Control of microbial population using of nanocomposite film in vitro and in vivo tests

Active and intelligent packaging devices and systems represent emerging technologies that may have profound implications on the quality, safety and integrity of packaged food and beverage products. It is well known that the photocatalyst titanium dioxide (TiO2), being a wide band gap (3.2 eV) semiconductor under UV illumination, generates energy-rich electron-hole pairs that can be transferred to the surface of TiO2, and promotes reactivity with the surface-absorbed molecules leading to the production of active radicals. These active radicals oxidize C-H bonds resulting in degradation of the organic molecules TiO2 photocatalyst has been used to degrade organic pollutants and inactivate a wide spectrum of microorganisms. To the best of our knowledge, there is no report on the photocatalytic disinfection properties of low density polyethylene (LDPE)-TiO2 nanocomposite produced by extrusion. Therefore, in this study LDPE-TiO2 nanocomposite film was prepared by a film blowing machine. The antimicrobial activity of the new packaging film against Pseudomonas spp. and Rhodotorula mucilaginosa, representing the main microorganisms on fruit and vegetable crops, was examined in vitro under UVA light. The antimicrobial property of the developed active film was also tested in a food application
LDPE -TiO2 nanocomposite was prepared by the melt blending method. Modified TiO2 (M-TiO2) powder obtained by mixing modified anatase and rutile phases in a weight ratio of 7:3 (total of 30 g), LDPE granules (935 g), PE-MA (30 g) and glycerol (5 g) were blended for 1 h using a mixer. The mixture was extruded by a Brabender twin-screw compounder (model DSE 20, Germany) for incorporating nanoparticles into the LDPE matrix. For LDPE and its nanocompounds, a constant temperature of 130 °C was used in all the zones of the extruder and the speed of the central screw was set to 120 rpm. The extrudate was cooled down in air at 23 ± 3 °C and pelletized. A composite LDPE-TiO2 film with a thickness of 30 ± 3 µm was finally obtained by a film-blowing machine. The resulting film had a milky whitish appearance.
In vitro and in vivo antimicrobial activity test of film
Each test film (6 cm diameter) was placed in sterilized petri dishes under aseptic conditions. One mL of each microorganism stock solution (containing approximately 108 and 107 CFU/mL for Pseudomonas spp. and R. mucilaginosa, respectively) was pipetted onto each test piece in its petri dish. Test samples were placed at a distance of 25 cm from six 8-WUVA black light bulbs Samples were taken in three replicates at 60 min intervals for 3 h. Then, 9 mL of sterile saline solution was added to the petri dishes containing the test and polyethylene films and shaken for 180 s on a universal small shaker (IKA MS 3 digital, Germany). One milliliter of solution was withdrawn at each sampling event and diluted to 1/10, 1/100, 1/1000, and 1/10,000 with sterile saline solution. A volume of 0.1 mL of the undiluted and diluted solutions was plated over appropriate media. Pseudomonas agar base (PAB) was incubated at 25 °C for 48 h for Pseudomonas spp. and Sabouraud dextrose agar for R. mucilaginosa and the colony-forming units (CFU) were counted. For in vivo test, every 6 days, approximately 20 g of packed apricot pieces with nanocompsite films, were collected randomly and placed into 180 mL of saline solution and agitated in a stomacher bag for 120 s. Decimal dilutions were made in sterile saline solution and 0.1 mL of the undiluted and diluted solutions were plated.
This work describes a TiO2 nanocomposite thin film with biocidal capacity for food packaging that was prepared by the extrusion method. The film caused inactivation of Pseudomonas spp., R. mucilaginosa and mesophilic bacteria in saline solution and on apricot when exposed to UVA light. The number of microorganisms on LDPE-TiO2 nanocomposite film plus UVA light was lower than that on LDPE-TiO2 nanocomposite film without UVA light and LDPE film exposed to UVA light. These results suggest that the TiO2 nanoparticles were responsible for the antimicrobial effect when exposed to UVA light illumination. The higher antimicrobial activity of the composite films under UV light is due to the photocatalytic reaction of the TiO2 nanoparticles in the matrix. Thus, the prepared TiO2 nanocomposite films are effective in diminishing live microorganisms and are promising as antimicrobial packages.