Lysozyme and nisin are both antimicrobial proteins effective against gram-positive bacteria. The use of these antimicrobials in combination with chelating agents displays increased effectiveness against gram-negative bacteria. Packaging films with lysozyme or nisin incorporated into the film structure were tested separately for inhibition against Lactobacillus plantarum. Both lysozyme and nisin were used in combination with EDTA in films and were evaluated for inhibition against Escherichia coli. Two packaging film-forming methods were used to incorporate lysozyme or nisin into biodegradable protein films to determine if antimicrobial properties could be induced in the film. Heat-press and casting methods were used to produce films made from soy protein and corn zein. Circular samples were cut from the finished films, which were then placed on a bacterial lawn, incubated, and measured for any zones of inhibition. Both cast and heat-press films with added lysozyme or nisin formed excellent films and exhibited inhibition of bacterial growth. The lysozyme and nisin retained their bacteriocidal properties throughout both the heat-press and cast film-forming processes. The cast films exhibited larger inhibitory zones, as compared to the heat-press films, when the same levels of lysozyme or nisin were incorporated. L. plantarum was inhibited by films containing nisin or lysozyme. The addition of EDTA increased the inhibitory effect of films against E. coli.

The effect of the addition of lauric acid and nisin to corn zein films on the water permeability and inhibition of bacterial growth was examined using two methods. A zone of inhibition test on solid media and a log reduction assay with the film exposed to a bacterial culture (Lactobacillus plantarum) in liquid media (cell count method) for six hours were conducted. the corn zein cast films contained nisin (5.0 mg nisin/g film and lauric acid at 0, 4, and 8% (w/w). the highest level of lauric acid addition (8%) significantly lowered the film water permeability compared to the 4 and 0% levels. the zone of inhibition test on solid media showed no clear inhibitory zones for films containing lauric acid alone, but did produce clear zones for films with nisin with or without lauric acid. Zone size decreased as the level of lauric acid increased or with decreased water permeability. Cell counts in liquid media exposed to film were reduced by the addition of lauric acid alone to the film. the nisin alone and in combination with lauric acid in the film reduced cell numbers in the cell count as well as increased zone size in the zone assay. No additional cell reductions were found when lauric acid and nisin were combined in the films compared to films containing lauric acid alone. Nisin alone was not as effective in reducing cell counts as 8% lauric acid. Log reductions for the nisin only treatment were 2 logs after 1 h and 3 logs after 4 h. However, while the lauric acid only treatment achieved the same ultimate 5.5 log reduction, the 1 and 2 h reductions were 0 and 1 logs, respectively.