Evaluation of characterization and disinfection efficacy of chlorocresol nanoemulsion disinfectant

Abstract

The aim of this study is to evaluate the characterization and disinfection efficacy of chlorocresol nanoemulsion disinfectant (CND). The characterization of CND was examined by observing the appearance with the naked eye, identifying the structure type with dye tests and dilution tests, investigating the morphology with transmission electron microscopy, and measuring droplet size with a laser particle size analyzer. Conductivity, viscosity and pH values were separately measured and the stability was assessed by centrifugation tests and accelerated tests. The disinfection efficacy of CND was investigated with quantitative bactericidal tests of a bacteria suspension of E. coli 8099 and on-the-spot disinfection tests of the object surface. The results showed that the appearance of CND was transparent and no phase separation occurred after centrifuging, the structure type was an O/W nanoemulsion, the morphology was spherical droplets and the mean droplet size was 27.43 nm with normal distribution. The conductivity, viscosity and pH values were 479.67 μs cm⁻¹, 204.33 cp and 2.52, respectively. CND showed good stability and could be stored for 2 years. In addition, CND exhibited strong bactericidal activity increasing with the increase of the disinfectant concentrations and disinfection time. Moreover, the bactericidal ability of CND was stronger than that of chlorocresol aqueous solution (P < 0.01). In conclusion, the results suggested that CND was an effective novel nanoemulsion disinfectant.

---

Introduction

Chlorocresol, a chlorinated phenolic derivative, is used as a disinfectant and preservative because it shows a strong bactericidal effect against gram positive and gram negative bacteria, fungi and mycobacterium tuberculosis. It has been used for disinfection of animal houses, vehicles, implements and environmental surfaces in recent years. Furthermore, it exhibits also bacteriostatic activity at the concentration (0.1–0.2%) normally employed in pharmaceutical products.¹ Chlorocresol not only has a bactericidal effect by penetrating and disrupting bacterial cytomembrane, and then leading to leakage of cellular inclusion and protein denaturation, but also bacteriostatic action by inactivating enzymatic activity of bacterial dehydrogenase and oxidase.² Obviously, chlorocresol has great potential disinfection value, but its poor water solubility and heavy phenol smell have seriously limited its applications. Hence, it is important to find a suitable delivery system for improving the solubility and masking the smell of chlorocresol to gain the reasonable disinfection efficacy.

Nanoemulsion is an isotropic and thermodynamically stable colloidal system with a mean droplet size in range of 10–100 nm.³ As an advanced mode of drug delivery system,⁴ nanoemulsion has been widely used in pharmaceutical systems in recent years. Acting as a drug carrier, nanoemulsion has incomparable advantages to other drug carriers: ① nanoemulsion belongs to thermodynamic stability systems, its stability is good and preparation and preservation are also easy. ② It can improve physical stability of drugs. ③ It has an ability of solubilizing water-insoluble drugs.⁵ ④ Drug encapsulated in nanoemulsion droplets is free from air, light and hard environment. Therefore, nanoemulsion can not only protect them from oxidation and hydrolysis but also possess an ability of sustained release.⁶ ⑤ It can intensify bioavailability of drugs^7,8 by dispersing them into nanoscale droplets having greater surface area providing greater absorption. ⑥ Because of small droplet size, nanoemulsion is transparent and uniform. It can greatly increase dispersity and permeability of drugs. ⑦ The most important application of nanoemulsion is to mask the disagreeable taste of some drugs.⁴ As a result, nanoemulsion has been considered as a promising ideal drug carrier. Many applications of nanoemulsion have been reported in disinfectant formulas.^9–12 In our previous study, chlorocresol nanoemulsion disinfectant (CND) had been prepared with nanoemulsion as a drug carrier, chlorocresol as a model drug. The disinfectant possesses high solubility, weak phenol smell and simple production process. Now the preparation method of CND has been applied for Chinese invention patent.¹³ In present paper, the characterization of CND was confirmed and its disinfection efficacy was investigated for future clinical applications.

Experimental

Test bacteria

E. coli 8099 was provided by animal pharmacology laboratory, Henan Institute of Science and Technology, China.

Preparation of CND

The blank nanoemulsion was formulated using four ingredients: non-ionic surfactant (polyoxyethylene castor oil-40, EL-40), glacial acetic acid, oil phase (isopropyl myristate, IPM) and distilled water. Firstly, four ingredients and chlorocresol were weighed respectively according to the amount in the recipe. In this recipe, mass ratio of EL-40 to glacial acetic acid and the prepared mixture to IPM was 3 : 2 and 9 : 1, respectively. After EL-40, glacial acetic acid and IPM were mixed evenly at room temperature, distilled water was dropwise added to the mixed liquid and kept stirring at the same time until transparent and uniform blank nanoemulsion carrier was formed. At last, CND was obtained after a certain amount of chlorocresol was thoroughly dissolved in the blank nanoemulsion. The nanoemulsion disinfectant can be diluted to the required concentration by distilled water and used in the following experiments.

Evaluation of characterization of CND

Identification of appearance. The appearance of CND was observed with the naked eyes. The disinfectant was centrifuged at 15 000 rpm for 15 min to test whether or not the phase separation (flocculation, creaming and cracking) existed according to the method previously described.¹⁴ The CND was represented good stability if phase separation was not observed.

Identification of structure type. Structure types of nanoemulsion, including oil-in-water (O/W), water-in-oil (W/O) and bi-continuous type, were confirmed by dye and dilution tests.⁴
Dye test. The type of nanoemulsion was identified by dye test with observing diffusion velocity of staining agents including water-soluble dye methylthionine chloride (blue) and oil-soluble dye Sudan red (red).¹⁵ If blue dye spread faster than red one, the disinfectant was O/W type nanoemulsion. Conversely, the disinfectant was water-in-oil (W/O) type.
Dilution test. An O/W nanoemulsion can be diluted with water, whereas a W/O nanoemulsion can be diluted with oil.⁴ In order to identify the type of CND, dilution test was carried out by water or oil added into the disinfectant.

Morphology observation

The morphology of CND droplets was observed by transmission electron microscopy (TEM, Hitachi, Japan). For negative staining, one drop of the disinfectant was placed on carbon-coated copper grids and air-dried. Then, the samples were stained immediately with 2% phosphotungstic acid for 15 min, washed three times with ultrapure water and dried naturally.

Mean droplet size and polydispersity index (PDI)

PDI indicates the uniformity of droplet size distribution in the nanoemulsion system. The higher the value of polydispersity, lower will be uniformity of droplet size of nanoemulsion.⁴ Mean droplet size and PDI of CND was measured by a laser scattering analyzer (Malvern, UK).

Conductivity, viscosity and pH measurements

Because high concentration of CND needed to be diluted by water when it was used in clinical, characteristic parameters such as conductivity, viscosity and pH of 10 times diluent were assessed. The conductivity was measured using a conductivity meter (DDS-307A, Shanghai Jingke, China). The viscosity was measured by a viscometer (RVA-TecMaster, Newport). The pH was determined using a digital pH meter (MP511, Shanghai Curui, China).

Stability assessment

Centrifugation test. CND was performed centrifuging test at 10 000 rpm for 2 h and followed by an examination for phase.⁶

Accelerated test. The accelerated test method was used for stability assessment of CND. The sealed CND was kept for 3 months at 37 °C. The drug contents were separately detected and the change in color was observed for 0 and 3 months. When more than 15% drug content decreased, the stability of the disinfectant did not conform to requirement.¹⁶

Evaluation of disinfection efficacy of CND. Disinfection efficacy of CND was evaluated according to “Technical standard for disinfection” (2008 edition) of China.¹⁶

Neutralizer screened test

Effective neutralization of chemical biocide is the first step in the accurate evaluation of disinfectants to avoid overestimation of the biocide activity.¹⁷ Hence the bactericidal evaluations of disinfectants require the use of an appropriate neutralizer. In the present study, the neutralizer was selected from three candidates including 3% (w/v, the same below) Tween-80 in PBS, 0.5% sodium thiosulfate in PBS and 1% Tween-80 + 1% lecithin + 0.5% sodium thiosulfate in PBS. And 5 × 10³ to 3 × 10⁴ cfu mL⁻¹ (colony forming units, cfu) of the bacteria suspension, the third generation of E. coli 8099, and 3.0% (w/v) bovine serum albumin were mixed at volume ratio 1 : 1 for using as test bacteria of the neutralizer screened test. The test was divided into eight groups (Table 1) and carried out with 0.20% of CND according to the suspension quantitative evaluation test procedures of neutralizer.¹⁶ The selected neutralizer and its concentration were determined appropriateness if deviation rate of colony count among group 3, 4 and 5 was not more than 15% and bacteria did not grow in 6, 7, 8 group. The screened test was performed in triplicate.

Table 1 Groups of neutralizer screened test

Group	Composition	Group	Composition
Group 1	Disinfectant + bacterial suspension	Group 5	Diluent + bacterial suspension
Group 2	(Disinfectant + bacterial suspension) + neutralizer	Group 6	Culture medium
Group 3	Neutralizer + bacterial suspension	Group 7	Diluent
Group 4	(Disinfectant + neutralizer) + bacterial suspension	Group 8	Neutralizer

---

Quantitative bactericidal test of bacteria suspension

The quantitative bactericidal test of bacteria suspension of E. coli 8099 was carried out at 20 °C ± 1 °C. The required different concentrations (0.05%, 0.06%, 0.08%, and 0.10%) of the disinfectant were prepared with sterile standard hard water. After 0.5 mL bacteria suspension (1 × 10⁸ to 5 × 10⁸ cfu mL⁻¹) and 0.5 mL 3.0% (w/v) bovine serum albumin as an organic interfering substance were mixed in sterile tubes, 4 mL disinfectant was added into tubes, quickly blended and timed immediately. When the reaction reached the scheduled time (5, 10, and 15 min), 0.5 mL sample solution was removed into the tube containing 4.5 mL of selected neutralizer and neutralized for 10 min. This sample was finally inoculated on the agar plate at 37 °C for 24 h to monitor viable bacteria counts. The killing log value of the bacteria was calculated with the following equation:

Killing log value (KL) = N₀ − N_x

where N₀ was the initial log value of the average viable bacteria concentration (cfu mL⁻¹), and N_x was the log value of the disinfectant group.

The bactericidal activity of the disinfectant was regulated to be qualified when KL was more than or equal to 5. Meanwhile, the parallel test as positive control was conducted with sterile standard hard water instead of disinfectant and negative control was also conducted. And the negative controls were performed with residual neutralizer solution, PBS diluent and culture medium, respectively. The test was performed in triplicate.

Comparison test of bactericidal effect

A comparison test of bactericidal effect was carried out between the same concentration (0.06%) of CND and chlorocresol aqueous solution according to the above-mentioned method of quantitative bactericidal test of bacteria suspension of E. coli 8099.

On-the-spot disinfection test of henhouse ground

In order to prove clinical disinfection efficacy, on-the-spot disinfection test of object surface was performed on henhouse ground of experimental animal center from Henan Institute of Science and Technology. The method of the test was as follows: ① on the ground, multiple adjacent plots were randomly blocked with 5.0 cm × 5.0 cm space. One of the plots was used as share sampling control plot before disinfecting, and the others were used as sampling treated plots after disinfecting. ② The sterile cotton swabs soaked and wiped in the tubes containing 10 mL PBS diluent were served for swabbing sampling in sampling control plot, which was used as a share positive control sample. ③ After different concentrations disinfectant (0.1%, 0.2%, 0.5%, 1.0%, and 2.0%) disinfected for different time (5, 10, and 15 min) in treated plots, the sterile cotton swabs soaked and wiped in the tubes containing 10 mL selected neutralizer solution were used for swabbing sampling. Afterwards, sampling ends of cotton swabs were cut into the tubes containing selected neutralizer solutions, which were served as samples of treated plots after disinfecting. ④ Samples from positive control plot and treated plots were all counted viable bacteria inoculated culture at 37 °C for 24 h and done 30 times. Meanwhile, the negative controls were conducted with residual neutralizer solution, PBS diluent, culture medium, respectively. When KL was more than or equal to 1, the bactericidal activities of the disinfectant against national bacteria of henhouse ground was qualified.

Statistical analysis

In comparison test of bactericidal effect, experiments were conducted in triplicate and data of KL on E. coli after disinfecting for different time (5, 10 and 15 min) were expressed as mean ± standard deviation (SD). Statistical analysis was performed by Student's t-test in SPSS 10.0 (SPSS Inc., Chicago, Illinois, USA). Significant differences were accepted at P < 0.05.

Results and discussion

Characterization of CND

Appearance of CND. The prepared CND was found to be clear, transparent and uniform in appearance with faint yellow color. After centrifugation for 15 min at 15 000 rpm, any flocculation, creaming, cracking, or other phase separation did not exist, indicating a good phase stability of CND.

Structure type of CND. In the dye test, the diffusion velocity of blue dye (methylene blue) was faster than that of red dye (Sudan red) in CND. Moreover, CND could be easily diluted with distilled water and remained stable in the dilution test. The two performed tests illustrated that CND was O/W nanoemulsion.

Morphology, mean droplet size and PDI of CND. The morphology of CND under transmission electron microscope was spherical droplets (Fig. 1). The size distribution, one of the most important physical characteristics of a nanoemulsion, analyzed by intensity was shown in Fig. 2. The mean diameter of droplet size was 27.43 nm with normal distribution, and PDI was 0.230 indicating the uniformity of droplet size of CND.

Fig. 1 Transmission electron micrograph image of CND (×100 000).

Fig. 2 Size distribution of CND.

Conductivity, viscosity and pH of CND. The characteristic parameters such as conductivity, viscosity and pH of CND were shown in Table 2. When CND was diluted by water, parameter values changed. Conductivity and pH of 5% CND were all lower than that of 0.5% CND. However, viscosity value of 5% CND was higher than that of 0.5% CND. The conductivity and viscosity studies suggested that nanoemulsion formulation was O/W type.¹⁸

Table 2 Conductivity, viscosity and pH of CND (mean ± SD, n = 3)

Different concentrations of CND	Conductivity (μs cm^−1)	Viscosity (cp)	pH
5% CND (w/w)	479.67 ± 1.53	204.33 ± 4.51	2.52 ± 0.04
0.5% CND (w/w)	724.33 ± 3.06	169.00 ± 5.00	2.83 ± 0.05

---

Stability of CND. After centrifuging at 10 000 rpm for 2 h, any creaming, cracking, flocculation or phase separation was not found, and CND was still clear and transparent indicating an excellent phase stability of the nanoemulsion.

CND showed good stability showing no phase separation, either flocculation or precipitation for 3 months at 37 °C. However, color was changed from faint yellow to yellow. The stability percentage was 93.17% chlorocresol in CND which indicated that less than 15% effective component decreased. According to the rule of accelerated test,¹⁶ CND could be stored for 2 years.

Disinfection efficacy of CND

Screened neutralizer of CND. Complete neutralization of disinfectants is important for the accuracy of a bactericidal assay as bactericidal activity is commonly measured as survivors with time and inhibition of microbial growth by low levels of residual disinfectants would lead to exaggerated measures of bactericidal activity.¹⁹ The selected neutralizer should not only be able to completely inactivate all of the bacteriostatic activity of the residual antimicrobial agent likely to be carried over into recovery media, but also be inherently non-bactericidal to the test organisms.²⁰ In the present study, when 3% Tween-80 in PBS served as the neutralizer, colony counts were similar among group 3, 4, and 5, and the deviation rate of colony count was 12.1% (<15%), whereas deviation rates using other two neutralizers were all more than 15% (Table 3). These data indicated that 3% Tween-80 in PBS could effectively neutralize the residual bactericidal activity of CND against test bacteria, and neutralizer and its neutralizing products had no adverse effects on bacteria and culture medium. On these grounds, 3% Tween-80 in PBS was selected as the appropriate neutralizer for the bactericidal evaluation of CND.

Table 3 Results of neutralizer screened test of CND against E. coli

Neutralizer	Colony count of each group/cfu mL^−1								Deviation rate of colony count among group 3, 4 and 5/%
	1	2	3	4	5	6	7	8
3% Tween-80 in PBS	0	0	80	60	80	0	0	0	12.1
0.5% sodium thiosulfate in PBS	0	0	160	20	80	0	0	0	56.3
1% Tween-80 + 1% lecithin + 0.5% sodium thiosulfate in PBS	0	0	100	40	80	0	0	0	30.3

---

Bactericidal activity of CND. In the quantitative bactericidal test of CND, log value of viable bacteria concentration decreased, and the corresponding KL increased after disinfecting for different time (Table 4), which indicated that bactericidal activity of CND enhanced with the increase of the disinfectant concentrations and disinfection time. KL was all more than 5 indicating the disinfection qualifications when 0.06% and 0.08% CND severally disinfected for 10 and 5 min. Furthermore, 100% of the bacteria were killed as 0.06%, 0.08% and 0.10% of the disinfectant acted for 15, 10 and 5 min, respectively.

Table 4 Results of the quantitative bactericidal test of CND against E. coli 8099^a

Groups	Log value of viable bacteria concentration after disinfecting for different time			KL on E. coli after disinfecting for different time
	5 min	10 min	15 min	5 min	10 min	15 min
a Log value of viable bacteria concentration was 7.43 in the positive control group, and no bacterial growth occurred in the negative control groups. No meant that no viable bacteria were found.
0.05% CND	4.40	3.23	3.00	3.02	4.02	4.43
0.06% CND	2.61	1.30	No	4.82	6.13	≥7.43
0.08% CND	1.00	No	No	6.43	≥7.43	≥7.43
0.10% CND	No	No	No	≥7.43	≥7.43	≥7.43

---

Comparison of bactericidal effect. As shown in Table 5, KL of 0.06% CND were all more than 5 after disinfecting respectively for 10 and 15 min, while KL of 0.06% chlorocresol aqueous solution were all less than 5 indicating disinfection disqualifications. Further, KL of CND after disinfecting for different time were all higher than that of chlorocresol aqueous solution (P < 0.01), which the bactericidal effect of CND was stronger than that of chlorocresol aqueous solution. The result suggested that nanoemulsified disinfectant could improve efficacy against microorganism more than unemulsified disinfectant, which was in accordance with a research reported by Ramalingam K. et al.²¹ The improvements in the efficacy of nanoemulsified drugs have also been reported.^22–24 In addition, it was reported that nanoemulsion structure itself had also extensive bactericidal activity.^9,25

Table 5 Comparison of bactericidal effect^a

Groups	KL on E. coli after disinfecting for different time
	5 min	10 min	15 min
a Log value of viable bacteria concentration was 7.47 in the positive control group, and no bacterial growth occurred in the negative control groups. Data is presented as mean ± standard deviation.b P < 0.01 (highly significant) compared between CND and chlorocresol aqueous solution.
0.06% CND	4.277 ± 0.031^b	5.137 ± 0.015^b	≥7.480 ± 0.000^b
0.06% chlorocresol aqueous solution	3.333 ± 0.025	3.823 ± 0.021	4.880 ± 0.026

---

On-the-spot disinfection efficacy of CND. In the on-the-spot disinfection test of CND, the viable bacteria concentration decreased until no growth of natural bacteria, and the corresponding KL increased with the increase of the disinfectant concentrations and action time (Table 6), which revealed the bactericidal activity of CND against natural bacteria on henhouse ground increased. KL was all more than 1 stating that the disinfection was qualified when 0.1% and 0.2% CND separately contacted for 15 and 5 min. Moreover, the killing rates were all 100% as 1.0% and 2.0% of CND disinfected for 15 and 5 min, respectively.

Table 6 Results of on-the-spot disinfection test of CND against natural bacteria on henhouse ground^a

Groups	Log value of viable bacteria concentration after disinfecting for different time			KL on natural bacteria after disinfecting for different time
	5 min	10 min	15 min	5 min	10 min	15 min
a Log value of viable bacteria concentration was 4.64 in the positive control group, and no bacterial growth occurred in the negative control groups. No meant that no viable bacteria were found.
0.1% CND	3.72	3.66	3.60	0.93	0.98	1.04
0.2% CND	3.56	3.58	3.38	1.09	1.06	1.26
0.5% CND	3.48	3.30	2.78	1.17	1.34	1.87
1.0% CND	2.90	1.60	No	1.74	3.04	≥4.64
2.0% CND	No	No	No	≥4.64	≥4.64	≥4.64

---

Nanoemulsion has been reported to have broad biocidal efficacy against bacteria, enveloped viruses, and fungi by disruption of their outer membranes. Moreover, because the mechanism of action of nanoemulsion appears to be the nonspecific disruption of bacterial cell membranes, nanoemulsion would not result in the development of resistant strains.²¹ Nanoemulsion is a new form of disinfectant composed of detergents and vegetable oil suspended in water,¹² and the application of nanoemulsion as disinfectant is a new and promising innovation. In recent years, some nanoemulsion disinfectants have been reported.^21,26,27 One research revealed that waterline biofilms exposed to cetylpyridinium chloride-containing nanoemulsion disinfectant for 1, 6, 12, 24, 48, and 72 h showed high reduction of colonies, and very low counts after 12 and 24 h were observed. In addition, the nanoemulsion employed improves efficacy against microorganism more than that of unemulsified components.²¹ EcoTru had been reported as a nanoemulsion disinfectant which is non-toxic, biodegradable, safe and no harmful impact on people and the environment.²⁶ Chepurnov et al. reported that the nanoemulsion ATB is an effective disinfectant for Ebola virus.¹² A compound germicidal nanoemulsion prepared by Wei et al. was a novel compound skin disinfectant containing chlorhexidine, antimicrobial nanomaterial and other ingredients. The killing rates for S. aureus, E. coli and C. albicans on cloth strips respectively exposed to this new disinfectant for 3, 3, and 5 min were all >99.90%. Moreover, the killing rates for three microorganism separately exposed for 5, 5, and 10 min were all >100%.²⁷ In the present study, the disinfection of the quantitative bactericidal test was qualified when 0.06% and 0.08% CND disinfected for more than 10 and 5 min, respectively. And in on-the-spot disinfection test of henhouse ground, the qualified disinfection was exhibited as 0.1% and 0.2% CND separately contacted for more than 15 and 5 min. Furthermore, the bactericidal effect of CND was stronger than that of chlorocresol aqueous solution (P < 0.01). These data clearly showed that the prepared CND was a novel nanoemulsion disinfectant with strong bactericidal activity, which suggested that it had potential as an effective disinfectant for use in animal housing facilities and environment. However, a few issues have yet to be addressed. Firstly, only E. coli 8099 as test bacteria was used in bactericidal activity, which was representative of clinically important microbes. More species have to be included in future studies to better define the nanoemulsion disinfectant anti-bacterial spectrum. Secondly, microbe species have not been identified in on-the-spot disinfection test. Currently, we are in the process of investigating these issues.

Conclusions

The prepared CND in this study has characterizations of transparent appearance, O/W type, spherical droplets, and 27.43 nm of mean droplets diameter with normal distribution. The conductivity, viscosity and pH values were 479.67 μs cm⁻¹, 204.33 cp and 2.52, respectively. It had good stability. CND exhibited the strong bactericidal activity increasing with the increase of the disinfectant concentrations and disinfection time. Moreover, the bactericidal ability of CND was stronger than that of chlorocresol aqueous solution. The results suggested that CND was an effective novel nanoemulsion disinfectant in clinical practice.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This study was supported by the grant of Characteristic Innovation Project (Grant No. 2015ZD09) and Undergraduate Innovative Experiment Projects (Grant No. 2014CX040) of Henan Institute of Science and Technology, and Development of Science and Technology Plan Projects of Xinxiang, Henan Province (Grant No. 15NY04).

References

1. The European Agency for the Evaluation of Medicinal Products. Committee for veterinary medicinal products: Chlorocresol, EMEA/MRL/074/96-FINAL, 1996, 1–3.
2. Z. L. Chen, Veterinary pharmacology, China Agriculture Press, Beijing, 2009, in Chinese.
3. M. J. Tsai, Y. S. Fu, Y. H. Lin, Y. B. Huang and P. C. Wu, PLoS One, 2014, 9, e102850.
4. M. Jaiswal, R. Dudhe and P. K. Sharma, Biotechnology, 2015, 5, 123–127.
5. Y. Liu, D. Zhang, D. Zou, Y. Wang, C. Duan, L. Jia, P. Xie, F. Feng, F. Wang, X. Zhang and Q. Zhang, J. Biomed. Nanotechnol., 2011, 7, 621–631.
6. Y. Zhang, Z. H. Shang, C. H. Gao, M. Du, S. X. Xu, H. W. Song and T. T. Liu, AAPS PharmSciTech, 2014, 15, 1000–1008.
7. Y. H. Gong, Y. K. Wu, C. L. Zheng, L. Y. Fan, F. Xiong and J. B. Zhu, AAPS PharmSciTech, 2012, 13, 961–966.
8. L. C. Chang, C. L. Wu, C. W. Liu, W. H. Chuo, P. C. Li and T. R. Tsai, J. Biomed. Nanotechnol., 2011, 7, 558–567.
9. T. Hamouda, A. Myc, B. Donovan, A. Y. Shih, J. D. Reuter and J. R. Baker Jr, Microbiol. Res., 2001, 156, 1–7.
10. A. Myc, T. Vanhecke, J. J. Landers, T. Hamouda and J. R. Baker Jr, Mycopathologia, 2001, 155, 195–201.
11. P. C. Teixeira, G. M. Leite, R. J. Domingues, J. Silva, P. A. Gibbs and J. P. Ferreira, Int. J. Food Microbiol., 2007, 118, 15–19.
12. A. A. Chepurnov, L. F. Bakulina, A. A. Dadaeva, E. N. Ustinova, T. S. Chepurnova and J. R. Baker Jr, Acta Trop., 2003, 87, 315–320.
13. X. F. Yang, R. F. Li, X. J. Ren, D. Y. Liu, Y. W. Sun, Z. L. Wang, H. H. Zhang, J. H. Hu, S. Y. Mu, Y. Z. Bai and Y. L. Shi, CHN Pat., 201510074247.2, 2015, in Chinese.
14. G. X. Liu, J. Y. Zhang, P. X. Wu, J. Y. Li, Y. Liu, X. Z. Zhou, X. J. Wei, J. R. Niu and B. Li, Sci. Agric. Sin., 2009, 42, 3328–3333.
15. H. O. Ho, C. C. Hsiao and M. T. Sheu, J. Pharm. Sci., 1996, 85, 138–143.
16. Ministry of Health of the People's Republic of China. Technical standard for disinfection. 2008. (in Chinese).
17. N. S. Sheraba, A. S. Yassin, A. Fahmy and M. A. Amin, Afr. J. Microbiol. Res., 2012, 6, 6565–6571.
18. M. R. Patel, R. B. Patel, J. R. Parikh and B. G. Patel, J. Pharm. Invest., 2013, 3, 1–12.
19. M. E. Eissa, M. S. El-Din Ashour and M. S. Mansy, World Appl. Sci. J., 2012, 20, 823–831.
20. W. Sheikh, Antimicrob. Agents Chemother., 1981, 19, 429–434.
21. K. Ramalingam, N. C. Frohlich and V. A. Lee, J. Dent. Sci., 2013, 8, 333–336.
22. L. J. Danielli, M. D. Reis, M. Bianchini, G. S. Camargo, S. A. L. Bordignon, I. K. Guerreiro, A. Fuentefria and M. A. Apel, Ind. Crops Prod., 2013, 50, 23–28.
23. Y. J. Kim, S. J. Houng, J. H. Kim, Y. R. Kim, H. G. Ji and S. J. Lee, J. Nutr. Biochem., 2012, 23, 186–191.
24. S. Vatsraj, K. Chauhan and H. Pathak, J. Nanosci., 2014, 2014, 1–7.
25. T. Hamouda, M. M. Hayes, Z. Cao, R. Tonda, K. Johnson, D. C. Wright, J. Brisker and J. R. Baker Jr, J. Infect. Dis., 1999, 180, 1939–1949.
26. http://www.infectioncontroltoday.com/news/2003/05/u-s-environmental-protection-agency-approves-envi.aspx, accessed August 2015.
27. Q. H. Wei, W. F. Zhang, C. D. Wang, R. Lu, J. Y. Wang, M. Zhang and X. G. Ji, Chin. J. Disinfect., 2004, 21, 1–4.

---

Footnote

† These authors contributed equally to this work.

---