Lactobacillus helveticus as a tool to change proteolysis and functionality in Swiss-type cheeses.

Lactobacillus helveticus exhibits a great biodiversity in terms of protease gene content, with 1 to 4 cell envelope proteinases. Among them, proteinases PrtH and PrtH2 were shown to have different cleavage specificity on pure α(s1)-casein. The aim of this work was to investigate the proteolytic activity of 2L. helveticus strains in cheese matrix: ITGLH77 (PrtH2 only) and ITGLH1 (at least 2 proteinases, PrtH and PrtH2). Cell viability, proteolysis, autolysis, and stretchability of experimental Emmental cheeses were measured during ripening. The peptides identified by mass spectrometry showed very different profiles in the 2 cheeses. Regardless of the casein origin, the number of different peptides containing more than 20 amino acids was greater in cheeses manufactured with strain ITGLH77. This accumulation of large peptides, including those from α(s1)- and α(s2)-caseins, was in agreement with the lower overall extent of proteolysis obtained in ITGLH77 cheeses, which can be attributed to the presence of one cell envelope proteinase of the lactobacilli strains or lesser release of intracellular peptidases into the cheese aqueous phase. In parallel, stretchability was measured throughout ripening time. Emmental strands observed by confocal laser scanning microscopy showed microstructure similar to that of mozzarella strands. Stretchability was correlated with a specific type of peptide (hydrophobic), as shown by principal component analysis, and with a lower degree of proteolysis.

Actinides and decay products in selected produce and bioindicators in the vicinity of a uranium plant.

Cypress needles collected at the edge of the Malvési uranium facility (SW France) exhibit enhanced activities of actinides and some decay products (uranium, americium, plutonium, (230)Th, (226)Ra) compared to a remote site. These enhanced activities resulted from the release of U via smokestacks and passive release from former artificial ponds located inside the nuclear site. Enhanced activities are also observed in selected produce (wheat, lettuce, fruits) sampled from the edge of the site. However, excess actinides measured in wheat grains in 2007 are inconsistent with the activities and the uranium ratio measured in the soils. This result suggests that the studied annual crops were contaminated mainly through the short-term release of airborne actinides, and that other transfer pathways, such as, uptake through the roots or adhesion of soil particles, were negligible.

Simultaneous presence of PrtH and PrtH2 proteinases in Lactobacillus helveticus Strains improves breakdown of the pure alphas1-casein.

Lactobacillus helveticus can possess one or two cell envelope proteinases (CEPs), called PrtH2 and PrtH. The aim of this work was to explore the diversity of 15 strains of L. helveticus, isolated from various origins, in terms of their proteolytic activities and specificities on pure caseins or on milk casein micelles. CEP activity differed 14-fold when the strains were assayed on a synthetic substrate, but no significant differences were detected between strains possessing one or two CEPs. No correlation was observed between the proteolytic activities of the strains and their rates of acidification in milk. The kinetics of hydrolysis of purified α(s1)- and ß-casein by L. helveticus whole cells was monitored using Tris-Tricine sodium dodecyl sulfate (SDS) electrophoresis, and for four strains, the peptides released were identified using mass spectrometry. While rapid hydrolysis of pure ß-casein was observed for all strains, the hydrolysis kinetics of α(s1)-casein was the only criterion capable of distinguishing between the strains based on the number of CEPs. Fifty-four to 74 peptides were identified for each strain. When only PrtH2 was present, 22 to 30% of the peptides originated from α(s1)-casein. The percentage increased to 41 to 49% for strains in which both CEPs were expressed. The peptide size ranged from 6 to 33 amino acids, revealing a broad range of cleavage specificities, involving all classes of amino acids (Leu, Val, Ala, Ile, Glu, Gln, Lys, Arg, Met, and Pro). Regions resistant to proteolysis were identified in both caseins. When strains were grown in milk, a drastic reduction in the number of peptides was observed, reflecting changes in accessibility and/or peptide assimilation during growth.

Isotopic evidence of natural uranium and spent fuel uranium releases into the environment.

Uranium and plutonium isotopes were measured in soils, sediments and waters in an area subject to the past and present discharges from the uranium conversion plant of Malvési (France). The isotopes (236)U and (239)Pu are well known activation products of uranium and they prove to be powerful tracers of spent fuel releases in soils and sediments. On the other hand (234)U and (238)U activities measured in waters can be used to distinguish between releases and background uranium sources. Such findings contribute to improve the monitoring of the actinides releases by nuclear fuel facilities (mining sites, conversion, enrichment and fuel plants, reprocessing plants).

Inhibition of Listeria monocytogenes by in situ produced and semipurified bacteriocins of Carnobacterium spp. on vacuum-packed, refrigerated cold-smoked salmon.

Listeria monocytogenes inhibition by Carnobacterium strains and crude bacteriocins on sterile and commercial vacuum-packed cold-smoked salmon stored at 4 degrees C and 8 degrees C was investigated. Carnobacterium piscicola V1 was bactericidal against L. monocytogenes at the two temperatures, whereas Carnobacterium divergens V41 presented a bacteriostatic effect. C. piscicola SF668 delayed L. monocytogenes growth at 8 degrees C and had a bacteriostatic effect at 4 degrees C. Listeria growth was not affected by a non-bacteriocin-producing C. piscicola. Crude extracts of piscicocins were bactericidal at 4 degrees C and 8 degrees C. Listeria growth was delayed by divercin V41 at 8 degrees C and was inhibited at 4 degrees C. Nisin delayed Listeria growth at 8 degrees C and was bacteriostatic at 4 degrees C. The present study demonstrates that L. monocytogenes growth could be prevented on vacuum-packed cold-smoked salmon by Carnobacterium and associated bacteriocins at chilled temperatures. Moreover, no product spoilage could be observed with the use of such bacteriocin-producing strains as demonstrated by good sensorial analyses and low biogenic amine production.

Increasing incidence and comparison of nalidixic acid-resistant Salmonella enterica subsp. enterica serotype typhimurium isolates from humans and animals.

We determined the resistance to quinolone of 309 Salmonella enterica subsp. enterica serotype Typhimurium strains isolated from humans and animals (cattle, pigs, or poultry) in 1995 or 1996. Nalidixic acid resistance increased from 8.5% in 1995 to 18.6% in 1996. The highest resistance levels correlated with a mutation at Ser-83 (or Asp-82). All strains remained ciprofloxacin susceptible. Human and animal isolates were compared by pulsed-field gel electrophoresis, and the banding patterns of the human isolates most closely matched those of the bovine isolates.

[Growing incidence of nalidixic acid resistance and sensitivity to quinolones in Salmonella typhimurium strains isolated from man or animal]. / Incidence croissante de la résistance à l'acide nalidixique et sensibilité aux quinolones des souches de Salmonella typhimurium isolées chez l'homme ou l'animal.

To determine the prevalence of quinolone resistance in Salmonella typhimurium strains from humans or animals (cattle, poultry, swine), the S. typhimurium strains isolated at a teaching hospital and at the central veterinary laboratory of the same district between January 1, 1995, and December 31, 1996 were studied. Susceptibility to nalidixic acid was determined using the disk diffusion method. Strains with decreased susceptibility to nalidixic acid were subjected to minimal inhibitory concentration (MIC) determination for pefloxacin, ofloxacin, ciprofloxacin, norfloxacin, levofloxacin, and grepafloxacin. Decreased susceptibility to nalidixic acid was demonstrated for 41 of the 309 strains studied and increased from 8.5% in 1995 to 18.6% in 1996. MIC90 values of fluoroquinolones for strains with decreased susceptibility to nalidixic acid were lower than 1 mg/L, which is the cutoff above which a strain is classified as susceptible, but were higher than for strains that were susceptible to nalidixic acid. These low levels of resistance may be the first step in selection of mutant strains with high levels of resistance to fluoroquinolones. This warrants continued monitoring of resistance of Salmonella to fluoroquinolones.

[Effect of hygiene measures, water sanitation and oral rehydration therapy on diarrhea in children less than five years old in the south of Ivory Coast]. / Effet de l'observance des mesures d'hygiène, d'approvisionnement en eau et de la thérapie par voie orale sur les diarrhées chez les enfants de moins de 5 ans dans le sud de la Côte d'Ivoire.

The purpose is to assess the impact of compliance with measures of hygiene and water supply and oral rehydration on the diarrhoea with under 5 years of age in four villages of southern Côte d'Ivoire. The method used is to compare morbidity and mortality of children, firstly between two groups of villages without such measures, secondly in two villages before and after implementing them. Initially, an exhaustive survey has determined the incidence rate of diarrhoea, the proportion of deaths resulting from such diarrhoea as well as the mortality rate 1988. Two similar survey were made in 1990 and 1992. The results show a 50% reduction of the incidence rate of diarrhoea and a 85% reduction of the proportion of deaths related to diarrhoea in the villages with intervention. The mortality rate to diarrhoea was likewise reduced by 85%. These results show the importance of the improvement and accessibility to drinkable water and hygiene in the prevention of diarrhoea in areas children.

[Impact of improved sanitary conditions and domestic hygiene on the incidence of ascaridiasis and ancylostomiasis in children two to four years old in the rural zones of Ivory Coast]. / Impact de l'assainissement et de l'hygiène domestique sur l'incidence de l'ascaridiose et de l'ankylostomose chez les enfants de 2 à 4 ans dans les zones rurales de Côte d'Ivoire.

The purpose of this study is to assess the impact of improvement measures for hygiene and water supply, installation of latrines and health education on the incidence of ascaridiasis and ankylostomiasis with 2 to 4 years old children in some villages of southern Côte d'Ivoire. The method used is the comparison of children carrying parasites before and after intervention. Initially in 1988, the stools of all children aged from 2 to 4 were collected and examined in order to look for the presence of parasites in the stools. Two identical inquiries were made in 1990 and 1992. The results show a reduction of the rate of incidence for ascaridiasis by 75% and ankylostomiasis by 82%. They demonstrate the importance of an appropriate evacuation of excreta, of an education of mothers and of domestic hygiene in the process of prevention of children parasitosis.

E3N, a French cohort study on cancer risk factors. E3N Group. Etude Epidémiologique auprès de femmes de l'Education Nationale.

This paper describes the design of E3N, a prospective cohort study conducted in France on risk factors for female cancers. The cohort comprises 100,000 women, aged 40-65 years at baseline in 1990. Participants were asked to complete questionnaires every 18 months. The main hypotheses studied concern the relationship between diet and cancer and between hormonal treatments and cancer. All cancers diagnosed are registered, together with other diseases (cardiovascular diseases, diabetes, osteoporosis). The study population and the follow-up procedure are described.

Lipase and Esterase Activities of Propionibacterium freudenreichii subsp. freudenreichii.

The lipase and esterase activities of eight strains of dairy Propionibacterium freudenreichii subsp. freudenreichii were studied. A lipase activity was detected on whole cells and in the culture supernatant. The highest activity was expressed at 45 degrees C and pH 6.8. An esterase activity was also detected in the culture medium. The electrophoresis of the intracellular fractions of the cells revealed from three to six different esterase activities. Two esterases were common to all the strains. The substrate specificity was dependent on each esterase, but no activity was revealed, in our experimental conditions, on ester substrates with a chain length longer than that of butyrate.

Fractionation of the multiple forms of bovine gastric aspartic proteases by chromatofocusing.

By extending the chromatofocusing technique to a very acidic pH range (down to pH 2.0) a method which, in a single-step procedure, allows separation of the three main aspartic proteases secreted by the bovine abomasal mucosa i.e., chymosin (EC 3.4.23.4), gastricsin (EC 3.4.23.3), and pepsin A (EC 3.4.23.1), has been developed. Starting materials for separation were crude commercial milk-clotting extracts or abomasal juices. A multistep procedure, using narrower pH gradients, enabled the fractionation of these proteases into their multiple forms. Chymosins A and B, which are known to differ only by a single amino acid substitution (Asp/Gly), were completely resolved. Their elution pHs, 3.75 and 3.80, respectively, though far from their "normal" pIs (around 4.7 in isoelectric focusing), demonstrate the resolving power of such a technique. Multiple forms of bovine pepsin A, which differ in their organic phosphate content (0-3 phosphate group(s) per molecule of enzyme) and whose pIs are lower than 2.5, were also separated using 15-20 mM glycine buffer, pH 2.0, as eluent. Although many attempts to get a linear gradient remained unsuccessful within this pH range, resolution appeared quite satisfactory, as judged from analytical isoelectric focusing patterns. In particular, the two subcomponents of bpA1, which presumably have a different site of post-translational phosphorylation, were resolved in this way.

Study of the heterogeneity of bovine pepsin A by pseudo isoelectric focusing.

Pseudo isoelectric focusing (PIEF), i.e. focusing stopped before the equilibrium is reached, has been successfully applied to analyse bovine pepsins A obtained from abomasum mucosa extracts and from 3 individual abomasal juices. Whole bovine pepsin A from mucosa extracts, was resolved in PIEF, using an acidic pH gradient(2-4), into five major and active components. Its fractionation on hydroxyapatite only gave four components which were not homogeneous in PIEF, suggesting that interactions between the different components occur during the chromatographic procedure. After treatment with potato acid phosphatase, whole bovine pepsin A showed only one band in PIEF, displaying enzymic activity and with a mobility identical to that of the less anodic band in untreated pepsin. These results, together with organic phosphate determinations, obviously confirm that the heterogeneity of bovine pepsin A is due to the phosphate content which appears to range between 0 and 3 mol./mol. for the 5 components. Two of them, the phosphate content of which appears to be 1 mol./mol., seem to differ in the location of this phosphate group in the molecule. Whole bovine pepsin A preparations from abomasal juices shared the same pattern in PIEF, identical to that observed with bovine pepsin A from mucosa extracts, thus excluding that dephosphorylation might be involved in the secretory process, as well as disproving the theory that dephosphopepsinogen might be an intermediate in the synthesis of pepsinogen, as it was previously suggested.

["In vitro" effect of medium size molecules on lymphocyte proliferation (author's transl)]. / Effet in vitro des moyennes molécules sur la prolifération lymphocytaire

Eluates from uremic sera were obtained by chromatography on sephadex G 25. They were assayed on lymphocytes cultured in the presence of various stimulants. Lymphocyte proliferation induced by mitogens or allogeneic cells was markedly inhibited in the presence of medium size molecules (MM), or of larger molecules (MW greater than 5000).

Inhibitory effect of medium sized molecules from patients with renal failure on lymphocyte stimulation by phytohemagglutinin.

Middle molecular weight molecules were separated from uremic sera by chromatography on Sephadex G25 fine and assayed on lymphocyte cultures stimulated by phytohemagglutinin. They were found to inhibit markedly the cell proliferation by this mitogen, and this inhibition was still noticeable when cells were incubated for only one hour with eluate, before the culture. These molecules, as well as methylguanidine, might play a part in the cell-mediated immunodeficiency secondary to renal failure.