Conclusions

5.1.1 Effect of Film Processing Method and Additives

Several series of LDPE films containing thymol or carvacrol were successfully prepared using both compression moulding and extrusion film blowing. A laboratory-scale compression moulding press was used to prepare AM films to investigate the following effects: AM agent concentration; compounding an additive polymer (PEG and/or EVA) with LDPE; and mixtures of AM agents in the films. Antimicrobial films prepared by pilot-scale extrusion film blowing were used for further evaluation of AM activity and films characterisation. The retention of AM agents in the extruded films was significantly higher than the retention of thymol and carvacrol in the moulded films.

The addition of EVA in the LDPE matrix showed a significant effect on the retention of AM agents during thermal processing. Moreover, with the increasing concentration of EVA in the formulation, the retention of AM agents increased with a more pronounced effect on the retention of thymol than that of carvacrol. In the absence of EVA, PEG did not improve the retention of the AM agents in the polymer matrix. The results of AM activity for films containing various combinations of EVA and PEG, however, suggest

that PEG acts in binding the AM agent resulting in slowing the release of AM agent in the short-term.

5.1.2 AM Activity In Vitro

Optimum AM Concentration

Compression moulded films containing thymol and carvacrol at varying concentrations showed AM activity against bacteria via an agar disc diffusion assay. The relationship between the AM activity and the concentration in the films using three principal phases were obtained using a modified Gompertz function. The concentrations between the minimum inhibitory and maximum effective concentrations were identified as the optimum concentrations for AM activity. According to the inhibitory concentrations, Gram positive bacteria were found to be more sensitive to the AM activity of films containing thymol or carvacrol in comparison with Gram negative bacteria. Films containing thymol showed considerably higher antibacterial activity on agar media than did carvacrol. Accurate determination of the optimum AM agent concentration can enable a balance between the sensory acceptability, safety and AM efficacy.

AM Activity of Extruded Films

Inhibition of bacterial growth on solid media by AM films was confined to the area underneath the film. This suggests the need to have direct contact between the food surface and the AM films for effective control of microorganisms. The effect of AM films on the bacterial inhibition in liquid media was successfully modelled by both the Gompertz and the Baranyi models. Significantly different quantitative changes in growth profiles by AM films compared to that of the control film suggests three main effects of AM films on bacterial growth in liquid media: extension in lag period; reduction in growth rate; and/or reduction in the maximum population density. These effects, however, depend on the type of bacteria, initial bacterial cell concentration, type of AM agent and the concentration of AM agent. The thymol films inhibited bacterial growth more effectively than the carvacrol films. The difference between the AM films was most pronounced at higher AM concentrations and lower inoculum level of bacteria. Although the results on AM activity from in vitro studies are highly dependent

on the experimental conditions, their application allows a broad comparison of different film preparations. Ideally, all films should be subjected to a standard protocol involving a non-food medium to reduce the complexity of the assays.

5.1.3 Effect of AM Combinations

According to the conventional models, FIC and effect additivity, the films containing various combinations of thymol and carvacrol show mainly an antagonistic effect on inhibition of bacteria on agar media. Interaction effects of films containing AM combinations moved towards a positive effect with an increasing concentration of thymol in the combination and with increasing the incubation time. Interpretation of these results shows that it is difficult to anticipate the effects or to explain observed activity when considering binary mixtures of the natural antimicrobials used in this study. Furthermore, the conclusions on interaction effects may vary depending on the model used to interpret the results. Although the conventional models may be useful for initial screening, more robust and informative models may be required to understand the interaction effects of AM combinations in films.

5.1.4 Film Characterisation and Migration of AM agents

Tensile and Thermal Properties

Incorporation of thymol or carvacrol into the LDPE/EVA films did not significantly change either the MD or TD tensile properties of the film. In addition, there was no significant difference in the thermal properties of the AM films in comparison with the control film.

Migration of AM Agents from Films to Atmosphere

The retention of AM agent in the extruded films was examined under three different storage conditions: exposed to air at room temperature; covered in foil and stored at room temperature; and covered in foil and stored in a refrigerator. During short-term storage, a significantly higher retention of AM agents was obtained when the films were

covered in foil and covered in foil and refrigerated compared to that of open air storage. The AM retention, however, did not significantly increase with the reduced temperature of refrigeration during storage when the films were covered in foil. The effect of foil on the retention of carvacrol was higher than that of thymol. Under long-term storage, however, films covered in foil and refrigerated had a significantly higher retention of AM agents than the films covered in foil and stored at room temperature.

The release of the AM agents from film to atmosphere can be satisfactorily described by diffusion and first-order kinetics equations. From the kinetics analyses, it was revealed that the release of AM agents to atmosphere occurs very rapidly with the release of carvacrol from AM films being faster than that of thymol. Moreover, films containing AM agents at higher initial concentrations released the agent at a faster rate than films containing a lower initial concentration. Thus, carvacrol with higher volatility and at higher initial concentrations can be expected to volatilize rapidly into the package headspace and reach the food surface more rapidly than thymol.

Release of AM Agents into food simulants

The release of the AM agents from films into food simulants can be adequately and consistently described by short-term and long-term migration equations. Moreover, adequate fits to first-order kinetics were obtained. It is suggested that the release of AM agents from films to simulants is more complex and needs more detailed approaches towards the analysis. It can be inferred from the results that the release of the AM agents into isooctane was consistently high and was followed by release into 95% (v/v) ethanol/water whereas the release into 10% (v/v) ethanol/water was considerably slower. The release of carvacrol was found to be higher than that of thymol at any given condition. Moreover, an increase in the rate of release of AM agent with an increase in temperature was observed. The temperature dependency of the kinetic parameters was well described by the Arrhenius equation. A higher energy barrier for the release of AM agents was observed in aqueous solvents. Diffusion of AM agents from films containing thymol and carvacrol can also be modeled successfully using an extension of the model derived for the case of an idealized system involving an infinite "sheet" of polymer immersed in an infinite volume of food simulant. The extension technique involves

shifting the boundary condition separating the short-term and long-term diffusion data to 0.5 instead of the usual value of 0.6. Such a shift in the boundary condition can be theoretically made with little consequence to the numerical accuracy of any subsequent diffusion analysis and in some cases can improve the fit of the data.

5.1.5 Application of AM Films on Cheddar Cheese

Challenge Test

In the challenge test, the inactivation of E. coli and S. aureus inoculated on Cheddar cheese by AM films were successfully modeled by the first-order biphasic model. Compared to the control films, AM films showed a significant inhibitory action against both bacteria depending on the type of AM agent and its concentration. The inactivation kinetics nevertheless suggests that the biphasic nature of inactivation is a cause of effective AM concentration that changes over the period of microbial exposure. Furthermore, the challenge tests were found to be much more suitable than the in vitro

experiments in the prediction of the AM activity of films for food applications.

Effect on Quality of Cheddar Cheese

The AM films had an effect on the microbial and physio-chemical attributes of Cheddar cheese under actual storage conditions. The AM films had both positive and negative effects on the microbiological quality by delaying the onset of YM growth, reducing the TAB counts and affecting the functional bacteria of Cheddar cheese. In the sensory studies, the presence of thymol was more readily perceived by the panelists than that of carvacrol and the panellists had the ability to identify the odour of AM agents at very low concentrations. Sensory analysis based on a triangle test was able to differentiate between the cheeses packaged in AM films containing higher concentrations of AM agents from those packaged in control films. The addition of higher concentrations of AM agents clearly imparted a noticeable odour to Cheddar cheese during storage. Significant differences in the odour attributes ‘Cheddary’ and ‘spicy’ were perceived on cheeses packaged in different AM films. Thymol had a more pronounced effect than carvacrol on the odour of Cheddar cheese. The acceptance of cheeses packaged in

control film was significantly higher than the cheeses packaged in AM films during the first week. However, after a few weeks of storage, the difference for acceptance became insignificant. Moreover, the intensity of acceptance scores revealed that the spicy odour detected on Cheddar cheeses packaged in AM films was not necessarily perceived as off-odour.

Effect on Shelf life of Cheddar Cheese

In the present study, among all of the quality parameters that may change during the storage of Cheddar cheese, the microbial and sensorial qualities in particular were considered the most representative in building the shelf-life model. The microbial quality of Cheddar cheese was affected by the type of AM agent and was proportional to the concentration of AM agent in the films. A better microbial quality was observed for cheeses packaged in thymol films. Regardless of the higher negative effect on functional bacteria, films with higher AM concentrations had a more pronounced effect on microbiological quality. The sensory quality progressively decreased with an increasing concentration of AM agent in the films while thymol films had a more negative effect. The shelf-life extension of Cheddar cheese by AM films was affected by the type and concentration of the AM agent. However, the overall quality did not necessarily increase with an increasing concentration of AM agent in the films. According to these results, the optimum AM concentration in films should be established with reference to the quality, safety and functionality of the product in its application.