- Email: [email protected]
Controlled release of nisin from polyvinyl alcohol - Alyssum homolocarpum seed gum composite films: Nisin kinetics
Food Bioscience 28 (2019) 133–139
Contents lists available at ScienceDirect
Food Bioscience journal homepage: www.elsevier.com/locate/fbio
Controlled release of nisin from polyvinyl alcohol - Alyssum homolocarpum seed gum composite films: Nisin kinetics
T
Leila Monjazeb Marvdashti, Masoud Yavarmanesh∗, Arash Koocheki Department of Food Science and Technology, Faculty of Agriculture, Ferdowsi University of Mashhad (FUM), Mashhad, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: Active packaging Diffusion coefficient Nisin Power law Weibull model
Antimicrobial biodegradable films based on PVA-AHSG incorporated with nisin were prepared. These films were characterized for their ability to control the overall release rate of nisin into foods, which would be needed for active food packaging. The objectives of this study were to assay the biodegradable films for nisin release, and to investigate their antimicrobial activity against L. innocua. The release kinetics of nisin from the films were described using Fick's Second Law, mass ratio, partition coefficient, desorption coefficient and the Weibull model. All PVA-AHSG films showed inhibition zones that increased with increasing nisin concentration. Nisin release increased as its concentration in the film matrix increased. Release of nisin was faster at higher concentrations, and the diffusion coefficients increased significantly with increasing concentration. The partition coefficient (k) values obtained were 1.42, 0.61 and 0.64 for 3000, 5000 and 10,000 IU of nisin, respectively. According to the Weibull parameters, nisin is released following pseudo-Fickian diffusion. Results showed that the PVA-AHSG composite films showed promise for developing controlled release applications with nisin in aqueous solutions.
1. Introduction
(Monjazeb Marvdashti, Yavarmanesh, & Koocheki, 2016). However, blending the AHSG with polyvinyl alcohol (PVA) gave a film with greater strength and flexibility (Monjazeb Marvdashti et al., 2016, 2017). Nisin is an antimicrobial peptide produced by strains of Lactococcus lactis subsp. Lactis (de Arauz, Jozala, Mazzola, & Penna, 2009). Nisin has a wide range of activity against Gram-positive bacteria and is commonly used as a natural food preservative. The efficiency of the antimicrobial activity of nisin depends on several parameters, such as pH, temperature, salt and fat concentration (Dean & Zottola, 1996; Jung, Bodyfelt, & Daeschel, 1992). These parameters have an important role in nisin bioactivity, solubility, stability, rate of desorption from films and diffusion into food. Release kinetics of antimicrobial agents used in food packaging matrices have not been widely studied, whereas the release of active agents from drugs has received more attention (Buonocore, Del Nobile, Panizza, Corbo, & Nicolais, 2003; Galdámez, Serna, Larry Duda, & Danner, 2007). Fick's second law has been used to describe the release kinetics of antimicrobial agents from packaging material to food simulants or food matrixes (Buonocore et al., 2003; Han & Floros, 1998; Kim et al., 2002). The modeling of nisin diffusion from a packaging material into the food system can help to increase the capacity and efficiency of packaging using an antimicrobial carriers. In packaging
Packaging extends the shelf life and safety of food products (Bastarrachea, Dhawan, Sablani, & Powers, 2010). Antimicrobial agents can be added to food systems for controlling contaminations. However the antimicrobial activity was rapidly lost due to interactions with food components (Kim, An, Park, Park, & Lee, 2002). This problem can be reduced with antimicrobial active packaging because antimicrobial packaging establishes a more efficient protection of the food as controlled release involves a more convenient, steady and gradual delivery of active agents (Guiga et al., 2010). Alyssum homolocarpum seed gum (AHSG) is a galactan-type polysaccharide. Using a sample similar to the current sample, the seed gum contained 83.0% galactose, 5.70% glucose, 5.04% rhamnose, 2.72% xylose, 3.04% mannose, and 0.530% arabinose with ∼3.7 × 105 g/mol average molecular weight (Alaeddini, Koocheki, Mohammadzadeh Milani, Razavi, & Ghanbarzadeh, 2018; Hesarinejad, Razavi, & Koocheki, 2015; Khoshakhlagh, Koocheki, Mohebbi, & Allafchian, 2017; Koocheki & Kadkhodaee, 2011; Koocheki & Razavi, 2009). This food hydrocolloid can be used as a thickening/gelling agent, fat replacer and stabilizer (Koocheki et al., 2009a,b, 2010). Previous work showed that films made with AHSG were a good oxygen barriers and had an acceptable appearance with weak mechanical properties ∗
Corresponding author. E-mail address: [email protected] (M. Yavarmanesh).
https://doi.org/10.1016/j.fbio.2019.01.010 Received 2 November 2017; Received in revised form 14 January 2019; Accepted 21 January 2019 Available online 28 January 2019 2212-4292/ © 2019 Elsevier Ltd. All rights reserved.
Food Bioscience 28 (2019) 133–139
L.M. Marvdashti et al.
Nisin was purchased from Sigma Aldrich (Saint Louis, MO, USA), and was prepared by dissolving 0.1 g of nisin powder in 2 mL of a 0.01 M HCl solution (pH = 2). This solution was filtered through a nominal 0.2 mm pore size Millipore filter (Nalgene, Rochester, NY, USA) and kept at 4 °C for maximum of 1 wk.
Dickinson, Sparks, MD, USA) containing 15% glycerol at −80 °C. One loopful of stock was inoculated onto the modified Oxford agar (MOX: Difco), incubated at 37 °C for 24 h and eventually stored at 4 °C until use (a maximum of 1 month). A single colony of L. innocua was inoculated into 9 mL of TSB plus 1% yeast extract (TSBYE: Difco) and incubated for 24 h at 37 °C. The determination of the inhibition zone was carried out using the method of Tramer and Fowler (1964): the semi-soft agar was prepared using the TSBYE plus 0.7% agar (Difco) and 1% Tween 20 (Fischer Scientific, Fair Lawn, NJ, USA). Then, 100 mL of the semi-soft agar medium was seeded with 1 mL of the L. innocua strain to give an approximate concentration of 106 CFU/mL. The Tween 20 was used to enhance the diffusion of nisin through the agar medium. Approximately 4 mL of the seeded semisoft agar was poured over the petri dishes with TSA (Sparks, MD, USA). The film discs with nisin were placed over the semi-soft agar. The plates containing the film discs were kept at 4 °C for 24 h and were afterwards incubated at 37 °C for another 24 h. The refrigeration step was important to allow an optimum release of nisin, prior to the growth of bacteria. The refrigeration step subsequently led to larger inhibition zones (Neetoo, Ye, & Chen, 2007). After the incubation, the diameters of the inhibition zones were measured to the nearest 0.01 mm with an electron digital caliper (Fisher Scientific, Pittsburgh, PA, USA). Four measurements were done of each inhibition zone. All experiments were repeated at least three times.
2.2. Preparation of AHSG powder
2.5. Nisin quantification
AHSG powder was prepared using the method developed by Monjazeb Marvdashti et al. (2017). In brief, A. homolocarpum seeds were soaked in distilled water (seed to water ratio of 1:40). The pH of the water was adjusted to 4 using NaOH (10 M). The slurry was mixed at 40 °C for 1 h. The gum was extracted using an extractor (Parskhazar, Tehran, Iran), precipitated with ethanol (96%), dried in a freeze drier (Martin Christ Freeze Dryer, Osterode am Harz, Germany), milled using a laboratory hummer mill and sieved to an average particle size of 100 μm, packed in an air tight glass jar and stored at 4 °C for a maximum of 20 wk (Koocheki et al., 2010).
A colorimetric method, namely the bicinchoninic acid (BCA) assay, was used to determine nisin concentration. Briefly, a 150 μL volume of sample and 150 μL of BCA solution were mixed together. Briefly, BCA solution was prepared by mixing the BCA and copper (II) sulfate in a ratio of 49:1 (v/v) (Zufferey, 2016). This solution was incubated for 1 h at 60 °C. The absorbance was measured at 580 nm with a spectrophotometer (UV-2100, UNICO, Shanghai, China). The nisin contents in solution were calculated from the standard curve. The line of the standard curve had the following equation:
films, antimicrobials can be characterized in terms of their ability to extend the shelf-life of food products. The antimicrobial activity can be modeled using attributes such as the diffusion coefficient (D), desorption coefficient (Kd), partition coefficient (K) and mass ratio (α) (Franssen, Rumsey, & Krochta, 2004; Imran, Klouj, Revol-Junelles, & Desobry, 2014). The Weibull model considers the non-linear behavior of several factors. In recent years, the Weibull model has been commonly applied in studying the kinetics of chemical changes and microbiological destruction (Van Boekel, 2008). The objective of this study was to investigate the release kinetics of nisin from a biodegradable PVA-AHSG composite film at three nisin concentrations. The kinetics of nisin release were also studied. Antibacterial activities of the different films with nisin were measured using Listeria innocua. 2. Materials and methods 2.1. Preparation of nisin standard solution
Absorbance (580nm) = 1.75 × ⎡Nisin ⎢ ⎣
2.3. Preparation of active PVA-AHSG composite films Films were obtained by using a solution casting procedure using the method of Monjazeb Marvdashti et al. (2017). To prepare the film, AHSG powder (1.5% w/w) was dissolved in distilled water for 20 min at room temperature (23 ± 2 °C). The solutions were then allowed to hydrate for 24 h at 4 °C. Thereafter, the solution was homogenized using a rotor-stator homogenizer (Ultraturrax D125, Janke and Kunkel, Staufen im Breisgau, Germany) at 13,500 rpm for 1 min. PVA (1.5% w/ w) was dissolved in distilled water at 95 °C for 60 min. After the PVA solution cooled, it was added into the previously hydrated AHSG solutions (60:40 PVA to AHSG ratio). Glycerol was also added (0.75%). The nisin solution was then added to the film solution to give various levels (3000, 5000 and 10,000 IU). The International Units (IU) for nisin activity is the amount of nisin required to inhibit one cell of Streptococcus agalactiae in 1 mL of broth. Finally, PVA-AHSG solutions were stirred for 30 min and centrifuged in a Sigma 3K30 centrifuge (Sigma Co, Osterode am Harz, Germany) at 7000 g for 10 min at room temperature to remove air bubbles. Film forming dispersions were spread over petri dishes (diameter 15 cm). Films were dried in an air convection heat oven (Soroush oven, Tehran, Iran) at 40 °C for 24 h, and then were peeled off and stored at 4 °C for a maximum of 14 days.
⎤
2
R ( mg ml ) ⎥ ⎦
= 0.98
(1)
2.6. Experimental design The PVA-AHSG active films were floated on the surface of a desorption solution (100 mL of different NaCl solutions). These solutions were stirred for 52 h. A volume of 200 μL was periodically taken from this solution to quantify the desorbed nisin. The impact of three parameters on desorption kinetics was studied, including the pH (between 3.8 and 6.3), NaCl concentration (0.8 and 3.2%) and the concentration of nisin in the active films (3000, 5000 and 10,000 IU). 2.7. Kinetic of nisin release The power law is applied for investigating the diffusion mechanism (Yoshida, Bastos, & Franco, 2010).
mt = kt n m∞
(2)
Where mt is the amount of nisin released at time t, m∞ is the amount of nisin released at equilibrium; ‘k’ is a constant that is associated with the network of macromolecules and ‘n’ is a diffusion exponent attribute of the release mechanism. With this law, for n = 0.5, the mechanism of diffusion was considered as Fickian transport; for n = 1, the solute diffusion directly correlates with time; for 0.5 < n < 1.0, the principal mechanism is anomalous or non-Fickian transport; for n > 1.0, the solute is released in subsequent stages (Crank, 1975; Yoshida et al., 2010).
2.4. Test of antimicrobial activity of the films The L. innocua (ATCC 33090) was provided by the Iranian Research Organization for Science and Technology (Tehran, Iran). The stock culture was maintained in tryptic soy broth (TSB: Difco, Becton 134
Food Bioscience 28 (2019) 133–139
L.M. Marvdashti et al.
Fick's second law was used to find and describe the diffusion pattern of nisin as a bioactive agent, into the release solution.
solution and the PVA-AHSG is equal to 1, due to the high solubility of PVA-AHSG film in the water.
∂CF (x , t ) ∂C (x , t ) =D F 2 ∂t ∂x
2.11. Release kinetics
(3)
Where CF(x, t) is the nisin concentration in the film at position x and time t, and D is the diffusion coefficient of the nisin through the film (cm2 s−1). The diffusion coefficient (D) of nisin was obtained from a film with the following assumptions: (1) the initial nisin concentration was homogeneous throughout the film, (2) the initial concentration of nisin in the simulated food solution was zero, and (3) the nisin amount diffused in water was the same as the amount released from the film (Crank, 1975).
Mt 8 =1− 2 M∞ π
∞
∑ n=0
−2(n + 1)2π 2Dt ⎤ 1 exp ⎡ 2 ⎥ ⎢ (2n + 1) L2 ⎦ ⎣
The kinetic data for the release of nisin from the PVA-AHSG composite films into the release solution was also determined using the Weibull model (Van Boekel, 2008):
Ln
2.12. Statistical analyses
Where Mt and M∞ are the amount of nisin released from the film at time t and at infinite time, respectively, n is the number of terms in a Taylor series and L is the film thickness. For short durations of time (Mt/M∞ < 2/3), Equation (4) can be simplified to Equation (5). Therefore, the D value can be calculated using Equation (6) (Crank, 1975).
All the experiments were replicated at least three times using a completely randomized design. Analysis of variance (ANOVA) was carried out and the results were divided into the Duncan's Multiple Range test (p < 0.05) using Statistical Package for the Social Sciences (SPSS) software Version 19 (IBM SPSS, Armonk, NY, USA). The difference between treatments were identified using Duncan's multiple range test and were considered significant when p < 0.05. The model calculations were done using MATLAB (R2012a) software (Math Works, Natick, MA, USA).
(5)
S × L ⎞2 D=⎛ ×π ⎝ 2 ⎠
(11)
Where MF,t and MF,0 are the amounts of nisin in the film at time t and 0, respectively. In this model, ‘n’ is the shape factor and ‘b’ is the scale parameter.
(4)
Mt Dt 1/2 = 4⎛ 2 ⎞ M∞ ⎝ 4L π ⎠
MF , t = −(bt )n MF , 0
(6)
where S is the slope of the plot of Mt/M∞ against
3. Result and discussion
t 1/2 .
3.1. Desorption solution 2.8. Partition coefficient The desorption solution has an important role in the diffusion of an active agent from a matrix to surrounding media. The desorption coefficients (kd) of nisin from the PVA-AHSG composite film indicated that with increasing concentration of NaCl and pH in the desorption solution, the release of nisin decreased (Table 1). Generally, the molecular weight of a migrant and its affinity for the matrix and/or desorption solution can effect desorption (Sebti, Blanc, Carnet-Ripoche, Saurel, & Coma, 2004). In 3.2% NaCl, as pH increased from 3.8 to 8.8, a significant decrease in kd was observed. This might be due to decreased nisin solubility at higher pH (Xiao, Go¨mmel, Davidson, & Zhong, 2011). Results also showed that the kd of the solutions increased with decreasing NaCl concentration due to enhanced intermolecular interactions between macromolecules (Table 1). As a result, slower diffusion occurs at higher NaCl because of weaker electrostatic repulsions and stronger hydrophobic attractions (Zhong, Jin, Davidson, & Zivanovic, 2009). Similar results have been reported by Kim et al. (2002) for vinyl acetate-ethylene. The desorption solution with 0.8% of NaCl at pH 3.8 was chosen as an optimal desorption condition for further experiments.
In general, the partition coefficient is described as the ratio of the migrant in the food stimulant (Cs, ∞) to the migrant in the film polymer (CF, ∞) at equilibrium (Bastarrachea et al., 2010).
K=
Cs, ∞ CF , ∞
(7)
2.9. Mass ratio The mass ratio between the amount of migrant in the solution and the amount in the film at equilibrium is defined as α. It is calculated as follows (Bastarrachea et al., 2010):
α=
Cs, ∞ CF ,0 − CF , ∞ = CF ,0 − Cs, ∞ CF , ∞
(8)
2.10. Desorption coefficients The mass balance for desorption of nisin from the PVA-AHSG composite films when placed on top of the desorption solution was studied using the following equation (Guiga et al., 2010).
dC ∗ ) Vsol sol = AK d (Csol − Csol dt
3.2. Nisin release kinetics As shown in Fig. 1, the release of nisin from the PVA-AHSG Table 1 Desorption coefficients obtained from the analysis of the experimental design.
(9)
The resolution of Equation (9) gives:
pH
%NaCl
Kd (×10−2) m s−1
(10)
3.8
Equation (10) could be solved with the following boundary conditions:
6.8
0.8 3.2 0.8 3.2 0.8 3.2
2.8 ± 0.1a 2.1 ± 0.1b 2.1 ± 0.1 b 1.7 ± 0.1 c 2.3 ± 0.1 a 0.8 ± 0.1d
∗ Csol = Csol + (Csol −
AK d ⎞t −⎛ ∗ Csol )e ⎝ V ⎠
8.8
t = 0 Ct 0, sol = 0 For all t> 0 Csol = KEQ × CPVA − AHSG
Values within each column with different letters are significantly different (p < 0.05).
The equilibrium partition constant KEQ between the desorption 135
Food Bioscience 28 (2019) 133–139
L.M. Marvdashti et al.
Fig. 1. Release profile of nisin.
composite film showed a concentration-dependence. The release profiles of nisin films led to an increased release as the concentration of nisin in the film matrix increased. This might be due to the enhanced porosity of the film with increasing concentration of nisin (Monjazeb Marvdashti et al., 2017). The results also showed that initial release rate for all concentration of nisin were faster and decreased until equilibrium was obtained. The concentration of nisin in the solution at equilibrium was 301, 326 and 715 IU/mL for 3000, 5000, and 10,000 IU, respectively (data not shown).
3.3. Power law A power law model (Equation (2)) was used to determine if the diffusion of nisin from the films was Fickian or non-Fickian. The diffusion exponents (n) and diffusion constants (k) were calculated from the plot of “Mt/M∞” vs “t” for the films. A good fit was obtained between experimental data and the model (Fig. 2). With increasing nisin concentration, a significant increase in the power law scale parameter, K, was obtained, indicating that the WVP and moisture affinity of the polymer matrix was increased (Table 2). Similar results were also observed by Yoshida et al. (2010) for release of potassium sorbate from chitosan films (Yoshida et al., 2010). The value of “n” was 0.19, 0.24 and 0.42 for 3000, 5000 and 10,000 IU of nisin, respectively. It indicated pseudo-Fickian diffusion with increasing nisin concentration in the PVA-AHSG composite matrix film, the release mechanism was constant. Similar results were reported for release of nisin from polylactic acid and chitosan matrix (Imran et al., 2014). Fig. 2. Curves of Mt/M0 versus t for active films containing 3000 IU (A), 5000 IU (B), and 10000 IU (C) nisin, showing the observed data (●) and fitted with the Power law equation (▬).
3.4. Diffusion coefficient (D) Nisin diffusivity through the PVA-AHSG composite films was concentration dependent (Table 3). With increasing nisin concentration, D was significantly increased due to raising the hydrophilicity of the composite film (Monjazeb Marvdashti et al., 2017). Similar results have been reported by Ozdemir and Floros (2001), and Miltz and RosenDoody (1984) for the diffusion of potassium sorbate and styrene from whey protein films and polystyrene packaging materials, respectively. These results were also confirmed by solubility and water vapor permeability data. With increasing nisin concentration, the solubility and water vapor permeability of the films were increased due to the increased hydrophilicity of the active films (Monjazeb Marvdashti et al., 2016). The D values obtained were higher than those determined for the release of nisin from hydroxypropyl methylcellulose by Imran et al. (2014) and indicated that nisin had a stronger interaction with hydroxypropyl methylcellulose than the PVA-AHSG matrix.
Table 2 Power law parameters for different nisin concentrations. Nisin concentration (IU)
k
n
R2
3000 5000 10,000
0.070 ± 0.01c 0.15 ± 0.01b 0.28 ± 0.03a
0.19 ± 0.03c 0.24 ± 0.04b 0.42 ± 0.03a
0.98 0.82 0.98
Values within each column with different letters are significantly different (p < 0.05).
3.5. Partition coefficient (K) K was significantly (p < 0.05) affected by nisin concentration in the film matrix (Table 3). The K values for all concentrations of nisin in the film, was below 1 except for 3000 IU. The K≪1 for active films 136
Food Bioscience 28 (2019) 133–139
L.M. Marvdashti et al.
Table 3 Diffusion coefficient (D), Partition Coefficient (K) and Mass Ratio (α) for different nisin concentrations. Nisin concentration (IU)
D (cm2/s)×10−11
K
α
3000 5000 10,000
3.1 ± 0.1b 4.69 ± 0.03ab 5.2 ± 0.1a
1.4 ± 0.05a 0.61 ± 0.08b 0.64 ± 0.03b
2.4 ± 0.1a 1.6 ± 0.1b 1.6 ± 0.1b
Values within each column with different letters are significantly different (p < 0.05).
indicated that nisin had a better affinity for polymers than the release solutions. Many factors, such as chemical nature, solubility, polarity and affinity of a diffusing agent to the polymer matrix affect K (Franz & Störmer, 2008; Suppakul, Miltz, Sonneveld, & Bigger, 2003). Since, the PVA-AHSG composite films with 5000 and 10,000 IU had less compact structures and higher polarity and solubility (Monjazeb Marvdashti et al., 2017), the nisin can easily diffuse from the matrix into the surrounding medium. As a result, it is suggested that nisin showed a higher affinity for the film matrix than to release solution. 3.6. Mass ratio The mass ratio (α) of nisin in the release solution is shown in Table 3. The value of α, significantly increased with increasing nisin concentration up to 5000 IU. This might be due to fewer intermolecular interactions between polymers in the film matrix (Monjazeb Marvdashti et al., 2017). However, with increasing nisin concentration from 5000 to 10,000 IU, the α value was almost constant. Similar results have been also obtained by Bastarrachea et al. (2010) regarding the release of nisin from poly (butylene adipate-co-terephthalate) films into water (Bastarrachea et al., 2010). These results were in accordance with the antimicrobial activity of PVA-AHSG composite films during the antimicrobial test. As a result, with increasing nisin concentration, the inhibitory effect of films on microbial population increased. Films with 10,000 IU nisin had the greatest inhibition with incubation time in the liquid medium due to its higher nisin release (Monjazeb Marvdashti et al., 2016). 3.7. Kinetics of nisin release Weibull is an empirical model which is commonly applied for both the rapid and the extended release of active agents (Azadi, Hamidi, & Rouini, 2013). Application of the Weibull model for the fitness of experimental data for pertaining to the release of nisin is shown in Fig. 3, indicating a good predictive power. With increasing nisin concentration, the parameter b of Weibull's model was significantly increased (Table 4). In general, a higher value of b is an indicator of a faster release at the beginning (Mateus, Lindinger, Gumy, & Liardon, 2007). It indicated that initial release of nisin was faster for 5000 IU nisin. The values for the shape factor (n) ranged from 0.25 to 0.7. The n value of the Weibull model describes the shape of the release curve. The n value for all conditions was lower than 1, which showed that the release curves have an upward concavity. All release conditions indicated a pattern of Fickian diffusion according to the b values. However, contrary to the n value in the power law model, the Weibull shape factor (n) does not discriminate between Fickian and pseudo-Fickian diffusion (Papadopoulou, Kosmidis, Vlachou, & Macheras, 2006). After 52 h, a similarity is found among the release of nisin from films with concentrations of 3000 and 5000 IU. Probably the effect of film thickness on nisin release is more evident at low concentrations of nisin.
Fig. 3. Release kinetics of nisin at 3000 IU (A), 5000 IU (B), and 10000 IU (C) over 52 h, showing the observed data (♦) and fitted with the Weibull's model (▬). Table 4 Weibull model parameters for different nisin concentrations. Nisin concentration (IU)
b (h−1)
n
R2
3000 5000 10,000
0.0004 ± 0.00001c 0.02 ± 0.0003a 0.005 ± 0.0001b
0.22 ± 0.08b 0.29 ± 0.04b 0.48 ± 0.06a
0.96 0.81 0.98
Values within each column with different letters are significantly different (p < 0.05).
different concentration of nisin was determined using the disc diffusion method. As shown in Fig. 4, with increasing nisin concentration, the inhibition zone against L. innocua significantly increased. The films without nisin had no antimicrobial activity. The diameters of the inhibition zones were 7.7, 9.8 and 13.3 mm for 3000, 5000 and 10,000 IU
3.8. Antimicrobial activity The antimicrobial activity of the PVA-AHSG composite films with 137
Food Bioscience 28 (2019) 133–139
L.M. Marvdashti et al.
References Alaeddini, B., Koocheki, A., Mohammadzadeh Milani, J., Razavi, S. M. A., & Ghanbarzadeh, B. (2018). Steady and dynamic shear rheological behavior of semi dilute Alyssum homolocarpum seed gum solutions: influence of concentration, temperature and heating–cooling rate. Journal of the Science of Food and Agriculture, 98(7), 2713–2720. de Arauz, L. J., Jozala, A. F., Mazzola, P. G., & Penna, T. C. V. (2009). Nisin biotechnological production and application: a review. Trends in Food Science & Technology, 20(3), 146–154. Azadi, A., Hamidi, M., & Rouini, M.-R. (2013). Methotrexate-loaded chitosan nanogels as ‘Trojan Horses’ for drug delivery to brain: preparation and in vitro/in vivo characterization. International Journal of Biological Macromolecules, 62, 523–530. Bastarrachea, L., Dhawan, S., Sablani, S. S., & Powers, J. (2010). Release kinetics of nisin from biodegradable poly (butylene adipate-co-terephthalate) films into water. Journal of Food Engineering, 100(1), 93–101. Buonocore, G., Del Nobile, M., Panizza, A., Corbo, M., & Nicolais, L. (2003). A general approach to describe the antimicrobial agent release from highly swellable films intended for food packaging applications. Journal of Controlled Release, 90(1), 97–107. Cao, L., Grégoire, L., Chaine, A., & Waché, Y. (2010). Importance and efficiency of indepth antimicrobial activity for the control of Listeria development with nisin-incorporated sodium caseinate films. Food Control, 21(9), 1227–1233. Crank, J. (1975). The mathematics of diffusion. New York: Oxford University Press ISBN:019-853411-6. Dean, J., & Zottola, E. (1996). Use of nisin in ice cream and effect on the survival of Listeria monocytogenes. Journal of Food Protection, 59(5), 476–480. Franssen, L., Rumsey, T., & Krochta, J. (2004). Whey protein film composition effects on potassium sorbate and natamycin diffusion. Journal of Food Science, 69(5), C347–C350. Franz, R., & Störmer, A. (2008). Migration of plastic constituents. In O. G. Piringer, & A. L. Baner (Eds.). Plastic Packaging: Interactions With Food and Pharmaceuticals (pp. 349– 416). Darmstadt, Germany: Wiley-VCH. Galdámez, J. R., Serna, L. V., Larry Duda, J., & Danner, R. P. (2007). Effect of blend composition on diffusivity and solubility of small molecules in polystyrene/poly (vinyl methyl ether) blends. Journal of Polymer Science Part B: Polymer Physics, 45(15), 2071–2082. Guiga, W., Swesi, Y., Galland, S., Peyrol, E., Degraeve, P., & Sebti, I. (2010). Innovative multilayer antimicrobial films made with Nisaplin or nisin and cellulosic ethers: physico-chemical characterization, bioactivity and nisin desorption kinetics. Innovative Food Science & Emerging Technologies, 11(2), 352–360. Han, J. H., & Floros, J. D. (1998). Simulating diffusion model and determining diffusivity of potassium sorbate through plastics to develop antimicrobial packaging films. Journal of Food Processing and Preservation, 22(2), 107–122. Hesarinejad, M. A., Razavi, S. M., & Koocheki, A. (2015). Alyssum homolocarpum seed gum: dilute solution and some physicochemical properties. International Journal of Biological Macromolecules, 81, 418–426. Imran, M., Klouj, A., Revol-Junelles, A.-M., & Desobry, S. (2014). Controlled release of nisin from HPMC, sodium caseinate, poly-lactic acid and chitosan for active packaging applications. Journal of Food Engineering, 143, 178–185. Jung, D.-S., Bodyfelt, F. W., & Daeschel, M. A. (1992). Influence of fat and emulsifiers on the efficacy of nisin in inhibiting Listeria monocytogenes in fluid milk. Journal of Dairy Science, 75(2), 387–393. Khoshakhlagh, K., Koocheki, A., Mohebbi, M., & Allafchian, A. (2017). Development and characterization of electrosprayed Alyssum homolocarpum seed gum nanoparticles for encapsulation of d-limonene. Journal of Colloid and Interface Science, 490, 562–575. Kim, Y. M., An, D. S., Park, H. J., Park, J. M., & Lee, D. S. (2002). Properties of nisin‐incorporated polymer coatings as antimicrobial packaging materials. Packaging Technology and Science, 15(5), 247–254. Koocheki, A., & Kadkhodaee, R. (2011). Effect of Alyssum homolocarpum seed gum, Tween 80 and NaCl on droplets characteristics, flow properties and physical stability of ultrasonically prepared corn oil-in-water emulsions. Food Hydrocolloids, 25(5), 1149–1157. Koocheki, A., Kadkhodaee, R., Mortazavi, S. A., Shahidi, F., & Taherian, A. R. (2009a). Influence of Alyssum homolocarpum seed gum on the stability and flow properties of O/W emulsion prepared by high intensity ultrasound. Food Hydrocolloids, 23(8), 2416–2424. Koocheki, A., Mortazavi, S. A., Shahidi, F., Razavi, S., Kadkhodaee, R., & Milani, J. M. (2010). Optimization of mucilage extraction from Qodume shirazi seed (Alyssum homolocarpum) using response surface methodology. Journal of Food Process Engineering, 33(5), 861–882. Koocheki, A., Mortazavi, S. A., Shahidi, F., Razavi, S. M. A., & Taherian, A. (2009b). Rheological properties of mucilage extracted from Alyssum homolocarpum seed as a new source of thickening agent. Journal of Food Engineering, 91(3), 490–496. Koocheki, A., & Razavi, S. M. (2009). Effect of concentration and temperature on flow properties of Alyssum homolocarpum seed gum solutions: assessment of time dependency and thixotropy. Food Biophysics, 4(4), 353–364. Mateus, M.-L., Lindinger, C., Gumy, J.-C., & Liardon, R. (2007). Release kinetics of volatile organic compounds from roasted and ground coffee: online measurements by PTR-MS and mathematical modeling. Journal of Agricultural and Food Chemistry, 55(25), 10117–10128. Miltz, J., & Rosen-Doody, V. (1984). Migration of styrene monomer from polystyrene packaging materials into food simulanta. Journal of Food Processing and Preservation, 8, 151–161. Monjazeb Marvdashti, L., Koocheki, A., & Yavarmanesh, M. (2017). Alyssum
Fig. 4. Inhibition zone (mm) against Listeria innocua using PVA-AHSG active films after 24 h incubation at 37 °C. Values with different letters are significantly different (p < 0.05).
of nisin, respectively, in the films. When nisin was added to the PVAAHSG composite films, it showed significant nisin release in a concentration dependent manner. Similar sizes of inhibition zones were reported by Cao, Grégoire, Chaine, and Waché (2010) with sodium caseinate films containing nisin concentrations of 1000 IU/cm2. The results for antimicrobial activity were in accordance with those obtained for the diffusion coefficient. The zone of inhibition produced by nisin diffusion showed that nisin is effective as an antimicrobial agent when it is incorporated into PVA-AHSG composite films. 4. Conclusion Fick's second law, the partition coefficient, mass ratio and the Weibull model were used to describe the release kinetic of nisin from PVA-AHSG composite films with different concentration of nisin. The study of the nisin release into the different desorption solutions showed that the release of nisin was significantly affected by NaCl concentration and pH of the surrounding media. The release kinetics can be predicted using the Weibull based models. This research showed that the antimicrobial activity of the PVA-AHSG composite films against L. innocua depended on the concentration of released nisin. Results also showed that PVA-AHSG composite films had a good potential to be used as a controlled release matrix. Nisin concentration in the PVA-AHSG composite films significantly determined affected the release process. Based on the experimental results for diffusion and antimicrobial activity, the nisin-PVA-AHSG films showed a release pattern that is well controlled, and a strong antimicrobial activity against L. innocua was observed. PVA-AHSG composite film with lower nisin concentration can be used as a controlled release matrix. Conflict of Interest It is declared that there is no conflict of interest in publication of this work. Acknowledgements The authors gratefully acknowledge the Department of Food Science and Technology, Ferdowsi University of Mashhad for financial support of this work. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fbio.2019.01.010. 138
Food Bioscience 28 (2019) 133–139
L.M. Marvdashti et al. homolocarpum seed gum-polyvinyl alcohol biodegradable composite film: physicochemical, mechanical, thermal and barrier properties. Carbohydrate Polymers, 155, 280–293. Monjazeb Marvdashti, L., Yavarmanesh, M., & Koocheki, A. (2016). The effect of different concentrations of glycerol on properties of blend films based on polyvinyl alcoholAllysum homolocarpum seed gum. Iranian Food Science and Technology Research Journal, 12(5), 663–677. Neetoo, H., Ye, M., & Chen, H. (2007). Effectiveness and stability of plastic films coated with nisin for inhibition of Listeria monocytogenes. Journal of Food Protection, 70(5), 1267–1271. Papadopoulou, V., Kosmidis, K., Vlachou, M., & Macheras, P. (2006). On the use of the Weibull function for the discernment of drug release mechanisms. International Journal of Pharmaceutics, 309(1), 44–50. Ozdemir, M., & Floros, J. D. (2001). Analysis and modeling of potassium sorbate diffusion through edible whey protein films. Journal of Food Engineering, 47(2), 149–155. Sebti, I., Blanc, D., Carnet-Ripoche, A., Saurel, R., & Coma, V. (2004). Experimental study and modeling of nisin diffusion in agarose gels. Journal of Food Engineering, 63(2), 185–190.
Suppakul, P., Miltz, J., Sonneveld, K., & Bigger, S. W. (2003). Active packaging technologies with an emphasis on antimicrobial packaging and its applications. Journal of Food Science, 68(2), 408–420. Tramer, J., & Fowler, G. (1964). Estimation of nisin in foods. Journal of the Science of Food and Agriculture, 15(8), 522–528. Van Boekel, M. A. (2008). Kinetic modeling of food quality: a critical review. Comprehensive Reviews in Food Science and Food Safety, 7(1), 144–158. Xiao, D., Gömmel, C., Davidson, P. M., & Zhong, Q. (2011). Intrinsic Tween 20 improves release and antilisterial properties of co-encapsulated nisin and thymol. Journal of Agricultural and Food Chemistry, 59(17), 9572–9580. Yoshida, C. M., Bastos, C. E. N., & Franco, T. T. (2010). Modeling of potassium sorbate diffusion through chitosan films. Food Science and Technology, 43(4), 584–589. Zhong, Q., Jin, M., Davidson, P. M., & Zivanovic, S. (2009). Sustained release of lysozyme from zein microcapsules produced by a supercritical anti-solvent process. Food Chemistry, 115(2), 697–700. Zufferey, R. (2016). In vitro assay to measure phosphatidylethanolamine methyltransferase activity. Journal of Visualized Experiments, 107, 1–16.
139
Contents lists available at ScienceDirect
Food Bioscience journal homepage: www.elsevier.com/locate/fbio
Controlled release of nisin from polyvinyl alcohol - Alyssum homolocarpum seed gum composite films: Nisin kinetics
T
Leila Monjazeb Marvdashti, Masoud Yavarmanesh∗, Arash Koocheki Department of Food Science and Technology, Faculty of Agriculture, Ferdowsi University of Mashhad (FUM), Mashhad, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: Active packaging Diffusion coefficient Nisin Power law Weibull model
Antimicrobial biodegradable films based on PVA-AHSG incorporated with nisin were prepared. These films were characterized for their ability to control the overall release rate of nisin into foods, which would be needed for active food packaging. The objectives of this study were to assay the biodegradable films for nisin release, and to investigate their antimicrobial activity against L. innocua. The release kinetics of nisin from the films were described using Fick's Second Law, mass ratio, partition coefficient, desorption coefficient and the Weibull model. All PVA-AHSG films showed inhibition zones that increased with increasing nisin concentration. Nisin release increased as its concentration in the film matrix increased. Release of nisin was faster at higher concentrations, and the diffusion coefficients increased significantly with increasing concentration. The partition coefficient (k) values obtained were 1.42, 0.61 and 0.64 for 3000, 5000 and 10,000 IU of nisin, respectively. According to the Weibull parameters, nisin is released following pseudo-Fickian diffusion. Results showed that the PVA-AHSG composite films showed promise for developing controlled release applications with nisin in aqueous solutions.
1. Introduction
(Monjazeb Marvdashti, Yavarmanesh, & Koocheki, 2016). However, blending the AHSG with polyvinyl alcohol (PVA) gave a film with greater strength and flexibility (Monjazeb Marvdashti et al., 2016, 2017). Nisin is an antimicrobial peptide produced by strains of Lactococcus lactis subsp. Lactis (de Arauz, Jozala, Mazzola, & Penna, 2009). Nisin has a wide range of activity against Gram-positive bacteria and is commonly used as a natural food preservative. The efficiency of the antimicrobial activity of nisin depends on several parameters, such as pH, temperature, salt and fat concentration (Dean & Zottola, 1996; Jung, Bodyfelt, & Daeschel, 1992). These parameters have an important role in nisin bioactivity, solubility, stability, rate of desorption from films and diffusion into food. Release kinetics of antimicrobial agents used in food packaging matrices have not been widely studied, whereas the release of active agents from drugs has received more attention (Buonocore, Del Nobile, Panizza, Corbo, & Nicolais, 2003; Galdámez, Serna, Larry Duda, & Danner, 2007). Fick's second law has been used to describe the release kinetics of antimicrobial agents from packaging material to food simulants or food matrixes (Buonocore et al., 2003; Han & Floros, 1998; Kim et al., 2002). The modeling of nisin diffusion from a packaging material into the food system can help to increase the capacity and efficiency of packaging using an antimicrobial carriers. In packaging
Packaging extends the shelf life and safety of food products (Bastarrachea, Dhawan, Sablani, & Powers, 2010). Antimicrobial agents can be added to food systems for controlling contaminations. However the antimicrobial activity was rapidly lost due to interactions with food components (Kim, An, Park, Park, & Lee, 2002). This problem can be reduced with antimicrobial active packaging because antimicrobial packaging establishes a more efficient protection of the food as controlled release involves a more convenient, steady and gradual delivery of active agents (Guiga et al., 2010). Alyssum homolocarpum seed gum (AHSG) is a galactan-type polysaccharide. Using a sample similar to the current sample, the seed gum contained 83.0% galactose, 5.70% glucose, 5.04% rhamnose, 2.72% xylose, 3.04% mannose, and 0.530% arabinose with ∼3.7 × 105 g/mol average molecular weight (Alaeddini, Koocheki, Mohammadzadeh Milani, Razavi, & Ghanbarzadeh, 2018; Hesarinejad, Razavi, & Koocheki, 2015; Khoshakhlagh, Koocheki, Mohebbi, & Allafchian, 2017; Koocheki & Kadkhodaee, 2011; Koocheki & Razavi, 2009). This food hydrocolloid can be used as a thickening/gelling agent, fat replacer and stabilizer (Koocheki et al., 2009a,b, 2010). Previous work showed that films made with AHSG were a good oxygen barriers and had an acceptable appearance with weak mechanical properties ∗
Corresponding author. E-mail address: [email protected] (M. Yavarmanesh).
https://doi.org/10.1016/j.fbio.2019.01.010 Received 2 November 2017; Received in revised form 14 January 2019; Accepted 21 January 2019 Available online 28 January 2019 2212-4292/ © 2019 Elsevier Ltd. All rights reserved.
Food Bioscience 28 (2019) 133–139
L.M. Marvdashti et al.
Nisin was purchased from Sigma Aldrich (Saint Louis, MO, USA), and was prepared by dissolving 0.1 g of nisin powder in 2 mL of a 0.01 M HCl solution (pH = 2). This solution was filtered through a nominal 0.2 mm pore size Millipore filter (Nalgene, Rochester, NY, USA) and kept at 4 °C for maximum of 1 wk.
Dickinson, Sparks, MD, USA) containing 15% glycerol at −80 °C. One loopful of stock was inoculated onto the modified Oxford agar (MOX: Difco), incubated at 37 °C for 24 h and eventually stored at 4 °C until use (a maximum of 1 month). A single colony of L. innocua was inoculated into 9 mL of TSB plus 1% yeast extract (TSBYE: Difco) and incubated for 24 h at 37 °C. The determination of the inhibition zone was carried out using the method of Tramer and Fowler (1964): the semi-soft agar was prepared using the TSBYE plus 0.7% agar (Difco) and 1% Tween 20 (Fischer Scientific, Fair Lawn, NJ, USA). Then, 100 mL of the semi-soft agar medium was seeded with 1 mL of the L. innocua strain to give an approximate concentration of 106 CFU/mL. The Tween 20 was used to enhance the diffusion of nisin through the agar medium. Approximately 4 mL of the seeded semisoft agar was poured over the petri dishes with TSA (Sparks, MD, USA). The film discs with nisin were placed over the semi-soft agar. The plates containing the film discs were kept at 4 °C for 24 h and were afterwards incubated at 37 °C for another 24 h. The refrigeration step was important to allow an optimum release of nisin, prior to the growth of bacteria. The refrigeration step subsequently led to larger inhibition zones (Neetoo, Ye, & Chen, 2007). After the incubation, the diameters of the inhibition zones were measured to the nearest 0.01 mm with an electron digital caliper (Fisher Scientific, Pittsburgh, PA, USA). Four measurements were done of each inhibition zone. All experiments were repeated at least three times.
2.2. Preparation of AHSG powder
2.5. Nisin quantification
AHSG powder was prepared using the method developed by Monjazeb Marvdashti et al. (2017). In brief, A. homolocarpum seeds were soaked in distilled water (seed to water ratio of 1:40). The pH of the water was adjusted to 4 using NaOH (10 M). The slurry was mixed at 40 °C for 1 h. The gum was extracted using an extractor (Parskhazar, Tehran, Iran), precipitated with ethanol (96%), dried in a freeze drier (Martin Christ Freeze Dryer, Osterode am Harz, Germany), milled using a laboratory hummer mill and sieved to an average particle size of 100 μm, packed in an air tight glass jar and stored at 4 °C for a maximum of 20 wk (Koocheki et al., 2010).
A colorimetric method, namely the bicinchoninic acid (BCA) assay, was used to determine nisin concentration. Briefly, a 150 μL volume of sample and 150 μL of BCA solution were mixed together. Briefly, BCA solution was prepared by mixing the BCA and copper (II) sulfate in a ratio of 49:1 (v/v) (Zufferey, 2016). This solution was incubated for 1 h at 60 °C. The absorbance was measured at 580 nm with a spectrophotometer (UV-2100, UNICO, Shanghai, China). The nisin contents in solution were calculated from the standard curve. The line of the standard curve had the following equation:
films, antimicrobials can be characterized in terms of their ability to extend the shelf-life of food products. The antimicrobial activity can be modeled using attributes such as the diffusion coefficient (D), desorption coefficient (Kd), partition coefficient (K) and mass ratio (α) (Franssen, Rumsey, & Krochta, 2004; Imran, Klouj, Revol-Junelles, & Desobry, 2014). The Weibull model considers the non-linear behavior of several factors. In recent years, the Weibull model has been commonly applied in studying the kinetics of chemical changes and microbiological destruction (Van Boekel, 2008). The objective of this study was to investigate the release kinetics of nisin from a biodegradable PVA-AHSG composite film at three nisin concentrations. The kinetics of nisin release were also studied. Antibacterial activities of the different films with nisin were measured using Listeria innocua. 2. Materials and methods 2.1. Preparation of nisin standard solution
Absorbance (580nm) = 1.75 × ⎡Nisin ⎢ ⎣
2.3. Preparation of active PVA-AHSG composite films Films were obtained by using a solution casting procedure using the method of Monjazeb Marvdashti et al. (2017). To prepare the film, AHSG powder (1.5% w/w) was dissolved in distilled water for 20 min at room temperature (23 ± 2 °C). The solutions were then allowed to hydrate for 24 h at 4 °C. Thereafter, the solution was homogenized using a rotor-stator homogenizer (Ultraturrax D125, Janke and Kunkel, Staufen im Breisgau, Germany) at 13,500 rpm for 1 min. PVA (1.5% w/ w) was dissolved in distilled water at 95 °C for 60 min. After the PVA solution cooled, it was added into the previously hydrated AHSG solutions (60:40 PVA to AHSG ratio). Glycerol was also added (0.75%). The nisin solution was then added to the film solution to give various levels (3000, 5000 and 10,000 IU). The International Units (IU) for nisin activity is the amount of nisin required to inhibit one cell of Streptococcus agalactiae in 1 mL of broth. Finally, PVA-AHSG solutions were stirred for 30 min and centrifuged in a Sigma 3K30 centrifuge (Sigma Co, Osterode am Harz, Germany) at 7000 g for 10 min at room temperature to remove air bubbles. Film forming dispersions were spread over petri dishes (diameter 15 cm). Films were dried in an air convection heat oven (Soroush oven, Tehran, Iran) at 40 °C for 24 h, and then were peeled off and stored at 4 °C for a maximum of 14 days.
⎤
2
R ( mg ml ) ⎥ ⎦
= 0.98
(1)
2.6. Experimental design The PVA-AHSG active films were floated on the surface of a desorption solution (100 mL of different NaCl solutions). These solutions were stirred for 52 h. A volume of 200 μL was periodically taken from this solution to quantify the desorbed nisin. The impact of three parameters on desorption kinetics was studied, including the pH (between 3.8 and 6.3), NaCl concentration (0.8 and 3.2%) and the concentration of nisin in the active films (3000, 5000 and 10,000 IU). 2.7. Kinetic of nisin release The power law is applied for investigating the diffusion mechanism (Yoshida, Bastos, & Franco, 2010).
mt = kt n m∞
(2)
Where mt is the amount of nisin released at time t, m∞ is the amount of nisin released at equilibrium; ‘k’ is a constant that is associated with the network of macromolecules and ‘n’ is a diffusion exponent attribute of the release mechanism. With this law, for n = 0.5, the mechanism of diffusion was considered as Fickian transport; for n = 1, the solute diffusion directly correlates with time; for 0.5 < n < 1.0, the principal mechanism is anomalous or non-Fickian transport; for n > 1.0, the solute is released in subsequent stages (Crank, 1975; Yoshida et al., 2010).
2.4. Test of antimicrobial activity of the films The L. innocua (ATCC 33090) was provided by the Iranian Research Organization for Science and Technology (Tehran, Iran). The stock culture was maintained in tryptic soy broth (TSB: Difco, Becton 134
Food Bioscience 28 (2019) 133–139
L.M. Marvdashti et al.
Fick's second law was used to find and describe the diffusion pattern of nisin as a bioactive agent, into the release solution.
solution and the PVA-AHSG is equal to 1, due to the high solubility of PVA-AHSG film in the water.
∂CF (x , t ) ∂C (x , t ) =D F 2 ∂t ∂x
2.11. Release kinetics
(3)
Where CF(x, t) is the nisin concentration in the film at position x and time t, and D is the diffusion coefficient of the nisin through the film (cm2 s−1). The diffusion coefficient (D) of nisin was obtained from a film with the following assumptions: (1) the initial nisin concentration was homogeneous throughout the film, (2) the initial concentration of nisin in the simulated food solution was zero, and (3) the nisin amount diffused in water was the same as the amount released from the film (Crank, 1975).
Mt 8 =1− 2 M∞ π
∞
∑ n=0
−2(n + 1)2π 2Dt ⎤ 1 exp ⎡ 2 ⎥ ⎢ (2n + 1) L2 ⎦ ⎣
The kinetic data for the release of nisin from the PVA-AHSG composite films into the release solution was also determined using the Weibull model (Van Boekel, 2008):
Ln
2.12. Statistical analyses
Where Mt and M∞ are the amount of nisin released from the film at time t and at infinite time, respectively, n is the number of terms in a Taylor series and L is the film thickness. For short durations of time (Mt/M∞ < 2/3), Equation (4) can be simplified to Equation (5). Therefore, the D value can be calculated using Equation (6) (Crank, 1975).
All the experiments were replicated at least three times using a completely randomized design. Analysis of variance (ANOVA) was carried out and the results were divided into the Duncan's Multiple Range test (p < 0.05) using Statistical Package for the Social Sciences (SPSS) software Version 19 (IBM SPSS, Armonk, NY, USA). The difference between treatments were identified using Duncan's multiple range test and were considered significant when p < 0.05. The model calculations were done using MATLAB (R2012a) software (Math Works, Natick, MA, USA).
(5)
S × L ⎞2 D=⎛ ×π ⎝ 2 ⎠
(11)
Where MF,t and MF,0 are the amounts of nisin in the film at time t and 0, respectively. In this model, ‘n’ is the shape factor and ‘b’ is the scale parameter.
(4)
Mt Dt 1/2 = 4⎛ 2 ⎞ M∞ ⎝ 4L π ⎠
MF , t = −(bt )n MF , 0
(6)
where S is the slope of the plot of Mt/M∞ against
3. Result and discussion
t 1/2 .
3.1. Desorption solution 2.8. Partition coefficient The desorption solution has an important role in the diffusion of an active agent from a matrix to surrounding media. The desorption coefficients (kd) of nisin from the PVA-AHSG composite film indicated that with increasing concentration of NaCl and pH in the desorption solution, the release of nisin decreased (Table 1). Generally, the molecular weight of a migrant and its affinity for the matrix and/or desorption solution can effect desorption (Sebti, Blanc, Carnet-Ripoche, Saurel, & Coma, 2004). In 3.2% NaCl, as pH increased from 3.8 to 8.8, a significant decrease in kd was observed. This might be due to decreased nisin solubility at higher pH (Xiao, Go¨mmel, Davidson, & Zhong, 2011). Results also showed that the kd of the solutions increased with decreasing NaCl concentration due to enhanced intermolecular interactions between macromolecules (Table 1). As a result, slower diffusion occurs at higher NaCl because of weaker electrostatic repulsions and stronger hydrophobic attractions (Zhong, Jin, Davidson, & Zivanovic, 2009). Similar results have been reported by Kim et al. (2002) for vinyl acetate-ethylene. The desorption solution with 0.8% of NaCl at pH 3.8 was chosen as an optimal desorption condition for further experiments.
In general, the partition coefficient is described as the ratio of the migrant in the food stimulant (Cs, ∞) to the migrant in the film polymer (CF, ∞) at equilibrium (Bastarrachea et al., 2010).
K=
Cs, ∞ CF , ∞
(7)
2.9. Mass ratio The mass ratio between the amount of migrant in the solution and the amount in the film at equilibrium is defined as α. It is calculated as follows (Bastarrachea et al., 2010):
α=
Cs, ∞ CF ,0 − CF , ∞ = CF ,0 − Cs, ∞ CF , ∞
(8)
2.10. Desorption coefficients The mass balance for desorption of nisin from the PVA-AHSG composite films when placed on top of the desorption solution was studied using the following equation (Guiga et al., 2010).
dC ∗ ) Vsol sol = AK d (Csol − Csol dt
3.2. Nisin release kinetics As shown in Fig. 1, the release of nisin from the PVA-AHSG Table 1 Desorption coefficients obtained from the analysis of the experimental design.
(9)
The resolution of Equation (9) gives:
pH
%NaCl
Kd (×10−2) m s−1
(10)
3.8
Equation (10) could be solved with the following boundary conditions:
6.8
0.8 3.2 0.8 3.2 0.8 3.2
2.8 ± 0.1a 2.1 ± 0.1b 2.1 ± 0.1 b 1.7 ± 0.1 c 2.3 ± 0.1 a 0.8 ± 0.1d
∗ Csol = Csol + (Csol −
AK d ⎞t −⎛ ∗ Csol )e ⎝ V ⎠
8.8
t = 0 Ct 0, sol = 0 For all t> 0 Csol = KEQ × CPVA − AHSG
Values within each column with different letters are significantly different (p < 0.05).
The equilibrium partition constant KEQ between the desorption 135
Food Bioscience 28 (2019) 133–139
L.M. Marvdashti et al.
Fig. 1. Release profile of nisin.
composite film showed a concentration-dependence. The release profiles of nisin films led to an increased release as the concentration of nisin in the film matrix increased. This might be due to the enhanced porosity of the film with increasing concentration of nisin (Monjazeb Marvdashti et al., 2017). The results also showed that initial release rate for all concentration of nisin were faster and decreased until equilibrium was obtained. The concentration of nisin in the solution at equilibrium was 301, 326 and 715 IU/mL for 3000, 5000, and 10,000 IU, respectively (data not shown).
3.3. Power law A power law model (Equation (2)) was used to determine if the diffusion of nisin from the films was Fickian or non-Fickian. The diffusion exponents (n) and diffusion constants (k) were calculated from the plot of “Mt/M∞” vs “t” for the films. A good fit was obtained between experimental data and the model (Fig. 2). With increasing nisin concentration, a significant increase in the power law scale parameter, K, was obtained, indicating that the WVP and moisture affinity of the polymer matrix was increased (Table 2). Similar results were also observed by Yoshida et al. (2010) for release of potassium sorbate from chitosan films (Yoshida et al., 2010). The value of “n” was 0.19, 0.24 and 0.42 for 3000, 5000 and 10,000 IU of nisin, respectively. It indicated pseudo-Fickian diffusion with increasing nisin concentration in the PVA-AHSG composite matrix film, the release mechanism was constant. Similar results were reported for release of nisin from polylactic acid and chitosan matrix (Imran et al., 2014). Fig. 2. Curves of Mt/M0 versus t for active films containing 3000 IU (A), 5000 IU (B), and 10000 IU (C) nisin, showing the observed data (●) and fitted with the Power law equation (▬).
3.4. Diffusion coefficient (D) Nisin diffusivity through the PVA-AHSG composite films was concentration dependent (Table 3). With increasing nisin concentration, D was significantly increased due to raising the hydrophilicity of the composite film (Monjazeb Marvdashti et al., 2017). Similar results have been reported by Ozdemir and Floros (2001), and Miltz and RosenDoody (1984) for the diffusion of potassium sorbate and styrene from whey protein films and polystyrene packaging materials, respectively. These results were also confirmed by solubility and water vapor permeability data. With increasing nisin concentration, the solubility and water vapor permeability of the films were increased due to the increased hydrophilicity of the active films (Monjazeb Marvdashti et al., 2016). The D values obtained were higher than those determined for the release of nisin from hydroxypropyl methylcellulose by Imran et al. (2014) and indicated that nisin had a stronger interaction with hydroxypropyl methylcellulose than the PVA-AHSG matrix.
Table 2 Power law parameters for different nisin concentrations. Nisin concentration (IU)
k
n
R2
3000 5000 10,000
0.070 ± 0.01c 0.15 ± 0.01b 0.28 ± 0.03a
0.19 ± 0.03c 0.24 ± 0.04b 0.42 ± 0.03a
0.98 0.82 0.98
Values within each column with different letters are significantly different (p < 0.05).
3.5. Partition coefficient (K) K was significantly (p < 0.05) affected by nisin concentration in the film matrix (Table 3). The K values for all concentrations of nisin in the film, was below 1 except for 3000 IU. The K≪1 for active films 136
Food Bioscience 28 (2019) 133–139
L.M. Marvdashti et al.
Table 3 Diffusion coefficient (D), Partition Coefficient (K) and Mass Ratio (α) for different nisin concentrations. Nisin concentration (IU)
D (cm2/s)×10−11
K
α
3000 5000 10,000
3.1 ± 0.1b 4.69 ± 0.03ab 5.2 ± 0.1a
1.4 ± 0.05a 0.61 ± 0.08b 0.64 ± 0.03b
2.4 ± 0.1a 1.6 ± 0.1b 1.6 ± 0.1b
Values within each column with different letters are significantly different (p < 0.05).
indicated that nisin had a better affinity for polymers than the release solutions. Many factors, such as chemical nature, solubility, polarity and affinity of a diffusing agent to the polymer matrix affect K (Franz & Störmer, 2008; Suppakul, Miltz, Sonneveld, & Bigger, 2003). Since, the PVA-AHSG composite films with 5000 and 10,000 IU had less compact structures and higher polarity and solubility (Monjazeb Marvdashti et al., 2017), the nisin can easily diffuse from the matrix into the surrounding medium. As a result, it is suggested that nisin showed a higher affinity for the film matrix than to release solution. 3.6. Mass ratio The mass ratio (α) of nisin in the release solution is shown in Table 3. The value of α, significantly increased with increasing nisin concentration up to 5000 IU. This might be due to fewer intermolecular interactions between polymers in the film matrix (Monjazeb Marvdashti et al., 2017). However, with increasing nisin concentration from 5000 to 10,000 IU, the α value was almost constant. Similar results have been also obtained by Bastarrachea et al. (2010) regarding the release of nisin from poly (butylene adipate-co-terephthalate) films into water (Bastarrachea et al., 2010). These results were in accordance with the antimicrobial activity of PVA-AHSG composite films during the antimicrobial test. As a result, with increasing nisin concentration, the inhibitory effect of films on microbial population increased. Films with 10,000 IU nisin had the greatest inhibition with incubation time in the liquid medium due to its higher nisin release (Monjazeb Marvdashti et al., 2016). 3.7. Kinetics of nisin release Weibull is an empirical model which is commonly applied for both the rapid and the extended release of active agents (Azadi, Hamidi, & Rouini, 2013). Application of the Weibull model for the fitness of experimental data for pertaining to the release of nisin is shown in Fig. 3, indicating a good predictive power. With increasing nisin concentration, the parameter b of Weibull's model was significantly increased (Table 4). In general, a higher value of b is an indicator of a faster release at the beginning (Mateus, Lindinger, Gumy, & Liardon, 2007). It indicated that initial release of nisin was faster for 5000 IU nisin. The values for the shape factor (n) ranged from 0.25 to 0.7. The n value of the Weibull model describes the shape of the release curve. The n value for all conditions was lower than 1, which showed that the release curves have an upward concavity. All release conditions indicated a pattern of Fickian diffusion according to the b values. However, contrary to the n value in the power law model, the Weibull shape factor (n) does not discriminate between Fickian and pseudo-Fickian diffusion (Papadopoulou, Kosmidis, Vlachou, & Macheras, 2006). After 52 h, a similarity is found among the release of nisin from films with concentrations of 3000 and 5000 IU. Probably the effect of film thickness on nisin release is more evident at low concentrations of nisin.
Fig. 3. Release kinetics of nisin at 3000 IU (A), 5000 IU (B), and 10000 IU (C) over 52 h, showing the observed data (♦) and fitted with the Weibull's model (▬). Table 4 Weibull model parameters for different nisin concentrations. Nisin concentration (IU)
b (h−1)
n
R2
3000 5000 10,000
0.0004 ± 0.00001c 0.02 ± 0.0003a 0.005 ± 0.0001b
0.22 ± 0.08b 0.29 ± 0.04b 0.48 ± 0.06a
0.96 0.81 0.98
Values within each column with different letters are significantly different (p < 0.05).
different concentration of nisin was determined using the disc diffusion method. As shown in Fig. 4, with increasing nisin concentration, the inhibition zone against L. innocua significantly increased. The films without nisin had no antimicrobial activity. The diameters of the inhibition zones were 7.7, 9.8 and 13.3 mm for 3000, 5000 and 10,000 IU
3.8. Antimicrobial activity The antimicrobial activity of the PVA-AHSG composite films with 137
Food Bioscience 28 (2019) 133–139
L.M. Marvdashti et al.
References Alaeddini, B., Koocheki, A., Mohammadzadeh Milani, J., Razavi, S. M. A., & Ghanbarzadeh, B. (2018). Steady and dynamic shear rheological behavior of semi dilute Alyssum homolocarpum seed gum solutions: influence of concentration, temperature and heating–cooling rate. Journal of the Science of Food and Agriculture, 98(7), 2713–2720. de Arauz, L. J., Jozala, A. F., Mazzola, P. G., & Penna, T. C. V. (2009). Nisin biotechnological production and application: a review. Trends in Food Science & Technology, 20(3), 146–154. Azadi, A., Hamidi, M., & Rouini, M.-R. (2013). Methotrexate-loaded chitosan nanogels as ‘Trojan Horses’ for drug delivery to brain: preparation and in vitro/in vivo characterization. International Journal of Biological Macromolecules, 62, 523–530. Bastarrachea, L., Dhawan, S., Sablani, S. S., & Powers, J. (2010). Release kinetics of nisin from biodegradable poly (butylene adipate-co-terephthalate) films into water. Journal of Food Engineering, 100(1), 93–101. Buonocore, G., Del Nobile, M., Panizza, A., Corbo, M., & Nicolais, L. (2003). A general approach to describe the antimicrobial agent release from highly swellable films intended for food packaging applications. Journal of Controlled Release, 90(1), 97–107. Cao, L., Grégoire, L., Chaine, A., & Waché, Y. (2010). Importance and efficiency of indepth antimicrobial activity for the control of Listeria development with nisin-incorporated sodium caseinate films. Food Control, 21(9), 1227–1233. Crank, J. (1975). The mathematics of diffusion. New York: Oxford University Press ISBN:019-853411-6. Dean, J., & Zottola, E. (1996). Use of nisin in ice cream and effect on the survival of Listeria monocytogenes. Journal of Food Protection, 59(5), 476–480. Franssen, L., Rumsey, T., & Krochta, J. (2004). Whey protein film composition effects on potassium sorbate and natamycin diffusion. Journal of Food Science, 69(5), C347–C350. Franz, R., & Störmer, A. (2008). Migration of plastic constituents. In O. G. Piringer, & A. L. Baner (Eds.). Plastic Packaging: Interactions With Food and Pharmaceuticals (pp. 349– 416). Darmstadt, Germany: Wiley-VCH. Galdámez, J. R., Serna, L. V., Larry Duda, J., & Danner, R. P. (2007). Effect of blend composition on diffusivity and solubility of small molecules in polystyrene/poly (vinyl methyl ether) blends. Journal of Polymer Science Part B: Polymer Physics, 45(15), 2071–2082. Guiga, W., Swesi, Y., Galland, S., Peyrol, E., Degraeve, P., & Sebti, I. (2010). Innovative multilayer antimicrobial films made with Nisaplin or nisin and cellulosic ethers: physico-chemical characterization, bioactivity and nisin desorption kinetics. Innovative Food Science & Emerging Technologies, 11(2), 352–360. Han, J. H., & Floros, J. D. (1998). Simulating diffusion model and determining diffusivity of potassium sorbate through plastics to develop antimicrobial packaging films. Journal of Food Processing and Preservation, 22(2), 107–122. Hesarinejad, M. A., Razavi, S. M., & Koocheki, A. (2015). Alyssum homolocarpum seed gum: dilute solution and some physicochemical properties. International Journal of Biological Macromolecules, 81, 418–426. Imran, M., Klouj, A., Revol-Junelles, A.-M., & Desobry, S. (2014). Controlled release of nisin from HPMC, sodium caseinate, poly-lactic acid and chitosan for active packaging applications. Journal of Food Engineering, 143, 178–185. Jung, D.-S., Bodyfelt, F. W., & Daeschel, M. A. (1992). Influence of fat and emulsifiers on the efficacy of nisin in inhibiting Listeria monocytogenes in fluid milk. Journal of Dairy Science, 75(2), 387–393. Khoshakhlagh, K., Koocheki, A., Mohebbi, M., & Allafchian, A. (2017). Development and characterization of electrosprayed Alyssum homolocarpum seed gum nanoparticles for encapsulation of d-limonene. Journal of Colloid and Interface Science, 490, 562–575. Kim, Y. M., An, D. S., Park, H. J., Park, J. M., & Lee, D. S. (2002). Properties of nisin‐incorporated polymer coatings as antimicrobial packaging materials. Packaging Technology and Science, 15(5), 247–254. Koocheki, A., & Kadkhodaee, R. (2011). Effect of Alyssum homolocarpum seed gum, Tween 80 and NaCl on droplets characteristics, flow properties and physical stability of ultrasonically prepared corn oil-in-water emulsions. Food Hydrocolloids, 25(5), 1149–1157. Koocheki, A., Kadkhodaee, R., Mortazavi, S. A., Shahidi, F., & Taherian, A. R. (2009a). Influence of Alyssum homolocarpum seed gum on the stability and flow properties of O/W emulsion prepared by high intensity ultrasound. Food Hydrocolloids, 23(8), 2416–2424. Koocheki, A., Mortazavi, S. A., Shahidi, F., Razavi, S., Kadkhodaee, R., & Milani, J. M. (2010). Optimization of mucilage extraction from Qodume shirazi seed (Alyssum homolocarpum) using response surface methodology. Journal of Food Process Engineering, 33(5), 861–882. Koocheki, A., Mortazavi, S. A., Shahidi, F., Razavi, S. M. A., & Taherian, A. (2009b). Rheological properties of mucilage extracted from Alyssum homolocarpum seed as a new source of thickening agent. Journal of Food Engineering, 91(3), 490–496. Koocheki, A., & Razavi, S. M. (2009). Effect of concentration and temperature on flow properties of Alyssum homolocarpum seed gum solutions: assessment of time dependency and thixotropy. Food Biophysics, 4(4), 353–364. Mateus, M.-L., Lindinger, C., Gumy, J.-C., & Liardon, R. (2007). Release kinetics of volatile organic compounds from roasted and ground coffee: online measurements by PTR-MS and mathematical modeling. Journal of Agricultural and Food Chemistry, 55(25), 10117–10128. Miltz, J., & Rosen-Doody, V. (1984). Migration of styrene monomer from polystyrene packaging materials into food simulanta. Journal of Food Processing and Preservation, 8, 151–161. Monjazeb Marvdashti, L., Koocheki, A., & Yavarmanesh, M. (2017). Alyssum
Fig. 4. Inhibition zone (mm) against Listeria innocua using PVA-AHSG active films after 24 h incubation at 37 °C. Values with different letters are significantly different (p < 0.05).
of nisin, respectively, in the films. When nisin was added to the PVAAHSG composite films, it showed significant nisin release in a concentration dependent manner. Similar sizes of inhibition zones were reported by Cao, Grégoire, Chaine, and Waché (2010) with sodium caseinate films containing nisin concentrations of 1000 IU/cm2. The results for antimicrobial activity were in accordance with those obtained for the diffusion coefficient. The zone of inhibition produced by nisin diffusion showed that nisin is effective as an antimicrobial agent when it is incorporated into PVA-AHSG composite films. 4. Conclusion Fick's second law, the partition coefficient, mass ratio and the Weibull model were used to describe the release kinetic of nisin from PVA-AHSG composite films with different concentration of nisin. The study of the nisin release into the different desorption solutions showed that the release of nisin was significantly affected by NaCl concentration and pH of the surrounding media. The release kinetics can be predicted using the Weibull based models. This research showed that the antimicrobial activity of the PVA-AHSG composite films against L. innocua depended on the concentration of released nisin. Results also showed that PVA-AHSG composite films had a good potential to be used as a controlled release matrix. Nisin concentration in the PVA-AHSG composite films significantly determined affected the release process. Based on the experimental results for diffusion and antimicrobial activity, the nisin-PVA-AHSG films showed a release pattern that is well controlled, and a strong antimicrobial activity against L. innocua was observed. PVA-AHSG composite film with lower nisin concentration can be used as a controlled release matrix. Conflict of Interest It is declared that there is no conflict of interest in publication of this work. Acknowledgements The authors gratefully acknowledge the Department of Food Science and Technology, Ferdowsi University of Mashhad for financial support of this work. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fbio.2019.01.010. 138
Food Bioscience 28 (2019) 133–139
L.M. Marvdashti et al. homolocarpum seed gum-polyvinyl alcohol biodegradable composite film: physicochemical, mechanical, thermal and barrier properties. Carbohydrate Polymers, 155, 280–293. Monjazeb Marvdashti, L., Yavarmanesh, M., & Koocheki, A. (2016). The effect of different concentrations of glycerol on properties of blend films based on polyvinyl alcoholAllysum homolocarpum seed gum. Iranian Food Science and Technology Research Journal, 12(5), 663–677. Neetoo, H., Ye, M., & Chen, H. (2007). Effectiveness and stability of plastic films coated with nisin for inhibition of Listeria monocytogenes. Journal of Food Protection, 70(5), 1267–1271. Papadopoulou, V., Kosmidis, K., Vlachou, M., & Macheras, P. (2006). On the use of the Weibull function for the discernment of drug release mechanisms. International Journal of Pharmaceutics, 309(1), 44–50. Ozdemir, M., & Floros, J. D. (2001). Analysis and modeling of potassium sorbate diffusion through edible whey protein films. Journal of Food Engineering, 47(2), 149–155. Sebti, I., Blanc, D., Carnet-Ripoche, A., Saurel, R., & Coma, V. (2004). Experimental study and modeling of nisin diffusion in agarose gels. Journal of Food Engineering, 63(2), 185–190.
Suppakul, P., Miltz, J., Sonneveld, K., & Bigger, S. W. (2003). Active packaging technologies with an emphasis on antimicrobial packaging and its applications. Journal of Food Science, 68(2), 408–420. Tramer, J., & Fowler, G. (1964). Estimation of nisin in foods. Journal of the Science of Food and Agriculture, 15(8), 522–528. Van Boekel, M. A. (2008). Kinetic modeling of food quality: a critical review. Comprehensive Reviews in Food Science and Food Safety, 7(1), 144–158. Xiao, D., Gömmel, C., Davidson, P. M., & Zhong, Q. (2011). Intrinsic Tween 20 improves release and antilisterial properties of co-encapsulated nisin and thymol. Journal of Agricultural and Food Chemistry, 59(17), 9572–9580. Yoshida, C. M., Bastos, C. E. N., & Franco, T. T. (2010). Modeling of potassium sorbate diffusion through chitosan films. Food Science and Technology, 43(4), 584–589. Zhong, Q., Jin, M., Davidson, P. M., & Zivanovic, S. (2009). Sustained release of lysozyme from zein microcapsules produced by a supercritical anti-solvent process. Food Chemistry, 115(2), 697–700. Zufferey, R. (2016). In vitro assay to measure phosphatidylethanolamine methyltransferase activity. Journal of Visualized Experiments, 107, 1–16.
139