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theses

Although thymol is a natural antimicrobial with broad-range activities, it is not suitable to be added directly as an ingredient into the food formulation (a delivery mode known as “instant addition”), because effective microbial inhibition requires high concentrations of thymol, while its strong aroma is undesirable to consumers. Meanwhile, high concentrations mean high cost, which discourages food manufacturers from substituting synthetic preservatives with thymol. In order to expand the applications of thymol as a food antimicrobial, an immediate challenge is to reduce the required concentration to below sensory and economical threshold. This challenge may be addressed by exploring novel delivery modes instead of the traditional instant addition.

Controlled release packaging (CRP) is an innovative technology, which releases active compounds from packaging material at targeted rates in a controlled manner over extended storage, in order to enhance the quality and safety of foods. Previously, controlled release of the antimicrobial nisin has been reported to be more effective in inhibiting the growth of Micrococcus luteus than instant addition. It has also been proposed that there’s an optimum range of release profiles, called “target release profile”, which exhibits effective antimicrobial activity using as little antimicrobial as possible.

This study aims to further understand the relationship between release profiles, the resulting concentration profiles and microbial inhibition, and to adopt the unique characteristics of controlled release to extend the potential applications of thymol as a food antimicrobial.
In the first part of the study, liquid-phase thymol was released via different modes of delivery in order to compare their effectiveness, and the microbial growth kinetics were measured to link back to the release kinetics, in order to further unveil the underlying mechanism for the dependence of inhibitory effects on delivery modes.

Since most studies on the antimicrobial activity of liquid-phase thymol were conducted via instant addition, there is a lack of knowledge about how different modes of delivery affect the inhibitory effect of thymol, especially when a combination of two or more delivery modes is used, such as instant addition plus controlled release. The rationale behind using a combined mode of delivery lies within a practical consideration: in practice, due to technical limitations, a single delivery mode may not be able to suffice the requirement of effective inhibition, while a combined mode provides more flexibility. Therefore, the objective of the first part is to compare the inhibitory effects of liquid-phase thymol via three delivery modes (instant addition, controlled release, and combined mode) on the growth of Escherichia coli DH5α, and to find the relationship between the release (and the resulting concentration) profile and the corresponding inhibitory effect, in order to propose basic requirements for an effective release profile and to narrow down the range of the optimum release profile.

The results showed that via controlled release and combined mode, the minimum inhibitory concentrations of thymol were lowered by 25% and 50% respectively, as compared to instant addition. It could be proposed that in order to effectively inhibit microbial growth, an instant inhibitory effect must be achieved in the beginning, followed by sustained release at later time to maintain the effect. Combined mode of delivery in the study was able to satisfy both requirements, making it the most effective. Specifically, a minimum of 123 mg/L thymol must be released during the inherent lag phase (1.14 hours), in order to provide an instant inhibitory effect as proposed. This minimum dosage can be achieved using a combination of instant addition and controlled release. Based on the amount instantly added at time zero, a subsequent release profile can be designed using Crank’s diffusion model. By varying the amount instantly added, various release profiles can be generated, to accommodate the variable conditions in real-life situations and to best guarantee effective inhibition throughout the shelf life of foods.

In the second part of the study, vapor-phase thymol was released via instant addition and controlled release, and the correlation between the actual concentration profile and microbial growth was investigated, followed by linking back to the release profile.

Few previous studies have shown that via instant addition, thymol and thyme oil in vapor phase are effective against bacteria and fungi, utilizing less concentration than those in liquid and solid phase. However, current evidence is still lacking, and more research is needed to substantiate the prominent antimicrobial activity of vapor-phase thymol. Moreover, upon addition, thymol’s vapor concentration is subject to constant change. There is little knowledge of thymol’s actual concentration profile upon its addition, and a lack of understanding of how the profile may affect microbial growth over time. It is plausible to hypothesize that concentration profiles of varying shapes will affect the inhibitory effect differently. Therefore, it is important to verify the antimicrobial activity of vapor-phase thymol, as well as to identify the actual concentration profile and correlate it with the antimicrobial behavior over time.

Furthermore, controlled release of a deliberate design has rarely been exploited in vapor-phase antimicrobials, leaving a knowledge gap concerning the effects of vapor-phase antimicrobials via controlled release. Thus, it is of necessity to test the feasibility of controlled release using vapor-phase thymol. It can be hypothesized that by controlling the release of vapor-phase thymol, it may be possible to generate an effective concentration profile with lower amount used, thereby enhancing the possibility of thymol as a food antimicrobial. Therefore, the objective of the second part is to investigate the inhibitory effect of vapor-phase thymol against Escherichia coli DH5α via instant addition and controlled release.

The results showed that via instant addition, vapor-phase thymol was more effective against Escherichia coli DH5α than liquid- or solid-phase thymol, with a MID of 8 mg/L headspace, as compared to a MIC of 500 mg/L or 320 mg/L agar, respectively. The results also showed that the headspace concentration, although at a much lower level in comparison to the added dose, was the main contributor to thymol’s antimicrobial activity, and a growth phase of 4 hours in the profile when the headspace concentration keeps increasing is necessary for effective inhibition. Controlled release of vapor-phase thymol was able to achieve the same inhibitory effect with only 41% of the amount used compared to instant addition. The underlying reason for its higher effectiveness is that by continuously supplementing the lost portion, controlled release created a concentration profile that satisfied the 4-hour growth phase requirement and thus effectively inhibited microbial growth with lower amount added.

To conclude, this work identified the important role that delivery modes play in affecting the inhibitory effect of an antimicrobial. It further enhanced our understanding of the relationship between release profile, concentration profile, and microbial inhibition. It provided implications for the design of CRP, using the natural antimicrobial thymol as an alternative to synthetic preservatives.

ETD_10429

Ph.D.

Includes bibliographical references

doi:10.7282/t3-3dx8-9b31

ETD doctoral

The author owns the copyright to this work.