Quantification of Leuconostoc populations in mixed dairy starter cultures using fluorescence in situ hybridization

AIMS: Development of a rapid method to identify and quantify Leuconostoc populations in mesophilic starter cultures. METHODS AND RESULTS: 16S rRNA-targeted oligonucleotide probes were used in a whole cell in situ hybridization assay for the identification of the genus Leuconostoc and an undescribed Leuconostoc ribospecies. The probes were fluorescently labelled and used to quantify the Leuconostoc populations in five different mixed starter cultures. CONCLUSIONS: There was a good correlation between the results obtained using fluorescence in situ hybridization (FISH) with that of standard plate counting methods. SIGNIFICANCE AND IMPACT OF THE STUDY: To develop a FISH method capable of identifying and quantifying the Leuconostoc population in starter cultures within 1 day.     

Monitoring of <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> DRC starter in fermented vegetable by random integration of chloramphenicol acetyltransferase gene

In 2004, Leuconostoc mesenteroides DRC was first used as a starter culture for achieving higher organoleptic effects in Korean kimchi manufacture. For a better understanding of starter growth in a mixed culture system, and for predicting starter predominance in kimchi, a monitoring system for the starter was established. The chloramphenicol resistance marker gene (cat) was randomly integrated into chromosomal DNA of L. mesenteroides DRC using a viral transposon and transposase. The DRC mutant, tDRC2, had a similar growth pattern to the host strain, with no major alteration in phenotypic characteristics. The mutant strain was inoculated into real kimchi, and monitoring of the starter population was successfully achieved. The overall predominance of Leuconostoc in kimchi inoculated with DRC followed the general growth pattern of this genus during kimchi fermentation. Our results also demonstrate the competitive ability of the DRC starter against Leuconostoc from natural flora, maintaining its predominance above 88% during the whole fermentation period. Based on this experiment, the random gene integration method using a transposon was shown to be of utility in transferring any commercial starter into a selectable and monitorable strain for simulation purposes.     

Detection of dairy Leuconostoc strains using the polymerase chain reaction

This paper reports the design of a Leuconostoc -specific oligonucleotide based on 16S rRNA sequence data. When this oligonucleotide was used in a polymerase chain reaction (PCR) in conjunction with an oligonucleotide to a conserved region of the 16S rRNA sequence, a Leuconostoc -specific PCR product of approximately 470 bp was produced. The use of a second oligonucleotide to a conserved region allowed the production of an approximately 350 bp product in all PCRs, acting as a positive control. The PCR procedure described was particularly useful for detecting the presence of Leuconostoc in mixed mesophilic starter cultures. The Leuconostoc -specific oligonucleotide was used also as a specific hybridization probe.     

Monitoring of Leuconostoc population during sauerkraut fermentation by quantitative real-time polymerase chain reaction

A real-time PCR assay method was established to monitor Leuconostoc spp. populations via specific amplification of the dextransucrase gene. Quantification of L. mesenteroides B-512F using both genomic DNA and cell suspensions yielded a log-linear correlation spanning approximately 5 log units. By using this method, monitoring changes of Leuconostoc spp. during sauerkraut fermentation was successfully accomplished with accuracy after inoculation of starter and sugars (sucrose and maltose).     

Malolactic fermentation in Chardonnay: growth and sensory effects of commercial strains of Leuconostoc oenos

Samples of fermenting Chardonnay juice were inoculated with five commercial cultures of Leuconostoc oenos to promote malolactic fermentation. Controls were not inoculated with malolactic starter cultures; one was held under the same conditions as the juice inoculated with malolactic starter cultures and the other was held under conditions in which malolactic fermentation was inhibited. Bacterial growth and chemical composition of the wines were monitored for eight weeks after the wines were inoculated with the yeast starter culture. The five strains of L. oenos differed in growth kinetics and rates of malic acid degradation. Significant differences were detected among the finished wines subjected to sensory evaluation.     

Isolation and characterization of bacteriophages from fermenting sauerkraut

This paper presents the first report of bacteriophage isolated from commercial vegetable fermentations. Nine phages were isolated from two 90-ton commercial sauerkraut fermentations. These phages were active against fermentation isolates and selected Leuconostoc mesenteroides and Lactobacillus plantarum strains, including a starter culture. Phages were characterized as members of the Siphoviridae and Myoviridae families. All Leuconostoc phages reported previously, primarily of dairy origin, belonged to the Siphoviridae family.     

Inhibition of bacterial growth and malolactic fermentation in wine by bacteriophage

Bacteriophage present in wine can attack bacterial starter cultures and inhibit the malolactic fermentation. The possibility of starter culture failure due to phage attack was studied in a commercial dry red wine of pH 3·23, inoculated with a multiple strain starter culture. During two stages of malolactic fermentation, bacterial growth and malate degradation in the wine were inhibited. A phage capable of lysing isolates of Leuconostoc oenos was isolated from the wine. The isolated phage had an icosahedral head of 42–45 nm diameter and a flexible, regularly cross-striated tail 197–207 nm long with a small baseplate. The results confirm that phage can attack bacterial starter cultures in wine at low pH.     

The use of mesophilic cultures in the dairy industry

The use of mesophilic starter cultures, containing group N Streptococcus and Leuconostoc species, in the dairy industry is examined. Bacteriophage attack is identified as the main cause of culture inhibition and criteria used to select stable mixed-strain starter cultures and phage-insensitive defined strains are established. The key aspects of culture propagation and storage are highlighted, as well as bulk-culture protection based on physical exclusion of phage and the use of phage-inhibitory media. Developments in the application of starter culture systems in different countries are examined and show that significant process control and elimination of phage problems can be achieved.     

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.     

Quantifying the significance of phage attack on starter cultures: a mechanistic model for population dynamics of phage and their hosts isolated from fermenting sauerkraut

We investigated the possibility of using starter cultures in sauerkraut fermentation and thereby reducing the quantity of salt used in the process. This, in turn, would reduce the amount of waste salt that would enter in our water resources. Phage, naturally present in sauerkraut fermentation, could potentially affect the starter cultures introduced. Thus, a mechanistic mathematical model was developed to quantify the growth kinetics of the phage and starter cultures. The model was validated by independent experiments with two Leuconostoc mesenteroides strains isolated from sauerkraut and their corresponding phage. Model simulations and experimental evidence showed the presence of phage-resistant cell populations in starter cultures which replaced phage-sensitive cells, even when the initial phage density (P(0)) and multiplicity of infection (MOI) were low (P(0) < 1 x 10(3) PFU/ml; MOI < 10(-4)) in the MRS media. Based on the results of model simulation and parameter optimization, it was suggested that the kinetic parameters of phage-host interaction, especially the adsorption rate, vary with the initial phage and host densities and with time. The model was validated in MRS broth. Therefore, the effects of heterogeneity and other environmental factors, such as temperature and pH, should be considered to make the model applicable to commercial fermentations.     

Enhancing acid tolerance of <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> with glutathione

Leuconostoc mesenteroides is a commercially important lactic acid bacterium currently used as a starter for kimchi and kefir. However, its sensitivity to acid stress limits its performance. L. mesenteroides was grown in a medium supplemented with 3.2 or 6.4 mM glutathione (GSH), and cell survival rates were measured during a long-term mild acid challenge (pH 4.0). As a result, GSH was imported by the cells and protected against acid stress; thereafter it was consumed as a nutrient. Acid stress resistance of starter cultures of this bacterium can thus be improved by cultivating it in media supplemented with GSH.     

Pilot scale production and preservation of a new malolactic culture Leuconostoc oenos 44.40 For use in secondary wine fermentation

Pilot scale production of a starter culture Leuconostoc oenos 44.40 for the secondary fermentation of wine has been demonstrated. Cultures harvested in mid-log phase gave maximum viability after lyophilization. Lyophilized cultures retained their viability best when packaged under nitrogen and stored in dry cool conditions.     

Improved enumeration of lactic acid bacteria in mesophilic dairy starter cultures by using multiplex quantitative real-time PCR and flow cytometry-fluorescence in situ hybridization

Nucleic acid-based assays were developed to enumerate members of the three taxa Lactococcus lactis subsp. cremoris, L. lactis subsp. lactis, and Leuconostoc spp. in mesophilic starter cultures. To our knowledge the present is the first study to present a multiplex quantitative PCR (qPCR) strategy for the relative enumeration of bacteria. The multiplex qPCR strategy was designed to quantify the target DNA simultaneously relative to total bacterial DNA. The assay has a high discriminatory power and resolves concentration changes as low as 1.3-fold. The methodology was compared with flow cytometric fluorescence in situ hybridization (FLOW-FISH) and 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-Gal)-calcium citrate agar-based plate counting. For enumeration by FLOW-FISH, three new probes having the same specificity as the qPCR assay were designed and established. A combination with flow cytometry greatly reduced the time consumed compared to manual enumeration. Both qPCR and FLOW-FISH yielded similar community compositions for 10 complex starter cultures, with all detected subpopulations being highly significantly correlated (P < 0.001). Correlations between X-Gal-calcium citrate agar-based CFU and qPCR-derived counts were highly significant (P < 0.01 and P < 0.001, respectively) for the number of acidifiers versus L. lactis subsp. cremoris and for Leuconostoc spp. as quantified by the two techniques, respectively. This confirmed that most acidifiers in the studied PROBAT cultures are members of L. lactis subsp. cremoris. Quantitative real-time PCR and FLOW-FISH were found to be effective and accurate tools for the bacterial community analysis of complex starter cultures.     

Differential viable count of mixed starter cultures of lactic acid bacteria in doughs by using modified Chalmers medium

The modified Chalmers medium appeared as quite suitable for counting a mixed population consisting of different species of lactic acid bacteria used as starter in breadmaking. Selected strains of Lactobacillus plantarum, Leuconostoc mesenteroides subsp. mesenteroides, Lactobacillus sanfranciscensis, Enterococcus faecalis together with Saccharomyces cerevisiae could be easily differentiated and counted with an acceptable recovery in comparison with reference media.     

Multifactorial diversity sustains microbial community stability

Maintenance of a high degree of biodiversity in homogeneous environments is poorly understood. A complex cheese starter culture with a long history of use was characterized as a model system to study simple microbial communities. Eight distinct genetic lineages were identified, encompassing two species: Lactococcus lactis and Leuconostoc mesenteroides. The genetic lineages were found to be collections of strains with variable plasmid content and phage sensitivities. Kill-the-winner hypothesis explaining the suppression of the fittest strains by density-dependent phage predation was operational at the strain level. This prevents the eradication of entire genetic lineages from the community during propagation regimes (back-slopping), stabilizing the genetic heterogeneity in the starter culture against environmental uncertainty.     

Synbiotic synthesis of oligosaccharides during milk fermentation by addition of leuconostoc starter and sugars

Synthesis of oligosaccharides during milk fermentation was attempted by inoculating Leuconostoc citreum with Lactobacillus casei, Lb. delbrueckii subsp. bulgaricus, and Streptococcus thermophilus as starters. Dextransucrase of Ln. citreum worked as a catalyst for the transglycosylation reaction of sugars; sucrose was added as the glucose donor, and lactose or maltose acted as the acceptor compound for the reaction. When 4% sucrose was added in milk, glucosyl-lactose was synthesized (about 1%, w/v) after 1-2 days of fermentation at 15 or 25 degrees C. Alternatively, when sucrose and maltose (2% each, w/v) were added, panose (about 1%, w/v) and other isomaltooligosaccharides were made in a day at 15-35 degrees C. Growth patterns of lactobacilli and streptococci starters were not affected by the coculture of leuconostoc starter, but the rate of acid synthesis was slightly slowed at every temperature. Addition of sugars in milk did not give any adverse effect on the lactate fermentation. Accordingly, the use of leuconostoc starter and addition of sugars in milk allowed the production of oligosaccharides-containing fermented milk, and application of this method will facilitate the extensive development of synbiotic lactate foods.     

Quantifying the Significance of Phage Attack on Starter Cultures: a Mechanistic Model for Population Dynamics of Phage and Their Hosts Isolated from Fermenting Sauerkraut

We investigated the possibility of using starter cultures in sauerkraut fermentation and thereby reducing the quantity of salt used in the process. This, in turn, would reduce the amount of waste salt that would enter in our water resources. Phage, naturally present in sauerkraut fermentation, could potentially affect the starter cultures introduced. Thus, a mechanistic mathematical model was developed to quantify the growth kinetics of the phage and starter cultures. The model was validated by independent experiments with two Leuconostoc mesenteroides strains isolated from sauerkraut and their corresponding phage. Model simulations and experimental evidence showed the presence of phage-resistant cell populations in starter cultures which replaced phage-sensitive cells, even when the initial phage density (P₀) and multiplicity of infection (MOI) were low (P₀ < 1 × 10³ PFU/ml; MOI < 10⁻⁴) in the MRS media. Based on the results of model simulation and parameter optimization, it was suggested that the kinetic parameters of phage-host interaction, especially the adsorption rate, vary with the initial phage and host densities and with time. The model was validated in MRS broth. Therefore, the effects of heterogeneity and other environmental factors, such as temperature and pH, should be considered to make the model applicable to commercial fermentations.     

Genomic analysis of Oenococcus oeni PSU-1 and its relevance to winemaking

Oenococcus oeni is an acidophilic member of the Leuconostoc branch of lactic acid bacteria indigenous to wine and similar environments. O. oeni is commonly responsible for the malolactic fermentation in wine and due to its positive contribution is frequently used as a starter culture to promote malolactic fermentation. In collaboration with the Lactic Acid Bacteria Genome Consortium the genome sequence of O. oeni PSU-1 has been determined. The complete genome is 1,780,517 nt with a GC content of 38%. 1701 ORFs could be predicted from the sequence of which 75% were functionally classified. Consistent with its classification as an obligately heterofermentative lactic acid bacterium the PSU-1 genome encodes all the enzymes for the phosphoketolase pathway. Moreover, genes related to flavor modification in wine, such as malolactic fermentation capacity and citrate utilization were readily identified. The completion of the O. oeni genome marks a significant new phase for wine-related research on lactic acid bacteria in which the physiology, genetic diversity and performance of O. oeni starter cultures can be more rigorously examined.     

Controlling Wine Malolactic Fermentation with Nisin and Nisin-Resistant Strains of Leuconostoc oenos

The polypeptide nisin (100 U/ml) prevented malolactic fermentation in wines by indigenous or intentionally added lactic acid bacteria. Nisin (100 U/ml)-resistant mutants of Leuconostoc oenos were obtained and used with nisin in wine to carry out a pure-culture malolactic fermentation in the presence or absence of other lactic acid bacteria. Nisin degradation by mutants was not observed, and residual nisin was detectable in wines 4 months after it was added. Results indicated that nisin or nisin with resistant bacterial starter cultures can be used to control malolactic fermentation in wines.     

Polyphasic screening, homopolysaccharide composition, and viscoelastic behavior of wheat Sourdough from a Leuconostoc lactis and Lactobacillus curvatus exopolysaccharide-producing starter culture

After isolation from different doughs and sourdoughs, 177 strains of lactic acid bacteria were screened at the phenotypic level for exopolysaccharide production on media containing different carbohydrate sources. Two exopolysaccharide-producing lactic acid bacteria (Lactobacillus curvatus 69B2 and Leuconostoc lactis 95A) were selected through quantitative analysis on solid media containing sucrose and yeast extract. The PCR detection of homopolysaccharide (gtf and lev) and heteropolysaccharide (epsA, epsB, epsD and epsE, and epsEFG) genes showed different distributions within species and strains of the lactic acid bacteria studied. Moreover, in some strains both homopolysaccharide and heteropolysaccharide genes were detected. Proton nuclear magnetic resonance spectra suggest that Lactobacillus curvatus 69B2 and Leuconostoc lactis 95A produced the same exopolysaccharide, which was constituted by a single repeating glucopyranosyl unit linked by an α-(1→6) glycosidic bond in a dextran-type carbohydrate. Microbial growth, acidification, and viscoelastic properties of sourdoughs obtained by exopolysaccharide-producing and nonproducing lactic acid bacterial strains were evaluated. Sourdough obtained after 15 h at 30°C with exopolysaccharide-producing lactic acid bacteria reached higher total titratable acidity as well as elastic and dissipative modulus curves with respect to the starter not producing exopolysaccharide, but they showed similar levels of pH and microbial growth. On increasing the fermentation time, no difference in the viscoelastic properties of exopolysaccharide-producing and nonproducing samples was observed. This study suggests that dextran-producing Leuconostoc lactis 95A and Lactobacillus curvatus 69B2 can be employed to prepare sourdough, and this would be particularly useful to improve the quality of baked goods while avoiding the use of commercially available hydrocolloids as texturizing additives.