Characterization of nisin-resistant variants of Pediococcus acidilactici UL5, a producer of pediocin

Four spontaneous nisin-resistant variants R1, R1M, T5 and T7 of Pediococcus acidilactici UL5, a pediocin producer, were isolated on a nisin gradient. The minimum inhibitory concentration of Ped. acidilactici UL5 using an agar diffusion test was 0·25 ng, while that of R1, R1M, T5 and T7 were 10, 25 and more than 32·5 μg for the two latter, respectively. Nisin resistance phenotype was stable after 60 generations in MRS nisin-free liquid media and 10 consecutive transfers in solid medium. Pediococcus acidilactici UL5 and its nisin-resistant variants exhibited the same total DNA profile, level of production of pediocin and adsorption of nisin on the cell surface. The specific growth rate (μ) decreased with the level of resistance of the culture. Nisin-resistant variants and parental strain UL5 showed differences in sensitivity to antibiotics in which some act on the cell surfaces. Moreover, the fatty acid composition of the cell wall in nisin-resistant variants, compared with UL5, was different, particularly in C16:1 and C18:1. Results suggest that a change in structure/composition of nisin-resistant variants might be associated with nisin resistance.     

Improvement of plasmid stability of recombinant Bacillus-subtilis cells in continuous immobilized cultures

Abstract: The immobilization of recombinant Bacillus subtilis in K-carrageenan gel beads has been performed in order to study the growth conditions inside the gel beads and to improve plasmid stability. Bacterial colonies showing high cell density were studied using scanning electron microscopy. A series of continuous cultures of free and immobilized B. subtilis MT119 (pHV1431, pIL252 and pIL252 Kpn) have been developed without selection pressure. In the free-cell systems, it was found that a loss of plasmid vectors occurred after a short period. In contrast, in the immobilized cell systems, plasmid-free segregants were not detected in any of the cases during the first 80 h of the culture.     

Simple method of purification and sequencing of a bacteriocin produced by Pediococcus acidilactici UL5

H. DABA, C. LACROIX, J. HUANG, R.E. SIMARD AND L. LEMIEUX. 1994. A bacteriocin produced by a strain of Pediococcus acidilactici was successfully purified sequentially by acid extraction (at pH 2) and reverse-phase high-performance liquid chromatography (HPLC). Cell extracts of derivative strains deficient in bacteriocin production exhibited a similar HPLC elution profile to the active extracts except for the two peaks containing bacteriocin activity. The sequence of the antibacterial peptide consisted of 44 amino acid residues of which 42 were identified, and its molecular weight was 4624 Da, as determined by mass spectrometry. Moreover, according to the molecular weight of the peptide, the unidentified residues in the bacteriocin sequence must correspond to two tryptophan residues, confirming that the peptide isolated from Ped. acidilactici UL5 is pediocin PA-1. However, oxidized forms of the bacteriocin produced during storage also showed bacteriocin activity and resulted in a second peak with activity in the chromatograms. HPLC chromatograms of cell surface preparations from the active and from the deficient strains were confirmed by capillary electrophoresis. The purification method used is simple and effective in comparison with traditional methods, permitting a selective recovery of cell-associated bacteriocin at low pH, and its isolation in pure form for sequencing.     

Pediocin production in milk by Pediococcus acidilactici in co-culture with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus

The production of pediocin in milk by Pediococcus acidilactici was evaluated in co-culture with the dairy fermentation cultures Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. The cultures were tested singly and in different combinations in milk (0 or 2% fat content) during incubation at 40°C for up to 10 h. Cell-free milk samples taken every 60 min were tested for bacteriocin activity against Listeria monocytogenes. Pediocin activity was not detectable when P. acidilactici was inoculated into milk as a monoculture. When P. acidilactici was grown in combination with the yogurt starter cultures S. thermophilus and Lb. delbrueckii ssp. bulgaricus, pediocin concentration reached 3,200–6,400 units ml⁻¹ after 8 h of incubation. The results showed that pediocin producing pediococci may be useful adjunct components in mixed cultures of S. thermophilus and Lb. delbrueckii ssp. bulgaricus to amplify the bioprotective properties of fermented dairy foods against Listeria contamination.     

Pediocin production by Pediococcus acidilactici in solid state culture on a waste medium: process simulation and experimental results

The production of pediocin by Pediococcus acidilactici was comparatively studied in submerged and solid-state culture, using polyurethane foam particles soaked in commercial (MRS) and waste media with various supplements, where product concentrations were 15 times higher in MRS medium. For the solid state analysis, cultures were treated by successive compression and refilling of tubular minireactors equipped with a piston, without the need for reinoculation. This method was found to be simple, reproducible, and easily controllable, allowing culture productivity to be maintained for long periods of time without alterations in the basic properties of the system. In addition, yields were found to be superior compared to those from submerged culture. The system kinetics were modeled on the basis of widely accepted assumptions with a good fit to the experimental results and observed biomass fluctuations less evident than those predicted by the kinetic model.     

Conjugal transfer of a plasmid encoding bacteriocin production and immunity in Pediococcus acidilactici H

Previous investigations indicated that curing of a 7.4-Md plasmid (pSMB74) resulted in concomitant loss of bacteriocin activity and immunity in Pediococcus acidilactici H. Transfer of pSMB74 to a gentamicin-neomycin resistant (Gm^rNm^r) derivative of P. acidilactici LB42, which was devoid of any plasmid DNA, required cell-to-cell contact on a solid mating surface and converted the strain to Bac⁺Bac^r phenotype. Gene transfer processes such as transduction and transformation were ruled out from the experiment. Treatment of donor cells with chloroform did not allow the appearance of recombinant clones, confirming that viable cells were essential for this particular mechanism of genetic transfer. Transconjugants obtained from selective agar surface were subjected to plasmid isolation and agarose gel electrophoresis. Each of them exhibited plasmid size corresponding to pSMB74 of donor strain. All results suggested this genetic transfer similar to conjugation, and provided presumptive evidence for plasmid-encoded bacteriocin activity and immunity in P. acidilactici H.     

Continuous production of pediocin by immobilized Pediococcus acidilactici PO2 in a packed-bed bioreactor

 A continuous bioreactor packed with a fibrous matrix was set up. Cells of Pediococcus acidilactici PO2 were inoculated and MRS broth was fed gradually until cell growth and immobilization were achieved. Kinetics of fermentation and production of bacteriocin were investigated at dilution rates ranging from 0.63 day^-1 to 1.58 day^-1 and at pH values that varied between 4.0 and 5.5. A maximum bacteriocin activity of 6400 AU/ml was detected when the medium was fermented at dilution rates of at least 1.19 day^-1 and the pH controlled at 4.5. The maximum bacteriocin productivity was 1.0×10⁷ AUl^-1 day^-1 at a dilution rate of 1.58 day^-1 and pH 4.5. At this high dilution rate, 1.21 g cells/l medium was produced, 95.9% of the glucose in MRS broth was utilized, and 15.1 g lactic acid/l accumulated in the bioreactor effluent. The bioreactor was operated continuously for 3 months without encountering any clogging, degeneration, or contamination problems, indicating good long-term stability of the bioreactor for bacteriocin production. About 94% of the cells in the bioreactor were immobilized, and the remainder were suspended in the medium. According to scanning electron microscopic observations, cell immobilization in the fibrous matrix was attained by natural attachment to fiber surfaces and entrapment in the void volume within the fibrous matrix. In conclusion, conditions for the optimum continuous production of pediocin were defined; this may facilitate the development of large-scale industrial processes for production of this bacteriocin. Received: 25 September 1995/Received revision: 30 November 1995/Accepted: January 1996     

Pediocin SA-1, an antimicrobial peptide from Pediococcus acidilactici NRRL B5627: production conditions, purification and characterization

Fermentation broths of Pediococcus acidilactici NRRL B5627 exhibited a certain antimicrobial activity due to a bacteriocin produced during early growth and until the stationary phase of growth was reached (at optimum of 60% dissolved oxygen saturation). Its size was determined by electrospray ionization mass spectrometric analysis as 3.660 kDa. N-terminal sequencing showed that the bacteriocin had 19 amino acid residues in the order KYYGXNGVXTXGKHSXVDX. The purified bacteriocin is similar to pediocins isolated by various Pediococci and therefore, it belongs to the class IIa of bacteriocins and is thus designated pediocin SA-1. Sensitivity of the purified pediocin to various storage temperatures and enzyme treatments was examined. Purified pediocin SA-1 is heat stable for up to 60 min at 121 °C. Pediocin SA-1 is inhibitory to several food-borne pathogens and food spoilage bacteria. It appears to be significantly more effective against Listeria spp. compared to pediocin PD-1 produced by P. damnosus. The mode of action of the purified bacteriocin appears to be bactericidal.     

Optimized culture conditions for bacteriocin production by Pediococcus acidilactici LAB 5 and its characterization

A strain of Pediococcus acidilactici LAB 5 was isolated from vacuum-packed fermented meat product, in order to obtain a novel bacteriocin from food-grade organisms. Optimized culture conditions for bacteriocin production in different media (viz., MRS, TGE, TGE + buffer, TGE + Tween 80, and TGE + Tween 80 + buffer) and at different temperatures and pH conditions were reported. TGE + Tween 80 + buffer medium was found to be most effective for bacteriocin production (about 2400 AU/ml) by this strain, when incubated at 37 degrees C for 24 h. Bacteriocin, partially purified by adsorption-desorption method showed molecular mass of 10.3 kDa and produced prominent inhibition zone in activity gel. It showed significant storage stability both at high as well as in low temperatures for up to 6 months and retained its activity in a number of organic solvents, except in 2-mercaptoethanol. The treatment with amylase and lysozyme did not change its activity, but it lost its activity on proteinase K treatment. Antibacterial efficacy of bacteriocin was proved against some food spoilage and human pathogenic bacteria like Enterococcus, Leuconostoc, Listeria, Staphylococcus and Streptococcus.     

Exopolysaccharide production by free and immobilized microbial cultures

The batch production of different exopolysaccharides (alginate, xanthan, pullulan, dextran) by free and immobilized microbial cultures was investigated. First, conventional free-cell cultures were performed to obtain control fermentation parameters and macromolecular characteristics of exopolysaccharides. Then microbial cultures were immobilized in composite agar layer/microporous membrane structures and tested for polysaccharide production. The immobilized-cell system proved unsuitable for xanthan and pullulan production. Owing to the fouling of the microporous membrane by the polysaccharide, dextran production by immobilized Leuconostoc mesenteroides also was inefficient. More promising results have been obtained with immobilized Azotobacter vinelandii cultures. The amount of alginate produced by immobilized A. vinelandii represented about 60% of that recovered from a free-cell culture, whereas the polysaccharide yield reached 35% instead of 9% for the free counterpart. These results are compared to the macromolecular characteristics of exopolysaccharides.     

Influence of Growth Conditions on the Production of a Bacteriocin, Pediocin AcH, by Pediococcus acidilactici H

The influence of growth parameters on the production of pediocin AcH by Pediococcus acidilactici H was studied. This strain produced large quantities of pediocin AcH in TGE broth (Trypticase [1%], glucose [1%], yeast extract [1%], Tween 80 [0.2%], Mn²⁺ [0.033 mM], Mg²⁺ [0.02 mM] [pH 6.5]) within 16 to 18 h at 30 to 37°C (final pH, 3.6 to 3.7). Pediocin AcH production was negligible when the pH of the medium was maintained at 5.0 or above, even in the presence of high cell mass.     

Agitation rate effects on plasmid stability in immobilized and free-cell continuous cultures of recombinant E. coli.

Escherichia coli B/pTG201 recombinant cells were immobilized by entrapment in a carrageenan gel and cultivated in nonselective media to investigate the effect of agitation rate on plasmid stability, biomass concentration, and enzyme productivity. These parameters were studied in continuous cultures for free and immobilized cells, respectively. Immobilized recombinant cells exhibit an increase in the stability of the plasmid pTG201 compared to free cells, even under conditions where the tendency of plasmid stability for free cells decreased generally more rapidly under a higher agitation rate. Intensive agitation, resulting also in a strong shear stress, greatly reduced cell concentration within gel beads throughout the course of growth. Higher enzyme expression of catechol 2–3, dioxygenase was also obtained in leaked cells due to better maintenance of plasmid stability and higher plasmid copy number with regard to free cells. Enzyme productivity of leaked and free cells in minimal medium decreased with the increase in agitation rate, due to decreased plasmid stability; however, in LB medium, it increased in the presence of higher agitation rate related to important cell concentration.     

Catabolism of Serine by Pediococcus acidilactici and Pediococcus pentosaceus

The ability to produce diacetyl from pyruvate and l-serine was studied in various strains of Pediococcus pentosaceus and Pediococcus acidilactici isolated from cheese. After being incubated on both substrates, only P. pentosaceus produced significant amounts of diacetyl. This property correlated with measurable serine dehydratase activity in cell extracts. A gene encoding the serine dehydratase (dsdA) was identified in P. pentosaceus, and strains that showed no serine dehydratase activity carried mutations that rendered the gene product inactive. A functional dsdA was cloned from P. pentosaceus FAM19132 and expressed in Escherichia coli. The purified recombinant enzyme catalyzed the formation of pyruvate from l- and d-serine and was active at low pH and elevated NaCl concentrations, environmental conditions usually present in cheese. Analysis of the amino acid profiles of culture supernatants from dsdA wild-type and dsdA mutant strains of P. pentosaceus did not show differences in serine levels. In contrast, P. acidilactici degraded serine. Moreover, this species also catabolized threonine and produced alanine and α-aminobutyrate.     

Batch and continuous production of lactic acid from salt whey using free and immobilized cultures of lactobacilli

Salt whey permeate was used as a substrate for lactic acid production by different strains of homofermentative lactobacilli. An isolate from Egyptian Cheddar cheese proved to be the most effective lactic acid producer. The salt whey permeate was optimized by addition of yeast extract and minerals to enable exponential growth of organisms. The lactic acid productivity of free and immobilized cells was compared and fermentation conditions were improved. Continuous lactic acid fermentation from salt whey permeate with cells immobilized in agarose beads was successfully carried out in a chemostat with a steady lactic acid concentration of 33.4 mg/ml.     

Complete nucleotide sequence of pSMB 74, a plasmid encoding the production of pediocin AcH in Pediococcus acidilactici

Several Pediococcus acidilactici strains produce a plasmid-encoded bacteriocin, pediocin AcH. Previous studies have shown that this plasmid, designated as pSMB 74, encodes genes associated with the production of prepediocin, its post-translation processing to pediocin AcH, transmembrane translocation of these molecules, and immunity of producer cells against pediocin AcH. We report here the complete nucleotide sequence of pSMB 74. The plasmid has a total of 8877 bp. Four genes have been located on pSMB 74. The genes are arranged in a gene cluster of 3500 bp and share a common promoter and rho-independent stem-loop terminator. The four genes, each with independent ribosome binding sites (rbs), initiation and termination codons and spacer sequences in between, were designated as pap A, pap B, pap C and pap D and encode respectively for proteins of 62, 112, 174 and 724 amino acids. The results of this study can be useful either to introduce a suitable marker at a unique restriction site in pSMB 74 and use it as a vector or to clone the pap gene cluster in a suitable plasmid and transform desirable strains for pediocin AcH production. The gene sequence has been submitted to Gene Bank (Acc. No. U02482).     

Exopolysaccharide production during batch cultures with free and immobilized Lactobacillus rhamnosus RW-9595M

AIMS: Exopolysaccharides (EPS) were produced by Lactobacillus rhamnosus RW-9595M during pH-controlled batch cultures with free cells and repeated-batch cultures with cells immobilized on solid porous supports (ImmobaSil). METHODS AND RESULTS: Cultures were conducted in supplemented whey permeate (SWP) medium containing 5 or 8% (w/w) whey permeate. For free-cell batch cultures in 8% SWP medium, very high maximum cell counts (1.3 x 10(10) CFU ml(-1)) and EPS production (2350 mg l(-1)) were measured. A high EPS production (1750 mg l(-1)) was measured after four cycles for a short incubation period of only 7 h. Several methods for immobilized biomass determination based on analysis of biomass components (proteins, ATP and DNA) were tested. The DNA analysis method proved to be the most appropriate under these circumstances. This method revealed a high maximum immobilized biomass of 8.5 x 10(11) CFU ml(-1) support during repeated immobilized cell cultures in 5% SWP. The high immobilized biomass increased maximum EPS volumetric productivity (250 mg l(-1) h(-1) after 7 h culture) compared with free-cell batch cultures (110 mg l(-1) h(-1) after 18 h culture). CONCLUSIONS: High EPS productions were achieved during batch cultures of Lact. rhamnosus RW-9595M in SWP medium, exceeding 1.7 g EPS per litre. Repeated-batch cultures with immobilized cells resulted in increased EPS productivity compared with traditional free-cell cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: The study clearly shows the high potential of the strain Lact. rhamnosus RW-9595M and immobilized cell technology for production of EPS as a functional food ingredient.     

Agitation rate effects on plasmid stability in immobilized and free-cell continuous cultures of recombinant E. coli

Escherichia coli B/pTG201 recombinant cells were immobilized by entrapment in a carrageenan gel and cultivated in nonselective media to investigate the effect of agitation rate on plasmid stability, biomass concentration, and enzyme productivity. These parameters were studied in continuous cultures for free and immobilized cells, respectively. Immobilized recombinant cells exhibit an increase in the stability of the plasmid pTG201 compared to free cells, even under conditions where the tendency of plasmid stability for free cells decreased generally more rapidly under a higher agitation rate. Intensive agitation, resulting also in a strong shear stress, greatly reduced cell concentration within gel beads throughout the course of growth. Higher enzyme expression of catechol 2–3, dioxygenase was also obtained in leaked cells due to better maintenance of plasmid stability and higher plasmid copy number with regard to free cells. Enzyme productivity of leaked and free cells in minimal medium decreased with the increase in agitation rate, due to decreased plasmid stability; however, in LB medium, it increased in the presence of higher agitation rate related to important cell concentration.     

Ascorbic acid-induced loss of a pediocin-encoding plasmid in Pediococcus acidilactici CFR K7

A strain of Pediococcus acidilactici CFR K7 grown in presence of 1 mm ascorbate for 18 h resulted in 35% colony-forming units (CFU) irreversibly losing their ability to produce pediocin. Agarose gel electrophoresis of plasmid DNA and dot-blot hybridization showed concomitant loss of a 7.8 kb plasmid coding for pediocin from these colonies. Sensitivity of these colonies to nitrofurantoin was also observed after ascorbate treatment. Since ascorbic acid is a readily available and non-hazardous compound in contrast to other conventional plasmid curing agents, the possibility of its use in determining plasmid-encoded traits in food grade lactic acid bacteria is also proposed. This revised version was published online in July 2006 with corrections to the Cover Date.