- Email: [email protected]
Development of immobilized lysozyme based active film
Journal of Food Engineering 78 (2007) 741–745 www.elsevier.com/locate/jfoodeng
Development of immobilized lysozyme based active film A. Conte a, G.G. Buonocore b, M. Sinigaglia a, M.A. Del Nobile b
a,*
a Department of Food Science, University of Foggia, Via Napoli, 25 – 71100 Foggia, Italy Institute of Composite and Biomedical Materials, National Research Council, P.le Tecchio, 80 – 80125 Naples, Italy
Received 21 April 2005; accepted 16 November 2005 Available online 20 January 2006
Abstract The aim of this work is to develop an active packaging material in which the active compound, lysozyme, is completely immobilized onto the polymeric material and acts directly from the film without being released into the packed foodstuff. Cross-linked films of polyvinyl alcohol, differing in the amount of both antimicrobial and binding agents, were prepared. In order to determine the amount of active substance completely bonded to the polymer backbone, lysozyme release tests were run. The antimicrobial activity of the developed active films was investigated in order to verify their effectiveness in controlling microbial spoilage. A modified version of Gompertz equation was used to quantitatively determine the antimicrobial activity of the films. Results indicate the developed active films are effective in inhibiting the growth of selected microorganism, and that the antimicrobial activity of the investigated films increases as the amount of enzyme incorporated increases. 2005 Elsevier Ltd. All rights reserved. Keywords: Active packaging; Lysozyme; Immobilization
1. Introduction In recent years research interest in active food packaging concepts increased considerably, this is due to the fact that ‘‘active materials’’ provide additional functions to food packaging with respect to the traditional passive ones, which only protect the packaged food against external deteriorative factors, such as humidity and oxygen. Several attempts have been made in developing active packaging systems in which antimicrobial agents are incorporated into the polymeric material and are slowly released on the food surface (Devlieghere, Vermeiren, & Debevere, 2004; Quintavalla & Vicini, 2002; Vermeiren, Devlieghere, & Debevere, 2002). But it has been proven that the application of the active substance directly onto the food surface as a coating layer can cause rapid diffusion into the bulk of food from the surface (Siragusa & Dickson, 1992; Torres, Motoki, & Karel, 1985). In order to overcome *
Corresponding author. Tel.: +39 881 589 242; fax: +39 881 740 211. E-mail address: [email protected] (M.A. Del Nobile).
0260-8774/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2005.11.013
the limited benefits of this kind of systems, active films incorporating antimicrobial compound into/onto the polymeric structure were developed. They are aimed to control or even prevent the growth of undesired bacterial species responsible for the packed foodstuff degradation (Bezemer et al., 2000; Cha, Choi, Chinnan, & Park, 2002; Ouattara, Simard, Piette, Begin, & Holley, 2000; Padgett, Han, & Dawson, 1998). Several works are reported in the literature dealing with the study of controlled and uncontrolled release systems (Chen, Yen, & Chiang, 1996; Chung, Chikindas, & Yam, 2001; Chung, Papadakis, & Yam, 2001; Han & Floros, 1998). As far as the release of compounds from the packaging material to the packaged foodstuff is concerned, at the present, in Europe, there is still a lack of legislation. Due to this reason the use of antimicrobial substances completely bound into the polymeric material, which do not need to migrate into the food to be effective, would be desirable (Appendini & Hotchkiss, 1997; Scannell et al., 2000). Natural compounds such as organic acid, bacteriocins, enzymes, fungicides and spice extracts have been studied
742
A. Conte et al. / Journal of Food Engineering 78 (2007) 741–745
as food preservatives in place of the chemical ones, because of their ability to prolong the shelf life of packed food and their potential safety for human consumption (Chen et al., 1996; Ha, Kim, & Lee, 2001; Han, 2000; Lee, An, Park, & Lee, 2003; Lee, Hwang, & Cho, 1998). A promising antimicrobial compound is lysozyme, single peptide protein, which possesses enzymatic activity against beta 1–4 glycosidic linkages between N-acetylmuramic acid and N-acetylglucosamine found in peptidoglycan of the bacteria. In this work the immobilization of lysozyme onto polyvinyl alcohol (PVOH) matrix is addressed. The antimicrobial activity of lysozyme and its effectiveness against a selected microorganism was investigated in order to determine the optimal concentration of binding agent needed to completely immobilize the enzyme.
As control, films with glutaraldehyde and glacial acetic acid, without lysozyme were also prepared and it will be referred to as Film A. 2.2. Film washing Each active film (20 · 13.5 cm) was immersed into 4.5 L of distilled water, under continuous stirring at room temperature. The amount of lysozyme eventually released from the film in the washing solution was evaluated by monitoring its concentration, by means of an HPLC, until the attainment of equilibrium conditions. For each film the tests to determine the amount of lysozyme released during washing were run in triplicate. 2.3. Lysozyme determination
2. Materials and methods 2.1. Film preparation The films studied in this paper were obtained using polyvinyalcohol (PVOH) (MW 70,000–100,000 SigmaAldrich, Gallarate, Italy) as polymeric matrix, lysozyme (MW 14,000 Da, Sigma-Aldrich, Gallarate, Italy) as antimicrobial active compound, glyoxal (40% Riedel de Haen, Gallarate, Italy) as cross-linking agents and glutaraldehyde (50% Aldrich, Gallarate, Italy) as binding agents. The films were produced by casting technique. A 13% (w/v) PVOH solution (3.25 g of polymer matrix in 25 mL of water) was autoclaved for 15 min and then cooled to room temperature. After dissolution, the PVOH was cross-linked by adding 5 lL of glyoxal and hydrochloric acid (0.2 mL) was used as reaction catalyst. After homogenizing at a speed of 150 rpm, lysozyme and glutaraldehyde were added to the solution in different amounts. The resulting solution was homogenized at a speed of 150 rpm to uniformly distribute the antimicrobial compound and the binding agent. Two mL of glacial acetic acid (Sigma-Aldrich, Gallarate, Italy) were also added as reaction catalyst. After homogenizing, the cross-linked solution was cast onto a plexiglass plate and the obtained film was dried at ambient condition until the solvent was completely evaporated and then further dried under vacuum for one day. In this way, it was possible to obtain films differing in the amount of both antimicrobial and bounding agent. Films had a thickness of ca 100 lm. The obtained active films are listed as follows: Film B: 500 mg glutaraldehyde. Film C: 100 mg glutaraldehyde. Film D: 50 mg glutaraldehyde. Film E: 20 mg glutaraldehyde.
of lysozyme and 0.025 mL of of lysozyme and 0.005 mL of of
lysozyme
and 0.005 mL of
of
lysozyme
and
0.005 mL
of
The amount of lysozyme released in water during either washing or release tests was determined by means of an HPLC (Agilent Mod. 1100). A C18 Reverse phase column was used (250 · 4 mm, 5 lm) and a gradient elution with water-acetonitrile gradients (1 mL/min) containing 0.1% trifluoracetic acid (TFA) was used. The calibration curve was constructed for peak area against lysozyme concentration of standard solutions from 6 to 300 ppm, with five replicate samples for each lysozyme concentration. 2.4. Lysozyme release The films were washed according to the procedure reported above. The washed samples (270 cm2) containing the immobilized lysozyme were brought in contact with 610 mL of water in order to create a ratio volume/surface of 2:1. The amount of lysozyme eventually released was evaluated by monitoring, by means of an HPLC, the concentration of the antimicrobial compound in the surrounding solution. The time interval during which the concentration lysozyme in the outer water solution was monitored is equal to the time interval used to for the antimicrobial activity tests. Also in this case the release tests were run in triplicate. 2.5. Antimicrobial activity As reported by Appendini and Hotchkiss (1997), lysozyme activity can be determined by measuring the decrease in absorbance of the Micrococcus lysodeikticus incubated with the film in buffer. The M. lysodeikticus was selected because of its high susceptibility to lysozyme antimicrobial activity. A suspension of lyophilized M. lysodeikticus cells (Sigma-Aldrich) were incubated at room temperature in 610 mL of 0.1 M phosphate buffer, pH 6.8, (absorbance at 450 nm) to reach a cell concentration of 107 organisms/mL. Antimicrobial films were washed according to the above mentioned procedure and then brought in contact with the obtained suspension. The ratio between the volume of solution and the active surface of the film
A. Conte et al. / Journal of Food Engineering 78 (2007) 741–745
was 2:1. The absorbance at 450 nm of the suspension, which was continuously stirred, contacted with the antimicrobial film was monitored until a constant value was reached. Each test was conducted in triplicate. As control the decrease of absorbance at 450 nm of a microbial suspension in phosphate buffer without film was also measured. 2.6. Antimicrobial efficacy determination The Gompertz equation as modified by Zwietering was used to quantitatively determine the film antimicrobial activity (Zwietering, Jongenburger, Roumbouts, & Van’t Riet, 1990): IðtÞ ¼ K þ A kt exp exp ðlmax 2:7182Þ þ1 A
ð1Þ
where IðtÞ is the normalized intensity at time t obtained by dividing the intensity at time t by the initial intensity, K is the initial value of IðtÞ, and, as expected, is always about 1, A is the maximum decrease in the normalized intensity, lmax is the maximal decrease rate (expressed as s1), k is the lag time (expressed as s). Eq. (1) was fitted to the experimental data, and the value of lmax was taken as a measure of the film antimicrobial activity.
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3. Results and discussion As reported above the aim of this work is to develop an active packaging material in which the active compound is completely immobilized into the polymeric materials, and acts directly from the film without being released into the packaged foodstuff. In order to achieve this goal, it is necessary to address the following aspects: (1) to study the influence of the binding agent concentration on the amount of active substance completely bonded to the polymer backbone; (2) to study the effectiveness of the obtained ‘‘active materials’’ in slowing down the growth of selected microorganism. As far as the first point is concerned, five films were prepared according to the procedure reported above. The films differ in the ratio between amount of bounding agent and lysozyme. As reported in Section 2, each film was washed in 4.5 L of distilled water in order to remove the active substance not bonded to the polymer network. The concentration of lysozyme into the washing solution was monitored during time until the attainment of an equilibrium value. In Fig. 1 the release kinetics for each of the investigated film are reported. As can be inferred from the above data, Films D and E do not release any lysozyme, whereas Films B and C release a different amount of active substance, each film with a different kinetic. The washing time needed to completely release the not-bonded lysozyme, for each film, was determined from the above results. The results obtained along with the amount of enzyme released at
2.7. Statistical analysis All analyses were carried out in triplicate. The average values and their standard deviation were calculated. Also the parameters listed in the tables are the average of all repetitions. The confidence intervals of model’s parameters were evaluated as follows: first, a fit was run with the original data; then, using the data points standard deviation 100 additional fits were run on artificial data sets, which were generated by randomly varying the data around the fitted function. From these additional fits, a distribution of values for each parameter was obtained. The sets of data obtained for each parameter was statistically treated to obtain the 95% confidence interval. The unpaired Student t-tests with unequal variance were run using the Kaleidagraph software (Synergy Software, Reading, PA, USA).
Fig. 1. Evolution of the amount of lysozyme released during washing tests. (j) Film B, () Film C, (d) Film D, (s) Film E.
Table 1 Results obtained from washing tests Sample
Lysozyme initially added to the film (mg)
Releasing time (h)
Lysozyme released into the washing solution (mg)
Lysozyme immobilized into the film (mg)
Film Film Film Film
500 100 50 20
30 5 – –
316.58 47.59 0 0
183.42 52.41 50 20
B C D E
The values of washing time needed to reach an asymptotic value and the asymptotic amount of lysozyme released are reported.
E% lag
[2.0752e6, 2.8935e6] [1.4370e4, 2.2709e4] [8.2079e5, 1.1215e4] [3.0262e5, 3.9815e5] [1.5121e5, 1.9055e5] 2.4316e6 1.8243e4 3.7026e5 3.4678e5 1.6996e5
lmax A
5.1186e2 [4.6744e2, 5.5938e2] 0.9818 [0.8318, 1.1186] 1.1865 [0.8143, 1.0840] 0.9468 [0.8472, 1.1812] 0.7994 [0.7722, 0.8380] [1.0047, 1.0087] [0.882, 1.1811] [0.8769, 1.1474] [0.9389, 1.0607] [0.9882, 1.0146]
k
1.0065 1.0396 1.1139 0.9845 0.9998 A B C D E
Sample
Table 2 Values of the parameters obtained fitting Eq. (1) to the experimental data
Fig. 2. Effectiveness of the different active films against M. lysodeikticus suspension. Fitting of modified Gompertz equation to the experimental data: (h) absorbance of Film A as a function of time; (—–) fitting to the data obtained from Film A. () Absorbance of Film B as a function of time; (– – –) fitting to the data obtained from Film B. (j) Absorbance of Film C as a function of time; (- - -) fitting to the data obtained from Film C. (s) Absorbance of Film D as a function of time; (- Æ -) fitting to the data obtained from Film D. (d) Absorbance of Film E as a function of time; ( ) fitting to the data obtained from Film E.
7718.1180 [5594.2453, 9546.0032] 7.7187e14 [6.2335e14, 9.1049e14] 1.2851e11 [0.00, 2785.1624] 6271.1255 [1985.3767, 8781.1931] 1.1205e4 [8998.7288, 1.2899e+4]
equilibrium are reported in Table 1. As one would expect the amount of bonded lysozyme increases as the ratio between the amount of loaded lysozyme and amount of bounding agent decreases. Unfortunately, it is not easy to quantitatively interpret the data shown in Table 1 as glutaraldehyde acts both as cross-linking agent for the polymeric matrix and as bounding agent for lysozyme. However, it is worth noting that for films D and E, the lysozyme is completely bounded to the polymeric matrix. As far as the antimicrobial activity of the proposed films is concerned, each film was washed for the proper time and then brought in contact with a suspension of lyophilized M. lysodeikticus cells (cell concentration of 107 organisms/mL). As described in Section 2, in order to evaluate the effectiveness of the film, the decrease of absorbance of the suspension during time was monitored for each sample. Results are reported in Fig. 2. For the sake of comparison, the decrease in the absorbance of the microbial suspension in phosphate buffer in contact with control film (Film A) is also reported. As can be inferred from the above data, when the suspension is in contact with control film a slight decrease is observed, probably due to a certain degree of mortality of the M. lysodeikticus itself. Active films, instead, show a high effectiveness against the investigated microbial cells. As expected, as the amount of lysozyme increases, the antimicrobial activity seems to be higher. In order to quantify the film antimicrobial activity, Eq. (1) was fitted to the experimental data. The fitting curves obtained are also reported in Fig. 2. The goodness of fit was evaluated by calculating the relative percent difference E% (Boquet, Chirifie, & Iglesias, 1978). The values of the E% and the fitting parameters are listed in Table 2. As reported by Zwietering et al. (1990), the most meaningful parameter with respect to the antimicrobial activity is lmax.
0.2539 1.7503 1.6153 8.7611 1.1697
A. Conte et al. / Journal of Food Engineering 78 (2007) 741–745
Film Film Film Film Film
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A. Conte et al. / Journal of Food Engineering 78 (2007) 741–745
Fig. 3. Antimicrobial activity of lysozyme plotted as function of immobilized lysozyme.
Thus, the values of lmax for each film were reported as a function of the actual amount of immobilized lysozyme onto each respective film (data are reported in Fig. 3). As can be inferred from the data shown in the above figure, the films effectiveness increases as the amount of immobilized lysozyme increases. In order to find out if the antimicrobial activity of the washed active film can be ascribed to active compound released into the microbial solution, release tests were run. Results show that no lysozyme was released from the investigated washed films. Results suggest that the effectiveness of the investigated films is related exclusively to the immobilized lysozyme. 4. Conclusions An active packaging material in which the active compound is immobilized into the polymeric material was proposed. Five cross-linked films differing in the ratio between amount of bounding agent and lysozyme were prepared and investigated. The amount of active substance completely bonded to the polymer backbone and the effectiveness of the obtained active films in slowing down the growth of M. lysodeikticus were determined. The results obtained indicate that the proposed films are effective in inhibiting the growth of the investigated microorganism, and that the antimicrobial activity of the investigated films must be ascribed to the immobilized enzyme. Results also indicate that the antimicrobial efficacy of the proposed film increases as the amount of bond enzyme increases. References Appendini, P., & Hotchkiss, J. H. (1997). Immobilization of lysozyme on food contact polymers as a potential antimicrobial films. Packaging Technology and Science, 10, 271–279. Bezemer, J. M., Radersma, R., Grijpma, D. W., Dijkstra, P. J., Feijen, J., & Van Blitterswijk, C. A. (2000). Zero-order release of lysozyme from
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poly(ethylene glycol)/poly(butyene terephthalate) matrices. Journal of Controlled Release, 64, 179–192. Boquet, R., Chirifie, J., & Iglesias, H. A. (1978). Equations for fitting water sorption isotherms of foods. II. Evaluation of various twoparameters models. Journal of Food Technology, 13, 319–327. Cha, D. S., Choi, J. H., Chinnan, M. S., & Park, H. J. (2002). Antimicrobial films based on Na-alginate and K-carrageenan. Lebensmittel-Wissenschaft und-Technologie, 35, 715–719. Chen, M. C., Yen, G. H. C., & Chiang, B. H. (1996). Antimicrobial and physicochemical properties of methylcellulose and chitosan films containing a preservative. Journal of Food Processing and Preservation, 20, 379–390. Chung, D., Chikindas, M. L., & Yam, K. L. (2001). Inhibition of Saccharomyces cerevisiae by slow release of propyl paraben from a polymer coating. Journal of Food Protection, 64, 1420–1424. Chung, D., Papadakis, S. P., & Yam, K. L. (2001). Release of propyl paraben from a polymer coating into water and food simulating solvents for antimicrobial packaging applications. Journal of Food Processing and Preservation, 25, 71–87. Devlieghere, F., Vermeiren, L., & Debevere, J. (2004). New preservation technologies: Possibilities and limitations. International Dairy Journal, 14, 273–285. Ha, J. U., Kim, Y. M., & Lee, D. S. (2001). Multilayered antimicrobial polyethylene films applied to the packaging of ground beef. Packaging Technology and Science, 14, 55–62. Han, J. H. (2000). Antimicrobial food packaging. Food Technology, 54, 56–65. Han, J. H., & Floros, J. D. (1998). Simulating diffusion model and determining diffusivity of potassium sorbate through plastic to develop antimicrobial packaging films. Journal of Food and Preservation, 22, 107–122. Lee, C. H., An, D. S., Park, H. J., & Lee, D. S. (2003). Wide spectrum antimicrobial packaging materials incorporating nisin and chitosan in the coating. Packaging Technology and Science, 16, 99–106. Lee, D. S., Hwang, Y. I., & Cho, S. H. (1998). Developing antimicrobial packaging film for curled lettuce and soybean sprouts. Food Science Biotechnology, 7, 117–121. Ouattara, B., Simard, R. E., Piette, G., Begin, A., & Holley, R. A. (2000). Diffusion of acetic and propionic acids from chitosan-based antimicrobial packaging films. Journal of Food Science, 65, 768– 773. Padgett, T., Han, I. Y., & Dawson, P. L. (1998). Incorporation of foodgrade antimicrobial compounds into biodegradable packaging films. Journal of Food Protection, 61, 1330–1335. Quintavalla, S., & Vicini, L. (2002). Antimicrobial food packaging in meat industry. Meat Science, 62, 373–380. Scannell, A. G. M., Hill, C., Ross, R. P., Marx, S., Hartmeier, W., & Arendt, E. K. (2000). Development of bioactive food packaging materials using immobilised bacteriocins Lacticin 3147 and Nisaplin. International Journal of Food Microbiology, 60, 241–249. Siragusa, G. A., & Dickson, J. S. (1992). Inhibition of Listeria monocitogenens on beef tissue by application of organic acids immobilized in a calcium alginate gel. Journal of Food Science, 57, 293–296. Torres, J. A., Motoki, M., & Karel, M. (1985). Microbial stabilization of intermediate moisture food surfaces. I. Control of surface preservative concentration. Journal of Food Processing and Preservation, 9, 75–92. Vermeiren, L., Devlieghere, F., & Debevere, J. (2002). Effectiveness of same recent antimicrobial packaging concepts. Food Additives and Contaminants, 19, 163–171. Zwietering, M. H., Jongenburger, F. M., Roumbouts, M., & Van’t Riet, K. (1990). Modeling of the bacterial growth curve. Applied and Environmental Microbiology, 56, 1875–1881.
Development of immobilized lysozyme based active film A. Conte a, G.G. Buonocore b, M. Sinigaglia a, M.A. Del Nobile b
a,*
a Department of Food Science, University of Foggia, Via Napoli, 25 – 71100 Foggia, Italy Institute of Composite and Biomedical Materials, National Research Council, P.le Tecchio, 80 – 80125 Naples, Italy
Received 21 April 2005; accepted 16 November 2005 Available online 20 January 2006
Abstract The aim of this work is to develop an active packaging material in which the active compound, lysozyme, is completely immobilized onto the polymeric material and acts directly from the film without being released into the packed foodstuff. Cross-linked films of polyvinyl alcohol, differing in the amount of both antimicrobial and binding agents, were prepared. In order to determine the amount of active substance completely bonded to the polymer backbone, lysozyme release tests were run. The antimicrobial activity of the developed active films was investigated in order to verify their effectiveness in controlling microbial spoilage. A modified version of Gompertz equation was used to quantitatively determine the antimicrobial activity of the films. Results indicate the developed active films are effective in inhibiting the growth of selected microorganism, and that the antimicrobial activity of the investigated films increases as the amount of enzyme incorporated increases. 2005 Elsevier Ltd. All rights reserved. Keywords: Active packaging; Lysozyme; Immobilization
1. Introduction In recent years research interest in active food packaging concepts increased considerably, this is due to the fact that ‘‘active materials’’ provide additional functions to food packaging with respect to the traditional passive ones, which only protect the packaged food against external deteriorative factors, such as humidity and oxygen. Several attempts have been made in developing active packaging systems in which antimicrobial agents are incorporated into the polymeric material and are slowly released on the food surface (Devlieghere, Vermeiren, & Debevere, 2004; Quintavalla & Vicini, 2002; Vermeiren, Devlieghere, & Debevere, 2002). But it has been proven that the application of the active substance directly onto the food surface as a coating layer can cause rapid diffusion into the bulk of food from the surface (Siragusa & Dickson, 1992; Torres, Motoki, & Karel, 1985). In order to overcome *
Corresponding author. Tel.: +39 881 589 242; fax: +39 881 740 211. E-mail address: [email protected] (M.A. Del Nobile).
0260-8774/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2005.11.013
the limited benefits of this kind of systems, active films incorporating antimicrobial compound into/onto the polymeric structure were developed. They are aimed to control or even prevent the growth of undesired bacterial species responsible for the packed foodstuff degradation (Bezemer et al., 2000; Cha, Choi, Chinnan, & Park, 2002; Ouattara, Simard, Piette, Begin, & Holley, 2000; Padgett, Han, & Dawson, 1998). Several works are reported in the literature dealing with the study of controlled and uncontrolled release systems (Chen, Yen, & Chiang, 1996; Chung, Chikindas, & Yam, 2001; Chung, Papadakis, & Yam, 2001; Han & Floros, 1998). As far as the release of compounds from the packaging material to the packaged foodstuff is concerned, at the present, in Europe, there is still a lack of legislation. Due to this reason the use of antimicrobial substances completely bound into the polymeric material, which do not need to migrate into the food to be effective, would be desirable (Appendini & Hotchkiss, 1997; Scannell et al., 2000). Natural compounds such as organic acid, bacteriocins, enzymes, fungicides and spice extracts have been studied
742
A. Conte et al. / Journal of Food Engineering 78 (2007) 741–745
as food preservatives in place of the chemical ones, because of their ability to prolong the shelf life of packed food and their potential safety for human consumption (Chen et al., 1996; Ha, Kim, & Lee, 2001; Han, 2000; Lee, An, Park, & Lee, 2003; Lee, Hwang, & Cho, 1998). A promising antimicrobial compound is lysozyme, single peptide protein, which possesses enzymatic activity against beta 1–4 glycosidic linkages between N-acetylmuramic acid and N-acetylglucosamine found in peptidoglycan of the bacteria. In this work the immobilization of lysozyme onto polyvinyl alcohol (PVOH) matrix is addressed. The antimicrobial activity of lysozyme and its effectiveness against a selected microorganism was investigated in order to determine the optimal concentration of binding agent needed to completely immobilize the enzyme.
As control, films with glutaraldehyde and glacial acetic acid, without lysozyme were also prepared and it will be referred to as Film A. 2.2. Film washing Each active film (20 · 13.5 cm) was immersed into 4.5 L of distilled water, under continuous stirring at room temperature. The amount of lysozyme eventually released from the film in the washing solution was evaluated by monitoring its concentration, by means of an HPLC, until the attainment of equilibrium conditions. For each film the tests to determine the amount of lysozyme released during washing were run in triplicate. 2.3. Lysozyme determination
2. Materials and methods 2.1. Film preparation The films studied in this paper were obtained using polyvinyalcohol (PVOH) (MW 70,000–100,000 SigmaAldrich, Gallarate, Italy) as polymeric matrix, lysozyme (MW 14,000 Da, Sigma-Aldrich, Gallarate, Italy) as antimicrobial active compound, glyoxal (40% Riedel de Haen, Gallarate, Italy) as cross-linking agents and glutaraldehyde (50% Aldrich, Gallarate, Italy) as binding agents. The films were produced by casting technique. A 13% (w/v) PVOH solution (3.25 g of polymer matrix in 25 mL of water) was autoclaved for 15 min and then cooled to room temperature. After dissolution, the PVOH was cross-linked by adding 5 lL of glyoxal and hydrochloric acid (0.2 mL) was used as reaction catalyst. After homogenizing at a speed of 150 rpm, lysozyme and glutaraldehyde were added to the solution in different amounts. The resulting solution was homogenized at a speed of 150 rpm to uniformly distribute the antimicrobial compound and the binding agent. Two mL of glacial acetic acid (Sigma-Aldrich, Gallarate, Italy) were also added as reaction catalyst. After homogenizing, the cross-linked solution was cast onto a plexiglass plate and the obtained film was dried at ambient condition until the solvent was completely evaporated and then further dried under vacuum for one day. In this way, it was possible to obtain films differing in the amount of both antimicrobial and bounding agent. Films had a thickness of ca 100 lm. The obtained active films are listed as follows: Film B: 500 mg glutaraldehyde. Film C: 100 mg glutaraldehyde. Film D: 50 mg glutaraldehyde. Film E: 20 mg glutaraldehyde.
of lysozyme and 0.025 mL of of lysozyme and 0.005 mL of of
lysozyme
and 0.005 mL of
of
lysozyme
and
0.005 mL
of
The amount of lysozyme released in water during either washing or release tests was determined by means of an HPLC (Agilent Mod. 1100). A C18 Reverse phase column was used (250 · 4 mm, 5 lm) and a gradient elution with water-acetonitrile gradients (1 mL/min) containing 0.1% trifluoracetic acid (TFA) was used. The calibration curve was constructed for peak area against lysozyme concentration of standard solutions from 6 to 300 ppm, with five replicate samples for each lysozyme concentration. 2.4. Lysozyme release The films were washed according to the procedure reported above. The washed samples (270 cm2) containing the immobilized lysozyme were brought in contact with 610 mL of water in order to create a ratio volume/surface of 2:1. The amount of lysozyme eventually released was evaluated by monitoring, by means of an HPLC, the concentration of the antimicrobial compound in the surrounding solution. The time interval during which the concentration lysozyme in the outer water solution was monitored is equal to the time interval used to for the antimicrobial activity tests. Also in this case the release tests were run in triplicate. 2.5. Antimicrobial activity As reported by Appendini and Hotchkiss (1997), lysozyme activity can be determined by measuring the decrease in absorbance of the Micrococcus lysodeikticus incubated with the film in buffer. The M. lysodeikticus was selected because of its high susceptibility to lysozyme antimicrobial activity. A suspension of lyophilized M. lysodeikticus cells (Sigma-Aldrich) were incubated at room temperature in 610 mL of 0.1 M phosphate buffer, pH 6.8, (absorbance at 450 nm) to reach a cell concentration of 107 organisms/mL. Antimicrobial films were washed according to the above mentioned procedure and then brought in contact with the obtained suspension. The ratio between the volume of solution and the active surface of the film
A. Conte et al. / Journal of Food Engineering 78 (2007) 741–745
was 2:1. The absorbance at 450 nm of the suspension, which was continuously stirred, contacted with the antimicrobial film was monitored until a constant value was reached. Each test was conducted in triplicate. As control the decrease of absorbance at 450 nm of a microbial suspension in phosphate buffer without film was also measured. 2.6. Antimicrobial efficacy determination The Gompertz equation as modified by Zwietering was used to quantitatively determine the film antimicrobial activity (Zwietering, Jongenburger, Roumbouts, & Van’t Riet, 1990): IðtÞ ¼ K þ A kt exp exp ðlmax 2:7182Þ þ1 A
ð1Þ
where IðtÞ is the normalized intensity at time t obtained by dividing the intensity at time t by the initial intensity, K is the initial value of IðtÞ, and, as expected, is always about 1, A is the maximum decrease in the normalized intensity, lmax is the maximal decrease rate (expressed as s1), k is the lag time (expressed as s). Eq. (1) was fitted to the experimental data, and the value of lmax was taken as a measure of the film antimicrobial activity.
743
3. Results and discussion As reported above the aim of this work is to develop an active packaging material in which the active compound is completely immobilized into the polymeric materials, and acts directly from the film without being released into the packaged foodstuff. In order to achieve this goal, it is necessary to address the following aspects: (1) to study the influence of the binding agent concentration on the amount of active substance completely bonded to the polymer backbone; (2) to study the effectiveness of the obtained ‘‘active materials’’ in slowing down the growth of selected microorganism. As far as the first point is concerned, five films were prepared according to the procedure reported above. The films differ in the ratio between amount of bounding agent and lysozyme. As reported in Section 2, each film was washed in 4.5 L of distilled water in order to remove the active substance not bonded to the polymer network. The concentration of lysozyme into the washing solution was monitored during time until the attainment of an equilibrium value. In Fig. 1 the release kinetics for each of the investigated film are reported. As can be inferred from the above data, Films D and E do not release any lysozyme, whereas Films B and C release a different amount of active substance, each film with a different kinetic. The washing time needed to completely release the not-bonded lysozyme, for each film, was determined from the above results. The results obtained along with the amount of enzyme released at
2.7. Statistical analysis All analyses were carried out in triplicate. The average values and their standard deviation were calculated. Also the parameters listed in the tables are the average of all repetitions. The confidence intervals of model’s parameters were evaluated as follows: first, a fit was run with the original data; then, using the data points standard deviation 100 additional fits were run on artificial data sets, which were generated by randomly varying the data around the fitted function. From these additional fits, a distribution of values for each parameter was obtained. The sets of data obtained for each parameter was statistically treated to obtain the 95% confidence interval. The unpaired Student t-tests with unequal variance were run using the Kaleidagraph software (Synergy Software, Reading, PA, USA).
Fig. 1. Evolution of the amount of lysozyme released during washing tests. (j) Film B, () Film C, (d) Film D, (s) Film E.
Table 1 Results obtained from washing tests Sample
Lysozyme initially added to the film (mg)
Releasing time (h)
Lysozyme released into the washing solution (mg)
Lysozyme immobilized into the film (mg)
Film Film Film Film
500 100 50 20
30 5 – –
316.58 47.59 0 0
183.42 52.41 50 20
B C D E
The values of washing time needed to reach an asymptotic value and the asymptotic amount of lysozyme released are reported.
E% lag
[2.0752e6, 2.8935e6] [1.4370e4, 2.2709e4] [8.2079e5, 1.1215e4] [3.0262e5, 3.9815e5] [1.5121e5, 1.9055e5] 2.4316e6 1.8243e4 3.7026e5 3.4678e5 1.6996e5
lmax A
5.1186e2 [4.6744e2, 5.5938e2] 0.9818 [0.8318, 1.1186] 1.1865 [0.8143, 1.0840] 0.9468 [0.8472, 1.1812] 0.7994 [0.7722, 0.8380] [1.0047, 1.0087] [0.882, 1.1811] [0.8769, 1.1474] [0.9389, 1.0607] [0.9882, 1.0146]
k
1.0065 1.0396 1.1139 0.9845 0.9998 A B C D E
Sample
Table 2 Values of the parameters obtained fitting Eq. (1) to the experimental data
Fig. 2. Effectiveness of the different active films against M. lysodeikticus suspension. Fitting of modified Gompertz equation to the experimental data: (h) absorbance of Film A as a function of time; (—–) fitting to the data obtained from Film A. () Absorbance of Film B as a function of time; (– – –) fitting to the data obtained from Film B. (j) Absorbance of Film C as a function of time; (- - -) fitting to the data obtained from Film C. (s) Absorbance of Film D as a function of time; (- Æ -) fitting to the data obtained from Film D. (d) Absorbance of Film E as a function of time; ( ) fitting to the data obtained from Film E.
7718.1180 [5594.2453, 9546.0032] 7.7187e14 [6.2335e14, 9.1049e14] 1.2851e11 [0.00, 2785.1624] 6271.1255 [1985.3767, 8781.1931] 1.1205e4 [8998.7288, 1.2899e+4]
equilibrium are reported in Table 1. As one would expect the amount of bonded lysozyme increases as the ratio between the amount of loaded lysozyme and amount of bounding agent decreases. Unfortunately, it is not easy to quantitatively interpret the data shown in Table 1 as glutaraldehyde acts both as cross-linking agent for the polymeric matrix and as bounding agent for lysozyme. However, it is worth noting that for films D and E, the lysozyme is completely bounded to the polymeric matrix. As far as the antimicrobial activity of the proposed films is concerned, each film was washed for the proper time and then brought in contact with a suspension of lyophilized M. lysodeikticus cells (cell concentration of 107 organisms/mL). As described in Section 2, in order to evaluate the effectiveness of the film, the decrease of absorbance of the suspension during time was monitored for each sample. Results are reported in Fig. 2. For the sake of comparison, the decrease in the absorbance of the microbial suspension in phosphate buffer in contact with control film (Film A) is also reported. As can be inferred from the above data, when the suspension is in contact with control film a slight decrease is observed, probably due to a certain degree of mortality of the M. lysodeikticus itself. Active films, instead, show a high effectiveness against the investigated microbial cells. As expected, as the amount of lysozyme increases, the antimicrobial activity seems to be higher. In order to quantify the film antimicrobial activity, Eq. (1) was fitted to the experimental data. The fitting curves obtained are also reported in Fig. 2. The goodness of fit was evaluated by calculating the relative percent difference E% (Boquet, Chirifie, & Iglesias, 1978). The values of the E% and the fitting parameters are listed in Table 2. As reported by Zwietering et al. (1990), the most meaningful parameter with respect to the antimicrobial activity is lmax.
0.2539 1.7503 1.6153 8.7611 1.1697
A. Conte et al. / Journal of Food Engineering 78 (2007) 741–745
Film Film Film Film Film
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Fig. 3. Antimicrobial activity of lysozyme plotted as function of immobilized lysozyme.
Thus, the values of lmax for each film were reported as a function of the actual amount of immobilized lysozyme onto each respective film (data are reported in Fig. 3). As can be inferred from the data shown in the above figure, the films effectiveness increases as the amount of immobilized lysozyme increases. In order to find out if the antimicrobial activity of the washed active film can be ascribed to active compound released into the microbial solution, release tests were run. Results show that no lysozyme was released from the investigated washed films. Results suggest that the effectiveness of the investigated films is related exclusively to the immobilized lysozyme. 4. Conclusions An active packaging material in which the active compound is immobilized into the polymeric material was proposed. Five cross-linked films differing in the ratio between amount of bounding agent and lysozyme were prepared and investigated. The amount of active substance completely bonded to the polymer backbone and the effectiveness of the obtained active films in slowing down the growth of M. lysodeikticus were determined. The results obtained indicate that the proposed films are effective in inhibiting the growth of the investigated microorganism, and that the antimicrobial activity of the investigated films must be ascribed to the immobilized enzyme. Results also indicate that the antimicrobial efficacy of the proposed film increases as the amount of bond enzyme increases. References Appendini, P., & Hotchkiss, J. H. (1997). Immobilization of lysozyme on food contact polymers as a potential antimicrobial films. Packaging Technology and Science, 10, 271–279. Bezemer, J. M., Radersma, R., Grijpma, D. W., Dijkstra, P. J., Feijen, J., & Van Blitterswijk, C. A. (2000). Zero-order release of lysozyme from
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