Carbohydrate Polymers 127 (2015) 64–71

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Kinetics and functional effectiveness of nisin loaded antimicrobial packaging film based on chitosan/poly(vinyl alcohol) Hualin Wang a,c,∗ , Ru Zhang a , Heng Zhang a , Suwei Jiang a , Huan Liu a , Min Sun a , Shaotong Jiang b,c a

School of Chemistry and Chemical Technology, Hefei University of Technology, Hefei 230009, Anhui, People’s Republic of China School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009, Anhui, People’s Republic of China c Anhui Institute of Agro-Products Intensive Processing Technology, Hefei 230009, Anhui, People’s Republic of China b

a r t i c l e

i n f o

Article history: Received 12 February 2015 Received in revised form 10 March 2015 Accepted 12 March 2015 Available online 30 March 2015 Keywords: Nisin–chitosan/poly(vinyl alcohol) Antimicrobial packaging film Kinetics Thermodynamics Functional effectiveness

a b s t r a c t The aim of this study was to evaluate the kinetics and functional effectiveness of Nisin loaded chitosan/poly(vinyl alcohol) (Nisin–CS/PVA) as an antibacterial packaging film. The films were prepared by coating method and Staphylococcus aureus (S. aureus, ATCC6538) was used as test bacterium. The intermolecular hydrogen bonds between CS and PVA molecules were confirmed. The elasticity of films was significantly improved by the incorporation of PVA, and the film could also bear a relative high tensile strength at 26.7 MPa for CS/PVA = 1/1. As CS/PVA ratio decreased, the water vapor permeability (WVP) decreased and reached its minimum value 0.983 × 10−10 g m−1 s−1 at CS/PVA = 1/1, meanwhile, oxygen permeability (OP) increased but still lower than 0.91 cm3 ␮m m−2 d−1 kPa−1 for CS/PVA = 1/1 as the CS/PVA ratio was above 1:1. The initial diffusion of nisin (Mt /M∞ < 2/3) from CS/PVA film could be well described by the Fickian diffusion equation. Owing to the positively charged nisin at pH below isoelectric point (pI, 8.8) and its increasing dissolubility in water as the pH reduced, the diffusion of nisin from the films strongly depended on pH and ionic strength besides CS/PVA ratio and temperature. Moreover, the thermodynamic parameters suggested the spontaneous and endothermic diffusion of nisin from the films. The resulting data can provide some valuable information for the design of film in structure and ingredient. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Recently, a growing attention has been paid to antibacterial packaging, which was widely investigated as a response to consumer demands or industrial production trends toward mildly preserved, fresh, tasty and convenient food products with extended shelf-life and controlled quality (Aider, 2010). As antimicrobial packaging films have been showed more efficient in controlling migration of antimicrobials into the food and keeping antimicrobial activity over time during the transport and storage (Dutta, Tripathi, Mehrotra, & Dutta, 2009; Park, Daeschel, & Zhao, 2004), it could play a profound role in food security assurance. The principle action of antimicrobial packaging is based on the release of antimicrobial

∗ Corresponding author at: Hefei University of Technology, School of Chemical Technology, Tunxi 193, Hefei 230009, Anhui, People’s Republic of China. Tel.: +86 551 62901450; fax: +86 551 62901450. E-mail address: [email protected] (H. Wang). http://dx.doi.org/10.1016/j.carbpol.2015.03.058 0144-8617/© 2015 Elsevier Ltd. All rights reserved.

entities in film, hence, the entity carrier film and entity diffusion in film are the two key factors. As a deacetylated derivative of chitin, chitosan (CS) has shown a great promise for its application in food preservation by the virture of non-toxic, biodegradable, bio-functional and biocompatible properties. On account of its excellent oxygen barrier and film-forming ability, CS has been used in bio-packaging for food preservation (Agulló, Rodríguez, Ramos, & Albertengo, 2003; Dutta et al., 2009). Nevertheless, the brittleness and moisture barrier limited the application of pure CS film in packaging. Poly(vinyl alcohol) (PVA) is a non-toxic water-soluble polymer and has good flexibility and film-forming ability. Furthermore, it is convenient to prepare CS/PVA composite film by solution blend method (Chen, Wang, Mao, & Yang, 2007). At present, serials of investigations were concerned on CS/PVA composite film in preparation (Chen, Wang, Mao, Liao, & Hsieh, 2008), mechanical, thermal and surface properties (El-Hefian, Nasef, & Yahaya, 2011), compatibility (Kumar et al., 2010), physicochemical and bioactivity (Tripathi, Mehrotra, & Dutta, 2009) and hydrogel property (Yang, Su, & Yang, 2004). However, no literature was reported on the diffusion behavior of

H. Wang et al. / Carbohydrate Polymers 127 (2015) 64–71

antimicrobial entities in CS/PVA composite film in antibacterial packaging to the best of our knowledge at present. As an antibacterial entity, the release and antibacterial behaviors of nisin have been investigated in films such as methylcellulose-hydroxypropyl methylcellulose (Grower, Cooksey, & Getty, 2004), starch-based (Sanjurjo, Flores, Gerschenson, & Jagus, 2006), cellulose (Nguyen, Gidley, & Dykes, 2008) and heat-pressed and cast films (Cha, Cooksey, Chinnan, & Park, 2003). The diffusivity of antimicrobial entity in film can provide important information for the design of film in structure and ingredient. Much valuable work has been done on determining the diffusivity of antimicrobial entities in antimicrobial films in food systems such as acetic and propionic acids from chitosan-based (Ouattara, Simard, Piette, Begin, & Holley, 2000), linalool and methylchavicol from polyethylene-based (Suppakul, Sonneveld, Bigger, & Miltz, 2011) and potassium sorbate from ␬-carrageenan-based (Choi et al., 2005) antimicrobial packaging films. Thermodynamic parameters, including enthalpy (H0 ), entropy (S0 ) and Gibbs free energy (G0 ), can provide additional information regarding the inherent energetic changes associated with the diffusion. However, few attentions were paid on the diffusion of antimicrobial entities from films. In the present work, we aimed to evaluate the kinetics and functional effectiveness of nisin loaded chitosan/poly(vinyl alcohol) (Nisin–CS/PVA) as an antibacterial packaging film and focused on the diffusion of nisin from the film by kinetics and thermodynamics, and the effects of CS/PVA ratio, temperature, pH and ionic strength on the diffusion efficiency. To this aim, an operationally simple coating method was used to prepare the films by an automatic film applicator. The mechanical property, structure, water vapor permeability (WVP) and oxygen permeability (OP) of the CS/PVA films as well as the antimicrobial activity of Nisin–CS/PVA films were assessed. Nisin was used as a model antimicrobial entity and Staphylococcus aureus (S. aureus, ATCC6538) as test bacterium.

2. Experimental 2.1. Materials

65

PE film and vacuum dried at 60 ◦ C for 24 h to remove the residue water and acetic acid. Nisin–CS/PVA films were prepared on the AFA-III automatic film applicator and the parameters were the same as that of CS/PVA films. Required nisin (1, 3, 5, 8, 10 and 20%, w/w, based on the weight of CS/PVA) was gradually added into the CS/PA solutions and stirred continuously at room temperature for 4 h before coating on the substrate film. 2.3. Measurement and characterization Film thickness was measured with a hand-held micrometer (BC Ames Co., Waltham, MA, USA). Nine film thickness measurements were taken from along the length of each specimen and the mean value was used in calculating tensile strength and diffusion coefficient. The tensile strength (TS) and elongation at break (EB) of specimens were conducted on a TA-XTPlus Texture Analyser (Stable Micro Systems, Co., UK). All the dry films were cut into rectangular blocks (1 cm × 10 cm in width and length). The morphologies of films were estimated by scanning electron microscopy (SEM; SU8020, Hitachi, Japan) under an accelerating voltage of 15 kV, and all of the specimens were sputter-coated with a layer of gold. Fourier transform infrared (FTIR) spectroscopy was conducted with a Nicolet 6700 spectrometer (Thermo Nicolet, USA) with KBr pellets. 2.4. Permeability of films Water vapor permeability (WVP) were measured using a modified method as described by Limpan, Prodpran, Benjakul, and Prasarpran (2010). The specimens, sealed on beakers, containing silica gel (0% RH) were placed in incubator containing distilled water. The chamber of incubator was provided with a psychrometer for relative humidity, and the temperature of incubator was maintained at 30 ◦ C. The moisture absorbed was estimated by weighing of beakers at 3 h intervals during 3 days. WVP (g m−1 s−1 Pa−1 ) was determined for three replicate specimens each type, as follows: WVP =

w×x A × t × P

(1)

Chitosan (CS, Mw 300 kDa, viscosity 100 MPa s, DD 95%) was purchased from Zhejiang Aoxing Biochemical CO., Ltd., (Zhejiang, Chain). Polyvinyl alcohol (PVA, DP 1750 ± 50) was supplied from Sinopharm Chemical Reagent CO., Ltd., (Shanghai China). The 2.5% nisin preparation was a powdered product from MP Biomedicals, LLC (Solon, Ohio, USA). The product was labeled with a nisin concentration of 1000 IU mg−1 solids. Staphylococcus aureus (S. aureus, ATCC6538) was provided by China Center of Industrial Culture Collection (Beijing, China). All the other chemical reagents used were of analytical grade and available from Sinopharm Chemical Reagent Co. Ltd., (Shanghai, China).

OP = OTR × thickness

2.2. Preparation of samples

The thickness and open testing area of each sample in three parallel measurements were approximately 100 ␮m and 50 cm2 .

Chitosan was dissolved into 2% (v/v) acetic acid to obtain 1 wt.% CS solution. A PVA aqueous solution (5 wt.%) was prepared by dissolving PVA powder into distilled water at 85 ◦ C. Afterwards, serials of CS/PVA blend solutions with different ratio of CS/PVA (3:1, 2:1, 1:1, 1:2 and 1:3, w/w) were prepared by mixing. Correspondingly, the films prepared were defined as CS/PVA = 3/1, CS/PVA = 2/1, CS/PVA = 1/1, CS/PVA = 1/2 and CS/PVA = 1/3, respectively. All the films were preformed on an AFA-III automatic film applicator (Hefei Kejing Material Technology Co., Ltd., China). The CS/PVA blend solution was coated onto a substrate polyethylene (PE) film. After drying at 35 ◦ C for 72 h, the films were peeled from

where w is the weight gain of the beaker (g), x is the film thickness (m), A is the area of exposed film (m2 ), t is the time of weight gain (s), and P is the water vapor partial pressure difference (Pa) across the two sides of film calculated on the basis of relative humidity. Oxygen transmission rate (OTR, according to ASTMD1434) of films was determined at 23 ◦ C and 0% RH on a N500 gas permeameter (Guangzhou Biaoji packaging equipment Co., Ltd., Guangzhou, China). Oxygen permeability (OP) was calculated from OTR (cm3 m−2 d−1 kPa−1 ) by the following equation: (2)

2.5. Antibacterial activity assay The antibacterial activity evaluation of film against S. aureus was performed in liquid culture medium (Wang et al., 2013). The concentration of S. aureus for bacterial suspension after rejuvenating was set as 1.0, and the absorbance at 600 nm for each sample with concentration of S. aureus at 0.05, 0.1, 0.15, 0.2 and 0.25 was measured. On the basis of experimental data, the linear regression equation from the calibration curve was determined as following: Y = 0.8196X − 0.00406(R2 = 0.99925)

(3)

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H. Wang et al. / Carbohydrate Polymers 127 (2015) 64–71

where the X is the relative concentration of S. aureus and Y is the absorbance. 1 ml rejuvenated bacterial suspension was transferred into 9 ml KMB broth, then 30 mg Nisin–CS/PVA film was soaked into liquid culture medium containing S. aureus after sterilization by ultraviolet light. The culture media were propagated for 24 h on a shaker platform (SHZ-82, Changzhou Guohua Electric Appliance Co., Ltd., Jiangsu, China) at 160 rpm and 37 ◦ C. The absorbance at 600 nm was measured using a spectrophotometer (UV-754PC, Shanghai Jinghua Technology Instruments Co., Ltd., Shanghai, China) and relative concentration of S. aureas was calculated based on Eq. (3).

2.7. Statistical analysis Each experiment was repeated three times. Statistical analysis was performed using the unpaired Student’s t-test, and the results were expressed as the means ± standard deviation (SD). A value of p < 0.05 was considered to be statistically significant. 3. Results and discussion 3.1. Structure and mechanical property of CS/PVA film FTIR spectroscopy was used to investigate the interaction between CS and PVA and the spectra were shown in Fig. 1. In the spectrum of PVA, characteristic peaks for PVA were present at: 3352 ( OH stretching); 2944 ( CH anti-symmetric stretching); 1430, 1335 ( CH angular deformation) and 1090, 1142 cm−1 (C O stretching, band at 1142 cm−1 was mostly attributed to the crystallinity of PVA) (Wang et al., 2014). In the spectrum of CS, characteristic peaks of its saccharide structure were present at 902, 1020 and 1083 cm−1 (skeletal vibrations referring to the C O stretching), and 1151 cm−1 (anti-symmetric stretching of C O C bridge, glycosidic band) (Ávila, Bierbrauer, Pucci, López-González, & Strumia, 2012; Li, Hu, Ren, Worley, & Huang, 2013). The absorption bands at 1654, 1596 and 1390 cm−1 were assigned to the stretching vibration of C O (amide I), bending vibration of NH2 (amide II) and bending vibration of CH2 , respectively, while the absorption band at 2875 cm−1 corresponded to the bending vibration of CH in CH3 from NHCOCH3 . In addition, the strong and broad band at around 3430 cm−1 was due to the stretching vibration of OH, extension vibration of NH and intermolecular hydrogen bonds of the polysaccharide moieties (Sajomsang, Ruktanonchai, Gonil, & Warin, 2010). As compared to the spectra of CS and PVA, the band at around 3430 cm−1 concerned with OH and NH was broadened and obviously shifted to a lower wave number around 3330 cm−1 in the spectrum of CS/PVA film, indicating the formation of intermolecular hydrogen bonds between CS and PVA molecules (Fig. 1B). Tensile strength (TS) and elongation at break (EB) can reflect strength and elasticity of film. Fig. 2 demonstrated the results of TS and EB of the films. For pure CS film, it had a high TS (46.8 MPa), but very low EB (21.6%), while pure PVA film exhibited better elasticity and the EB reached 222.8%. As expected, its EB was sharply improved by incorporating PVA into CS matrix, and the film could also bear a relative high TS at 26.7 MPa for CS/PVA = 1/1. The reason was likely that CS had a high molecular weight and hard backbones as compared to PVA. Similar trend was reported by Srinivasa, Ramesh, Kumar, and Tharanathan (2003). To better understand the

2.6. Diffusion test Series of nisin solutions (50, 100, 150, 200, 250 and 300 ␮g ml−1 ) were prepared by dissolving nisin into distilled water in Erlenmeyer flasks. 1 ml above solution was stained with 2 ml Coomassie brilliant blue G250 solution. The absorbance at 595 nm was measured for each solution (1 ml distilled water and 2 ml Coomassie brilliant blue G250 blend solution as blank). On the basis of experimental data, the regression line from the calibration curve was expressed as following: Y = 0.00142X + 0.09179(R2 = 0.99548)

(4)

where X (␮g ml−1 ) is the concentration of nisin, and Y is the absorbance. Films were cut into 4 cm squares, then the square film was covered with aluminum foil tape on one side and immersed in a Erlenmeyer flak containing 100 ml distilled water. The flaks were shaken on the shaker platform (100 rpm, 25 ◦ C) and achieved diffusion equilibration. The absorbances at 595 nm were measured with the spectrophotometer. The concentrations of nisin at different time and diffusion equilibration were determined based on Eq. (4). The accumulative release percentage of nisin from the films was calculated using the following equation: Accumulative release(%) =

Mt × 100% M0

(5)

where Mt (␮g) is the released nisin at time t, M0 (␮g) is the total trapped or entrapped nisin. The pH values of diffusion solutions were adjusted by adding a thimbleful of 0.01 M HCl or 0.01 M NaOH solution. Most experiments were carried out at pH 6.5 ± 0.1. According to our experimental data, the diffusion percentage is higher if the solution pH is lower, while the diffusion percentage is lower if the pH is higher. Thus, a pH approximately 6.5 is representative.

Tranmittance

(A)

(B)

PVA

3352

2944

H

1335 1090 1430 1139

CS

Intermolecular 665

1654 3430

1596 1390

2875

1151

O O

O O

O

1083 1022

H

O

NH2 r ula lec o ram Int

O

NH 2 Intermolecular

H H

O CH2 CH

3330

3500

HOH2C HOH2C

902

CS/PVA

4000

CH2 CH n O

3000

2500

2000

1500

1000

n

500

-1

Wave number(cm ) Fig. 1. (A) FT-IR spectra of samples; (B) proposed structure of CS/PVA (Wan Ngah, Kamari, & Koay, 2004). Intermolecular hydrogen bonds existed between CS and PVA molecules.

H. Wang et al. / Carbohydrate Polymers 127 (2015) 64–71

67

Fig. 2. Tensile properties of films in: (A) tensile strength and (B) elongation at break. Tensile strength of films decreased seriously (p < 0.05) by incorporating PVA into CS matrix, meanwhile, the corresponding elongation at break was sharply improved (p < 0.05). The data were representative of the results from repeated experiments (n = 3) and expressed as the mean ± SD.

Fig. 3. Cross-section SEM photographs of specimens at break. Cross-section of CS/PVA film showed lots of tensile cracks compared to CS and PLA films.

tensile properties of films, SEM study was performed at the crosssection of the corresponding pure CS, pure PVA and CS/PVA = 1/1 films at break (Fig. 3). The cross-section SEM photograph of pure CS (Fig. 3a) and PVA film (Fig. 3c) at break showed a smooth surface and no obvious tensile cracks. However, lots of cracks appeared on surface of CS/PVA = 1/1 film at break (Fig. 3b), suggesting the incorporation of PVA into CS matrix improved elasticity of the film.

3.2. Water vapor and oxygen permeability As could be seen from WVP results of the films (Fig. 4A), the WVP decreased with the decrease of CS/PVA ratio and then increased again after reached the minimum value 0.983 × 10−10 g m−1 s−1 Pa−1 for CS/PVA = 1/1. The reason may be attributed to the high crosslink effects formed by intermolecular hydrogen bonds between CS and PVA molecules for CS/PVA = 1/1,

Fig. 4. Effect of CS/PVA ratio on (A) water vapor permeability and (B) oxygen permeability. With decreasing CS/PVA ratio, water vapor permeability of films decreased and then increased after reached the minimum value (p < 0.05), meanwhile, the corresponding oxygen permeability improved significantly (p < 0.05). The data were representative of the results from repeated experiments (n = 3) and expressed as the mean ± SD.

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H. Wang et al. / Carbohydrate Polymers 127 (2015) 64–71

Relative concentration of S. aureus

which makes the film structure become more compact (Zhang, Xu, & Jiang, 2012). Furthermore, the WVP of the pure PVA film was slightly higher than that of pure CS film owing to the higher hydrophilicity toward CS. Fig. 4B illustrated OP of pure and blended films. Pure CS and PVA had OP values of 0.05 and 3.13 cm3 ␮m m−2 d−1 kPa−1 . As non-polar molecules, oxygen molecules would dissolve to a greater extent in less polar polymers, thus, a less polar film would have higher OP due to higher sorption toward oxygen. When PVA was incorporated into CS matrix, the polarity of matrix was weakened owing to the decrease in amount of NH2 and OH. As a result, the blended films showed a decrease in oxygen barrier with decreasing CS/PVA ratio, but OP value was still lower than 0.91 cm3 ␮m m−2 d−1 kPa−1 for CS/PVA = 1/1 as the CS/PVA ratio was above 1:1. Based on the mechanical property, WVP and OP results, the CS/PVA ratios were chosen at 1:1 and above to prepare Nisin–CS/PVA antimicrobial films.

1.0 0.8 0.6 0.4 0.2 0.0 0

5

10

15

20

Nisin content (%) 3.3. Antimicrobial activity evaluation in liquid culture medium

Fig. 5. Relative concentrations of S. aureus of Nisin–CS/PVA film (CS/PVA = 1:1, propagated 24 h, 37 ◦ C). The relative concentration of S. aureus decreased significantly compared to CS/PVA film without nisin (p < 0.05). The data were representative of the results from repeated experiments (n = 3) and expressed as the mean ± SD.

As it is known, antimicrobial activity is inversely proportional to the relative concentration of S. aureus. Fig. 5 depicted the relative concentration of S. aureus for Nisin–CS/PVA films in liquid culture medium after incubation for 24 h at 37 ◦ C. It was found that the relative concentration of S. aureus decreased sharply from 100% to 11.65% when nisin content increased from 0 to 10%, and then decreased gradually. The releasing dosage of nisin into bacterial suspension increased in quantity with the increase of the nisin content in films, therefore, the growth of S. aureus was inhibited effectively. While nisin content was above 10%, the increase of nisin content had no obvious effect on the antimicrobial activity.

suggesting a good controlled release behavior for Nisin–CS/PVA film (Selvam, Manikandan, Kennedy, & Vijaya, 2013). It was noteworthy that the accumulative release percentage at diffusion equilibrium decreased with the decrease of CS/PVA ratio. The diffusion of nisin on or near surface of film resulted in the initial burst release under the diffusion driving force by nisin content (Moditsi, Lazaridou, Moschakis, & Biliaderis, 2014; Ozdemir & Floros, 2001). While the nisin being trapped into the inner core of the matrix would take longer time to be released owing to longer diffusion pathway and showed an increase in accumulative release. Nisin was an amphiphilic molecule simultaneously displaying hydrophobic and hydrophilic behaviors, however, it had the better affinity toward hydrophilic materials (Karam et al., 2013a, 2013b). Therefore, the incorporation of CS into PVA matrix increased the hydrophobicity of film, resulting in that the accumulative release percentage at equilibrium increased with the increase of CS/PVA ratio.

3.4. Diffusion kinetics of nisin from Nisin–CS/PVA film 3.4.1. Release of nisin For assessing the release behavior and acquiring basic information for diffusion kinetics, the accumulative release percentages of nisin from films with CS/PVA ratios at 3:1, 2:1 and 1:1 were calculated by Eq. (5) and plotted versus time in Fig. 6A. Each curve showed initial burst release phenomenon, and reached a plateau after a significantly increase in accumulative release percentage,

(A)

70

(B)

50

1.0 0.8

40

Mt /M

Accumulative release(Mt/M0,%

60

30

0.6 0.4

20 10

CS/PVA=1:1 CS/PVA=2:1 CS/PVA=3:1

0

0.2 0.0

-10 0

3000

6000

t (min)

9000

12000

15000

0

3000

6000

9000

12000

15000

t (min)

Fig. 6. (A) Accumulative release of nisin from Nisin–CS/PVA films (T = 25 ◦ C, pH = 6.5 ± 0.1, nisin 10 wt.%). Each curve showed initial burst release phenomenon, and reached a plateau after a significantly increase (p < 0.05); (B) fractional diffusion of nisin in films Mt /M∞ versus time t, inset: linear regression of Mt /M∞ versus square root of time t1/2 (T = 25 ◦ C, pH = 6.5 ± 0.1, nisin 10 wt.%). Mt /M∞ increased with the increase of CS/PVA ratio, and each curve showed a sharp increase before a plateau (p < 0.05). The initial diffusion of nisin (Mt /M∞ < 2/3) from films could be well described by the Fickian diffusion equation. The data (mean ± SD) are results from three independent experiments.

H. Wang et al. / Carbohydrate Polymers 127 (2015) 64–71

3.4.2. Determination of diffusivity of nisin An initial mathematical model for analysis of nisin diffusion from Nisin–CS/PVA film could be derived from Fick’s second law (Crank, 1975):

69

-26.5

-27.0

2

∂C ∂ C =D ∂t ∂x2

(6)

4C0  1 C= exp  2n + 1 ∞

n=0



D(2n + 1)2 2 t − h2

 sin

(2n + 1) x h

(7)

where C0 (kg m−3 ) is the initial content of nisin in film, h (m) is the thickness of Nisin–CS/PVA film. Eq. (7) integrated over the thickness of the film could be expressed in linear form (Choi et al., 2005; Moditsi et al., 2014; Yoshida, Bastos, & Franco, 2010).

 Mt 8 =1− exp M∞ (2n + 1)2 2 ∞



n=0

D(2n + 1)2 2 t − h2

 (8)

where Mt (␮g) is the amount of nisin diffused at time t, M∞ (␮g) is the amount of nisin diffused at equilibrium. For Mt /M∞ < 2/3, Mt /M∞ could be calculated by the following formula (Crank, 1975; Malley, Bardon, Rollet, Taverdet, & Vergnaud, 1987; Moditsi et al., 2014; Peppas, 1985; Yoshida et al., 2010):



Mt Dt =4 M∞ h2

1/2

= kt 1/2

(9)

where k (1/s1/2 ) is the slope of the linear regression of Mt /M∞ versus t1/2 . Consequently, the diffusivity could be counted by the following equation: D=

 kh 2 4



-28.5

CS/PVA=1:1 CS/PVA=2:1 CS/PVA=3:1 -29.0

0.0031

CS/PVA ratio

Temperature (◦ C)

D (×10−13 m2 /s)a

3:1

5 25 45 5 25 45 5 25 45

8.071 14.644 27.366 4.252 9.197 18.814 2.468 6.489 16.074

± ± ± ± ± ± ± ± ±

0.26 0.65 0.80 0.14 0.54 0.73 0.15 0.23 0.68

R2 b 0.99925 0.99971 0.99910 0.99827 0.99942 0.99916 0.99903 0.99992 0.99831

a Nisin diffusivity was calculated using Eq. (10). The data (mean ± SD) are results from three independent experiments. b Given correlation coefficient (R2 ) was the largest one among replications (n = 3).

0.0032

0.0033

0.0034

0.0035

0.0036

-1

1/T(K ) Fig. 7. Temperature effects on diffusion coefficient (CS/PVA = 1:1, pH = 6.5 ± 0.1, nisin 10 wt.%). The effect of temperature on diffusion coefficient could be described by Arrhenius activation model. The data (mean ± SD) are results from three independent experiments.

cases, linearity with respect to t1/2 predicted by Eq. (9) for the initial portion of the curve (Mt /M∞ < 2/3) was strong, indicating that the diffusion mechanism could be well described by Fickian diffusion. The linear regressions of Mt /M∞ versus t1/2 for CS/PVA films at different ratios were shown in the inset of Fig. 6B, correspondingly, the diffusion coefficient D calculated by Eq. (10) was listed in Table 1. Similarly, the D values for CS/PVA films at 5 ◦ C and 45 ◦ C were obtained and summarized in Table 1. The high correlation coefficient R2 > 0.998 indicated that the predominant diffusion was Fickian diffusion. As expected, Table 1 showed a decrease in D value with the decrease CS/PVA ratio at each given temperature due to the higher affinity of nisin toward PVA, and higher temperature caused an increase in D value for the same CS/PVA films. 3.4.3. Temperature dependence of diffusivity The temperature effect on diffusivity is generally described by the Arrhenius activation energy mode.

 E  a

(10)

Table 1 Diffusivity of nisin from Nisin–CS/PVA films (pH = 6.5 ± 0.1, nisin 10 wt.%).

1:1

-28.0

D = D0 exp −

Based on Fig. 6A, the whole curves of Mt /M∞ versus time were demonstrated in Fig. 6B. As could be seen from Fig. 6B, Mt /M∞ depended on film composition at given time and increased with the increase of CS/PVA ratio owing to the lower affinity of nisin toward CS as compared to PVA. Moreover, each curve showed a similar shape by increasing sharply before reaching a plateau. In all

2:1

ln D

where C (kg m−3 ) is the nisin concentration in the film, D (m2 s−1 ) is the diffusivity or diffusion coefficient, x (m) is the coordinate measured in the direction of transport, and t (s) is the time. The following assumptions were made: (1) The film prepared was plane sheet. (2) The dispersion of nisin in film was uniform and the nisin concentration in aqueous medium was zero. (3) The diffusion of nisin was regarded as one-dimensional diffusion (As mentioned in experimental, the specimen was covered with aluminum foil tape on one side) and a non-steady state phenomenon of nonconcentration-dependent diffusion. Based on these assumptions, a classical solution was obtained (Crank, 1975):

-27.5

(11)

RT

where D0 (m2 s−1 ) is a constant, Ea (J mol−1 ) is activation energy of the diffusivity, R (J mol−1 K−1 ) is universal gas constant and T (K) is absolute temperature. The Arrhenius plots were derived from logarithmic transform of Eq. (11) (Fig. 7). From the slope of line, the corresponding Ea were summarized in Table 2. The high correlation coefficient values (R2 > 0.999) implied that the effect of temperature on diffusion coefficient could be described by Arrhenius activation model. The greater the Ea value, the more sensitive the diffusivity to temperature changes. In addition, Ea might predict the molecular interactions between the diffusate and diffusion medium. The Table 2 Activation energy of diffusion for nisin from Nisin–CS/PVA films (pH = 6.5 ± 0.1, nisin 10 wt.%). CS/PVA ratio

Ea (kJ/mol)a

R2 b

3:1 2:1 1:1

21.997 ± 0.813 26.843 ± 1.342 33.813 ± 1.276

0.99178 0.99838 0.99798

a Ea was calculated using Eq. (11). The data (mean ± SD) are results from three independent experiments. b Given correlation coefficient (R2 ) was the largest one among replications (n = 3).

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H. Wang et al. / Carbohydrate Polymers 127 (2015) 64–71

higher Ea value indicated a stronger interaction between nisin and CS/PVA matrix, because more work in the form of energy was needed to overcome the energetic barrier. As mentioned above, nisin had higher affinity toward PVA as compared to CS. Hence, the decrease of CS/PVA ratio caused an increase in Ea value at same temperature.

repulsion suppressed by the increasing ionic strength, meanwhile, a higher ionic strength could also enhance the hydrophobicity of nisin (Zhong, Jin, Davidson, & Zivanovic, 2009). However, no distinguish in M∞ /M0 was observed in NaCl solutions owing to the poor solubility of nisin at pH > 8.8. 3.5. Thermodynamic parameter

3.4.4. Effects of pH and ionic strength on diffusion There was a significant difference in the diffusion percentage at equilibrium (M∞ /M0 ) as a function of pH for the diffusion of nisin from film in NaCl solutions (Fig. 8A), and each curve could be divided into three regions. As pH reduced, M∞ /M0 showed a slightly increase in region I (pH > 8.8), a significantly increase in region II (pH 4.5–8.8) and a high level at pH < 4.5 (region III). Nisin was positively charged at pH below the isoelectric point (pI, 8.8) (Xiao, Gömmel, Davidson, & Zhong, 2011), and the dissolubility in water was improved as the pH reduced (Bani-Jaber, McGuire, Ayres, & Daeschel, 2000). On the other hand, CS was also positively charged in acidic situation and the quantity of amino groups protonated increased at a reduced pH (Jiang, Wang, Li, Jiang, & Lu, 2005; Katas & Alpar, 2006). In region I, M∞ /M0 depended on the solubility of nisin and showed a slightly increase at a reduced pH. Nevertheless, the electrostatic repulsion from the positively charged nisin and CS in acidic situation favored nisin to diffuse from film to aqueous solution. As a result, a significantly increase in M∞ /M0 appeared in region II (especially at pH < 7.0) owing to the enhancing electrostatic repulsion at a reduced pH. As shown in Fig. 8A, M∞ /M0 also depended on NaCl concentrations at each given pH. An increase in NaCl concentration resulted in a lower M∞ /M0 at pH < 8.8. This behavior was most likely due to the Debye length and the effective distance of electrostatic

/M0,%)

(A) 100

region III

M∞ m · M0 − M∞ V

(12)

where V (ml) was the volume of the solution and m (g) was the weight of each film. The enthalpy change (H0 ) and entropy change (S0 ) for the diffusion of nisin from the film were calculated by the intercept and slope of the plot of lnKd versus 1/T (Fig. 8B) based on temperaturedependent distribution coefficient relationship: ln Kd =

S 0 H 0 − R RT

(13)

where R (8.314 J mol−1 K−1 ) is the ideal gas constant, and T (K) is the temperature in Kelvin. Meanwhile, the corresponding G0 was obtained by the general expression: G0 = H 0 − TS 0

(14)

Thermodynamic parameters based on Eqs. (13) and (14) were summarized in Table 3. The positive H0 suggested the diffusion was endothermic, because kinetic energy was needed for the entrapped nisin to travel through the CS/PVA matrix. The positive

CS/PVA=1:1 CS/PVA=2:1 CS/PVA=3:1

2.1

80 region I

70 60

1.8

ln Kd(g/L)

Diffusion percentage(M

Kd =

(B) 2.4

region II

90

A distribution coefficient (Kd ) associated with the total entrapped nisin M0 in film and the amount of nisin diffused at equilibrium M∞ was adapted:

50 40

1.00M NaCl 0.10M NaCl 0.01M NaCl 0 M NaCl

30 20

1.5 1.2 0.9 0.6

10

0.3

0 2

4

6

8

10

0.0031

0.0032

0.0033

0.0034

0.0035

0.0036

-1

1/T (K )

pH

Fig. 8. (A) Diffusion percentage at equilibrium (M∞ /M0 ) as a function of pH in NaCl solutions (T = 25 ◦ C, CS/PVA = 1:1, nisin 10 wt.%). At given NaCl solution, M∞ /M0 showed a slightly increase at pH > 8.8, a significantly increase at pH 4.5–8.8 (p < 0.05) and a high level at pH < 4.5. Moreover, an increase in NaCl concentration resulted in a lower M∞ /M0 at pH < 8.8. (B) Plots of lnKd versus 1/T for the diffusion of nisin from the film (pH = 6.5 ± 0.1, nisin 10 wt.%). Plots showed a good linear correlation between d lnKd and 1/T. The data (mean ± SD) are results from three independent experiments. Table 3 Thermodynamic parameters of diffusion for nisin from Nisin–CS/PVA films (pH = 6.5 ± 0.1, nisin 10 wt.%). CS/PVA ratio

S0 (J (mol K)−1 )a

H0 (kJ mol−1 )a

3:1 2:1 1:1

84.929 ± 2.387 63.729 ± 2.874 34.632 ± 1.153

9.006 ± 0.279 16.260 ± 0.748 21.000 ± 0.861

a b

G0 (kJ mol−1 )b 278.15 K

298.15 K

318.15 K

−2.602 ± 0.082 −1.466 ± 0.063 −0.627 ± 0.018

−4.321 ± 0.116 −2.741 ± 0.092 −1.318 ± 0.064

−6.020 ± 0.125 −4.015 ± 0.103 −2.012 ± 0.092

S0 and H0 were calculated using Eq. (13). The data (mean ± SD) are results from three independent experiments. G0 were calculated using Eq. (14). The data (mean ± SD) are results from three independent experiments.

H. Wang et al. / Carbohydrate Polymers 127 (2015) 64–71

S0 might be related to the affinity of nisin toward CS/PVA matrix and the dispersion change of nisin in CS/PVA matrix. It was noteworthy that the value of G0 was negative and decreased with an increase in temperature, indicating that the diffusion of nisin in CS/PVA matrix was spontaneous and the spontaneity improved by increasing temperature. 4. Conclusion The present study reported Nisin–CS/PVA antibacterial films prepared by coating method. The interaction between CS and PVA molecules was confirmed to be intermolecular hydrogen bonds. The incorporation of PVA into CS matrix could sharply improve the elasticity of the film, and the film could also bear a relative high tensile strength at 26.7 MPa for CS/PVA = 1/1. With the decrease of CS/PVA ratio, the WVP decreased and reached the minimum value 0.983 × 10−10 g m−1 s−1 at CS/PVA = 1/1, at the same time, OP increased but was still lower than 0.91 cm3 ␮m m−2 d−1 kPa−1 for CS/PVA = 1/1 as the CS/PVA ratio was above 1:1. The initial diffusion of nisin (Mt /M∞ < 2/3) from CS/PVA film could be well described by the Fickian diffusion equation. M∞ /M0 showed a slightly increase in region I (pH > 8.8), a significantly increase in region II (pH 4.5–8.8) and a high level at pH < 4.5 (region III). The foreign ionic strength had significant influence on the diffusion of nisin in CS/PVA films at pH < 8.8, and no distinguish pH > 8.8. The thermodynamic parameters indicated that the diffusion of nisin was endothermic and spontaneous. The experimental results showed that the films revealed well controlled release and better antimicrobial activity against S. aureus, which may have potential as an active film in food packaging. Acknowledgments Financial support from National Natural Science Foundation of China (31371859) is gratefully acknowledged. References Agulló, E., Rodríguez, M. S., Ramos, V., & Albertengo, L. (2003). Present and future role of chitin and chitosan in food. Macromolecular Bioscience, 3(10), 521–530. Aider, M. (2010). Chitosan application for active bio-based films production and potential in the food industry: Review. LWT—Food Science and Technology, 43(6), 837–842. Ávila, A., Bierbrauer, K., Pucci, G., López-González, M., & Strumia, M. (2012). Study of optimization of the synthesis and properties of biocomposite films based on grafted chitosan. Journal of Food Engineering, 109(4), 752–761. Bani-Jaber, A., McGuire, J., Ayres, J., & Daeschel, M. (2000). Efficacy of the antimicrobial peptide nisin in emulsifying oil in water. Journal of Food Science, 65(3), 502–506. Cha, D., Cooksey, K., Chinnan, M., & Park, H. (2003). Release of nisin from various heat-pressed and cast films. LWT—Food Science and Technology, 36(2), 209–213. Chen, C.-H., Wang, F.-Y., Mao, C.-F., Liao, W.-T., & Hsieh, C.-D. (2008). Studies of chitosan: II. Preparation and characterization of chitosan/poly(vinyl alcohol)/gelatin ternary blend films. International Journal of Biological Macromolecules, 43(1), 37–42. Chen, C. H., Wang, F. Y., Mao, C. F., & Yang, C. H. (2007). Studies of chitosan. I. Preparation and characterization of chitosan/poly(vinyl alcohol) blend films. Journal of Applied Polymer Science, 105(3), 1086–1092. Choi, J., Choi, W., Cha, D., Chinnan, M., Park, H., Lee, D., et al. (2005). Diffusivity of potassium sorbate in ␬-carrageenan based antimicrobial film. LWT—Food Science and Technology, 38(4), 417–423. Crank, J. (1975). The mathematics of diffusion (2nd ed.). New York: Oxford University Press. Dutta, P., Tripathi, S., Mehrotra, G., & Dutta, J. (2009). Perspectives for chitosan based antimicrobial films in food applications. Food Chemistry, 114(4), 1173–1182. El-Hefian, E. A., Nasef, M. M., & Yahaya, A. H. (2011). Preparation and characterization of chitosan/poly(vinyl alcohol) blended films: Mechanical, thermal and surface investigations. Journal of Chemistry, 8(1), 91–96. Grower, J., Cooksey, K., & Getty, K. (2004). Release of Nisin from methylcellulosehydroxypropyl methylcellulose film formed on low-density polyethylene film. Journal of Food Science, 69(4), FMS107–FMS111.

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poly(vinyl alcohol).

The aim of this study was to evaluate the kinetics and functional effectiveness of Nisin loaded chitosan/poly(vinyl alcohol) (Nisin-CS/PVA) as an anti...
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