Release Profile and Antimicrobial Activity of Nisin Control-released from Agar Gel Foods

B. thermosphacta was selected as the target microorganism to determine the antimicrobial activity of nisin released from the agar gel. All purpose Tween (APT, Difco, Becton Dickinson, Sparks, MD, USA) agar slants were used to maintain bacterial culture at 4 °C. B. thermosphacta (ATCC 11509) was cultured in brain heart infusion (BHI, Difco, Detroit, MI, USA) at 25 °C. Cells from 48 h-culture were collected by centrifugation (4,000×g, 15 min, 22 °C; GyroSpin, Gyrozen Co. Ltd., Seoul, Korea) and suspended in 0.1% (w/v) peptone water. The suspension (approximately 10⁹ CFU/mL) was diluted with 0.1% peptone water for inoculation.

Nisin (Sigma-Aldrich Co., St. Louis, MO, USA) powder (1 g) was dissolved in 10 mL distilled water in a beaker with a stirring bar. The nisin solution was covered and agitated overnight to make nisin dissolve thoroughly. After 24 h, agar powder (A-1296, Sigma-Aldrich Co.) were dissolved in distilled water to prepare 1, 2, or 3% (w/v) agar solutions. Agar solutions were autoclaved at 121 °C for 15 min and left in a water bath at 50 °C. After equilibrium, the nisin solution was slowly transferred to the agar solution. The nisin-agar solution was left overnight at 24±2ºC in the dark to form agar gel model foods. The concentration of nisin in the gel was 2% (w/ v) and those of agar were varied at 1, 2 and 3% (w/v). The gel thickness was 1 cm.

The scheme of diffusion experiment is illustrated in Fig. 1. Agar gel model food was trimmed into cylinders of 1.0 cm diameter by 3.0 cm length using a sterile cheese borer. Each gel without edge damage was carefully transferred to a volumetric beaker, containing 100 mL distilled water. The beakers were covered with paraffin films and placed on a shaker (Julabo SW22, Labortechnik GmbH, Seelbach, Germany) rotating at 60 rpm at 24±2ºC. The absorbance was measured at 280 nm every 1 h using a spectrophotometer (Ultraspec 2000, Pharmacia Biotech, Piscataway, NJ, USA) (Breukink et al., 1998). The concentration of nisin released into water was calculated from a nisin standard curve. The release rate of nisin was determined with the ratio of the level of nisin released to the initial level of nisin in the agar.

Fig. 1. Scheme of nisin release experiments.

Download Original Figure

The diffusion through the film was determined from the data obtained using the following solution for diffusion in a plane sheet, derived from Fick’s law (Crank, 1975). In this experiment, agar gel was assumed to be a homogeneous system containing nisin molecules uniformly in the gel matrix.

where,

Eq. (1) can be simplified to Eq. (2) by a log transformation using only the first term in the summation series (Crank, 1975):

When only the experimental data over 40% of the maximum release were used, the following linear regression can be used:

where

Diffusion coefficient (D) can be determined as follows (Han et al., 2000):(4)

Experiment was conducted with the set-up illustrated in Fig. 1 using the B. thermosphacta-inoculated APT broth instead of distilled water. The inoculum of B. thermosphacta at 8 log CFU/mL (OD₆₀₀ = 0.915) was transferred to 100 mL APT broth. Three experiments were conducted. First, the inhibitory effect of nisin in APT broth on the growth of B. thermosphacta in the broth was investigated. The concentrations of nisin in the broth were 0.1 and 0.5% (w/v). Secondly, the antimicrobial activities of the agar gel cylinders incorporating nisin (2%, w/ v) were evaluated in nisin free-APT broth. The concentrations of agar in the gels were 1, 2 and 3% (w/v). Lastly, the inhibitory effects of nisin released from agar gels with different concentrations of agar were studied in APT broth containing nisin at 0.1%. The cell density of the APT broth was measured at 600 nm. The measurement was made every hour with the samples in the shaker rotating at 60 rpm during incubation at 24±2ºC for 120 h.

All experiments were replicated in two times. Each observation within each replication was determined in triplicate. The slope of the data points for D determination was determined by fitting a straight line to the data using Microsoft Excel (Microsoft Corp., Redmond, WA, USA).

The highest release of nisin was shown in 1% agar gel, followed by 2% and 3%. The maximum release of nisin, demonstrated with 1% agar gel was 86%, while for 2% and 3% agar gels, the levels of release were 77% and 67%, respectively (Fig. 2). After about 30 h of release, nisin reached a steady state with its maximum release. At its steady-state, microorganisms could be continuously attacked by nisin which releases at maximum.

Fig. 2. The release profiles of nisin incorporated in the gel systems of agar at 1, 2 and 3%. Diamond (◇), square (□) and triangle (△) are the absorbance values of the gels containing agar at 1, 2 and 3%, respectively. The concentration of nisin in the gels was 2% (w/v).

Download Original Figure

The experimental data which are over 40% of the maximum release rate were fit by the linear regression model of Eq. (3) (Fig. 3). Fig. 3 shows that the steady-state of the release has a very good fit to the linear model. The high linearity of ln (1- M₀/M_∞) to time (Fig. 3) indicates that this linear regression model is adequate to describe nisin release profile from the agar gels. The parameters of the regression models, determined from the release profiles of the gels of 1, 2 and 3% agar, are summarized in Table 1. The results in Table 1 and Fig. 3 demonstrate that the diffusion efficacy of nisin decreases with the increase of agar concentration in the gel. This might be due to formation of a gel network that could hold more nisin at a higher agar concentration. Hence, nisin release rate from agar gels can be controlled by adjusting the agar concentration.

Fig. 3. The release profiles of nisin in the gel samples incorporating nisin (2%, w/v) during storage at 24ºC. The concentration of nisin in the gels was 2% (w/v). Experimental data and model predicted data are shown with symbols and a solid line, respectively. Diamond (◇), square (□) and triangle (△) represent the gel samples of agar at 1, 2 and 3%, respectively.

Download Original Figure

The D values decreased as the agar concentration increased, agreeing with the release results described previously. The difference between the D values obtained from 1 and 2% agar gels (0.0044) was higher than the difference between the values obtained from 2 and 3% agar gels (0.0018). No experiments were conducted with agar at the concentration >3% since solidification of agar gels became difficult to control and gel cylinders were easy to break, which is consistent with the report by Sebti et al. (2004).

Ripoche et al. (2006) investigated nisin diffusion in agar gels at 10ºC for 6 days and reported the D values for the diffusion in the gels containing agar at 3.2, 3.9, and 6.7% as 4.2×10^-7 , 3.4×10^-7 , and 2.5×10^-7 cm² /s, respectively. The D for nisin diffusion in hydroxylpropyl methylcellulose polymer film at 25ºC ranged from 6.5×10^-7 cm² /s to 3.3×10^-6 cm² /s in a 3%- agar gel containing fat at 5 or 30% (w/w) (Chollet et al., 2009). In the current study, the D values were 1.2×10^-2 , 8.2×10^-3 , and 6.4×10^-3 cm² /s with the gels containing agar at 1, 2 and 3%, respectively, and they are relatively high. The inconsistency of the D values might be due to various factors, including temperature, polymer morphologies including density, crystallinity, tortuosity, degree of crosslinking and branching, and glass transition temperature, and molecular interactions of the diffusants and host polymers (Bawa et al., 1985; Helmroth et al., 2002). The results suggest that the gel food system can be used as a nisin carrier for a controlled release of nisin, which is determined by the concentration of agar in the system.

Addition of nisin at 0.1 and 0.5% in the APT broth retarded the growth of B. thermosphacta in the broth for about 9 and >72 h, respectively (Fig. 4). Treatment with the agar gels incorporating nisin also inhibited the growth of B. thermosphacta (Fig. 4). Without nisin in liquid broth solution, the release of nisin affected B. thermosphacta growth, prolonging the lag phase of B. thermosphacta growth by about 26 h (Fig. 4). These results suggest that nisin-incorporating agar gel is capable of inhibiting the growth of B. thermosphacta. A longer inhibition of the growth was observed with the gel containing agar at 1% than the gel with 3% agar. This could be due to a higher release of nisin from the gel at a lower concentration of agar in the gels as described previously.

Fig. 4. Growth of B. thermosphacta in APT broth at 24ºC. Closed circles (● ) represent cultures grown in the absence of nisin (control) while closed squares (■) and closed triangles (▲) represent cultures grown in the presence of nisin at 0.1 and 0.5 % (w/v), respectively. Open squares (□), open circles (○) and cross symbols (+) represent cultures grown in the presence of the nisin (2%, w/v)-incorporating gels of agar at 1, 2 and 3% (w/v), respectively.

Download Original Figure

A simultaneous treatment of B. thermosphacta with the nisin (0.1%)-dissolved broth and the nisin-incorporating gel effectively inhibited the microbial growth in the medium, showing the initiation of logarithmic phase on about 95 h, irrespective of the concentration of the agar in the gels (Fig. 5). The effect of the concentration of agar in the gel formulation was not demonstrated in this result. This might be due to swelling of the gels induced by excessive storage of the gels in the liquid medium (115 h), which could destruct the nisin-hold gel networks at any concentration of agar. If the network were more resistant to moisture absorption, the microbial inhibition could be extended since potential uncontrolled outpouring of nisin into the broth, which can use up nisin immediately, could be prevented. The initiation point of the logarithmic phase of B. thermosphacta in the simultaneous treatment (90 h) was longer than the summation of the initiation points in the individual treatments of the nisin (0.1%)-dissolved broth (13 h) and the nisin-incorporating agar gel (30 h). The simultaneous treatment demonstrates its potential use as a means to control B. thermosphacta in liquid foods containing the gels as particulates.

Fig. 5. Growth of B. thermosphacta at 24ºC in APT broth in which nisin was dissolved and the agar gel containing nisin (2%, w/v) was placed. The symbols, *, △ and ◇ represent cultures exposed simultaneously to the nisin-dissolved broth and the nisin-incorporating gel containing agar at 1, 2 and 3%, respectively.

Download Original Figure