<i>Lactobacillus plantarum</i> (<i>Lb. plantarum</i>) NRRL B4496 and <i>Lactococcus lactis</i> (<i>Lc. lactis</i>) NRRL B1821 prolidase genes were isolated, cloned, and sequenced. The sequence-confirmed genes were subcloned into the expression systems. The recombinant prolidases from the pKK223-3 systems were purified through ammonium sulphate precipitation and anion-exchange column chromatography. Recombinant <i>Lb. plantarum prolidase</i>, however, demonstrated a loss of activity during the purification. The following characterization work was performed on purified recombinant <i>Lc. lactis prolidase</i>. <p>The mass spectroscopic result and the molecular modelling suggested a 80 kDa homodimer with two metal cations at the catalytic centre of the prolidase. The optimum temperature was 50 ºC and showed more than 50% activities between 40 and 55 ºC. The enzyme was most stable at 30 ºC and withstood 20 min of heat-treatment up to 60 ºC, however, lost activity over 70 ºC. Circular dichroism indicated a denaturation temperature of 67 ºC. The optimum pH was 6.5 for hydrolyzing Leu-Pro and the enzyme did not display any activity below pH 5.5 nor above pH 7 with this peptide. However, Phe-Pro was hydrolyzed the fastest at pH 7 and Arg-Pro had a maximum rate at pH 9. This metallopeptidase exhibited a broad range of metal cation preference, hydrolyzing Leu-Pro with Mn++, Co++, Zn++, Ca++, and Mg++. Further kinetic analysis showed unusual allostery of the enzyme (Hill coefficient: 1.3). The unique substrate intakes onGlu-Pro and tripeptides were observed while Val-Pro was not hydrolyzed. The molecular modelling of this prolidase suggested a difference in the substrate specificity resulting from a loop structure, L33 to R40, near the substrate binding site.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Enzyme concentrates are available for use in commercial wineries to aid in wine processing, or to enhance wine quality. However, pectolytic enzyme remains the sole product routinely used in most wineries. One disadvantage of some of the products currently available commercially is they contain enzymes sourced from microorganisms not usually associated with grape juice or wine, typically fungi such as Aspergillus species. As a result, enzymes are inefficient catalysts under the harsh oenological conditions. In addition, some products contain secondary, and potentially undesirable, contaminant enzyme activities. Clearly there is the potential to develop enzyme preparations specifically for use in grape juice and wine. A potential source of such enzymes are the lactic acid bacteria (LAB), the organisms more commonly associated with the conduct of the malolactic fermentation (MLF) during vinification. In this study, the production of cell-associated enzymes with potential oenological applications by LAB was investigated. A screening of 50 LAB isolates for the production of lipases, esterases, tannases, and polysaccharide-degrading enzymes revealed wine LAB can produce enzymes of oenological importance. In general, activity towards polysaccharide substrates was more frequent among the lactobacilli and pediococci strains. Lipase activity was observed in three lactobacilli, and all strains were found to have tannase activity. Similarly, all strains displayed some esterase activity, although the activity was markedly stronger among the Oenococcus oeni. On the basis of the initial screen, a more detailed characterisation of the esterase activity of selected LAB isolates was conducted. Esterase activity was examined across a range of pH, temperature, and ethanol concentrations - all important oenological parameters. In addition, substrate specificity was determined using six ester substrates. In general, activity was maximal at pH values close to 6.0, and temperatures close to 40°C, although exceptions were observed with some strains. Increases in ethanol concentration resulted in lower activity for most lactobacilli and pediococci, but stimulated the esterase activity of all O. oeni. Work conducted with dairy LAB isolates has suggested esterases may be capable of both hydrolysing and synthesising esters. In the wine industry, the results of some volatile-profiling studies tend to support this theory, with concentrations of esters being reported to both increase and decrease during MLF. Malolactic fermentation trials were conducted in wine with six strains of O. oeni and GCMS was used to quantify particular esters before and after MLF. Some esters were found to increase in concentration during MLF, while others were found to decrease. These findings suggest LAB esterases are in fact capable of both synthesising and hydrolysing ester substrates in wine. To further dissect the esterase make-up of selected LAB strains, attempts to clone and heterologously express three structural genes for these enzymes were made. Three putative esterase genes were identified in O. oeni and cloned. Sequencing was completed and alignment with published esterase sequences used to reveal theoretical proteins of the O. oeni genes with high homology with those from other organisms. Of note, key features, such as active site motifs, were conserved in each O. oeni sequence. Expression of the recombinant proteins in E. coli resulted in higher esterase activity in one of the clones compared to the host. These results indicate that the open reading frame of one esterase gene in 0. oeni has been identified. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1297212 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2007

Thesis (M. Sc.)--University of Alberta, 2009. / Title from pdf file main screen (viewed on Sept. 10, 2009). "A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Sciences, Agricultural, Food, and Nutritional Sciences, University of Alberta." Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1956. / Typescript. Abstracted in Dissertation abstracts, v. 16 (1956) no. 11, p. 2000. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 257-268).

Thesis (M.Sc.)(Food Science)--University of Pretoria, 2005. / Includes summary. Includes bibliographical references. Available on the Internet via the World Wide Web.

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Bibliography: leaves 200-214. / xi, 214 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Three structurally related compounds, 2-acetyltetrahydropyridine (ACTPY), 2-ethyltetrahydropyridine (ETPY) and N-heterocycle, 2-acetyl-1-pyrroline (ACPY), were quantified and found to be unique components of mousy wines. 35 lactic acid bacteria (LAB) were screened for the ability to produce mousy off-flavour. In addition to Lactobacillus brevis and L. cellobiosus, a diversity of LAB species, particularly heterofermentative Lactobacillus spp. and Oenococcus oeni exhibited this ability in a range of ethanolic and wine-based media. The substrates and metabolism of mousy compound formation by LAB were also investigated. A pathway for the formation of ACPY and ACTPY by heterofermentative LAB was proposed. / Thesis (Ph.D.)--University of Adelaide, Dept. of Horticulture, Viticulture & Oenology, 1999

Internally-pH-controlled, phosphate containing and non-phosphate containing Italian bulk starter media were compared to reconstituted nonfat dry milk and commercial bulk starter media for their ability to support the growth and activities of commercially frozen thermophilic lactic acid cultures. Cultures grown in internally-pH-controlled media demonstrated superior acid-production capability. The cheese made from cultures grown in internally-pH-controlled media was comparable to that made from the culture grown in commercial medium. However, the internally-pH-controlled media were not bacteriophage inhibitory, nor were the reconstituted nonfat dry milk or two of the three commercial bulk starter media. Hence, cheese whey and nonfat milk based, low solids, bacteriophage inhibitory bulk starter media were formulated for the cultivation of mixed cultures of Streptococcus thermophilus and Lactobacillus bulgaricus. The new media supported the growth of lactobacilli better than the commercial media. Even at low solids levels, the buffering capacities of the new media were comparable to commercial media. Late addition of magnesium hydroxide as a neutralizing agent to commercial as well as experimental bulk starter media resulted in increased growth and improved activities of rod-coccus cultures. The cultures also retained their activities longer under refrigerated storage. Late addition of magnesium hydroxide did not encourage the proliferation of bacteriophages in the growth media. / Graduation date: 1987

Starter cultures of different bacteria strains like lactic acid producing bacteria, Staphylococcus and Kocuria are used when making salami. Starter cultures give the sausage specific flavours and improve the quality and ripening of the final product. Probiotic strains can also be added during the production of salami. Studies have shown that probiotics are good for health and are therefore added to food, such as fermented sausages. In order to work as a probiotic strain, the bacteria have to survive during the production process, storage and through the whole human gastrointestinal tract. The aim of this study was to trace the probiotic strains Lactobacillus casei and Lactobacillus paracasei in salami samples to see if they had survived the production process. Methods used were DNA extraction, PCR, colony PCR and gel electrophoresis. Out of 100 samples in duplicate run in PCR, probiotics were found in only 3 of them. To see if screening of probiotics directly from plates was possible, a colony PCR was done. Colony PCR was made on colonies of two different strains of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus sakei. From each bacteria strain, 5 colonies were analysed. Result showed that colony PCR, to screen for probiotic is a possible method.