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A microscopic and voltammetric characterization of lactate oxidase- (LOx-) based bioanalytical platforms for biosensor applications is presented. In this context, emphasis is placed on amperometric biosensors based on LOx that have been immobilized by direct absorption on carbon surfaces, in particular, glassy carbon (GC) and highly ordered pyrolytic graphite (HOPG). The immobilized LOx layers have been characterized using atomic force microscopy (AFM) under liquid conditions and cyclic voltammetry. In addition, spatially resolved mapping of enzymatic activity has been carried out using scanning electrochemical microscopy (SECM). In the presence of lactate with hydroxymethylferrocene (HMF) as a redox mediator in solution, biosensors obtained by direct adsorption of LOx onto GC electrodes exhibited a clear electrocatalytic activity, and lactate could be determined amperometrically at 300 mV versus SSCE. The proposed biosensor also exhibits good operating performance in terms of linearity, detection limit, and lifetime.
The homofermentative lactic acid bacterium Tetragenococcus halophila showed mixed acid fermentation at low growth-rates under glucose limiting conditions and in the presence of 10% NaCl. Maximum growth yields in fermentors with cell retention were not affected by pH, but maintenance requirement was at pH 5.2 four times higher than at pH 7.0. Despite the high salt-concentration of the medium, maintenance requirements were low compared to other lactic acid bacteria. The possible causes of the observed differences in maintenance requirements are discussed.
Production of the anti-listerial bacteriocin, pediocin, by lactic acid bacteria (LAB) transformed with the cloning vector pPC418 (Ped+, 9.1 kb) was influenced by composition of media and incubation temperature. Maximum pediocin production, tested against Listeria innocua, by electrotransformants of Lactococcus lactis ssp. lactis was measured in tryptone/lactose/yeast extract medium after 24 h growth at 30 degrees C, while incubation at 40 degrees C was optimum for Ped+ transformants of Streptococcus thermophilus and Enterococcus faecalis. The amount of pediocin produced by S. thermophilus in skim milk and cheese whey supplemented with 0.5% yeast extract was estimated as 51,000 units ml(-1) and 25,000 units ml(-1), respectively. Pediocin production remained essentially unchanged in reconstituted skim milk or whey media diluted up to 10-fold. The results demonstrate the capacity of recombinant strains of LAB to produce pediocin in a variety of growth media including skim milk and inexpensive cheese whey-based media, requiring minimum nutritional supplementation.
Chitosan (0.1 g l(-1)), assayed in a simple medium, reduced the viability of four lactic acid bacteria isolated during the beer production process by 5 logarithmic cycles, whereas activity against seven commercial brewing yeasts required up to 1 g chitosan l(-1). Antimicrobial activity was inversely affected by the pH of the assay medium. In brewery wort, chitosan (0.1 g l(-1)) selectively inhibited bacterial growth without altering yeast viability or fermenting performance.
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1. The growth of Pseudomonas AM1 is much more sensitive to inhibition by sulphanilamide when methanol, rather than succinate, acts as the sole carbon and energy source; a sulphanilamide concentration of 1mm, which causes almost complete inhibition of growth on methanol, has little effect in a succinate medium. 2. Similar results have been obtained with sulphadiazine and sulphathiazole. Sulphanilic acid has little effect. 3. A similar differential sensitivity to sulphanilamide is shown by Protaminobacter ruber and Pseudomonas extorquens when grown on methanol media as compared with succinate. 4. Sulphanilamide inhibited the growth of Pseudomonas oxalaticus on formate, oxalate and succinate media to about the same extent. 5. Inhibition of growth of Pseudomonas AM1 by sulphanilamide is accompanied by an accumulation of glycine in the cells. 6. Inhibition of growth by sulphanilamide can be reversed by p-aminobenzoic acid. 7. Microbiological assays of the folate content of Pseudomonas AM1 have been performed after growth on both methanol and succinate, and the results are discussed in terms of differences in metabolism.
1. Metabolism of folate was studied in six ewes in an advanced state of vitamin B(12) deficiency as judged by voluntary food intake and in their pair-fed controls receiving vitamin B(12). A group of four animals that were maintained throughout the experiment at pasture was also studied. 2. After 34-40 weeks on the cobalt-deficient diet urinary excretion of formiminoglutamate by four deficient animals was about 3.2mmol/day and this was not significantly decreased by injection of three of them with about 4.5mug of [2-(14)C]folate/kg body weight per day for 5 days. Three days after the last injection retention of [2-(14)C]folate by the livers of the deficient animals (5.5% of the dose) was lower than that of their pair-fed controls (26% of the dose) but there was no evidence of net retention of injected folate in the livers of either group. Urinary excretion of (14)C indicated that renal clearance of folate may have been impaired in very severe vitamin B(12) deficiency. 3. As estimated by microbiological assays total folates in the livers of animals at pasture (12.9mug/g) included about 24% of 5-methyltetrahydrofolate as compared with about 72% of a total of 12.5mug/g in three further ewes fed on a stock diet of wheaten hay-chaff and lucerne-chaff. Liver folates of vitamin B(12)-deficient animals (0.5mug/g) included about 88% of 5-methyltetrahydrofolate as compared with about 51% of a total of 5.2mug/g in pair-fed animals treated with vitamin B(12). 4. Chromatography of liver folates of the pair-fed animals permitted quantitative estimates of the pteroylglutamates present. The results showed that the vitamin B(12)-deficient livers were more severely depleted of tetrahydrofolates and formyltetrahydrofolates than of methyltetrahydrofolates and that as the deficiency developed they were more severely depleted of the higher polyglutamates than of the monoglutamate within each of these classes. Results from animals injected with [2-(14)C]folate indicated an impairment of the exchange between pteroylmonoglutamates and pteroylpolyglutamates in the livers of deficient animals. 5. In vitamin B(12)-deficient animals with food intakes below 200g/day some of the liver folates were not completely reduced and some degradation of pteroylpolyglutamates was detected. The latter condition may have been associated with fatty liver. 6. The results are discussed in relation to current theories of vitamin B(12)-folate interactions.
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The evolution of the microflora of three Comté cheeses made in duplicate with raw milk from three different sources was followed during ripening. The same starter was used with each type of milk. The comparison of the cheeses did not reveal any significant difference in the development of the microflora. Starter lactic acid bacteria (Streptococcus thermophilus and Lactobacillus helveticus), which are added at the beginning of manufacture, decreased quickly in the first stages of ripening supporting the hypothesis of cell autolysis. Other microorganisms, i.e. homofermentative and heterofermentative lactobacilli (Lact. delbrueckii ssp. lactis, Lact. paracasei ssp. paracasei, Lact. rhamnosus and Lact. fermentum), pediococci, enterococci and propionibacteria grew in cheese from small numbers in fresh curd. The characterization of Strep. thermophilus by pulsed-field gel electrophoresis showed that wild strains were also able to grow in the curd. The values for the genome size of 11 Strep. thermophilus strains determined in this investigation were in the range of 1.8-2.3 Mbp. The potential role of starter and raw milk microflora in cheese flavour development was considered.
Six mice were immunized intraperitoneally (i.p.) with a chemically synthesized 9-mer fragment (PH1) designed from the N-terminal part of the bacteriocin pediocin PA-1 and conjugated to keyhole limpet haemocyanin (KLH). After three doses of the immunogen had been administered, serum-specific antibodies were detected by a competitive direct ELISA. Myeloma cells were injected i.p. into mice in order to obtain ascites polyclonal antibodies. Although four mice developed ascites, only mouse 2 had detectable specific antibodies in the ascites fluid. The serum and ascites antibodies were specific for PH1 but they did not recognize the whole pediocin PA-1 molecule. This is the first attempt to generate antibodies against bacteriocins with a chemically synthesized oligopeptide as immunogen. This approach still remains attractive for detection, quantification, mode of action studies and purification of bacteriocins, especially those for which the purification process is difficult or inefficient at present.