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A numerical taxonomic study of lactic acid bacteria from vacuum-packed beef, pork, lamb and bacon.

A numerical taxonomic study using 79 unit characters has been performed on 100 isolates of lactic acid bacteria from refrigerated vacuum-packed beef, pork, lamb and bacon. Three clusters were observed at 78% S which contained all the strains apart from three unidentifiable streptobacteria, one Leuconostoc, and one strain of Pediococcus pentosaceus. One cluster (III) consisted of only one strain of Leuc. paramesenteroides and six unidentifiable Leuconostoc strains. The two largest clusters (I and II) were both composed entirely of streptobacteria. Cluster I contained 31 strains (G + C content 33.2-36.9 moles%) which were not identifiable with any described species. Cluster II contained 57 strains (G + C content 40.7-43.7 moles %) which were provisionally identified with Lactobacillus sake or Lact. bavaricus according to the lactic acid isomer produced. The division of nearly all the streptobacteria into two clearly defined clusters has resolved problems which have existed in the classification of lactic acid bacteria from vacuum-packed meat.

Animals↗

Microflora of the aerobic preservation of directly brined green olives from Hojiblanca cultivar.

New procedures for the preservation stage of ripe olives from Hojiblanca cultivar were studied. An aerobic fermentative process was used with initial pH correction (0.3% acetic acid) and various NaCl concentrations: 6, 3 and 0% (w/v) in tap water. Treatments were carried out at industrial level and the spontaneous changes monitored. At initial salt concentrations of 6 and 3% (w/v) NaCl, pH rose progressively, reaching 4.3 at equilibrium maintaining during this period a constant free lactic acidity of around 0.4% (w/v). When the initial solution was tap waste, however, the pH decreased rapidly to stabilize at about 3.7, and lactic acidity increased continuously to reach values over 1% (w/v) at the end of the preservation process. In all treatments aeration effectively purged the carbon dioxide from the preservation brines, preventing shrivelling of olives. The microbial growth was strongly influenced by the initial NaCl concentration. At 6 and 3%, only yeasts grew, the most abundant being Pichia membranaefaciens, P. vini, P. fermentans and Hansenula polymorpha. However, when there was no NaCl, lactic acid bacteria colonized the solution. Lactobacillus plantarum and Pediococcus inopinatus were the only species found. In this case there was a co-existence between yeasts and lactic acid bacteria. As the treatment that supported lactic acid bacteria achieved the best final pH and acidity for olive stability, it may help to overcome the obstacles to a lactic fermentative process during the preservation stage of ripe olives from the Hojiblanca cultivar.

Food Microbiology↗

Detection of resistance of lactic acid bacteria to a mixture of the hop analogue compounds tetrahydroiso-alpha-acids by noninvasive measurement of intracellular pH.

AIMS: To examine the resistance of beer isolates of lactic acid bacteria (LAB) towards a mixture of tetrahydroiso-alpha-acids (Tetra) by growth experiments as well as by measurement of intracellular pH. METHODS AND RESULTS: Beer LAB isolates were identified to species level by SDS-PAGE of whole-cell proteins. Beer isolates of Lactobacillus brevis showed better ability for growth in the presence of Tetra than nonbeer isolates of the L. brevis or other species of LAB including beer and nonbeer isolates. The antimicrobial effect of Tetra was also examined by noninvasive measurement of intracellular pH by fluorescence ratio imaging microscopy for selected beer isolates of L. brevis and Pediococcus inopinatus. Strains of L. brevis showing limited decrease of intracellular pH during exposure to Tetra also showed better ability for growth in the presence of these compounds as well as in commercial beer products. CONCLUSIONS: It was possible to apply a method for noninvasive measurement of intracellular pH to predict the resistance of beer spoilage LAB towards the Tetra hop analogue compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the usability of a new rapid method for detecting hop-resistant variants of known beer spoilage LAB species.

Beer↗

Identification and production of a bacteriocin from Enterococcus mundtii QU 2 isolated from soybean.

AIMS: Identification of the bacteriocin produced by Enterococcus mundtii QU 2 newly isolated from soybean and fermentative production of the bacteriocin. METHODS AND RESULTS: The bacteriocin produced by Ent. mundtii QU 2 inhibited the growth of various indicator strains, including Enterococcus, Lactobacillus, Leuconostoc, Pediococcus and Listeria. The bacteriocin activity was stable at wide pH range and against heat treatment, but completely abolished by proteolytic enzymes. The bacteriocin was purified from the culture supernatant by the three-step chromatographic procedure. Mass spectrometry, amino acid sequencing and DNA sequencing revealed that the bacteriocin was similar to class IIa bacteriocins produced by other Ent. mundtii strains. The bacteriocin production decreased in the absence of glucose, nitrogen sources, or Tween 80 in MRS medium. Additionally, it was strongly suppressed by addition of Ca(2+) (CaCO(3) or CaCl(2)). In pH-controlled fermentations, the highest bacteriocin production was achieved at pH 6.0, whereas the highest cell growth was obtained at pH 7.0. CONCLUSIONS: Ent. mundtii QU 2 produced a class IIa bacteriocin. Some growth factors (e.g. Ca(2+) and pH) influenced the bacteriocin production. SIGNIFICANCE AND IMPACT OF THE STUDY: A new soybean isolate, Ent. mundtii QU 2 was found to be a class IIa bacteriocin producer. Factors influencing the bacteriocin production described herein are valuable for applications of the bacteriocins from Ent. mundtii strains.

Amino Acid Sequence↗

Protein modification by fermentation: effect of fermentation on the potential allergenicity of pea.

The effect of fermentation on components of potential significance for the allergenicity of pea was analyzed. Pea flour was fermented with three lactic acid bacteria, Pediococcus pentosaceus, Lactococcus raffinolactis, and Lactobacillus plantarum, and two fungi, Rhizopus microsporus, var. oligosporus and Geotrichum candidum. Residual antigenicity against antipea antibodies was reduced to 10% by the three lactic acid bacteria and R. microsporus. Reactions to anti-pea profilin and anti-Bet v 1 were still detectable after fermentation. The contents of lectin and pea protease inhibitor were not reduced by the microorganisms.

Animals↗

Biodiversity of Lactobacillus sanfranciscensis strains isolated from five sourdoughs.

AIMS: Five different sourdoughs were investigated for the composition of lactic acid bacteria (LAB) and the biodiversity of Lactobacillus sanfranciscensis strains. METHODS AND RESULTS: A total of 57 strains were isolated from five sourdoughs. Isolated strains were all identified by the 16S rDNA sequence and species-specific primers for L. sanfranciscensis. Results of identification showed that LAB strains were L. sanfranciscensis, Lactobacillus plantarum, Lactobacillus paralimentarius, Lactobacillus fermentum, Lactobacillus pontis, Lactobacillus casei, Weisella confusa and Pediococcus pentosaceus. A total of 21 strains were identified as L. sanfranciscensis and these isolates were detected in all five sourdoughs. Ribotyping was applied to investigate the relationship between intraspecies diversity of L. sanfranciscensis and sourdough. A total of 22 strains of L. sanfranciscensis including L. sanfranciscensis JCM 5668T were compared by ribotyping. The dendrogram of 21 ribotyping patterns showed four clusters, and L. sanfranciscensis JCM 5668T was independent of the others. The different biotypes of L. sanfranciscensis were present in two sourdoughs compared with other three sourdoughs. CONCLUSIONS: The LAB compositions of five sourdoughs were different and the relationship between intraspecies diversity of L. sanfranciscensis strains and five sourdoughs was shown by ribotyping. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that ribotyping was useful for distinguishing L. sanfranciscensis strains. A further important result is that the intra-species diversity of L. sanfranciscensis strains seems to be related to the sourdough preparation.

Fermentation↗

Isolation and characterization of lactic acid bacteria from dochi (fermented black beans), a traditional fermented food in Taiwan.

AIMS: To isolate, characterize and identify lactic acid bacteria (LAB) in dochi (fermented black beans), a traditional fermented food in Taiwan. METHODS AND RESULTS: A total of 30 samples were collected from three different dochi producers and analysed after different periods of storage. Fifty-two cultures of LAB were isolated from dochi samples and the isolates were divided into classes by phenotype and then into groups by restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Phenotypic and biochemical characteristics identified six different bacterial groups (A-F) and showed that the majority of the isolates were homofermentative LAB. Enterococcus faecium was the most abundant of the dochi-isolated LAB. All isolated LAB were able to grow in MRS broth containing 6% NaCl, but only Enterococcus, Pediococcus and Tetragenococcus species could grow in MRS broth containing 10% NaCl. Furthermore, antibacterial activities of isolates were determined, and four isolates showed inhibitory activities against the indicator strain Lactobacillus sakei JCM 1157(T). CONCLUSIONS: These results suggest that Ent. faecium is the main LAB present during the fermentation of dochi. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the distribution and varieties of LAB that exist in the dochi fermentation process.

Antibiosis↗

Vanillin production from simple phenols by wine-associated lactic acid bacteria.

AIMS: The ability of lactic acid bacteria (LAB) to metabolize certain phenolic precursors to vanillin was investigated. METHODS AND RESULTS: Gas chromatography-mass spectrometry (GC-MS) or HPLC was used to evaluate the biosynthesis of vanillin from simple phenolic precursors. LAB were not able to form vanillin from eugenol, isoeugenol or vanillic acid. However Oenococcus oeni or Lactobacillus sp. could convert ferulic acid to vanillin, but in low yield. Only Lactobacillus sp. or Pediococcus sp. strains were able to produce significant quantities of 4-vinylguaiacol from ferulic acid. Moreover, LAB reduced vanillin to the corresponding vanillyl alcohol. CONCLUSIONS: The transformation of phenolic compounds tested by LAB could not explain the concentrations of vanillin observed during LAB growth in contact with wood. SIGNIFICANCE AND IMPACT OF THE STUDY: Important details of the role of LAB in the conversion of phenolic compounds to vanillin have been elucidated. These findings contribute to the understanding of malolactic fermentation in the production of aroma compounds.

Benzaldehydes↗

Usefulness of length heterogeneity-PCR for monitoring lactic acid bacteria succession during maize ensiling.

The use of length-heterogeneity PCR was explored to monitor lactic acid bacteria succession during ensiling of maize. Bacterial diversity was studied during the fermentation of 30-day-old maize in optimal and spoilage-simulating conditions. A length heterogeneity PCR profile database of lactic acid bacteria isolated from the silage and identified by 16S rRNA gene sequencing was established. Although interoperonic 16S rRNA gene length polymorphisms were detected in some isolates, strain analysis showed that most of the lactic acid bacteria species thriving in silage could be discriminated by this method. The length heterogeneity PCR profiles of bacterial communities during maize fermentation were compared with those on a database. Under optimal fermentation conditions all the ecological indices of bacterial diversity, richness and evenness, deduced from community profiles, increased until day thirteen of fermentation and then decreased to the initial values. Pediococcus and Weissella dominated, especially in the first days of fermentation. Lactococcus lactis ssp. lactis and Lactobacillus brevis were mainly found after six days of fermentation. A peak corresponding to Lactobacillus plantarum was present in all the fermentation phases, but was only a minor fraction of the population. Unsuitable fermentation conditions and withered maize leaves in the presence of oxygen and water excess caused an enrichment of Enterococcus sp. and Enterobacter sp.

Base Sequence↗

Identification of the replication region of Lactobacillus acidophilus plasmid pLA103.

The structure of the region necessary for replication of the plasmid pLA103 from Lactobacillus acidophilus TK8912 has been characterized. Sequence analysis revealed that the replication region contained an open reading frame (OrfA) encoding a 282-amino acid peptide preceded by a 22-bp tandem repeat sequence region. The predicted OrfA protein showed homology to the replication protein of a plasmid from Pediococcus halophilus. The plasmid containing the repeat sequence region preceding OrfA was able to replicate in the Lactobacillus host when provided with OrfA in trans, suggesting that the repeat sequence region contains the origin sequence essential for the pLA103 replication.

Amino Acid Sequence↗

Purification and partial characterization of an antigen specific to Lactobacillus brevis strains with beer spoilage activity.

Certain Lactobacillus brevis strains are resistant to hop-derived compounds such as isohumulone and are able to grow in beer. In this study, we raised an antiserum against our beer spoilage laboratory strain L. brevis 578 which reacted with 23 of 24 beer spoilers and two of 13 non-spoilers in precipitation reactions using 0.5 M NaOH cell extracts. This specific antigen to the beer spoilage L. brevis strains (SABSL) was demonstrated to be located beneath the S-layer proteins by agglutination reactions using S-layer protein-stripped cells obtained by treatment with 0.1 M NaOH. SABSL was purified using an affinity column coupled with an antibody against SABSL. The purified antigen was hydrolyzed with 2 M HCl and the hydrolyzate was analyzed by thin-layer chromatography and enzymatic analysis. The results showed that SABSL contains glycerol, phosphate, glycerophosphate, D-galactose and D-glucose. D-Galactose and D-glucose accounted for 4.7% and 0.1% of the composition, respectively. Melibiose, but not mannose, inhibited the precipitation reaction. Intense precipitation reactions were obtained with fractions which did not bind to the ConA-column. These results indicate that the immunodominant component of the SABSL is galactose and the SABSL determinant is most probably a galactosylated glycerol teichoic acid. The antiserum raised against the beer spoilage strain L. brevis 578 could distinguish between Pediococcus beer spoilers and non-spoilers in precipitation reactions.

Animals↗

Enterococcal-type glycopeptide resistance genes in non-enterococcal organisms.

Although the emergence of vancomycin-resistant enterococci can be attributed, in part, to the increasing use of vancomycin in clinical practice, and glycopeptide use in animal husbandry, the origins of the enterococcal vancomycin resistance genes are not clear. The vancomycin resistance-associated genes in Enterococcus gallinarum, Enterococcus casseliflavus/flavescens, Lactobacillus spp., Leuconostoc spp., Pediococcus spp., and Erysipelothrix rhusiopathiae, are not the source of the high-level vancomycin resistance-associated genes in enterococci. There are, however, environmental organisms which have been found to have gene clusters homologous to the enterococcal vanA, vanB and vanC gene clusters; these include the biopesticide Paenibacillus popilliae, and, to a lesser extent, the glycopeptide-producing organisms Amycolatopsis orientalis and Streptomyces toyocaensis. Still, the exact sources of the enterococcal vancomycin resistance genes remain a mystery.

Anti-Bacterial Agents↗

A PCR-based method for identification of lactobacilli at the genus level.

We developed a polymerase chain reaction (PCR)-based method for the identification of lactobacilli at the genus level. One specific primer, LbLMA1-rev, was designed by analysing similarities between the nucleotide sequence of the spacer between the 16S and 23S rRNA genes in a number of Lactobacillus strains. Amplification with LbLMA1-rev and R16-1, a universal primer, generated a PCR product for 23 Lactobacillus species. Electrophoresis did not reveal any discrete bands when Escherichia coli, Lactococcus lactis, Leuconostoc mesenteroides, Streptococcus thermophilus, Carnobacterium pissicola, Pediococcus pentosaceus, Bifidobacterium bifidum, Weissella confusa, Enterococcus hirae, Staphylococcus aureus or Listeria monocytogenes DNA were used as template.

Base Sequence↗

Sparing of folinic acid by thymidine.

Thymidine, which is inactive when added alone to Pediococcus cerevisiae (Leuconostoc citrovorum) 8081, was found to increase its growth in the presence of folinic acid. The addition of thymidine increased both the sensitivity of the folinic acid assay and the total growth yield. The mechanism of the synergistic effect of thymidine is briefly discussed.

Folic Acid↗

Daptomycin susceptibility of unusual gram-positive bacteria: comparison of results obtained by the Etest and the broth microdilution method.

MICs of daptomycin, linezolid, and vancomycin against 212 isolates, including Listeria monocytogenes and Pediococcus, Leuconostoc, Rhodococcus, and Nocardia spp., were determined by the broth microdilution method; daptomycin MICs were also determined by the Etest. Except with those for Leuconostoc spp., daptomycin Etest MICs showed >90% agreement with MICs obtained by the broth microdilution method.

Culture Media↗

Dihydrofolate reductases in some folate-requiring bacteria with low trimethoprim susceptibility.

Dihydrofolate reductases from the folate-requiring strains Streptococcus faecalis ATCC 8043, Lactobacillus casei ATCC 7496, and Pediococcus cerevisiae ATCC 8081, as well as from Lactobacillus arabinosus, which is not dependent on exogenous folate, were isolated, and their properties were compared to reductases of Escherichia coli B, Staphylococcus aureus, and rat liver reductase. An inhibition profile with six different inhibitors revealed significant differences among all enzymes. All lactobacilli reductases are less sensitive to trimethoprim than the enzymes of E. coli and S. aureus, the reductase of P. cerevisiae requiring a concentration at least 1,000 times higher for 50% inhibition. Inhibition of growth of S. faecalis by pyrimethamine and 2,4-diamino-6,7-diisopropyl-pteridine was seen to be much stronger than was predicted from the enzymatic data.

Bacteria↗

In vitro activities of daptomycin and other antimicrobial agents against vancomycin-resistant gram-positive bacteria.

A comparative evaluation of daptomycin and eight other antimicrobial agents was performed by the agar dilution technique with 56 strains of vancomycin-resistant gram-positive bacteria, including Leuconostoc, Lactobacillus, and Pediococcus spp. Erythromycin, deptomycin, clindamycin, and gentamicin exhibited the greatest activities, whereas penicillin, ampicillin, and cefotaxime showed moderate activities. The organisms were all highly resistant to vancomycin and cefoxitin.

Anti-Bacterial Agents↗

In vitro activity of RP59500, an injectable streptogramin antibiotic, against vancomycin-resistant gram-positive organisms.

The in vitro activity of RP59500, a streptogramin antibiotic, against 146 clinical isolates of vancomycin-resistant gram-positive bacteria was examined. Five strains of the species Enterococcus casseliflavus and Enterococcus gallinarum, for which the MIC of vancomycin was 8 micrograms/ml, were also studied. Twenty-eight vancomycin-susceptible strains of Enterococcus faecalis and Enterococcus faecium were included for comparison. The drug was highly active against Leuconostoc spp., Lactobacillus spp., and Pediococcus spp. (MICs, < or = 2 micrograms/ml). RP59500 was more active against vancomycin-susceptible strains of E. faecium than E. faecalis (MICs for 90% of the strains [MIC90s], 1.0 versus 32 micrograms/ml). Vancomycin-resistant strains of E. faecalis were as resistant to RP59500 as vancomycin-susceptible strains (MIC90, 32 micrograms/ml), but some vancomycin-resistant E. faecium strains were relatively more resistant to the new agent (MIC90, 16; MIC range, 0.5 to 32 micrograms/ml) than were vancomycin-susceptible organisms of this species.

Drug Resistance, Microbial↗