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

S haw B. G. & H arding C harmaigne D. 1984. A numerical taxonomic study of lactic acid bacteria from vacuum packed beef, pork, lamb and bacon. Journal of Applied Bacteriology 56 , 25–40.
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.     

Bacteriophages induced by mitomycin C treatment of Leuconostoc oenos strains from Portuguese wines

Twenty-two Leuconostoc oenos strains from Portuguese wines and seven strains from other sources were induced with mitomycin C. Bacteriophages were detected in the supernatants of 19 different cultures. Analysis of their host-range and plating efficiencies suggests that phage typing could be useful for strain identification in Leuc. oenos.     

Sugar and organic acid metabolism in mixed cultures of Pediococcus pentosaceus and Leuconostoc oenos isolated from wine

Pediococcus pentosaceus 12p and Leuconostoc oenos X₂L isolated from Argentinian wine were examined for growth and changes in the concentrations of glucose, fructose, sucrose and mannitol and malic, citric, acetic and lactic acids in pure and mixed cultures. In mixed cultures a mutualistic growth response and a change in the balance of end-products of sugar and organic acid metabolism were observed. The production of mannitol and acetic acid was lower while D(-) and L(+) lactic acids were detected in higher levels than in pure cultures. Malic and citric acids were metabolized simultaneously, but the amount of citric acid consumed was lower than in pure culture of Leuc. oenos.     

Effect of pH and age of culture on cellular fatty acid composition of Leuconostoc oenos

Z. DRICI-CACHON, J.F. CAVIN AND C. DIVIÈS. 1996. This study is concerned with the fatty acid composition of three strains of Leuconostoc oenos grown at different p^H. The most abundant fatty acids were C18: 1 w ⁹, C19: 0 cy( w ^9,10) and C16:0, followed by C16: 1 w ⁹ and C14: 0. The p^H considerably modified the fatty acid distribution in Lo107 (an acidophilic strain) and Lo8413 (a moderately acidophilic strain). However, moderate changes occurred for LoATCC 23277 (a less acidophilic strain). At p^H 2.9, Lo107 has a remarkably high level of C19: 0 cy-( w ^9,10) and C19:0 cy( w ^11,12). Proportions of C18: 1 and C19:0 cyclo acids varied mainly with the p^H of the medium and also as a function of growth phase. The degree of unsaturation of fatty acids also varied with p^H.     

Lactic acid bacteria found in fermented fish in Thailand

Forty-seven strains of homofermentative rod-shaped and 5 heterofermentative sphere-shaped lactic acid bacteria were isolated from 4 kinds of fermented fish (pla-ra, pla-chom, kung-chom, and hoi-dong) in Thailand. These bacteria were separated into four groups by phenotypic and chemotaxonomic characteristics, including fluorometric DNA-DNA hybridization. Five strains (Group I) contained meso-diaminopimelic acid in the cell wall. Four strains were identified as Lactobacillus pentosus, and one strain was L. plantarum. Tested strains of this group produced DL-lactic acid. The rest of the rod-shaped bacteria, 23 strains (Group II) and 19 strains (Group III), lacked meso-diaminopimelic acid in the cell wall and were identified as L. farciminis and Lactobacillus species, respectively. The tested strains of these groups produced L-lactic acid. The amount of cellular fatty acids of C16:0 and C18:1, and the DNA base compositions were significant for differentiating the strains in Groups II and III. Five strains of cocci in chains (Group IV) produced gas from glucose. The tested strains of this group produced d-lactic acid. They were identified as a Leuconostoc species. The distribution of these bacteria in fermented fish in Thailand is discussed.     

Factors affecting the induction of malolactic fermentation in red wines with Leuconostoc oenos

Malolactic fermentation was induced in red wines by inoculation with several strains of Leuconostoc oenos . The progress of Malolactic Fermentation was monitored by following the kinetics of bacterial growth and degradation of malic acid. These kinetics varied significantly depending on the strain of Leuc. oenos inoculated, the strain of Saccharomyces cerevisiae used to conduct the alcoholic fermentation, and the wine properties of pH and concentrations of ethanol and sulphur dioxide. Rapid, predictable malolactic fermentation was achieved by inoculating a high density (> 10⁶ cfu/ml) of Leuc. oenos , whereby malic acid degradation was not connected to the growth of the bacterial cells. Wines after malolactic fermentation were not bacteriologically stable and supported the growth of Leuc. oenos inoculated into the wines.     

Utilization of glucose, fructose and malic acid by malolactic bacteria: effect of ethanol and formation of mannitol and volatile acids

Five lactic acid bacteria capable of carrying out the malolactic fermentation were studied at 15°C and 25°C. Results indicated that at 15°C hardly any glucose, fructose and l -malic acid was utilized over a 9d period. After 9d at 25°C very little glucose and fructose and most (if not all) of the l -malic acid was degraded. The addition of ethanol markedly affected the l -malic acid utilization by the four Leuconostoc oenos strains at 25°C. Large differences in their ability to convert l -malic acid were found amongst the four strains in media containing 5% and 10%(v/v) ethanol.     

Specific enumeration of lactic acid bacteria in fermenting grape must and wine by colony hybridization with non-isotopic DNA probes

A. LONVAUD-FUNEL, A. JOYEUX AND O. LEDOUX. 1991. Total DNA extracted from lactic acid bacteria commonly found in musts and wines was randomly labelled with digoxigenin. It was assayed for the detection of several species by dot-blot hybridization. The method proved to be specific as there was no cross-hybridization between most of the species belonging to the genera Leuconostoc, Pediococcus and Lactobacillus , homofermentative and heterofermentative ( Lact. plantarum, Lact. casei, Leuc. mesenteroides, Leuc. oenos, Ped. damnosus, Ped. pentosaceus ). However, it failed for some Lact. brevis strains which strongly hybridized with Lact. hilgardii.
Colony hybridization was performed directly on plates soon after enumeration. Eight probes of the most common species were used; it was possible to follow the evolution of each species during the vinification of two red wines. According to the phase of alcoholic fermentation, then malolactic fermentation, the predominance or regression of bacilli and cocci could be established.     

Inter-strain relationships among wine leuconostocs and their divergence from other Leuconostoc species, as revealed by low frequency restriction fragment analysis of genomic DNA

Thirty Leuconostoc oenos strains, representing 28 different isolates, were distributed into 20 genomic groups according to PFGE patterns of restriction digests. The 8 bp-specific enzymes Sfi I, Not I and Asc I cleaved the Leuc. oenos DNA in a mean of 17, 11 and four fragments respectively and Sma I produced more than 50 fragments per genome. The strain differentiating capacity of the four enzymes was similar; only two related genomic groups failed to be distinguished by Asc I or Not I. Genomic relationships between Leuc. oenos strains were quantified by numerical analysis of Not I and Sfi I banding patterns. More than half of the strains, including the starters ML34 and PSU-1, formed a major cluster. The average size of the Leuc. oenos genome was estimated as 1.86 Mb. Although similar values were obtained for the genomes of Leuc. mesenteroides, Leuc. pseudomesenteroides, Leuc. gelidum and Leuc. citreum, a significant divergence between wine and non-wine species was inferred from comparisons of genome cleavage frequencies, determined with five different enzymes.     

Mixed culture of Lactobacillus hilgardii and Leuconostoc oenos isolated from Argentine wine

Growth, sugar and organic acid metabolism of Lactobacillus hilgardii X₁B and Leuconostoc oenos X₂L isolated from Argentinian wine were examined in pure and mixed cultures. In mixed culture Leuc. oenos X₂L did not grow, no viable cells were detected after 24 h, but the consumption of glucose and fructose by Lact. hilgardii was higher. An increase of mannitol and acetic acid production was detected during the early stages of growth. Over 12 h, higher levels of d (-) lactic acid and slightly increased levels of l (+) lactic acid were observed. Citric and malic acid were simultaneously metabolized, but a slight increase in citric acid consumption was observed in mixed culture.     

Phenotypic diversity of lactic acid bacteria isolated from fermented sausages produced in Basilicata (Southern Italy)

AIMS: to evaluate the evolution of lactic acid bacteria (LAB) populations in traditional fermented sausages (salsiccia and soppressata) produced in artisanal and industrial plants in Basilicata (Southern Italy). METHODS AND RESULTS: Four hundred and fourteen lactic acid bacteria (LAB) cultures were isolated from samples of sausages at different stages of ripening. A phenotypic characterization of the isolates was carried out using a set of 28 tests, and 34 clusters were identified at the 80% similarity level using hierarchical cluster analysis. Of the isolates 50% were identified as Lactobacillus sakei (with several biotypes), 22% as Pediococcus spp. (mainly Ped. pentosaceus), 7% as Leuconostoc (Leuc. carnosum, Leuc. gelidum, Leuc. pseudomesenteroides), 6% as Lact. plantarum, 1% as Lact. curvatus. Other lactobacilli, including unidentified species, were present in lower numbers. CONCLUSION: The phenotypic diversity and composition of the LAB flora varied as a function of the production plant, product type and ripening time. SIGNIFICANCE AND IMPACT OT THE STUDY: A new procedure based on bootstrapping and Multidimensional Scaling was successfully used to obtain a graphical representation of the evolution of the LAB populations.     

Effect of pH and the bacteriocin carnocin 54 on growth and cell morphology of two Leuconostoc strains

Leuconostoc carnosum LA54A produces carnocin 54, a bacteriocin inhibitory to Listeria and closely related lactic acid bacteria. The effects of the pH of cell-free LA54 culture supernatants on the antibacterial activity of carnocin 54 was assessed using Leuc. mesenteroides DSM 20343 and TA10C as indicator strains. Carnocin 54 showed greatest activity against both strains at pH 4.5. At pH 6.5, activity was reduced, especially against Leuc. mesenteroides TA10C. Scanning electron microscopy showed irregular and rough surfaces on bacteriocin-treated cells at both pH values.     

Stimulation of the growth of leuconostoc oenos by tomato juice

Summary Tomato juice contains a growth factor (TJF) active for some strains of Leuconostoc oenos but not required by other lactic acid bacteria. TJF does not appear to be identical with any known growth factor, is of limited distribution in natural products and is destroyed by the growth of most strains of Leuconostoc oenos and by Lactobacillus plantarum and Pediococcus cerevisiae.TJF is essential for the growth of Leuconostoc oenos NCDO 1674 when organic acids are in a medium with an initial pH of 4.8 and incubated at 22° C and also with an initial pH of 6.0 and incubated at 30° C. Under the first conditions only and in the absence of organic acids Tween 80 is essential for growth if tomato juice is absent.     

Analysis of cellular fatty acids in Orientia tsutsugamushi as taxonomic markers

Six Orientia strains including 3 prototype strains such as Gilliam, Karp, and Kato, and 3 strains (Boryong, Pajoo, and Yongworl) isolated in Korea, were studied for the profiles of their cellular fatty acids. All tested strains contained octadecenoic acid C (18: 1) omega 9 c(57.3 +/- 3.5%), octadecanoic acid C (18: 0) (15.3 +/- 1.5%), and hexadecanoic acid C (16: 0) (12.7 +/- 1.7%) as major components; however, interestingly, eicosenoic acid C (20: 1) omega 9 c(2.6 +/- 0.6%) was found in all strains except the Yongworl strain. Furthermore none of the strains contained 3-hydroxy fatty acids. The ratios of total saturated fatty acid (SFA) to total unsaturated fatty acid (UFA) were within the range of 0.34 to 0.54. These results showed that the cellular fatty acid profile should provide more reliable information for the identification of these bacteria.     

Stability of Lactic Acid Bacteria to Freezing as Related to Their Fatty Acid Composition

The viability of Streptococcus lactis and Lactobacillus sp. A-12 after freezing at -17°C for 48 h was better preserved when the cells were grown in medium supplemented with oleic acid or Tween 80 (polyoxyethylene sorbitan monooleate). A pronounced change in the cellular fatty acid composition was noted when the bacteria were grown in the presence of Tween 80. In S. lactis the ratio of unsaturated to saturated fatty acids increased from 1.18 to 2.55 and in Lactobacillus sp. A-12 it increased from 0.85 to 1.67 when Tween 80 was added to the growth medium. The antibiotic cerulenin markedly inhibited the growth of lactic acid bacteria in tomato juice (TJ) medium but had almost no effect on the growth of the bacteria in TJ medium containing Tween 80 (or oleic acid). The antibiotic inhibited markedly the incorporation of [1-¹⁴C]acetate but had no inhibitory effect on the incorporation of exogenous [1-¹⁴C]oleate (or [1-¹⁴C]palmitate) into the lipid fractions of lactic acid bacteria. Thus, the fatty acid composition of lactic acid bacteria, inhibited by the antibiotic cerulenin, can be modulated by exogenously added oleic acid (or Tween 80) without the concurrent endogenous fatty acid synthesis from acetate. The data obtained suggest that cerulenin inhibits neither cyclopropane fatty acid synthesis nor elongation of fatty acid acyl intermediates. The radioactivity of cells grown in the presence of [1-¹⁴C]oleate and cerulenin was associated mainly with cyclopropane Δ19:0, 20:0 + 20:1, and 21:0 acids. As a consequence, cerulenin caused a decrease in the ratio of unsaturated to saturated fatty acids in lactic acid bacteria as compared with cells grown in TJ medium plus Tween 80 but without cerulenin. Cerulenin caused a decrease in the viability of S. lactis and Lactobacillus sp. A-12 after freezing at -17°C for 48 h only when Tween 80 was present in the growth medium. We conclude that the sensitivity of lactic acid bacteria to damage from freezing can be correlated with specific alterations in the cellular fatty acids.     

Rapid GC analysis of cellular fatty acids for characterizing Lactobacillus sake and Lact. curvatus strains of meat origin

A rapid procedure based on the gas chromatographic analysis of cellular fatty acids was used to differentiate between strains of Lactobacillus sake and Lact. curvatus isolated from dry salami. All strains had very similar fatty acid profiles except four of them which lacked C19 cycl acid, but neither this feature nor other differences in single fatty acid contents could be successfully correlated with the biochemical discrimination of Lact. sake from Lact. curvatus . When, however, strains were compared on the basis of the total content of fatty acids with 18 carbon atoms divided by that with 16 carbon atoms, a very good correlation with strain characterization by classical methods was achieved. It was concluded that selected cellular fatty acid ratios might be useful for characterizing phylogenetically related strains of lactic acid bacteria.     

Bacteriocin production by lactic acid bacteria isolated from Rioja red wines

Forty-two lactic acid bacteria (LAB) of the genera Lactobacillus (32), Leuconostoc (6), Pediococcus (3) and Lactococcus (1), isolated from Rioja red wines, were tested for antimicrobial activity. All these strains, as well as 18 Leuconostoc oenos and 19 yeast strains were used as indicators. Only nine strains showed antimicrobial activity, and all were of the species Lactobacillus plantarum, which constitutes the predominant microflora in Rioja red wines after alcoholic fermentation. Lact. plantarum strain J-51 showed the widest range of action, inhibiting the growth of 31 strains of the four studied LAB genera. Lact. plantarum J-51 antimicrobial activity was lost after treatment with proteases, suggesting a proteinaceous nature for this activity. It was found to be stable between pH 3 and 9 and under strong heating conditions (100 degrees C for 60 min). Polymerase chain reaction (PCR) analysis of Lact. plantarum J-51 genome revealed the presence of the plnA gene that encodes the plantaricin precursor PlnA. A 366-bp fragment was sequenced and showed 95% identity with pln locus of Lact. plantarum C-11. The deduced precursor peptide sequence showed one mutation (Gly7 to Ser7) at the double glycine leader peptide, and the three putative 26-, 23- and 22-residue active peptides remain identical to those of Lact. plantarum C-11. Therefore, antimicrobial peptides constitute a potent adaptation advantage for those strains that dominate in a medium such as wine, and can play an important role in the ecology of wine microflora.     

Using specific polyclonal antibodies to study the malolactic enzyme from Leuconostoc oenos and other lactic acid bacteria

Specific polyclonal antibodies directed against the malolactic enzyme of Leuconostoc oenos were obtained. Despite the homologies between the malolactic enzymes from Leuc. oenos and Lactococcus lactis , no immunological relationship was detected with the L. lactis malolactic enzyme, suggesting differences in their structural organization. The use of the antiserum also demonstrated that the problem of heterologous expression occurring in the recombinant Escherichia coli strain (Labarre et al. 1996a) resulted in a low synthesis of the malolactic enzyme from Leuc. oenos . Moreover, a small amount of the protein was found to be peripherally associated to the membrane of Leuc. oenos.     

Numerical taxonomy of some leuconostocs and related bacteria isolated from Scotch whisky distilleries

Forty-six presumptive Leuconostoc strains and 13 lactobacilli isolated from malt and grain whisky fermentations together with 14 reference strains of Leuconostoc and Lactobacillus were examined for 99 characters. Data were analysed using a variety of similarity coefficients and clustering algorithms. Two aggregate clusters representing Leuconostoc and Lactobacillus were consistently recovered. Many of the presumptive leuconostocs from whisky fermentations were identified as Leuc. mesenteroides. A second large cluster of strains could not be positively identified. Although recovered within the Leuconostoc aggregate cluster, some of these strains hydrolysed arginine, a characteristic of lactobacilli. The study demonstrates a more diverse flora of lactic acid bacteria existing in the distillery fermentation than had been previously recognized.     

Characterization of temperate bacteriophages of Leuconostoc oenos and evidence for two prophage attachment sites in the genome of starter strain PSU-1

The close relatedness between 17 Leuconostoc oenos bacteriophages, induced with mitomycin C from strains isolated in different geographic regions, was inferred from their morphology, DNA homology and protein composition. The genome of all the phages had cohesive end termini and ranged in size from 36.4 to 40.9 kb. According to the restriction patterns obtained by digestion with five enzymes, the phages were divided in six groups. Lysogenization of a spontaneous phage-cured derivative of Leuc. oenos strain PSU-1 was achieved with 16 phages and the analysis of the lysogens showed that the phage DNA integrates in the host chromosome in one or two sites. The att B loci were located on the macrorestriction Asc I and Not I fragments of the recipient strain. A survey of Leuc. oenos strains with a phage DNA probe confirmed the lysogenic nature of several, but not all of the original phage hosts. These results are discussed in the light of evidence for the instability of some lysogenic PSU-1 derivatives.