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Psychrotrophic bacteria, such as Pseudomonas fluorescens B52, are a major cause of milk spoilage at refrigeration temperature due to the production of lipolytic and proteolytic enzymes. Regulatory mechanisms controlling the production of lipase and protease by the B52 lipA and aprX genes were investigated. Transposon mutagenesis identified the possible involvement of a poly-A polymerase enzyme which destabilises mRNA by 3' polyadenylation. A homologue of the E. coli EnvZ/OmpR two-component sensor/regulator system was identified by transposon mutagenesis and shown to repress lipase and protease production. This system responds to Na+ and K+ concentration in E. coli and these ions were also shown to repress lipase and protease expression in B52, however the EnvZ/OmpR system is not solely responsible for this. Assays of translational lacZ fusions with aprX and lipA were used to speculate on the mechanism by which Na+ and EnvZ/OmpR repress the aprX-lipA operon. A membrane-bound sensor, MspA, which regulates protease production in P. fluorescens LS107d<SUB>2</SUB>, was shown to exist in B52 but mutagenesis of the B52 mspA gene had no effect on lipase and protease expression. A homologue of the P. fluorescens CHA0 rsmA gene, encoding an RNA-binding translation repressor, was found in B52. Although aprX and possibly lipA contain consensus sequences for RsmA, mutagenesis of rsmA had no significant effect on lipase and protease expression. Repression of lipase and protease expression by Na+ was increased by expression of the P. fluorescens M114 pbrA sigma-factor gene in B52.

Field pea is the most produced and exported pulse crop in Canada, and makes a major contribution to Western Canadian agricultural diversification programs. Canada is now the world largest exporter of pea, lentil and chickpea and is fourth in dry bean. The demand for Canadian pulse products is steadily rising and the export market would continue to rise with the expected increased in production. Field pea exported to countries with tropical climates is at particular risk due to rapid loss of quality. It is therefore important to develop practical strategies for safe storage of feed pea. Knowledge on the moisture adsorption and spoilage characteristics of pea stored in adverse storage conditions is important in the transportation and storage of this export commodity. <p> This study was initiated to examine the conditions that lead to quality losses in storage and transport of pea. Tropical and subtropical conditions were simulated in airtight chambers. Relative humidities (RH) of 60, 70, 80 and 90% were created by saturated salt solutions in airtight chambers at temperatures of 10, 20 and 30°C, while the same range of humidity was provided by dilute sulphuric acid in airtight chambers at 40°C in environmental cabinets. The four RH levels at each temperature for both whole and feed-grade pea were tested in duplicate. The samples were observed for changes in moisture content (MC), mold appearance and RH in specific time intervals. The amount of produced carbon dioxide (CO2) was measured in airtight chambers during storage to control the condition existing in sealed airtight chambers. Also, all components of feed-grade pea were exposed to RH of 90% and temperature of 40°C in separate airtight chambers to find the effect of each component on mold appearance. Molds were identified after appearance on the samples in order to pinpoint potential toxicity. <p> Both feed and whole sound peas became molded after a short time of storage at high temperatures and high RH, but those stored at 70% and below did not develop mold after 175 days at 30 and 40°C (experiment duration) and 216 days at 10 and 20°C (experiment duration). Molds were identified mostly as species of Aspergillus and Penicillium. The amount of CO2 in the airtight chambers showed almost no difference from the ambient CO2 except at high temperature and high RH when samples had gone molded. <p> Moisture adsorption equations were developed based on the moisture adsorption data in dynamic environment. Although the Page model showed to fit the data better, the exponential model was chosen to fit the data because its parameters can be better expressed as a function of temperature and RH of the storage environment. <p> The mold-free days for both feed pea and clean pea were modeled at temperatures of 10, 20, 30 and 40°C and RH of 80 and 90%.

Field pea is the most produced and exported pulse crop in Canada, and makes a major contribution to Western Canadian agricultural diversification programs. Canada is now the world largest exporter of pea, lentil and chickpea and is fourth in dry bean. The demand for Canadian pulse products is steadily rising and the export market would continue to rise with the expected increased in production. Field pea exported to countries with tropical climates is at particular risk due to rapid loss of quality. It is therefore important to develop practical strategies for safe storage of feed pea. Knowledge on the moisture adsorption and spoilage characteristics of pea stored in adverse storage conditions is important in the transportation and storage of this export commodity. <p> This study was initiated to examine the conditions that lead to quality losses in storage and transport of pea. Tropical and subtropical conditions were simulated in airtight chambers. Relative humidities (RH) of 60, 70, 80 and 90% were created by saturated salt solutions in airtight chambers at temperatures of 10, 20 and 30°C, while the same range of humidity was provided by dilute sulphuric acid in airtight chambers at 40°C in environmental cabinets. The four RH levels at each temperature for both whole and feed-grade pea were tested in duplicate. The samples were observed for changes in moisture content (MC), mold appearance and RH in specific time intervals. The amount of produced carbon dioxide (CO2) was measured in airtight chambers during storage to control the condition existing in sealed airtight chambers. Also, all components of feed-grade pea were exposed to RH of 90% and temperature of 40°C in separate airtight chambers to find the effect of each component on mold appearance. Molds were identified after appearance on the samples in order to pinpoint potential toxicity. <p> Both feed and whole sound peas became molded after a short time of storage at high temperatures and high RH, but those stored at 70% and below did not develop mold after 175 days at 30 and 40°C (experiment duration) and 216 days at 10 and 20°C (experiment duration). Molds were identified mostly as species of Aspergillus and Penicillium. The amount of CO2 in the airtight chambers showed almost no difference from the ambient CO2 except at high temperature and high RH when samples had gone molded. <p> Moisture adsorption equations were developed based on the moisture adsorption data in dynamic environment. Although the Page model showed to fit the data better, the exponential model was chosen to fit the data because its parameters can be better expressed as a function of temperature and RH of the storage environment. <p> The mold-free days for both feed pea and clean pea were modeled at temperatures of 10, 20, 30 and 40°C and RH of 80 and 90%.

The effects of irradiation dose (0, 0.5 and 1.0 kGy), various gas atmospheres and storage temperature (5, 15 and 25$ sp circ$C) on the physical, chemical, microbiological, and organoleptic changes in fresh pork were studied using factorial design experiments. The effects on toxin production by Clostridium botulinum were also investigated using challenge studies. Shelf life could be extended to 21 d when product was packaged in 0% O$ sb2$, irradiated at 1.0 kGy and stored at 5$ sp circ$C compared to 4 d for control samples. While the presence of O$ sb2$ in the package headspace enhanced the antimicrobial effects of low-dose irradiation, it adversely affected the organoleptic qualities of pork. Botulinum toxin was detected after only 2 d in all inoculated treatments stored at 25$ sp circ$C. At 15$ sp circ$C, toxin was produced faster when pork was initially packaged with O$ sb2$ or low levels of CO$ sb2$ (15-30%) as compared to 100% N$ sb2$. Higher levels of CO$ sb2$ (45-75%) delayed toxin production. In most treatments, spoilage preceded toxigenesis. Models were developed relating the above factors to the time until toxin production and to the probability of toxigenesis. Temperature, initial O$ sb2$ and irradiation were all significant factors.

Listeria monocytogenes is a pathogenic, psychrotrophic microorganism that is ubiquitous in nature. L. monocytogenes has been isolated from numerous meat products, both fresh and processed, the incidence of contamination varying greatly. The ability of Listeria to grow in meats depends on temperature, pH, water activity (a$ sb{ rm w}$), nutrients, species and numbers of competing microorganisms, gaseous conditions, and levels of additional barriers. Therefore, methods to control the growth of L.monocytogenes are of great importance to food processors since this organism can grow under a wide range of environmental and storage conditions. Two methods of control, in conjunction with temperature, were studied in this project: (i) modified atmosphere packaging (MAP) and (ii) chitosan, to determine the optimum levels of these "hurdles" needed to effectively control the outgrowth of L.monocytogenes in both model broth and agar systems and in fresh pork loin. On the basis of these preliminary studies, a combination of chitosan as a dipping solution and modified atmosphere packaging were investigated to control the growth of L.monocytogenes in fresh pork loin. Pork loin samples were dipped in a 0.2% chitosan solution for 60 seconds and packaged under various atmospheres in Cryovac bags and stored at 5, 10 and 15$ sp circ$C up to 28 days. Samples were monitored for physical, chemical and microbiological changes throughout the storage period. Optimum control over the growth of L. monocytogenes was achieved using a combination of 100%N$ sb2$ + an Ageless FX oxygen absorbent and dipping in a 0.2% chitosan solution. Based on these studies, a combination of 0.2% chitosan and MAP could be used to extend the shelf life of pork without adversely affecting color, odor and exudate loss while inhibiting the growth of the pathogenic microorganism, L.monocytogenes. (Abstract shortened by UMI.)

During storage of grain, there may have significant damage to its quality due to unfavorable physical and biological interactions and thus requires continuous monitoring. Therefore, an easy, cost-effective and environmentally friendly method is necessary for efficient monitoring of stored-grain. Arrays of sensors are being used for classifying liquors, perfumes, quality of food products mimicking mammalian olfactory systems. Monitoring of stored grain is a new application of sensor arrays. The main objective was to fabricate a carbon black polymer sensor array which can easily monitor incipient grain spoilage by detecting spoiling stored grain volatiles (benzene derivatives and aliphatic hydrocarbon derivatives) with minimum interference from relative humidity. Various aspects of a good sensor were analyzed using statistical analysis (RSD, LDA, PCA, t-test). The developed sensor array can identify red flour beetle-infected and uninfected wheat and fungal volatiles at ambient conditions as well as some stored grain conditions (MC 16%, RH 52%).

Previous results indicated that lactic acid bacteria (LAB) and Bacillus spores were the predominant spoilage microorganisms in minimally processed products after 35 days storage at 15$ sp circ$C. Furthermore, most packages were swollen due to carbon dioxide production by the LAB. The fact that spores survived mild heat treatment is of concern since it implies that other more pathogenic spores, e.g. Clostridium botulinum would also survive and may pose a public health problem in products stored at 15$ sp circ$C. As such, the influence of combination treatments (viz., mild heat processing treatment, pH, water activity, storage temperature, lactic acid bacteria) were evaluated through challenge studies with selected strains of C. botulinum type A, B, and E spores. The shelf life and safety of the products following a 13D process at 75$ sp circ$C, could be extended to $ ge$42 days by a combination of pH $ le$ 5.0 and storage at 5$ sp circ$C. Other combination treatments were also effective in controlling growth of, and toxin formation in the products by C. botulinum. For instance, botulinum toxin was not detected until day 35 in inoculated spaghetti and meat sauce products (pH 5.25 and a$ sb{w}$ 0.992) with type A and B spores and stored at 15$ sp circ$C. Shelf life extension and inhibition of toxin production was also possible through reductions in a$ sb{w}$ levels achieved by addition of 1-3% salt (w/w) to the products. Water activity levels of $ le$0.983 prevented botulinum toxin production in the minimally processed spaghetti and meat sauce product for $ ge$42 days during storage at 15$ sp circ$C. / Similar trends were observed in sous-vide rice and salmon products. Furthermore, when sodium lactate was used as the humectant to lower the a$ sb{w}$ of this product, toxin production was also delayed, but not to the same extent as achieved with NaCl. When two strains of bacteriocin-producing lactic acid bacteria were co-inoculated separately in the sous-vide rice and salmon products that had been challenged with C. botulinum type E spores, toxin formation was only slightly delayed in products with added NaCl (1%), while toxin formation was neither delayed nor prevented in similarly lactic acid bacteria-treated samples with no added NaCl. Furthermore, in most of the products in which botulinum toxin was detected, spoilage preceded toxigenesis, however, in some samples stored at 5$ sp circ$C toxigenesis preceded spoilage. Addition of $ alpha$-2-macroglobulin at a level of 2.7 ppm did not delay or prevent toxin formation in the product. Microwave heating of products at half-power or full power (800 Watts) for 5 to 10 min proved effective in inactivating all of the pre-formed toxin in toxic samples. (Abstract shortened by UMI.)

Proteolysis of myofibrillar proteins in Pacific whiting surimi occurs when the 50- 70°C temperature range is reached during standard cooking procedures (e.g. 90°C for 15 min). This proteolytic activity results in the softening of surimi gels. Bovine plasma protein (BPP) is the most effective of the food-grade inhibitors used to prevent this reaction, and enhance gel strength in PW surimi. The objective of this study was to determine the effective components of bovine plasma that enhance gel strength in PW surimi. Five bovine plasma fractions were evaluated for components that contribute to gel strength enhancement in PW surimi. Fraction I, which consists mostly of fibrinogen and albumin, was found to also contain plasma transglutaminase (PTGase) activity. Part of fraction I gel-enhancing ability may be attributed to an unknown component which inhibited papain independently of Ca²⁺ and inhibited 40% of surimi proteolytic activity. Fibrinogen or albumin did not inhibit papain activity or enhance gel strength of surimi. For fraction I-S, which is a more concentrated PTGase fraction, gel-enhancement of PW surimi was completely dependent on the presence of Ca²⁺. Autolytic inhibitory activity of fraction I-S in surimi was completely eliminated by the presence of Zn²⁺. Fraction II+III (1%) inhibited over 50% of surimi autolytic activity and displayed a small amount of PTGase activity. Fraction II+III (1%) gel enhancing abilities were low when compared to the other fractions and BPP, and only slightly effected by EGTA. Fraction IV (1%), which contains approximately 50% albumin and 15% α₂-macroglobulin, inhibited over 70% of surimi autolytic activity. It enhanced gel strength at a 1% (w/w) concentration when set for 20 hr at 4°C before cooking, and was not affected by EGTA. This fraction displayed no apparent PTGase activity. Fraction IV-1 (1%), which contains approximately 20-30% α₂-macroglobulin, gel strength enhancement surpassed the other fractions and BPP when set for 20 hr at 4°C and 2 hr at 25°C before being cooked at 90°C for 15 min. The gel strength enhancing abilities of fraction IV-1 were significantly affected by EGTA. Fraction IV-1 (1%) inhibited over 80% of surimi proteolytic activity. The gel strength of 1 mM (0.03%) E-64, which is a cysteine protease inhibitor, was equivalent to that of BPP (1%) after setting at 4°C for 20 hrs before cooking. E-64 (1 mM) inhibited 83% of the autolytic activity of PW surimi and BPP (1%) inhibited 78%. These data indicate that a cysteine protease inhibitor can increase gel strength, and suggests that BPP is acting as a cysteine protease inhibitor. Ca²⁺ dependent gel strength enhancement was attributed to transglutaminase (TGase) activity, both added PTGase and endogenous TGase. Gel strength enhancement that was Ca²⁺ independent was attributed to cysteine protease inhibitors, specifically α₂-macroglobulin. Overall, it was determined that gel strength in PW surimi was greatly enhanced by both concentrated PTGase (I and I-S) and concentrated α₂-macroglobulin (IV-1) fractions, with a combination of these fractions being most effective in gel strength enhancement, when the surimi is first set at 4°C or 25°C before cooking at 90°C for 15 min. These data suggest that the mechanisms of gel strength enhancement of BPP are from cysteine protease inhibition, possibly from α₂-macroglobulin, and from crosslinking of myosin in surimi from both added (PTGase) and endogenous TGase activity. / Graduation date: 1996

Dissertation (PhD)--University of Stellenbosch, 2007. / Bibliography. Also available via the Internet.