LC Strains Research Articles

Influence of fermentation temperature and autolysis on ACE-inhibitory activity and peptide profiles of milk fermented by selected strains of Lactobacillus helveticus and Lactococcus lactis

Bovine milk was fermented with single strains of Lc. lactis or Lb. helveticus at three temperatures around the optimum for each species. Fermentation was stopped at pH 4.6 and 4.3/4.0, and the products were stored at 5 °C for 1 and 7 days. The extent of lysis was examined by release of X-prolyldipeptidyl aminopeptidase activity, and the angiotensin-I-converting enzyme- (ACE-) inhibitory activity and peptide profiles of the products were determined. Fermentation temperature significantly influenced the bacterial growth, extent of lysis, and ACE-inhibitory activity. ACE-inhibitory activity was high at all temperatures, and slightly higher at the optimal growth temperature, whereas the extent of lysis was highest at a suboptimal growth temperature. Peptide profiles were marginally affected by temperature and cell lysis. The major peptides were identified, including a number of known ACE-inhibitory peptides. Our results suggest that the cell wall proteinase was the primary catalyst in release of ACE-inhibitory peptides.

A component of polysaccharide peptidoglycan complex on Lactobacillus induced an improvement of murine model of inflammatory bowel disease and colitis‐associated cancer

Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signals play key roles in the pathogenesis of inflammatory bowel disease (IBD). We previously described that both intact cells and a cell wall-derived polysaccharide-peptidoglycan complex (PSPG) in a strain of lactobacillus [Lactobacillus casei Shirota (LcS)] inhibited IL-6 production in lipopolysaccharide (LPS)-stimulated lamina propria mononuclear cells (LPMCs) isolated from murine IBD. Diets with LcS improve murine IBD by suppression of IL-6 synthesis in LPMCs. Moreover, LcS supplementation with fermented milk ameliorates disease activity in patients with active ulcerative colitis. Here, we focused on the specific roles of PSPG in LcS concerning their anti-inflammatory actions. PSPG derived from LcS, and no other strain of lactobacilli, inhibited IL-6 production in LPS-stimulated murine IBD LPMCs. Purified PSPG-I from LcS inhibited IL-6 synthesis in LPS-stimulated murine IBD LPMCs through the inhibition of nuclear factor-kappaB. The anti-IL-6 action of LcS PSPG was abrogated by masking with monoclonal anti-PSPG-I. Furthermore, PSPG-I-negative L. casei strains (PSPG-I-negative mutant LcS: LC(DeltaPSPG-I), L. casei ATCC 334) did not inhibit IL-6 production. Finally, we confirmed the effects of PSPG-I on LcS in the models of both IBD and colitis-associated cancer (CAC). In the IBD model, ingestion of LcS improved ileitis and inhibited activation of IL-6/STAT3 signaling, while ingestion of the LC(DeltaPSPG-I) strain did not. In the CAC model, treatment with LcS, but not the LC(DeltaPSPG-I) strain, showed tumour-suppressive effects with an inhibition of IL-6 production in the colonic mucosa. These results suggested that a specific polysaccharide component in an L. casei strain plays a crucial role in its anti-inflammatory actions in chronic intestinal inflammatory disorders.

Gordonia polyisoprenivorans from groundwater contaminated with landfill leachate in a subtropical area: characterization of the isolate and exopolysaccharide production

A strain of Gordonia sp. (strain Lc), from landfill leachate-contaminated groundwater was characterized by polyphasic taxonomy and studied for exopolysaccharide (EPS) production. The cells were Gram-positive, catalase-positive, oxidase-negative and non-motile. The organism grew both aerobically and, in anoxic environment, in the presence of NaNO3. Rods occured singly, in pairs or in a typical coryneform V-shaped. The organism had morphological, physiological and chemical properties consistent with its assignment to the genus Gordonia and mycolic and fatty acid pattern that corresponded to those of G. polyisoprenivorans DSM 44302T. The comparison of the sequence of the first 500 bases of the 16S rDNA of strain Lc gave 100% similarity with the type strain of Gordonia polyisoprenivorans DSM 44302T. Experiments conducted in anaerobic conditions in liquid E medium with either glucose or sucrose as the main carbon source showed that sucrose did not support the growth of Lc strain and that on glucose the maximum specific growth rate was 0.17h-1, representing a generation time of approximately 4 hours. On glucose, a maximum of total EPS was produced during the exponential phase (126.17 ± 15.63 g l-1). The production of free EPS exceeded that of capsular and the free/capsular EPS ratio increased from 1.9 during the exponential phase to 7.8 during the stationary phase. At present, six strains of G. polyisoprenivorans have been isolated from various environments. Lc is the sixth strain of G. polyisoprenivorans described, the second strain detected in the landfill leachate-contaminated groundwater and the first that is being studied for EPS production.

Rapid discrimination of lactic acid bacteria by matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was applied to the rapid discrimination of four lactic acid bacteria (Lactobacillus acidophilus, Lactococcus lactis subsp. hordniae, and two strains of Lc. lactis subsp. lactis). Specific biomarker peaks generated from intact bacteria cell were observed on the mass spectra over the range m/z 4000∼12000. High reproducibility for the relative peak intensities between three culture plates under the same experimental conditions was confirmed by analysis of variance (ANOVA). The growth temperature strongly affected the peak distribution of the biomarkers, whereas the difference in the culture media (MRS and LB media) gave similar mass spectra. Under the optimized conditions, four lactic acid bacteria with different genus, species, or strains could be discriminated.

Preliminary observations on proteolysis in Manchego cheese made with a defined-strain starter culture and adjunct starter (Lactobacillus plantarum) or a commercial starter

Different authors have demonstrated the potential of adding lactobacilli as adjunct cultures to pasteurized milk used in cheese manufacture. The aim of this work was to observe the effect of the use of a defined-strain starter culture and the addition of an adjunct culture (Lactobacillus plantarum) to cheesemilk in order to determine their effect on the ripening of Manchego cheese. Manchego cheeses were manufactured using one of the following starter culture systems: a defined starter consisting of Lactococcus lactis ssp. lactis and Leuconostoc mesenteroides ssp. dextranicum; a defined starter, as described above, and Lb. plantarum, which were isolated from a good quality Manchego cheese made from raw milk, or a commercial starter comprised of two strains of Lc. lactis. The cheeses were sampled at 15, 45, 90 and 150 d of ripening. Principal component analysis of peak heights of reversed-phase HPLC chromatograms of 70% (v/v) ethanol-insoluble and -soluble fractions distributed the samples according to the starter used in their manufacture. Quantitative differences in several peptides were evident between the three cheeses. Cheeses made with the defined-strain starters received higher scores for the flavour quality and intensity and for overall impression than the cheeses made with the commercial starter.

Sreening of Bacteriocin-Producing Bacteria from Various Sources

In this study, 1,602 bacterial strains were isolated from various sources and 18 isolates including 12 strains of Lactobacillus, five of Lactococcus lactis and one of Pseudomonas fluorescens, showed bacteriocin-like activity. These isolates were all from Kimchi and Tsukemono. Five strains of Lc. lactis inhibited Listeria monocytogenes, Clostridium difficile, C. perfringens and vancomycin resistant Enterococcus, while P. fluorescens inhibited methicillin resistant Staphylococcus aureus and Salmonella Enteritidis. However, Lactobacillus strains showed no activity against pathogens among these isolates.

122. The use of risk pathways and HACCP to assess CBPP control by use of veterinary cordon fences in Botswana

Rapid and specific detection of organisms belonging to the Mycoplasma mycoides cluster, among them Mycoplasma (M.) mycoides subsp. mycoides SC, the agent of contagious bovine pleuropneumonia, is an important condition for effective control of the respective animal diseases. In an effort to improve diagnosis, a PCR identification scheme for these mycoplasmas was developed. A set of primer combinations derived from the CAP-21 genomic regions of member organisms of the Mycoplasma mycoides cluster was selected by means of which complete differentiation within the cluster can be accomplished. Nested PCR involving cluster-specific amplification at the first stage and group-specific amplification using internal primers at the second stage was shown to be applicable for identification of all six groups forming the cluster. For example, external primers P1/P2 and internal primers P6/P7 were used to distinguish M. mycoides subsp. mycoides SC from LC strains. Using the present PCR procedure, identification of mycoplasmas could be considerably accelerated in comparison to conventional methods (5 h vs. one week) and specificity was also improved.

Screening for exopolysaccharide-producing bacteria from sub-tropical polluted groundwater.

A selection of exopolysaccharide (EPS)--producing bacterial strains was conducted in groundwater adjacent to an old controlled landfill in the City of São Carlos (São Paulo, Brazil). The strains were isolated in P and E media under aerobic and microacrophilic conditions at 25 degrees C. A total of 26 strains were isolated and based on the mucoid mode of the colonies, 6 were selected and their morphological, physiological and biochemical aspects were characterized. All strains presented pigmentation, ranging from yellow to orange and from pink to salmon, with a shiny glistening aspect in all tested media. Strains Lb, Lc and Lg, which excelled the others with regard to the mucoid mode of the colonies, were selected to be cultured in E medium with alternate sucrose and glucose as carbon sources in anaerobiosis at 25 degrees C to analyze the production of EPS. Strains Lc and Lg were classified as being of order Actinomycelates, suborder Corynebacterineae. Lg strain was identified as Gordonia polyisoprenivorans and Lc strain did not correspond to a known description and therefore a more detailed study is under preparation. Considering all ecological aspects and the metabolic potential associated with the microorganisms of the environment studied, as well as the capacity to produce pigment and EPS, and the presence of G. polyisoprenivorans, a rubber degrader bacterium, the potential of the groundwater analyzed is evident as a source of microorganisms to be utilized in studies related to environmental remediation.

Accumulation of casein-derived peptides during growth of proteinase-positive strains of Lactococcus lactis in milk: their contribution to subsequent bacterial growth is impaired by their internal transport.

To explain the limited nutritional value of milk cultured with proteinase-positive (Prt+) strains of Lactococcus lactis for the subsequent growth of dairy lactococci, we investigated further the time courses of modifications in the free amino acid and peptide contents of cultured milk. When growing in milk for up to 24 h, Prt+ strains of Lc. lactis progressively accumulated amino acids and casein-derived peptides. The growth of proteinase-negative (Prt-) wild-type strains and peptide transport mutants of Lc. lactis in cultured milk showed that casein-derived peptides could sustain growth up to 5 x 10(8) cfu/ml, depending on the extent of casein degradation during the preliminary growth of Prt+ strains and the Prt- strains. Of the casein-derived oligopeptides, < 25% were transported into the cell and used for Lc. lactis growth. However, they played a prominent role, contributing 90% to growth. In contrast, di- and tripeptides did not contribute to growth, suggesting that either few were released from caseins or they did not supply essential amino acids.

Evolution of Lactococcus strains during ripening in Brie cheese using Fourier transform infrared spectroscopy

The diversity of the Lactococcus flora during maturation of soft cheese, produced using one of 2 different starter cultures (S A and S B ), was determined using Fourier transform infrared spectroscopy (FTIR). Identification of Lactococcus sp. was achieved using a model composed of the 6 strains of Lc. lactis ssp. lactis and the 2 strains of Lc. lactis ssp. cremoris, present in the starter cultures S A and S B , as well as a reference strain for each of the subspecies considered. For the cheeses made with the starter culture S A , the proportion of strains within the cheeses was relatively homo- geneous throughout maturation, while in the cheeses made with the starter culture S B , one strain predominated. However, with both starters the strains of Lc. lactis ssp. cremoris failed to develop in the milk culture and in the cheeses. Results demonstrated that FTIR spectroscopy is a rapid and robust method for the qualitative analysis of cheese flora.

Genomic and antigenic differences between the European and African/Australian clusters of Mycoplasma mycoides subsp. mycoides SC The GenBank accession numbers for the nucleotide sequences determined in this work are: AF165134 for the 3·4 kb HindIII fragment from M. mycoides subsp. mycoides SC strain L2; AF165135 for the analogous locus in strain Afadé (containing lppB and IS1634); and AF1651136 for the

Mycoplasma mycoides subsp. mycoides small-colony type (SC), the aetiological agent of contagious bovine pleuropneumonia (CBPP), can be grouped into two major, epidemiologically distinct, clusters. One cluster contains strains isolated from different European countries since 1980 and a second cluster contains African and Australian strains collected over the last 50 years. Genetic analysis of representative strains from the two clusters revealed a genomic segment of 8.84 kb, located close to a copy of IS1296, which is present in all strains of the African cluster but lacking in all strains of the European cluster. This segment contains a copy of IS1634, a gene for a potential lipoprotein, IppB, open reading frames encoding a putative surface-located membrane protein and a hypothetical proline-rich membrane protein, and two open reading frames showing similarity to putative ABC transporters. The product of the IppB gene, lipoprotein B (LppB), has an apparent molecular mass of 70 kDa and was shown to be surface located. It is detected with monospecific antibodies in all strains of the African cluster tested, but not in European-cluster strains. DNA sequence analysis of the splicing site at which European strains differ from African-cluster strains by the lack of the 8.84 kb segment showed that the European cluster has arisen by deletion from a strain of the African cluster. Hence, M. mycoides subsp. mycoides SC strains isolated in different European countries from the newly reemerging outbreaks of CBPP, which occurred after the eradication of the epizootic in Europe in the middle of the 20th century, represent a phylogenetically newer cluster that has been derived from a strain of the older cluster of M. mycoides subsp. mycoides SC which is still endemic on the African continent.

Nutritional value of the non-protein N that accumulates during growth of proteinase-positive strains of Lactococcus lactis in milk for dairy lactococcal and leuconostoc isolates

To estimate the suitability of cultured milk for the subsequent growth of dairy lactic acid bacteria in cheese manufacturing, control milk was first cultured until the end of the exponential phase with one of four proteolytic (Prt+) strains of Lactococcus lactis differing in their proteolytic enzymes, and pasteurized after readjusting the pH to its initial value. Nineteen non-proteolytic (Prt−) strains of Lc. lactis and nine Leuconostoc mesenteroides strains of dairy origin were then grown in the precultured milk until the stationary phase and their growth was compared with that in control milk. Despite the accumulation of non-protein N (NPN) during preculture, the growth of most Prt− strains of Lc. lactis in precultured milk was either reduced or unchanged whereas that of Ln. mesenteroides strains was unchanged or slightly stimulated. This reduction in growth was reversed by adding an NPN source to precultured milk, indicating that it was due to the exhaustion of assimilable NPN in precultured milk. Thus, preculturing milk with Prt+ strains of Lc. lactis could not be recommended for promoting the subsequent growth of starter cultures in cheese manufacturing.

Plasmids with Homology to pFX3 and pCI3340 in Starter Lactococci and Lactobacilli and Electroporation of Selected Strains with these Vectors

An important consideration in the genetic manipulation of lactic acid bacteria is whether or not strains retain their normal industrial properties after transformation with plasmid DNA. A survey of plasmid types in various lactic acid bacteria showed that pE194-type rolling circle (RCR) plasmids were present in 24% of 70 commercial Lactococcus lactis cheese starters but not in any of 38 strains representing nine species of Lactobacillus. By contrast, theta plasmids showing homology to the pCI305 rep gene were present in most (though not all) lactococcal strains and not in any of the lactobacilli. Plasmids not belonging to either group were also present in some Lc. lactis and Lactobacillus strains. Six selected Lc. lactis subsp. cremoris strains were transformable with pFX3 (a 4.5 kb pE194-type rolling circle vector), yet four of these strains had significantly lower or zero transformation frequencies with pCI3340 (a 5.7 kb pCI305-based theta vector), possibly due to plasmid incompatibility. However, all transformants maintained full milkcoagulating activity. In addition to Lc. lactis, strains of Lc. garviae, Enterococcus faecalis and Lactobacillus reuteri were able to be transformed with pCI3340, making this a potentially useful vector for these species.

A PCR scheme for differentiation of organisms belonging to the Mycoplasma mycoides cluster

Rapid and specific detection of organisms belonging to the Mycoplasma mycoides cluster, among them Mycoplasma (M.) mycoides subsp. mycoides SC, the agent of contagious bovine pleuropneumonia, is an important condition for effective control of the respective animal diseases. In an effort to improve diagnosis, a PCR identification scheme for these mycoplasmas was developed. A set of primer combinations derived from the CAP-21 genomic regions of member organisms of the Mycoplasma mycoides cluster was selected by means of which complete differentiation within the cluster can be accomplished. Nested PCR involving cluster-specific amplification at the first stage and group-specific amplification using internal primers at the second stage was shown to be applicable for identification of all six groups forming the cluster. For example, external primers P1/P2 and internal primers P6/P7 were used to distinguish M. mycoides subsp. mycoides SC from LC strains. Using the present PCR procedure, identification of mycoplasmas could be considerably accelerated in comparison to conventional methods (5 h vs. one week) and specificity was also improved.

Genetic properties of Mycoplasma mycoides subspecies mycoides LC strains isolated from a goat in Japan in 1991.

Field isolated Mycoplasma strains LG1 and SF1 were isolated from the lung and leg joint fluid of a goat, respectively in Japan in 1991. These strains were closely related and had the following biochemical properties: glucose was metabolized, arginine and urea were not hydrolyzed, tetrazolium was reduced, phosphatase produced, and casein and coagulated serum were digested. Serologically, these strains cross-reacted with three Mycoplasma species [M. mycoides subsp. mycoides (Mmm), M. mycoides subsp. capri, and M. sp. Group 7]. From the above results, the field isolates were considered to be Mmm. In addition, DNA-DNA relatedness between the field isolates and other Mycoplasma species called the "mycoides cluster" was determined, and a recently developed PCR method was also used for mycoplasmal identification. From these results, the field isolates were identified as Mmm LC type.

Xenia and Maternal Effects on Maize Agronomic Traits at Three Plant Densities

Kernel mass is a primary yield component in maize (Zea mays L.) governed by both xenia and maternal effects. Because kernel growth depends on assimilate supply, we wanted to learn if kernel‐weight genes also affect plant size and agronomic characters. We reciprocally crossed three strains known to differ for rates of grain filling [high (HC), random (RC), and low (LC) R‐nj color] with two kernel‐weight strains [large (LG), and small (SM)] to produce 12 hybrid strains. For 2 yr at two locations, we grew plants of each hybrid at 24 000, 48 000, and 72 000 plants ha−1 in plots arranged so that wind pollinated silks with either LG or SM pollen. Leaf number, leaf width, leaf length, and time to silk were measured to compute leaf area and average leaf expansion rate (ALER). We also measured grain yield and grain moisture concentration at harvest. In one of four environments, hybrids pollinated by the LG strain had 34% higher yields and 24 g kg−1 lower harvest moisture than hybrids pollinated by the SM strain. Increasing plant density delayed silking, increased leaves per plant, and decreased leaf width. Hybrid plants from the LG strain yielded 10% more than hybrid plants from the SM strain. They also had 12% higher grain moisture at harvest, 11% greater leaf surface area (mainly from more and wider leaves), and silked 0.6 d later than SM plants. Hybrid plants from the HC strain produced higher grain yields and greater leaf surface areas than plants from the RC or the LC strains. Leaf surface area was correlated (r = 0.80, P &lt; 0.01) with grain yield. Kernel growth genes influenced yield via xenia effects but mainly by genes in the plant. Although R‐nj color genes only increased yields of specific reciprocal hybrids, in properly planned hybrids these genes may complement genes that increase yield via larger kernels.

Grain filling of R-nj color-selected maize strains divergently selected for kernel weight

Grain-fill characteristics in maize (Zea mays L.) affect yield by changing kernel weight. The objective of this study was to learn how divergent tandem selection for R-nj color expression and kernel weight affected rate of dry matter accumulation (RDMA), effective grain-filling period (EFPD), and lag phase duration (LAGP). We studied development of apical, mid-ear, and basal kernels in two genetic backgrounds. We derived 12 maize strains by tandem selection within each of two early-maturing synthetics. Mass selection of synthetics NDSF and NDSD for four cycles for high (HC), random (RC), and low (LC) R-nj color expression produced six substrains. Then, four cycles of divergent mass selection for kernel weight within each color-derived substrain produced 12 substrains for study. Using the 12 strains, we conducted field experiments using a completely random experimental design within each of 2 yr at Fargo, ND. Sequential kernel samples of individual ears within each strain provided data to estimate RDMA, EFPD, LAGP, and five-kernel weight (KWT). We sampled at 3- to 4-d intervals during the linear phase of grain-filling and at maturity. Selection for HC increased RDMA but tended to decrease EFPD compared to LC strains in both NDSF and NDSD. Selecting heavier kernels increased KWT of basal and mid-ear kernels by increasing RDMA. Direct and correlated responses to R-nj color selection were evident after four subsequent cycles of divergent tandem selection for kernel weight. Therefore, R-nj expression was not a temporary maternal effect. Kernel weight selection responses differed among the color strains and synthetics. Kernel weight seemed mainly determined by RDMA that was affected by selection for R-nj color expression and for kernel mass. Key words:Zea mays L., aleurone color, mass selection, correlation, yield components

Antagonism between lactic acid bacteria of wines: inhibition of Leuconostoc oenos by Lactobacillus plantarum and Pediococcus pentosaceus

In both grape juice and culture broth, lactic acid bacteria belonging to all the species found in wines interacted. Cultivation modified growth media in such a way that when added to fresh media they inhibited subsequent growth. Two strains, a Lactobacillus plantarum and a Pediococcus pentosaceus exhibited the most significant inhibitory effect against all the bacteria tested, especially Leuconostoc oenos. This effect was due to accumulation in the medium of some unidentified compounds. Added to the culture broth, the extracts prepared from L. plantarum and P. pentosaceus cultivated media did decrease the Lc. oenos growth rate and the final cell density, but they did not kill the bacteria and completely inhibit growth. They were not strictly strain-specific, several strains of Lc. oenos were inhibited. Some properties of the active substances were investigated. These substances were small, less than 1000 Da and thermostable. Their activity was altered by proteolytic treatments and heating.

Taxonomic significance of differences in DNA methylation within the ‘Mycoplasma mycoides cluster’ detected with restriction endonucleases MboI and DpnI

DNA from 22 strains belonging to the ‘Mycoplasma mycoides cluster’ was tested for methylation of adenine in GATC sequences by its resistance or susceptibility to digestion by the restriction endonucleases MboI, which is inhibited by the methylation, or DpnI, which requires the methylation. Strains of bovine serogroup 7, M. mycoides subsp. mycoides SC, and some M. mycoides subsp. capri strains plus M. capricolum strain Cal Kid lacked the methylation, whereas other strains of M. mycoides subsp. capri and of M. capricolum, plus the F38-like and M. mycoides subsp. mycoides LC strains, possessed it. We conclude that this simple test could provide a valuable criterion in identifying these mycoplamas.

Cytotoxicity of Mycoplasma mycoides Subspecies mycoides for Cultured Endothelial Cells

Toxic effect of 5 SC- and 4 LC-type strains of Mycoplasma mycoides subspecies mycoides has been demonstrated in cultured endothelial cells. The SC strains are agents of contagious bovine pleuropneumonia and presumably specific for cattle although occasionally isolated from goats. The LC strains produce an acute septicemic disease in goats and a few strains have been reported in cattle. The tissue culture cytotoxic dose causing 75% cell death (TCCD75) was calculated for each strain. Cytotoxicity ranged from 0.9 x 109 to 12.0 x 10 9 when strains were tested in bovine endothelial cells with the SC strains being twice as cytotoxic as the LC strains on average. Strains G175/78 and D44 representing the most cytotoxic SC and LC strains respectively, were selected for comparative experiments using bovine, caprine and porcine endothelial cells, bovine embryonic lung fibroblast cells (BELF) and the bovine cell line Madin-Darby kidney cells (MDBK). Strain G175/78 was significantly more cytotoxic for bovine endothelial cells than caprine and porcine, suggesting that the cytotoxicity reflects specificity for the bovine species. This strain was also cytotoxic for BELF but not for MDBK cells indicating that not all bovine cells are susceptible. Conversely, cytotoxicity of strain D44 was not significantly different in any of the endothelial cells tested, although cytotoxicity for BELF was significantly different, the cytotoxicity of the LC strains seems to be of less specificity.