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Kot W.,Copenhagen University | Hansen L.H.,Copenhagen University | Hansen L.H.,University of Aarhus | Neve H.,Max Rubner Institute | And 6 more authors.
International Journal of Food Microbiology | Year: 2014

Bacteriophages attacking Leuconostoc species may significantly influence the quality of the final product. There is however limited knowledge of this group of phages in the literature. We have determined the complete genome sequences of nine Leuconostoc bacteriophages virulent to either Leuconostoc mesenteroides or Leuconostoc pseudomesenteroides strains. The phages have dsDNA genomes with sizes ranging from 25.7 to 28.4. kb. Comparative genomics analysis helped classify the 9 phages into two classes, which correlates with the host species. High percentage of similarity within the classes on both nucleotide and protein levels was observed. Genome comparison also revealed very high conservation of the overall genomic organization between the classes. The genes were organized in functional modules responsible for replication, packaging, head and tail morphogenesis, cell lysis and regulation and modification, respectively. No lysogeny modules were detected. To our knowledge this report provides the first comparative genomic work done on Leuconostoc dairy phages. © 2014 Elsevier B.V.


PubMed | University of Aarhus, Clerici Sacco Group, Technical University of Denmark, Copenhagen University and Max Rubner Institute
Type: | Journal: International journal of food microbiology | Year: 2014

Bacteriophages attacking Leuconostoc species may significantly influence the quality of the final product. There is however limited knowledge of this group of phages in the literature. We have determined the complete genome sequences of nine Leuconostoc bacteriophages virulent to either Leuconostoc mesenteroides or Leuconostoc pseudomesenteroides strains. The phages have dsDNA genomes with sizes ranging from 25.7 to 28.4 kb. Comparative genomics analysis helped classify the 9 phages into two classes, which correlates with the host species. High percentage of similarity within the classes on both nucleotide and protein levels was observed. Genome comparison also revealed very high conservation of the overall genomic organization between the classes. The genes were organized in functional modules responsible for replication, packaging, head and tail morphogenesis, cell lysis and regulation and modification, respectively. No lysogeny modules were detected. To our knowledge this report provides the first comparative genomic work done on Leuconostoc dairy phages.


Schoster A.,Copenhagen University | Kokotovic B.,Technical University of Denmark | Permin A.,Technical University of Denmark | Pedersen P.D.,Clerici Sacco Group | And 2 more authors.
Anaerobe | Year: 2013

Probiotics have gained importance in human and veterinary medicine to prevent and control clostridial enteric disease. Limited information is available on the ability of different probiotic bacteria used in food products to inhibit Clostridium difficile and Clostridium perfringens. The objective of this study was to examine the in vitro inhibitory effects of selected commercial bacterial strains on pathogenic clostridia and their growth characteristics under simulated gastrointestinal conditions.The inhibitory effects of 17 commercial strains of Lactobacillus (. n = 16) and Bifidobacterium (. n = 1) on the reference strains of C. difficile and C. perfringens were assessed by an agar well diffusion assay and by a broth culture inhibition assay using cell-free supernatant harvested at different growth phases, with and without pH neutralization. To study growth characteristics, probiotic strains were cultivated in different acid and bile environments, and growth in the modified media was compared to growth in standard medium.In the agar well diffusion assay, supernatant obtained from two probiotic strains inhibited the growth of both reference and clinical strains of C. perfringens. This effect as seen when supernatant was assessed with and without pH neutralization. Supernatants obtained from 10 probiotic strains inhibited C. difficile only when supernatant was added without pH neutralization. In the broth culture inhibition assay, growth of C. perfringens and C. difficile was inhibited by supernatant without pH neutralization from 5 and 10 probiotic strains, respectively. All potential probiotic strains were able to grow at pH 4.0 and in the presence of 0.15% and 0.3% bile but none were able to grow or survive at pH 2.0.Altogether five probiotic strains [. Lactobacillus plantarum (. n = 2), Lactobacillus rhamnosus (. n = 2), Bifidobacterium animalis lactis (. n = 1)] were shown to inhibit all strains of C. difficile and C. perfringens. The inhibitory effect was probiotic strain-specific. Two strains showed a pH-independent inhibitory effect likely due to production of either antibiotics or bacteriocins inhibiting C. perfringens only. These strains have favourable growth characteristics for use as probiotics and their efficacy as prophylactic or therapeutic measures against clostridial enteric disease should be further evaluated by clinical trials in animals. © 2013 Elsevier Ltd.


Vigentini I.,University of Milan | Antoniani D.,University of Milan | Roscini L.,University of Perugia | Comasio A.,University of Milan | And 7 more authors.
Food Microbiology | Year: 2014

Candida milleri, together with Candida humilis, is the most representative yeast species found in type I sourdough ecosystems. In this work, comparison of the ITS region and the D1/D2 domain of 26S rDNA gene partial sequences, karyotyping, mtDNA-RFLP analysis, Intron Splice Site dispersion (ISS-PCR) and (GTG)5 microsatellite analyses, assimilation test of different carbohydrates, and metabolome assessment by FT-IR analysis, were investigated in seventeen strains isolated from four different companies as well as in type strains CBS6897T and CBS5658T. Most isolates were ascribed to C.milleri, even if a strong relatedness was confirmed with C.humilis as well, particularly for three strains. Genetic characterization showed a high degree of intraspecific polymorphism since 12 different genotypes were discriminated. The number of chromosomes varied from 9 to 13 and their size ranged from less than 0.3 to over 2Mbp. Phenotypic traits let to recognize 9 different profiles of carbon sources assimilation. FT-IR spectra from yeast cells cultivated in different media and collected at different growth phases revealed a diversity of behaviour among strains in accordance with the results of PCR-based fingerprinting. A clear evidence of the polymorphic status of C.milleri species is provided thus representing an important feature for the development of technological applications in bakery industries. © 2014 Elsevier Ltd.


Malvisi M.,University of Milan | Stuknyte M.,University of Milan | Magro G.,University of Milan | Minozzi G.,University of Milan | And 3 more authors.
Journal of Dairy Science | Year: 2016

Twenty-nine strains of mastitis pathogens were used to study the antibacterial activity of the cell-free supernatants (CFS) of 25 strains of Lactococcus lactis ssp. lactis. Out of the tested strains, only the CFS of L. lactis LL11 and SL153 were active, inhibiting and killing most of the pathogens. By means of ultra-performance liquid chromatography/high resolution mass spectrometry, they were shown to produce nisin A, a class I bacteriocin. A variable sensitivity to nisin A-containing CFS was observed among Streptococcus uberis and Enterococcus faecalis strains. Nonetheless, Streptococcus agalactiae, Strep. uberis, and E. faecalis displayed high minimum inhibitory concentration values, reaching 384 arbitrary units/mL. Interestingly, the minimum inhibitory values and the bactericidal concentrations were almost identical among them for each of the 2 stains, LL11 and SL153. Staphylococci were, on average, less sensitive than streptococci, but the 2 CFS inhibited and killed, at different dilutions, strains of methicillin-resistant Staphylococcus aureus. The immune response to nisin A-containing CFS was tested using the bovine mammary epithelial cell line BME-UV1. Application of CFS did not damage epithelial integrity, as demonstrated by the higher activity of N-acetyl-β-. d-glucosaminidase (NAGase) and lysozyme inside the cells, in both treated and control samples. On the other hand, the increase of released NAGase after 15 to 24 h of treatment with LL11 or SL153 live cultures demonstrated an inflammatory response of epithelial cells. Similarly, a significantly higher lysozyme activity was detected in the cells treated with LL11 live culture confirming the stimulation of lysosomal activity. The treatment of epithelial cells with SL153 live culture induced a significant tumor necrosis factor-α downregulation in the cells, but did not influence IL-8 expression. The control of tumor necrosis factor-α release could be an interesting approach to reduce the symptoms linked to clinical intramammary infections. Due to their antibacterial activity and to the stimulation of lysosomal activity of mammary epithelial cells, the L. lactis strains SL153 and LL11 could be of interest for the development of alternative intramammary treatments to control cow mastitis. © 2016 American Dairy Science Association.


Minervini F.,University of Bari | Siragusa S.,University of Bari | Faccia M.,University of Bari | Dal Bello F.,Clerici Sacco Group | And 2 more authors.
Journal of Dairy Science | Year: 2012

This work aimed to select heat-resistant probiotic lactobacilli to be added to Fior di Latte (high-moisture cow milk Mozzarella) cheese. First, 18 probiotic strains belonging to Lactobacillus casei, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus reuteri were screened. Resistance to heating (65 or 55°C for 10min) varied markedly between strains. Adaptation at 42°C for 10min increased the heat resistance at 55°C for 10min of all probiotic lactobacilli. Heat-adapted L. delbrueckii ssp. bulgaricus SP5 (decimal reduction time at 55°C of 227.4min) and L. paracasei BGP1 (decimal reduction time at 55°C of 40.8min) showed the highest survival under heat conditions that mimicked the stretching of the curd and were used for the manufacture of Fior di Latte cheese. Two technology options were chosen: chemical (addition of lactic acid to milk) or biological (Streptococcus thermophilus as starter culture) acidification with or without addition of probiotics. As determined by random amplified polymorphic DNA-PCR and 16S rRNA gene analyses, the cell density of L. delbrueckii ssp. bulgaricus SP5 and L. paracasei BGP1 in chemically or biologically acidified Fior di Latte cheese was approximately 8.0 log 10cfu/g. Microbiological, compositional, biochemical, and sensory analyses (panel test by 30 untrained judges) showed that the use of L. delbrueckii ssp. bulgaricus SP5 and L. paracasei BGP1 enhanced flavor formation and shelf-life of Fior di Latte cheeses. © 2012 American Dairy Science Association.


PubMed | University of Perugia, Clerici Sacco Group and University of Milan
Type: | Journal: Food microbiology | Year: 2014

Candida milleri, together with Candida humilis, is the most representative yeast species found in type I sourdough ecosystems. In this work, comparison of the ITS region and the D1/D2 domain of 26S rDNA gene partial sequences, karyotyping, mtDNA-RFLP analysis, Intron Splice Site dispersion (ISS-PCR) and (GTG)5 microsatellite analyses, assimilation test of different carbohydrates, and metabolome assessment by FT-IR analysis, were investigated in seventeen strains isolated from four different companies as well as in type strains CBS6897(T) and CBS5658(T). Most isolates were ascribed to C.milleri, even if a strong relatedness was confirmed with C.humilis as well, particularly for three strains. Genetic characterization showed a high degree of intraspecific polymorphism since 12 different genotypes were discriminated. The number of chromosomes varied from 9 to 13 and their size ranged from less than 0.3 to over 2Mbp. Phenotypic traits let to recognize 9 different profiles of carbon sources assimilation. FT-IR spectra from yeast cells cultivated in different media and collected at different growth phases revealed a diversity of behaviour among strains in accordance with the results of PCR-based fingerprinting. A clear evidence of the polymorphic status of C.milleri species is provided thus representing an important feature for the development of technological applications in bakery industries.


PubMed | Clerici Sacco Group and University of Milan
Type: Journal Article | Journal: Journal of dairy science | Year: 2016

Twenty-nine strains of mastitis pathogens were used to study the antibacterial activity of the cell-free supernatants (CFS) of 25 strains of Lactococcus lactis ssp. lactis. Out of the tested strains, only the CFS of L. lactis LL11 and SL153 were active, inhibiting and killing most of the pathogens. By means of ultra-performance liquid chromatography/high resolution mass spectrometry, they were shown to produce nisin A, a class I bacteriocin. A variable sensitivity to nisin A-containing CFS was observed among Streptococcus uberis and Enterococcus faecalis strains. Nonetheless, Streptococcus agalactiae, Strep. uberis, and E. faecalis displayed high minimum inhibitory concentration values, reaching 384 arbitrary units/mL. Interestingly, the minimum inhibitory values and the bactericidal concentrations were almost identical among them for each of the 2 stains, LL11 and SL153. Staphylococci were, on average, less sensitive than streptococci, but the 2 CFS inhibited and killed, at different dilutions, strains of methicillin-resistant Staphylococcus aureus. The immune response to nisin A-containing CFS was tested using the bovine mammary epithelial cell line BME-UV1. Application of CFS did not damage epithelial integrity, as demonstrated by the higher activity of N-acetyl--d-glucosaminidase (NAGase) and lysozyme inside the cells, in both treated and control samples. On the other hand, the increase of released NAGase after 15 to 24h of treatment with LL11 or SL153 live cultures demonstrated an inflammatory response of epithelial cells. Similarly, a significantly higher lysozyme activity was detected in the cells treated with LL11 live culture confirming the stimulation of lysosomal activity. The treatment of epithelial cells with SL153 live culture induced a significant tumor necrosis factor- downregulation in the cells, but did not influence IL-8 expression. The control of tumor necrosis factor- release could be an interesting approach to reduce the symptoms linked to clinical intramammary infections. Due to their antibacterial activity and to the stimulation of lysosomal activity of mammary epithelial cells, the L. lactis strains SL153 and LL11 could be of interest for the development of alternative intramammary treatments to control cow mastitis.

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