The effects of process conditions and growth kinetics on the production of the bacteriocin sakacin P by Lactobacillus sakei CCUG 42687 have been studied in pH-controlled fermentations. The fermentations could be divided into phases based on the growth kinetics, phase one being a short period of exponential growth, and three subsequent ones being phases of with decreasing specific growth rate. Sakacin P production was maximal at 20 degrees C. At higher temperatures (25-30 degrees C) the production ceased at lower cell masses, when less glucose was consumed, resulting in much lower sakacin P concentrations. With similar media and pH, the maximum sakacin P concentration at 20 degrees C was seven times higher than that at 30 degrees C. The growth rate increased with increasing concentrations of yeast extract, and the maximum concentration and specific production rate of sakacin P increased concomitantly. Increasing tryptone concentrations also had a positive influence upon sakacin P production, though the effect was significantly lower than that of yeast extract. The maximum sakacin P concentration obtained in this study was 20.5 mg l(-1). On the basis of the growth and production kinetics, possible metabolic regulation of bacteriocin synthesis is discussed, e.g. the effects of availability of essential amino acids, other nutrients, and energy.

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Campylobacter jejuni is a major cause of diarrheal disease and food-borne gastroenteritis. The main reservoir of C. jejuni in poultry is the cecum, with an estimated content of 6 to 8 log 10 CFU/g. If a flock is infected with C. jejuni, the majority of the birds in that flock will harbor the bacterium. Diagnostics at the flock level could thus be an important control point. The aim of the work presented here was to develop a complete quantitative PCR-based detection assay for C. jejuni obtained directly from cecal contents and fecal samples. We applied an approach in which the same paramagnetic beads were used both for cell isolation and for DNA purification. This integrated approach enabled both fully automated and quantitative sample preparation and a DNA extraction method. We developed a complete quantitative diagnostic assay through the combination of the sample preparation approach and real-time 5-nuclease PCR. The assay was evaluated both by spiking the samples with C. jejuni and through the detection of C. jejuni in naturally colonized chickens. Detection limits between 2 and 25 CFU per PCR and a quantitative range of >4 log 10 were obtained for spiked fecal and cecal samples. Thirty-one different poultry flocks were screened for naturally colonized chickens. A total of 262 (204 fecal and 58 cecal) samples were analyzed. Nineteen of the flocks were Campylobacter positive, whereas 12 were negative. Two of the flocks contained Campylobacter species other than C. jejuni. There was a large difference in the C. jejuni content, ranging from 4 to 8 log 10 CFU/g of fecal or cecal material, for the different flocks tested. Some issues that have not yet promoted much attention are the prequantitative differences in the ability of C. jejuni to colonize poultry and the importance of these differences for causing human disease through food contamination. Understanding the colonization kinetics in poultry is therefore of great importance for controlling human infections by this bacterium.

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Two hundred strains of Listeria monocytogenes collected from food and the food industry were analyzed for susceptibility to the class IIa bacteriocins sakacin P, sakacin A, and pediocin PA-1 and the class I bacteriocin nisin. The individual 50% inhibitory concentrations (IC 50 ) were determined in a microtiter assay and expressed in nanograms per milliliter. The IC 50 of sakacin P ranged from 0.01 to 0.61 ng ml ؊1 . The corresponding values for pediocin PA-1, sakacin A, and nisin were 0.10 to 7.34, 0.16 to 44.2, and 2.2 to 781 ng ml ؊1 , respectively. The use of a large number of strains and the accuracy of the IC 50 determination revealed patterns not previously described, and for the first time it was shown that the IC 50 of sakacin P divided the L. monocytogenes strains into two distinct groups. Ten strains from each group were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and amplified fragment length polymorphism. The results from these studies essentially confirmed the grouping based on the IC 50 of sakacin P. A high correlation was found between the IC 50 of sakacin P and that of pediocin PA-1 for the 200 strains. Surprisingly, the correlation between the IC 50 of the two class IIa bacteriocins sakacin A and sakacin P was lower than the correlation between the IC 50 of sakacin A and the class I bacteriocin nisin.

Aims: To evaluate the potential of sakacin P and sakacin P-producing Lactobacillus sakei for the inhibition of growth of Listeria monocytogenes in chicken cold cuts, by answering the following questions. (i) Is sakacin P actually produced in food? (ii) Is sakacin P produced in situ responsible for the inhibiting effect? (iii) How stable is sakacin P in food? Methods and Results: Listeria monocytogenes, a Lact. sakei strain and/or the bacteriocin sakacin P were added to chicken cold cuts, vacuum packed and incubated at 4 or 10°C for 4 weeks. Each of two isogenic Lact. sakei strains, one producing sakacin P and the other not, had an inhibiting effect on the growth of L. monocytogenes. The effect of these two isogenic strains on the growth of L. monocytogenes was indistinguishable, even though sakacin P was produced in the product by one of the two Lact. sakei strains. The addition of purified sakacin P had an inhibiting effect on the growth of L. monocytogenes. A high dosage of sakacin P (3AE5 lg g )1 ) had a bacteriostatic effect throughout the storage period of 4 weeks, while a low dosage (12 ng g )1 ) permitted initial growth, but at a slow rate. After 4 weeks of storage, the number of L. monocytogenes in the samples with a low dosage of sakacin P was 2 logs below that in the untreated control. When using a high dosage of sakacin P, the bacteriocin was detected in samples stored for up to 6 weeks. Conclusions: (i) Sakacin P is produced by a Lact. sakei strain when growing on vacuumpacked chicken cold cuts. (ii) Inhibiting effects of Lact. sakei, other than sakacin P, are active in inhibiting the growth of L. monocytogenes growing on chicken cold cuts. (iii) Sakacin P is stable on chicken cold cuts over a period of 4 weeks. Significance and Impact of the Study: Both sakacin P and Lact. sakei were found to have potential for use in the control of L. monocytogenes in chicken cold cuts.

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A system for efficient heterologous expression of class II bacteriocins is described that is based on introducing two plasmids in a bacteriocin-negative Lactobacillus sake strain. The first plasmid (pSAK20) contains the genes necessary for transcriptional activation of the Sakacin A promoter as well as export and processing of bacteriocin precursors. The second plasmid (a pLPV111 derivative) contains the structural and immunity genes for the bacteriocin of interest fused to the sakacin A promoter. Using this system, various bacteriocins were produced at levels equal to or higher than those obtained with the corresponding wild-type producer strains.