Food Research International 39 (2006) 749754 www.elsevier.

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Study of the performance of nisin supported in edible lms

Karen Sanjurjo a, Silvia Flores
b

b,c

, La Gerschenson

b,d,* ,

Rosa Jagus

a Departamento de Ingeniera Qumica, Facultad de Ingeniera, Universidad de Buenos Aires, Ciudad Universitaria, 1428 Buenos Aires, Argentina Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, 1428 Buenos Aires, Argentina c Fellow of Consejo Nacional de Investigaciones, Cientcas y Tecnicas de la Republica (CONICET), Argentina d Member of Consejo Nacional de Investigaciones, Cientcas y Tecnicas de la Republica (CONICET), Argentina

Received 25 January 2006; accepted 28 January 2006

Abstract The antimicrobial activity of nisin supported in edible lms prepared with suspensions of tapioca starch containing glycerol, was studied. Films were prepared by casting the systems after gelatinization. The eect of the edible lm as antimicrobial barrier to external hazard as well as the diusional characteristics of the nisin and its release characteristics were studied in parallel to antimicrobial inactivation. Studies were performed with L. innocua, after equilibration of edible lms at a relative humidity (RH) of 57.5% and at 25 C. Results obtained showed that nisin supported in starch-based lms is active and that the lm is a useful barrier to further product contamination. Gradual release of the antimicrobial from the edible lm can also help to preclude microorganism proliferation better than nisin directly added because it seems to counterbalance, at least partially, the inactivation of nisin. 2006 Elsevier Ltd. All rights reserved.
Keywords: Tapioca-starch; Nisin; Edible lms; Antimicrobial eect

1. Introduction Edible lms are not expected to totally replace synthetic packaging. They can control moisture, gases, lipid migration as well as be used as carriers of additives and nutrients (Baker, Baldwin, & Nisperos-Carriedo, 1994; Garca, Mar tino, & Zaritzky, 1998). Cellulose, starch, proteins and lipids can be used to formulate edible lms which must be completely neutral with reference to color, taste and odor. One important component of edible lms is the plasticizer which is required to overcome lm brittleness and improve its exibility and extensibility (McHugh & Krochta, 1994). In the last years much research has been done concerning the use of edible lms as a way of supporting antimicrobials in food products as well as to provide the slow release of the antimicrobial. They have been studied as carriers of
Corresponding author. Tel./fax: +54 11 45763366. E-mail addresses: lia@di.fcen.uba.ar, pauladanher@ciudad.com.ar (L. Gerschenson). 0963-9969/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2006.01.016
*

natamycin and potassium sorbate for antimicrobial surface application (Franssen, Rumsey, & Krochta, 2002), for slow release of lysozime and nisin (Buonocore et al., 2003a, Buonocore, Del Nobile, Panizza, Corbo, & Nicolais, 2003b; Dawson, Hirt, Rieck, Acton, & Sotthibandhu, 2003; Ko, Janes, Hettiarachchy, & Johnson, 2001; Padgett, Han, & Dawson, 2000; Sebti, Blanc, Carnet-Ripoche, Saurel, & Coma, 2004), for developing sorbate or benzoate carrier lms (Chen, Yeh, & Chiang, 1996), for slow release of propylparaben (Chung, Chikindas, & Yan, 2001). In general, lost of activity of the antimicrobial due to storage conditions, reaction with food matrix and/or degradation pathways as well as antimicrobial eect on lm performance when applied to food products, has not been well established in the bibliography. Starches are a renewable resource widely available and can be obtained from dierent by products of harvesting and industrialization. They are widely used in the food industry and can be modied to enhance their functional properties (Zobel, 1994). The Food and Agriculture Orga-

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nization (FAO) highlighted recently that tapioca is a good commercial cash crop and a major source of food security, and that it needs a competitive edge to thrive in the global starch market. Due to shortage or high price of traditional starch sources, such as wheat and corn, the tapioca starch is viewed as an alternative by the food companies for use as an ingredient (Anonymous, 2004). Nisin is an antibacterial peptide produced by Lactococcus lactis that eectively inhibits Gram-positive bacteria and also the outgrowth of spores of Bacilli and Clostridia (Cleveland, Montville, Nes, & Chikindas, 2001; Hurst, 1981; Hurst & Hoover, 1993). It is the rst antimicrobial peptide with a generally recognized as safe status in the United States for use in processed cheese (Food & Drug Administration, 1998). In addition, its use in various food products is allowed in several countries (Delves-Broughton, 1990). According to previously mentioned facts it is important to clarify nisin behavior when supported in edible lms with the object of analyzing usefulness of lms as a hurdle for food preservation. The objective of this research was to study the eect on L. innocua growth of nisin supported in edible lms prepared with suspensions of tapioca starch containing glycerol. 2. Materials and methods 2.1. Film preparation Mixtures of starch, glycerol and water (5.0:2.5:92.5 in weight) or starch, glycerol, nisin and water were prepared. In the case of lms containing nisin, 15 g of water were replaced by a solution of nisin of a concentration such that each milliliter of nal system contained 2000, 3000 or 5000 IU/ml of the antimicrobial. The pH of the systems was adjusted to 4.0 with citric acid solution (50%, w/w). Gelatinization was performed at a constant rate of 1.8 C/min for @30 min. The temperature of gelatinization was @7075 C. Sample nal temperature was @82 C. After gelatinization, vacuum was applied to remove air from the systems before casting. The mixtures were casted over glass plates and dried at 50 C during 2 h. Drying was nished at 25 C over calcium chloride. In the case of systems containing nisin, the antimicrobial was added after gelatinization to preclude aecting nisin activity. Addition was accomplished under agitation to assure system homogeneity. Once formed, lms were peeled from glass plates and conditioned at 25 C, over saturated solution of NaBr (water activity, aW @ 0.575) during 7 days. Nisin content was calculated after lm stabilization taking into account the change of weight of the systems. The nal concentration of the antimicrobial in the lms resulted to be 881, 1322 or 2204 IU/cm2, for an initial content of 2000, 3000 or 5000 IU nisin/ml of each system, respectively.

2.2. Materials used z Starch was provided by Industrias del Ma S.A. (Argentina). Glycerol and citric acid used were of analytical grade (Mallickrodt, Argentina). A stock solution of nisin (1 105 IU/ml) was prepared by dissolving Nisaplin (Aplin & Barret Ltd., Dotset, UK) in sterile distilled water. The pH was adjusted to 2.0 with 0.1 N HCl to ensure high bacteriocin solubility and solution was stored at 20 C. 2.3. Strains and growth conditions L. innocua (CIP 8011, CCMA 29) was grown in 150 ml Tryptic Soy broth enriched with 0.6% (w/v) yeast extract (TSBYE), in a continuously agitated temperature-controlled shaker at 28 C overnight. Three to ve milliliters were inoculated in 150 ml of fresh TSBYE and agitated for 12 h, as previously explained, to obtain the nal desired concentration of cells. 2.4. Agar diusion technique The agar diusion test was used for determining the antimicrobial eect of lms on L. innocua. 25 ml portions of TSYE agar were poured in Petri dishes. An aliquot of the culture was then transferred to the agar (nal count @ 1.5 1010 CFU/ml) obtaining the assay plate. Films with (1322 IU/cm2) or without nisin were then cut in 7 mm diameter disks, brought in contact with agar, maintained at 7 C during 48 h and incubated at 37 C for 48 h. The antimicrobial eect of the lm was determined by observing the existence of inhibition zones at the contact area as well as around disks. 2.5. Barrier to microbial contamination Petri dishes containing TSYE agar with water activity mica Argencontrolled to 0.94 with dextrose (Merck Qu tina, Buenos Aires, Argentina) according to Chirife, Ferro Fontan, and Benmergui (1980) and pH 5.0 (citric acid, 50%, w/w) were used to resemble a food product. Disks of 1 cm diameter were cut from lms with (881; 1322 or 2204 IU/cm2) or without nisin and brought in contact with the surface of the agar. Then, 10 ll of a culture of L. innocua containing 2 109 CFU/ml, were dispensed on the disks. Samples were incubated at 28 C during 4 h and periodically sampled, to test bacterial viability. 2.6. Release to liquid medium and nisin inactivation Tryptic soy broths with dierent pHs (5.5, 6.5 or 7.2) were used for preservative release studies. Three disks of the lms of 1.4 cm diameter, with 1322 IU/cm2 or without nisin (control) were immersed in each glass tube containing 20 ml of broth with L. innocua culture in exponential phase to yield approximately 104 CFU/ml. Systems were incu-

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bated at 28 C with agitation, at 200 rpm in an orbital Vicking shaker (Vicking S.A., Buenos Aires, Argentina) along 24 h. The cultures were sampled (1 ml) over time of incubation to determine the concentration of bacterial cells. Dilution drops (20 ll) of each sample were spotted in duplicate onto TSYE agar. The number of viable cells (CFU/ml) was determined after incubation at 37 C for 48 h. To test the eect of the content of nisin in the lms, the experiment was also performed with lms containing 881, 1322 or 2204 IU/cm2 (Tryptic soy broth, pH: 5.5) for 10 h. Assays performed with lms without the antimicrobial helped to analyse the eect of other components of the lm on results obtained. 2.6.1. Release in the absence of microorganisms The amount of nisin released from the lm (2204 IU nisin/cm2 and pH 5.5) to the liquid medium was tested performing an assay with tubes containing TSYE broth without microorganisms. Three circular disks of 1.4 cm diameter, with or without nisin were introduced in each tube. Tubes containing lms without nisin allowed to evaluate the eect of other components of lm on bacteria growth. The release experiment was performed at 28 C and with an agitation of 200 rpm in an orbital shaker (Vicking, model Shaker Pro, Argentina) for 12 h. The broth was sampled (1 ml) at dierent time periods and samples were left in contact for 30 min at 7 C with a L. innocua pellet (1 107 CFU/ml). Then, they were centrifuged for 20 min at 10,000 rpm (Presvac centrifuge, model EPF-12, Argentina). The pellet was resuspended in peptone water, serially diluted and tested in Petri dishes containing TSYE agar for the assessment of bacteria survival through the spot technique. Plates were incubated for 48 h at 37 C before counting. 2.6.2. Nisin inactivation To study the loss of nisin activity, known quantities of the antimicrobial were dispensed in tubes containing 20 ml of TSYE broth attaining concentrations of 0, 25, 50, 75 or 150 IU/ml. The pH of the systems was adjusted to 5.5 with citric acid solution (50%, w/w). Tubes were incubated at 28 C with agitation (200 rpm) for 9 h and sampled at dierent periods of time. Samples were brought in contact with a pellet of L. innocua (1 107 CFU/ml). After 30 min of contact at 7 C, cells were harvested by centrifugation (10,000 RPM, 20 min). Subsequently, the pellet was suspended, serially diluted in 0.1% peptone water and plated in Petri dishes containing TSYE agar for the assessment of L. innocua survival through the spot technique. Plates were incubated for 48 h at 37 C before counting. 2.7. Data treatment All the experiments were performed in duplicate. Data are reported in the gures as average and standard error of log N, being N the number of colony forming units.

3. Results and discussion 3.1. Diusivity in solid medium The antimicrobial properties of the lms were evaluated by noting whether there was inhibition of bacterial growth at the lm/medium interface, along with the existence of clear inhibitory zones around the lm after incubation. The results of the research revealed that the starch based lm itself could not inhibit the microbial growth at the lm/medium interface. On the contrary, the starch lm containing nisin yielded a low density of growth at the lm/medium interface and a clear but narrow inhibitory zone around the lm disk (total diameter of inhibition: 9 mm for an initial disk diameter of 7 mm), showing antimicrobial diusion to the solid medium. Similar results were observed by Padgett et al. (2000) when studying the eect of nisin contained in corn zein lms. It must be stated that edible lm swelling, which determined lm diameter increase along experimental work might have aected results obtained. The diusion observed is useful for microbial inhibition but it might result in an increase in diusion rate from the surface into the foodstu due to the high concentration gradient, not allowing to maintain a high concentration of the antimicrobial in the surface (Chen et al., 1996). 3.2. Films as barriers to contamination Recent studies have focused on the application of edible lms on the food surface preventing the diusion of preservative into the food and inhibiting surface microbial growth (Franssen et al., 2002; Ozdemir & Floros, 2001; Sebti et al., 2004; Vodjani & Torres, 1989a, 1989b). One of the major potentials of this hurdle lies in the storage of semi-moist foods (Chen et al., 1996). According to this, an experiment was designed with the object of testing the ecacy of studied lms as microbial barriers. Results obtained showed (Fig. 1) a rapid decrease of L. innocua viable counts, followed by an additional slow inac8 7 6

Log N

5 4 3 2 1 0 0 30 60 90 120 150 180 210 240

time (min)

Fig. 1. Tapioca starch lm as barrier to microbial contamination. N: Number of colony forming units; X: control; r: 881 IU /cm2; j: 1322 IU/ cm2; m: 2204 IU/cm2.

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tivation during the period evaluated. The antimicrobial eect increased with lm nisin content, showing a reduction of 4 log cycle during 240 min of contact with a lm with 2204 IU/cm2. On the other hand, lms without the natural antimicrobial did not aect initial counts of studied bacteria. 3.3. Release to liquid medium 3.3.1. Eect of pH Nisin molecule is cationic but its net charge depends on pH (Thomas, Clarkson, & Delves-Broughton, 2000). At neutral pH, the positive charge diminishes due to loss of hystidine protons and at acidic pH the charge is retained due to the high pK of lysine (Rollema, Kuipers, Both, de Vos, & Siezen, 1995). Nisin is, in general, more eective at acidic pHs and has a maximum activity at pH 5.5 (Dykes, Hancock, & Hatings, 1998). According to Ganzle, Weber, and Hammes (1999), the pH mediated changes that occur in the secondary structure of nisin inuence the interaction between nisin and microbial membrane aecting antimicrobial activity. For the purpose of studying nisin performance when supported in the edible starch lms, samples with 1322 IU/cm2 of nisin were immersed in broth media of different pHs. After 6 h of immersion, lms studied swelled as visually determined through the increase in their diameter. This behavior, probably aected starch network organization and nisin release as previously stated by Buonocore et al. (2003b). As can be observed in Fig. 2, L. innocua growth was smaller when nisin was present in the lms. Cell reduction produced by nisin release in the rst 2 h of the assay was similar for pH 6.5 (b) and 7.2 (a) and higher when medium pH was 5.5 (c). From then on, cells contained in the higher pH broths continued their growth while that contained in broth with pH 5.5, only increased slightly their number maintaining this behavior until 6.5 h. After 23 h of assay, counts were similar for all pH assayed. The counts for control tubes containing lms without nisin increased along the assay except for pH 5.5 tubes which showed a delay of 6 hours; the decrease of pH from 7.2 to 5.5 depressed L. innocua growth for times shorter than 6.5 h of assay when nisin was absent. Anyhow, it is clearly observed that lms with nisin were more inhibitory for studied microorganism than lms without the antimicrobial, being nisin active at all pH evaluated. 3.3.2. Eect of dierent nisin concentrations The eect of dierent nisin contents on L. innocua growth at pH 5.5 was studied. As can be observed in Fig. 3, the increase in nisin content produced a decrease in microbial growth. In general, the greatest eect was observed at, approximately, 12 h of contact and then, cells continued their growth with dierent rates, being smallest the one observed in the media with lms containing 2204 IU/cm2 of nisin. This trend seem to indicate a high

(a)

log N

8 6 4 2 0 0 2 4 6 8 10 12 14 16 18 20 22 24

time (h)
16 14 12

(b)

log N

10 8 6 4 2 0 0 2 4 6 8 10 12 14 16 18 20 22 24

time (h)
14 12 10

(c)

Log N

8 6 4 2 0 0 2 4 6 8 10 12 14 16 18 20 22 24

time (h)
Fig. 2. Release assay. Eect of nisin and media pH on L. innocua growth. N: Number of colony forming units. (a) pH 7.2 ; (b) pH 6.5; (c) pH 5.5. X: Control; j: 1322 IU /cm2.

initial release of preservative. This release of preservative could inhibit the microbial growth at the early stage of storage. Chen et al. (1996) observed a rapid release when working with edible lms constituted by methylcellulose and chitosan and containing sorbate or benzoate as antimicrobial and stated that such high diusion rate might in turn reduce the antimicrobial eect of the lm for long term storage. 3.3.3. Nisin liberation in the absence of microorganism The release of nisin from edible lms (2204 IU/cm2) at pH 5.5 was also studied in the absence of microorganisms and compared with nisin behavior described in Fig. 3. Through the study of a control system, it was observed that L. innocua showed adequate viability (Fig. 4). An hour after the beginning of the experience, lms studied produced 2.6 log cycles reduction in microbial population. Samples exposed for longer times showed similar eect

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6 5
7 9 8

753

Log N

3 2 1 0 0 1 2 3 4 5 6 7 8 9 10

Log N

5 4 3 2 1

time (h)

0 0 2 4 6 8 10

time (h)

Fig. 3. Nisin release to pH 5.5 liquid systems from lms containing dierent antimicrobial concentrations. N: Number of colony forming units; X: control; r: 881 IU/cm2; j: 1322 IU/cm2; m: 2204 IU/cm2.

Fig. 5. Response of L. innocua to direct addition of nisin. N: Number of colony forming units; X: Control; s: 25 IU/ml; }: 50 IU/ml; h: 75 IU/ml; n: 150 IU/ml.

9 8 7 6

5 4 3 2 1 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

According to Chung et al. (2001) slow release might not be as eective as direct addition of the antimicrobial when the initial concentration of the microorganism is rather high. However, the expected microbial population in a preserved food must be low. Anyhow, a major advantage of slow release over direct addition is:  Continuous microbial inhibition by slow delivery to food during an extended period fact that can help to reduce cross contamination during food use and storage rather than during preservation.  Reduced destruction of the antimicrobial due to protection provided by lm matrix. Anyhow, it must be remembered that the matrix might form complexes that can inhibit antimicrobial from acting (Kurup, Wan, & Chan, 1995; Ofman, Campos, & Gerschenson, 2004). As can be observed through comparison of Figs. 4 and 5, although lm performance in the rst 12 h of contact was comparable to the direct presence of 50 IU/ml, the eect of gradual delivery of nisin from lm during more than 8 hours produced a more ecient eect over L. innocua growth than direct addition. This fact can help to reduce contamination during storage. 4. Conclusions Presence of nisin in edible lms formulated with tapioca starch and glycerol reduced L. innocua growth, producing count decrease and acting as a barrier to contamination after processing. The study of nisin performance through the agar diusion technique was not satisfactory due to the competence between rate of nisin diusion, diameter increase due to swelling and microbial growth. However, studied bacteria showed a decreased population under the lm with nisin which also produced clear inhibitory zones. The initial release of nisin to the liquid medium produced an important inhibition of L. innocua, specially when

Log N

time (h)

Fig. 4. Nisin liberation to TSYE broth in the absence of microorganisms. N: Number of colony forming units; X: Control; m: 2204 IU/cm2.

on microbial population, a fact that can be attributed to liberation coupled with reduction in nisin activity or to no additional liberation of the antimicrobial. It is interesting to remark that a greater population reduction was observed when liberation was studied in the absence of L. innocua (compare Figs. 3 and 4 for equal nisin concentration). This trend might be attributed to the increase in external resistance to nisin diusion when microorganisms are present during antimicrobial release. 3.3.4. Nisin inactivation To clarify nisin behavior in the conditions of the assay, the eect of direct addition of nisin on microbial growth as a function of time was evaluated. Results show (Fig. 5) a maximum activity (log N = 2.7), for a nisin concentration of 150 IU/ml for times shorter than 1 h. Afterwards, and for all concentrations assayed, it was observed that population tended to increase probably due to nisin inactivation by combination with TSYE broth. As a consequence, we can conclude that nisin released from lms can also suer loss of activity and that the constance in microbial population observed in Fig. 4 when lms with antimicrobial were present might be ascribed to gradual liberation of nisin that compensated inactivation of the antimicrobial.

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K. Sanjurjo et al. / Food Research International 39 (2006) 749754 Dawson, P. L., Hirt, D. E., Rieck, J. R., Acton, J. C., & Sotthibandhu, A. (2003). Nisin release from lms is aected by both protein type and lm-forming method. Food Research International, 36, 959968. Delves-Broughton, J. (1990). Review: nisin and its application as a food preservative. Journal of the Society of Dairy Technology, 43, 7376. Dykes, G. A., Hancock, R. E. W., & Hatings, J. W. (1998). Structural variations in nisin associated with dierent membrane mimicking and pH environments. Biochemical and Biophysical Research Communications, 247, 723727. Food and Drug Administration (1998). Nisin preparation: armation of GRAS status as a direct human food ingredient. FDA, Federal Regulation, 53, 11247. Franssen, L., Rumsey, T., & Krochta, J. M. (2002). Modelling of natamycin and potassium sorbate diusion in whey protein isolate lms to application to cheddar cheese. 2002 Annual Meeting and Food Expo. Anaheim, CA. Session 28. Available from http://ift.confex.com/ ift/2002/techprogram/paper_11523.htm. Ganzle, M. G., Weber, S., & Hammes, W. P. (1999). Eect of ecological factors on the inhibitory spectrum and activity of bacteriocins. International Journal of Food Microbiology, 46, 207217. a, Garc M., Martino, M., & Zaritzky, N. (1998). Starch-based coatings: eect on refrigerated strawberry quality. Journal of the Science of Food and Agriculture, 76, 411420. Hurst, A. (1981). Nisin. Advances in Applied Microbiology, 27, 85123. Hurst, A., & Hoover, D. G. (1993). Nisin. In P. M. Davidson & A. L. Branen (Eds.), Antimicrobials in food (pp. 369394). New York: Marcel Dekker. Ko, S., Janes, M. E., Hettiarachchy, N. S., & Johnson, M. G. (2001). Physical and chemical properties of edible lms containing nisin and their action against Listeria Monocytogenes. Journal of Food Science, 66(7), 10061011. Kurup, T. R. R., Wan, L. S., & Chan, L. W. (1995). Interaction of preservatives with macromolecules. Part II. Cellulose derivatives. Pharmaceutica Acta Helvetiae, 70, 187193. McHugh, T. H., & Krochta, J. M. (1994). Sorbitol vs glycerol-plasticized whey protein edible lms: integrated oxygen permeability and tensile property evaluation. Journal of Agricultural and Food Chemistry, 42(4), 841845. Ofman, M., Campos, C., & Gerschenson, L. (2004). Eect of preservatives on the functional properties of tapioca starch: analysis of interactions. Lebensmittel Wissenschaft und Technologie, 37, 355360. Ozdemir, M., & Floros, J. D. (2001). Analysis and modeling of potassium sorbate diusion through edible whey protein lms. Journal of Food Engineering, 47, 149155. Padgett, T., Han, I., & Dawson, P. L. (2000). Eect of lauric acid addition on the antimicrobial ecacy and water permeability of corn zein lms containing nisin. Journal of Food Processing Preservation, 24, 423432. Rollema, H. S., Kuipers, O. P., Both, P., de Vos, W. M., & Siezen, R. J. (1995). Improvement of solubility and stability of the antimicrobial peptide nisin by protein engineering. Applied Environmental Microbiology, 61, 28732878. Sebti, I., Blanc, D., Carnet-Ripoche, A., Saurel, R., & Coma, V. (2004). Experimental study and modeling of nisin diusion in agarose gel. Journal of Food Engineering, 63, 185190. Thomas, L. V., Clarkson, M. R., & Delves-Broughton, J. (2000). Nisin. In A. S. Naidu (Ed.), Natural food antimicrobial systems (pp. 463524). Boca Raton: CRC Press. Vodjani, F., & Torres, J. A. (1989a). Potassium sorbate permeability of polysaccharide lms: chitosan, methylcellulose and hydroxypropyl methylcellulose. Journal of Food Processing Engineering, 12, 3337. Vodjani, F., & Torres, J. A. (1989b). Potassium sorbate permeability of methylcellulose and hydroxypropyl methylcellulose lms: eect of fatty acids. Journal of Food Processing Engineering, 13, 417421. Zobel, H. F. (1994). Starch granule structure. In R. J. Alexander & H. F. Zobel (Eds.), Developments in carbohydrate chemistry (pp. 136). St. Paul, MN: The American Association of Cereal Chemists.

liquid had a pH 5.5. This inhibition increased with nisin content of the lms. Although the nisin released from lms suered loss of activity, the gradual delivery of nisin still contained in lms controlled growth of L. innocua, helping to reduce contamination along storage better than direct addition. It can be concluded that antimicrobial activity of the lms starch-based and containing nisin describe this hurdle as adequate to act as a barrier to further product contamination. Gradual release can also help to preclude microbial proliferation better than nisin directly present in the media. Additional studies must be performed to determine if starch modication or additional changes in composition (i.e. mixture of starch and lipids) might modify antimicrobial activity and release producing changes in the possibilities brought about by this hurdle. Acknowledgements We acknowledge the nancial support from Agencia Nacional de Promocion Cientca y Tecnologica (ANPCyT), Universidad de Buenos Aires, Consejo Nacional de Investigaciones Cientcas y Tecnicas de la Republica Argentina (CONICET) and Fundacion Antorchas. We also thank Mr. Armando Aguirre (AMG S.R.L., Buenos Aires, Argentina) for the donation of Nisaplin samples. References
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