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M Garriga

Publications and source records attributed to M Garriga.

At least 19 recordsLinked to original sources
PubMed2006

Safety properties and molecular strain typing of lactic acid bacteria from slightly fermented sausages.

AIM: To evaluate the biodiversity of lactobacilli from slightly fermented sausages (chorizo, fuet and salchichon) by molecular typing, while considering their safety aspects. METHODS AND RESULTS: Species-specific PCR, plasmid profiling and randomly amplified polymorphic DNA (RAPD)-PCR were used to characterize 250 lactic acid bacteria (LAB) isolated from 21 low acid Spanish fermented sausages. Lactobacillus sakei was the predominant species (74%) followed by Lactobacillus curvatus (21.2%) and Leuconostoc mesenteroides (4.8%). By plasmid profiling and RAPD-PCR 144 different strains could be differentiated, 112 belonging to Lact. sakei, 23 to Lact. curvatus and 9 to Leuc. mesenteroides. Ion-pair high performance liquid chromatography was used to detect biogenic amine production. Tyramine and phenylethylamine were produced by 14.4 and 12.4% of the isolates, respectively, all belonging to the species Lact. curvatus. The production of tyramine was stronger than that of phenylethylamine. Partial sequencing of the tyrosine decarboxylase gene from Lact. curvatus was achieved. A specific PCR assay to detect the Lact. curvatus tyramine-producers was designed. The disc diffusion test was used to detect antibiotic resistance among the isolates. Most isolates displayed resistance to vancomycin and gentamicin. Only four strains were resistant to most of the antibiotics tested. None of the isolates were resistant to erythromycin. CONCLUSIONS: Lactobacillus sakei would be the species of choice for further use as starter culture in fermented sausage production. Strain typing and characterization of biogenic amine production together with antibiotic susceptibility testing for the selection of starter cultures could help to increase the quality and safety of the products. SIGNIFICANCE AND IMPACT OF THE STUDY: Species-specific PCR, RAPD and plasmid profiling proved to be efficient at typing LAB at species and strain level. Information on biogenic amine production and transferable antibiotic resistance is important in order to avoid selection of strains with undesirable properties as starter cultures.

Animals↗
PubMed2005

Molecular, technological and safety characterization of Gram-positive catalase-positive cocci from slightly fermented sausages.

The population of Gram-positive catalase-positive cocci from slightly fermented sausages was characterized at species and strain level by molecular techniques and some technological and hygienic aspects were also considered. Staphylococcus xylosus was the predominant species (80.8%) followed by Staphylococcus warneri (8.3%), Staphylococcus epidermidis (5.8%) Staphylococcus carnosus (4.6%), and Kocuria varians (0.4%). Proteolytic activity was observed in 23% of the isolates. The species with the highest percentage of proteolytic strains was S. warneri. Lipolytic activity was found in 45.8% of the isolates and S. xylosus was the species with the highest percentage of lipolytic isolates. Biogenic amine production was not widely distributed (only 14.6% of the isolates). Tyramine was the most intense amine produced, although by only 4.6% of the isolates. Phenylethylamine was more frequently detected (10.8% of isolates) but at lower levels. Some strains also produced putrescine (3.3%), cadaverine (2.9%), histamine (1.3%) and tryptamine (0.4%). All isolates were susceptible to linezolid and vancomicin and over 70% were resistant to penicillin G, ampicillin and sulphonamides. Most of the mecA+ strains (only 4.6% of isolates) also displayed resistance to multiple antibiotics. A reduced enterotoxigenic potential was found. Only 3.3% of isolates showed staphylococcal enterotoxins genes, all identified as entC gene. The combination of RAPD-PCR and plasmid profiling allowed the discrimination of 208 different profiles among the 240 Gram-positive catalase-positive cocci characterized, indicating a great genetic variability.

Animals↗
PubMed2005

Genetic diversity and safety aspects of enterococci from slightly fermented sausages.

AIMS: To determine the biodiversity of enterococci from slightly fermented sausages (chorizo and fuet) at species and strain level by molecular typing, while considering their safety aspects. METHODS AND RESULTS: Species-specific PCR and partial sequencing of 16S rRNA and sodA genes were used to identify enterococcal population. Enterococcus faecium was the most frequently isolated species followed by E. faecalis, E. hirae and E. durans. Randomly amplified polymorphic DNA (RAPD)-PCR revealed species-specific clusters and allowed strain typing. Sixty strains of 106 isolates exhibited different RAPD profiles indicating a high genetic variability. All the E. faecalis strains carried virulence genes (efaAfs, esp, agg and gelE) and all E. faecium isolates carried efaAfm gene. Enterococcus faecalis showed higher antibiotic resistance than the other species. Only one E. faecium strain showed vanA genotype (high-level resistance to glycopeptides) and E. gallinarum and E. casseliflavus/flavescens isolates showed vanC1 and vanC2/C3 genotypes (low-level resistance only to vancomycin) respectively. CONCLUSIONS: E. faecalis has been mainly associated with virulence factors and antimicrobial multi-resistance and, although potential risk for human health is low, the presence of this species in slightly fermented sausages should be avoided to obtain high quality products. SIGNIFICANCE AND IMPACT OF THE STUDY: The enterococcal population of slightly fermented sausages has been thoroughly characterized. Several relevant safety aspects have been revealed.

Base Sequence↗
PubMed2004

Quantification of Listeria monocytogenes in fermented sausages by MPN-PCR method.

AIMS: To combine the principles of most-probable-number (MPN) statistics and the conventional PCR technique to enumerate Listeria monocytogenes in fermented sausages. METHODS AND RESULTS: A simple method to enumerate L. monocytogenes in fermented sausages was developed and compared with direct plating in Palcam agar. Species-specific MPN-PCR, but not direct plating, made the enumeration of L. monocytogenes possible in all assayed samples. CONCLUSIONS: MPN-PCR proved to be a rapid and reliable method for enumerating L. monocytogenes in fermented sausages, including low contaminated samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This MPN-PCR technique may facilitate the enumeration of L. monocytogenes for routine analyses in fermented sausages without excessive work.

Animals↗
PubMed2003

Functionality of enterococci in meat products.

The presence of enterococci in meat fermentation is a constant as reported in the literature. Despite the concern about pathogenicity of enterococci, recent studies point out that food and meat enterococci, especially Enterococcus faecium have a much lower pathogenicity potential than clinical strains. Enterococci possess a competitive advantage over other microbiota in meat fermentations, and many enterococci isolated from sausages have the ability to produce enterocins harbouring antimicrobial activity against pathogens and spoilage microorganisms of meat concern. The application of enterocins producing enterococci or their purified metabolites, as extra hurdles for preservation in sausage fermentation and in sliced-vacuum packed cooked meat products can be beneficial, preventing the outgrowth of Listeria monocytogenes and slime-producing lactic acid bacteria. Enterocins and bacteriocinogenic enterococci hold considerable promise as alternatives to traditional chemical preservatives and they could be exploited for the control of emergent pathogens in meat products. Their inhibitory effect can be increased when used in conjunction with particular physical and chemical processes, but current regulation is hampering the application of purified bacteriocins.

Bacteriocins↗
PubMed2003

Microbial quality and direct PCR identification of lactic acid bacteria and nonpathogenic Staphylococci from artisanal low-acid sausages.

Detection of six species of lactic acid bacteria and six species of gram-positive catalase-positive cocci from low-acid fermented sausages (fuets and chorizos) was assessed by species-specific PCR. Without enrichment, Lactobacillus sakei and Lactobacillus curvatus were detected in 11.8% of the samples, and Lactobacillus plantarum and Staphylococcus xylosus were detected in 17.6%. Enriched samples allowed the detection of L. sakei and S. xylosus in all of the samples (100%) and of Enterococcus faecium in 11.8% of the sausages. The percentages of L. curvatus, L. plantarum, Staphylococcus carnosus, and Staphylococcus epidermidis varied depending on the sausage type. L. curvatus was detected in 80% of fuets and in 57% of chorizos. L. plantarum was found in 50% of fuets and 100% of chorizos. S. epidermidis was detected in only 11.8% of fuets, and S. carnosus was detected in only 5.9% of chorizos. Lactococcus lactis, Staphylococcus warneri, and Staphylococcus simulans were not detected in any sausage type. From a microbiological point of view, 70.6% of the samples could be considered of high quality, as they had low counts of Enterobacteriaceae and did not contain any of the food-borne pathogens assayed.

Base Sequence↗
PubMed2000

Effect of sausage ingredients and additives on the production of enterocin A and B by Enterococcus faecium CTC492. Optimization of in vitro production and anti-listerial effect in dry fermented sausages.

Enterocin A and B in Enterococcus faecium CTC492 were co-induced by the different factors assayed in this study (r = 0.93) and followed primary metabolic kinetics. Enterocin production was significantly inhibited by sausage ingredients and additives, with the exception of nitrate. The addition of sodium chloride and pepper decreased production 16-fold. The temperature and pH influenced enterocin production, with optima between 25 and 35 degrees C, and from 6.0 to 7.5 of initial pH. The maximum activity was achieved, under favourable growth conditions, with MRS supplemented with sucrose (2%) plus glucose (0.25%) and Tween-80 (1%). MRS concentration, NaCl plus pepper addition, absence of Tween-80 in the growth medium, incubation at 45 degrees C and an initial pH under 5.5 were detrimental to bacteriocin production. Stress conditions did not favour enterocin production. Desadsorption was Tween-dependent. Enterocin A activity in the crude extracts stored at -80 degrees C was better preserved than enterocin B (when tested against their specific indicator strain), but anti-listerial activity remained intact. Applied as anti-listerial additives in dry fermented sausages, enterocins significantly diminished Listeria counts by 1. 13 log (P < 0.001), while Enterococcus faecium CTC492 added as starter culture did not significantly reduce Listeria counts (P > 0. 1) compared with the standard starter culture (Bac-). Enterocins A and B could be considered as extra biopreservative hurdles for listeria prevention in dry fermented sausages.

Bacteriocins↗
PubMed2000

Application of enterocins as biopreservatives against Listeria innocua in meat products.

The antilisterial effect of enterocins A and B in meat and meat products (cooked ham, minced pork meat, deboned chicken breasts, pâté, and slightly fermented sausages [espetec]) have been shown. An infective dose of 5 to 10 most probable numbers (MPN)/g to simulate the counts of Listeria generally found in meat products was used. Enterocins at 4,800 AU/g reduced the numbers of Listeria innocua by 7.98 log cycles in cooked ham and by 9 log cycles in pâté when stored at 7 degrees C for 37 days. In deboned chicken breasts stored at 70 degrees C for 7 days, 4,800 AU/cm2 of enterocins diminished the L. innocua counts in 5.26 log cycles when compared to the control batch. In minced pork meat held at 7 degrees C for up to 6 days, 1,600 AU/g kept L. innocua counts under 3 MPN/g, while the control batch reached 50 CFU/g. In espetec sausages, 648 AU/g diminished the number of L. innocua under 50 CFU/g from the fifth day until the end of the process (12 days) while the control batch kept the initial counts (3 x 104 CFU/g). This is the first report on enterocins showing an antilisterial effect in different types of meat products.

Animals↗
PubMed1999

Neuronal expression of the rat M1 muscarinic acetylcholine receptor gene is regulated by elements in the first exon.

Muscarinic acetylcholine receptor genes are members of the G-protein coupled receptor superfamily. Each member of this family studied to date appears to have a distinct expression profile, however the mechanisms determining these expression patterns remain largely unknown. We have previously isolated a genomic clone containing the M1 muscarinic receptor gene and determined its gene structure [Pepitoni, Wood and Buckley (1997) J. Biol. Chem. 272, 17112-17117]. We have now identified DNA elements responsible for driving cell specific expression in transient transfection assays of immortalized cell lines. A region of the gene spanning 974 nucleotides and containing 602 nucleotides of the first exon is sufficient to drive specific expression in cell lines. Like the M4 and M2 gene promoters, the M1 promoter contains an Sp1 motif which can recruit transcription factor Sp1 and at least one other protein, although this site does not appear to be functionally important for M1 expression in our assay. We have identified a region within the first exon of the M1 gene that regulates expression in cell lines, contains several positive and negative acting elements and is able to drive expression of a heterologous promoter. A polypyrimidine/polypurine tract and a sequence conserved between M1 genes of various species act in concert to enhance M1 transcription and are able to activate a heterologous promoter. We show that DNA binding proteins interact in vitro with single-stranded DNA derived from these regions and suggest that topology of the DNA is important for regulation of M1 expression.

5' Untranslated Regions↗
PubMed1999

Lactobacillus salivarius CTC2197 prevents Salmonella enteritidis colonization in chickens.

A rifampin-resistant Lactobacillus salivarius strain, CTC2197, was assessed as a probiotic in poultry, by studying its ability to prevent Salmonella enteritidis C-114 colonization in chickens. When the probiotic strain was dosed by oral gavage together with S. enteritidis C-114 directly into the proventriculus in 1-day-old Leghorn chickens, the pathogen was completely removed from the birds after 21 days. The same results were obtained when the probiotic strain was also administered through the feed and the drinking water apart from direct inoculation into the proventriculus. The inclusion of L. salivarius CTC2197 in the first day chicken feed revealed that a concentration of 10(5) CFU g(-1) was enough to ensure the colonization of the gastrointestinal tract of the birds after 1 week. However, between 21 and 28 days, L. salivarius CTC2197 was undetectable in the gastrointestinal tract of some birds, showing that more than one dose would be necessary to ensure its presence till the end of the rearing time. Freeze-drying and freezing with glycerol or skim milk as cryoprotective agents, appeared to be suitable methods to preserve the probiotic strain. The inclusion of the L. salivarius CTC2197 in a commercial feed mixture seemed to be a good way to supply it on the farm, although the strain showed sensitivity to the temperatures used during the feed mixture storage and in the chicken incubator rooms. Moreover, survival had been improved after several reinoculations in chicken feed mixture.

Administration, Oral↗
PubMed1998

Selection of lactobacilli for chicken probiotic adjuncts.

During inhibitory activity screening of 296 strains of lactic acid bacteria from the gastro-intestinal tract of chicks, 77 strains showed inhibition against enteric indicator strains (Salmonella enteritidis and Escherichia coli). Eight different strains identified as Lactobacillus salivarius were selected for the following attributes: their ability to inhibit all the indicator strains; a high adhesion efficiency to the epithelial cells of chickens and also their resistance to a number of antibiotics, monensin, bile salts and pH 3.0. The inhibitory action was not affected by the addition of catalase and no inhibition was detected after neutralizing the supernatant culture fluid. The competitiveness of the most promising strains, Lact. salivarius CTC2183 and CTC2197, was assessed in chicken feed mixture and in vivo. It was concluded that both strains were capable of becoming predominant over the indigenous flora in the incubated chicken feed mixture. In vivo tests showed that Lact. salivarius CTC2197 was able to colonize and overcome Lact. salivarius CTC2183 and the indigenous flora in the crop and caecum of the inoculated chicks.

Animal Feed↗
PubMed1998

Carnimonas nigrificans gen. nov., sp. nov., a bacterial causative agent for black spot formation on cured meat products.

Nine different strains, CTCBS1T or CTCBS9, were identified to be the causative agents of black spots on the surface of raw cured meat products. The formation of black spots under aerobic conditions is reproducible upon reinoculation of meat products with any of these strains, indicating that they are the causative agent. The strains were Gram-negative, catalase-positive and obligately aerobic rods. The G+C content of DNA of strain CTCBS1T is 56.0 +/- 0.3 mol%. The content of non-polar main fatty acids were 16:0, 16:1, 18:1 and 19:0 cyc. Its phylogenetic position was elucidated by comparative sequence analysis of the 16S rRNA gene. Overall sequence similarity to other bacteria does not exceed 93.3%. Isolate CTCBS1T clustered phylogenetically within the gamma-subclass of the Proteobacteria and is closely related to members of Halomonas (90:5-91.9%) and to Zymobacter palmae (93.3%). A genetic homogeneity of the nine strains was demonstrated by M13 random amplified polymorphic DNA-PCR, whereas differentiation from other genera, e.g. Zymobacter and Pseudomonas, could easily be achieved by their chemotaxonomic characteristics. Taxonomic data revealed the status of a separate genus for which the name Carnimonas gen. nov., sp., nov. is proposed. Despite chemotaxonomic and physiological similarities, the new genus is at present not a member of the family Halomonadacease because of the lack of two out of 15 descriptive 16S rRNA signature sequences. The first member of the new genus is Carnimonas nigrificans. The use of a specific, 16S rRNA-targeted oligonucleotide primer allowed the identification of all nine strains of C. nigrificans in a PCR assay. Toxicological studies showed no pathogenic potential for C. nigrificans strain CTCBS1T (CECT 4437T).

Animals↗
PubMed1996

Technological and sensorial evaluation of Lactobacillus strains as starter cultures in fermented sausages.

The performance of several lactobacilli strains isolated from naturally fermented sausages as starter cultures was evaluated. Microbiological, physical and chemical changes, in addition to sensorial aspects were studied. From the 12 different strains tested, 10 were capable of leading the fermentation in every batch throughout the process. The monitoring of the inoculated strains was easily carried out by plasmid profiles and by checking the pH rate drop of the sausages, which was slower for the non-inoculated lots. Treatment using natural fermentation resulted in a product where hydrogen sulphide odours, which could be related to the higher content of Enterobacteriaceae throughout the ripening process, diminished its overall acceptability. The lots seeded with different L. sake strains were found to be low in acid in sensory evaluation, correlating with a low lactic acid content. In contrast, the L. plantarum lot gave rise to an overacidified product related to having the highest amount of lactic acid at the end of the process. As a general rule lactobacilli strains isolated from meat origins are good candidates as starter cultures in the manufacture of dry-fermented sausages and produce satisfactory products depending on the specific strains used more than on the species.

Fermentation↗
PubMed1996

Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins.

A new bacteriocin has been isolated from an Enterococcus faecium strain. The bacteriocin, termed enterocin A, was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and mass spectrometry analysis. By combining the data obtained from amino acid and DNA sequencing, the primary structure of enterocin A was determined. It consists of 47 amino acid residues, and the molecular weight was calculated to be 4,829, assuming that the four cysteine residues form intramolecular disulfide bridges. This molecular weight was confirmed by mass spectrometry analysis. The amino acid sequence of enterocin A shared significant homology with a group of bacteriocins (now termed pediocin-like bacteriocins) isolated from a variety of lactic acid-producing bacteria, which include members of the genera Lactobacillus, Pediococcus, Leuconostoc, and Carnobacterium. Sequencing of the structural gene of enterocin A, which is located on the bacterial chromosome, revealed an N-terminal leader sequence of 18 amino acid residues, which was removed during the maturation process. The enterocin A leader belongs to the double-glycine leaders which are found among most other small nonlantibiotic bacteriocins, some lantibiotics, and colicin V. Downstream of the enterocin A gene was located a second open reading frame, encoding a putative protein of 103 amino acid residues. This gene may encode the immunity factor of enterocin A, and it shares 40% identity with a similar open reading frame in the operon of leucocin AUL 187, another pediocin-like bacteriocin.

Amino Acid Sequence↗
PubMed1993

Biochemical characterization of lactobacilli from dry fermented sausages.

The characterization of 254 strains of lactobacilli isolated from dry spontaneously fermented sausages from 15 different producers at two different stages of ripening time is reported. The species identified were Lactobacillus sake 55%, L. curvatus 26%, L. bavaricus 11% and L. plantarum 8%. The main criteria for the identification of isolates to species level were: production of lactic acid isomers, presence of mDpm acid in cell walls, deamination of arginine and fermentation of mannitol and melibiose. The composition of the populations of lactobacilli were the same for the two stages of ripening. The deamination of arginine was tested in aerobic and anaerobic cultures and in different media by checking the production of ammonia and detecting the production of citrulline. In 94% of strains tested both methods gave identical results. In two L. sake strains arginine catabolism was dependent on culture media; for two other L. sake strains the deamination of arginine only occurred when oxygen was scarce.

Animals↗
PubMed1993

Bacteriocinogenic activity of lactobacilli from fermented sausages.

During the screening of the inhibitory activity of 254 strains of lactobacilli isolated from fermented sausages at different times of ripening, 22% of the strains showed inhibition that was not related to acid or hydrogen peroxide, towards one or more indicator strains. Not all the strains were capable of secreting the inhibitory compound in the supernatant fluid. The characterization of the inhibitory compound from three strains showed that they were bacteriocins with a bactericidal mode of action and a molecular weight exceeding 10,000 Da. Lactobacillus plantarum CTC 305, CTC 306 and Lact. sake CTC 372 inhibited Listeria monocytogenes. Lactobacillus sake CTC 372 was cured of two plasmids of 84.8 kbp and 41.3 kbp, losing the production and the immunity of a bacteriocin as well as the ability to ferment lactose.

Bacteriocins↗