Conformational and functional changes in trout hemoglobin and cod myosin were investigated at low and high pH and after subsequent refolding. Hemoglobin cooperatively unfolded at low pH to a “molten globular” state with different levels of structure depending on how low the pH was and if 500 mM NaCl was present. At low pH the heme lost its contact with the protein but was not released but the protein was fully dissociated. Alkaline pH had little effect on hemoglobin but the heme environment suggested a strong heme-distal histidine link at pH 10.5. Longer unfolding time, lower pH and higher ionic strength made it more difficult to refold hemoglobin. The more misfolded the protein was the more hydrophobic it became and the lower its solubility after refolding it to pH 5.5. The presence of salt greatly facilitated the hydrophobic aggregation. The more unfolded or misfolded the protein was the more pro-oxidative it became, maybe as a result of more exposed heme, heme and subunit dissociation and hemoglobin-membrane interactions. High pH completely suppressed pro-oxidative activity of hemoglobin, possibly due to a strong heme-distal histidine link preventing autoxidation. Helical structure of myosin rods was little affected at low and high but the head group was substantially unfolded. The protein was possibly fully dissociated at low pH while at high pH only half of the light chains dissociated. The myosin head was misfolded on refolding with increased hydrophobicity, reactive −SH groups, loss in ATPase activity and lower conformational stability. The heavy chains may have fully reassembled on refolding from acid pH but light chains failed to reassemble to the protein. Refolded myosin had almost identical solubility as native myosin at pH 7.5 while cod myofibrillar proteins had increased solubility after acid and alkali treatment compared to the untreated proteins. Myosin and myofibrillar proteins exhibited improved emulsification activity and stability at pH 7.5 after pH treatment. Myosin gelled at a lower temperature and formed stronger gels at pH 7.5 in 600 mM KCl. The pH treated myofibrillar proteins also gelled at a lower temperature than native myosin but had a different gelling mechanism on heating.

Vibrio vulnificus is a Gram-negative estuarine bacterium that can cause wound infection, primary septicemia and gastroenteritis in susceptible individuals. Since it is a notably lethal pathogen associated with seafood, rapid detection and quantification of V. vulnificus is an important issue for the protection of consumer health and the seafood industry. With conventional PCR the minimum detection level with a new nucleic acid dye (GelStar stain) was 16 CFU per PCR reaction for pure culture compared to 40 CFU with ethidium bromide (EB). With real time PCR, the detection limit was 100 CFU/g of tissue with a linear detection range of 102 to 108 CFU/g without enrichment and 1 CFU with a detection range of 1 to 106 CFU/g of tissue after 5h enrichment. A 508-bp DNA fragment was used as an internal standard for competition with target DNA in competitive PCR, by which the detection sensitivity was 220 CFU of pure culture per PCR reaction, 2700 CFU/PCR with shellfish tissue without enrichment, and 7 CFU/PCR with shellfish tissue after 10 h enrichment. Geobacillus stearothermophilus and hemoglobin were used as a reference strain and a PCR inhibitor respectively, to establish comparative real time PCR, by which the minimum detection sensitivity was 102 CFU per ml of pure culture and 103 CFU per gram of clam tissue. The equation (Ct, T0 = Ct,TX - ΔCt) can be used for enumeration of V. vulnificus in tissue in conjunction with a standard curve relating Ct values to Log genomic targets in the absence of PCR inhibitors via comparative RT-PCR. Amplification of DNA from heat killed cells in a mixture with viable cells was successfully inhibited by treatment of the cells with 2.5 μg/ml of ethidium bromide monoazide (EMA) followed by 15 min photolysis, whereas the DNA from viable cells present was successfully amplified by real time PCR. Using this technique, the detection sensitivity for viable bacterial cells was 10 CFU per ml of pure culture and 100 CFU/g of clam tissue.

An $\alpha$-amylase (EC 3.2.1.1) secreted by Clostridium perfringens NCTC 8679 Type A was purified to homogeneity and characterized. It was isolated from concentrated cell-free culture medium by ion exchange and gel permeation chromatography. The pH optimum and pI of the enzyme were 6.5 and 4.75 respectively. The estimated molecular weight of the purified enzyme was 76 000. Calcium (5 mM) increased the optimal temperature for activity of the purified enzyme from 30 to 40$\sp\circ$C. The purified enzyme was inactivated between 35 and 40$\sp\circ$C which increased to between 45 and 50$\sp\circ$C in the presence of calcium. The purified enzyme produced a mixture of oligosaccharides as major endproducts of starch hydrolysis indicating $\alpha$-amylase activity. The effect of glucose and other sugars on sporulation and extracellular amylase production by Clostridium perfringens NCTC 8679 type A in a defined medium was also studied. Cells grown in the presence of glucose and mannose yielded the highest levels of amylase activity while disaccharides such as lactose, maltose and sucrose resulted in moderate amylase production. Little amylase activity was detected in the medium in the presence of ribose or galactose. The concentration of each sugar resulting in highest amylase production was between 6 and 10 mM except for fructose (25 mM). Levels of heat resistant spores decreased as sugar concentrations increased. The addition of even small amounts of glucose to the medium before exponential growth suppressed sporulation but maximized amylase activity. The addition of glucose after the initiation of sporulation did not inhibit spore formation. However, its addition to 3-h amylase-producing cells did inhibit subsequent sporulation but promoted the continued excretion of amylase. The extracellular amylase produced by C. perfringens exhibited eight major extracellular amylolytic activity bands when examined by activity staining following polyacrylamide gel electrophoresis (PAGE). Extracellular amylolytic enzymes released in sporulation process were different from those released during vegetative growth (extracellular amylase accumulation) whereas their intracellular amylolytic activities remained identical. A significant increase of an endo-acting amylolytic activity which cleaved both $\alpha$-1,4- and $\alpha$-1,6-glucosidic linkage was observed when C. perfringens sporulated. The different response to glucose between sporulating cells and amylase-producing cells suggests that the mechanisms of catabolite repression of extracellular amylase production and sporulation are distinct in C. perfringens whereas regulation of amylolytic activity during sporulation is possibly mediated by sporulation-related, amylolytic enzyme modification.

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Sammanfattning  Syftet med studien är att undersöka betydelsen av mellanmålets innehåll och hur det påverkar elevers konsumtion av mellanmål i fritidshem, samt om elever upplever att de är delaktiga i att påverka mellanmålets innehåll och miljö. Frågeställningarna i studien är; ·         Hur upplever elever att innehållet av mellanmål påverkar konsumtionen av mellanmål på fritidshem? ·         Hur upplever elever att miljön där mellanmålet intas påverkar konsumtionen av mellanmål på fritidshemmet?  ·         Hur beskriver elever deras delaktighet beträffande mellanmålets innehåll och miljö på fritidshem? Uppsatsen är en kvalitativ studie som utgår från barns perspektiv. Med hjälp av semistrukturerade fokusgruppsintervjuer har nio elever intervjuats på tre olika fritidshem. I resultatet beskriver elever vad de äter på fritidshem såsom smörgås med diverse pålägg och mjölk eller vatten att dricka. Vidare redogör eleverna i resultatet vad de helst skulle vilja äta beroende på tycke och smak, allt från gröt till sushi. Eleverna delger även för miljön där de intar mellanmålet. Vidare i resultatet redogörs elevernas kännedom om deras möjlighet till delaktighet i var mellanmålet serveras och vad som serveras.

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The multibillion dollar agricultural industry is an important part of the United States economy, and the management of factors that affect crop and human health is imperative to maintaining this economic sector. The fungi Botrytis cinerea, Fusarium pallidoroseum, and Fusarium moniliforme are the causative agents of several plant diseases and can cause significant crop loss both before and after harvest in commodities such as strawberries, lettuce, citrus, and grains. Fungicides are employed to control these phytopathogens, but the use of these chemicals has led to an increase in fungicide resistance and may negatively affect the environment and human health. In addition to plant pathogens, foodborne pathogens also have a substantial impact on the agricultural industry. Foodborne disease outbreaks involving Listeria monocytogenes, Salmonella, and Escherichia coli O157:H7 not only cause considerable economic losses, but can also result in devastating health problems for consumers. The increase in fungicide resistance and number of produce-related foodborne disease outbreaks warrants investigation into additional methods of microbial control for use in the agricultural industry. Many bacterial species, including Lactic Acid Bacteria (LAB) and Bacillus species, produce antifungal and antimicrobial compounds, thus the use of biological control agents pre- and postharvest could augment current methods of pathogen management. The purpose of this study was to screen 22 bacterial isolates for inhibitory activity against the fungal phytopathogens Botrytis cinerea, Fusarium pallidoroseum, and Fusarium moniliforme and the foodborne pathogens Listeria monocytogenes, Salmonella, and Escherichia coli O157:H7 in vitro, then evaluate antimicrobial efficacy of select isolates against the foodborne pathogens on fresh produce. To evaluate antifungal activity, the bacterial isolates were individually spot-inoculated onto Tryptic Soy Agar, Potato Dextrose Agar, or MRS agar, depending on isolate growth requirements and then a plug of fungal-colonized agar was placed onto the center of the isolate-inoculated plate. Plates were incubated at 24°C for 10 days; fungal growth was evaluated daily, beginning on Day 3. Nine of the 22 isolates screened inhibited all three fungi; inhibition by these isolates ranged from 51-62% for B. cinerea, 60-68% for F. pallidoroseum, and 40-61% for F. moniliforme. Isolates were also screened for biosurfactant activity using the drop-collapse test. Biosurfactant production was detected in seven of the nine isolates. Bacillus megaterium, Bacillus coagulans, Bacillus thuringiensis BT2 and three Bacillus amyloliquefaciens isolates demonstrated strong biosurfactant activity and suppression of all three fungi, and therefore are recommended for further study. Antimicrobial activity of the isolates was assessed using two methods: LAB isolates were screened using a seeded-overlay method and all other isolates were evaluated by spot inoculating the isolate on pathogen-seeded TSA. Three LAB isolates and six Bacillus isolates suppressed L. monocytogenes, Salmonella, and E. coli O157:H7 in vitro. Based on the results of the screening, three LAB isolates—Lactobacillus plantarum, Pediococcus acidilactici, and Pediococcus pentosaceus—were selected for further evaluation and use in challenge studies on fresh produce. The role of organic acids in pathogen inhibition was evaluated by incubating L. monocytogenes, Salmonella, and E. coli O157:H7 cultures in the cell-free supernatant (CFS; pH 3.81-4.27) or the neutralized cell-free supernatant (pH adjusted to 6.5 -7.0) of each isolate. When neutralized, the antimicrobial activity of the CFS of the three LAB isolates was greatly diminished, illustrating the role of lactic acid in the inhibition of pathogen growth. To assess antimicrobial efficacy on Iceberg lettuce, a cocktail of the three LAB isolates (7-8 log CFU/g) was sprayed onto lettuce spot-inoculated with L. monocytogenes (2-3 log CFU/g); lettuce was incubated at 10°C for 14 d. L. monocytogenes levels were 1.84 log lower on LAB-treated lettuce than on untreated lettuce at the end of incubation. Because the LAB cocktail suppressed the growth of L. monocytogenes on lettuce, testing on fresh produce continued using DF1, which was a powdered product comprised of the three LAB isolates and media components. Because DF1 caused substantial browning of Iceberg lettuce after 2 d, Gala apples were chosen to evaluate the antimicrobial activity of DF1 against L. monocytogenes, Salmonella, and E. coli O157:H7. The effect of DF1 on L. monocytogenes, Salmonella, and E. coli O157:H7 on Gala apples was determined by spraying a Gala apple spot-inoculated with pathogen (6-7 log CFU/plug) with approximately 3 mL of a 20% DF1 solution, then incubating at 20°C for 5 d. After 5 d incubation, L. monocytogenes, Salmonella, and E. coli O157:H7 levels on DF1-treated apples were approximately 4, 2, and 2 log higher than the control, respectively. Based on the results of these experiments, DF1 is not the optimal formulation for the biocontrol of foodborne pathogens on fresh produce. This study identified several bacterial isolates with potential for use in the biocontrol of plant and foodborne pathogens. Further investigation is required to assess possible use in the agricultural industry, including characterization of bioactive compounds, optimization of biocontrol product formulation, and evaluation of the commercial viability of the biocontrol product

Thesis: M.S., Massachusetts Institute of Technology, Department of Nutrition and Food Science, 1972 / Cataloged from the official PDF version of thesis. / Includes bibliographical references (pages 143-155). / Carbon recycle by means of physicochemically synthesized carbohydrates has been proposed. These artificial sugars can be used to generate single cell protein. However, it is not known what effects the unnatural components will have on the yield, productivity, and metabolic regulation of the or­ganisms used. We have obtained from natural populations, a number of organisms which utilize L-glucose as sole carbon source. Of the twelve organisms isolated, five are gram-negative aerobic rods, one is a gram positive coccus, two are thermophilic bacilli, three are yeasts, and one is a mycelial form. Pre­liminary taxonomy was done on these organisms. When fully adapted to growth on L-glucose, one pseudomonad grows ex­ponentially with a doubling time of 14 to 16 hours with 5 g/L L-glucose in the medium. Cell yields are about 0.46 g dry cells/g L-glucose, and cell densities as high as 2.8 g/L have been acheived in shake flasks. The ap­parent maximum growth rate is 0.0506 hr.⁻¹ and the apparent overall K[subscript m] for growth is 0.14 g/L L-glucose. However, substrate inhibition sets in at about 4.5 g/L L-glucose. L-glucose transport takes place by facilitated diffusion at V[subscript max] = 2.63 x 10⁻³ mg L-glucose/(mg cells-min) and K[subscript m]= 0.65 g/L L-glucose. The organism probably utilizes the entire L-glucose molecule. There is evidence that carbon 1 is eliminated as CO₂ and subsequently reassimilated from the medium. One or more growth factors appear to be necessary for L­ glucose utilization. They are made by the organism under good growth con­ditions and one appears to be excreted into the medium. A hypothetical mechanism of L-glucose utilization consistent with the growth kinetics is proposed. This mechanism involves a catabolic sequence with at least two limiting reactions. The first is incipient transport limitation and the second is inhibition by an intracellular metabolite derived from L-glucose. / by Robert C.J. Fewkes. / M.S. / M.S. Massachusetts Institute of Technology, Department of Nutrition and Food Science

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