Recombinant Lactococcus lactis fails to secrete bovine chymosine.

Bovine chymosin is an important milk-clotting agent used in the manufacturing of cheeses. Currently, the production of recombinant proteins by genetically modified organisms is widespread, leading to greatly reduced costs. Lactococcus (L.) lactis, the model lactic acid bacterium, was considered a good candidate for heterologous chymosin production for the following reasons: (1) it is considered to be a GRAS (generally regarded as safe) microorganism, (2) only one protease is present on its surface, (3) it can secrete proteins of different sizes, and (4) it allows for the direct production of protein in fermented food products. Thus, three genetically modified L. lactis strains were constructed to produce and target the three different forms of bovine chymosin, prochymosin B, chymosin A and chymosin B to the extracellular medium. Although all three proteins were stably produced in L. lactis, none of the forms were detected in the extracellular medium or showed clotting activity in milk. Our hypothesis is that this secretion deficiency and lack of clotting activity can be explained by the recombinant protein being attached to the cell envelope. Thus, the development of other strategies is necessary to achieve both production and targeting of chymosin in L. lactis, which could facilitate the downstream processing and recovery of this industrially important protein.

Anti-inflammatory effects of Lactococcus lactis NCDO 2118 during the remission period of chemically induced colitis.

BACKGROUND: Many probiotic bacteria have been described as promising tools for the treatment and prevention of inflammatory bowel diseases (IBDs). Most of these bacteria are lactic acid bacteria, which are part of the healthy human microbiota. However, little is known about the effects of transient bacteria present in normal diets, including Lactococcus lactis. METHODS: In the present study, we analysed the immunomodulatory effects of three L. lactis strains in vitro using intestinal epithelial cells. L. lactis NCDO 2118 was administered for 4 days to C57BL/6 mice during the remission period of colitis induced by dextran sodium sulphate (DSS). RESULTS: Only one strain, L. lactis NCDO 2118, was able to reduce IL-1ß-induced IL-8 secretion in Caco-2 cells, suggesting a potential anti-inflammatory effect. Oral treatment using L. lactis NCDO 2118 resulted in a milder form of recurrent colitis than that observed in control diseased mice. This protective effect was not attributable to changes in secretory IgA (sIgA); however, NCDO 2118 administration was associated with an early increase in IL-6 production and sustained IL-10 production in colonic tissue. Mice fed L. lactis NCDO 2118 had an increased number of regulatory CD4(+) T cells (Tregs) bearing surface TGF-ß in its latent form (Latency-associated peptide-LAP) in the mesenteric lymph nodes and spleen. CONCLUSIONS: Here, we identified a new probiotic strain with a potential role in the treatment of IBD, and we elucidated some of the mechanisms underlying its anti-inflammatory effect.

An improved method for characterization of the mutation associated to porcine stress syndrome by PCR amplification followed by restriction analysis / Um método melhorado para caracterização da mutação associada à síndrome do estresse suíno por amplificação por PCR seguido de análise de restrição

A mutation in the gene coding for the ryanodine receptor 1 (RYR1), also known as halothane (hal) gene or swine stress gene, is associated to the porcine stress syndrome (PSS). Detection of the mutation is normally accomplished by PCR amplification of an 81bp fragment of the hal gene, followed by digestion with the HhaI restriction endonuclease. Wild-type allele (N) is cut in two fragments, whereas the mutant allele (n) is not digested by the restriction enzyme. Electrophoresis of the digested DNA on agarose gel and ethidium bromide staining allows the reading of the result. The correct interpretation is difficult due to the small size of the DNA fragments. In this study we designed a new set of primers for amplification of a 144bp fragment that facilitates the reading of the result. In addition, we optimized the PCR reaction to allow amplification from a single hair bulb, added directly into the PCR mix without previous treatment. This improved method was used to genotype 165 sows and boars used in a breeding program. Forty-nine percent of the animals had the NN genotype, whereas 50% were Nn and only 1% was nn.

Uma mutação no gene que codifica o receptor ryanodine 1 (RYR1), também conhecido como gene do halotano (hal) ou gene do estresse suíno, está associada à Síndrome do Estresse Suíno (PSS). A mutação é geralmente detectada por PCR, a partir da amplificação de um fragmento de 81pb do gene hal, seguida por digestão com a endonuclease de restrição HhaI. O alelo normal (N) é cortado em dois fragmentos, enquanto que o alelo mutado (n) não é digerido pela enzima de restrição. A eletroforese do DNA digerido em gel de agarose corado com brometo de etídio permite a leitura do resultado. A interpretação correta é difícil devido ao pequeno tamanho dos fragmentos. Neste estudo, foi projetado um novo par de iniciadores para a amplificação de um fragmento de 144pb, o que facilita a leitura do resultado. Adicionalmente, foi otimizada a reação de PCR para permitir a amplificação a partir de um único bulbo capilar, acrescentado diretamente na mistura de PCR, sem tratamento prévio. Esse método foi usado para genotipar 165 reprodutores utilizados em granjas produtoras de matrizes. Quarenta e nove porcento dos animais apresentaram genótipo NN, 50% Nn e apenas 1% nn.