Antifungal activity and humidity sensitivity of quaternized cellulose synthesized in NaOH/urea aqueous solution
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- Zhang, D., Xie, J., Yu, P. et al. Cellulose (2012) 19: 189. doi:10.1007/s10570-011-9626-8
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In this work we report the fabrication of cellulose-based humidity responsive material with antifungal activity. The quaternized cellulose (QC) derivatives with low degree of substitution (DS) values of 0.08–0.37 were synthesized in NaOH/urea aqueous solution. Water insoluble QC membranes (c-QCM) were prepared by casting from QC aqueous solutions, followed by crosslinking with glutaraldehyde. The c-QCMs were disintegrated in acid solutions, but were able to keep membrane shape in neutral and mild basic solutions with pH value of 7.2 and 9.7. The equilibrium water adsorption ratios of c-QCMs were in the range of 66–98%, depending on the DS values of quaternary ammonium groups and the pH value of the aqueous solutions. The antifungal activity of QC was evaluated and found that QC could effectively inhibit the reproduction of Rhizopus stolonifer, Aspergillusflavus and Penicillium digitatum, with minimum inhibitory concentration of 5, 10, and 7.5 mg/mL, respectively. The resistivity of the c-QCM changed for about 65–134 times corresponding to the change of environmental relative humidity from 20 to 99%; and the performance of c-QCM as a resistive-type humidity responsive material was consistent in the cycling of relative humidity.
KeywordsQuaternized celluloseAntifungal activitySwelling propertyHumidity response
Cellulose is the most abundant natural polymer on earth. Due to its good mechanical properties, biocompatibility and biodegradability (Cunha and Gandini 2010; Schurz 1999), cellulose has been developed into many types of environmentally friendly materials such as fibers, films and powders, with wide applications in areas of textiles, filtration, packaging, food additives and cosmetics, etc. In this work, a novel application as a humidity responsive material with antifungal activity was developed for quaternized cellulose derivative.
Contamination and diseases caused by moulds are of great concern in many areas, such as food packaging, food storage, building materials and so on. The conventional antifungal agents such as sorbic acid, potassium sorbate, imazalil and sodium ortho-phenylphenate, have been broadly applied as fungicides for fruits (Mehyar et al. 2011; Cabras et al. 1999; Montesinos-Herrero et al. 2011). However, significant concerns about the impact of these small molecule antifungal agents on the human health and the environment, and low durability are remained (Montesinos-Herrero et al. 2011). Because of the lower toxicity and long antifungal activity, antifungal polymers like polymeric quaternary ammonium compounds and polymeric N-halamine have been developed. For example, polymeric N-halamines were synthesized and used in antimicrobial paints, which provided antimicrobial efficiency against bacteria, fungi, and viruses (Cao and Sun 2009). The main concerns of polymeric N-halamines are its non-biodegradability after end use and the recontamination problem by the halogen (Hogan et al. 1979).
Due to the abundance, relative low cost, few allergy problems and biodegradability, naturally-occurring polymers such as chitosan and cellulose based antibacterial agents have been attracted many research focuses, especially in the age of seeking alternative sources of fossil. For example, quaternized chitosan derivatives show strong antibacterial efficacy against E. coli and S. aureus (Sajomsang et al. 2009; Ignatova et al. 2007). Sun et al. grafted 2-amino-4-chloro-6-hydroxy-s-triazine (ACHT) and methacrylamide (MAA) onto cotton cellulose (CC) and then the ACHT-immobilized and MMA-grafted cotton fabrics were chlorinated. The functionalized CC showed effective antimicrobial activities (Chen et al. 2007a; Luo and Sun 2006). Zhou et al. demonstrated that quaternized cellulose (QC) showed antimicrobial activity against Gram-negative bacteria (E. coli) and Gram-positive (S. aureus). The minimum inhibitory concentration (MIC) values of QC with DS value of 0.74 were 0.025 and 0.0125 wt% against E. coli and S. aureus, respectively (Song et al. 2010, 2008). However, the antifungal performance of these functional cellulose and chitosan derivatives has been rarely reported. In this study, QC is developed as a novel polymeric fungicide to inhibit the reproduction of the fungi Aspergillus flavus (A. flavus), Rhizopus stolonifer (R. stolonifier) and Penicillium digitatum (P. digitatum). The QC has stable antifungal functional groups, good water solubility, biocompatibility and biodegradability (Rodríguez et al. 2001). Hence it is a very promising green antifungal agent or component in antifungal paints, food packaging and food storage.
Polymers containing quaternary ammonium groups have been fabricated into potential humidity sensor materials. Lv et al. (2009) applied UV irradiated crosslinking approach to obtain a water-resistant resistive-type humidity sensitive film of poly(dimethyl-butyl-(ethyl-methacrylate)ammonium bromide) with high sensitivity (impedance of 103–107 Ω corresponding to the humidity range of 22–97%). Another humidity sensor prepared from an interpenetrating network of poly(dimethylaminoethyl methacrylate) and poly(glycidyl methacrylate) (Li et al. 2007) displayed impedance of 103–106 Ω corresponding to a humidity range of 20–97% and quick response time (<30 s). The quaternized cellulose is a quaternary ammonium salt and thus it may also display humidity responsive ability. Compared to other quaternary ammonium cationic polyelectrolytes synthesized by a bottom-up polymerization method in organic solvents (Lv et al. 2009; Li et al. 2007), the QC is easily fabricated by introducing quaternary ammonium groups onto cellulose in NaOH/urea aqueous solution under mild conditions. The QC is crosslinked by glutaraldehyde at 25 °C to obtain a water-insoluble resistive-type humidity responsive material. In combination of the antifungal and antibacterial efficiency, humidity sensitivity, and facile synthesis in an aqueous solution, the applications of the cellulose derivative QC will definitely be expanded to more fields.
The following materials were used: cellulose powder (CF11, Mw: 3.46 × 104, Whatman Ltd., UK); 3-chloro-2-hydroxypropyltrimethylammonium chloride powder (CHPTAC, Shanxi Dasheng Chemical Co., Ltd., Shanxi, China) was used without further purification; glutaraldehyde (25 wt% aqueous solution, Tianjin Fuchen Chemical Agent Co., Ltd., Tianjin, China); dialysis bag (MWCO: 7000, Shanghai Green Bird Science and Technology Development Co., Ltd., Shanghai, China). The reagents were analytical grade and used as received. The fungi A. flavus, R. stolonifer and P. digitatum were kindly provided by College of Life Science of Fujian Normal University, Fujian, China.
Quaternized cellulose derivatives by chemical modification
Nitrogen and carbon contents, DS value of quaternized cellulose and original cellulose
Mole ratio of CHPTAC/AHG
DS values obtained from N%
Preparation of crosslinked QC membranes
0.5 g of QC was added into 14.5 g of distilled water to make a 3.3 wt% QC aqueous solution. The solution was cast on a glass plate, and was air-dried at 26 °C to obtain the QC membrane (QCM).
To fabricate a crosslinked QCM, the QCM was immersed into a 7 wt% glutaraldehyde methanol solution with a composition: 7 wt% glutaraldehyde, 1 wt% hydrochloric acid, and 92 wt% methanol. The crosslinking reaction was performed at 25 °C for 4 h on a shaking bath. Then the QCM was thermally treated in an oven at 50 °C for 10 h to obtain the crosslinked QC membrane coded as c-QCM. The c-QCM was rinsed with distilled water to completely remove the uncrosslinked QC and the residual reactants. It was then dried in a 80 °C vacuum oven for 8 h, and stored in a desiccator before use.
Antifungal activity of QC
The fluid nutrient medium method was used to study the antifungal efficiency of QC. Firstly, a potato was peeled and shredded. 150 g of potato fragments were put into 500 mL distilled water and boiled for 2 h. The supernatant was collected by filtration. Into it 5 g of glucose was added and the solution was boiled for 1 h. The fluid nutrient medium (FNM) was obtained. Secondly, into FNM, varied volume of 1 wt% QC aqueous solution was added to reach QC concentration of 2.5, 5.0, 7.5, and 10.0 mg/mL. In such a way, the antifungal fluid nutrient medium (AFNM) was obtained. Thirdly, 20 μL of 1–2 × 104 colony-forming unit (CFU)/mL fungal aqueous suspension was added into 1.5 mL finger tub, followed by adding AFNM to reach a total volume of 1.5 mL. After inoculation, the fungal was incubated at 26.5 °C for 30 h in an incubator (Valencia-Chamorro et al. 2008; Clausen and Yang 2007). Inoculation of 1–2 × 104 CFU/mL fungal aqueous suspensions in FNM was run as blank control. After a certain time interval of incubation, the fungal proliferation on FNM was observed on a light microscopy(OLYMPUSBX51-DP70). From the hyphae filament density on the FNM, the antifungal efficiency can be determined. MIC is defined as the lowest QC concentration required to inhibit the reproduction of a fungal, at this concentration no obvious hyphae filaments are observed. The colonies were counted and the MIC values were obtained. Each sample was tested in triplicates.
Water absorption ratio of c-QCMs
Measurement of humidity response of c-QCM
Results and discussion
Antifungi activity of QC
Similar to the antimicrobial mechanism of quaternary ammonium compounds (Franklin and Snow 1981), the antifungal mechanism of QC compounds is supposed to be triggered by the adsorption of QC onto the fungal cell walls under the attracting forces such as electrostatic force (positive QC-negative fungal pairs), hydrogen bonding (QC-fungal proteins pairs) and hydrophobic interaction (QC-fungal proteins pairs). This binding to the cytoplasmic membrane blocks the permeable channels for the mass transportation therefore inhibiting the reproduction of fungal cells. It has been proved that high positive charge density on the antibacterial polymers may enhance the driving force for their binding to cytoplasmic membrane, and hence strengthening their antibacterial activity (Chang et al. 2010). This well explains that QC-5 with the highest DS of quaternary ammonium group shows the best antifungi activity. With respect to the renewability and biodegradability, QC based fungicide would be more favorable over petroleum based ones like quaternary ammonium polystyrenes (Chen et al. 2007b).
Water absorption ratio of c-QCM
Humidity sensitivity of c-QCM
The quaternized cellulose (QC) derivatives with low DS values in the range of 0.08–0.37 were synthesized in NaOH/urea aqueous solution. The QC samples displayed effective antifungal activities against R. stolonifer, P. digitatum and A.flavus, with minimum inhibitory concentration of 5, 10 and 7.5 mg/mL, respectively. QCs were crosslinked with glutaraldehyde to render them water-resistant. The membrane morphology of crosslinked QC membranes was maintained in neutral and mild basic solutions, but their water adsorption ratios were up to 166–198% of their original weight. The equilibrium water adsorption ratios were highly dependant on the DS values of the quaternary ammonium group and the pH values of the aqueous solutions. The crosslinked QC membranes were fabricated into resistive-type humidity responsive materials. They showed stable performance and high sensitivity to the environmental humidity. For example, the impedance of QC (DS = 0.25) decreased from 695.7 to 5.2 Ωm as the humidity increased from 20 to 99%. Due to the low DS value of quaternary ammonium groups, the current QCs showed slow response.
This work is supported by the National Basic Research Program of China (2010CB732203), the National Natural Science Foundation of China (No. 50973019, 50843030), the Natural Science Foundation of Fujian Province (2010J06017).