A food-grade process for isolation and partial purification of bacteriocins of lactic acid bacteria that uses diatomite calcium silicate.

Bacteriocins, including nisin, pediocin PO2, brevicin 286, and piscicolin 126, were extracted from fermentation media by adsorption onto Micro-Cel (a food-grade diatomite calcium silicate anticaking agent) and subsequent desorption. The optimal conditions for desorption of piscicolin 126 were determined and applied to other bacteriocins, and the relative purities of the desorbed preparations were compared. Piscicolin was not successfully desorbed from Micro-Cel at pH 1.0 to 12.0, with organic solvents, or by increase of ionic strength up to 1 M NaCl. However, 25 and 75% of the bacteriocin activity was desorbed by using 1% sodium deoxycholate and 1% sodium dodecyl sulfate (SDS), respectively. Higher levels (up to 100%) of desorption were achieved by repeated elution or by an increase in surfactant concentration. Desorption of piscicolin with 1/10 volume of SDS solution resulted in a preparation with 10 times concentration in activity, equivalent to that of ammonium sulfate preparations (409,600 to 819,200 activity units/ml). Determination of organic nitrogen (N) content revealed that the desorbed piscicolin preparations were substantially free of proteinaceous substances (approximately 92 to 99%) compared with original culture supernatants and ammonium sulfate preparations. Nisin, pediocin, and brevicin were also desorbed with 1% SDS with a similar level of purification.     

Novel expression system for large-scale production and purification of recombinant class IIa bacteriocins and its application to piscicolin 126

Piscicolin 126 is a class IIa bacteriocin isolated from Carnobacterium piscicola JG126 that exhibits strong activity against Listeria monocytogenes. The gene encoding mature piscicolin 126 (m-pisA) was cloned into an Escherichia coli expression system and expressed as a thioredoxin-piscicolin 126 fusion protein that was purified by affinity chromatography. Purified recombinant piscicolin 126 was obtained after CNBr cleavage of the fusion protein followed by reversed-phase chromatography. Recombinant piscicolin 126 contained a single disulfide bond and had a mass identical to that of native piscicolin 126. This novel bacteriocin expression system generated approximately 26 mg of purified bacteriocin from 1 liter of E. coli culture. The purified recombinant piscicolin 126 acted by disruption of the bacterial cell membrane.     

Efflux of ions and ATP depletion induced by pediocin PA-1 are concomitant with cell death in Listeria monocytogenes Scott A

Domination of Carnobacterium divergens LV13 by a bacteriocin-producing (bac⁺) organism Carnobacterium piscicola LV17 was dependent on the level of inoculum of the producer strain and its bacteriocin production. When C. piscicola LV17 was grown in APT broth from an initial inoculum of α-10⁴ cfu ml^-1, bacteriocin was not produced (bac^-) although maximum population was reached. The culture remained bac^- during subsequent inoculation at 10²-10⁷ cfu ml^-1 unless it was first grown on solid medium or if heat-treated supernatant fluids from a bac⁺ culture of C. piscicola LV17, LV17A or LV17B were added to the culture prior to the stationary phase of growth. Use of purified carnobacteriocins from C. piscicola LV17A and LV17B confirmed their role in regulation of the bac⁺ phenotype. The need for induction might account in part for differences in bacteriocin production by cultures in liquid and on solid growth media.     

Production of bacteriocin jenseniin G by Propionibacterium is pH sensitive

Bacteriocin jenseniin G is detected typically in 50-fold concentrated 10 d cultures of the producer, Propionibacterium thoenii ( jensenii ) P126 at low concentrations (32–64 AU ml⁻¹). The effect of pH on production was examined by growing the producer in sodium lactate broth, both statically and in stirred fermenters, with or without pH control. Activity was detected in unconcentrated static producer cultures at day 7 through to day 14. Maximal jenseniin G activity (21 AU ml⁻¹) was observed in concentrated supernates at day 9, remaining relatively constant through to day 14. Producer cultures grown in stirred fermenters without pH control yielded detectable activity in 50-fold concentrated supernates at day 3 and maximal activity at day 11. Producer growth in stirred fermenters at controlled pH yielded maximum production at pH 6·40 and maximum activity (160 AU ml⁻¹) at day 13. Jenseniin G activity at pH 6·40 represented a fivefold increase over previous reports. Medium pH was critical to jenseniin G production.     

Production of piscicolin 126 by Carnobacterium maltaromaticum UAL26 is controlled by temperature and induction peptide concentration

Carnobacterium maltaromaticum UAL26 produces the antimicrobial peptides (bacteriocins) piscicolin 126, first isolated from C. maltaromaticum JG126, and carnobacteriocin BM1, first isolated from C. maltaromaticum LV17. C. maltaromaticum UAL26 is especially inhibitory to strains of Listeria monocytogenes. Bacteriocin activity is not observable in the supernatant of cultures of UAL26 grown in liquid media at 25°C, but at temperatures less than 19°C bacteriocin activity can be detected. In contrast to JG126, the piscicolin 126 operon is downregulated in UAL26 at higher temperature, and piscicolin 126 mRNA is not detected when UAL26 is grown at 25°C. Bacteriocin production in UAL26 grown at 15°C can be induced by addition of 10⁻¹⁰ M of chemically synthesized piscicolin 126 induction peptide (PisN). However, induction of bacteriocin production in UAL26 grown at 25°C requires 10⁻⁷ M of PisN. The sequence of the piscicolin 126 operon in UAL26 contains 34 single nucleotide differences compared with the piscicolin 126 operon in JG126, including single nucleotide differences in the immunity, histidine kinase, dedicated ABC-transporter and accessory genes, as well as a single nucleotide deletion in the transport accessory gene. This deletion causes a frameshift, resulting in truncation of the PisE transport accessory protein in UAL26.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Optimization and biochemical characterization of a bacteriocin from a newly isolated Bacillus subtilis strain 14B for biocontrol of Agrobacterium spp. strains

Aims:  The identification of a new compound active against Agrobacterium tumefaciens .
Methods and Results:  The culture conditions of a newly isolated Bacillus subtilis strain, designed 14B, were optimized, as a first step, to produce its bacteriocin (termed Bac 14B) for the biocontrol of Agrobacterium spp., the causal agents of the crown gall disease. Bac 14B was then partially purified and biochemically characterized. Bacillus subtilis 14B was observed to produce an antibacterial compound having a protinaceous nature. As estimated by sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE), the semi-purified bacteriocin substance was found to be a monomeric protein with a molecular weight of 21 kDa. While the latter's antimicrobial activity was completely stable during exposure to a temperature range of up to 100°C for 2 h, its initial activity was totally lost at 121°C for 20 min. The maximum bacteriocin production (4096 AU ml⁻¹) was recorded after 96 h-incubation in an optimized Luria Bertani medium supplemented with 10 g l⁻¹ glucose, 15 g l⁻¹ K₂HPO₄ and 5 g l⁻¹ MgSO₄ 7H₂O at 30°C in a shaking flask culture. Interestingly, the B. subtilis 14B culture supernatant that contained the bacteriocin under study was proved efficient in reducing both the percentage of galled plants and the number of galls in tomato.
Conclusion:  The findings revealed that B. subtilis 14B and its bacteriocin are efficient in reducing the percentage of infections in plants caused by Ag. tumefaciens .
Significance and Impact of the Study:  The results could be useful for the nurserymen who are particularly interested in the biocontrol of the crown gall disease.     

Partial purification and characterization of bacteriocin from Yersinia kristensenii

A raw milk bacterial isolate, identified as Yersinia kristensenii was found to produce a bacteriocin which was inhibitory to Yersinia enterocolitica but not to other selected species of Yersinia or Gram-negative bacteria. Maximum production of bacteriocin was obtained when the organism was grown in shake culture at 28°C. Mitomycin C at a concentration of 0.5 μg ml^-1 induced bacteriocin production. The bacteriocin was partially purified and characterized by ammonium sulphate fractionation and gel filtration. The bacteriocin was completely inactivated when treated with proteolytic enzymes (trypsin and chymotrypsin). Bacteriocin activity was heat-resistant and it retained some of its activity after 5 min at boiling temperature. A total of 15 bacteriocin sensitive-suspected food isolates were further identified biochemically as Yersinia enterocolitica and a non-sensitive isolate was identified as Yersinia intermedia.     

ADSORPTION OF FULVIC ACID ON ALGAL SURFACES AND ITS EFFECT ON CARBON UPTAKE

Adsorption of Suwannee River fulvic acid (SRFA) to algal surfaces of three green algae was studied at environmentally relevant pH values (4 –7) and SRFA concentrations (5–100 mg·L ^{− 1}). The influence of adsorbed SRFA on carbon uptake of Scenedesmus subspicatus Chodat was also examined. Although no adsorption was observed at neutral pH values (pH 6 and 7), at pH 4 up to 31 mg SRFA·m ^{− 2} and at pH 5 up to 4 mg SRFA·m ^{− 2} was adsorbed to the algal surfaces. Electrophoretic mobility measurements of S. subspicatus demonstrated an increase in the negative surface charge of the alga in the presence of SRFA at pH 4. The adsorbed SRFA also influenced ¹⁴C uptake in S. subspicatus; in this case, enhanced carbon uptake could be related to the amount of adsorbed SRFA. The binding of humic substances by algal surfaces was interpreted as the result of hydrogen bonding and hydrophobic interactions.     

Adsorption of DNA on clay minerals: protection against DNaseI and influence on gene transfer

Abstract Numerous authors have investigated DNA relationships with sandy soil. A model composed of various DNAs adsorbed on montmorillonite clay was developed to assay enzyme (DNaseI) activity on clay-adsorbed nucleic acids. The extent of DNA adsorption was affected by the concentration and valency of the cations used (Mg²⁺, Ca²⁺, Na⁺), indicating a charge-dependent process. Calf thymus DNA was found to be highly adsorbed by smectite (up to 30 mg g⁻¹ of dry clay). Adsorbed DNA was shown to be more resistant to degradation by DNaseI than free DNA. Experimental data with plasmid and short linear amplified (through polymerase chain reaction) DNA showed that protection against nucleases was only partial. Nevertheless, clay-adsorbed DNA was found to be still able, even after a strong DNaseI treatment, to artificially transform competent Escherichia coli cells. The results show that persistance of DNA and gene transfer by genetic transformation may occur in soil.     

Inhibition and inactivation of pathogenic serogroups of Yersinia enterocolitica by a bacteriocin produced by Yersinia kristensenii

Growth of Yersinia enterocolitica strains representing serogroups O: 3, O: 5, 27, O:6, 30, O:8, O:9 (human isolates) and O:6, 31 (food isolate) were inhibited in the presence of a bacteriocin produced by Yersinia kristensenii at high initial cell count of 10⁶ ml^-1. Complete (100%) inactivation of most Y. enterocolitica cells of different serotypes was observed within 24 h at low initial cell counts of 10⁴ ml^-1. Complete injury of the cells was observed within 4–8 h, with all the serotypes at 10°C and 28°C. The degree of susceptibility to the injury and the recovery of cells from the injury varied from serogroup to serogroup.     

The influence of pH and growth rate on production of the bacteriocin, bavaricin MN, in batch and continuous fermentations

Bavaricin MN, a bacteriocin produced by Lactobacillus bavaricus MN, reached titres of 2000 AU ml^-1 in APT broth maintained at pH 6.0, 30°C in a batch fermenter. Levels of bavaricin MN at pH 5.5 and 6.5 were lower despite comparable levels of producer cells. The addition of 3.0 g l^-1 beef extract to APT broth resulted in increases in both the growth rate of the culture and the production of bavaracin MN. The titre of bavaricin MN in batch fermenters controlled at pH 6.0 in APT broth plus 3 g l^-1 beef extract reached 3200 AU ml^-1 at 30°C. This level was reduced to 800 AU ml^-1 by 76 h. Glucose-limited continuous culture of Lact. bavaricus MN under the same conditions resulted in an increase in the titre of bavaricin MN to 6400 AU ml^-1. This level was maintained, independent of growth rate, for 345 h. Growth rates of 0.205, 0.118, 0.169 and 0.058 h^-1 were examined.     

Incorporation of nisin into a meat binding system to inhibit bacteria on beef surfaces

In two separate experiments, the bacteriocin, nisin, was incorporated into a commercially available meat binding system (Fibrimex^®) and applied to meat surfaces as a way of inhibiting the meat spoilage organism, Brochothrix thermosphacta during extended refrigerated storage. In experiment 1, pre-rigor lean beef carcass tissue (BCT) was inoculated with B. thermosphacta , left untreated (U), treated with 10 μg ml⁻¹ nisin (N), Fibrimex^® (F) or Fibrimex^® containing 10 μg ml⁻¹ nisin (FN), held aerobically at 4 °C for up to 7 d, and populations of B. thermosphacta and nisin activity determined. Experiment 2 determined the effects of the same treatments but on post-rigor, frozen and thawed lean BCT that was inoculated, vacuum-packaged, and stored at 4 °C for up to 14 d. In both experiments, N- and FN-treated tissues exhibited significantly lower populations of B. thermosphacta compared to U- and F-treated tissues, for the duration of refrigerated storage. Nisin activity was detected up to 7 d in N- and FN-treated samples from experiment 1. However, activity was detected only to days 0 and 2 in FN- and N-treated samples, respectively, from experiment 2. These studies indicate that the addition of a bacteriocin to a meat binding system and application to meat surfaces may be useful in reducing undesirable bacteria in restructured meat products.     

Changes in Ion Content in Heat-Treated Tetrahymena Cells

SYNOPSIS Changes in the amounts of Na⁺, K⁺, Mg²⁺ and Ca²⁺ were determined in the supernates of homogenized samples of Tetrahymena cells which were exposed to 7 heat shocks. The amounts of the same ions were also determined in the pH 4.5-soluble fractions after dialysis. During the last shock, i.e., 6.5 hr after the start of heat treatment, there was a change in the ion balance characterized by a gain in Na⁺, Ca²⁺ and non-dialyzable Mg²⁺ and a loss of K⁺. The change was not in phase with the synchronous cell division.     

Purification and some properties of streptococcal NAD-glycohydrolase

Abstract NAD-glycohydrolase (NADase) was purified from culture supernatant fluids of group C streptococci by adsorption on silica gel, chromatography on hydroxyapatite and ion exchange on Mono S column. After inactivation of a chymotrypsin-like protease, a homogeneous enzyme was isolated with an N-terminal sequence of VSGKEGKKSDVKYEMTKVMEANATSS-KEDKHVMHTLDKVM. According to serological methods, the purified enzyme of group C streptococci was identical to the group A enzyme showing a specific activity of 10000000 U mg⁻¹. It did not attack NADH, NADP or NADPH. In addition, a streptodornase was isolated having an N-terminal sequence of KTVSVNQTYGE.     

Changes in the Activities of H+, K+-ATPase and Na+, K+-ATPase in Cultured Cells Derived from Micromeres of Sea Urchin Embryos with Special Reference to Their Roles in Spicule Rod Formation

In cultured cells derived from micromeres isolated at the 16-cell stage of sea urchin embryos, the activity of H⁺, K⁺-ATPase became detectable after 15 hr of culture, when the cells started to form spicules, and then increased reaching a plateau from 25 hr of culture. The Na⁺, K⁺-ATPase activity of isolated micromeres increased to a maximum at 20 hr of culture and thereafter decreased gradually. Allylisothiocyanate, an inhibitor of H⁺, K⁺-ATPase, caused a decrease in intracellular pH (pHi) accompanied by blockage of ⁴⁵Ca deposition in spicule rods in spicule-forming cells at 30 hr of culture. Ouabain and amiloride had scarcely any effect on the pHi or ⁴⁵, deposition. In cultured cells exposed to nifedipine, which blocked ⁴⁵Ca deposition in spicule rods, allylisothiocyanate did not cause any decrease in pHi. These results show that H⁺, which is generated in the overall reaction to produce CaCO₃ from Ca²⁺ and HCO₃⁻, is probably released from the cells mainly in the reaction catalyzed by H⁺, K⁺-ATPase to maintain successive production of CaCO₃.     

Selection of strains cured of the Rhizobium leguminosarum Sym-plasmid pRL1JI by using small bacteriocin

Abstract Fast growing Rhizobia are usually insensitive to a low M _r (small) bacteriocin. Introduction of the Sym-plasmid pRL1JI into these strains results in sensitivity towards small bacteriocin. Of such strains, small bacteriocin insensitive mutants (Sbs⁻) were selected.
A high percentage of these Sbs⁻ mutants appeared to be cured of pRL1JI. This selection of cured strains was feasible as well for the wild-type plasmid pRL1JI, as for a variety of transposon marked derivatives in several bacterial backgrounds.     

Reduction of Brochothrix thermosphacta on beef surfaces following immobilization of nisin in calcium alginate gels

Lean and adipose beef carcass tissues inoculated with Brochothrix thermosphacta (BT) (approx. 4.50 log₁₀ cfu cm⁻²) were left untreated (U) or treated with 100 μg ml⁻¹ nisin (N), calcium alginate (A) or 100 μg ml⁻¹ nisin immobilized in a calcium alginate gel (AN). Tissue samples were refrigerated after treatments and bacterial populations and nisin activity were determined at 0, 1, 2 and 7 d. U, A and N treatments of lean and adipose tissues did not suppress bacterial growth (>6 log₁₀ cfu cm⁻² by day 7) while treatments of lean and adipose tissues with AN suppressed bacteria (>2.42 log₁₀ cfu cm⁻² by day 7). Bacteriocin titres from both tissues were higher in AN vs N samples after the 7 d incubation. This study demonstrates that immobilization of nisin in a gel may be a more effective delivery system of a bacteriocin to the carcass surface than direct application.     

Actin filament barbed-end capping activity in neutrophil lysates: the role of capping protein-beta 2.

A barbed-end capping activity was found in high speed supernates of neutrophils lysed in submicromolar calcium. In dilute supernate (> or = 100-fold dilution of cytoplasm), this activity accounted for most of the inhibition of barbed-end elongation of pyrenyl-G-actin from spectrin-F-actin seeds. Pointed-end elongation from gelsolin-capped F-actin seeds was not inhibited at comparable concentrations of supernate, thus excluding actin monomer sequestration as a cause of the observed inhibition. Most of the capping activity was due to capping protein-beta 2 (a homologue of cap Z). Thus, while immunoadsorption of > or = 95% of the gelsolin in the supernate did not decrease capping activity, immunoadsorption of capping protein-beta 2 reduced capping activity proportionally to the amount of capping protein-beta 2 adsorbed. Depletion of > 90% of capping protein-beta 2 from the supernate removed 90% of its capping activity. The functional properties of the capping activity were defined. The dissociation constant for binding to barbed ends (determined by steady state and kinetic analyses) was approximately 1-2 nM; the on-rate of capping was between 7 x 10(5) and 5 x 10(6) M-1 s-1; and the off-rate was approximately 2 x 10(-3) s-1. The concentration of capper free in the intact cell (determined by adsorption of supernate with spectrin-actin seeds) was estimated to be approximately 1-2 microM. Thus, there appeared to be enough high affinity capper to cap all the barbed ends in vivo. Nevertheless, immediately after lysis with detergent, neutrophils contained sites that nucleate barbed-end elongation of pyrenyl-G-actin. These barbed ends subsequently become capped with a time course and concentration dependence similar to that of spectrin-F-actin seeds in high speed supernates. These observations suggest that, despite the excess of high affinity capper, some ends either are not capped in vivo or are transiently uncapped upon lysis and dilution.     

Dose responses of tsetse flies (Glossina) to carbon dioxide, acetone and octenol in the field

ABSTRACT Studies were conducted in Zimbabwe of the catch of Glossina pallidipes Austen from an electric net plus target baited with mixtures of acetone plus carbon dioxide or 1-octen-3-ol (octenol) plus carbon dioxide. For acetone dispensed alone at 5–50, 000 mg h^-1, ten-fold increments in the dose increased the catch 1.7 times. For carbon dioxide dispensed alone, dose increments from 12 to 1201 h^-1 doubled the catch, but the catch was not further increased by dispensing carbon dioxide at 600–1200 1 h^-1. For mixtures of these two odours, ten-fold increments in the dose of carbon dioxide between 12 and 12, 0001 h^-1 increased the catch c . 2.5 times if acetone was also dispensed at >50 mg h^-1; changes in the dose of acetone between 50 and 50 000 mg h^-1 did not affect the catch. The addition of octenol (0.05 mg h^-1) to carbon dioxide (12–12001 h^-1) doubled the catch. Ten-fold increments in the dose of octenol between 0.05 and 5 mg h^-1 did not increase the catch significantly and the catch was independent of changes in the dose of carbon dioxide between 120 and 12001 h^-1. The behavioural basis of the dose-response curves was investigated using an incomplete ring of electric nets to assess the flight orientation of tsetse in different odours. Upwind flight was not elicited by acetone or octenol alone, or by carbon dioxide unless it was at very high doses, however, mixtures of carbon dioxide with acetone or octenol elicited upwind flight. It is suggested that the attractiveness of mixtures of acetone and carbon dioxide is a function of the region of overlap of these two odours at above threshold concentration. Acetone and octenol on their own appear to increase the responsiveness of flies to visual cues.     

Characteristics of ATPase from sugarcane protoplast and vacuole membranes

Characteristics of membrane-associated ATPase from commercial Hawaiian varieties of sugarcane ( Saccharum spp. hybrids) were investigated in preparations from sugarcane cell suspension culture and from stalk tissues of the intact plant. In order to examine comparable preparations, protoplasts and vacuoles, in turn, were obtained from both sources. ATPase from preparations of crude protoplast membranes and tonoplast had a pH optimum of 6 to 6.5. The relative effectiveness of divalent cations in stimulating ATPase was Mg²⁺ > Mn²⁺≥ Co²⁺ > Ca²⁺≥ Zn²⁺. Enzyme activity was not stimulated by K⁺, nor by other monovalent cations. Protoplasts and vacuoles from both sources showed significant acid phosphatase activity. Acid phosphatase activity was inhibited by molybdate, but ATPase activity was unaffected. Membrane preparations from protoplasts contained inorganic pyrophosphatase, but enzyme activity was low or not present in tonoplast preparations. Cell suspension and stalk tissue preparations hydrolyzed a large number of nucleoside di- and triphosphates. The hydrolysis is most likely due to a series of enzymes rather than a single enzyme. ATPase from protoplast and tonoplast preparations was inhibited 30–50% by diethylstilbestrol and sodium ortho-vanadate and was unaffected by ionophores. This study illustrates the complexity of phosphohydrolase activities in membrane preparations from sugarcane. The study, however, also illustrates substantial similarity in the behavior of these enzymes, whether they are derived from the plant itself or from cell cultures originating from comparable tissues of the plant.