Leuconostoc Strains Research Articles

Method for the selection of Lactococcus lactis mutants producing excess carbon dioxide.

One of the characteristics of Roquefort cheese is the presence of irregularly shaped openings. Although many factors affect the development of opening in blue-veined cheeses (Martley & Crow, 1996), the limiting step in the case of Roquefort cheese is the production of CO2 by lactic acid bacteria (J.-P. Reverbel, pers. comm.). Most of the opening occurs after moulding; the process is difficult to control and many manufacturing runs result in cheeses with an insufficient opening. Concentrated suspensions of Leuconostoc strains are used to increase the production of CO2 (Devoyod & Muller, 1969), but it would clearly be useful to have microorganisms that produce larger quantities of the gas. McKay & Baldwin (1974) isolated a spontaneous mutant of Lactococcus lactis that produced more acetoin and CO2 than the parent strain. This mutant was lactate dehydrogenase (LDH)-deficient, which favoured the conversion of pyruvate into end products other than lactate. Following nitrosoguanidine mutagenesis, we isolated three Lc. lactis mutants whose LDH activities were reduced to varying extents and which produced varying amounts of CO2 (Boumerdassi et al. 1997). Subsequent work showed that these mutants were unstable on successive subculture in milk or synthetic broth (El Attar et al. 2000).The aim of our current work was to select a large number of Lc. lactis mutants producing excess CO2. This would increase the probability of selecting stable mutants and also provide a collection of strains with differing gas production activities. Currently available screening methods are, however, unsuitable for processing large numbers of mutants, which is why we have developed an improved screening method.

Lactobacillus farciminis MD, a newer strain with potential for bacteriocin and antibiotic assay.

A native isolate Lactobacillus farciminis MD isolated from fermenting mushroom exhibited a high degree of sensitivity to the majority of the bacteriocins produced by strains of lactobacilli, leuconostoc and pediococci. Also, the efficacy of Lact. farciminis MD as a sensitive strain for antibiotic assay was established against different antibiotics including ampicillin, cefazoline, chloramphenicol and nitrofurantoin at concentrations of 30 microg each, showing an inhibition zone of 30 mm diameter. The high degree of sensitivity towards bacteriocins and antibiotics provide potential for the exploitation of Lact. farciminis MD in establishing very well-defined bacteriocin producers.

Specific detection of Leuconostoc mesenteroides subsp. mesenteroides with DNA primers identified by randomly amplified polymorphic DNA analysis.

Randomly amplified polymorphic DNA analysis using primer 239 (5' CTGAAGCGGA 3') was performed to characterize Leuconostoc sp. strains. All the strains of Leuconostoc mesenteroides subsp. mesenteroides (with the exception of two strains), two strains formerly identified as L. gelidum, and one strain of Leuconostoc showed a common band at about 1.1 kb. This DNA fragment was cloned and sequenced in order to verify its suitability for identifying L. mesenteroides subsp. mesenteroides strains.

Development and PFGE monitoring of dominance among spoilage lactic acid bacteria from cured meats

Strains of lactic acid bacteria (LAB), isolated from intact vacuum-packaged cured meats obtained at retail, were subjected to single inhibitory factor and mixed-culture dominance tests during challenge with different conditions of pH and temperature in the presence of NaCl and NaNO2to study reasons for strain dominance. Bacteria were checked for bacteriocin production. Both initial pH and temperature had a significant effect on the growth of spoilage LAB isolates in modified MRS broth. Lactobacilli generally grew better than Lc. mesenteroides at pH 5·5. It appeared that L. curvatus and L. sakei grew faster at elevated temperature (12°C) than Lc. mesenteroides. It was also noted that there was a similar response for strains within the same species to the challenges of NaCl, NaNO2, pH and temperature. When present in mixed cultures, Leuconostoc strains did not grow well at 2°C and at an initial pH of 5·5 compared with lactobacilli, but performed better at this low temperature as pure cultures than when mixed with lactobacilli. At normal cured meat pH (6·0 and 6·5) and higher temperatures (6°C and 12°C), dominant bacteria always grew from the originally larger bacterial population. When leuconostocs and lactobacilli were present in equal initial numbers, leuconostocs generally did not grow as well as lactobacilli. Lc. mesenteroides grew faster than L. curvatus at 6°C but did not out-compete L. sakei at this temperature (pH ≥ 6·0). Pulsed field gel electrophoresis (PFGE) of SmaI digested genomic DNA successfully distinguished all the strains under study. Difficulties associated with enumeration of LAB having similar biochemical properties were addressed by the development of a composite-simultaneous PFGE method for interspecies qualification using single colonies from agar plates to generate digested DNA for analysis.

Improved screening procedure for biogenic amine production by lactic acid bacteria

An improved screening plate method for the detection of amino acid decarboxylase-positive microorganisms (especially lactic acid bacteria) was developed. The suitability and detection level of the designed medium were quantitatively evaluated by confirmation of amine-forming capacity using an HPLC procedure. The potential to produce the biogenic amines (BA) tyramine, histamine, putrescine, and cadaverine, was investigated in a wide number of lactic acid bacteria (LAB) of different origin, including starter cultures, protective cultures, type strains and strains isolated from different food products. Also, several strains of Enterobacteriaceae were examined. Modifications to previously described methods included lowering glucose and sodium chloride concentrations, and increasing the buffer effect with calcium carbonate and potassium phosphate. In addition, pyridoxal-5-phosphate was included as a codecarboxylase factor for its enhancing effect on the amino acid decarboxylase activity. The screening plate method showed a good correlation with the chemical analysis and due to its simplicity it is presented as a suitable and sensitive method to investigate the capacity of biogenic amine production by LAB. Tyramine was the main amine formed by the LAB strains investigated. Enterococci, carnobacteria and some strains of lactobacilli, particularly of Lb. curvatus, Lb. brevis and Lb. buchneri, were the most intensive tyramine formers. Several strains of lactobacilli, Leuconostoc spp., Weissella spp. and pediococci did not show any potential to produce amines. Enterobacteriaceae were associated with cadaverine and putrescine formation. No significant histamine production could be detected for any of the strains tested.

Selection of dairy Leuconostoc isolates for important technological properties

Twenty-four dairy Leuconostoc strains isolated from French commercial starters and four reference strains were screened for 58 morphological and biochemical characters. Hierarchical clustering analysis was performed on the results of dextran production and carbohydrate fermentation tests. Most strains fitted into three clusters at a similarity level of 65% and were identified as belonging to the species lactis or mesenteroides. Fourteen strains were then selected and further characterized for their technological properties. They were examined for growth, acidification kinetics and proteolytic capacity when growing in milk, and peptidase activities and diacetyl production after growth in MRS broth. Principal component analysis was carried out to group strains and compare their technological properties. This allowed selection of strains of dairy interest. Special attention was given to the flavouring capacity of Leuconostoc lactis isolates that could be useful in designing commercial starters.

Differentiation of dextran-producing leuconostoc strains by a modified randomly amplified polymorphic DNA protocol

Seven dextran-producing Leuconostoc strains were differentiated by using a modified randomly amplified polymorphic DNA (RAPD) protocol that incorporated specific primers designed from conserved regions of dextransucrase genes. RAPD profiles showed intraspecies differences among the Leuconostoc mesenteroides strains tested. This modified RAPD protocol will aid in the differentiation of polymer-producing leuconostocs, which are currently distinguished by time-consuming analyses of the dextrans they synthesize.

Biogenic amine production by wild lactococcal and leuconostoc strains.

Two qualitative and one quantitative HPLC methods were evaluated for the detection of biogenic amine producers among wild dairy lactococcal and leuconostoc strains. High tyramine producers ranging from 370 to 807 mg l-1 were detected by qualitative methods and confirmed by HPLC analysis. Tyramine levels detected throughout the incubation time depended on the concentration of the amino acid precursor available and no tyramine production was observed when strains were grown in milk. However, increasing amounts of tyramine were detected in cultures grown in milk supplemented with different concentrations of tyrosine. Qualitative methods failed to detect weak producers so that tryptamine production (< 7 mg l-1) could only be determined by HPLC. None of the tested strains was able to produce histamine. Simultaneous production of different amines was observed by HPLC although no colour change was observed in the specific decarboxylase media. Thus, it was concluded that the amine forming ability should be taken into account when selecting starters for milk fermentations. Qualitative methods could be used as a first screening step to eliminate the highest amine producers while the quantitative methods would detect any producing strain.

Growth and metabolic activity of a cheese starter in CO 2 -acidified and non-acidified refrigerated milk

The growth and activity of two Lactococcus strains and one Leuconostoc strain in CO2-acidified and non-acidified refrigerated milk were evaluated separately and as a mixed culture to determine their suitability for use as a starter in the manufacture of Afuega’l Pitu, an acid-coagulated Spanish cheese, from refrigerated CO2-acidified milk. The growth of the strains studied and their production of organic acids were similar in CO2-acidified and non-acidified refrigerated or fresh milk, indicating that CO2 treatment does not affect the metabolic activity of the strains. However, refrigeration enhanced the production of acetaldehyde, ethanol and diacetyl in CO2-acidified and non-acidified milk. The level of diacetyl was also greater in refrigerated CO2-acidified milk than in refrigerated non-acidified milk. It was concluded that refrigerated milk acidified with CO2 can be satisfactorily used in the manufacture of Afuega’l Pitu cheese, and that this technique can be also used in the production of other acid-coagulated cheeses.

A comparative study of mannitol production by two lactic acid bacteria

Two different lactic acid bacteria, designated Lactobacillus sp. Y-107 and Leuconostoc sp. Y-002, were isolated during the fermentation of kimchi, a Korean fermented food product, and found to possess the capability of mannitol production. To optimize the culture conditions and compare the mannitol formation characteristics of the two strains, various fermentation conditions were investigated. Of several sugars tested, only fructose and sucrose were utilized as substrates for mannitol formation. With both strains, maximal mannitol production was obtained with fructose as the sole carbon source. Under the optimal culture conditions, the final mannitol concentrations produced by the Lactobacillus and Leuconostoc strains were 73 and 26 g/ l from 100 g/ l fructose with indicated yields of 86 and 65% based on the fructose consumed, respectively. Neither isolate produced other polyols such as glycerol and sorbitol as by-products. The two organisms exhibited very similar physiological behavior in mannitol biosynthesis apart from their metabolism of some sugars.

Detection and partial characterization of a bacteriocin produced by Carnobacterium piscicola 213.

BLIS 213, is a bacteriocin-like inhibitory substance produced by Carnobacterium piscicola 213. It is active against Carnobacterium, Enterococcus and Listeria spp. No activity was observed against tested Lactobacillus, Lactococcus, Leuconostoc and Pediococcus strains, nor against Gram-negative bacteria. The BLIS 213 activity was inactivated by several proteolytic enzymes. It was heat resistant (121 degrees C for 20 min), and stable over a pH range of 2-8. Activity was determined by a dilution micromethod; it was increased after SDS treatment. A mutant strain which lacks bacteriocin production was isolated and designated as Carnobacterium piscicola 213a. It had the same phenotypic and biochemical properties as the parent strain, and was not sensitive to bacteriocin activity. The apparent molecular weight of the bacteriocin in the crude extract was greater than 10 kDa. It was about 6 kDa after SDS-PAGE of a partially purified bacteriocin by adsorption on producer cells. The isoelectric point of the BLIS 213 was around 9.3.

Proteolysis and volatile components of reduced-fat cheeses made from ultrafiltered milk and different starters supplemented with lactobacilli and Lac-Prt-lactococci

Summary - Proteolysis and volatile components of serni-hard cheeses manufactured from a mixture of cow's, ewe's and goat's milk, with reduced fat content and concentrated by ultrafiltration, were folJowed during ripening. A starter culture (IFPL), consisting of several strains of Lactococcus, Lactobacillus and Leuconostoc, selected previously for its high proteolytic activity, and the same starter with the addition of a concentrate of Lactococcus lactis subsp lactis TI, lacking the ability to utilize lactose and casein (Lac Prr), were tested with the aim of accelerating cheese ripening as compared with a commercial starter (FD). Casein hydrolysis was not affected by the type of starter culture employed, whereas appreciable differences were found in the non-volatile water-soluble nitrogen fraction. The peptide pattern allowed a clear distinction among cheeses made with different bacterial cultures. IFPL cheeses exhibited, after 1 month of ripening, a higher content of total free amino acids than FD cheeses after 3 months of ripening. Furthermore, the addition of L lactis TI to the IFPL culture noticeably increased the release of total free ami no acids during ripening, as well as the content of total volatile compounds. reduced-fat cheese / proteolysis / starter / volatile component / LacrPrt lactococci Resume - Proteolyse et composes volatils de fromages a teneur reduite en matiere grasse prepares avec du lait ultrafiltre et differents levains additionnes de lactobacilles et de lactocoques Lac-Prt-. La proteolyse et les composes volatils ont ete suivis pendant l'affinage de fromages a teneur reduite en matiere grasse, prepares a partir d'un melange de lait (vache, brebis, chevre) ultrafiltre. Pour accelerer l'affinage, un levain (IFPL) contenant plusieurs souches de Lactococcus, Lactobacillus et Leuconostoc (prealablement selectionnes pour leur activite proteolytique elevee), et le

Bacteriocin-like substance of Aeromonas hydrophila.

The genus Aeromonas comprises Gram negative rods found mainly in aquatical environments that may infect humans and animals (JM Janda 1991 Clin Microbiol Rev 4: 397-410). In humans, some Aeromonas species have been associated with intestinal and extraintestinal infections and enterotoxins, cytotoxins as well as invasive mechanisms have been incriminated in the development of illness in the host (Janda loc. cit.). Bacteriocin-like substances (BLS) are protein compounds produced by some bacteria (G Ivanovics 1962 Bacteriol Rev 26: 108-118) showing antagonic activity against their own species (isoinhibitory activity IA) or other non-related species (heteroinhibitory activity HA). The use of the expression BLS is recommended to nominate bacterial products showing antagonic activity though not characterized (K Sandhu et al. 1983 J Clin Microbiol 17: 511-515). These substances have been widespread utilized in epidemiological studies as specific marker properties of bacteria, in the regulation of population dynamics in bacterial ecosystems and clinical treatment (V Fantinato & F Zelante 1991 Rev Microbiol 22: 49-51). As BLS have not been currently described in Aeromonas species, the purpose of this study was to investigate their production in strains isolated from animal, clinical and environmental sources. The assays for the production of BLS were performed according to Sandhu et al. (loc. cit.). The strains used as BLS producers and BLS indicators are listed in Table. Our results showed that among 32 Aeromonas strains, the BLS could be only demonstrated in a strain of A. hydrophila isolated from a water tank containing alligators. This strain demonstrated heteroinhibitory activity against four Staphylococcus aureus strains (one ATCC 6538 and three methicillin-resistant MRSA). The heteroinhibitory activity was demonstrated after an incubation of 48 hr at 37°C and not at 25°C, conditions also observed for the BLS production in Serratia, Pseudomonas, Leuconostoc and Enterococcus strains (JD Foulds & D Shemin 1969 J Bacteriol 99: 655-660, JRW Govan & G Harris 1985 J Clin Microbiol 22: 490-494, F Mathleu et al. 1993 J Appl Bacteriol 74: 372-379, F Villani 1993 J Appl Bacteriol 74: 380-387). The heteroinhibitory activity was demonstrated by inhibitory zones ranging from 17 to 27mm. JR Govan (1986 Scand J Infec Dis 49: 31-37) proposed that different inhibition zone diameters may depend on the potency either of BLS action or they may be correlated with the number of receptors for BLS in the bacterial surface. In spite of these results, further research is necessary to better understand the bacteriocin-like activity in Aeromonas species as well as to investigate the chemical nature of this substance and its pharmacological usefulness.

Esterase activities of cell-free extracts from ‘wild’ strains of leuconostocs and heterofermentative lactobacilli isolated from traditional Greek cheese

Cell-free extracts from strains of leuconostocs and heterofermentative lactobacilli were tested for esterase activities using synthetic substrates. Results showed that esterases from both lactic acid bacteria groups preferentially degraded short-chain fatty acids. Moreover, even though some similarities between strains of the same species were recorded, the observed differences were not strong enough to distinguish between species and therefore commend this technique as a tool for taxonomy of either leuconostocs or heterofermentative lactobacilli.

Citrate Utilization and Diacetyl Production by Various Strains of Leuconostoc mesenteroides ssp. cremoris

Sixty Leuconostoc strains from different culture collections were studied for taxonomic identification. Based on morphological and biochemical tests, 21 strains, including 1 from the American Type Culture Collection, were typed as Leuconostoc mesenteroides ssp. cremoris. The objective of this identification and of an additional phenotypic characterization of Leu. mesenteroides ssp. cremoris strains was to select the strains that were most useful for producing flavor compounds in dairy fermentations. Strains were examined for citrate utilization, production of diacetyl and acetoin under neutral and acidic conditions, diacetyl reductase activity, and plasmid profiles. Considerable variability within strains was observed. Most strains utilized citrate under neutral conditions, pH 6.5, without a concomitant production of diacetyl or acetoin. Although addition of yeast extract to milk stimulated bacterial growth and citrate utilization, no diacetyl or acetoin was detected in these cultures. Only the addition of citric acid after a preincubation period resulted in significant production of diacetyl and acetoin by pure cultures of Leu. mesenteroides ssp. cremoris. Strains were found that utilized citrate to produce diacetyl and acetoin with low diacetyl reductase activity, and these strains were compatible with acid-producing lactococci. Such strains are most valuable for use as starters for cultured dairy products that rely on the presence of diacetyl for maximum quality.

A highly vancomycin-resistant laboratory mutant of Staphylococcus aureus.

A resistant mutant with vancomycin MIC of 100 micrograms/ml was isolated relatively easily through step pressure in the laboratory from a Staphylococcus aureus strain with initial MIC of 1.5 micrograms/ml for the antibiotic. Upon addition of vancomycin (50 micrograms/ml) to the growth medium mass increase of the culture and peptidoglycan synthesis continued but cell division (daughter cell separation), cell wall turnover and autolysis were inhibited, resulting in the production of multicellular clumps of bacteria. Parallel with the increase of culture density, the concentration of vancomycin measured both by biological activity and by HPLC gradually declined in the culture medium. Cell division and wall turnover of the culture resumed with the production of cells of normal morphology at the time when the concentration of the drug in the medium decreased below 0.5-1.0 micrograms/ml. There was no detectable change in the antibiotic concentration in the culture medium during growth of a vancomycin-resistant (vanA-positive) strain of Enterococcus faecium and an intrinsically vancomycin-resistant strain of Leuconostoc. The vancomycin-resistant staphylococcal mutant gave no signal with the vanA or vanB DNA probes and contained no detectable D-lactate terminating cell wall precursors. The biochemical mechanism and clinical significance of such glycopeptide-resistant mutants remained to be established.

A highly vancomycin-resistant laboratory mutant of Staphylococcus aureus

A resistant mutant with vancomycin MIC of 100 μg/ml was isolated relatively easily through step pressure in the laboratory from a Staphylococcus aureus strain with initial MIC of 1.5 μg/ml for the antibiotic. Upon addition of vancomycin (50 μg/ml) to the growth medium mass increase of the culture and peptidoglycan synthesis continued but cell division (daughter cell separation), cell wall turnover and autolysis were inhibited, resulting in the production of multicellular clumps of bacteria. Parallel with the increase of culture density, the concentration of vancomycin measured both by biological activity and by HPLC gradually declined in the culture medium. Cell division and wall turnover of the culture resumed with the production of cells of normal morphology at the time when the concentration of the drug in the medium decreased below 0.5–1.0 μg/ml. There was no detectable change in the antibiotic concentration in the culture medium during growth of a vancomycin-resistant ( vanA-positive) strain of Enterococcus faecium and an intrinsically vancomycin-resistant strain of Leuconostoc. The vancomycin-resistant staphylococcal mutant gave no signal with the vanA or vanB DNA probes and contained no detectable d-lactate terminating cell wall precursors. The biochemical mechanism and clinical significance of such glycopeptide-resistant mutants remains to be established.

The putative immunity protein of the gram-positive bacteria Leuconostoc mesenteroides is preferentially located in the cytoplasm compartment.

Immunity proteins are though to protect bacteriocin-producing bacterial strains against the bactericidal effects of their own bacteriocin. The immunity protein which protects the lactic acid bacterium Leuconostoc mesenteroides against mesentericin Y105(37) bacteriocin was detected and localized by immunofluorescence and electron microscopy, using antibodies directed against the C-terminal end of the predicted immunity protein. The antibodies recognized the immunity proteins of various strains of Leuconostoc, including Leuconostoc mesenteroides and Leuconostoc gelidum. This study demonstrated that immunity proteins produced by Leuconostoc mesenteroides accumulated in the cytoplasmic compartment of the bacteria. This is in contrast with other known immunity proteins, such as the colicin immunity proteins, which are integral membrane proteins possessing three to four transmembrane domains.

The putative immunity protein of the Gram-positive bacteria Leuconostoc mesenteroides is preferentially located in the cytoplasm compartment

Immunity proteins are thought to protect bacteriocin-producing bacterial strains against the bactericidal effects of their own bacteriocin. The immunity protein which protects the lactic acid bacterium Leuconostoc mesenteroides against mesentericin Y105 37 bacteriocin was detected and localized by immunofluorescence and electron microscopy, using antibodies directed against the C-terminal end of the predicted immunity protein. The antibodies recognized the immunity proteins of various strains of Leuconostoc, including Leuconostoc mesenteroides and Leuconostoc gelidum. This study demonstrated that immunity proteins produced by Leuconostoc mesenteroides accumulated in the cytoplasmic compartment of the bacteria. This is in contrast with other known immunity proteins, such as the colicin immunity proteins, which are integral membrane proteins possessing three to four transmembrane domains.

Effect of lactic starter cultures on the organic acid composition of milk and cheese during ripening—analysis by HPLC

The major function of lactic starter cultures in cheese making is to produce lactic and other organic acids from the carbohydrates present in milk. The activity of six starter cultures consisting of two Lactococcus lactis ssp. lactis, two Lactococcus lactis ssp. lactis biovar. diacetylactis and two Leuconostoc strains, was tested by monitoring the evolution of the organic acid composition of milk by a modified HPLC method. In addition, their performance as cheese starters was also tested. The HPLC method developed proved to be a precise tool to monitor the organic acid content. Thus, it can be used to follow the fermentation ability of starter cultures, providing information about the type of fermentation. The use of any of the six starters assayed is suggested for manufacturing Afuega'l Pitu cheese.