Lactis MG Research Articles

Suppression of lactate production by aerobic fed-batch cultures of Lactococcus lactis

Aerobic fed-batch cultures were studied as a means of suppressing the production of lactate, which inhibits the growth of lactic acid bacteria (LAB). LAB produce lactate via lactate dehydrogenase (LDH), regenerating nicotinamide adenine dinucleotide (NAD+) consumed during glycolysis. Therefore, we focused on NADH oxidase (NOX), employing oxygen as an electron acceptor, as an alternative pathway to LDH for NAD+ regeneration. To avoid glucose repression of NOX and NAD+ consumption by glycolysis exceeding NAD+ regeneration by NOX, glucose was fed gradually. When Lactococcus lactis MG 1363 was aerobically fed at a specific growth rate of 0.2 h-1, the amount of lactate produced per amount of grown cell was reduced to 12% of that in anaerobic batch cultures. Metabolic flux analysis revealed that in addition to NAD+ regeneration by NOX, ATP acquisition by production of acetate and NAD+ regeneration by production of acetoin and 2,3-butanediol contributed to suppression of lactate production.

Preparation and characterization of antimicrobial cationized peptides from barley (Hordeum vulgare L.) proteins

In the current study barley protein hydrolysate was cationized using polyethyleneimine and the physicochemical and antimicrobial properties of the modified peptides were investigated. Degree of substitution and zeta potential analysis verified the high proportion of positively charged components. Antimicrobial assay showed that cationized peptides effectively inhibited non-beneficial bacterial growth (Escherichia coli JM109 and Micrococcus luteus (ATCC 4698)) at low concentration (98–195 μg/mL), while higher concentration levels (390–1560 μg/mL) were required to inhibit the growth of beneficial lactic acid bacteria (Lactobacillus reuteri (ATCC 23272) and Lactococcus lactis MG 1363). Structural studies revealed the unordered open structures as the major structural elements of cationized peptides which resulted in conformational flexibility. Highly exposed hydrophobic segments accompanied by the chain flexibility could positively contribute to adsorption and penetration of cationized peptides into bacterial membrane. These unique structural characteristics of barley proteins in combination with positively charged side chains confer potential antimicrobial activity to barley-derived peptides which can be applied to enhance the shelf life stability of food, feed and other products.

CsiA is a bacterial cell wall synthesis inhibitor contributing to DNA translocation through the cell envelope

Conjugation is a widely spread mechanism allowing bacteria to adapt and evolve by acquiring foreign DNA. The chromosome of Lactococcus lactis MG 1363 contains a 60 kb conjugative element called the sex factor capable of high-frequency DNA transfer. Yet, little is known about the proteins involved in this process. Comparative genomics revealed a close relationship between the sex factor and elements found in Gram-positive pathogenic cocci. Among the conserved gene products, CsiA is a large protein that contains a highly conserved domain (HCD) and a C-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) domain in its C-terminal moiety. Here, we show that CsiA is required for DNA transfer. Surprisingly, increased expression of CsiA affects cell viability and the cells become susceptible to lysis. Point mutagenesis of HCD reveals that this domain is responsible for the observed phenotypes. Growth studies and electron microscope observations suggest that CsiA is acting as a cell wall synthesis inhibitor. In vitro experiments reveal the capacity of CsiA to bind d-Ala-d-Ala analogues and to prevent the action of penicillin binding proteins. Our results strongly suggest that CsiA sequesters the peptidoglycan precursor and prevents the final stage of cell wall biosynthesis to enable the localized assembly of the DNA transfer machinery through the cell wall.

Mutational and biochemical analyses of the endolysin Lys gaY encoded by the Lactobacillus gasseri JCM 1131 T phage φgaY

The Lys gaY of Lactobacillus gasseri JCM 1131 T phage φgaY endolysin was purified to homogeneity using the Escherichia coli/His·Tag system. Zymographic and spectrophotometric assays showed that Lys gaY lysed over 20 heated Gram-positive bacterial species as the substrates, including lactobacilli, lactococci, enterococci, micrococci, and staphylococci. The enzymatic activity had the pH and temperature optima of about 6.5 and 37°C, respectively. Amino-acid substitution analysis revealed that 13 residues of Lys gaY were involved in cell-lytic activity: in the β/α gaY domain, G10, D12, E33, D36, H60, Y61, D96, E98, V124, L132, and D198; in the SH3b gaY domain, Y272 and W284. In addition, deletion analysis demonstrated that the β/α gaY domain of N-terminal 216 residues is the core enzyme portion, although the cell-lytic ability is lower than that of Lys gaY. These mutational experiments suggested that β/α gaY (in which two acidic residues of D12 and E98 likely act as catalytic residues) is responsible for cell-lytic activity, and SH3b gaY promotes β/α gaY possibly through cell-wall binding function. The purified His-tagged SH3b gaY domain containing 94 residues from S217 to K310 (i) bound to Gram-positive bacteria susceptible to Lys gaY, (ii) induced aggregation of exponentially growing cells of L. gasseri JCM 1131 T, L. casei IAM 1045, Lactococcus lactis C2, L. lactis MG 1363, and Enterococcus hirae IAM 1262 by forming thread-like chained cells, (iii) inhibited lytic activity of Lys gaY, and (iv) impeded autolysis of L. gasseri JCM 1131 T in buffer systems. A truncated protein HΔSH3b gaY lacking in N-terminal 21 residues (from S217 to E237) of SH3b gaY and an amino-acid substituted protein HSH3b gaYG (W284G) lost the activities of HSH3b gaY, showing that the N-terminal region and W284 probably play important roles in the SH3b gaY function(s).

Investigations of the Antibacterial Properties of Ciprofloxacin@SiO2

The antibacterial activity of ciprofloxacin-encapsulated silica nanoshells synthesized from gold@silica core-shell nanoparticles has been investigated. The minimum inhibitory concentration of the material was found using the agar dilution method, and it showed better antibacterial activity compared to free ciprofloxacin in the case of Escherichia coli DH5, whereas the same activity was found for Lactococcus lactis MG 1363. Hydrophobicity measurements carried out in an octanol-water mixture suggested that ciprofloxacin@SiO2 is distributed almost equally in the aqueous and nonaqueous phases. The kinetics of the uptake of ciprofloxacin@SiO2 was compared with that of free ciprofloxacin. Fluorescence imaging studies carried out using fluorescein isothiocyanate@SiO2 showed that the nanoshells enter the bacterial cell. The uptake of silica shells has been probed by transmission electron microscopy also.

Combined high pressure and temperature induced lethal and sublethal injury of Lactococcus lactis—Application of multivariate statistical analysis

It was the aim of this work to determine the combined effects of pressure, temperature, and co-solutes on Lactococcus lactis, and to detect correlations between culture-dependent and culture-independent methods for assessment of cellular viability and sublethal injury. Therefore, the pressure induced inactivation of L. lactis MG 1363 was investigated in buffer and in buffer with 1.5 M sucrose or 4 M NaCl at a pressure range of 0.1 to 500 MPa and a temperature range of 5 to 50 degrees C. The inactivation was characterised by viable cell counts, stress resistant cell counts, membrane integrity, metabolic activity, and the activity of the multi-drug-resistance transport enzyme LmrP. L. lactis was most resistant to pressure application at 20-30 degrees C. Sucrose protected towards inactivation at any temperature, NaCl provided protection at high temperatures only. By using Principal Component Analysis, correlations were detected between viable cell counts and metabolic activity as well as stress resistant cell counts and LmrP activity. In conclusion, the pressure-inactivation of L. lactis is strongly temperature dependent, baroprotection by sucrose occurs at any temperature but the baroprotective effects of NaCl is temperature dependent. Further on, a combination of two experimental methods fully describe lethal and sublethal injury of pressure treated cells. These simplification of data acquisition and model development facilitates the establishment of pressure processes in food technology.

Non‐linear modelling and statistical anlaysis of multi‐stage high‐pressure inactivation of lactic acid bacteria

AbstractIt was the aim of this work to determine the combined effects of pressure, temperature, and co‐solvents on Lactococcus lactis , and to detect correlations between culture‐dependent and culture‐independent methods for assessment of cellular viability and sublethal injury. Therefore, the pressure induced inactivation of L. lactis MG 1363 was investigated in 21 buffer systems at a pressure range of 0.1 MPa to 600 MPa and a temperature range of 5 to 50 Grad Celsius. The inactivation was characterised by viable cell counts, stress resistant cell counts, membrane integrity, metabolic activity, and LmrP activity. By using Principal Component Analysis, correlations were detected between viable cell counts and metabolic activity as well as stress resistant cell counts and LmrP activity. Based on these correlations, a fuzzy logic model was formulated. The model uses two of the five physiological states as autonomous output variables. Input variables are pressure, temperature, application time and food systems. Latter fully describe the inactivation process of L. lactis correctly. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI.

A new food-grade cloning vector for lactic acid bacteria was constructed using the nisin immunity gene nisI as a selection marker. The food-grade plasmid, pLEB 590, was constructed entirely of lactococcal DNA: the pSH 71 replicon, the nisI gene, and the constitutive promoter P45 for nisI expression. Electroporation into Lactococcus lactis MG 1614 with 60 international units (IU) nisin/ml selection yielded approximately 10(5) transformants/ micro g DNA. MG 1614 carrying pLEB 590 was shown to be able to grow in medium containing a maximum of 250 IU nisin/ml. Plasmid pLEB 590 was successfully transformed into an industrial L. lactis cheese starter carrying multiple cryptic plasmids. Suitability for molecular cloning was confirmed by cloning and expressing the proline iminopeptidase gene pepI from Lactobacillus helveticus in L. lactis and Lb. plantarum. These results show that the food-grade expression system reported in this paper has potential for expression of foreign genes in lactic acid bacteria in order to construct improved starter bacteria for food applications.

Pharmacokinetics of Lactobacillus plantarum NCIMB 8826, Lactobacillus fermentum KLD, and Lactococcus lactis MG 1363 in the human gastrointestinal tract.

Genetically modified lactic acid bacteria may be a way to deliver vaccinal epitopes in the gastrointestinal tract. Three strains of lactic acid bacteria were studied for their pharmacokinetics in the human gastrointestinal tract. The survival of the strains was studied up to the ileum in six subjects each, after ingestion of 150 g of fermented milk. The strains and their concentrations in the products were Lactobacillus fermentum KLD (107 cfu/g), Lactobacillus plantarum NCIMB 8826 (108 cfu/g), and Lactococcus lactis MG 1363 (108 cfu/g). Ileal fluid was aspirated by intestinal intubation and immediately cultured. L. plantarum NCIMB 8826, which was found in high concentrations in the ileum, was studied for its survival in the faeces after consumption of 150 g of fermented milk three times daily for 7 days. Faecal samples were collected for culture. The concentration of L. plantarum NCIMB 8826 in the ileum reached 108 cfu/mL after a single dose, with a survival of 7%. L. fermentum KLD and Lc. lactis MG 1363 had lower (0.5 and 1.0%, respectively) and shorter (4 h) survival in the ileum. During the 7-day ingestion period, L. plantarum NCIMB 8826 reached high concentrations (108 cfu/g) in the faeces, with a survival of 25 +/- 29%. None of the strains colonized. L. plantarum NCIMB 8826 has a promising pharmacokinetic profile as a candidate vaccine vehicle.

PepN-like aminopeptidase from Lactohacillus curvatus DPC2024: purification and characterization

An enzyme with a PepN-like aminopeptidase activity was purified 127-fold with 4.3 % recovery from a cell-free extract of Lactobacillus curvatus DPC2024, a component of the non-starter lactic acid bacterial flora of Cheddar cheese. The purified enzyme exhibited maximum activity on leucyl-p-nitroanilide (Leu-pNA) at pH 7.0 and 40 oc.The enzyme had a molecular mass of -98 kDa as estimated by gel permeation chromatography, and showed one band corresponding to a molecu- lar mass of -95 kDa on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), indicating that the native enzyme existed as a monomer. The aminopeptidase appeared to be a met- alloenzyme since it was strongly inhibited by ethylenediaminetetraacetic acid (EDTA) and o-phenan- throline at 0.1 mmol-Lr ' concentration and was partially reactivated by Zn2+ and C02+. The enzyme was also partially inhibited by p-chloromercuribenzoate and iodoacetic acid, suggesting the involve- ment of su1phydryl groups in the reaction mechanism. The enzyme had a broad substrate specificity, hydrolysing a number of p-nitroanilide derivatives of amino acids and peptides and di-, tri-, tetra- and pentapeptides. The N-tenninal amino acid sequence of the first 20 amino acid residues was determined (AELMRFYQSFQPEHYQVFLD), and showed 40-55 % homology with Zn2+-dependent aminopep- tidases from Streptococcus thermophilus NCDu53, Lactobacillus delbrueckii subsp. lactis DSM7290, Lactococcus lactis subsp. lactis MG 1363 and Lactococcus lactis subsp. cremoris W g2. © Inra/Elsevier, Paris.

Functional analysis of promoters in the nisin gene cluster of Lactococcus lactis

The promoters in the nisin gene cluster nisABTCIPRKFEG of Lactococcus lactis were characterized by primer extension and transcriptional fusions to the Escherichia coli promoterless beta-glucuronidase gene (gusA). Three promoters preceding the nisA, nisR, and nisF genes, which all give rise to gusA expression in the nisin-producing strain L. lactis NZ9700, were identified. The transcriptional autoregulation of nisA by signal transduction involving the sensor histidine kinase NisK and the response regulator NisR has been demonstrated previously (0. P. Kuipers, M. M. Beerthuyzen, P. G. G. A. de Ruyter, E. J. Luesink, and W. M. de Vos, J. Biol. Chem. 270: 27299-27304, 1995), and therefore the possible nisin-dependent expression of gusA under control of the nisR and nisF promoters was also investigated. The nisR promoter was shown to direct nisin-independent gusA expression in L. lactis MG 1363, which is a nisin-transposon- and plasmid-free strain. L. lactis NZ9800, which does not produce nisin because of a deletion in the nisA gene, containing the nisF-gusA fusion plasmid, gave rise to beta-glucuronidase production only after induction by nisin. A similar regulation was found in L. lactis NZ3900, which contains a single copy of the nisR and nisK genes but no other genes of the nisin gene cluster. In contrast, when the nisK gene was disrupted, no beta-glucuronidase activity directed by the nisF promoter could be detected even after induction with nisin. These results show that, like the nisA promoter, the nisF promoter is nisin inducible. The nisF and nisA promoter sequences have significant similarities and contain a conserved region that could be important for transcriptional control.