Anrong Zeng,Yangtao Wang,Dajun Li,Juedong Guo,Qiaowen Chen

College of Materials and Chemical Engineering,Liming Vocational University,Quanzhou 362000,China

Keywords:Chitosan Antibacterial properties Blend Polymers Mechanical properties Quaternized chitosan

ABSTRACT This article is a preliminary study on antibacterial blends of polycaprolactone,chitosan and quaternized chitosan by melt processing.Blends were characterized,mechanical test and antibacterial evaluation against Escherichia coli and Staphylococcus aureus,were conducted.Results showed that the antibacterial potential of chitosan was limited in blends and polycaprolactone/chitosan did not show significant antibacterial effect compared with neat polycaprolactone (PCL).Inhibition rates of polycaprolactone/quaternized chitosan were 39.2%–99.9% against Escherichia coli,while inhibition rate was 40.9%–99.9%against Staphylococcus aureus.When quaternized chitosan (QCTS) content was up to 20%,blends exhibited 99.9% inhibition rates against both two types of bacteria.

1.Introduction

Chitosan (CTS) is a linear polysaccharide consisting of (1,4)-linked 2-amino-deoxy-β-d-glucan.It is a deacetylated derivative of chitin,which is the second most abundant polysaccharide in nature [1,2].It was attractive due to its numerous properties such as biodegradable,non-toxic,biocompatible,and antibacterial properties[3].It has been widely used in food reservation,water treatment,drug delivery,cosmetics,etc.

The antibacterial property as well as the biodegradability make CTS a suitable candidate for antibacterial food packaging [4].CTS exhibited antibacterial properties against a wide range of bacteria,yeasts,and fungi [5],which could help to extend the shelf life of food products.Enzymatic degradation property of CTS also makes it a suitable material for food packaging purposes.The hydrolyzes enzymes which degrade CTS are widely distributed in nature [4].When the role of CTS is over,it could go back to nature.

Various methods have been developed to prepare CTS films,such as direct casting,coating,layer-by-layer assembly,and melt processing [4,6].CTS could be used alone as antibacterial films[7,8],but mostly it displayed relatively weak mechanical properties as well as weak processability.Blend films with plastic matrix could be a better option.Solution blending or solvent casting were easily adopted in lab.However,they might not be welcome in industrial production.Solvent removal such as evaporation required complicated process and consumed a lot of energy.Solvent residue also raised safety concern.Melt processing of CTS films draw more interest,since it was of high yield,less need of space,and greater control of the final product’s characteristics[9,10].For example,blends of CTS with polyolefin [3,5,9,11–14],or polyester[15,16]were studied.Composites could combine each component’s advantages.Plastic matrix could exhibit good mechanical properties,processability,stability,while additives could exhibit antibacterial properties.More efforts could be devoted to exploring more blends of CTS with synthetic polymers by melt processing.

However,there was still a lot of room for improvement about CTS blend films.Firstly,the antibacterial performance of CTS was sensitive to pH.One of the proposed antibacterial mechanism was the interaction between the cationic group of CTS and the anionic bacterial cell wall peptidoglycans[4].However,CTS is hard to dissolve in neutral or alkaline environment and the amino groups cannot be protonated.It was reported that the antibacterial properties were low or even negative in neutral or alkaline environment[17].If CTS in films was not exposed to acid environment,its antibacterial performance might be lower.Secondly,high processing temperatures might lead to degradation.It is necessary to take the processing temperature into account to preserve the maximum antibacterial properties when selecting film matrix.

Therefore,it was proposed that CTS derivatives with high antibacterial activities in neutral or alkaline condition to be added into films.Quaternized CTS (QCTS),which has high antibacterial performance [18,19] and low toxicity [20,21],could be used to replace CTS.Also,polycaprolactone (PCL) could be selected as matrix.PCL is a kind of synthetic polymers which is non-toxic[22,23] and environmentally friendly [24,25].It is widely used in tissue engineering,wound dressing,or food packing due to its degradability,biocompatibility,superior rheological properties,and viscoelastic properties [26].It is thermoplastic and its low melting point (around 60 °C) made it a perfect melting matrix for CTS and its derivatives.

Many attempts were made to blend PCL with CTS.Most of them used solution blending method [27–29].PCL and CTS were dissolved in solvents to prepare homogenous mixture and then blends were obtained by casting or evaporation.As for melt process,several attempts were reported.Correlo [15] prepared CTS blends with several aliphatic polyester including PCL.Thermal properties,mechanical properties and compatibility were evaluated.Results showed that the content of CTS by mass added to PCL could be up to 50%.CTS displayed intermediate adhesion to the polyester matrix.PCL/CTS blends displayed fibrous appearances at the fractured surface.However,the antibacterial performance of the final blends was not tested.Wu [30] blended acrylic acid grafted PCL(PCL-g-AA)with CTS in molten state.PCL-g-AA improved the compatibility and mechanical properties of the blend.Antibacterial ability was not determined neither.No study about PCL and derivatives of CTS by melt process was reported.

In this study,we conducted preliminary trail to prepare blends of PCL and QCTS.PCL/CTS blends were prepared as well for comparison.Blends were characterized with Fourier transform infrared spectroscopy(FTIR),1H nuclear magnetic resonance spectroscopy(1H NMR),X-ray photoelectron spectroscopy(XPS),thermogravimetric analysis(TGA)-differential thermal analysis(DTA)and scanning electron microscope(SEM).Tensile properties and antibacterial properties were also tested.This study could be helpful to explore more antibacterial plastics.

2.Materials and Methods

2.1.Materials

CTS in powder form was purchased from Sinopharm Chemical Reagent Co.,Ltd.PCL,CAPA-6800,was provided by Perstorp,with molecular weight (MW) of around 80000,melting temperature of 58–60 °C,and melt flow index of 7.3 g﹒min-1at 190 °C,according to specifications from the manufacturer.Gentamicin was produced by Fujian Huitian Bio-pharma Co.,Ltd.All other reagents were provided by Xilong Scientific,Co.,Ltd.,and used as received.

2.2.QCTS synthesis

QCTS was obtained in lab by referring literature [31].The reaction formula was showed in Fig.1.5 g CTS was immersed in 100 ml isopropanol in a flask,stirred sufficiently,heated,and refluxed in 75°C water bath.25 g 2,3-epoxypropyltrimethyl ammonium chloride was dissolved in 40 ml deionized water and was added dropwise into CTS/isopropanol mixture.After reaction for 8 h,the solution was poured into ethyl alcohol to get precipitates.Precipitates were rinsed with ethyl alcohol for 3 times.They were further dissolved in deionized water and the solution was dialyzed for 48 hours to remove unreacted reactants.Then the solution was concentrated with 60 °C rotatory evaporator under vacuum.Finally,concentrates were lyophilized to get the final product QCTS.The yield,which was defined as the ratio of quaternized chitosan and added chitosan [32],was around 60%–70%.

2.3.PCL blends preparation

Formulations of each blend were demonstrated in Table 1.PCL,CTS and QCTS were dried in a vacuum drier at 40 to 50 °C to remove moisture.Blends were prepared in a HAPRO RM-200A torque rheometer (China).PCL was firstly fed into the batch mixing chamber and melted.CTS or QCTS was further fed into the mixer and blended with PCL thoroughly in the following conditions:mixing temperature of 110 °C and the rotational speed of 20 r﹒min-1.When the torque value was stable,the mixing was continued for another 15–20 min.Then blends were removed from the chamber and further compressed to sheets.Sheets with thickness of 2.0 mm and 0.5 mm were prepared.Compressing pressure was set at 10 MPa,temperature was set at 85 °C,and pressing time was set at 10 min.Dumbbell specimens were cut from sheets of 2.0 mm thickness and tested for mechanical properties.Sheets of 0.5 mm were cut into 26 mm × 26 mm and were used for antibacterial properties.

Table 1 Mass (%) of each component in blends

Table 2 The carbon,oxygen,nitrogen,and chlorine atomic concentration of CTS and QCTS

2.4.Characterization

2.4.1.Degree of deacetylation (DD) determination of CTS

DD of CTS was 80%–95% according to specification and was reevaluated in lab with acid-base titration method [33].0.15 g CTS was dissolved in 20 ml standard solution of HCl(0.1 mol﹒L-1)with magnetic stirring.The mixture was further diluted with 20 ml deionized water.Excess HCl was back titrated with standard solution of NaOH(0.1 mol﹒L-1).Two drops of 0.1%methyl orange-aniline blue (1:2,v/v) water solution were used as the indicator.The titration ended when the color of the solution changed from purple to blue-green.The titrations were performed in triplicates.Water content of CTS was measured under 105 °C and repeated 3 times to obtain the average.DD was calculated by Eq.(1):

Fig.1.The reaction formula of QCTS.

where C1and C2were concentrations of the standard solution of HCl and NaOH (mol﹒L-1),respectively.V1was the volume of HCl (ml),and V2was the volume of NaOH at the end point of titration (mL).0.016(g)was amino group content equal to 1 ml HCl standard solution(1 mol﹒L-1).0.0994 was the ideal amino group content of chitin.G was the CTS sample mass (g).W was the average water content(%).

2.4.2.MW determination of CTS

MW of CTS was determined by viscometric method [9].CTS powder was dried under vacuum at 60 °C to remove moisture and 0.5 g CTS was accurately weighed at room temperature.Then CTS was dissolved in a solution with 0.2 mol﹒L-1sodium acetate and 0.3 mol﹒L-1acetic acid.Five concentrations of CTS solutions were prepared:0.25,0.50,0.75,1.00 and 1.25 mg﹒mL-1,respectively.The relative viscosity was measured in a constant temperature water bath ((25 ± 0.5) °C) by using an Ubbelohde capillary viscometer.Intrinsic viscosity [?] of CTS was obtained by extrapolating the reduced viscosity versus concentration data to zero concentration,and the intercept on the ordinate was the [?] value.MW was calculated based on the Mark-Houwaink equation as follows:

where[?]was the intrinsic viscosity,M was the average MW,K and α were empirical constants.Values of K and α were 3.04×10-5and 1.26,respectively.This determination was tested for 3 times to get the average.

2.4.3.Degree of substitution (DS) of quaternary ammonium groups determination

DS of QCTS was determined by precipitation titration method[34,35].QCTS was dried at 60 °C under vacuum and 0.1 g QCTS was accurately weighed.QCTS was then dissolved in 50 ml deionized water with magnetic stirring.Two drops of 5% (w/w) K2CrO4solution were used as indicator.AgNO3(0.02 mol﹒L-1) solution was used as a titration reagent.The titration was ended when brick red precipitates were generated.The volume of the consumed AgNO3solution was recorded and marked as V.The blank test with 50 ml deionized water only was tested as well,and the volume of the consumed AgNO3solution was marked as V0.DS(%)was calculated according to Eq.(3).where V and V0were the volumes of AgNO3solution (L) consumed during titration of QCTS and blank sample,respectively.C was the concentration of AgNO3solution(mol﹒L-1).W was the mass of QCTS(g).M1was the molar mass of CTS structural unit(161.16 g﹒mol-1),while M2was the molar mass of QCTS structural unit(313.80 g﹒mol-1).This determination was performed 3 times to get the average.

2.4.4.FTIR analysis

FTIR of CTS and QCTS were compared by using KBr pressed disks.ATR-FTIR was applied to examine blends (PCL,PCL/CTS-20,PCL/QCTS-20).All tests were performed with NICOLET 5700 FT-IR Spectrometer and scanned from 4000 cm-1to 400 cm-1.

2.4.5.1H NMR analysis

1H NMR spectra of CTS (in CF3COOD) and QCTS (in D2O) were obtained on Bruker Advance AV 400 MHz (Switzerland) at room temperature.

2.4.6.XPS analysis

X-ray photoelectron spectroscopy (XPS,PHI 5000 Versaprobe-II) analyses were conducted and Monochromatic Al Kα X-ray was employed.The atomic concentration of chlorine was adopted to calculate the DS of QCTS according to Eqs.(4) and (5).

where CI (%) was the atomic concentration of chlorine of QCTS.NQCTSwas the number of atoms without Hydrogen in QCTS,which was 20 in total.NCTSwas the number of atoms without Hydrogen in CTS,which was 11 in total.

2.4.7.Thermal analysis

Thermogravimetric analysis (TGA) was carried out on a Q600(TA instruments,USA).Samples were taken and heated upto 800°C at a heating rate of 10°C﹒min-1under nitrogen flow.Blends including PCL,PCL/CTS-20,and PCL/QCTS-20 were tested.

2.4.8.Scanning electron microscopy (SEM)

A typical specimen from each composition was selected and cryogenically fractured in liquid nitrogen.Fracture surface was then gold-sputtered and studied with scanning electron microscope (SEM,Hitachi S4800,Japan).

2.4.9.Solubility determination

Solubilities of CTS and QCTS were determined as follows [32]:Dried CTS (1 g) was dispersed in 10 ml of 0.5% (v/v) acetic acid solution with magnetic stirring.Excess amount of acetic acid solution was added to dissolve the sample completely at room temperature.Dried QCTS (1 g) was dispersed in 10 ml of distilled water and 10 ml of 0.5% (v/v) acetic acid solution,respectively.Excess amount of solvent was added to dissolve the sample completely at room temperature.

2.5.Mechanical analysis

Dumbbell specimens for mechanical tests were cut from sheets of 2.0 mm.Tensile properties were measured using CMT6203 universal testing machine (USA).Samples were conditioned at room temperature for at least 48 h before testing.A constant stretching rate of 100 mm﹒min-1was applied.Mechanical properties were determined from the average of at least 6 measurements.

2.6.Antibacterial activity evaluation

The antibacterial activities were evaluated as follows.Minimum inhibitory concentration(MIC)and Minimum bactericidal concentration (MBC) were adopted to evaluate CTS and QCTS solution.Shake flask method were used to test samples by referring Chinese national standard GB/T 20944.3-2008.

Escherichia coli (ATCC 29522) and Staphylococcus aureus (ATCC 6538) were used and provided by Shanghai Luwei Microbial Sci.&Tech Co.Ltd.Bacterial suspensions were prepared according to GB/T20944.3-2008 and were further used for sample inoculation.Nutrient broth (beef extract 3 g,peptone 5 g,and distilled water 1000 ml) and nutrient agar medium (beef extract 3 g,peptone 5 g,agar powder 15 g,and distilled water 1000 ml)were prepared.Unless otherwise specified,all glassware was autoclaved at 121°C for 25 min prior to used.

2.6.1.MIC and MBC of CTS and QCTS

The concentrations of CTS and QCTS for MIC and MBC were set at 1000,900,800,700,600,500,400,300,200,and 100 μg﹒ml-1.Samples were divided into 5 groups.In group 1,different amounts of CTS powder were suspended in nutrient broth (neutral) to obtain CTS suspensions.In group 2,CTS was dissolved to different concentrations with nutrient broth (with 0.5% acetic acid).Similarly,QCTS was dissolved in nutrient broth (neutral) in group 3,while QCTS in group 4 was dissolved in nutrient broth (with 0.5%acetic acid).Nutrient broth with 0.5% (v/v) acetic acid was treated as the negative control.200 μl of 40000 μg﹒ml-1gentamicin was added into 2 ml sterile nutrient broth and treated as the positive control.Nutrient broth alone was treated as a blank sample.All samples were sterilized in 121 °C autoclave for at least 25 min and then cooled in clean benches for further test.3 repeats for each sample with different concentrations were conducted.

MIC and MBC against Escherichia coli and Staphylococcus aureus was evaluated by referring previous reports [36,37].A single colony of bacteria were individually taken using a platinum loop and were inoculated into 20 ml of nutrient broth.The suspension was incubated at 37 °C for 24 h.Then this inoculum was diluted with 0.03 mol﹒L-1phosphate buffer solution(PBS)and the concentration was adjusted to 1.5×108CFU﹒ml-1(determined by McFarland standard).The suspension was further diluted to 1.5 × 107CFU﹒ml-1with nutrient broth and used for MIC and MBC test.

2 ml of each solution from all groups was added with 100 μl bacteria suspension in tubes.The tubes were incubated at 37 °C for 24 h.The tubes were then examined for visible signs of turbidity.The lowest concentration of CTS and QCTS that inhibited the growth of bacteria was considered as MIC.

To determine the MBC for each group,500 μl from each tube without visible growth was transferred to agar plates(2 replicates per tube),and signs of growth after 24 h incubation at 37 °C were recorded.If no growth was observed,the original tube contained no living bacteria and the CTS and QCTS considered being bactericidal at that concentration (MBC).

2.6.2.Shake flask method

Shake flask method was conducted according to Chinese national standard GB/T 20944.3-2008.Each type of blends(0.5 mm in thickness) for this method were randomly cut into pieces with 26 mm × 26 mm in area.The mass of 2 pieces was approximately equal to (0.75 ± 0.05) g as required.Blend samples were sterilized with 75% alcohol and transferred to clean benches with UV light sterilization for another 20 min for each side to reduce possible bacteria contamination.

Samples for this method were divided into 3 groups.Group 1 was neat PCL.Group 2 contained PCL/CTS and PCL/QCTS blends with different compositions.Group 3 was set as blank group,using sterile water.Each sample had 3 repeats.As required by the standard,blank controls(group 3)were used to observe the activities of test bacteria during test period and prevent virtual test results caused by its own attenuation.

Samples were prepared,sterilized as above,and immersed into 250 ml conical flasks containing 70 ml PBS solution.Each flask was inoculated with 5 ml bacteria culture at the concentration of 3 × 105to 4 × 105CFU﹒ml-1.Immediately,Group 1 and 3 were put in a shaking incubator (250 r﹒min-1) at 24 °C for 1 min.After that,dilutions at 10-2from each flask were prepared.1 ml of 10-2dilution was evenly deposited on a petri dish (Diameter of 90 mm) and approximately 15 ml nutrient agar (50 °C) was poured into the dish and cooled to room temperature.2 agar plates were prepared for each dilution.All agar plates were marked as ‘‘0-Hour”and placed in an incubator at 37 °C for 24 h.Finally,CFU of each plate were counted and recorded as‘‘0-Hour”data.

Group 1,2,3 were then put in a shaking incubator(150 r﹒min-1)at 24°C for 18 h.After that,4 dilutions at 10-1,10-2,10-3and 10-4of each culture were prepared.2 agar plates were prepared for each dilution.All agar plates were marked as ‘‘18-Hour”and placed in an incubator at 37°C for 24 h.Then CFU of each plate were counted and recorded as ‘‘18-Hour”contact data.

The inhibition rate for each sample was calculated according to Eq.(6).

where R was inhibition rate of each sample.Wtwas the average bacteria concentration in group 1 after 18 h incubation (CFU﹒ml-1).Qtwas the average bacteria concentration in group 2 after 18 h incubation (CFU﹒ml-1).

The validation of antibacterial test was conducted according to Eq.(7) as mentioned in GB/T 20944.3-2008.When F ≥1.5,as well as the bacteria concentrations in group 3 were increased after 18 h,the test could be determined to be valid.

Fig.2.FTIR spectra of CTS and QCTS.

Fig.3.ATR-FTIR spectra of neat PCL and different blends.

Fig.4.1H NMR of CTS (a) and QCTS (b).

where F was the Log increase of bacterial load in group 1 in 18 h incubation.Wtwas the average bacteria concentration in group 1 after 18 h incubation(CFU﹒ml-1).W0was the average bacteria concentration in group 1 of 0 h (CFU﹒ml-1).

2.7.Statistical analysis

Data collected in mechanical and antibacterial analysis were expressed as mean ± SD.Data were determined using one-way analysis of variance (ANOVA).The level of significance was set at P=0.05.

3.Results and Discussion

3.1.Characterization

3.1.1.DD of CTS

Average DD of the raw chitosan was 93.4% ± 0.5% (n=3) and was within the range provided by the manufacturer (80%–95%).

3.1.2.MW of CTS

Intrinsic viscosity of CTS was(452±10)ml﹒mg-1(n=3),and the estimated MW was 493000 ± 8000 calculated by Mark-Houwaink equation.

3.1.3.DS of QCTS

DS were related to the average number of substituted quaternary ammonium groups per glucosamine unit.DS obtained from precipitation titration was(80.5±3.0)%(n=3).The atomic concentration of chlorine of XPS was also adopted to calculate the DS of QCTS (Table 2).The calculated DS% was around 69.3%.Since XPS only analyzed the surface,identified the composition on a selected area,and did not consider Hydrogen element in CTS and QCTS,we took DS calculated by XPS results as supplement data only.

3.1.4.FTIR

FTIR spectra were showed in Fig.2.The broad peak at 3600–3200 cm-1was the stretching vibration of O—H and N—H [38].Peaks at 2920 cm-1and 2870 cm-1may belong to antisymmetric and symmetric stretching vibration respectively of C—H in —CH3and —CH2— groups [39,40],which could be observed in both CTS and QCTS.For CTS,peaks at 1597 cm-1was the characteristic bands of the N—H bending vibration and belonged to the —NH2[39,40].While for QCTS,this N—H bending disappeared,indicating the substitution of the primary amine groups [20].Peaks at 1483 cm-1of QCTS indicated the existence of—CH2—N+and could be also assigned to the quaternary ammonium salt group [41].Characteristic peaks of the hydroxyl and second hydroxyl group of chitosan were almost unchanged comparing QCTS and CTS:Peaks at 1148 cm-1,1068 cm-1,and 1026 cm-1could be attributed to C—O—C bonds of QCTS,and were similar with peaks at 1156 cm-1,1092 cm-1,1030 cm-1in CTS.Therefore,it could be concluded that the substitution was mainly occurred at the amino groups of CTS.

Fig.5.XPS of CTS (a) and QCTS (b).

ATR-FTIR was applied to compare PCL,PCL/CTS-20,and PCL/QCTS-20.Results were showed in Fig.3.An intense peak at 1720 cm-1was observed in neat PCL,which could be ascribed to the C—O stretching of the ester carbonyl group in PCL [22].Peaks at 2943 cm-1(C—H asymmetric stretching vibration),2866 cm-1(C—H symmetric stretching vibration),1469 cm-1(C—H bending vibration) and 1419 cm-1(C—H shear vibration) were also the characteristic peaks of PCL and could be observed for all tested blends.For blends with CTS and QCTS,1597 cm-1and 1531 cm-1could be assigned to —NH2.Since ATR-FTIR examined the functional groups on surface,it might result in little difference between PCL/CTS-20 and PCL/QCTS-20.

3.1.5.1H NMR analysis

1H NMR spectra of CTS and QCTS were showed in Fig.4 and analyzed by referring literature[42,43].For CTS,chemical shifts at 4.54 could be assigned to [H1],chemical shifts at 3.50–4.20 could be assigned to [H3]–[H6],and shifts at 2.79 could be assigned to[H2].For QCTS,an obvious characteristic peak at 3.14 could be ascribed to trimethy ammonium groups.Shifts at 3.56–4.23 could be assigned to [H3]–[H6].Shifts at 2.46 could be assigned to [H2],[Ha]and[Hc].Shifts at 4.46 could be assigned to[H1]and[Hb].1H NMR spectra also indicated that the substitution was successful.

3.1.6.XPS analysis

XPS was applied to examine the chemical structure of QCTS.XPS wide scan spectra of CTS and QCTS were showed in Fig.5.In case of CTS,carbon (C1s),oxygen (O1s),and nitrogen (N1s) were detected.In case of QCTS,a new peak of chlorine (Cl2p) was detected.

Fig.6.TGA (a) and DTG (b) curves of neat PCL and different blends.

Table 2 listed the atomic concentration according to their peak areas and sensitivity factors.Compared with CTS,nitrogen increased from 6.43% to 9.43% for QCTS.The existence of chlorine and the increase of nitrogen concentration could be attributed to quaternary ammonium groups.XPS results also indicated that QCTS was prepared successfully.

3.1.7.Thermal analysis

Thermal stability of blends was evaluated by TGA.Fig.6 showed TGA and DTG diagrams of blends with different compositions.Thermogravimetric data were listed in Table 3.The onset temperature of neat PCL degradation was observed at around 300 °C.For PCL/CTS-20 and PCL/QCTS-20,the onset temperatures were around 231 and 230°C respectively,which might be the start point of CTS and QCTS degradation.

Table 3 TGA-DTG results of neat PCL and different blends

Table 4 Solubility of CTS and QCTS

Table 5 Tensile strength (a) and elongation at break (b) of neat PCL and blends (n=6 or 7)

According to DTG curves,neat PCL exhibited thermal decomposition stage ranging from 300 to 484°C,and the maximum decomposition temperature of 404 °C.The weight at around 484 °C was less than 1%,indicating PCL was almost degraded.It was reported that PCL has two-step mechanism of degradation [44]:In the first step,pyrolysis of the ester groups which was associated with random chain scission leads to release of CO2,H2O and hexanoic acid.In the second step(400–530°C),ε-caprolactone was constituted as a product of an unzipping depolymerization process.

PCL/CTS-20 and PCL/QCTS-20 exhibited two decomposition stages.The first decomposition stage of PCL/CTS-20 happened around 231–328 °C,and the second happened around 328–508 °C.For PCL/QCTS-20,the two stages were 230–328 °C and 324–486°C,respectively.For these two blends,the first stage could be ascribed to CTS or QCTS degradation,while the second stagemight be ascribed to PCL decomposition.PCL decomposition range was not changed significantly.

Fig.7.SEM of neat PCL and different blends.

3.1.8.SEM analysis

SEM results of neat PCL and blends were demonstrated in Fig.7.Neat PCL in Fig.5 (a) was homogeneous.As for blends,the morphologies were heterogenic and sea-island structures were formed.In Fig.7 (e),lamellar CTS could be observed.Cavities were also observed at the boundary of PCL matrix and CTS or QCTS,possibly due to week adhesion between the two phases.PCL and CTS/QCTS were immiscible.Similar observations [15,45] were reported in polyester/CTS blends.

3.1.9.Solubility determination

Dried CTS and QCTS were dispersed in different solvents.Results were given in Table 4.QCTS was soluble in water.Solubility of QCTS in 0.5% (v/v) acetic acid solution was higher than that of CTS.

3.2.Mechanical properties

Table 5 showed tensile strength and elongation at break data of neat PCL and blends.Tensile strength of neat PCL was 19.3 MPa.For PCL/CTS groups,though 5%,10% and 15% CTS addition decreased tensile strengths of final blends,the decrease was not significant compared with neat PCL.When CTS composition was upto 20%,significant decrease was observed,and its tensile strength wasabout 15.6 MPa.For PCL/QCTS blends,5%QCTS did not significantly lowered tensile strength(18.8 MPa)of the blend.However,tensile strengths of blends were significantly lowered as the composition of QCTS increased.When QCTS was upto 20%,tensile strength of blend was about 11.8 MPa.According to SEM results,the decreasing tensile strengths may be attributed to cavities observed along phases boundary.Elongation at break of PCL was also influenced by the addition of CTS or QCTS with similar trend.More CTS or QCTS addition would lead to lower elongation at break.The trend of mechanical results were similar to the work of Correlo [15].In their study,the tensile strength of PCL was(27.3±0.8)MPa,while that of PCL/CTS(50:50 by mass)was(21.1±0.9)MPa.The elongation of neat PCL was(674±36)%and decreased to(5±0.28)%when CTS content was upto 50% by mass.

Table 6 MIC (μg﹒ml-1) and MBC (μg﹒ml-1) of CTS and QCTS against Escherichia coli and Staphylococcus aureus in water and 1% (v/v) acetic acid

Table 7 Bacteria inhibition rates in presences of PCL blends (n=3)

3.3.Antibacterial activity evaluation

3.3.1.MIC and MBC of CTS and QCTS

Table 6 showed the MIC and MBC against Escherichia coli and Staphylococcus aureus in water and 0.5% (v/v) acetic acid respectively.CTS/water suspension did not show significant inhibiting effect against bacteria in given concentration compared to CTS/acetic acid solutions.MIC and MBC value of CTS in acetic acid solutions were 400 μg﹒ml-1and 600 μg﹒ml-1respectively against two types of bacteria.QCTS showed antibacterial effect in both water and acetic acid medium.Acidic QCTS solution showed lower MIC and MBC values than acidic CTS,indicating QCTS with quaternary ammonium salt exhibited higher antibacterial effect.Meanwhile,QCTS showed lower MIC and MBC values in acetic acid medium than those in water medium.This could be explained by the protonation of unsubstituted—NH2groups on the glucosamine residues[32].

3.3.2.Antibacterial performance by shake flask method

In this paper,Chinese national standard GB/T 20944.3-2008 was adopted to evaluate the antibacterial performance of PCL blends,and the data were demonstrated in Table 7.PCL did not exhibit antibacterial effect.Though the increasing CTS content could increase the inhibition rate against both two types of bacteria,there was no significant difference compared with neat PCL.The number of CFU in petri dishes decreased considerably by exposing the bacteria to PCL/QCTS films,particularly at higher QCTS concentrations.When QCTS content was upto 20%,no colony observed on the agar plate of each dilution after 18-h inoculation.PCL/QCTS-20 showed reduction above 99.9% of the original inoculum for both bacteria.Overall,PCL/QCTS group showed higher inhibition rates than those of PCL/CTS group.

Proposed antibacterial mechanism[9]of CTS was that the direct interaction between positively charged amino groups of CTS and microbial membrane with negative charge.QCTS,with positive quaternary ammonium groups,could also bind to negative microbial membrane.QCTS can also chelate the essential trace metals required for microbial metabolism and hence decrease their availability to the microbes leading to inhibition of their metabolic machinery and ultimately their death [4].In this study,PCL/QCTS blends showed better antibacterial activities than PCL/CTS.The possible explanation was that CTS was subjected to neutral environment and it was hard for them to be swelled,soluble or protonated.The inhibition rates in CTS groups were low and were not significant different from that of neat PCL.Therefore,the molecular conformation in the blend may limit the amount of free amine groups and restrain the antibacterial potential of CTS.QCTS with long chain quaternary ammonium group,was soluble in humid or water environment.QCTS were prepared with higher solubility and antibacterial performance not just in acidic environment.Quaternary ammonium groups of QCTS were electropositive and QCTS showed better antibacterial effect than CTS in other report [19],and this might be used to explain the better antibacterial performance of PCL/QCTS.

4.Conclusions

In this study,CTS and QCTS were added as antibacterial agents to PCL to prepared antibacterial material.Preliminary studies including characterization (FTIR,1H NMR,XPS,TGA/DTG,and SEM),mechanical test and antibacterial evaluation were conducted.Specially,the antibacterial potential of CTS might be prohibited in PCL blend,while QCTS played important role in antibacterial modification of PCL.This blend could be potentially applied as packing materials.More QCTS blends with plastics could be prepared to explore more possibilities.

Acknowledgements

The present study was supported by the Science Foundations from Department of Education,Fujian Province,China (JZ180899).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.