Medium components effecting bacteriocin production by two strains of Lactobacillus plantarum ST414BZ and ST664BZ isolated from boza

Bacteriocins ST414BZ and ST664BZ, produced by Lactobacillus plantarum, inhibited the growth of a number of lactic acid bacteria, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacter cloacae. Optimal production of bacteriocin ST664BZ (12 800 AU/mL) was recorded in MRS broth with an initial pH of 6.0 and 6.5. Bacteriocin ST414BZ was produced in MRS broth at lower pH values, ranging from 6.5 to 5.0. Low levels of bacteriocin activity were produced in BHI, M17, 10% (w/v) soy flour and 10% (w/v) molasses, suggesting that specific nutrients are required for optimal production. Bacteriocin ST414BZ production doubled (from 12 800 to 25 600 AU/mL) in MRS broth with tryptone as sole nitrogen source, or when glucose was replaced with maltose. Bacteriocin ST664BZ production, on the other hand, was less influenced by changes in nitrogen content, but increased two-fold (to 25 600 AU/mL) when glucose was replaced with sucrose, maltose or mannose, or when MRS broth was supplemented with 2.0 g/L KH₂ PO₄. Enrichment of MRS broth with vitamins B₁₂, B₁ or C did not stimulate production of the two bacteriocins. Growth in the presence of DL-6,8-thioctic acid increased bacteriocin ST664BZ production to 25 600 AU/mL. Concluded from these results, optimal levels of bacteriocins ST414BZ and ST664BZ will be produced in boza enriched with tryptone and maltose.     

Enzymatic properties of lipase and characteristics production byLactobacillus delbrueckii subsp.bulgaricus

Some properties of an extracellular lipase produced byLactobacillus delbrueckii subsp.bulgaricus were studied. Maximum enzyme activity was found against olive and butter oil as enzyme substrates. Addition of 9% acacia gum, 0.1% Na-deoxycholate and 0.01 M CaCl₂ to the enzyme reaction mixture increased-lipase activity from 5.3 to 14.5 (FFA/mg protein/minute) at pH 6.0 and at 40° C. Maximum lipase production was reached in the presence of glucose as a sole source of carbon, wheat bran as nitrogen source, olive oil as a sole lipid source and butyric acid as fatty acid supporting the growth medium. An initial pH value of the culture medium of 6.0 and a temperature of 35° C gave the highest lipolytic activity.     

Factors affecting the adsorption of bacteriocins ST194BZ and ST23LD to Lactobacillus sakei and Enterococcus sp

Bacteriocins ST194BZ and ST23LD, produced by Lactobacillus plantarum, inhibit Gram-positive and Gram-negative bacteria. Images obtained by atomic force microscopy showed clear signs of membrane damage of Lactobacillus sakei, accompanied by the leakage of DNA and beta-galactosidase. Adsorption of the bacteriocins to cells was increased when cells were treated with buffers at pH values above neutral. An increase in bacteriocin ST194BZ adsorption to cells of Enterococcus sp. and L. sakei was observed with an increase in incubation temperatures, but at different rates for the two species. Treatment of the two species with various inorganic salts and solvents gave different results regarding the adsorption of the two bacteriocins. In general, pre-treatment of the two sensitive cells with Triton X-100, Triton X-114 and chloroform increased the adsorption of the two bacteriocins. Increased adsorption of bacteriocin ST23LD to L. sakei was recorded when the cells were pre-treated with Tris and NH4-citrate. Treatment of Enterococcus sp. and L. sakei with Na-EDTA and SDS decreased the adsorption of the two bacteriocins. Variable results were recorded with inorganic salts.     

Effect of age,amount of inoculum and inoculation medium composition on lactic acid production from glucose byLactobacillus casei subsp.rhamnosus

Effect of composition of the medium used for the inoculum cultivation, of the age and amount of the inoculum was investigated using a 3-L glass fermentor with a working volume of 1 L. The highest productivity of the culture was obtained when using a 20% (V/V) 1-d inoculum grown in the MRS medium. Yields of lactic acid were 88-97%, while the L(+)-isomer represented about 80% of the total product.     

Effect of lactic acid on diacetyl and acetoin production byLactobacillus casei subsp.rhamnosus ATCC 7469

Lactic acid or its acidity apparently play an important role in the regulation of the biosynthesis of flavor compounds inLactobacillus casei subsp.rhamnosus ATCC 7469. In pyruvate-containing media,L. casei produces lactic acid, acetoin, and diacetyl. A specific pH-dependent system is necessary for both the use of pyruvate and the induction of acetoin and diacetyl production. In cell extracts ofL. casei, lactic acid inhibits the enzymatic activity of acetolactate decarboxylase (ALD) and acetolactate synthetase (ALS); this effect does not occur in whole cells under standard physiological conditions. Lactic acid prevents the use of pyruvate, and the induction of acetoin and diacetyl production. When pyruvate-containing media are used, the pH must be kept close to 6.0 in order to obtain the best production of acetoin and diacetyl.     

Characteristics and identification of bacteriocins produced by Lactococcus lactis subsp. lactis 194-K

The Lactococcus lactis subsp. lactis 194-K strain has been established to be able to produce two bacteriocins, one of which was identified as the known lantibiotic nisin A, and the other 194-D bacteriocin represents a polypeptide with a 2589-Da molecular mass and comprises 20 amino acid residues. Both bacteriocins were produced in varying proportions in all of the studied culture media, which support the growth of the producer. Depending on the cultivation medium, the nisin A content was 380- to 1123-fold lower in the 194-K stain culture broth than that of the 194-D peptide. In comparision to nisin A Bacteriocin 194-D possessed a wide range of antibacterial activity and suppressed the growth of both Gram-positive and Gram-negative bacteria. An optimal medium for 194-D bacteriocin synthesis was shown to be a fermentation medium which contained yeast extract, casein hydrolysate, and potassium phosphate. The biosynthesis of bacteriocin 194-D by the 194-K strain in these media occurred parallel to producer growth, and its maximal accumulation in the culture broth was observed at14–20 h of the strain’s growth.     

Influence of temperature and pH on production of two bacteriocins by Leuconostoc mesenteroides subsp. mesenteroides FR52 during batch fermentation

The influence of temperature and pH on growth of Leuconostoc mesenteroides subsp. mesenteroides FR52 and production of its two bacteriocins, mesenterocin 52A and mesenterocin 52B, was studied during batch fermentation. Temperature and pH had a strong influence on the production of the two bacteriocins which was stimulated by slow growth rates. The optimal temperature was 20 °C for production of mesenterocin 52A and 25 °C for mesenterocin 52B. Optimal pH values were 5.5 and 5.0 for production of mesenterocin 52A and mesenterocin 52B respectively. Thus, by changing the culture conditions, production of one bacteriocin can be favoured in relation to the other. The relationship between growth and specific production rates of the two bacteriocins, as a function of the culture conditions, showed different kinetics of production and the presence of several peaks in the specific production rates during growth. Received: 13 February 1998 / Received revision: 27 May 1998 / Accepted: 1 June 1998     

Study of two bacteriocins produced by Enterococcus casseliflavus and Ent. faecalis

AIMS: The antimicrobial activity of two plasmid-borne bacteriocins produced by Enterococcus casseliflavus IM 416K1 and Ent. faecalis IM 388C and their mating transferability were studied. METHODS AND RESULTS: Both bacteriocins showed antibacterial activity against taxonomically related micro-organisms and Listeria monocytogenes but differ for heat sensitivity, antimicrobial titre, molecular size and class of affiliation. The transferability by mating of the antibacterial properties from producers to Enterococcus faecalis JH2-2 revealed that the bacteriocin-phenotype was linked in both strains to genes located on a 34 MDa plasmid. This result was confirmed by loss of antibacterial activity and immunity after curing treatment. CONCLUSIONS: Restriction analysis has shown a different profile of the two conjugative plasmids. Enterocin 416K1 and Enterocin 388C could represent natural antilisterial agents to use in food technology. SIGNIFICANCE AND IMPACT OF THE STUDY: The transferability of the 34 MDa conjugative plasmids might be considered a possibility for the study of bacteriocins expression in bacterial hosts different from the native strains.     

Stabilization of freeze-dried Lactobacillus paracasei subsp. paracasei JCM 8130T with the addition of disaccharides,polymers, and their mixtures

Although freeze-drying is a widely used dehydration technique for the stabilizing of unstable lactic acid bacteria, Lactobacillus paracasei subsp. paracasei JCM 8130^T (L. paracasei) is destabilized after freeze-drying and subsequent storage. In order to improve the stability of freeze-dried L. paracasei, effects of disaccharides (sucrose and trehalose), polymers (maltodextrin; MD and bovine serum albumin; BSA), and their mixtures on the survival rate of freeze-dried L. paracasei were investigated. The survival rate of non-additive sample decreased slightly after freeze-drying but decreased drastically after subsequent storage at 37 °C for 4 weeks. The reduction was diminished by the addition of disaccharides and polymers. The stabilizing effect of disaccharides was not affected by the co-addition of MD. In contrast, the disaccharide–BSA mixtures had a synergistic stabilizing effect, and the survival rates were largely maintained even after storage. It is suggested that the synergistic effect originates from the conformational stabilization of the dehydrated bacteria.     

Purification and N-terminal amino acid sequence determination of the cell-wall-bound proteinase from Lactobacillus paracasei subsp. paracasei.

The cell-wall-bound proteinase from Lactobacillus paracasei subsp. paracasei NCDO 151 was purified to homogeneity by anion-exchange and hydrophobic-interaction chromatography, chromatofocusing and gel-filtration. The purification resulted in a 600-700-fold increase in specific activity of the proteinase and the final yield was approximately 20%. Upon chromatofocusing, two proteolytically active components, termed pro135 and pro110, were detected. pro135 had an isoelectric point of 4.2. It had an Mr of about 300,000 as determined by gel-filtration and 135,000 as judged by SDS-PAGE, indicating that it may exist as a dimer in its native state. pro110 had an isoelectric point of 4.4, and an Mr of about 150,000 as determined by gel-filtration and 110,000 as judged by SDS-PAGE. pro110 appears to be a degradation product of pro135 as they have the same N-terminal amino acid sequence. The first N-terminal amino acid was ambiguous for both components, whereas the sequence from the second to the ninth amino acid was Ala-Lys-Ala-Asn-Ser-Met-Ala-Asn. This is identical to the corresponding sequence of the lactococcal cell-wall-bound proteinases. Although the Lactobacillus proteinase was a little smaller than the lactococcal proteinase, their purification characteristics were very similar, suggesting that these proteinases are related.     

Bioconversion of squid pen by Lactobacillus paracasei subsp paracasei TKU010 for the production of proteases and lettuce growth enhancing biofertilizers

A protease-producing bacterium, strain TKU010, was isolated from infant vomited milk and identified as Lactobacillus paracasei subsp. paracasei. A surfactant-stable protease, purified 64-fold from the third day culture supernatant to homogeneity in an overall yield of 11%, has a molecular weight of about 49,000. The enzyme degraded casein and gelatin, but did not degrade albumin, fibrin, and elastin. The enzyme activity was increased about 1.5-fold by the addition of 5 mM Ba²⁺. However, Fe²⁺ and Cu²⁺ ions strongly inhibited the enzyme. The enzyme was maximally active at pH 10 and 60 °C and retained 94% and 71% activity in the presence of Tween 20 (2% w/v) and SDS (2 mM), respectively. The result of identification of TKU010 protease showed that nine tryptic peptides were identical to Serratia protease (serralysin) (GenBank accession number gi999638) with 35% sequence coverage. In comparison with the tryptic peptides of L. paracasei subsp. paracasei TKU012 protease, TKU010 protease possessed two additional peptides with sequences of AATTGYDAVDDLLHYHER and QTFTHEIGHALGLSHPGDYNAGEGNPTYR. The fourth day culture supernatant of TKU010 showed maximal activity of about 5-fold growth enhancing effect on lettuce weight, which was not shown with L. paracasei subsp paracasei TKU012.     

Cloning, sequencing and expression of the gene encoding the cell-envelope-associated proteinase from Lactobacillus paracasei subsp. paracasei NCDO 151.

The gene encoding the cell-envelope-associated proteinase of Lactobacillus paracasei subsp. paracasei NCDO 151 (formerly Lactobacillus casei NCDO 151) was cloned and sequenced. The gene was located on the chromosome and encoded a polypeptide of 1902 amino acids. The proteinase is N-terminally cleaved upon maturation. It shows extensive homology to the Lactococcus lactis subsp. cremoris Wg2 proteinase. Similar to the situation in Lactococcus, a maturation gene was found upstream of the proteinase gene. The cloned proteinase gene was expressed in Lactobacillus plantarum. However, no expression was observed when the gene was cloned in Lactococcus lactis.     

Effect of modified MRS medium on production and purification of antimicrobial peptide ST4SA produced by Enterococcus mundtii

Highest antimicrobial activity of peptide ST4SA (51,200 AU/mL) was recorded after 14 h of growth in MRS broth with optimal production at pH 6.0 or 6.5. Growth of strain ST4SA in the presence of tryptone, yeast extract, or a combination of the two, yielded 102,400 AU/mL. An increase in production of peptide ST4SA to 102,400 AU/mL was recorded in the presence of 20.0 g/L fructose, but decreased to 25,600 AU/mL in the presence of lactose (20.0 g/L) or mannose (20.0 g/L) as sole carbon source. Lower activity (25,600 AU/mL) was recorded when 2.0 g/L K₂HPO₄ was replaced by 2.0 g/L KH₂PO₄ in MRS broth. An increase of K₂HPO₄ to 10.0 g/L and 20.0 g/L resulted in higher activity (102,400 AU/mL). Addition of glycerol to MRS broth had a negative effect on peptide ST4SA production. Production of peptide ST4SA required the presence of magnesium sulphate, manganese sulphate and 5.0 g/L sodium acetate. Exclusion of tri-ammonium citrate from the medium resulted in reduction of activity to 3,200 AU/mL. Maximum activity (102,400 AU/mL) was recorded in MRS supplemented with 1.0 ppm Vit. C, DL-6,8-thioctic acid or thiamine, respectively. Growth of Listeria ivanovii susbp. ivanovii ATCC 19119 in the presence of peptide ST4SA (12,800 AU/mL) resulted in 99% cell lysis after 18 h. Improved production of peptide ST4SA was recorded in MRS broth (Biolab) pre-treated with Amberlite XAD-1180. Precipitation with ammonium sulphate, followed by gel filtration chromatography, yielded the highest level of peptide ST4SA. This paper describes the partially deproteination of growth medium to facilitate peptide ST4SA purification.     

Structural characterisation and enzymic modification of the exopolysaccharide produced by Lactococcus lactis subsp. cremoris B39

Lactococcus lactis subsp. cremoris B39 grown on whey permeate produced an exopolysaccharide containing L-Rha, D-Gal and D-Glc in a molar ratio of 2:3:2. The polysaccharide was modified using an enzyme preparation from Aspergillus aculeatus, resulting in the release of Gal and a polymer with approximately the same hydrodynamic volume as the native polysaccharide. Linkage analysis and 1H NMR studies of both the native and modified exopolysaccharides elucidated that terminally linked Gal was released during modification and that the chemical structure of the branches within the repeating units is: beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->. 2D NMR experiments (both 1H-1H and 1H-13C) revealed that exopolysaccharide B39 consists of a branched heptasaccharide repeating unit with the following structure: [structure: see text].     

Structural characterisation and enzymic modification of the exopolysaccharide produced by Lactococcus lactis subsp. cremoris B891

Lactococcus lactis subsp. cremoris B891 grown on whey permeate produced an exopolysaccharide containing D-Gal and D-Glc in a molar ratio of 2:3. The polysaccharide was partially O-acetylated. By means of HF solvolysis, O-deacetylation, enzymic modification, sugar linkage analysis and ID/2D NMR studies the exopolysaccharide was shown to be composed of repeating units with the following structure: [structure: see text].     

Effect of medium components on bacteriocin production by Lactobacillus plantarum strains ST23LD and ST341LD, isolated from spoiled olive brine

Bacteriocin ST23LD levels of 2930AU/OD were recorded in MRS broth (pH of 6.5) and in the presence of tryptone and yeast extract as sole nitrogen sources. Growth in MRS broth at an initial pH of 6.0 yielded only 1460AU/OD bacteriocin ST23LD. Activities of 5861AU/OD were recorded with maltose (20, 30 and 40 g/l) as sole carbon source and 9036AU/OD with the addition of 2.0-10.0 g/l KH2PO4. Bacteriocin ST341LD levels of 2850 and 2841AU/OD were recorded in MRS broth at an initial pH of 6.0 or 5.5, respectively. Only 709AU/OD was recorded in the same medium with an initial pH of 6.5. Bacteriocin ST341LD production was stimulated by the presence of tryptone. However, glucose at 10 and 40 g/l, or the presence of 5.0 or 10.0 g/l K2HPO4, resulted in a 50% reduction of bacteriocin activity. Glycerol in the growth medium repressed bacteriocin production. No increased bacteriocin production was recorded in medium supplemented with vitamins.     

Comparative studies of the degradation of grass fructan and inulin by strains of Lactobacillus paracasei subsp. paracasei and Lactobacillus plantarum

Four strains of Lactobacillus paracasei subsp. paracasei and Lact. plantarum are investigated within 16 d in order to determine the formation of metabolites during the degradation of grass fructan and inulin as well as the subsequent fermentation to lactic acid. The decrease of the total content of fructans throughout the entire time of investigation shows differences specific for strains as for either fructan substrate. The strain Lact. plantarum V 54/6 completely degrades the grass fructan and inulin within no longer than 13 d. The utilization of fructan by the other strains is temporally delayed, and in a smaller degree of degradation, especially remarkable for inulin cleavage. The structural modifications of decomposed fructans are characterized by a noticeable shift of the mean DP from approximately 80 to the oligomeric range analysed by anion exchange chromatography. Additionally, a newly formed series of peaks of oligomeric saccharides was detected during the degradation of grass fructan and inulin. Part of the fructose that is derived from cleavage of fructans is fermented immediately by the LAB strains into differently high amounts of lactic acid. The abundance of formed fructose is enriched in the medium to a varying extent, depending on the strain as well as the substrate used. From these results a number of fructan degradative enzymes in lactobacilli have been concluded to possibly vary their modes of regulation: strain specific exo- and endohydrolases with different activities against β-2,1 and β-2,6 linked fructan.     

The effect of yeast extract supplementation on the production of lactic acid from whey permeate byLactobacillus helueticus

Summary Batch and continuous two-stage cultures have been conducted in order to determine the effect of yeast extract (YE) on the homolactic fermentation of whey permeate byLactobacillus helveticus. Supplementation with YE had a significant effet on lactic acid concentration, volumetric productivity, and substrate conversion, but not on lactic acid yield. Volumetric productivity in the first stage increased from 2 to 9 g l^–1 per hour by increasing the YE concentration from 1.5 to 25 g l^–1 At the same time conversion improved from 22% to 93% at a dilution rate of 0.2 h^–1. The second stage demonstrated the effect of YE at a lower dilution rate (0.14 h^–1. A high system conversion (97%) and a high final lactic acid concentration (40 g l^–1) were achieved with 10 g l^–1 YE.     

Molecular cloning and characterisation of the two DNA components of tomato golden mosaic virus.

We report the molecular cloning of the tomato golden mosaic virus (TGMV) genome in the E. coli plasmid pAT 153. The results of this work conclusively show that TGMV DNA consists of two components (designated A and B) of almost, but not exactly, the same size. Four different recombinant plasmids are described, two containing component A in opposite orientation and two containing component B in opposite orientation. Southern blot analysis revealed little sequence homology between A and B and showed both components to be equally represented in viral and intracellular DNA forms. Detailed restriction maps of the cloned DNAs are presented, and a comparison of these with digests of intracellular viral dsDNA indicates that the former are full-length faithful copies of TGMV DNA. This is the first report of the cloning of a geminivirus genome.