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The influence of free base of quinolinilporphyrin and its Zn complex on infectivity of lactophages E3, E5 and E17 has been studied. The results of our investigations show that inhibition of lactophage activity by Zn complex of quinolinilporphyrin at concentration 10 μM and 20 μM was in the range 53–62% and 65–85%, respectively. The presence of this porphyrin in nutrient medium prevents the propagation of bacteriophage infection in Lactococcus lactis, but does not affect the phage adsorption process. The free base of quinolinilporphyrin slightly inhibits the activity of lactophages.
Purified proline-specific amino peptidases from Lactobacillus curvatus and from Lactococcus lactis were active on both X-proline dipeptidyl aminopeptidase (PepX) substrates, Gly-Pro-AMC or Gly-PropNA and on proline endopepetidase (PEP) substrates Suc-Gly-Pro-Leu-Gly-Pro, Suc-Gly-Pro-AMC, Z-Gly-Pro-AMC or Suc-Gly-Pro-pNA, however; activity on PEP substrates was markedly less than that on PepX substrates. The enzymes from Lactobacillus and Lactococcus hydrolyzed a number of oligopeptides containing 7-11 amino acids residues and proline at the penultimate position from N-terminus, but hydrolysis of natural PEP oligopeptide substrates containing proline residues at internal positions was negligible. The two proline-specific enzymes were strongly stimulated by NaCl and inhibited by phenylmethylsulfonyl fluoride and organic solvents.
Eleven strains of Lactococcus lacťis synthesising substances of the properties antagonistic toward the strains of the same species were examined. The effect of the strains on pathogenic bacteria: Escherichia coli, Salmonella sp., Staphylococcus aureus and Listeria, monocytogenes was assessed by spot-diffusion method and on a basis of synthesis of inhibitory substance in a liquid medium. Seven out of eleven strains were found to inhibit the growth of the pathogenic bacteria. In the liquid medium only one strain, however, produced the inhibitory substance in the concentration required to inhibit the growth of L. monocytogenes and S. aureus, and, in the same conditions, three strains synthesised the substance of activity against only L. monocytogenes. These substances showed the character of bacteriocins since they did not inhibit the growth of gram-negative bacteria, lost the activity after incubation with proteolytic enzymes, were considerably thermostable in acid and neutral medium, and were inactivated after heating in basic medium. The antibacterial activity of bacteriocins found in this work limits their potential use for preventing the growth of pathogenic L. monocytogenes and S. aureus.
We present here the results of an exploration of the bacteriophage content of dairy wheys collected from milk plants localized in various regions of Poland. Thirty-three whey samples from 17 regions were analyzed and found to contain phages active against L. lactis strains. High phage titer in all whey samples suggested phage-induced lysis to be the main cause of fermentation failures. In total, over 220 isolated phages were examined for their restriction patterns, genome sizes, genetic groups of DNA homology, and host ranges. Based on DNA digestions the identified phages were classified into 34 distinct DNA restriction groups. Phage genome sizes were estimated at 14-35 kb. Multiplex PCR analysis established that the studied phages belong to two out of the three main lactococcal phage types - c2 and 936, while P335-type phages were not detected. Yet, analyses of bacterial starter strains revealed that the majority of them are lysogenic and carry prophages of P335-type in their chromosome. Phage geographical distribution and host range are additionally discussed.
In this work we present cloning and overexpression of lactococcal CcpA protein in Escherichia coli Xllblue strain as a fusion with 6xHis tag. A high yield of the CcpA protein was obtained when the cells were cultured in liquid medium LB with 100 ug/ml ampicillin at 37°C and subsequently for 4 h after induction by IPTG. The proce­dure let us obtain 5 mg of homogenous CcpA protein. Glutaraldehyde crosslinking analysis indicated the formation of dimer or tetramer forms of the CcpA protein.
Experimental Allergic Encephalomyelitis (EAE) is the animal model of Multiple Sclerosis (MS), human chronic and progressive autoimmunological disease that lead to neurodegeneration in Central Nervous System (CNS). Although there are some hypothesis, like genetic, environmental or viral factors involvement, cause and patophysiology of MS remains still unknown, and that is the reason why there is no sufficient MS treatment so far. Most common MS therapy is use of immunosupressive drugs, but that is not very effective and costs number of health complications. Also new targeted therapies are burdened with the risk of side effects, which may be even lethal. Therefore, the efficient alternative treatment is urgently needed. The autoimmune base of the disease directed treatment searching into immunological mechanisms. Few years ago we proposed application of animal spinal cord hydrolysate for inducing oral tolerance, which effect lies in reducing of immunoresponse for previously fed antigen. We presented the effectiveness of this type of treatment in EAE rat model. The success of oral tolerance with mixture of peptides stimulates us to development bacteria that may express active peptide related to myelin fragment. As far as it is known, that the dose of fed antigen is crucial in evoking oral tolerance, the aim of our study, was to investigate which dose or doses of Lactococcus lactis expressing myelin peptides is sufficient for EAE treatment. We used autolising strain of Lactococcus lactis, producing one of three myelin peptides, which are considered to be crucial in MS developement: Myelin Basic Protein (MBP aa85-97), Proteolipid Protein (PLP aa139- 151) or Myelin Oligodendrocyte Protein (MOG aa35-55). We mixed all three peptide variants, and made whole-cell extracts. For our experiments we used female Lewis rats (180–200 g), which were fed with ball-pointed needle with mixed bacteria extracts for 20 days. Doses of preparations ranged from 101 to 108 cells/rat/feeding suspended in 0.5 ml PBS. At the 10th day of feeding, EAE was evoked by hind paw injection of guinea pig spinal cord homogenate in Freund Adjuvant with Mycobacterium tuberculosis. During the whole experiment animals were weighted, and clinical symptomes were observed. The obtained results demonstrated, that the sufficient doses of Lactococcus lactis expressing myelin peptides, given orally to animals are 103 and 106 cells/rat/feeding. Further experiments including cytokine level measurement and microscopic observation of rats spinal cord are in progress.
Zapotrzebowanie na produkty żywnościowe o cechach świeżych surowców jest przyczyną rozwoju technologii żywności o małym stopniu przetworzenia. Dla utrwalenia wymaga ona stosowania łagodnego ogrzewania lub zastosowania nietermicznych metod utrwalania. W artykule przedstawiono nietermiczne techniki przedłużania trwałości żywności wraz z niektórymi metodami pakowania.
During this study His-tagged CcpA protein purified under native conditions to obtain a biologically active protein was used for molecular analysis of CcpA-dependent regulation. Using electrophoretic mobility shift assays it was demonstrated that CcpA of L. lactis can bind DNA in the absence of the HPr-Ser-P corepressor and exhibits DNA-binding affinity for nucleotide sequences lacking cre sites. However, purified HPr-Ser-P protein from Bacillus subtilis was shown to slightly increase the DNA-binding capacity of the CcpA protein. It was also observed that CcpA bound to the cre box forms an apparently more stable complex than that resulting from unspecific binding. Competition gel retardation assay performed on DNA sequences from two PEP:PTS regions demonstrated that the ybhE, bglS, rheB, yebE, ptcB and yecA genes situated in these regions are most probably directly regulated by CcpA.
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