Antagonistic effect of enterocin CCM 4231 from Enterococcus faecium on "bryndza", a traditional Slovak dairy product from sheep milk

"Bryndza" is a traditional Slovak dairy product (type of soft cheese) made from sheep cheese which was ripened for 14 days. Because its manufacture, transporting and/or storing represent conditions which facilitate contamination, the effect of enterocin CCM4231 in "bryndza" was investigated with the aim to reduce the contaminant agents. "Bryndza" was divided into equal portions (50 g). The experimental sample (ES) as well as the control sample one (C1) were inoculated with Listeria innocua Li1 strain. The other control samples C2 and C3 were without Li1 strain. C3 control was selected as a reference control. ES and C2 portions were treated with purified enterocin CCM4231 in a concentration of 6400 AU/ml. Before the experimental inoculation, "bryndza" was checked for the presence of contaminant agents. The experiment lasted 1 week and the samples were stored in the refrigerator at 4 degrees C. Sampling was performed on day 1, on day 4 and on day 7. The control samples C2 and C3 were checked only on day 1 and then after 1 week. The following contaminant agents were detected in "bryndza" before its experimental inoculation with L. innocua Li1 strain: Escherichia coli in the amount 10(3) cfu/ml/g, Staphylococcus aureus (10(2) cfu/ml/g) and enterococci (10(4) cfu/ml/g). In the control sample C2, the number of E. coli was reduced to 10(2) cfu/ml/g. Enterococci and staphylococci were totally eliminated there. Concerning C3 control, natural decrease of bacteria was found and/or their unchanged counts. The value of pH (5) was stable during the whole experiment. In the experimental sample inoculated with Li1 strain, its counts were decreased immediately after enterocin CCM4231 addition approximately by one order of magnitude. This inhibitory effect was also detectable on day 4 by the difference of one order of magnitude between ES and C1. On day 7, 10(3) cfu/ml/g of Li1 strain were detected in both samples (ES, C1). The difference by one order of magnitude indicated, an inhibitory effect of enterocin CCM4231 in "bryndza". However, bacteriocin activity was not determined by laboratory analyses.     

The use of enterocin CCM 4231 in soy milk to control the growth of Listeria monocytogenes and Staphylococcus aureus

The inhibitory effect of enterocin CCM 4231 (concentration 3200 AU ml-1) was used to control the growth of Listeria monocytogenes Ohio and Staphylococcus aureus in soy milk. The growth and bacteriocin (enterocin) production of producer strain CCM 4231 in soy milk was also checked. Bacteriocin production by CCM 4231 strain in soy milk was first detected after 2 h from the beginning of cultivation (100 AU ml-1). The stationary phase for CCM 4231 was reached after 6 h reaching 10.38 cfu ml-1 (log10) with a slight increase up to 24 h (10.43 cfu ml-1, log10), and the maximum bacteriocin production in soy milk (200 AU ml-1) was noted after 8 h of the beginning of cultivation with stability up to 24 h. The addition of enterocin CCM 4231 at 3200 AU ml-1 to a growing indicator strain, L. monocytogenes Ohio, in soy milk resulted in inhibition for 24 h. The high inhibitory effect of enterocin was found after 1 h and 2 h of its addition (in 5 h-6 h of cultivation), the difference between the experimental and the control samples (ES, CS) being 4.96 log cycles at 5 h and 5.15 log cycles at 6 h. Staphylococcus aureus was not fully inhibited, although a difference of 3.55 log cycles was found when ES and CS were compared at the end of cultivation (24 h). The pH was not influenced by enterocin addition. The inhibitory effect of enterocin CCM 4231 against L. monocytogenes Ohio in soy milk was probably bacteriocidal; while Staph. aureus was influenced bacteriostatically. In general, the observed inhibitory activity confirmed the possibility for further application of bacteriocins in food environments as the protective agents. Of course, legislation problems must be solved.     

Photosynthetic characteristics of the economic brown seaweed <Emphasis Type="BoldItalic">Hizikia fusiforme</Emphasis> (Sargassaceae,Phaeophyta), with special reference to its “leaf” and receptacle

Photosynthetic responses to irradiance and temperature of “leaves” and receptacles were compared in February (vegetative stage) and May (reproductive stage) in the seaweed, Hizikia fusiforme (Harvey) Okamura (Sargassaceae, Phaeophyta) from Nanao Island, Shantou, China. Irradiance-saturated photosynthesis (P_max) was significantly higher in receptacles than in “leaves” on a fresh weight basis, and that of “leaves” was greater in May than in February at ambient seawater temperatures. The optimum temperature for P_max was 30^∘C for both “leaves” and receptacles, being 5–10^∘C higher than the ambient seawater temperature. The apparent photosynthetic efficiencies were greater in receptacles than in “leaves” within the tested temperature range of 10–40^∘C. The irradiance for saturating photosynthesis for both “leaves” and receptacles was temperature-dependent, with the highest values (about 200μmolphotonsm⁻²s⁻¹) at 30^∘C.     

Effect of enterocin CCM 4231 on Listeria monocytogenes in Saint-Paulin cheese

The bacteriocin production byEnterococcus faecium strain in cheese milk and cheese was demonstrated. Purified enterocin CCM 4231 exhibited an anti-listerial effect during Saint-Paulin cheese manufacture. During cheese production the strain grew to a final concentration of 10.1±0.01 log CFU per mL per g in cheese. Then only a slight decrease of the cell concentration was noticed during ripening and was almost stable for 8 weeks. No significant differences in pH were observed between the experimental and reference cheeses. Bacteriocin production during cheese manufacture was detected only in milk samples and curd, reaching a level of 100 AU/mL. After addition of purified enterocin CCM 4231 (concentration 3200 AU/mL) into the experimental cheese, the initial concentration of 6.7±0.06 log CFU per mL ofListeria monocytogenes Ohio was reduced up to 1.9±0.01 log CFU per mL per g. After 6 weeks and at the end of the experiment the difference of surviving cells ofL. monocytogenes Ohio in ECH was only one or 0.7 log cycle compared to the control cheese. Although enterocin CCM 4231 partially inhibitedL. monocytogenes in Saint-Paulin cheese manufacture, an inhibitory effect of enterocin added was shown in 1-week cheese; however, it was not possible to detect bacteriocin activity by the agar spot test. The traditional fermentation and ripening process was not disturbed, resulting in acceptable end-products, including sensory aspects.     

Hong Kong’s worst “red tide”—causative factors reflected in a phytoplankton study at Port Shelter station in 1998

From March through April 1998, a massive “red tide” occurred in coastal waters of south China, including Hong Kong. The “red tide” rapidly killed various species of caged fish and affected coral fishes, killing a few of them, and caused great economic loss and ecological damage. Samples collected from a permanent station located in Port Shelter revealed a new dinoflagellate species, Karenia digitata which was suspected to be the causative species of this “red tide”. Species composition and abundance analysis revealed that an algal bloom persisted in Port Shelter during this entire period. Diatoms and dinoflagellates were the two main groups which dominated the phytoplankton and, in general, when there was an increase in the density of diatoms there was a decline in the density of dinoflagellates, and vice versa. The suspected “red tide” causative species together with other dinoflagellate species started to bloom in late February and reached their highest density on 18 March, when fish kills were first reported at Crooked Island, a semi-enclosed bay to the northeast of Hong Kong. During a 16-week period, dinoflagellate species dominated three times, and coincided with low wind speeds. Constant salinity and a continuing increase in sea surface water temperature suggested warm water intrusion into Hong Kong’s coastal waters during this “red tide” bloom period. Various nutrient elements, e.g. NH₄-N, total Kjeldahl nitrogen (TKN), and PO₄-P were high at the beginning of the bloom but experienced a sharp decrease thereafter. It is suggested that this early 1998 massive “red tide” in Hong Kong waters might have been triggered by a synchronous appearance of optimal climatic, nutritional and hydrographic conditions.     

Acid dissociation constants and pH values for standard “bes” and “tricine” buffer solutions in 30, 40, and 50 mass% dimethyl sulfoxide/water between 25 and −25 °C

Information is given concerning two standard buffer solutions suitable as pH references in 30, 40, and 50 mass% dimethyl sulfoxide (DMSO)/H₂O mixed solvents at subzero temperatures from −20 to 0 °C, with the intention of establishing a pH (designated pH^*) scale. The two buffers selected were the ampholytes N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid (“bes”) and N-tris(hydroxymethyl)methylglycine (“tricine”), and the reference standard consisted of equal molal quantities of the buffer and its respective sodium salt. The assignment of pH^* values was based on measurements of the emf of cells without liquid junction of the type: Pt;H₂(g,1 atm) ¦Bes, Na Besate, NaCl ¦ AgCl;Ag and Pt;H₂(g,1 atm) ¦Tricine, Na Tricinate, NaCl ¦AgCl;Ag and the pH^* was derived from a determination of K₂, the equilibrium constant for the dissociation process (Buffer)^±/ai (Buffer)⁻ + H⁺.     

Hydrology of winter–spring “red tides” in Bahía de Mazatlán, Sinaloa, México

“Red tide” events are frequent and periodical in Bahía de Mazatlán, Sinaloa, México. Yet, the ones observed from 4 February to 4 June 2000, showed some distinctive features: First, the dinoflagellates Prorocentrum balticum (85%), P. mexicanum (5%), and Ceratium furca (5%), dominated the composition of the blooms; Second, the average cell abundance by date was 1.3×10⁶ cells l⁻¹, with a range of 3.5×10³ to 24,500 × 10³ cells l⁻¹, well above previous records; Third, the temperature registered at 10–20 m deep was unusually cold (19 °C), below the normal average of 21.5 °C observed over the last 10 years. Salinity was high (35.9 psu) and showed very little influence on the water density gradient. A mean thermal stratification index (TSI), of 3.4, with a maximum of 7 °C, was observed throughout the period, in spite of a weak upwelling activity and intermittent strong NW winds which were unable to break up water column stratification. Temperature fluctuations at the surface and at the bottom layers showed a 30-day periodicity, suggesting some association with the lunar cycle. To explain the characteristics of the “red tides” registered in Bahía de Mazatlán during the winter–spring period of year 2000, it is proposed that the temperature and density stratification, stabilized further by internal waves that compensated for the weak upwelling activity and provided the necessary nutrients to the surface layer, favored the persistence and intensity of the harmful algal bloom events then observed.     

Selected microbial consortium of raw and digested pig slurry and its susceptibility to enterocins

A consortium of bacterial genera from raw and digested pig slurry (pig farm at Figa, Slovakia; input and output samples) was counted from February to October 2000. The total counts of enterococci and staphylococci were well-balanced in input samples, with visible reduction of cells in May (3.22 and/or 4.21 log c.f.u./ml). Among organisms important from a sanitary perspective only a slight reduction after standard slurry treatment was found between input and output samples (2.0 log cycles), with no effect in April and May. However, their counts were high (8.1–9.01 log c.f.u./ml). Yersinia sp. were detected in rather high counts (6.47; 6.39 log c.f.u./ml). But these species, as well as pseudomonads and Aeromonas sp. were very effectively reduced by standard slurry treatment. Enterocins (CCM4231, V24 and EC24) produced by our own isolates of enterococci were used to determine the susceptibility of selected microbial strains from slurry to those enterocins. For quantifying the inhibitory activity of enterocins, the titre (expressed in activity-arbitrary units [AU/ml]), corresponding to the reciprocal of the highest dilution showing a distinct inhibition zone of the indicator, was determined. Under the conditions used, enterocins used were active against the selected microbial consortium by activity from 100 up to 800 AU/ml. Moreover, enterocin V24 reduced the growth of Enterobacter cloacae ECL751 as well as Pseudomonas sp. minimally with differences of 1. 54 and 2.2 log cycles.     

Glucose biosensor based on nano-SiO2 and “unprotected” Pt nanoclusters

The “unprotected” Pt nanoclusters (average size 2 nm) mixed with the nanoscale SiO₂ particles (average size 13 nm) were used as a glucose oxidase immobilization carrier to fabricate the amperometric glucose biosensor. The bioactivity of glucose oxidase (GOx) immobilized on the composite was maintained and the as-prepared biosensor demonstrated high sensitivity (3.85 μA mM⁻¹) and good stability in glucose solution. The Pt–SiO₂ biosensor showed a detection limit of 1.5 μM with a linear range from 0.27 to 4.08 mM. In addition, the biosensor can be operated under wide pH range (pH 4.9–7.5) without great changes in its sensitivity. Cyclic voltammetry measurements showed a mixed controlled electrode reaction.     

Cells of <Emphasis Type="BoldItalic">Candida utilis</Emphasis> for <Emphasis Type="BoldItalic">in vitro</Emphasis> (<Emphasis Type="BoldItalic">R</Emphasis>)-phenylacetylcarbinol production in an aqueous/octanol two-phase reactor

(R)-Phenylacetylcarbinol (PAC), a pharmaceutical precursor, was produced from benzaldehyde and pyruvate by pyruvate decarboxylase (PDC) of Candida utilis in an aqueous/organic two-phase emulsion reactor. When the partially purified enzyme in this previously established in vitro process was replaced with C. utilis cells and the temperature was increased from 4 to 21 °C, a screen of several 1-alcohols (C4–C9) confirmed the suitability of 1-octanol as the organic phase. Benzyl alcohol, the major by-product in the commercial in vivo conversion of benzaldehyde and sugar to PAC by Saccharomyces cerevisiae, was not formed. With a phase volume ratio of 1:1 and 5.6 g C. utilis l⁻¹ (PDC activity 2.5 U ml⁻¹), PAC levels of 103 g l⁻¹ in the octanol phase and 12.8 g l⁻¹ in the aqueous phase were produced in 15 h at 21 °C. In comparison to our previously published process with partially purified PDC in an aqueous/octanol emulsion at 4 °C, PAC was produced at a 4-times increased specific rate (1.54 versus 0.39 mg U⁻¹ h⁻¹) with simplified catalyst production and reduced cooling cost. Compared to traditional in vivo whole cell PAC production, the yield on benzaldehyde was 26% higher, the product concentration increased 3.9-fold (or 6.9-fold based on the organic phase), the productivity improved 3.1-fold (3.9 g l⁻¹ h⁻¹) and the catalyst was 6.9-fold more efficient (PAC/dry cell mass 10.3 g g⁻¹).*Dedicated with gratitude to Prof. Dr. Franz Lingens – “Theo”.     

“Action potentials” in NEUROSPORA CRASSA, a mycelial fungus

Occasional spontaneous “action potentiais” are found in mature hyphae of the fungus Neurospora crassa. They can arise either from low-level sinusoidal oscillations of the membrane potential or from a linear slow depolarization which accelerates into a rapid upstroke at a voltage 5–20 mV depolarized from the normal resting potential (near − 180 mV). The “action potentiais” are long-lasting, 1–2 min and at the peak reach a membrane potential near −40 mV. A 2− to 8−fold increase of membrane conductance accompanies the main depolarization, but a slight decrease of membrane conductance occurs during the slow depolarization. Two plausible mechanisms for the phenomenon are (a) periodic increases of membrane permeability to inorganic ions, particularly H⁺ or Cl^- and (b) periodic decreases in activity of the major electrogenic pump (H⁺) of the Neurospora membrane, coupled with a nonlinear (inverse sigmoid) current-voltage relationship.Identification of action potential-like disturbances in fungi means that such behavior has now been found in all major biologic taxa which have been probed with suitable electrodes. As yet there is no obvious function for the events in fungi.     

Studies on fungal polysaccharide XVII. A new glucuronan “protuberic acid” produced by a fungus Kobayashi Nipponica

A water-soluble glucuronan “protuberic acid”, [α]_d²² −83.6° and purified from Kobayashia Nipponica, and its physicochemical properties were investigated.The purified protuberic acid was homogeneous as shown by zone electrophoresis, gel filtration over Sepharose 4B, and ultracentrifugation. The sedimentation coefficient was 1.8 S and its intrinsic viscosity was 1.1 dl/g. By gel filtration the molecular weight was estimated to be about 170 000. The results of periodate oxidation, methylation analysis, and partial acid hydrolysis indicated that this acidic polysaccharide has a linear structure of mainly 1,4-linkages and containing an acid-labile linkage. Reduced protuberic acid, [α]_d²² −44°, is also described.     

Expression and Purification of a Recombinant “Small” Sialidase fromClostridium perfringensA99

A 1.4-kb gene encoding the “small” sialidase isoenzyme ofClostridium perfringensA99, including its own promoter, was previously cloned in and expressed byEscherichia coliJM 101. Since all attempts to purify this enzyme to homogeneity were unsuccessful, a new strategy was developed. The structural gene was amplified by means of a PCR technique and inserted into the plasmid vector pQE-10, transferring a six-histidine affinity tag (His₆) to the N-terminus of the protein. In order to minimize proteolytic degradation of the sialidase protein, the gene was subcloned into theEscherichia colistrain BL21(DE3)pLys S with reduced protease activity. The sialidase production was increased about 2.5-fold when compared with that of the original clone. The enzyme, released by lysozyme treatment of the bacterial cells, was purified by metal chelate chromatography on Ni–nitrilo-triacetic acid agarose to apparent homogeneity in SDS–PAGE. The 42-kDa protein was enriched 62-fold with a yield of 82% and a specific activity of 280 U mg⁻¹. A total amount of 1 mg sialidase was obtained from 1 liter of bacterial culture. For future studies, including crystallization experiments, the histidine affinity tag was removed from the sialidase enzyme by aminopeptidase K. The sialidase was then separated from aminopeptidase K by ion-exchange chromatography, resulting in an overall yield of 83% and a specific activity of 305 U mg⁻¹using 4-methylumbelliferyl-α- -N-acetylneuraminic acid under standard conditions. The two forms (with or without the histidine tag) of sialidase exhibited similar kinetic properties when compared to the wild-type enzyme.     

Control of Pythium damping-off of sugar beet by seed treatment with crop straw powders and a biocontrol agent

Seed treatment with non-sterilized powdered straws from 39 crops was tested for the control of Pythium damping-off of sugar beet. Four straws, including flax, coriander, pea, and lentil were effective in controlling the disease in soil artificially infested with Pythium sp. “group G.” Sterilizing flax and pea straws eliminated the efficacy of these straws. Wheat straw powder coated on sugar beet seeds increased the incidence of Pythium damping-off but this effect was reversed by the co-inoculation of wheat straws with the biocontrol agent Pseudomonas fluorescens 708. Coating sugar beet seeds with P. fluorescens 708 and flax or pea straws also increased the efficiency of the bacterial strain for the control of Pythium damping-off. Pea straws and to a lesser extent lentil straws produced volatile substances that affected mycelial growth of Pythium sp. “group G” on potato dextrose agar in Petri plates when the straws were mixed with water and left to ferment for two days. Fermentation of pea straws led to the accumulation of volatile ammonia, which was produced by the reduction of the large amount of nitrate stored in the straw. Reduction of nitrate and therefore the release of volatile ammonia did not occur in sterilized pea straws. However, fermenting sterile pea straws with bacteria from different genera restored nitrate reduction and the release of volatile ammonia, suggesting that microorganisms associated with pea straws are responsible for the conversion of nitrate into volatile ammonia which in turn control Pythium damping-off disease in sugar beet.     

Simultaneous determination of α-tocopheryl acetate and tocopherols in aquatic organisms and fish feed

In aquaculture, α-tocopheryl acetate (α-TA) is the main source of vitamin E used to fortify fish feed. α-TA in fish is often determined indirectly, i.e., by alkaline hydrolysis, followed by quantitation of “total α-tocopherol” (α-T) and subtraction of the natively present α-T. The aim of this study was to develop an HPLC method for the simultaneous quantitative determination of α-TA and free tocopherols in aquatic organisms and fish feed. The assay consists of a simple extraction with methanol containing butylhydroxytoluene (BHT) as an antioxidant, followed by reversed-phase chromatography with consecutive UV and fluorescence detection of α-TA and tocopherols, respectively. The peak of the internal standard tocol in the fluorescence trace was used for quantitation. Linearity was achieved over the range of 0.2 to 4.2 μg α-TA per ml extract of Artemia nauplii, which would correspond to 30.7 to 614.4 μg/g dry mass. The within-run coefficient of variation was 1.9% at a level of 310 μg/g dry mass. The recovery of α-TA ranged from 97.7 to 100.8% (concentration=2.1 and 20.5 μg/ml, n=6). The detection limit was about 7 ng and the quantification limit on spiked samples was 0.2 μg/ml. This method was routinely applied to determine α-TA and α-, γ- and δ-tocopherol (α-T, γ-T, δ-T) simultaneously in Artemia, fish feed, shrimp eggs and various other aquatic organisms.     

Common Evolutionary Origin and Birth-and-Death Process in the Replication-Independent Histone H1 Isoforms from Vertebrate and Invertebrate Genomes

The H1 histone multigene family shows the greatest diversity of isoforms among the five histone gene families, including replication-dependent (RD) and replication-independent (RI) genes, according to their expression patterns along the cell cycle and their genomic organization. Although the molecular characterization of the RI isoforms has been well documented in vertebrates, similar information is lacking in invertebrates. In this work we provide evidence for a polyadenylation signature in the Mytilus “orphon” H1 genes similar to the polyadenylation characteristic of RI H1 genes. These mussel genes, together with the sea urchin H1δ genes, are part of a lineage of invertebrate “orphon” H1 genes that share several control elements with vertebrate RI H1 genes. These control elements include the UCE element, H1-box and H4-box. We provide evidence for a functional evolution of vertebrate and invertebrate RI H1 genes, which exhibit a clustering pattern by type instead of by species, with a marked difference from the somatic variants. In addition, these genes display an extensive silent divergence at the nucleotide level which is always significantly larger than the nonsilent. It thus appears that RI and RD H1 isoforms display similar long-term evolutionary patterns, best described by the birth-and-death model of evolution. Notably, this observation is in contrast with the theoretical belief that clustered RD H1 genes evolve in a concerted manner. The split of the RI group from the main RD group must therefore have occurred before the divergence between vertebrates and invertebrates about 815 million years ago. This was the result of the transposition of H1 genes to solitary locations in the genome.[Reviewing Editor:Dr. Yves Van de Peer]     

The “erythromycin-resistance” methylated sequence of Staphylococcus aureus ribosomal RNA

We have determined the sequence of an oligonucleotide from the large ribosomal subunit RNA of Staphylococcus aureus whose methylation renders the organism resistant to erythromycin and other antibiotics (the “MLS” phenotype). Analysis of RNase A digests of [³H]methyl-, ³²P-labeled RNA yielded the sequence GG · m₂⁶A · AAGACp, where m₂⁶A is an N⁶-dimethylated adenosine residue that in sensitive cells is unmethylated. Comparison with homologous sequences recently reported for Saccharomyces cerevesiae mitochondria indicates that an A to G mutation in this latter system mimics dimethylation in St. aureus with regard to functional consequences.     

Floral abnormalities in field grown “Tigrina” plants from xHaynaldoticum sardoum Meletti et Onnis (Gramineae)

A ‘tigrina’ mutant stock of xHaynaldoticum sardoum Meletti et Onnis grown under field conditions produced high frequency spikes with abnormal stamens and pistils. The abnormalities occurred mostly in the stamens, which frequently changed to either pseudoovaries, or leaf-shaped structures or, in extreme cases, did not form at all. The developmental abnormalities of the stamens resulted in male sterility in the whole or in part of the spike. It is proposed that the “tigrina” status changes the physiology of the plant (e.g. photosynthetic activity) in such a way that it responds with floral abnormalities brought about by environmental conditions which permit normal flower development in control (non-mutant) plants.     

Dose response curves after irradiation of Arabidopsis seeds: a possible explanation for the “saturation” in mutant frequency at high radiation doses

Mutant frequency “saturation” in Arabidopsis' “main” inflorescence after fast neutron or X-ray treatment of seeds was observed using cell populations with heterogeneous radiation-sensitivities. Mutant frequencies were determined in (1) morphologically normal and conspicuous plants, (2) main and lateral inflorescences, and (3) different M₁-fertility classes. At moderate radiation doses (47 Gy fast neutrons and 233 Gy X-rays) significant differences in mutant frequencies between different M₁-fertility classes were observed. This suggests that cell populations with heterogeneous sensitivities were studied. No significant differences were observed either between the mutant frequencies in main and lateral inflorescences or between the ones in inflorescences of morphologically normal and conspicuous plants. This suggests that these inflorescences were formed by cell populations with similar but heterogeneous sensitivities.