Cloning and expression of a novel lactococcal aggregation factor from Lactococcus lactis subsp. lactis BGKP1

<p>Abstract</p> <p>Background</p> <p>Aggregation may play a main role in the adhesion of bacteria to the gastrointestinal epithelium and their colonization ability, as well as in probiotic effects through co-aggregation with intestinal pathogens and their subsequent removal. The aggregation phenomenon in lactococci is directly associated with the sex factor and lactose plasmid co-integration event or duplication of the cell wall spanning (CWS) domain of PrtP proteinase.</p> <p>Results</p> <p><it>Lactococcus lactis </it>subsp. <it>lactis </it>BGKP1 was isolated from artisanal semi-hard homemade cheese and selected due to its strong auto-aggregation phenotype. Subsequently, non-aggregating derivative (Agg^-) of BGKP1, designated as BGKP1-20, was isolated, too. Comparative analysis of cell surface proteins of BGKP1 and derivative BGKP1-20 revealed a protein of approximately 200 kDa only in the parental strain BGKP1. The gene involved in aggregation (<it>aggL</it>) was mapped on plasmid pKP1 (16.2 kb), cloned and expressed in homologous and heterologous lactococci and enterococci. This novel lactococcal aggregation protein was shown to be sufficient for cell aggregation in all tested hosts. In addition to the <it>aggL </it>gene, six more ORFs involved in replication (<it>repB </it>and <it>repX</it>), restriction and modification (<it>hsdS</it>), transposition (<it>tnp</it>) and possible interaction with mucin (<it>mbpL</it>) were also located on plasmid pKP1.</p> <p>Conclusion</p> <p>AggL is a new protein belonging to the collagen-binding superfamily of proteins and is sufficient for cell aggregation in lactococci.</p

Use of Lactobacillus helveticus BGRA43 for Manufacturing Fermented Milk Products

Soj bakterije Lactobacillus helveticus BGRA43, izoliran iz crijeva čovjeka, ima antimikrobni učinak na sojeve patogenih mikroorganizama u hrani, a tijekom fermentacije u mlijeku oslobađa peptide protuupalnih svojstava. U ovom je radu potvrđeno da soj BGRA43 djeluje antimikrobno na patogene sojeve, kao što su: Yersinia enterocolitica, Shigella sonnei, S. flexneri i Streptococcus pneumoniae. Stanice soja BGRA43 mogu preživjeti u simuliranim uvjetima gastrointestinalnog sustava uz dodatak mlijeka, pri čemu zadržavaju dovoljnu brojnost. Osim toga, LC/MS/MS analizom potvrđeno je da stanice soja BGRA43 mogu hidrolizirati β-laktoglobulin. U prisutnosti prebiotika, tj. inulina ili koncentrata β-glukana iz zobenih mekinja (Nutrim®) povećao se rast stanica soja BGRA43, čak i kad je prebiotik upotrijebljen kao jedini izvor ugljika. Soj BGRA43 odlično je rastao i u punomasnom kravljem ili kozjem mlijeku, te mlijeku koje je sadržavalo inulin ili Nutrim. Primjenom probiotičkog soja BGRA43 kao starter kulture dobiveni su fermentirani mliječni proizvodi od kravljeg ili kozjeg mlijeka sa ili bez dodatka inulina ili Nutrima s otprilike 107 CFU/mL stanica probiotika. Svi su proizvodi bili homogeni i viskozni, a najbolju su senzorsku ocijenu dobili fermentirani napici proizvedeni od rekonstituiranog mlijeka, punomasnog kravljeg mlijeka te kozjeg mlijeka s dodatkom 1 % inulina.Lactobacillus helveticus BGRA43 isolated from human intestines shows antimicrobial activity against foodborne pathogens and during fermentation in milk releases peptides with demonstrated anti-inflammatory properties. In this study, it was found that strain BGRA43 exhibits antimicrobial activity against human pathogens Yersinia enterocolitica, Shigella sonnei, S. flexneri and Streptococcus pneumoniae. Strain BGRA43 was able to survive in simulated gastric juice containing milk and retained cell number stability during the incubation in simulated intestinal conditions. In addition, LC/MS/MS analysis showed the ability of BGRA43 to hydrolyze β-lactoglobulin. Abundant growth of strain BGRA43 occurred in the presence of prebiotics inulin or concentrated oat bran β-glucan (Nutrim®), even when used as the sole carbon source. Similarly, strain BGRA43 grew satisfactorily in pure cow\u27s or goat\u27s milk as well as in the milk containing inulin or Nutrim®. Using the probiotic strain BGRA43 as a single starter strain, fermented milk products obtained from cow\u27s or goat\u27s milk with or without inulin or Nutrim® contained about 107 CFU/mL. The products were homogeneous and viscous and the best sensory scores were observed for fermented milk beverage made from reconstituted skimmed milk, whole cow\u27s milk and whole goat\u27s milk supplemented with 1 % inulin

Antioxidant Activity of Pea Protein Hydrolysates Produced by Batch Fermentation with Lactic Acid Bacteria

Nine Lactobacillus strains known for surface proteinase activity were chosen from our collection and tested for their ability to grow in pea seed protein-based medium, and to hydrolyze purified pea proteins in order to produce peptides with antioxidant (AO) activity. Two strains, Lactobacillus rhamnosus BGT10 and Lactobacillus zeae LMG17315, exhibited strong proteolytic activity against pea proteins. The AO activity of the pea hydrolysate fraction, MW lt 10 kDa, obtained by the fermentation of purified pea proteins with Lactobacillus rhamnosus BGT10, was tested by standard spectrophotometric assays (DPPH, ABTS, Fe3+-reducing capacity) and the recently developed direct current (DC) polarographic assay. The low molecular weight fraction of the obtained hydrolysate was separated using ion exchange chromatography, while the AO activity of eluted fractions was determined by means of a sensitive DC polarographic assay without previous concentration of samples. Results revealed that the fraction present in low abundance that contained basic peptides possessed the highest antioxidant activity. Based on the obtained results, it can be concluded that Lactobacillus rhamnosus BGT10 should be further investigated as a candidate strain for large-scale production of bioactive peptides from legume proteins

Analysis of natural isolates of Lactobacilli resistant to bacteriocin nisin

Kolekcija bakterija mlečne kiseline (BMK) je napravljena od mikroorganizama izolovanih iz fermentisanih mlečno-kiselinskih proizvoda dobijenih na tradicionalan način. Iz kolekcije 51 izolat je identifikovan kao Lactobacillus sp. Svi izolovani laktobacili pripadaju grupi mezofilnih sojeva koji dobro rastu na temperaturama od 15°C i 30°C, a ne rastu na temperaturi od 45°C. Testiranje sposobnosti rasta u prisustvu nizina pokazalo je da su izolati BGCGK4, BGHN40, BGBUK2-8, BGBUK2-7 i BGBUK2-16 rezistentni na bakteriocin nizin. U eksperimentu određivanja minimalne inhibitorne koncentracije (MIK) za nizin pokazano je da je najrezistentniji izolat Lactobacillus sp. BGCGK4. Izolat BGBUK2-16, determinisan kao Lactobacillus paracasei subsp. paracasei, produkuje bakteriocin označen kao Bac217 i pokazuje rezistenciju na 8000 IU/ml. Čišćenjem plazmida iz soja BGBUK2-16 dobijena su 2 derivata označena kao BGBUK2-16/K2 i BGBUK2-16/K4. Derivat BGBUK2-16/K2 zadržao je rezistenciju na Bac217 i nizin, ali je izgubio sposobnost sinteze Bac217, dok je derivat BGBUK2-16/K4 pored gubitka sposobnosti sinteze Bac217 postao senzitivan na Bac217 i nizin. Prirodno rezistentni laktobacili se mogu iskoristiti za pripremanje starter kultura u kombinaciji sa nizinom kao konzervansom u cilju kontrolisane mlečno-kiselinske fermentacije.The collection of lactic acid bacteria (LAB) was made by isolation of microorganisms from fermented products traditionally manufactured in different geographical regions (high mountains, river valleys, seaside, etc). Among collected LAB, 51 isolates were identified as Lactobacillus sp. Results showed that all isolated lactobacilli were mesophilic strains, since they grew at 15°C and 30°C but not at 45°C. Testing the ability of isolated lactobacilli to grow in the presence of nisin revealed that Lactobacillus sp. isolates designed BGCGK4, BGHN40, BGBUK2-8, BGBUK2-7 and BGBUK2-16 were resistant to nisin. Determination of the minimal inhibitory concentrations (MIC) for nisin revealed that the most resistant isolate was Lactobacillus sp. BGCGK4. Isolate BGBUK2-16, determined as Lactobacillus paracasei subsp. paracasei, produces bacteriocin, named Bac217 and showed a resistance to 8000 IU/ml of nisin. Plasmid curing of BGBUK2-16 resulted in derivatives BGBUK2-16/K2 and BGBUK2-16/K4. Derivative BGBUK2-16/K2 retained resistance to Bac217 and nisin, but lost the ability to synthesise Bac217. Derivative BGBUK2-16/K4 lost concomitantly the resistance to both Bac217 and nisin

A successful use of a new shuttle cloning vector pA13 for the cloning of the bacteriocins BacSJ and acidocin 8912

The aim of this paper was to research the molecular cloning of genes encoding the novel bacteriocin BacSJ from Lactobacillus paracasei subsp. paracasei BGSJ2-8 by using a newly constructed shuttle cloning vector pA13. A new shuttle-cloning vector, pA13, was constructed and successfully introduced into Escherichia coli, Lactobacillus and Lactococcus strains, showing a high segregational and structural stability in all three hosts. The natural plasmid pSJ2-8 from L. paracasei subsp. paracasei BGSJ2-8 was cloned in the pA13 using BamHI, obtaining the construct pB5. Sequencing and in silico analysis of the pB5 revealed 15 open reading frames (ORF). Plasmid pSJ2-8 harbors the genes encoding the production of two bacteriocins, BacSJ and acidocin 8912. The combined N-terminal amino acid sequencing of BacSJ in combination with DNA sequencing of the bacSJ2-8 gene enabled the determination of the primary structure of a bacteriocin BacSJ. The production and functional expression of BacSJ in homologous and heterologous hosts suggest that bacSJ2-8 and bacSJ2-8i together with the genes encoding the ABC transporter and accessory protein are the minimal requirement for the production of BacSJ. Biochemical and genetic analyses showed that BacSJ belongs to the class II bacteriocins. The shuttle cloning vector pA13 could be used as a tool for genetic manipulations in lactobacilli and lactococci

Characterisation of the exopolysaccharide (EPS)-producing Lactobacillus paraplantarum BGCG11 and its non-EPS producing derivative strains as potential probiotics

Traditional fermented foods are the best source for the isolation of strains with specific traits to act as functional starters and to keep the biodiversity of the culture collections. Besides, these strains could be used in the formulation of foods claimed to promote health benefits, i.e. those containing probiotic microorganisms. For the rational selection of strains acting as probiotics, several in vitro tests have been proposed. In the current study, we have characterized the probiotic potential of the strain Lactobacillus paraplantarum BGCG11, isolated from a Serbian soft, white, homemade cheese, which is able to produce a >ropy> exopolysaccharide (EPS). Three novobiocin derivative strains, which have lost the ropy phenotype, were characterized as well in order to determine the putative role of the EPS in the probiotic potential. Under chemically gastrointestinal conditions, all strains were able to survive around 1-2% (10 6-10 7cfu/ml cultivable bacteria) only when they were included in a food matrix (1% skimmed milk). The strains were more resistant to acid conditions than to bile salts and gastric or pancreatic enzymes, which could be due to a pre-adaptation of the parental strain to acidic conditions in the cheese habitat. The ropy EPS did not improve the survival of the producing strain. On the contrary, the presence of an EPS layer surrounding the strain BGCG11 hindered its adhesion to the three epithelial intestinal cell lines tested, since the adhesion of the three non-ropy derivatives was higher than the parental one and also than that of the reference strain Lactobacillus rhamnosus GG. Aiming to propose a potential target application of these strains as probiotics, the cytokine production of peripheral blood mononuclear cells (PBMC) was analyzed. The EPS-producing L. paraplantarum BGCG11 strain showed an anti-inflammatory or immunosuppressor profile whereas the non-ropy derivative strains induced higher pro-inflammatory response. In addition, when PBMC were stimulated with increasing concentrations of the purified ropy EPS (1, 10 and 100μg/ml) the cytokine profile was similar to that obtained with the EPS-producing lactobacilli, therefore pointing to a putative role of this biopolymer in its immune response. © 2012 Elsevier B.V.This work was financed by the Spanish Ministry of Science and Innovation (MICINN) through the project AGL2009-09445 (FEDER European Union funds) and by the Ministry of Education and Science of the Republic of Serbia grant No. 173019.Peer Reviewe

Characterization and antimicrobial activity of vaginal lactobacillus isolate

The aim of this study was to investigate the probiotic potential of bacteriocin-producing lactobacilli strain Lactobacillus plantarum G2 isolated from the vaginal mucus of healthy women. The antimicrobial effect of G2 was confirmed in the mixed culture with pathogenic Escherichia coli, Staphylococcus aureus, Salmonella abony and Pseudomonas aeruginosa, while bacteriocine activity was detected against S. aureus and S. abony only. The strain showed an excellent survival rate in low pH and in the presence of bile salts. The percentage of adhered cells of L. plantarum G2 to hexadecane was 63.85±2.0 indicating the intermediate hydrophobicity

Probiotic features of two oral Lactobacillus isolates

In this study, we checked lactobacilli strains of human origin for their potential as probiotic. Samples were collected from oral mucosa of 16 healthy individuals, out of which twenty isolates were obtained and two of them were selected and identified as Lactobacillus plantarum (G1) and L. casei (G3). Both isolates exhibited antagonistic action towards pathogenic microorganisms such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella abony, and Clostridium sporogenes, but not on the growth of Candida albicans. The bacteriocin activity against Staphylococcus aureus ATCC 6358-P was shown only by L. plantarum G1. Moreover, the isolates G1 and G3 showed good viability in the acid gastric environment and in the gut environment containing bovine bile salts. The viability of G1 and G3 isolates in the gastrointestinal tract, and the adhesion to the intestinal mucosa were also confirmed in vivo. The biochemical tests of blood samples revealed lower levels of serum triglycerides and cholesterol, as well as reduced activity of alkaline phosphatase in all lactobacilli-treated Wistar rats, compared to control ones. No toxicity for NMRI Ham mice was observed. According to our experimental results, these findings imply that L. plantarum G1 and L. casei G3 could be characterized as potential probiotics

Exopolysaccharide production and ropy phenotype are determined by two gene clusters in putative probiotic strain Lactobacillus paraplantarum BGCG11

Lactobacillus paraplantarum BGCG11, a putative probiotic strain isolated from a soft, white, artisanal cheese, produces a highmolecular-weight heteropolysaccharide, exopolysaccharide (EPS)-CG11, responsible for the ropy phenotype and immunomodulatory activity of the strain. In this study, a 26.4-kb region originating from the pCG1 plasmid, previously shown to be responsible for the production of EPS-CG11 and a ropy phenotype, was cloned, sequenced, and functionally characterized. In this region 16 putative open reading frames (ORFs), encoding enzymes for the production of EPS-CG11, were organized in specific loci involved in the biosynthesis of the repeat unit, polymerization, export, regulation, and chain length determination. Interestingly, downstream of the eps gene cluster, a putative transposase gene was identified, followed by an additional rfb gene cluster containing the rfbACBD genes, the ones most probably responsible for dTDP-L-rhamnose biosynthesis. The functional analysis showed that the production of the high-molecular-weight fraction of EPS-CG11 was absent in two knockout mutants, one in the eps and the other in the rfb gene cluster, as confirmed by size exclusion chromatography analysis. Therefore, both eps and rfb genes clusters are prerequisites for the production of high-molecular-weight EPS-CG11 and for the ropy phenotype of strain L. paraplantarum BGCG11. © 2015, American Society for Microbiology.This study was financially supported by the Ministry of Education, Science and the Technological Development, Republic of Serbia (grant no. 173019). The short stay of Patricia Ruas Madiedo at Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, was covered by the bilateral collaboration project AIB2010SE-00386 between Spain and the Republic of Serbia.Peer Reviewe

Exopolysaccharide Production and Ropy Phenotype Are Determined by Two Gene Clusters in Putative Probiotic Strain Lactobacillus paraplantarum BGCG11

Lactobacillus paraplantarum BGCG11, a putative probiotic strain isolated from a soft, white, artisanal cheese, produces a high-molecular-weight heteropolysaccharide, exopolysaccharide (EPS)-CG11, responsible for the ropy phenotype and immunomodulatory activity of the strain. In this study, a 26.4-kb region originating from the pCG1 plasmid, previously shown to be responsible for the production of EPS-CG11 and a ropy phenotype, was cloned, sequenced, and functionally characterized. In this region 16 putative open reading frames (ORFs), encoding enzymes for the production of EPS-CG11, were organized in specific loci involved in the biosynthesis of the repeat unit, polymerization, export, regulation, and chain length determination. Interestingly, downstream of the eps gene cluster, a putative transposase gene was identified, followed by an additional rfb gene cluster containing the rfbACBD genes, the ones most probably responsible for dTDP-l-rhamnose biosynthesis. The functional analysis showed that the production of the high-molecular-weight fraction of EPS-CG11 was absent in two knockout mutants, one in the eps and the other in the rfb gene cluster, as confirmed by size exclusion chromatography analysis. Therefore, both eps and rfb genes clusters are prerequisites for the production of high-molecular-weight EPS-CG11 and for the ropy phenotype of strain L. paraplantarum BGCG11