A 'bacteriocin PCR array' for identification of bacteriocin-related structural genes in lactic acid bacteria

Bacteriocins have been identified in many strains of lactic acid bacteria (LAB) which are a source of natural food preservatives and microbial inhibitors. Our objectives were to use a PCR array of primers to identify bacteriocin structural genes in Bac⁺ LAB. DNA sequence homology at the 5′- and 3′-ends of the various structural genes indicated that non-specific priming may allow PCR amplification of heterologous bacteriocin genes. Successful amplification was obtained by real-time PCR and confirmed by melting curve and agarose gel analysis. Sequence information specific to targeted bacteriocin structural genes from the intra-primer regions of amplimers was compared to sequences residing in GenBank. The bacteriocin PCR array allowed the successful amplification of bacteriocin structural genes from strains of Lactobacillus, Lactococcus, and Pediococcus including one whose amino acid sequence was unable to be determined by Edman degradation analysis. DNA sequence analysis identified as many as 3 bacteriocin structural genes within a given strain, identifying ten unique bacteriocin sequences that were previously uncharacterized (partial homology) and one that was 100% identical to sequences in GenBank. This study provides a rapid approach to sequence and identify bacteriocin structural genes among Bac⁺ LAB using a microplate bacteriocin PCR array.     

Antimicrobial activity of lysostaphin and a Listeria monocytogenes bacteriophage endolysin produced and secreted by lactic acid bacteria

The expression and secretion signals of the Sep protein from Lactobacillus fermentum BR11 were used to direct export of two peptidoglycan hydrolases by Lb. fermentum BR11, Lactobacillus rhamnosus GG, Lactobacillus plantarum ATCC 14917 and Lactococcus lactis MG1363. The production levels, hydrolytic and bacteriocidal activities of the Listeria monocytogenes bacteriophage N-acetylmuramoyl-l-alanine amidase endolysin Ply511 and the glycylglycine endopeptidase lysostaphin were examined. Buffering of the growth media to a neutral pH allowed detection of Ply511 and lysostaphin peptidoglycan hydrolytic activity from all lactic acid bacteria. It was found that purified Ply511 has a pH activity range similar to that of lysostaphin with both enzymes functioning optimally under alkaline conditions. Supernatants from lactobacilli expressing lysostaphin reduced viability of methicillin resistant Staphylococcus aureus (MRSA) by approximately 8 log(10) CFU/ml compared to controls. However, supernatants containing Ply511 were unable to control L. monocytogenes growth. In coculture experiments, both Lb. plantarum and Lb. fermentum synthesizing lysostaphin were able to effectively reduce MRSA cell numbers by >7.4 and 1.7 log(10)CFU/ml, respectively, while lactic acid bacteria secreting Ply511 were unable to significantly inhibit the growth of L. monocytogenes. Our results demonstrate that lysostaphin and Ply511 can be expressed in an active form from different lactic acid bacteria and lysostaphin showed superior killing activity. Lactobacilli producing lysostaphin may have potential for in situ biopreservation in foodstuffs or for prevention of S. aureus infections.     

Honeybees and beehives are rich sources for fructophilic lactic acid bacteria

Fructophilic lactic acid bacteria (FLAB) are a specific group of lactic acid bacteria (LAB) characterized and described only recently. They prefer fructose as growth substrate and inhabit only fructose-rich niches. Honeybees are high-fructose-consuming insects and important pollinators in nature, but reported to be decreasing in the wild. In the present study, we analyzed FLAB microbiota in honeybees, larvae, fresh honey and bee pollen. A total of 66 strains of LAB were isolated from samples using a selective isolation technique for FLAB. Surprisingly, all strains showed fructophilic characteristics. The 66 strains and ten FLAB strains isolated from flowers in a separate study were genotypically separated into six groups, four of which being identified as Lactobacillus kunkeei and two as Fructobacillus fructosus. One of the L. kunkeei isolates showed antibacterial activity against Melissococcus plutonius, a causative pathogen of European foulbrood, this protection being attributable to production of an antibacterial peptide or protein. Culture-independent analysis suggested that bee products and larvae contained simple Lactobacillus-group microbiota, dominated by L. kunkeei, although adult bees carried a more complex microbiota. The findings clearly demonstrate that honeybees and their products are rich sources of FLAB, and FLAB are potential candidates for future bee probiotics.     

Isolation and characterization of fructophilic lactic acid bacteria from fructose-rich niches

Fourteen strains of fructophilic lactic acid bacteria were isolated from fructose-rich niches, flowers, and fruits. Phylogenetic analysis and BLAST analysis of 16S rDNA sequences identified six strains as Lactobacillus kunkeei, four as Fructobacillus pseudoficulneus, and one as Fructobacillus fructosus. The remaining three strains grouped within the Lactobacillus buchneri phylogenetic subcluster, but shared low sequence similarities to other known Lactobacillus spp. The fructophilic strains fermented only a few carbohydrates and fermented d-fructose faster than d-glucose. Based on the growth characteristics, the 14 isolates were divided into two groups. Strains in the first group containing L. kunkeei, F. fructosus, and F. pseudoficulneus grew well on d-fructose and on d-glucose with pyruvate or oxygen as external electron acceptors, but poorly on d-glucose without the electron acceptors. Strains in this group were classified as “obligately” fructophilic lactic acid bacteria. The second group contained three unidentified strains of Lactobacillus that grew well on d-fructose and on d-glucose with the electron acceptors. These strains grew on d-glucose without the electron acceptors, but at a delayed rate. Strains in this group were classified as facultatively fructophilic lactic acid bacteria. All fructophilic isolates were heterofermentative lactic acid bacteria, but “obligately” fructophilic lactic acid bacteria mainly produced lactic acid and acetic acid and very little ethanol from d-glucose. Facultatively fructophilic strains produced lactic acid, acetic acid and ethanol, but at a ratio different from that recorded for heterofermentative lactic acid bacteria. These unique characteristics may have been obtained through adaptation to the habitat.     

From physiology to systems metabolic engineering for the production of biochemicals by lactic acid bacteria

The lactic acid bacteria (LAB) are a functionally related group of low-GC Gram-positive bacteria known essentially for their roles in bioprocessing of foods and animal feeds. Due to extensive industrial use and enormous economical value, LAB have been intensively studied and a large body of comprehensive data on their metabolism and genetics was generated throughout the years. This knowledge has been instrumental in the implementation of successful applications in the food industry, such as the selection of robust starter cultures with desired phenotypic traits. The advent of genomics, functional genomics and high-throughput experimentation combined with powerful computational tools currently allows for a systems level understanding of these food industry workhorses. The technological developments in the last decade have provided the foundation for the use of LAB in applications beyond the classic food fermentations. Here we discuss recent metabolic engineering strategies to improve particular cellular traits of LAB and to design LAB cell factories for the bioproduction of added value chemicals.     

Natural populations of lactic acid bacteria associated with silage fermentation as determined by phenotype, 16S ribosomal RNA and recA gene analysis

One hundred and fifty-six strains isolated from corn (Zea mays L.), forage paddy rice (Oryza sativa L.), sorghum (Sorghum bicolor L.) and alfalfa (Medicago sativa L.) silages prepared on dairy farms were screened, of which 110 isolates were considered to be lactic acid bacteria (LAB) according to their Gram-positive and catalase-negative characteristics and, mainly, the lactic acid metabolic products. These isolates were divided into eight groups (A-H) based on the following properties: morphological and biochemical characteristics, γ-aminobutyric acid production capacity, and 16S rRNA gene sequences. They were identified as Weissella cibaria (36.4%), Weissella confusa (9.1%), Leuconostoc citreum (5.3%), Leuconostoc lactis (4.9%), Leuconostoc pseudomesenteroides (8.0%), Lactococcus lactis subsp. lactis (4.5%), Lactobacillus paraplantarum (4.5%) and Lactobacillus plantarum (27.3%). W. cibaria and W. confusa were mainly present in corn silages, and L. plantarum was dominant on sorghum and forage paddy rice silages, while L. pseudomesenteroides, L. plantarum and L. paraplantarum were the dominant species in alfalfa silage. The corn, sorghum and forage paddy rice silages were well preserved with lower pH values and ammonia-N concentrations, but had higher lactic acid content, while the alfalfa silage had relatively poor quality with higher pH values and ammonia-N concentrations, and lower lactic acid content. The present study confirmed the diversity of LAB species inhabiting silages. It showed that the differing natural populations of LAB on these silages might influence fermentation quality. These results will enable future research on the relationship between LAB species and silage fermentation quality, and will enhance the screening of appropriate inoculants aimed at improving such quality.     

A rapid method for differentiation of dairy lactic acid bacteria by enzyme systems

Summary A rapid and simple technique utilizing the APIZYM enzymatic patterns complemented with arginine dihydrolase and citratase was developed for species differentiation of 40 lactic acid bacteria relevant to the dairy industry.Streptococcus species in general produced no -galactosidase, except forStreptococcus thermophilus. Lactobacillus species showed strong aminopeptidases and galactosidases but contained no arginine dihydrolase and citratase. Among the group N-streptococci,Streptococcus diacetylactis produced citratase, whereasStreptococcus cremoris differed by the production of butyrate esterase.Streptococcus faecalis was readily distinguishable fromStreptococcus lactis by butyrate esterase activity that was the basis of the differential agar developed. Heterofermentative lactobacilli differed from homofermentative lactobacilli in possessing arginine dihydrolase and citratase but by not producing leucine-aminopeptidase.     

Molecular characterization of lactic acid bacteria recovered from natural fermentation of beet root and carrot Kanji

The lactic acid bacteria (LAB) play an important role in the fermentation of vegetables to improve nutritive value, palatability, acceptability, microbial quality and shelf life of the fermented produce. The LAB associated with beetroot and carrot fermentation were identified and characterized using different molecular tools. Amplified ribosomal DNA restriction analysis (ARDRA) provided similar DNA profile for the 16 LAB strains isolated from beetroot and carrot fermentation while repetitive extragenic palindromic PCR (rep-PCR) genotyping could differentiate the LAB strains into eight genotypes. Thirteen strains represented by five genotypes could be clustered in five distinct groups while three LAB strains exhibiting distinct genotypes remained ungrouped. These genotypes could be identified to be belonging to L. plantarum group by 16S rDNA sequencing. The recAnested multiplex PCR employing species-specific primers for the L. plantarum group members identified the LAB strains of six genotypes to be L. paraplantarum and the other two genotypes to be L. pentosus. Three genotypes of L. paraplantarum were consistently found on the third and sixth day of beetroot fermentation whereas a distinct genotype of L. paraplantarum and L. pentosus appeared predominant on the tenth day. From carrot Kanji two distinct genotypes of L. paraplantarum and one genotype of L. pentosus were identified. REP-PCR DNA fingerprinting coupled with 16S rDNA sequencing and recA-nested multiplex PCR could clearly identify as well as differentiate the diverse L. plantarum group strains involved in the fermentation.     

Isolation, characterization and evolution of a new thermophilic Bacillus licheniformis for lactic acid production in mineral salts medium

The high fermentation cost of lactic acid is a barrier for polylactic acid (PLA) to compete with the petrochemical derived plastics. In order to lower the cost of lactic acid, the industry needs a microorganism that can ferment various sugars at high temperature (50 °C) and at the same time using low cost mineral salts (MS) medium. One such bacterium, BL1, was isolated at 50 °C and identified as Bacillus licheniformis. BL1 can ferment glucose to optically pure l-lactate with a maximum specific productivity of 7.8 g/h l in LB medium and 0.7 g/h l in MS medium at 50 °C. BL1 can also consume 10% and 15% glucose in 20 and 48 h, respectively. After serial transfer of BL1 and BL2 in different concentrations of xylose and MS medium respectively, the final mutant BL3 could efficiently ferment glucose and xylose with specific productivity of 1.9 g/h l and 1.2 g/h l in strict MS medium.     

应用乳酸菌生物保护剂控制肉制品中单增李斯特菌的研究进展

肉制品营养丰富,极易被微生物污染,单增李斯特菌是污染肉制品主要病原菌之一。乳酸菌做为生物保护剂已经被广泛应用于食品中控制单增李斯特菌。本文首先分析了我国肉制品中单增李斯特菌的污染状况,总结了乳酸菌应用于肉制品安全控制的概况;然后进一步详细介绍了乳酸菌对单增李斯特菌的抑菌机理,着重探讨了乳酸菌对单增李斯特菌致病能力(生长、抗性和毒性)的影响;文章最后指出了乳酸菌在食品应用中存在的问题,并对未来的研究方向提供了建议,以期为乳酸菌在食品安全控制中的应用提供参考。     

Characterization and application of a native lactic acid bacterium isolated from tannery fleshings for fermentative bioconversion of tannery fleshings

Lactic acid bacteria (LAB) species isolated from limed and delimed tannery fleshings (TF) were evaluated for their fermentation efficiency and antibacterial property. The native LAB isolates efficiently fermented TF and resulted in a fermented mass with antioxidant properties, indicating their potential for effective eco-friendly bioconversion of TF. From among the LAB isolated, a proteolytic isolate showing better antimicrobial spectrum and reasonably good fermentation efficiency was identified as Enterococcus faecium HAB01 based on various biochemical and molecular tests. This isolate afforded a better degree of hydrolysis (81.36%) of TF than Pediococcus acidilactici (54.64%) that was previously reported by us. The bacteriocin produced by E. faecium was found to be antagonistic to several human pathogens including Listeria, Aeromonas, Staphylococcus and Salmonella. Further, E. faecium HAB01 bacteriocin was thermostable and had a molecular weight of around 5 kDa, apart from being stable at both acidic and alkaline conditions. The bacteriocin was unstable against proteases.     

Role of lipid rafts in E-cadherin-- and HGF-R/Met--mediated entry of Listeria monocytogenes into host cells

Listeria monocytogenes uptake by nonphagocytic cells is promoted by the bacterial invasion proteins internalin and InlB, which bind to their host receptors E-cadherin and hepatocyte growth factor receptor (HGF-R)/Met, respectively. Here, we present evidence that plasma membrane organization in lipid domains is critical for Listeria uptake. Cholesterol depletion by methyl-beta-cyclodextrin reversibly inhibited Listeria entry. Lipid raft markers, such as glycosylphosphatidylinositol-linked proteins, a myristoylated and palmitoylated peptide and the ganglioside GM1 were recruited at the bacterial entry site. We analyzed which molecular events require membrane cholesterol and found that the presence of E-cadherin in lipid domains was necessary for initial interaction with internalin to promote bacterial entry. In contrast, the initial interaction of InlB with HGF-R did not require membrane cholesterol, whereas downstream signaling leading to F-actin polymerization was cholesterol dependent. Our work, in addition to documenting for the first time the role of lipid rafts in Listeria entry, provides the first evidence that E-cadherin and HGF-R require lipid domain integrity for their full activity.     

Caseicin 80: purification and characterization of a new bacteriocin from Lactobacillus casei

When grown in complex or synthetic media, Lactobacillus casei B 80 synthesizes a mitomycin C-inducible polypeptide with very specific bactericidal activity against the sensitive strain Lactobacillus casei B 109. The amount of secreted bacteriocin in the culture solution was low, about 1 mg/l. The bacteriocin which we called caseicin 80, was also detectable in cell extracts, although only 2% of the total activity was retained intracellularly. Caseicin 80 was concentrated by ultrafiltration and purified by cation exchange chromatography with Cellulose SE-23 and Superose. The molecular weight was in the range of M _r=40,000–42,000 and the isoelectric point was pH 4.5.     

Purification, amino acid sequence and characterization of the class IIa bacteriocin weissellin A, produced by Weissella paramesenteroides DX

Weissella paramesenteroides DX has been shown to produce a 4450-Da class IIa bacteriocin, weissellin A, composed of 43 amino acids with the sequence KNYGNGVYCNKHKCSVDWATFSANIANNSVAMAGLTGGNAGN. The bacteriocin shares 68% similarity with leucocin C from Leuconostoc mesenteroides. Computational analyses predict that the bacteriocin is a hydrophobic molecule with a beta-sheet type conformation. Weissellin A exhibited various levels of activity against all gram-positive bacteria tested, but was not active against Salmonella enterica Enteritidis. The antimicrobial activity was not associated with target-cell lysis. The bacteriocin retained activity after exposure to 121 °C for 60 min or to −20 °C for 6 months, and to pH 2.0-10.0. It was not sensitive to trypsin, α-chymotrypsin, pepsin and papain, but was inactivated by proteinase K. At a dissolved oxygen concentration of 50%, weissellin A was produced with growth-associated kinetics. The properties of weissellin A make this bacteriocin a potentially suitable agent for food and feed preservation.     

Flour carbohydrate catabolism and metabolite production by sourdough lactic acid bacteria

The aim of this study was to assess the mode of carbohydrate catabolism by lactic acid bacteria isolated from traditional sourdoughs, as well as to study their effect on the metabolites produced. For this purpose, single cultures of the heterofermentative lactic acid bacteria Lactobacillus sanfranciscensis, Lactobacillus brevis, Weissella cibaria, and the homofermentative Lactobacillus paralimentarius and Pediococcus pentosaceus were grown in liquid media containing glucose, fructose, maltose and sucrose, either as a single carbon source or in combination with glucose. Carbon catabolism and the production of metabolites were determined by HPLC analysis. W. cibaria could ferment all carbon sources, L. sanfranciscensis, L. paralimentarius and P. pentosaceus could not ferment sucrose, while L. brevis could only ferment maltose. The presence of glucose did not influence the utilization of fructose and maltose by L. sanfranciscensis, while it repressed the fermentation of fructose, maltose and sucrose by W. cibaria, and fructose and maltose by L. paralimentarius and P. pentosaceus. Moreover, L. sanfranciscensis and L. brevis could obtain extra ATP through the reduction of fructose to mannitol, which favored the production of acetic acid against ethanol. The utilization of fructose as an electron acceptor has a decisive effect on the prevailing of L. sanfranciscensis and L. brevis in spontaneously fermented sourdough and in the scarce appearance of the other lactic acid bacteria studied.     

Characterization of lactic acid bacteria isolated from sourdoughs for Cornetto, a traditional bread produced in Basilicata (Southern Italy)

A total of 41 strains of lactic acid bacteria (LAB) isolated from durum wheat sourdoughs used to produce Cornetto di Matera bread, were identified by SDS-PAGE of whole cell proteins (WCP) and screened for acid production ability, antimicrobial activity and exopolysaccharide (EPS) production. The isolates were identified as Lactobacillus plantarum (49%), Leuconostoc mesenteroides (17%), Lactobacillus curvatus (15%), Lactobacillus paraplantarum (12%), Weissella cibaria (5%) and Lactobacillus pentosus (2%). Several strains of Lb. plantarum and Leuc. mesenteroides showed a high acid production ability. The antagonistic activity was tested using an agar-spot deferred antagonism assay against a set of five indicators. The species had different profiles of inhibition. Lb. plantarum had the largest spectrum of inhibition, while no isolates of W. cibaria and Leuc. mesenteroides showed antimicrobial activity. No strains had antimicrobial activity against Bacillus cereus. The inhibitory activity of five strains was confirmed to be sensitive to proteolytic enzymes and thus potentially due to bacteriocin production. All Leuc. mesenteroides and W. cibaria strains produced EPS from sucrose. Some Lb. plantarum and Lb. paraplantarum strains produced EPS from different sugars in solid media. EPS production in liquid media was different within the species, with the highest production in liquid media containing glucose and maltose. A defined strain starter culture (W. cibaria DBPZ1006, Lb. plantarum DBPZ1015 and S. cerevisiae MTG10) was selected on the basis of technological properties and tested in model sourdough fermentations.     

Evaluation of biodiversity of lactic acid bacteria microbiota in the calf intestinal tracts

Amplified fragment length polymorphism (AFLPs) were used to analyse the naturally occurring flora of lactic acid bacteria (LAB) in gastrointestinal tracts of two healthy 65-day-old calves. More than 1,000 of presumptive LAB were collected and cultured from the gastrointestinal tracts and, among the isolated colonies, a total of 311 strains were analysed and separated into eight clusters based on AFLP banding patterns. To precisely determine the species inside the clusters, partial sequences of fragments of the 16S ribosomal DNA gene were determined, and sequence homology searches were conducted through GenBank on few strains per cluster. The most representative genera of LAB were Lactobacillus (169 isolates, 54% of total) and Streptococcus (99 isolates, 32% of total), while the most frequent species was identified as L. mucosae with 86 different isolates (51% of the Lactobacillus spp. and 28% of the total). This report gives a first characterization of LAB strain biodiversity recovered directly from calf intestine and is the first account of the presence of the L. mucosae species in calves. Moreover it demonstrates that the AFLP is a robust and useful technique for characterizing the strain level of LAB microflora.     

Structure of a lactic acid ether-containing and glycerol phosphate-containing O-polysaccharide from Proteus mirabilis O40

An O-polysaccharide was isolated by mild acid hydrolysis from the lipopolysaccharide of Proteus mirabilis O40 and studied by NMR spectroscopy, including 2D 1H, 1H COSY, TOCSY, ROESY, and 1H, 13C HMQC experiments, along with chemical methods. The polysaccharide was found to contain an ether of GlcNAc with lactic acid and glycerol phosphate in the main chain and to have the following structure: --> 3)-beta-D-GlcpNAc4(R-Lac)-(1 --> 3)-alpha-D-Galp-(1 --> 3)-D-Gro-1-P-(O --> 3)-beta-D-GlcpNAc-(1 --> where D-GlcpNAc4(R-Lac) stands for 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose. This structure is unique among the known structures of the Proteus O-polysaccharides, which is in agreement with the classification of the strain studied into a separate O-serogroup. A serological relatedness of P. mirabilis O40 with some other Proteus strains was revealed and discussed in view of the O-polysaccharide structures.