Lactococci as probiotic strains: adhesion to human enterocyte-like Caco-2 cells and tolerance to low pH and bile

There have been few studies on the probiotic activity of Lactococcus strains although they are commonly used as starter bacteria in manufacturing many kinds of fermented dairy products. Nine strains of the genus Lactococcus were examined for their probiotic properties, such as adherence to human enterocyte-like Caco-2 cells and tolerance to acid and bile. Six strains were adhesive and the highest adhesion was observed with Lactcoccus lactis ssp. lactis NIAI527. This strain adhered to the microvilli of cells as observed by scanning electron microscopy and also tolerated low pH and bile. These properties should make strain 527 a potential new probiotic strain.     

Complete genome sequence of the prototype lactic acid bacterium Lactococcus lactis subsp. cremoris MG1363

Lactococcus lactis is of great importance for the nutrition of hundreds of millions of people worldwide. This paper describes the genome sequence of Lactococcus lactis subsp. cremoris MG1363, the lactococcal strain most intensively studied throughout the world. The 2,529,478-bp genome contains 81 pseudogenes and encodes 2,436 proteins. Of the 530 unique proteins, 47 belong to the COG (clusters of orthologous groups) functional category "carbohydrate metabolism and transport," by far the largest category of novel proteins in comparison with L. lactis subsp. lactis IL1403. Nearly one-fifth of the 71 insertion elements are concentrated in a specific 56-kb region. This integration hot-spot region carries genes that are typically associated with lactococcal plasmids and a repeat sequence specifically found on plasmids and in the "lateral gene transfer hot spot" in the genome of Streptococcus thermophilus. Although the parent of L. lactis MG1363 was used to demonstrate lysogeny in Lactococcus, L. lactis MG1363 carries four remnant/satellite phages and two apparently complete prophages. The availability of the L. lactis MG1363 genome sequence will reinforce its status as the prototype among lactic acid bacteria through facilitation of further applied and fundamental research.     

Ubiquity and diversity of multidrug resistance genes in Lactococcus lactis strains isolated between 1936 and 1995

The presence and the nucleotide sequence of four multidrug resistance genes, lmrA, lmrP, lmrC, and lmrD, were investigated in 13 strains of Lactococcus lactis ssp. lactis, four strains of Lactococcus lactis ssp. cremoris, two strains of Lactococcus plantarum, and two strains of Lactococcus raffinolactis. Multidrug resistance genes were present in all L. lactis isolates tested. However, none of them could be detected in the strains belonging to the species L. raffinolactis and L. plantarum, suggesting a different set of multidrug resistance genes in these species. The analysis of the four deduced amino acid sequences established two different variants depending on the subspecies of L. lactis. Either lmrA, or lmrP, or both were found naturally disrupted in five strains, while full-length lmrD was present in all strains.     

Effect of products of alphaS1-casein proteolysis on the activity of angiotensin-converting enzyme

The influence of alpha s1-casein proteolysis products of cow milk on the activity of angiotensin-converting enzyme (ACE) has been investigated. The peptide fraction has been obtained after incubation of alpha s1-casein with different strains Lactococcus lactis ssp. lactis and pepsin. The peptides with low molecular weight has been obtained with the help of a gel filtration. It is shown, that such peptides received with the help of some strains of Lactococcus lactis ssp. lactis and pepsin are capable to inhibit the activity of ACE. A conclusion about possibility of appearing antihypertensive peptides as a result of proteolitic processes in milk products has been made.     

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.     

超声波诱变选育乳链菌肽(Nisin)高产菌株

目的：从乳酸乳球菌中选育Nisin高产菌株。方法：利用超声波对乳酸乳球菌进行诱变,并用琼脂扩散法检测其效价。结果：获得一突变菌株S-1,该菌株的生物效价为343.53IU/mL,比原始菌株提高31.52%。结论：突变株S-1遗传性能稳定,为进一步菌种选育奠定基础。     

Additive Effects of Two Different Plasmid-Linked Restriction and Modification Systems in Lactococcus

Two plasmids, pND801 and pND802, encoding different restriction and modification systems were isolated from Lactococcus lactis ssp. lactis LL42-1 and Lactococcus lactis ssp. cremoris LC14-1, respectively. pND802 contained one Sphl restriction enzyme site and the whole plasmid was cloned into the Sphl site of the streptococcal/ E. coli shuttle vector pSA3 generating the plasmid pND803. pND803 was stably maintained in L.lactis MG1363 harbouring pND801. The combination of the two R/M systems within L.lactis MG1363 resulted in an additive resistance towards both isometric phage and prolate phage.     

Relationship between the presence of the citrate permease plasmid and high electron-donor surface properties of Lactococcus lactis ssp. lactis biovar. diacetylactis

Some strains of Lactococcus lactis subspecies possess a citrate permease that enables them to utilize citrate and to produce diacetyl. Such strains are classified as diacetylactis biovariants (L. lactis ssp. lactis biovar. diacetylactis). We investigated the electron-donor surface properties of L. lactis strains and observed that the diacetylactis biovariants presented increased adhesion to electron-acceptor solvents (microbial adhesion to solvents electron-donor characteristics of cells of <27% for L. lactis and about 50% for L. lactis ssp. lactis biovar diacetylactis). We investigated the properties of a pCitP- derivative and observed for a diacetylactis biovariant strain a loss of the electron-donor characteristics falling from 47% for a pCitP+ strain to 8% for its pCitP- derivative. This suggests that the presence of high electron-donor characteristics on the surface of L. lactis results to a large extent from the presence of the citrate permease plasmid.     

乳酸乳球菌作为黏膜免疫活载体疫苗传递抗原的研究进展

乳酸菌是人和动物肠道内的常见细菌,被公认为安全级(generally recognized as safe,GRAS)微生物。近年来,对于乳酸菌作为宿主菌表达外源蛋白或抗原的研究取得了一定进展。乳酸乳球菌(Lactococcus lactis)是乳酸菌的代表菌种,以其生长迅速、易于操作等优点成为表达外源抗原,作为黏膜免疫活载体疫苗的理想菌株。随着对乳酸乳球菌基因工程的研究逐渐深入,已发展了一系列组成型和诱导型乳酸乳球菌表达系统以及蛋白定位系统。破伤风毒素片段C、布氏杆菌L7/L12蛋白等多种病原微生物抗原已成功在乳酸乳球菌中表达,并已证明部分重组乳酸乳球菌作为黏膜免疫疫苗可以同时刺激局部黏膜免疫应答和系统免疫应答。目前,如何使活载体乳酸乳球菌以最佳方式向黏膜免疫系统提呈抗原继而诱导有效免疫反应是该领域的研究热点,也是巨大挑战。实现外源抗原在乳酸乳球菌中的准确定位及与细胞因子的共表达是未来研究的重要方向之一。乳酸乳球菌作为黏膜免疫活载体疫苗传递外源抗原具有广阔的应用前景。     

Comparative genome analysis of Lactococcus garvieae using a suppression subtractive hybridization library: discovery of novel DNA signatures

Lactococcus garvieae, the pathogenic species in the genus Lactococcus, is recognized as an emerging pathogen in fish, animals, and humans. Despite the widespread distribution and emerging clinical significance of L. garvieae, little is known about the genomic content of this microorganism. Suppression subtractive hybridization was performed to identify the genomic differences between L. garvieae and Lactococcus lactis ssp. lactis, its closest phylogenetic neighbor, and the type species of the genus Lactococcus. Twenty-seven clones were specific to L. garvieae and were highly different from Lactococcus lactis in their nucleotide and protein sequences. Lactococcus garvieae primer sets were subsequently designed for two of these clones corresponding to a pyrH gene and a novel DNA signature for application in the specific detection of L. garvieae. The primer specificities were evaluated relative to three previously described 16S rRNA gene-targeted methods using 32 Lactococcus and closely related strains. Both newly designed primer sets were highly specific to L. garvieae and performed better than did the existing primers. Our findings may be useful for developing more stable and accurate tools for the discrimination of L. garvieae from other closely related species.     

Identification and characterization of a cystathionine beta/gamma-lyase from Lactococcus lactis ssp. cremoris MG1363

This paper describes the nucleotide sequence of a gene encoding cystathionine beta/gamma-lyase from Lactococcus lactis ssp. cremoris MG1363, its overexpression in Escherichia coli and some functional characteristics of the purified recombinant protein.     

Minisatellite polymorphism as a tool to distinguish closely related Lactococcus lactis strains

Genome plasticity is considered as a means for bacteria to adapt to their environment. Plasticity in tandem repeat sequences on bacterial genomes has been recently exploited to trace the epidemiology of pathogens. Here, we examine the utility of minisatellite (i.e., a repeat unit of six nucleotides or more) typing in non-pathogenic food bacteria of the species Lactococcus lactis. Thirty-four minisatellites identified on the sequenced L. lactis ssp. lactis strain IL1403 genome were first analyzed in 10 closely related ssp. lactis strains, as determined by randomly amplified polymorphic DNA (RAPD). The selected tandem repeats varied in length, percent identity between repeats, and locations. We showed that: (i) the greatest polymorphism was in orfs encoding exported proteins or in intergenic regions; (ii) two thirds of minisatellites were little- or non-variable, despite as much as 90% identity between tandem repeats; and (iii) dendrograms based on either RAPD or minisatellite analyses were similar. Seven minisatellites identified in this study are potentially useful for lactococcal typing. We then asked whether tandem repeats in L. lactis were stable upon very long-term (up to two years) storage. Despite large rearrangements previously reported in derivative strains, just one of 10 minisatellites tested underwent an alteration, suggesting that tandem repeat rearrangements probably occur during active DNA replication. We conclude that multiple locus minisatellite analysis can be a valuable tool to follow lactococcal strain diversity.     

The dilemma of phage taxonomy illustrated by comparative genomics of Sfi21-like Siphoviridae in lactic acid bacteria

The complete genome sequences of two dairy phages, Streptococcus thermophilus phage 7201 and Lactobacillus casei phage A2, are reported. Comparative genomics reveals that both phages are members of the recently proposed Sfi21-like genus of Siphoviridae, a widely distributed phage type in low-GC-content gram-positive bacteria. Graded relatedness, the hallmark of evolving biological systems, was observed when different Sfi21-like phages were compared. Across the structural module, the graded relatedness was represented by a high level of DNA sequence similarity or protein sequence similarity, or a shared gene map in the absence of sequence relatedness. This varying range of relatedness was found within Sfi21-like phages from a single species as demonstrated by the different prophages harbored by Lactococcus lactis strain IL1403. A systematic dot plot analysis with 11 complete L. lactis phage genome sequences revealed a clear separation of all temperate phages from two classes of virulent phages. The temperate lactococcal phages share DNA sequence homology in a patchwise fashion over the nonstructural gene cluster. With respect to structural genes, four DNA homology groups could be defined within temperate L. lactis phages. Closely related structural modules for all four DNA homology groups were detected in phages from Streptococcus or Listeria, suggesting that they represent distinct evolutionary lineages that have not uniquely evolved in L. lactis. It seems reasonable to base phage taxonomy on data from comparative genomics. However, the peculiar modular nature of phage evolution creates ambiguities in the definition of phage taxa by comparative genomics. For example, depending on the module on which the classification is based, temperate lactococcal phages can be classified as a single phage species, as four distinct phage species, or as two if not three different phage genera. We propose to base phage taxonomy on comparative genomics of a single structural gene module (head or tail genes). This partially phylogeny-based taxonomical system still mirrors some aspects of the current International Committee on Taxonomy in Virology classification system. In this system the currently sequenced lactococcal phages would be grouped into five genera: c2-, sk1, Sfi11-, r1t-, and Sfi21-like phages.     

Application of the ligase chain reaction to the detection of nisinA and nisinZ genes in Lactococcus lactis ssp. lactis

Abstract This paper reports on the application of the ligase chain reaction (LCR) to the specific detection of variants of the nisin structural gene (nisinA and nisinZ) in nisin producing strains of Lactococcus lactis ssp lactis . The LCR assay was used to screen nisin producing strains to determine which form of the nisin structural gene they contained. This method of differentiating the nisin structural gene variants provides a useful alternative to the only other available genetic differentiation, that of sequencing the gene.     

Redox potential to discriminate among species of lactic acid bacteria

AIMS: To verify to what degree reducing capacity is a characterizing parameter of a species, and of the strains themselves within a given species, of lactic acid bacteria. METHODS AND RESULTS: Eighty-eight strains belonging to 10 species of lactic acid bacteria (LAB) isolated from traditional Italian cheeses were studied for their reduction activity: Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Streptococcus thermophilus, Lactococcus lactis ssp. lactis, Lactobacillus paracasei ssp. paracasei, Lactobacillus plantarum, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus helveticus and Pediococcus pentosaceus. It was observed that the lactococci reached minimum redox potential before the lactobacilli. The reduction rate of Enterococcus spp. and L. lactis ssp. lactis was higher than that of the streptococci and Lactobacillus spp. All the P. pentosaceus strains had poor reduction activity compared with the other species. CONCLUSIONS: The evolution of the redox potential in milk over a time span of 24 h has been found to be a parameter that characterizes a species: the different courses corresponding to the species in question are clearly evident, and interesting differences can also be noted within the same species. SIGNIFICANCE AND IMPACT OF THE STUDY: The reduction aptitude of strains might be used to select and adapt appropriate strains for use as starters for dairy products.