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With increasing world population, the demand for fish is growing thus there is a need to identify products with potential to increase the efficiency of fish production. Phytogenics are among the products being investigated as potential naturally derived growth promoters. The aim of this study was to identify phytogenic compounds and doses with growth-promoting effects in Nile tilapia and investigate relevant pathways underlying their growth promotion effects. The phytogenic compounds limonene, carvacrol and thymol, major constituents of essential oils from the plants citrus, oregano and thyme, respectively, were evaluated. Six Trials (Trials I, II, III, IV, V and VI) were carried out using diets supplemented with varying concentrations of the phytogenic compounds. In Trials I, II and III (Chapter 3), the effects of either limonene (Trial I), carvacrol (Trial II) or thymol (Trial III) on growth performance of Nile tilapia were investigated (objective 1) and performance parameters including final fish weight, daily growth coefficient, growth rate per metabolic body weight, percentage (%) weight gain, % survival, feed intake, feed conversion ratio and protein efficiency ratio were evaluated. Results from Trials I, II and III indicated that dietary supplementation of 400 and 500 ppm limonene and 750 ppm thymol had growth-promoting effects in Nile tilapia but the somatic growth was not associated with enhanced feed intake and feed utilisation efficiency. Trials IV and V (Chapter 4) investigated growth and nutritional physiology pathways in Nile tilapia regulated by individual phytogenic compounds (objective 2). This was accomplished by analysing the effects of limonene (Trial IV) and thymol (Trial V) supplemented diets on the expression of key genes participating in selected pathways of somatotropic axis-mediated growth, appetite regulation, nutrient digestion, absorption and transport, lipid metabolism, and antioxidant enzyme defence system. Limonene was supplemented in the diet at 0, 200, 400 and 600 ppm while thymol was supplemented at 0, 250 and 500 ppm. Trials IV and V found that growth-promoting effects of limonene (400 and 600 ppm) in Nile tilapia involved up-regulation of key genes within pathways including somatotropic axis-mediated growth, nutrient digestion, absorption and transport, lipid metabolism and antioxidant enzyme defence system. Dietary thymol at 250 and 500 ppm did not significantly enhance growth of Nile tilapia nor regulate the nutritional physiology pathways listed above. In Trial VI (Chapter 5), the effects of combined phytogenic compounds (limonene and thymol) on growth and nutritional physiology of Nile tilapia was tested (objective 3) to establish if the compounds had synergistic or additive effects on the growth of the fish as well as complementary effects on the selected nutritional physiology pathways. A candidate gene approach was also used for the selected pathways. Results from Trial VI showed that a diet supplemented with a combination of limonene (400 ppm) and thymol (500 ppm) has neither synergistic nor additive effects on the growth performance of Nile tilapia, with limonene mainly influencing the attained somatic growth. The analysed candidate genes involved in the pathways of nutrient digestion, absorption and transport, lipid metabolism, antioxidant enzymes and somatotropic axis growth also showed no synergistic or additive effects of a dietary combination of limonene and thymol in Nile tilapia. Overall, results from the study suggest approaches for developing functional diets for Nile tilapia using limonene and thymol growth promoters.

Little is known about the pharmacological activity of Monarda fistulosa L. essential oils. To address this issue, we isolated essential oils from the flowers and leaves of M. fistulosa and analyzed their chemical composition. We also analyzed the pharmacological effects of M. fistulosa essential oils on transient receptor potential (TRP) channel activity, as these channels are known targets of various essential oil constituents. Flower (MEOFl) and leaf (MEOLv) essential oils were comprised mainly of monoterpenes (43.1% and 21.1%) and oxygenated monoterpenes (54.8% and 77.7%), respectively, with a high abundance of monoterpene hydrocarbons, including p-cymene, γ-terpinene, α-terpinene, and α-thujene. Major oxygenated monoterpenes of MEOFl and MEOLv included carvacrol and thymol. Both MEOFl and MEOLv stimulated a transient increase in intracellular free Ca2+ concentration ([Ca2+]i) in TRPA1 but not in TRPV1 or TRPV4-transfected cells, with MEOLv being much more effective than MEOFl. Furthermore, the pure monoterpenes carvacrol, thymol, and β-myrcene activated TRPA1 but not the TRPV1 or TRPV4 channels, suggesting that these compounds represented the TRPA1-activating components of M. fistulosa essential oils. The transient increase in [Ca2+]i induced by MEOFl/MEOLv, carvacrol, β-myrcene, and thymol in TRPA1-transfected cells was blocked by a selective TRPA1 antagonist, HC-030031. Although carvacrol and thymol have been reported previously to activate the TRPA1 channels, this is the first report to show that β-myrcene is also a TRPA1 channel agonist. Finally, molecular modeling studies showed a substantial similarity between the docking poses of carvacrol, thymol, and β-myrcene in the binding site of human TRPA1. Thus, our results provide a cellular and molecular basis to explain at least part of the therapeutic properties of these essential oils, laying the foundation for prospective pharmacological studies involving TRP ion channels.

Spring wheat (Triticum aestivum L.) and oats (Avena sativa L.) are important crops due to their high consumption in our daily diet worldwide. They are the main food of consumption per capita due to the high amino-acid content as well of vitamins. However, grain production is limited due to the negative impact of the diseases that cause significant yield loss. Diseases in oats, such as crown rust caused by Puccinia coronata f. sp. avenae (Pca), and in wheat such as Fusarium head blight (FHB) caused by the fungus Fusarium graminearum. Foliar diseases such as tan spot incited by the fungi (Pyrenophora tritici-repentis), Septoria nodorum blotch (Parastagonospora nodorum), and spot blotch (Bipolaris Sorokiniana) are known to compromise grain yield and seed quality. The integrated pest management for these diseases is by using tolerant/resistant plant varieties, crop rotation, and mostly fungicide applications due to their high efficacy controlling fungal diseases. However, the overuse of synthetic fungicides has led to the development of pathogen resistance, and concerns in the environment and human health. Therefore, it is necessary to explore new products with lower toxicity and less negative impact on human health and the environment. Biopesticides such as essential oils (EOs) and pure bioactive compounds present in the EOs are substances with known antifungal properties, multiple mechanisms of action, and low toxicity in humans and environment. However, little is known about their effect on the fungal pathogens that affect small grains. The objectives of this study were: (1) To determine the potential EOs in the control of oat crown rust in vivo, (2) To determine the potential effect of EOs and bioactive compounds in the control of three important pathogens that cause foliar diseases in wheat in vitro, and (3) To determine in vitro and in vivo the potential as fungicide of two EOs, two commercial products with EOs, and two bioactive compounds presents in EOs, in the control of the fungal pathogen Fusarium graminearum. For the first objective, a two-year field study experiment, was conducted testing two EOs, neem and karanja, at two concentrations (52 and 72 %) and three rates (3.7, 7.5, 11.25 L/ha). The synthetic commercial fungicide Headline (Pyraclostrobin) and a nontreated (water) were used as checks. Applications of the above treatments were made at the flag leaf stage (Zadok’s- 37) in two oats cultivars, Horsepower and Goliath. Percentage of disease incidence (%DI), stem lodging percentage, normalized difference vegetation index (NDVI), test weight (TW), and yield were assessed for each year. In both years, the two essential oils showed a higher yield and a reduced %DI at the highest concentration and rate compared with the untreated check. The synthetic fungicide, Headline, showed a significantly higher yield (P

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.

The use of materials based on poly(lactic acid) (PLA) as alternatives to petroleum-based polymers for a range of applications has increased in recent years. In the case of food packaging in particular, PLA has experienced growth in combination with the use of a wide range of other materials and/or additives derived from natural and renewable resources. However, the initial costs associated with new technologies to produce PLA and/or other bio-based polymers are typically high, so new techniques are required to reduce costs without compromising material properties and biodegradability. Naturally sourced lignocellulose fibres such as kenaf (Hibiscus cannabinus L.) are often used as cost-reducing fillers and/or reinforcing agent for biopolymers such as PLA. This study explores the characteristics and antimicrobial (AM) activity of PLA and PLA/kenaf composites incorporated with thymol, a natural bio-active AM substance/agent. The production of PLA/kenaf composites containing thymol is intended for potential use in AM packaging applications such as rigid and/or flexible packaging and coatings. Composites of PLA incorporated with 5 to 40% w/w kenaf fibre loadings and thymol concentrations ranging from 5 to 30% w/w were prepared and compared with control systems containing either no kenaf or no thymol. Kenaf fibres can be treated by alkalisation to improve compatibility with polymer matrices. It was found that the PLA/kenaf composites containing treated kenaf possessed significantly higher tensile strength and stiffness than composites prepared with untreated kenaf. Micrograph images of the fracture surface revealed better adhesion between the treated kenaf fibres and the matrix, thereby resulting in improved reinforcement of the composite. Moreover, PLA/kenaf composites containing thymol exhibited lower tensile strength than those without thymol, suggesting a possible plasticizing effect in the presence of the additive.

In recent years, there have been a number of studies looking at inhibition of microorganisms by spices, herbs or their extracts.  Many of these products have been shown to have antimicrobial activity against foodborne pathogens.  The purpose of this research was to evaluate the antimicrobial activity of three essential oil (EO) compounds (thymol, eugenol, and trans-cinnamaldehyde) alone and in combination with three naturally-occurring apple aroma (AA) compounds (hexanal, trans-2-hexenal and 1-hexanol) to identify the minimum inhibitory concentrations necessary to inhibit E. coli O157:H7.  Three commercial apple juice flavoring mixtures (natural apple cinnamon, natural apple spice and natural red apple) were additionally tested alone for antimicrobial activity against E. coli O157:H7. The standard agar dilution method (SAD) and checkerboard assay were used to evaluate the efficacy of the nine compounds, alone and in combination against E. coli O157:H7.  In general, the EO compounds were significantly more effective against E. coli O157:H7 than the AA compounds (P