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    Measurement of minimum inhibitory concentration (MIC) of individual and combinations of essential oil volatiles in food : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
    (Massey University, 2015) Abdul Rahman, Mazidah
    The use of essential oil volatiles as natural food preservatives has received significant attention in recent years. Shelf life extension can be achieved through the appropriate design of active packaging systems that release volatiles into the product headspace at controlled rates. In some applications, the volatile concentrations required to delay or prevent spoilage can cause sensory changes to the product. The use of multiple volatiles and the potential for synergistic effects offer opportunities to minimise sensory effects in the pursuit of shelf life extension. The design of these systems requires a good knowledge of the target headspace volatile concentrations required to inhibit growth. The aim of this research was to analyse the methods for measurement of minimum inhibitory concentration (MIC) for individual and mixtures of essential oil volatiles in food systems. Carvacrol and thymol, the predominant phenolic constituents of Origanum vulgare and Thymus vulgaris were selected as example active agents. Both are known to have strong antimicrobial activity. The accuracy of the techniques normally applied to measure MICs against targeted microorganisms in the headspace is questionable. Volatile compounds released into the test headspace are absorbed by media and dish materials and lost into the environment. As such the MIC data collected are difficult to interpret and can even exceed saturated vapour pressures of the volatile compounds. To demonstrate this, the Petri dish reversed method headspace dynamics was characterised during standard MIC tests. Factors influencing the sorption of volatiles by the culture media (Potato Dextrose Agar) and other parts of the system were investigated. The concentrations of antimicrobial compounds in the headspace were quantified using gas chromatography-mass spectrometry (GC-MS). The study showed that very low concentrations were found during MIC measurement and that the concentrations changed dynamically during the incubation period. The results demonstrated that absorption of vapour by the Potato Dextrose Agar (PDA) strongly influenced the headspace dynamics and was the main reason for the low volatile concentrations in the headspace. The partitioning of carvacrol and thymol (KA/W values 5.94 x 10-5 and 2.58 x 10-4 respectively) strongly favour the solid phase, providing a basis for the design of a new method to enable better MIC measurement. A new method based on pre-mixing the volatile compound into the liquid media was developed. Testing showed that headspace volatile concentrations quickly stabilised and remained constant throughout the incubation period, making MIC determination easier. The potential of each compound and their binary combinations to inhibit growth were evaluated using the new MIC measurement method. This resulted in very repeatable results with much lower headspace concentrations than measured using traditional methods. To test for synergistic effects in the multiple volatile trials, an alternative data analysis approach was adopted. The inhibition time before growth observed in each sample was linearized and regressed against the thymol and carvacrol concentrations. This resulted in a simple model with a significant thymol/carvacrol interaction term, clearly demonstrating a synergistic, although minor effect. The study showed the measurement of stable and repeatable MIC values for individual and combinations of volatiles is possible using the new method. These findings strengthen the possibility of using natural essential oils as alternatives to chemicals to preserve food products. The key disadvantage of the new method is the requirement to mix the liquid essential oils directly into the liquid media before solidification. This prevents its application to solid food systems. For solid food systems, a system capable of delivering stable flows of air with volatiles at high concentrations in the presence of high relative humidity was designed. With this system, well-controlled and stable air compositions were achieved over two days, making the system suitable for measurement of the inhibitory effects on spoilage organism growth. Although further optimisation of the design and control of this system is required, it has the potential for collection of accurate target headspace conditions for controlled volatile release active packaging design.
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    Effects of high pressure processing and ethyl lauroyl arginate on the shelf-life of ready-to-eat chicken breast roast : a thesis submitted in partial fulfillment of the requirements for the degree of Master of Food Technology, Massey University, Albany, New Zealand
    (Massey University, 2011) Seemeen, Sadia; Seemeen, Sadia
    High pressure processing (HPP) is becoming increasingly popular in commercial food processing as it offers great potential within the food industry. The popularity of the technology is driven by the need to provide minimally processed foods which are safe, wholesome and have extended shelf-life that challenge traditional methods of food processing. High pressures of upto 900 MPa can be used to kill or inhibit microorganisms without changing the nutritional and sensory properties of the food. However, the inherent high resistances of bacterial endospores and food enzymes are the major challenges for the broader application of HPP. Therefore, a hurdle approach is almost axiomatic for significant widespread use of HPP in commercial food processing. Therefore, several antimicrobial compounds have been used in conjunction with HPP in a hurdle approach to improve the overall quality of the products. Ethyl lauroyl arginate (LAE) has not been investigated in combination with HPP. LAE is a novel antimicrobial compound derivative of lauric acid, L-arginine and ethanol, all of which are naturally occurring substances. LAE can extend the shelf-life of products due to its antimicrobial action on spoilage microorganisms during refrigerated storage. Therefore, the objective of this study was to investigate the effects of HPP and LAE on the shelf-life of ready-to-eat (RTE) cooked chicken breast roast during storage at 4°C for 16 weeks. The RTE cooked chicken breast roast was prepared using portions (samples) of freshly marinated chicken breasts, which were cooked to an internal temperature of 75°C for 5 minutes, and then cooled (4°C), sliced (60 mm) and vacuum-packaged. The study was conducted in two phases, each carried out for 16 weeks. The first phase comprised of fourteen unique treatments which were screened by microbial and instrumental analysis. Based on the results of the first phase, five treatments were selected for further work. Similar tests were carried on these treatments, in addition to sensory evaluation. The effects of HPP at 450 MPa and 600 MPa pressures at 1 min, 3.5 min and 5 min hold times respectively, on the shelf-life of RTE sliced chicken breast roast were studied for 16 weeks during storage at 4°C. HPP in combination with LAE (200 ppm) was also investigated using similar treatment pressures, hold times and storage conditions. The effects of LAE (200 ppm & 315 ppm) alone on the shelf life of RTE sliced chicken breast roast was studied for 16 weeks when stored at 4°C. RTE sliced chicken breast roast samples without any preservative and/or HPP treatment served as the controls. Aerobic plate counts (APCs), lactic acid bacteria (LAB) and yeasts and moulds (Y&M) were analyzed in five samples from each of the treatments at regular intervals for upto 16 weeks. Instrumental analyses of color and texture were also conducted on the samples to determine any significant changes during storage at 4°C. Five sample treatments were selected after screening and evaluated by consumer sensory analysis using a 9-point hedonic scale. Analyses for APCs, LAB, Y&M, color and texture were also conducted on the selected samples during refrigerated storage. Survival analysis methodology was used to estimate the consumer sensory shelf-life of the selected treatments at 25% and 50% rejection probability. The results showed the potential of using HPP to extend the microbiological and consumer sensory quality of the products. Samples treated with HPP alone, and HPP in combination with LAE (200 ppm) at 600 MPa inhibited the growth of APCs for 16 weeks when stored at 4°C. However, there was no significant (P>0.05) difference in the microbial shelf-life of samples treated with 200 ppm or 315 ppm LAE. No significant (P>0.05) changes in color and texture were detected in all the treatments. Further, no LAB or Y&M were detected in all the sample treatments for the entire storage period at 4°C. Samples treated with HP at 600 MPa for 1 min and 5 min, HPP+LAE (200 ppm) at 600 MPa for 1 min, LAE at 200 and 315 ppm were evaluated by a consumer sensory panel at different storage times. The results of the consumer sensory analysis showed no changes in color, texture, flavour and freshness of the HP-treated and HPP+LAE (200 ppm)-treated samples. LAE-treated (200 and 315 ppm) samples were not acceptable by a consumer panel at week 12. A maximum sensory shelf-life of >16 weeks at 50% and 13.8 weeks at 25% rejection probability was obtained for samples treated with HPP at 600 MPa for 1 min. Therefore, samples treated at 600 MPa for 1 min had stable sensory properties and were well-accepted by a consumer panel. Also, the samples had good microbiological quality.
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    A novel approach for controlling foodborne pathogens using modified atmosphere and Lactobacillus reuteri DPC16 : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Albany, New Zealand
    (Massey University, 2007) Lu, Guangjin
    The current trend of increasing demand for minimally processed food requires more effective preservation technologies than are presently used. In this study, an investigation has been made into a novel strategy to control some common foodborne pathogens, and therefore, to provide an alternative means for enhancing the safety and extending the shelf lives of food products. Modified atmosphere is able to extend the shelf life of seafood and meat products. In this study, a simulated controlled atmosphere (CA) broth system was used to investigate the potential of a modified atmosphere rich in CO2 at a concentration of 40%, supplemented with N2, to control common foodborne pathogens, such as Listeria monocytogenes, Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, Staphylococcus aureus and Vibrio parahaemolyticus. Controlled atmosphere significantly reduced the exponential growth rates of all tested pathogens, while the effects on other growth parameters (eg. lag phase duration and maximum population density) depended on the individual species and the specific growth conditions. The CA significantly extended the lag phase durations of S. aureus and V. parahaemolyticus at 20 degrees C at both pH 6.3 and 6.8, and that of L. monocytogenes at both 7 degrees C and 20 degrees C, and at both pH 6.3 and 6.8. The CA also significantly lowered the maximum population densities of S. aureus and V. parahaemolyticus at 20 degrees C, at pH 6.3 and 6.8, S. Typhimurium at pH 6.8, and L. monocytogenes at pH 6.3 and 7 degrees C. E. coli O157:H7 and S. Typhimurium were more resistant to the inhibitory effect of the CA, while S. aureus and V. parahaemolyticus were most sensitive. The inhibitory effect of CA was due mainly to the extensions of the lag phase duration and the reduction of the exponential growth rates of the test pathogens. This study confirms other studies that CA as a means for food preservation provides potential to control foodborne pathogens and therefore enhance the safety of a food product. The use of lactic acid bacteria (LAB) in controlling spoilage microorganisms and pathogens in foods has been a popular research theme worldwide. In this study, the antimicrobial effects of 18 lactic acid bacteria strains were evaluated in vitro, with emphasis on the most effective strain, the newly characterised Lactobacillus reuteri DPC16. The results demonstrated antagonistic effects of many strains against L. monocytogenes, E. coli O157:H7, S. Typhimurium and S. aureus. L. reuteri DPC16 showed the strongest antimicrobial activity against the tested pathogens including both Gram-positive and Gram-negative bacteria. Co-cultivation of L. reuteri DPC16, and co-incubation of its spent culture supernatant (DPC16-SCS), with the pathogens have demonstrated that the antimicrobial effect is bactericidal and valid at pH 4 - 6.5 and at a temperature as low as 10 degrees C. Further characterisation of the antimicrobial effect of L. reuteri DPC16 showed it to be mainly due to the presence of reuterin (ß-hydroxypropionaldehyde), although lactic acid may have also played a role. These characteristics of L. reuteri DPC16 and its metabolite reuterin make it an unique and potent candidate as a biopreservative to control both Gram-positive and Gram-negative bacteria in foods. The combination of L. reuteri DPC16 and CA was assessed for its inhibitory effect on L. monocytogenes using DPC16-SCS and the fermentative supernatant of L. reuteri DPC16 from a glycerol-water solution (DPC16-GFS). The results showed that both of these supernatants, at 25 AU/mL, in combination with CA (60% CO2:40% N2) had a combined inhibitory effect on L. monocytogenes which could not be achieved by any one of the individual factors alone. Analysis of the levels of expression of some stress response genes of L. monocytogenes, after growth in the presence of L. reuteri DPC16 supernatant and/or CA, showed that the expression of some genes was affected including genes betL, gbuA and opuCA responsible for osmosis adaptation and genes gadA, gadB and gadC responsible for acid tolerance. Induction of gbuA, gadB and gadC by the culture supernatant suggests activation of osmotic and acid adaptation and that these genes play a major role in the culture supernatant-induced stresses. An investigation was also carried out to determine if the changes in gene expression conferred a cross-protection to heat. The result showed that the survival of L. monocytogenes grown in the presence of the culture supernatant and CA was significantly increased after exposure to heat treatment at 56oC, suggesting that a cross-protection to thermal stress had been induced. Based on these findings it is proposed that a comprehensive novel strategy incorporating both L. reuteri DPC16 or its fermentative products and a modified atmosphere rich in CO2 could be developed to potentially control foodborne pathogens in food products.