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: email@example.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 274 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 276 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 277 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. 278 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. 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