Microencapsulation of Natural Antimicrobial Agents to Minimize Loss from Food Packaging Films

The inherent volatility and/or heat sensitivity of many natural antimicrobial (AM) additives can be detrimental to their widespread use in commodity polymer packaging film formulations. In this study, beta-cyclodextrin (β-CD) inclusion complexes with naturally-derived AM agents: thymol, carvacrol, and linalool were prepared using a co-precipitation technique. The complexes were optimised and then characterised by techniques including thermal analysis. They were then incorporated into low-density polyethylene (LDPE) films with AM agents added directly for comparison. The subsequent release of the AM agents into food simulants was studied followed by an investigation of the efficacy of the films in vitro against selected bacteria. The films were later tested on real foods to assess their potential for controlling microbial growth and lipid oxidation. In the initial experiments, conditions for synthesising the β-CD/AM agent complex including solvent composition, temperature, reaction time, and total solvent volume were investigated to optimise the inclusion efficiency (IE) and yield. Electrospray ionization mass spectrometry and gas chromatography were used to confirm the formation and quantify the amount AM agents that were encapsulated, absorbed onto the surface, or remaining in the filtered solvent. The systematic optimisation of the conditions improved both the yield of the complex and the IE of the AM agents. Using a 1:1 mole ratio of the AM agent to β-CD, the optimised parameters resulted in maximum yields of 87, 84 and 86% (w/w) for thymol, carvacrol and linalool respectively with IE’s close to 100% (w/w) for each agent. The kinetics of the thermal decomposition of the optimised β-CD and complexes of the three AM agents were then investigated using thermogravimetric analysis. Under a linear temperature ramp and in the degree of conversion, α, domain: 0.1 ≤ α ≤ 0.8, the major decomposition steps of the β-CD, and complexes with carvacrol, linalool and thymol occurred at ca. 300°C and followed Avrami-Erofeev kinetics with apparent activation energies, Ea, of: 156 ± 6, 107 ± 7, 96 ± 3 and 110 ± 3 kJ mol-1 respectively. Below ca. 300°C there were staged mass losses from each of the complexes that were not observed for the neat β-CD. These were attributed to lower energy binding interactions and accounted for a little over half of the available guest species in the complex in each case. Lower temperature mass losses for β-CD complexes with carvacrol (ca. 140 to 230°C) and linalool (ca. 95 to 150°C) were analysed and found to be adequately fitted by second-order kinetics with apparent Ea values of: 37 ± 1 and 69 ± 6 kJ mol-1 respectively. The results suggest the optimized complexes are generally thermally stable and would potentially be suitable for high-temperature extrusion processes with acceptably low losses. The next experiments involved the incorporation of the AM agents into LDPE films either directly or encapsulated in β-CD. Quantification of the AM agents was performed immediately following thermal processing, then six and thirty days after the film samples were stored in an open atmosphere. After six days, no AM agent was detected in the films where the agent was added directly to the film whereas the films containing encapsulated agents showed only small decreases in the concentrations of the agents up to 30 days. The migration of AM agents from LDPE films into 95% (v/v) ethanol/water mixtures food simulants at 4℃ was adequately described using first-order kinetics and Fick’s second law of diffusion. For the AM agents added directly to the film, the initial release rates were between four and eight times greater than those of the encapsulated agents. Similarly, the diffusion coefficients of the free agents were ca. four to five times greater than the encapsulated agents. The free and encapsulated natural AM agents incorporated into LDPE film were tested against Escherichia coli (ATCC 25922) in order to assess the potential of the AM inclusion complexes for use in food packaging films. The direct incorporation of the complexes in the film formulations resulted in little inhibition of the target bacterium as assessed by the agar diffusion method even with AM levels as high as 5% (w/w). In comparison, levels of 2% (w/w) of free thymol and carvacrol added directly to the film demonstrated inhibition. The addition of glycerol to the film formulations was investigated as a means of facilitating the AM agent from the complex. A concentration of 1% (w/w) of glycerol in the film formulation was found to result in microbial inhibition which increased with additional glycerol. The use of 2% (w/w) glycerol resulted in a more pronounced inhibition of targeted microorganism. Upon the addition of glycerol, all of the films showed AM activity against the target bacterium with the exception of those containing linalool in either the free or encapsulated forms. Upon the addition of 2% (w/w) of glycerol to the film formulation, encapsulated thymol at a concentration of 2% (w/w) was more effective than encapsulated carvacrol at a concentration of 3% (w/w) against E. coli with zones of inhibition of 30.70 ± 0.72 and 29.61 ± 0.86 mm respectively. In the final experiments, the LDPE films containing encapsulated thymol were tested on real food systems. The level of thymol was 1 to 3% (w/w) relative to the LDPE and glycerol was added in order to obtain the optimum controlled release. In the case of packaged minced beef inoculated with E. coli, no inhibition was observed when the concentration of encapsulated thymol was 3% (w/w) with 2% (w/w) glycerol, however, the same film reduced E. coli growth by 0.7 log10 CFU g-1 on chicken breast fillets compared with the control during storage at 4℃ for 12 days. The growth of E. coli was found to be affected by the temperature at 4℃ whereby the bacterial counts remained relatively low with slow growth over the test period under refrigeration. However, when the temperature increased to 10℃ it was also found that the presence of coliforms interfered with growth of E. coli and, in general, the films containing encapsulated thymol effectively reduced coliform growth. Analysis of the antioxidant (AO) activity of films using the diphenyl-picrylhydrazyl (DPPH) radical assay showed a 71% reduction in DPPH concentration for the LDPE/thymol/β-CD films containing 3% (w/w) thymol. Furthermore, a film comprised of 1% (w/w) thymol with glycerol stored at room temperature for 20 months showed a reduction by 23% in DPPH concentration confirming that the films are suitable for extended storage. Analysis of the formation of thiobarbituric acid reactive substances on packaged minced beef showed decreases in lipid oxidation of 60 and 75% for films containing 1% and 3% (w/w) thymol in the film respectively. The films therefore show promise for the dual purpose of AO and AM activity in order to prolong the shelf-life of selected food products.