Svetoslav D. Todorov

Boza, a natural source of probiotic lactic acid bacteria

Aims:u2002 To evaluate the probiotic properties of strains isolated from boza, a traditional beverage produced from cereals.

Bacteriocin production by Lactobacillus plantarum AMA-K isolated from Amasi, a Zimbabwean fermented milk product and study of the adsorption of bacteriocin AMA-K to Listeria sp.

Bacteriocin AMA-K produced by Lactobacillus plantarum AMA-K inhibits the growth of Enterococcus spp., Escherichia coli, Klebsiella pneumoniae and Listeria spp. Growth of strain AMA-K in BHI, M17, soy milk and molasses was similar to growth in MRS. The effect of organic nitrogen sources, carbohydrates, glycerol, K2HPO4 and KH2PO4, MgSO4, MnSO4, tri-ammonium citrate, Tween 80, vitamins and initial pH on bacteriocin AMA-K was determined. The mode of action of bacteriocin AMA-K was studied. The effect of bacteriocin AMA-K to actively growing Listeria innocua LMG13568, L. ivanovii subsp. ivanovii ATCC19119 and L. monocytogenes ScottA was determined. Adsorption of bacteriocin AMA-K to target cells at different temperatures, pH and in presence of Tween 20, Tween 80, ascorbic acid, potassium sorbate, sodium nitrate and sodium chloride were studied. Bacteriocin AMA-K shares high homology to pediocin PA-1.

Characterization of bacteriocins produced by two strains of Lactobacillus plantarum isolated from Beloura and Chouriço, traditional pork products from Portugal

Bacteriocins bacST202Ch and bacST216Ch, produced by Lactobacillus plantarum strains isolated from Beloura and Chouriço, respectively, inhibited the growth of a number of Gram-positive and Gram-negative meat spoilage bacteria. According to trycine-SDS-PAGE, bacST202Ch and bacST216Ch are approximately 3.5 and 10.0 kDa in size, respectively. Maximal activity of bacST202Ch (25,600 AU/ml) was recorded after 27 h of and bacST216Ch (102,400 AU/ml) after 22 h of growth. The mode of activity, as determined against Enterococcus faecium HKLHS, is bactericidal. Both peptides adsorb to the surface of the producer cells, but at very low concentrations. Both peptides remained active after 120 min at 100 degrees C and after 2 h of incubation at pH 2.0-12.0. Treatment for 120 min at 121 degrees C did not affect bacST216Ch activity. Activity of bacST202Ch and bacST216Ch was not affected by 1% Triton X-100, Tween 80, Tween 20, SDS, NaCl, urea and EDTA. Bacteriocin ST216Ch was deactivated in the presence of 1% Triton X-114. The nucleotide sequence of a 1044 bp DNA fragment amplified from L. plantarum ST202Ch is identical to the structural gene encoding pediocin PA-1, suggesting that the two bacteriocins are identical. Based on the broad spectrum of antibacterial activity, strains ST202Ch and ST216Ch may be used as starter cultures in the fermentation of meat products.

Bacteriocins from Lactobacillus plantarum production, genetic organization and mode of action

Bacteriocinas sao proteinas ou complexos proteicos biologicamente ativos que apresentam atividade bactericida contra especies relacionadas. Nas ultimas duas decadas, varias cepas de Lactobacillus plantarum produtoras de bacteriocinas foram isoladas de diferentes nichos ecologicos como carnes, peixes, frutas, vegetais e produtos lacteos e de cereais. Varias plantaricinas foram caracterizadas e suas sequencias de aminoacidos determinadas. Diferentes aspectos do modo de acao, otimizacao da fermentacao e organizacao genetica ja foram estudados. Entretanto, muitas bacteriocinas produzidas por diferentes cepas de Lactobacillus plantarum ainda nao foram completamente caracterizadas.Nesse artigo, apresenta-se uma breve revisao sobre a classificacao, genetica, caracterizacao, modo de acao, e otimizacao da producao de bacteriocinas de bacterias laticas em geral, e, quando apropriado, de bacteriocinas de Lactobacillus plantarum.Bacteriocins are biologically active proteins or protein complexes that display a bactericidal mode of action towards usually closely related species. Numerous strains of bacteriocin producing Lactobacillus plantarum have been isolated in the last two decades from different ecological niches including meat, fish, fruits, vegetables, and milk and cereal products. Several of these plantaricins have been characterized and the aminoacid sequence determined. Different aspects of the mode of action, fermentation optimization and genetic organization of the bacteriocin operon have been studied. However, numerous of bacteriocins produced by different Lactobacillus plantarum strains have not been fully characterized. In this article, a brief overview of the classification, genetics, characterization, including mode of action and production optimization for bacteriocins from Lactic Acid Bacteria in general, and where appropriate, with focus on bacteriocins produced by Lactobacillus plantarum, is presented.

The effect of prebiotics on production of antimicrobial compounds, resistance to growth at low pH and in the presence of bile, and adhesion of probiotic cells to intestinal mucus

Aims:u2002 Screening of five bile salt‐resistant and low pH‐tolerant lactic acid bacteria for inhibitory activity against lactic acid bacteria and bacterial strains isolated from the faeces of children with HIV/AIDS. Determining the effect of prebiotics and soy milk‐base on cell viability and adhesion of cells to intestinal mucus.

Probiotic properties of Lactococcus lactis ssp. lactis HV219, isolated from human vaginal secretions

Aims:u2002 To determine the resistance of Lactococcus lactis ssp. lactis HV219 to acids, bile, antibiotics, inflammatory drugs and spermicides, compare adsorption of the strain to bacteria and Caco‐2 cells under stress, and evaluate the antimicrobial activity of bacteriocin HV219.

Bacteriocin production by Lactobacillus pentosus ST712BZ isolated from boza

Bacteriocin ST712BZ (14.0kDa in size) inhibits the growth of Lactobacillus casei,Escherichia coli, Pseudomonas aeruginosa,Enterococcus faecalis, Klebsiella pneumoniae and Lactobacillus curvatus. Growth of strain ST712BZ in BHI, M17, soy milk and molasses was similar to growth in MRS, with optimal bacteriocin production (12800AU/mL) recorded in MRS after 24h. The same level of bacteriocin production (12800AU/mL) was recorded in MRS broth with an initial pH of 6.5, 6.0 and 5.5. However, MRS broth (pH 6.5) supplemented with 1mM EDTA, yielded only 6400AU/mL. Low levels of bacteriocin activity were recorded in MRS broth with an initial pH of 5.0 and 4.5. Of all media compositions tested, MRS supplemented with tryptone (20.0g/L), glucose (20.0 to 40.0g/L), mannose (20.0g/L), vitamin B12, or vitamin C yielded 12800AU/mL. Glycerol concentrations of 1.0g/L and higher repressed bacteriocin production. Maximal bacteriocin activity (25600AU/mL) was recorded in MRS supplemented with Vit. B1 or DL-6,8-thioctic acid.

Characterization of mesentericin ST99, a bacteriocin produced by Leuconostoc mesenteroides subsp. dextranicum ST99 isolated from boza

Lactic acid bacteria isolated from Boza, a cereal-fermented beverage from Belogratchik, Bulgaria, were screened for the production of bacteriocins. With the first screening, 13 of the 52 isolates inhibited the growth of Listeria innocua and Lactobacillus plantarum. The cell-free supernatant of one of these strains, classified as Leuconostoc mesenteroides subsp. dextranicum ST99, inhibited the growth of Bacillus subtilis, Enterococcus faecalis, several Lactobacillus spp., Lactococcus lactis subsp. cremoris, Listeria innocua, Listeria monocytogenes, Pediococcus pentosaceus, Staphylococcus aureus and Streptococcus thermophilus. Clostridium spp., Carnobacterium spp., L. mesenteroides and Gram-negative bacteria were not inhibited. Maximum antimicrobial activity, i.e. 6,400 arbitrary units (AU)/ml, was recorded in MRS broth after 24xa0h at 30°C. Incubation in the presence of protease IV and pronase E resulted in loss of antimicrobial activity, confirming that growth inhibition was caused by a bacteriocin, designated here as mesentericin ST99. No loss in activity was recorded after treatment with α-amylase, SDS, Tween 20, Tween 80, urea, Triton X-100, N-laurylsarcosin, EDTA and phenylmethylsulfonylfluoride. Mesentericin ST99 remained active after 30xa0min at 121°C and after 2xa0h of incubation at pHxa02 to 12. Metabolically active cells of L. innocua treated with mesentericin ST99 did not undergo lysis. Mesentericin ST99 did not adhere to the cell surface of strain ST99. Precipitation with ammonium sulfate (70% saturation), followed by Sep-Pack C18 chromatography and reverse-phase HPLC on a C18 Nucleosil column yielded one antimicrobial peptide.

Bacteriocin production by Pediococcus pentosaceus isolated from marula (Scerocarya birrea)

Strain ST44AM, isolated from marula, was identified as Pediococcus pentosaceus based on biochemical tests, sugar fermentation reactions (API 50CHL), PCR with species-specific primers and 16S rDNA sequencing. Strain ST44AM produces a 6.5 kDa class IIa bacteriocin, active against lactic acid bacteria, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Listeria innocua, Listeria ivanovii subsp. ivanovii and Listeria monocytogenes. The peptide is inactivated by proteolytic enzymes, but not when treated with alpha-amylase, Triton X-100, Triton X-114, SDS, Tween 20, Tween 80, urea, NaCl and EDTA. No change in activity was recorded after 2 h at pH values between 2.0 and 12.0, and after treatment at 100 degrees C for 120 min or 121 degrees C for 20 min. The mode of activity against L. ivanovii susbp. ivanovii ATCC19119 and Enterococcus faecium HKLHS is bactericidal, resulting in cell lyses and enzyme- and DNA-leakage. No significant differences in cell growth and bacteriocin production were observed when strain ST44AM was cultured in MRS broth at 26 degrees C, 30 degrees C and 37 degrees C for 24 h and tested against the same target strain. L. ivanovii subsp. ivanovii ATCC 19119 and E. faecium HKLHS did, however, differ in sensitivity to bacteriocin ST44AM (3.3x10(6) AU/mL and 2.6x10(4) AU/mL, respectively). Peptide ST44AM adsorbs at high levels (1600 AU/mL) to producer cells. Bacteriocin ST44AM may be a derivative of pediocin PA-1. This is the first report on the presence of P. pentosaceus in marula and a pediocin PA-1 derivative produced by this species. We are also the first to report on the synergetic effect ciprofloxacin has on a pediocin-like bacteriocin.

Phenotypic and genetic heterogeneity of lactic acid bacteria isolated from "Alheira", a traditional fermented sausage produced in Portugal.

The aim of this study was to evaluate the phenotypic and genetic heterogeneity of lactic acid bacteria (LAB) isolated from Alheira, a fermented sausage produced in Portugal. LAB were identified to genus and species level by phenotypic characteristics, using genus or species-specific primers and sequencing of the gene encoding 16S rRNA. Two-hundred and eighty-three isolates were grouped into 14 species. Lactobacillus plantarum was isolated from all sausages and Enterococcusfaecalis from most of the samples. Low numbers of Lactobacillus paraplantarum, Lactobacillus brevis, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus zeae, Lactobacillus paracasei, Leuconostoc mesenteroides, Pediococcus pentosaceus, Pediococcus acidilactici, Weissella cibaria, Weissella viridescens and Enterococcus faecium were recorded. The genetic heterogeneity of L. plantarum and E. faecalis strains were determined by numerical analysis of DNA banding patterns obtained by RAPD-PCR. Strains of L. plantarum and E. faecalis were different from different producers. This study forms the basis from which starter cultures could be selected for production of Alheira.