Leucocin A is a bacteriocin produced by Leucoconostoc gelidium UAL 187-22. Bacteriocin producer strains possess an immunity protein, which enables the strain to protect itself against its own bacteriocin. The immunity gene from Leucoconostoc gelidium was isolated via PCR from a recombinant clone pJF5.5. This fragment was cloned by amplifying the immunity gene from pJF5.5 and ligating it into pMALc2. The resulting recombinant plasmid pKP1 was then transformed into Escherichia coli strain JM103. The clone putative, was confirmed by DNA sequencing and southern blot hybridization using the primers EAL-2 and EAL-3. It was shown to contain an insert of 3.6 kb. Expression analysis showed the construct as an in frame malE fusion protein expressed within E. coli. The fusion construct was isolated by affinity chromatography. Leucocin A was purified to 90% purity, from the supernatant of Leucocnostoc gelidium UAL 187-22 by ion-exchange chromatography and HPLC. It was found to elute from a C18 reverse phase column at 55% actetonitrile, 0.1% TFA. Binding interaction and the stability of the immunity gene fusion protein were compared using a Biacore 2000. The supernatant and cytoplasmic extract isolated from Leucocnostoc gelidium UAL 187-22 were tested for interaction with the fusion construct. Surface Plasmon resonance studies indicated that there was no binding partner present in the supernatant which would influence the immunity process. However, a stable interaction was found between the immunity protein and an orphan ligand within the cytoplasm. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

Viren haben als Ursache lebensmittelassoziierter Infektionen eine große Bedeutung. Sie können vor allem über rohe oder unzureichend erhitzte Lebensmittel übertragen werden. In diesem Zusammenhang werden grüner Salat, Erdbeeren, Himbeeren, Frühlingszwiebeln, Muscheln, halbgetrocknete Tomaten, fäkal verunreinigtes Trinkwasser, Backwaren und Rohwürste als häufige Infektionsquellen genannt. Vor allem kurzgereifte Rohwürste gehören aus mikrobiologischer Sicht zu Risikoprodukten. Um eine gleichbleibende Qualität der Produkte zu gewährleisten, ist die Verwendung von Starterkulturen unerlässlich. Als sogenannte Schutzkulturen sollen sie gleichzeitig die Vermehrung unerwünschter bakterieller Pathogene unterbinden. Bisher ist allerdings nicht bekannt, inwieweit diese zur Virusinaktivierung in kurzgereiften Rohwürsten führen bzw. beitragen. Aus diesem Grund war es das Ziel dieser Arbeit, den Einfluss von rohwurstrelevanten Starter- und Schutzkulturen sowie deren Metabolite (Bacteriocine, Milchsäure) auf die Tenazität und Inaktivierungskinetik von Viren zu prüfen. Die Untersuchungen erfolgten mit dem murinen Norovirus (MNV) S 99 sowie dem humanen Influenzavirus H1N1 (A/WSN/33). Antivirale Effekte wurden zum einen anhand von in-vitro-Studien, zum anderen anhand von experimentell mit Viren kontaminierten kurzgereiften Rohwürsten (Mettwurst/Teewurst) geprüft. Die Bacteriocine Sakacin A und Nisin zeigten in phosphatgepufferter Salzlösung (PBS) keine viruzide Wirkung gegenüber MNV S 99 und H1N1 (pH 6,2; 24 °C; Exposition: 3 Tage). Weiterhin wurden anhand von in-vitro-Untersuchungen 29 verschiedene zellfreie Kulturüberstände [Milchsäurebakterien, Staphylococcus spp. (S.), Kocuria (K.) varians] hinsichtlich ihrer antiviralen Wirkung geprüft. Dabei konnte eine signifikante Titerreduktion von MNV S 99 bei Exposition mit dem Kulturüberstand eines Lactobacillus (Lb.) curvatus-Isolates festgestellt werden (p < 0,05). In mit dieser Kultur fermentiertem Tee- und Mettwurstbrät zeigte sich jedoch kein Effekt. Die Virustenazität von H1N1 und MNV S 99 konnte mit D,L-Milchsäure unter rohwurstrelevanten Bedingungen (pH 5,0 bis 6,2) sowohl in-vitro als auch im frischen Mettwurstbrät beeinflusst werden. In-vitro erzielte Titerreduktionen lagen bei 2,5 (H1N1) bzw. 3,25 log-Stufen (MNV S 99) nach drei Tagen (24 °C) Lagerung. Im Gegensatz dazu war MNV S 99 im Vergleich zu H1N1 im Mettwurstbrät stabiler. H1N1 konnte unterhalb von pH 5,5 bereits direkt nach dem Einmischen der Influenzaviren in das Wurstbrät nicht mehr nachgewiesen werden. MNV S 99 wurde hingegen erst nach einem Tag Lagerung (22 °C) maximal um 0,7 log-Stufen reduziert (pH 5,2). Die verwendeten Starter- und Schutzkulturen (Lb. sakei, Lb. curvatus, Lb. paracasei, Lb. plantarum, S. carnosus, S. xylosus, K. varians) zeigten im Mett- und Teewurstbrät im Vergleich zur Kontrolle (ohne Starterkultur) keinen zusätzlichen viruziden Effekt auf MNV S 99. Zunehmende Virustiterreduktionen konnten mit pH-Wert-Erniedrigung beobachtet werden. Nach der Reifung (1 Tag, 22 °C, pH 4,9) von Mettwurst mit Starterkulturen wurde das Virus um maximal 1,65 log-Stufen reduziert. In mit Einzel- beziehungsweise Mehrstamm-Mischkulturen fermentierter Teewurst (7 Tage, 22 °C, pH 4,9) betrug die Titerreduktion maximal 1,10 log-Stufen. Das Influenzavirus H1N1 konnte im Rohwurstbrät mit Starterkulturen auch nach Verwendung hoher Ausgangstiter bereits zu Beginn der Untersuchungen nicht mehr nachgewiesen werden. Aus den erzielten Daten kann geschlussfolgert werden, dass die Bacteriocine Sakacin A und Nisin nicht als antivirale Zusatzstoffe in Lebensmitteln (z. B. Rohwürste) geeignet sind. Das antivirale Potential von zellfreien Kulturüberständen war Bakterienstamm-spezifisch und nur in-vitro ersichtlich. Daher muss die Nutzung des Lb. curvatus 1-Stammes nicht anderen rohwurstrelevanten Starterkulturen vorgezogen werden. Die Verwendung von Milchsäure als Zusatzstoff im Rohwurstbrät eignet sich nur zum Ausschluss einer viralen Exposition im Zusammenhang mit H1N1. Frische Mettwurst muss allerdings hierzu adäquat gesäuert (pH < 5,5) werden. Neben dem antiviralen Effekt durch gebildete Säure, konnte keine weitere spezies-spezifische antivirale Wirkung verwendeter Starter- und Schutzkulturen auf MNV S 99 festgestellt werden. Die Säureleistung einzelner Kulturen ist demzufolge für eine Virusinaktivierung entscheidend. Das antivirale Potential verwendeter Starter- und Schutzkulturen in Rohwürsten ist im Zusammenhang mit MNV S 99 als gering einzuschätzen. Unter der Annahme, dass murine und humane Noroviren eine ähnliche Tenazität in kurzgereiften Rohwürsten aufweisen, sollten diese Produkte im Zusammenhang mit Noroviren als Risikoprodukte eingestuft werden.

This study examined the effects of 14 days of L-carnitine supplementation on blood and muscle lactate concentrations, and carnitine fractions, during high intensity sprint cycling exercise. Eight subjects performed three experimental trials - control I (CON I, 0 days), control II (CON II, 14 days), and L-carnitine (LCN, 28 days). Each trial consisted of a 4 min ride at 90% VO2max, followed by a rest period of 20 min, and then 5 x 1 min rides at 115% VO2max (2 min restbetween each). Following CON II, all subjects began dietary supplementation of L-carnitine for a period of 14 days (4 g/day). L-carnitine supplementation had no significant effect on either muscle carnitine or lactate concentrations following the 4 min 90% ride. Plasma total acid soluable and free carnitine concentrations were significantly higher at all time points following supplementation. Differences observed in blood hydrogen ion and lactate concentrations between CON I and CON II appear to be the result of an order effect. The data from the present investigation indicate that L-carnitine supplementation has no significant effect on blood or muscle lactate accumulation following high intensity sprint cycling exercise. / School of Physical Education

Lactic acid recovery from dilute fermentation broths is a growing requirement due to the increasing demand for pure lactic acid. Reactive extraction is proposed as an alternative to conventional methods of recovery, since the selectivity of separation is remarkably enhanced in reactive extraction. The aim of this study is to perform the equilibrium studies for the recovery of lactic acid from its synthetic aqueous solutions (not from real fermentation broths) by reactive extraction and investigate the effects of various parameters such as initial lactic acid concentration in the aqueous phase (0.25 - 1.3 M), initial pH of the aqueous phase (2 &ndash / 6), organic phase extractant concentration (0.1 &ndash / 0.5 M), type of the extractant (chloride, hydrogensulphate and hydroxide salts of tri-n-octylmethylammonium) and the type of diluent (oleyl alcohol or octanol). The results of the experiments showed that the degrees of extraction decreased with increasing use of diluent with the extractant and increasing initial lactic acid concentration of the aqueous phase. Highest degrees of extraction were achieved for undiluted extractants. The performance of the diluents were investigated by performing extraction experiments with solutions of TOMAC in oleyl alcohol or octanol at different pH values and it was observed that octanol had a higher solvating power than oleyl alcohol especially at lower aqueous phase pH values. Higher extraction efficiencies were obtained for TOMAC dissolved in octanol rather than oleyl alcohol. Initial aqueous pH of 6 was identified as the optimum pH for the extraction, also due to its being equal the average fermentation pH for the extractions with Lactobacillus species. Among the different salts of tri-n-octylmethylammonium, hydroxide salt exhibited the highest degrees of extraction (83% with undiluted TOMA(OH) and 78% with 0.5 M TOMA(OH) in octanol for the extraction of 0.316 M lactic acid solutions). The present work is the first step in the design of an industrial reactive extraction process that is going to attempt forward and backward extraction of lactic acid simultaneously in a hollow fiber membrane module that is going to be attached to the lactic acid fermentor to achieve continuous product recovery. The equilibrium data obtained from this study can be combined with the kinetic studies as the next step of designing a separation module.

iv The recovery of lactic acid from its dilute aqueous solutions is a major problem. The ester of lactic acid, namely, methyl lactate has a wide range of applications. The esterification of an aqueous solution of lactic acid with methanol is a reversible reaction. As excess of amount water is present in the reaction mixture, the conversion is greatly restricted by the chemical reaction equilibrium limitations. In this study the esterification kinetics of lactic acid with methanol both in the absence and presence of an ion exchange resin as a heterogeneous acid catalyst was investigated with isothermal batch experiments between 40 - 70 0 C and at atmospheric pressure. Self-polymerization of lactic acid was enlightened by considering the hydrolysis reaction of lactoyllactic acid at the reaction temperatures and at various initial concentrations. Both homogeneous and heterogeneous reaction rate constants were evaluated. Methyl lactate process development was also investigated. The process was based on the recovery of 10% lactic acid by reaction with methanol in a absorption column using ion-exchange resin Lewatit SPC-112 H+. The effect of various parameters including lactic acid concentration or reactant molar ratio, lactic acid feed flow rate, methanol and inert carrier rate on reactor performance were studied. The reaction of methyl lactate formation over the ion exchange resin catalyst was observed to be slower than the mass transfer rate whereas mass transfer of methanol in gas phase was the limiting step for methanol transfer to the liquid mixture. Mass transfer of water from liquid phase to the gas phase was controlled by the mass transfer resistance of liquid phase. Thus, it can be concluded that the counter-current gas-liquid reactors with acidic solid catalysts can be used as simultaneous reaction and separation equipment.

Lactic acid is a fermentation-derived organic acid used in a wide range of industries, such as food processing and pharmaceuticals. Its market is expected to expand due to the worldwide concern for the environment, as it is an essential feedstock for biodegradable polymers. However, fermentation product is a very dilute, multicomponent aqueous solution. Subsequent separation, purification and concentration of organic acids is difficult because of high affinity of the acids for water. Reactive extraction is a viable alternative to classical separation techniques. Amine extractants dissolved in organic diluents are suitable agents with reasonable ranges of viscosity and density of the solvent phase. The product is obtained in an organic phase after reactive extraction. The aim of this study is to obtain equilibrium data of back extraction of lactic acid into appropriate aqueous solutions from the organic phase. Aqueous solutions of NaCl, NaOH, Na2SO4, NaNO3 and Na2CO3 were examined as back extractant in various initial concentrations (0.005 &ndash / 3 M). The organic phase consists of tri-n-octylmethylammonium lactate (TOMA(La)) dissolved in either oleyl alcohol or octanol with initial concentrations between 0.1 and 0.3 M. According to results of the experiments, the level of back extraction generally increased with increasing initial salt concentration in aqueous phase and decreased with increasing initial TOMA(La) concentration in organic phase. For all salts investigated, considerable levels of back extraction were obtained. NaOH was considered as the most suitable back extractant among the salts investigated since it exhibits higher distribution coefficients, regenerates tri-n-octylmethylammonium hydroxide (TOMAOH) in the organic phase and has no adverse effect on fermentation medium when forward and backward extraction steps are coupled with the fermentation. The effect of diluent type of TOMA(La) was also investigated during the experiments and it was concluded that octanol is a better diluent since it gives higher equilibrium distribution coefficients in addition to its higher solvating power and lower viscosity. The present work is a part of a comprehensive research program aiming to collect data and develop knowledge for the design of an industrial reactive extraction process coupling forward and backward extraction of lactic acid in a single unit and integrating fermentation and product separation. The kinetic parameters should be obtained as the next step for the design of such a process.

In this study, the physico-chemical chances occurring in white cheese and possible effects of starter culture combinations to the ripening period during 30 days storage examinated. A total of thirty six lactic acid bacteria were isolated from a cheese made witohout using starter culture. For identification gram staining, catalase, gas production and coagulation tests were performed. For determination of species API50 CH (BioM&eacute / rieux) and partial 16S rDNA gene sequence analysis were used. Four cheese were produced, one by using commercial starter culture [Lyofast CMS (Lactococcus lactic subs. lactis and Lactococcus lactis subs. cremoris)]as control the other three by using different combinations of isolates [Lactococcus lactic subs. lactis (13%) +Lactobacillus brevis (40%)+Lactobacillus paracasei (47%) / Lactococcus lactic subs. lactis (36%)+Lactobacillus paracasei (64%) / Lactococus lactis subs. lactis (24,5%)+ Lactobacillus paracasei (28,5%) + Lactobacillus brevis (47%)] Cheese were ripened in 15% saline solution 4 C for 30 days.Samples were taken from each treatment and analyzed on 2nd,15th and 30th days Sensory ,microbiological and chemical properties of the cheese preparations as pH, acidity ,salt,fat,moisture,protein contents during storage period were determined. In this respect effect of using different starter culture combinations on quality of Turkish white chee was determined and Lactococcus lactis subs. lactis(13%)+Lactobacillus brevis (40%)+Lactobacillus paracasei(47%) combination was found as the best and can be suggested as ideal combination for white cheese production.

Several obstacles to widespread use of bacteriocins in food have been identified, including lack of specific, rapid quantitation methods, and little data on their efficacy in food systems. The first objective of this study was to develop a specific, rapid quantitation method for bacteriocins that did not rely on bioassays and their associated limitations. Phage display was chosen to reduce reliance on continued use of animals and produce antibodies to the bacteriocins leucocin A, piscicolin 126, and brochocin-A. Although the antibody libraries generated by phage display were not successful for antibody production, a strong immune response to leucocin A and piscicolin 126 was observed in mice. The second objective of the study was to determine the efficacy of brochocin-C against Clostridium botulinum in a model system and in a vacuum-packaged, chopped and formed pork product stored at refrigeration temperature. Group I Cl. botulinum was not controlled by brochocin-C, and was found to inactivate brochocin-C and several class IIa bacteriocins by proteolysis. Cell counts revealed that Group II Cl. botulinum was controlled by brochocin-C in a model meat system, but was not controlled in the chopped and formed pork product. Powdered smoke and NaCl in the pork product had a synergistic interaction against Group II Cl. botulinum, as shown by minimum inhibitory concentration testing. The choice of media for isolation of Cl. botulinum from the chopped and formed pork product was important, as the presence of background microflora isolated from the meat was found to impact growth of Group II Cl. botulinum on plating media. In the presence of the background microflora, which were identified by 16S rDNA sequencing as carnobacteria and staphylococci, inclusion of phosphate in the plating medium was found to allow growth of Cl. botulinum. Other nutrients such as magnesium, sulphur, or increased protein sources added to the medium had no effect on growth of Cl. botulinum. Two of the background microflora strains, Carnobacterium maltaromaticum MH3 and Staphylococcus pasteuri EIV-21, inhibited Cl. botulinum, while one strain, C. maltaromaticum MH2, stimulated growth of Cl. botulinum. / Food Science and Technology

The biocompatibility and biodegradability of poly(lactic acid) (PLA) facilitate its use in a variety of biomedical applications, ranging from sutures to drug delivery. However, uncontrolled interactions with cells and insufficient mechanical properties have prevented PLA from reaching its full potential as a scaffold for use in tissue engineering. Methods to improve the mechanical, chemical and biological properties of PLA are limited by the lack of functional groups along the backbone of the polymer. One possible approach towards overcoming these limitations involves the incorporation of functional groups into the backbone of the polymer through the copolymerization of monomers bearing protected functional groups. Deprotection and modification of these functional groups could provide the opportunity to direct the attachment of cells, and enhance to the physical properties of the polymer. We have developed a general methodology for the synthesis of lactide monomers substituted with protected functional groups (alcohols protected as benzyl ethers, amines protected as benzyl carbamates and carboxylic acids protected as benzyl esters). The monomers were homopolymerized, and copolymerized with lactide, and deprotected to give functional PLA copolymers with pendant hydroxyl, amine, and carboxyl groups. A thorough investigation of the chemical modification of PLA copolymers bearing functional groups along the polymer backbone was performed on a copolymer prepared by copolymerizarion of a dibenzyloxy-substituted lactide monomer with lactide followed by reductive debenzylation. Reaction of the resulting hydroxyl-substited PLA with succinic anhydride resulted in an acid-substituted PLA that is amenable to standard EDC/NHS coupling. The utility of this copolymer was illustrated by coupling with an amine derivative of biotin, and an RGD-containing peptide sequence. The preparation of the biodegradable polyester substituted with RGD, a ubiquitous adhesion peptide, provided us with control over cellular attachment to the hybrid material. We also explored approaches to make use of the pendant functional groups on PLA to enhance the physical properties of polymer foams. Copolymers with pendant photocrosslinkable cinnamate groups were prepared by reaction of the hydroxyl-substited PLA copolymers with cinnamoyl chloride. The copolymer was foamed using thermally-induced phase separation (TIPS), and photocrosslinked upon irradiation at 300 nm. Irradiation resulted in an increase in the compressive modulus of the foams. Crosslinking also led to a decrease in the rate of hydrolytic degradation of the foams, thereby demonstrating the potential for use of these strategies in the development of porous scaffolds for bioengineering. Another potential approach towards the preparation of robust polymer foams is the incorporation of a rigid polymer block which can phase separate during foam formation to provide additional structural integrity. Several poly(norbornene)-PLA diblock copolymer compositions were prepared by the ring-opening of lactide by a hydroxyl-terminated poly(norbornene) macroinitiator. The ability of the diblock copolymer to phase separate at elevated temperature was verified using small-angle x-ray scattering and wide-angle x-ray scattering.

The synthesis and characterization of a series of chiral indium complexes bearing a tridentate NNO ligand are reported. The ligand 2-[[[(dimethylamino)cyclohexyl]amino]methyl]- 4,6-bis(tert-butyl) phenol (H₂NNO) was synthesized via a previously published procedure and bound to indium by both a protonolysis and salt metathesis route. A dimethylated indium complex (NNO)InMe₂ (1) was isolated by reaction of InMe₃ with H₂NNO. A one-pot saltmetathesis route was used to produce a unique mixed-bridge dinuclear indium complex [(NNO)InCl] ₂(μ-OEt)(μ-Cl) (3) from a mixture of indium trichloride, potassium ethoxide and the monopotassiated salt of the ligand, KH(NNO). Direct reaction of KH(NNO) and indium trichloride resulted in the formation of (NNO)InCl₂ (4) which was carried forward to 3 by reaction with sodium ethoxide. The complex 3 is active for the ROP of β-butyrolactone ε-caprolactone and lactide and is the first reported indium-based catalyst for lactide or β-butyrolactone ROP. Kinetic studies of 3 for ROP of LA revealed that catalyst was well-behaved, and that the rate was first order with regard to lactide and catalyst. The enthalpy and entropy of activation for the ROP were experimentally determined. Polymer produced by ROP by 3 has narrow molecular weight distribution and a good correlation is seen between the observed moleular weight and monomer loading. A mechanism was proposed for 3 acting as a catalyst for the ROP of lactide; however further experiments are required to confirm this mechanism. Polymer samples isolated from the ROP of rac-lactide by rac-3 show isotactic enrichment. It is postulated that the chiral catalyst 3 is exerting stereocontrol via an enantiomorphic site control mechanism.