the lower electric conductivity of the cell culture treated with low concentration of electric conductivity during
the initial period of incubation. Lower concentration of polysaccharide did not destroy the cell membrane, but electrostatically combined with it (Rocklage & Quay, 1991). Thus, the number of ions in
the culture was reduced, that made the lower electric conductivity,
along with the increase of reaction time, the change of membrane
permeability was detected.
3.4. Cell wall permeability of E coli
Cell membrane plays an important role in bacterial metabolic
activity, transportation and selective permeated function. The
damage of cell membrane and the enzymes inactivation may seriously affect the cell metabolism (Smith et al., 2014). bgalactosidase, an induced enzyme in E coli, can hydrolyze ONPG
into yellow ortho-nitrophenol (ONP). Therefore, we can assay bgalactosidase activity with a colorimetric assay at 415 nm (Ishibe
et al., 1991). Effect of Cordyceps cicadae polysaccharide on the inner
membrane permeability of E. coli was showed in Fig. 4. The activity
change of b-galactosidasein cells was slow due to the protection of
cell membranes in normal cells. However, that of the samples treated with Cordyceps cicadae polysaccharide showed an obvious
increasing trendence. The b-galactosidase activity of the treated
sample increased over 30 min, ONPG immediately reacted with
b-galactosidase when the inner membrane of cells was destroyed.
These results indicated that Cordyceps cicadae polysaccharide could
permeate the inner membrane of E. coli, this was supported by the
observation of many researchers (Marri, Dallai, & Marchini, 1996;
Nakayama et al., 2013). Their results showed that the increase of
b-galactosidase activity in the presence of ceratotoxin A that could
enhance the permeability of the bacterial inner membrane. Similarly, these results were consistent with Epand’s study (Epand,
Pollard, Wright, Savage, & Epand, 2010), who reported that ceragenin CSA-13 permeated the inner membrane of E coli cells, as
measured by the activity change of b-galactosidase.
The anti-bacterial mechanisms of anti-bacterial compounds
included the damage of cell wall, change of the membrane permeability, change of proteins and nucleic acid molecules, inhibition
the actions of enzymes, inhibition of the synthesis of nucleic acid,
etc. (Balamayooran, Batra, Fessler, Happel, & Jeyaseelan, 2010).
According to the linear relationship between AKP concentration
and OD405, the standard equation is Y = 0.2165X + 0.0054
(R2 = 0.999). After incubation with Cordyceps cicadae polysaccharide for 4 h, AKP concentrations in the control and E coli medium,
were 0.127, 0.057 mM, respectively. AKP activity of polysaccharide
group was 127 U, almost 3-times as the control of 57 U. Therefore,
it indicated that Cordyceps cicadae polysaccharide could damage
the cell wall of E coli.
3.5. Effect of Cordyceps cicadae polysaccharide on the specific
conductance
Fig. 3 indicated the changes of the specific conductance of E coli
culture treated with Cordyceps cicadae polysaccharide. When treated with 2 MIC of Cordyceps cicadae polysaccharide in 60 min,
there was almost no difference between the experimental group
and the control. Afterwards, the experimental groups showed difference as the control. The specific conductance of E coli culture
solution showed higher, especially treated by 2 MIC of Cordyceps
cicadae polysaccharide. Changes of the specific conductance indicated the changes of membrane permeability. Additionally, the
membrane permeability was significantly depended on the concentration of Cordyceps cicadae polysaccharide (p < 0.05). What’s
more, we also found the higher electric conductivity of the culture
than that of the control. Therefore, Cordyceps cicadae polysaccharide could raise the membrane permeability and make ions infil-
10
3.7. Mycoproteins of Ecoli
Protein is an important part of bacterial structure, which is
involved in a variety of biochemical reactions for catalytic, protein
synthesis and expression and bacteria metabolism (Sonenberg &
Hinnebusch, 2009). Cho, Schiller, and Oh (2008) evaluated the
antimicrobial effects and biofilm formation inhibition of tea
polyphenols (TPP) extracted from Korean green tea (Camellia
sinensis L). The results showed the slightly raising of 3 kinds of
Control group
2×MIC
1×MIC
9.6
9.2
240
Enzyme activity (U)
Electric conductivity (ms/cm)
3.6. Cytoplasmic b-galactosidase activity of E coli
8.8
8.4
8
7.6
7.2
6.8
200
Control group
160
Experimental group
120
80
40
6.4
0
6
0
30
60
90
120 150 180 210 240 270 300 330 360 390
Time (min)
Fig. 3. Changes of electric conductivity of E coli treated with Cordyceps cicadae
polysaccharide.
10
40
70
100
130
Time (min)
Fig. 4. b-Galactosidase activity in the culture medium of E coli treated with
Cordyceps cicadae polysaccharide.
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Y. Zhang et al. / Journal of Functional Foods 38 (2017) 273–279
Fig. 5. SDS-Page patterns of proteins of E coli treated with Cordyceps cicadae polysaccharide. A: Whole cell proteins of E coli treated with Cordyceps cicadae polysaccharide. (1)
Protein Marker; (2) 2 MIC treated for 2 h; (3) 2 MIC treated for 4 h; (4) 1 MIC treated for 2 h; (5) 1 MIC treated for 4 h; (6) Control group (sterile water treated). B:
Membrane proteins of E coli treated with Cordyceps cicadae polysaccharide. (1) Protein Marker; (2) Control group (sterile water treated); (3) 4 MIC treated; (4) 2 MIC
treated; (5) 1 MIC treated.
protein expression and significantly decreasing of 14 kinds of protein expression, which led to the lower toxicity of bacterium. SDSPage of whole-cell proteins and protein concentration in the medium were assayed in our present study. The results showed that
total protein concentration of E coli cells decreased and soluble
proteins in the culture increased, which indicated the change of
membrane permeability, resulting in the consequence of growth
inhibition of E coli. SDS-Page patterns of total proteins of E coli
treated with Cordyceps cicadae polysaccharide (Fig. 5A), were light
dyed than the control (lane 6). Compared to lane 4 and lane 5 treated with 1 MIC of Cordyceps cicadae polysaccharide, the whole
cell protein patterns of lane 2 and lane 3 treated with 2 MIC were
light-colored. Moreover, mycoproteins treated for 4 h were light
dyed than that treated for 2 h. It indicated that the concentration
of whole-cell proteins was decreased after the treatment. The
decreasing was depended on the concentration of Cordyceps cicadae polysaccharide and incubation time. According to the linear
relationship between protein concentration in the medium and
OD562,
the
standard
equation
is
Y = 1822.6X 7.2015
(R2 = 0.9929). Protein concentration in the control medium was
2.48 mg/mL. Protein concentration of E. coli medium was
2.48 mg/mL after incubation with Cordyceps cicadae polysaccharide
for 4 h. The experimental group was obviously higher than the control. These results indicated that Cordyceps cicadae polysaccharide
could make E coli mycoprotein infiltrated into the culture.
patterns of lane 4 and lane 5 treated with 2 MIC and 1 MIC were
light-colored. This demonstrated that Cordyceps cicadae polysaccharide significantly destroyed the cell membrane, promoted the
extraction membrane proteins in the presence of Triton X-114.
The higher concentration Cordyceps cicadae polysaccharide caused
more significant damage to the cell membrane of E. coli. Our results
were consistent with the findings of other researchers. Nakayama
et al. (2013) found that epigallocatechin interacted with the outer
membrane porin protein of E coli cells, thereby inhibiting the
major function of porin. Kong et al. (2008) revealed that the targets
interacting with chitosan were likely proteins on the cell membrane. Moreover, Ibrahim et al. (2014)observed the differentiation
of proteins band and further concluded that chitosan effectively
destabilized the membrane proteins of Burkholderia cenocepacia.
4. Conclusions
Cordyceps cicadae polysaccharide had significant antibacterial
activity against Escherichia coli, Staphyloccocus aureus, Bacillus
subtilis, Salmonella paratyphi and Pseudomonas aeruginosa. MIC
to Escherichia coli was 0.10 mg/mL. Cordyceps cicadae polysaccharide could change the growth curve of E coli slightly. In addition,
Cordyceps cicadae polysaccharide could change the permeability
of cell wall and membrane, damage the membrane protein of E
coli, that resulted in the structural damage and release of cell components including proteins and electrolytes.
3.8. SDS-Page of membrane proteins of E coli
Membrane protein may play an important role in maintaining
the membrane permeability. Damage of membrane protein may
result in the damage of enzyme system integrity in bacteria membrane. The profiles of the control and E. coli cell proteins treated
with Cordyceps cicadae polysaccharide were showed in Fig. 5B.
The protein profiles of the control (lane 2) were blank. However,
the protein profiles of E coli treated with Cordyceps cicadae
polysaccharide were different. Moreover, compared to lane 3 treated with 4 MIC of Cordyceps cicadae polysaccharide, the protein
Acknowledgments
The study was supported financially by Xinmiao Talent Program
of Zhejiang Province (2016R423075) and Hangzhou Science and
Technology Development Plan (20150432B03).
Conflicts of interest
The authors declare no competing financial interests.
Y. Zhang et al. / Journal of Functional Foods 38 (2017) 273–279
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