International Journal of Biological Macromolecules 74 (2015) 76–84

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Synthesis and characterization of zinc oxide–neem oil–chitosan bionanocomposite for food packaging application S. Sanuja, A. Agalya, M.J. Umapathy ∗ Department of Chemistry, College of Engineering Guindy, Anna University, Chennai 600 025, India

a r t i c l e

i n f o

Article history: Received 17 September 2014 Received in revised form 16 November 2014 Accepted 25 November 2014 Available online 8 December 2014 Keywords: Chitosan Metal oxide Essential oil Bionanocomposite film Food packaging

a b s t r a c t Nano zinc oxide at different concentrations (0.1, 0.3 and 0.5%) and neem essential oil were incorporated into the chitosan polymer by solution cast method to enhance the properties of the bionanocomposite film. The functional groups, crystalline particle size, thermal stability and morphology were determined using FTIR, XRD, TGA and SEM, respectively. The results showed that 0.5% nano zinc oxide incorporated composite film have improved tensile strength, elongation, film thickness, film transparency and decreased water solubility, swelling and barrier properties due to the presence of neem oil and nano zinc oxide in the polymer matrix. Further antibacterial activity by well diffusion assay method was followed against Escherichia coli which were found to have good inhibition effect. In addition to this food quality application were carried against carrot and compared with the commercial film. © 2014 Published by Elsevier B.V.

1. Introduction Food packaging has become an icon in the packaging industry and plays a vital role in the modern society. The key functions of food packaging are to preserve, extend shelf life and deliver the food products until consumption. Apart from this there are some secondary functions of packaging, such as marketing, traceability, indication of tampering etc. [1–3]. Synthetic packaging materials have lots of short comings like costly, non-biodegradable and release of toxic gases. Technologist and researchers all across the globe have realized and opted for eco-friendly packaging materials availing the help of biopolymers and nanomaterials. Chitosan is a cationic polysaccharide with ␤-(1–4)-linked d-glucosamine (deacetylated unit) and N-acetyl-d-glucosamine (acetylated unit) distributed randomly. Chitosan is a good material with biocompatibility, biodegradability and antibacterial properties. However, its mechanical and barrier properties are not viable for food packaging applications and therefore to improve the above properties it has to be blended with nanomaterials [4–10]. Nanomaterials offer a spectrum of novel properties for the food packaging industries [11]. The nanomaterials includes, nanoclay, carbon nanoparticles, nanoscale metals and oxides [12,13].

∗ Corresponding author. Tel.: +91 044 22358669. E-mail addresses: [email protected] (S. Sanuja), mj [email protected] (M.J. Umapathy). http://dx.doi.org/10.1016/j.ijbiomac.2014.11.036 0141-8130/© 2014 Published by Elsevier B.V.

Incorporation of essential oil into the biofilms may prolong the antimicrobial activity and improve the safety of food materials [14–16]. Essential oil is a concentrated liquid which are produced by plants to protect themselves from pathogens and herbivores. They are organic in nature possessing volatile aromatic compounds which are usually extracted by steam distillation or solvent extraction process [17]. These essential oil are environmentally safe antimicrobial agent having good antimicrobial, fungicidal and insecticidal activities [18,19]. Neem is a medicinal plant having diverse chemical structure and biological effects. They are nontoxic, biodegradable, hydrophobic in nature and possess excellent antimicrobial property and therefore has multifarious application. Chitosan–silver oxide encapsulated film was fabricated by employing solution casting technique. Since silver oxide was used it revealed an excellent activity against pathogenic bacteria like Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Pseudomonas aeruginosa [20]. Active chitosan-polyvinyl alcohol films containing aqueous mint extract/pomegranate peel extract were prepared by Sweetie et al. Physical, mechanical, antimicrobial and antioxidant properties of the films incorporated with the extract were evaluated. It was found that these films exhibited increased protection against UV light containing the extracts. Apart from this the extracts introduced excellent antioxidation activities with improved mechanical properties. The film exhibited good antibacterial activity against gram-positive food pathogens [21]. Mehdi and his coworkers prepared a novel active bionanocomposite film inducted with rosemary essential oil and nanoclay into chitosan.

S. Sanuja et al. / International Journal of Biological Macromolecules 74 (2015) 76–84

It was found that addition of very low quantities of nanoclay and chitosan improved the physical and mechanical properties of the film significantly. Further these film possessed good antimicrobial properties and proved to be a good packaging material for food packaging [22]. Tooraj et al. have reported the fabrication of ediblechitosan composite film containing Thymus kotschyanus essential oil. This film exhibited good antibacterial and antioxidant properties [23]. Combining two different naturally occurring components with antimicrobial properties provide synergic effect on antimicrobial property. Packaging materials based on biopolymer nanocomposite have a huge potential in food packaging industries [24]. In this work, a new bionanocomposite film was fabricated using chitosan, nano zinc oxide and neem essential oil by solution cast method for food packaging application.

77

was washed thrice using distilled water to remove any impurities present in it. Later it was dried and calcined at 500 ◦ C for 2 h [25]. 2.2.3. Fabrication of bionanocomposites film by solution casting method To the 1% acetic acid solution, 1 g of chitosan was added and stirred constantly till it gets dissolved. To the above solution nano metal oxide (0.1, 0.3 and 0.5%) was dispersed into the solution for 2–3 h and it was sonicated for 15 min at 50 ◦ C. Later Tween 80 and 0.5 ml of neem essential oil were added and stirred to 24 h to get a homogeneous mixture. Finally the solution was cast onto the petri-plates and dried at 50 ◦ C in an oven overnight [22,26,27]. 2.3. Fourier-transform infrared spectra analysis [FTIR]

2. Materials and methods 2.1. Materials Hydrochloric acid, soluble starch and sodium hydroxide from Merck (Chennai) were used as received. Glacial acetic acid was purchased from Qualigens (Chennai). Zinc nitrate hexahydrate was obtained from Hi-Tech Enterprises Private Limited (Chennai). Crab shells and neem oil were purchased from import and export companies (Chennai) and from Cyrus Enterprises (Chennai), respectively.

FTIR spectrum was measured using a Perkin-Elmer spectrum RX1 instrument. Spectra were collected in the range of 4000–400 cm−1 . 2.4. X-ray diffraction analysis [XRD] XRD pattern were measured using PAN analytical Expert Pro instrument. The synthesized samples were scanned from 0 to 80 ◦ C at a scanning rate of 3◦ /min.

2.2. Methods

2.5. Thermo gravimetric analysis [TGA]

2.2.1. Synthesis of chitosan 2.2.1.1. Coarse purification. Crab shells of 250 g were taken, it was cleaned coarsely with water until impurities were removed and later it was dried in an oven at 110 ◦ C for overnight.

The thermal stability of synthesized film was measured using TG analyser (TGA, TG-2009 and Netzsch). The heating rate was 10 ◦ C/min in N2 with the flow rate of 20 ml/min. 2.6. Scanning electronic microscope [SEM]

2.2.1.2. Protein removal. The shells were made into powder. To this 2% of NaOH were added and heated at 60–70 ◦ C under constant stirring for half an hour. The process was repeated by filtering off with a strainer using distilled water till the solution become clear and colourless. This step was also known as deprotenization. 2.2.1.3. Calcium carbonate removal. To the shell powder 7% HCl was added until no gas escapes under stirring. As a check, HCl of 10 ml were added to the mixture. Later it was filtered off and washed well with distilled water. The product was dried at 60 ◦ C overnight as a result 50 g of chitin was obtained, this step was also known as demineralization

The morphological structures of prepared films were observed using a DXS-10ACKT scanning electronic microscope. 2.7. Mechanical property Tensile strength and elongation of the films were determined according to ASTM standard method with Universal Testing Machine (TNUM-5900). The film were cut into (7 × 3 cm) rectangular shapes and measured. 2.8. Water solubility test

2.2.1.4. Synthesis of chitosan from chitin by deacetylation. To the obtained chitin, 50% of NaOH was added and refluxed for 2 h at 125 ◦ C. After completion of reaction 100 ml of water was added and made to cool down overnight. The next day it was filtered and washed well with distilled water and dried in an oven at 60–70 ◦ C. In which 30 g of chitosan was obtained as a result from crabshells [4].

The solubility of the films (1 × 1 cm) were measured by immersing the pieces into 100 ml of distilled water and made to agitate in a mechanical shaker for 1 h at room temperature. Then the remaining samples were filtered and dried at 110 ◦ C for few hours and it was weighed as final dry weight. The initial dry weight was measured before immersing it in water by drying at 110 ◦ C [23,28,29].

2.2.2. Synthesis of zinc oxide (ZnO) nanoparticles by wet-chemical method Zinc oxide nanoparticles were synthesized using wet chemical method. To the 500 ml of 0.5% soluble starch solution, 0.1 mole of zinc nitrate hexahydrate was dissolved. To the above mixture 500 ml of (0.2 mol) of NaOH was added slowly under stirring condition till the mixture turned into milky white colloid solution. The reaction was continued for 2–3 h and it was made to settled down. Next day it was centrifuged at 1500 rpm for 15 min and the mixture

Solubility in water (%) =



initial dry weight − final dry weight initial dry weight



× 100

2.9. Swelling property test The synthesized films dried in an oven for specified temperature and time was preserved in a desiccator till it is weighed. The measured values are noted as dry weight. Further it was immersed in 100 ml of distilled water at room temperature. Later

78

S. Sanuja et al. / International Journal of Biological Macromolecules 74 (2015) 76–84

it was removed, patted dried and measured as wet weight. It was expressed in weight percentage. Swelling property test (%) =

 wet weight − dry weight  dry weight

× 100

2.10. Film thickness Thickness of the films were determined using the digital thickness measuring gauge to the nearest 0.001 mm. Measurements were taken using 5 different locations and its average were calculated [26]. 2.11. Film transparency Transparencies of the films were measured using UV spectrophotometer (Perkin Elmer, Lamda 35) by measuring the absorbance at particular wavelength of the different films. The films were cut into square pieces and inserted into the spectrophotometer test cell. An empty test cell was taken as the reference. The transparency of the films were calculated according to the formula Transparency = log (T wavelength range) X −1 where T wavelength range, is the transmittance at different wavelength of three various film and X is the thickness of the film (mm). According to this equation; when T has high values it indicates lower transparency and higher degree of opacity [23,30–32].

Fig. 1. FTIR of (a) chitosan, (b) nano ZnO, (c) chitosan/0.1% ZnO film, (d) chitosan/0.3% ZnO film, (e) chitosan/0.5% ZnO film, (f) chitosan/0.5% ZnO/neem oil film.

containing half its capacity of freshly dried silica gel. Immediately weigh the bottle along with sample and silica gel. Fix the bottle into the holder of machine and run the machine for not less than 7 h and not more than 16 h. Remove the bottle and weigh it. Note the time between two weighing [34,35].

Calculation :

water vapour permeability =

2.12. Anti-bacterial activity test Anti-bacterial activity was carried out by Well diffusion assay method [33]. Nutrient agar was prepared and poured in the sterile Petri dishes and allowed to solidify. The 24 h growing bacterial cultures (E. coli) were swabbed on it. The wells (10 mm diameter) were made by using cork borer. In which 25 and 50 ␮g concentration of the polymer, one negative control was loaded in the wells. The plates were then incubated at 37 ◦ C for 24 h. Later the plates were examined for any zone of growth inhibition. Inhibition zones were recorded as the diameter of growth free zones including the diameter of the well in mm at the end of incubation period. Percentage of inhibition was calculated by the formula 2.13. Water vapour permeability The test machine has a vertically mounted turntable with at least three stations to hold three pots. Its axis is parallel to the axis of rotation of turntable and is 67 ± 2 mm from the axis of rotation. The internal height of the pot is 80 ± 10 mm with circular clamping ring of pot of diameter 30 ± 10 mm. The speed of the machine is 75 ± 5 rpm. A paddle type fan with three blades inclined at 120◦ to each other and rotates at a speed of 1400 ± 100 rpm in a direction opposite to that of turn table. Cut a square piece of side 50 mm from the sampling location and buff the grain lightly as follows. Place the sample on a table with grain upwards. Press 180 grade emery paper against the sample under a load of 200 g and draw the emery paper across 10 times in various directions. Cut out circular test pieces of sample from the piece snuffed as above with diameter of approximately 34 mm. Fill a bottle of half its capacity with freshly dried silicagel (silicagel dried in an oven at 125 ± 5 ◦ C for at least 16 h and cool it for at least 8 h in a closed bottle). Clamp the test piece across the mouth of the bottle with grain inwards. Fix the bottle into the holder of water vapour permeability testing machine and start the motor. After the machine is run for more than 16 h and less than 24 h, remove the bottle and fix the sample to a second bottle

7640 × W D2 × t

where W = gain in weight in mg; D = mean internal diameter in mm of the neck of the bottle; t = time in minutes between the two weighing

2.14. Food quality analysis 2.14.1. Pretreatment of food sample 50 ml of sterile nutrient broth was prepared and E. coli was inoculated and allowed for overnight growth. Fine carrots were chosen and all pieces were cut and soaked for 5 min in nutrient broth. Those pieces were transferred to sterile plates and left undisturbed. The initial weight of the carrot was noted. The pieces were coated with the given standard and tested film, their weight was also noted. Further, serial dilution technique was carried out and spread plating was performed.

2.14.2. Isolation of bacteria Suspend 1 g of carrot in 10 ml of sterile distilled water to make 10−1 dilution. From 10−1 suspension, transfer 1 ml of sample to 9 ml of sterile water and subsequently serially dilute to range of 10−2 –10−9 . Take 0.1 ml suspension from the dilutions 10−3 , 10−5 and 10−7 dilutions and spread plated over the surface of sterile Nutrient Agar medium. Incubate the plates at 37 ± 2 ◦ C for 24–48 h.

2.14.3. Spread plate method The agar medium used for isolation is to be liquefied, cooled to 50 ◦ C, and poured into the bottom plate and gently rotated, so that it becomes evenly distributed. An aliquot (100 ␮l) of the diluted sample is to be placed onto the agar surface and spread uniformly with a sterile, bent glass rod. The plates are incubated at 37 ◦ C for 24 h and the plates were observed carefully.

S. Sanuja et al. / International Journal of Biological Macromolecules 74 (2015) 76–84

79

INTENSITY (a.u)

chitosan

nano- ZnO

0

10

20

30

40

50

60

70

80

90

2 THETA (DEGREE)

Fig. 3. TGA of (a) Chitosan, (b) nano ZnO, (c) Chitosan/0.5% ZnO/neem oil film.

Fig. 2. XRD of chitosan and nano-ZnO.

3. Result and discussion 3.1. FTIR Fig. 1a–f represents the FTIR spectrum of chitosan film, nano ZnO, chitosan/0.1% ZnO film, chitosan/0.3% ZnO film, chitosan/0.5% ZnO film and chitosan/0.5%ZnO/neem oil film, respectively. In Fig. 1a, the peak at 3600 cm−1 represents the OH stretching vibration and NH2 vibration of chitosan, respectively. The peak at 2892 cm−1 and 1639 cm−1 corresponds to the CH2 vibrations and H2 O bending mode, respectively. 1430 and 1375 cm−1 peaks represent CH2 bending vibrations [27]. In Fig. 1b, the peaks around 600 cm−1 indicates the presence of zinc oxide stretching mode and all other peaks are due to the presence of soluble starch. Fig. 1c, d, and e represents the 0.1%, 0.3% and 0.5% incorporated ZnO in chitosan matrix in which the N H bonded to the O H vibration shifted towards lower frequency from 3600 cm−1 to 3569 cm−1 , 3540 cm−1 and 3330 cm−1 , respectively, which is due to the reaction between the amine group in the chitosan and the metal oxide and the 0.5% incorporated ZnO film was found to be the best film due to the better reaction. In Fig. 1f chitosan/0.5% ZnO/neem oil nanocomposite film further shifted to 3204 cm−1 show that bonding has taken place effectively between the metal oxide, chitosan functional group and azadirachtin of phenol reactive group present in neem essential oil.

290 ◦ C was due to degradation of nano ZnO. In neem oil blended chitosan/0.5%ZnO/neem oil nanocomposites film three weight loss were noticeable. The weight loss at 150 ◦ C was because of moisture vapourization, 150–320 ◦ C due to degradation of chitosan and metal oxide and 550–720 ◦ C was due to degradation of neem oil. These thermal stability changes in the films were found due to the presence of ZnO and neem oil in chitosan polymer matrix and it proves that chitosan/0.5%ZnO/neem oil nanocomposite film was thermally more stable. 3.4. SEM The SEM images of pure chitosan film, chitosan–0.5% nano zinc oxide incorporated film and chitosan/0.5% ZnO–neem oil film were shown in Fig. 4a, b and c, respectively. The pure chitosan film morphology was non-uniform and rough due to undissolved chitosan particle present in it, while the 0.5% ZnO incorporated chitosan film showed improved homogeneous morphology which is due to the good blending of nano ZnO and chitosan materials. In Fig. 4c, the surface observes like hollow cavity which is due to the hydrophobic nature of neem essential oil present in the nanocomposite film. 3.5. Mechanical properties

3.3. TGA

3.5.1. Tensile strength Tensile strength of bionanocomposite films was presented in this Fig. 5a. It was observed that the tensile strength increases significantly by increasing the concentration of nanoparticles from 0.1% to 0.5% into the chitosan matrix. In which chitosan/0.5% ZnO nanocomposite films have higher tensile strength (51 MPa) when compared to the pure chitosan (30 MPa) and other two concentrations, this is because of maximum well dispersion of nano zinc oxide into chitosan polymer matrix which prevents material from coagulation. And further increased in essential oil incorporated in the chitosan/0.5% ZnO nanocomposite film (60 MPa) was found due to the good interaction between the metal oxide and chitosan as well as the strong interaction taking place between bio-active group present in neem oil and functional group present in chitosan–metal oxide composite material as earlier described in FTIR spectrum.

Thermogram of chitosan, nano ZnO, chitosan/0.5%ZnO/neem oil nanocomposites films were shown in Fig. 3a, b and c, respectively. The weight loss 100 ◦ C is due to the moisture vapourization and the weight loss around 210–390 ◦ C is due to the degradation of chitosan polymer [27]. In synthesized nano zinc oxide two weight losses were observed, in which first weight loss below 150◦ C was due to water vapourization and second loss around

3.5.2. Elongation Elongation percentage of the films was shown in Fig. 5b. In which pure chitosan film has 7% of elongation, low values when compared to other films. As we increase the concentration of nano zinc oxide from 0.1% to 0.5% in polymer matrix the percentage of elongation increased from 8 to 12.3%, respectively, which is due to the strong interaction between metal oxide and functional groups of chitosan.

3.2. XRD The XRD of synthesized chitosan and nano ZnO were shown in Fig. 2. The chitosan shows its characteristic peaks at 2 = 10◦ , 20◦ , 30◦ and 45◦ which is due to the hydrogen bonding of chitosan. It was observed from the peak that the synthesized chitosan is partially crystalline and partially amorphous in nature. While in prepared nano ZnO it was found to be highly crystalline in nature and their diffraction peaks were observed at 2 = 10◦ , 25◦ , 36◦ , 60◦ and 70◦ . From the Scherrer equation the crystalline particle size of nano ZnO and chitosan were found to be 27 nm and 70 nm, respectively.

80

S. Sanuja et al. / International Journal of Biological Macromolecules 74 (2015) 76–84

Fig. 4. (a) SEM of chitosan film. (b) SEM of chitosan–0.5% ZnO film. (c) SEM of chitosan–0.5% ZnO–neem oil film.

In oil incorporated film it increases further to 15.6% since oil present here reduces the brittleness of the film and increases the flexibility of the material and thus increases in elongation was found [22]. 3.6. Water solubility test Solubility of the synthesized films were shown in Fig. 6. The pure chitosan film showed higher (78.3%) affinity and maximum gets solubilized in water when compared to other films, since chitosan was more hydrophilic in nature it absorbs water rapidly. As we increases the zinc oxide concentration (0.1–0.5%) into the chitosan film water solubility decreases from 60.3 to 43.2% because it react more effectively with polymer matrix and reduces the number of hydrophilic group present in it as mentioned in WVP. Incorporation of oil into the chitosan based films shows further decrease in water solubility (30.11%) this phenomenon is due to the effect of more hydrophobic and many bioactive components present in the neem oil which restricts the uptaking of water and thus the chitosan/0.5% ZnO/neem oil film showing low affinity towards water [22,23]. 3.7. Swelling property test Swelling property of bionanocomposite films were shown in Fig. 7. The pure chitosan film swells up to 24.6% as it involved in hydrogen bonding quickly with water. As we increases the concentration of zinc oxide from 0.1 to 0.5% the swelling of the film decreases from (22.2% to 17.8%) due to strong interaction of metal oxide with chitosan functional groups as it reduces the hydrogen bonds involving in reaction. In neem oil incorporated film the swelling further decreased that it swells up to 15.4% only when compared to other films it is because the neem essential oil increases hydrophobicity of the film surface and reduces the moisture uptake, as it was described in water solubility.

3.8. Film thickness Film thickness of prepared nanocomposite films was shown in Fig. 8. As the concentration of nano zinc oxide increases from 0.1 to 0.5% the film thickness also varies from 0.22 to 0.45 mm, respectively. In oil incorporated film, the thickness increases further to 0.49 mm since the oil present in it react with the molecular chain of polymer by elaborating their network and thus increase in thickness was found [27]. 3.9. Film transparency Film transparencies of all films were shown in Fig. 9. It was observed that pure chitosan film transparency (2.312) was low which means it was more opaque when compared to other films. By increasing the concentration of ZnO in chitosan film as 0.1%, 0.3% and 0.5% its transparency increases (i.e., low opacity) from 4.065, 4.576 and 5.400, respectively, it is because of the well dispersion of nano metal oxide into the polymer matrix. In case of neem oil and nano zinc oxide incorporated chitosan film was found to be semi-transparent (3.699), which has higher transparency value (i.e., lower opacity) when compared to pure chitosan film and lower transparency value (i.e., higher opacity) when compared to metal oxide incorporated chitosan film which is due to the fact that though the metal oxide gets well dispersed into polymer matrix, addition of neem essential oil makes the film semi-transparent [23], this results in prevention of photo-degradation of material in case of light transmittance during storage periods. 3.10. Antibacterial activity Antibacterial activities of the prepared films were observed for E. coli, gram-negative bacteria were shown in Table 1. It was found

S. Sanuja et al. / International Journal of Biological Macromolecules 74 (2015) 76–84

19.92%

60

17.86%

C:0.3%Z

50

TENSILE STRENGTH (M Pa)

81

40

C:0.5%Z

C:0.5%Z:O

C:0.1%Z

15.4%

C

22.23%

30

20

24.59% 10

Fig. 7. Swelling property vs. film type.

0 C

C+0.1%Z

C+0.3%Z

C+0.5%Z

0.50

C+0.5%Z+Oil

FILM TYPE 0.45

FILM THICKNESS

a Tensile Strength Vs Film Type

16 14

0.40

0.35

0.30

ELONGATION (%)

12

0.25

10 8

0.20 C

6

C+0.1%Z

C+0.3%Z

C+0.5%Z

C+0.5%Z+Oil

FILM TYPE

4

Fig. 8. Film thickness vs. film type.

2 0 C

C+0.1%Z

C+0.3%Z

C+0.5%Z

C+0.5%Z+Oil

4

Fig. 5. (a) Tensile strength vs. film type. (b) Elongation vs. film type. 2

WATER SOLUBILITY (%)

80

C+

70

0

Z+ .5%

F IL

M

60

0

Oil

C+

0.5

TY

FILM TR A

b Elongation Vs Film Type

NSPAREN

CY

FILM TYPE

%Z

C+

PE

0.3

%Z

C+

0.1

%Z

C

Fig. 9. Film transparency vs. film types. 50

Table 1 Antibacterial activity of bio-nanocomposite films against Escherichia coli.

40

The inhibition zone (I), diameter in mm and the % of inhibition (I%) of polymer films against E. coli Film type

30 C

C+0.1%Z

C+0.3%Z

C+0.5%Z

FILM TYPE Fig. 6. Water solubility vs. film type.

C+0.5%Z+Oil

Chitosan Chitosan:0.1% ZnO Chitosan:0.3% ZnO Chitosan:0.5% ZnO Chitosan:0.5% ZnO: neem oil

I%

I 25 ␮g

50 ␮g

25 ␮g

50 ␮g

18 23 28 32 35

21 25 29 33 35.9

19.23 24.05 28.76 32.98 36.32

23.31 26.97 29.12 33.77 36.73

82

S. Sanuja et al. / International Journal of Biological Macromolecules 74 (2015) 76–84

Fig. 10. Photocopy of serial dilution technique and spread plate method.

that antibacterial activity increases as the concentration of zinc oxide increases, since zinc in general will acts as a good antibacterial agent. In chitosan/0.5% ZnO/neem oil nanocomposite films shows higher antibacterial activity against E. coli when compared to other two films. It is due to the fact the neem oil contains azadirachtin and other bioactive components such as nimbidin, nimbolin, mahmoodi, etc., in high composition in which phenol reactive group of azadirachtin and chitosan functional group together with nano ZnO will react with the cell membrane of the bacteria to inhibit the leakage through that membrane and cease the growth of microorganisms.

3.11. Water vapour permeability WVP is one of the major issues in food technology and science due to interaction of food and the packed film from the environment to provide safety and healthy food products. WVP for chitosan, chitosan/0.1% ZnO film, chitosan/0.3% ZnO film, chitosan/0.5% ZnO film and chitosan/0.5% ZnO/neem oil film were shown in Table 2. Chitosan film has higher WVP values (1.57 mg/cm2 /h) when compared to other films since chitosan has high inter and intramolecular hydrogen bonding ability with water [27]. In case of increasing the concentration of nano ZnO as 0.1, 0.3,

S. Sanuja et al. / International Journal of Biological Macromolecules 74 (2015) 76–84

83

Fig. 11. Photocopy of food quality test using Carrot.

0.5% in chitosan film the WVP values decreased from 1.23, 1.08 to 0.69 mg/cm2 /h, respectively, this is because, nano ZnO exhibit high barrier property as well as it gets well dispersed into the chitosan matrix so that it will reduce the hydrogen bonding interaction. In neem essential oil incorporated chitosan/0.5% ZnO nanocomposites film, WVP values further decreased from 0.67 to 0.41 mg/cm2 /h due to the reason that neem oil is more hydrophobic in nature, in addition reaction between functional groups in chitosan and OHgroup present in azadirachtin interact more effectively and reduces the number of hydrogen groups responsible for hydrophilic nature present in water vapour. Thus this film is disguising the path of water vapour penetration into the material.

Table 2 Water vapour permeability vs. film type. Film types

WVP-values (mg/cm2 /h)

Chitosan Chitosan:0.1% ZnO Chitosan:0.3% ZnO Chitosan:0.5% ZnO Chitosan:0.5% ZnO: neem oil

1.57 1.23 1.08 0.69 0.41

Table 3 Effect of the packaging film on sample weight. S.No.

Sample

1 2 3

Control Commercial CZN

3.12. Food quality test Quality test was carried out for the prepared chitosan/0.5% ZnO/neem oil film (CZN), commercial film and uncovered (control) film. Weight loss and colony forming unit for E. coli was determined before and after packing were shown in Figs. 10 and 11 and in Tables 3 and 4 it was observed that the CZN film have less weight loss during day 1 and 5 when compared to control and commercial. Colony forming unit was estimated by spread plate method, in which control film have 196 × 10−3 CFU & 440 × 10−3 CFU and commercial film have 65 × 10−3 CFU & 124 × 10−3 CFU for day 1 and 5, respectively. While (CZN), the synthesized film was found to have less CFU i.e., 58 × 10−3 and 95 × 10−3 for day 1 and day 5, respectively. This is due to the reason that the amino and hydroxyl functional group present in chitosan, azadirachtin bioactive component present in neem oil and metal oxide all together interact more effectively and increases the activity of a film. Thus the film acts as an eco-benign material and protects the food products from environmental hazards. Therefore the synthesized chitosan/0.5% ZnO/neem oil bio-nanocomposite film possesses best quality for active food packaging applications.

Table 4 Estimation of CFU after packaging.

Sample weight (g) Before

Day 1

Day 5

5.28 5.28 5.28

3.21 3.44 3.94

2.49 2.63 2.82

S.No.

Sample

1 2 3

Control Commercial CZN

CFU- colony forming units. Day 1

Day 5

196 × 10−3 65 × 10−3 58 × 10−3

440 × 10−3 124 × 10−3 95 × 10−3

84

S. Sanuja et al. / International Journal of Biological Macromolecules 74 (2015) 76–84

4. Conclusion This study demonstrated that the synthesized bionanocomposite film possess enhanced mechanical, physical, barrier and optical properties due to addition of both nanoparticles as well as essential oil into the polymer matrix. The FTIR revealed the good interaction among the materials. TGA and SEM describes the good thermal stability and morphology of the films, respectively. From the XRD Scherrer equation the crystalline particle size was found to be 27 nm for nano ZnO and 70 nm for chitosan materials. Further antibacterial activity against E. coli was investigated for all types of films, in which chitosan/0.5% zinc oxide/neem oil nanocomposites films shows good activity when compared to other films. Among the different concentration 0.5% nano zinc oxide incorporation was found to be best. So food quality test was carried out for chitosan/0.5% zinc oxide/neem oil nanocomposites film using carrot pieces and compared it with the commercial plastic film and it was found to have positive result on the test. Thus chitosan–ZnO containing neem essential oil may be a promising novel material for an active food packaging applications. Acknowledgements We thank the Department of Chemistry, College of Engineering Guindy, Anna University, Chennai for providing lab facilities and chemicals and we specially thank DST-FIST for providing instrumental facilities to carry out the research work. References [1] Y. Yokoyama, in: S. Hashimoto (Ed.), Package Design in Japan, Rikuyo-Sha Publishing, Japan, 1985, pp. 113–115. [2] A. Cagri, Z. Ustunol, E.T. Ryser, J. Food Protect. 67 (2004) 833–848. [3] S. Burt, Int. J. Food Microbiol. 94 (2004) 223–253. [4] F. Burrows, L. Clifford, A. Michael, O. Oghenekome, J. Agric. Environ. Sci. 2 (2007) 103–111. [5] C.M. Yeng, S. Husseinsyah, S.S. Ting, Polym.-Plast. Technol. 52 (2013) 1496–1502. [6] W.X. Du, C.W. Olsen, R.J. Avena-Bustillos, T.H. McHugh, C.E. Levin, M. Friedman, J. Agric. Food Chem. 56 (2008) 3082–3088. [7] A. Ghanem, D. Skonberg, J. Appl. Polym. Sci. 84 (2002) 405–413.

[8] Y. Ruiz-Navajas, M. Viuda-Martos, E. Sendra, J.A. Perez-Alvarez, J. FernándezLópez, Food Control 30 (2013) 386–392. [9] L. Wang, H. Wu, G. Qin, X. Meng, Food Control 41 (2014) 56–62. [10] M.C. Cruz-Romero, T. Murphy, M. Morris, E. Cummins, J.P. Kerry, Food Control 34 (2013) 393–397. [11] Y. Cho, C. Kim, N. Kim, C. Kim, B. Park, Biochip 2 (3) (2008) 183–185. [12] A.L. Brody, J. Food Technol. 60 (2006) 92–94. [13] Messina V. Paula, Verdinelli Valeria, Pieroni Olga, Manuel Ruso Juan, J. Colloid Polym. Sci. 291 (2013) 835–844. [14] Rotem Shemesh, Diana Goldman, Maksym Krepker, Yael Danin-Poleg, Yechezkel Kashi, Anita Vaxman, Ester Segal, J. Appl. Polym. Sci. 132 (41261) (2014) 1–8. [15] Saeedeh Shojaee-Aliabadi, Mohammad Amin Mohammadifar, Hedayat Hosseini, Abdorreza Mohammadi, Mehran Ghasemlou, Seyede Marzieh Hosseini, Mehrdad Haghshenas, Ramin Khaksar, Int. J. Biol. Macromol. 69 (2014) 282–289. [16] Saeedeh Shojaee-Aliabadi, Hedayat Hosseini, Mohammad Amin Mohammadifar, Abdorreza Mohammadi, Mehran Ghasemlou, Seyed Mahdi Ojagh, Seyede Marzieh Hosseini, Ramin Khaksar, Int. J. Biol. Macromol. 52 (2013) 116–124. [17] Natalia Dudareva, Florence Negre, Dinesh A. Nagegowda, Irina Orlova, Crit. Rev. Plant Sci. 25 (2006) 417–440. [18] Judith Reinhard, Mandyam V. Srinivasan, Shaowu Zhang, J. Nat. 427 (2004) 411. [19] Neeta Sharma, Abhishek Tripathi, World J. Microbiol. Biotechnol. 22 (2006) 587–593. [20] Shipra Tripathi, G.K. Mehrotra, P.K. Dutta, J. Bull. Mater. Sci. 34 (2011) 29–35. [21] Sweetie R. Kanatt, M.S. Rao, S.P. Chawla, Arun Sharma, Food Hydrocoll. 29 (2012) 290–297. [22] Mehdi Abdollahi, Masoud Rezaei, Gholamali Farzi, J. Food Eng. 111 (2012) 343–350. [23] Tooraj Mehdizadeh, Hossein Tajik, Seyed Mehdi Razavi Rohani, Abdol Rasso Oromiehie, Vet. Res. Forum 3 (2012) 167–173. [24] J.W. Rhim, P.K. Ng, J. Crit. Rev. Food Sci. 47 (4) (2007) 411–433. [25] A. Yadav, V. Prasad, A.A. Kathe, S. Raj, D. Yadav, C. Sundaramoorthy, N. Vigneshwaran, Bull. Mater. Sci. 29 (2006) 641–645. ´ Vera Lazic, ´ Jasna Gvozdenovic, ´ J. Process Energy Agric. 15 (2011) [26] Nevena Krkic, 165–168. [27] S. Sanuja, A. Agalya, M.J. Umapathy, Int. J. Polym. Mater. Polym. Biomater. 63 (2014) 733–740. [28] Maria Martelli Sílvia, Borges Laurindo Joao, Int. J. Polym. Mater. Polym. Biomater. 61 (2012) 17–29. [29] W. Tongdeesoontorn, L.J. Mauer, S. Wongruong, P. Sriburi, P. Rachtanapun, Int. J. Polym. Mater. Polym. Biomater. 61 (2012) 778–792. [30] Qiangxian Wu, Lina Zhang, J. Appl. Polym. Sci. 79 (2001) 2006–2013. [31] Tolga Gokkurt, Fehim Findık, Huseyin Unal, Abdullah Mimaroglu, Polym.-Plast. Technol. 51 (2012) 701–706. [32] Ubonrat Siripatrawan, Bruce R. Harte, J. Food Hydrocoll. 24 (2010) 770–775. [33] J.N. Eloff, Planta Med. 64 (1998) 711–713. [34] P.C. Srinivasa, M.N. Ramesh, R.N. Tharanathan, J. Food Hydrocoll. 21 (2007) 1113–1122. [35] D.W.S. Wong, F.A. Gastineau, K.S. Gregorski, S.J. Tillin, A.E. Paylath, J. Agric. Food Chem. 40 (1992) 540–544.

Synthesis and characterization of zinc oxide-neem oil-chitosan bionanocomposite for food packaging application.

Nano zinc oxide at different concentrations (0.1, 0.3 and 0.5%) and neem essential oil were incorporated into the chitosan polymer by solution cast me...
2MB Sizes 0 Downloads 4 Views