Purification, bacteriolytic activity, and specificity of beta-lytic protease from Lysobacter sp. IB-9374

Lysobacter sp. IB-9374, which was isolated from soil as a high lysyl endopeptidase-producing strain (Chohnanet al., FEMS Microbiol. Lett., 213, 13-20, 2002), was found to produce a beta-lytic protease capable of lysing gram-positive bacteria such as Staphylococcus aureus, Microccocuseus, and Bacillus subtilis. The Lysobacter strain secreted the beta-lytic protease into the culture medium at a 2.4-fold higher level than Achromobacter lyticus. The enzyme was highly purified through a series of six steps with a high yield. The enzyme was strongly inhibited by tetraethylene-pentamine and 1,10-phenanthroline. The purified enzyme lysed more efficiently almost all the gram-positive bacteria tested than lysozyme, lysostaphin, and mutanolysin. The enzyme was very similar to Achromobacter beta-lytic protease containing one zinc atom in terms of amino acid composition and N-terminal sequence. The nucleotide sequence revealed that the mature enzyme was composed of 179 amino acid residues with additional 198 amino acids at the amino-terminal end of the enzyme. The deduced amino acid sequence of the mature enzyme coincided with that of the Achromobacter enzyme, although the prepro-region showed a 41% sequence identity with the counterpart. These results indicate that Lysobacter sp. is a useful strain for an efficient large-scale preparation of beta-lytic protease capable of lysing bacteria.     

Simplex lattice mixture design approach on the rheological behavior of glucomannan based salep-honey drink mixtures: An optimization study based on the sensory properties

The rheological properties of salep drink sweetened with different honeys were measured using a controlled-stress rheometer. Mixture design experiments were used to study the effect of interactions among pine, flower and highland honeys on the rheological properties of salep-honey drink mixture (SHDM) samples. In addition, product optimization was carried out using the ridge analysis to determine the optimum mixture proportions based on sensory properties of SHDM samples. Flower honey was the component showing the highest effect on the consistency coefficient values of SHDM samples. The preference of panelists was more prominent for the SHDM samples including the higher concentrations of highland honey with respect to odour and overall preference parameters. Optimum values of pine, flower and highland honeys in the mixture were found to be 0–85%, 0–40% and 15–100%, respectively, with respect to sensory properties. In addition, ridge analysis results revealed that the SHDM should include 65% highland honey, 35% pine honey and no flower honey to obtain the maximum overall preference score (7.24). The consistency coefficient and flow behavior index values of the sample to get maximum overall preference score (7.24) were predicted to be 3.650 Pa sⁿ and 0.435, respectively.     

Ultrafine Wool and Cotton Powder and Their Characteristics

Micro powders of recycled wool and cotton from textile waste is prepared and characterized. Wool and cotton waste fibers were frozen in liquid nitrogen and milled for various period times to produce fine powders. The powders sizes distribution depends on the milling time. Scanning electron microscopy micrographs confirm that, the fine cotton and wool powders have average size around 60 μm, and that, there are powder particle with size even less than 20 μm. Differential scanning calorimeter (DSC) and thermal gravimetrical analysis (TGA) results show that as the powder particle size decreases, its thermal stability increases slightly. Fourier transform infrared spectroscopy, Raman spectroscopy, and amino acid analysis confirm that, the freeze milling technique is a safe technique to produce ultrafine powder, with no effect on the chemical structure of cotton and wool.     

Susceptibility of different life stages of saw-toothed grain beetle Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae) to modified atmospheres enriched with carbon dioxide

The susceptibility of the different life stages of the saw-toothed grain beetle Oryzaephilus surinamensis to different modified atmospheres (MAs) containing various concentrations of carbon dioxide (CO₂) was studied as an alternative to methyl bromide fumigation at 30 °C and 65 ± 5% relative humidity (r.h.). The tested MAs were 55%, 65%, 75% and 85% CO₂ gas in the air. Mortality (%) was recorded after exposure periods of 3, 6, 12, 24, 48, 72 and 96 h. Larvae and adults were more susceptible while eggs and pupae were more tolerant to CO₂. A two-day exposure period was adequate to completely kill larvae and adults under all tested MAs. All eggs and pupae were killed after four days of exposure to the high-CO₂ atmospheres (75% and 85%).     

Photostabilization of ascorbic acid with citric acid, tartaric acid and boric acid in cream formulations

This study involves the evaluation of the effect of certain stabilizers, that is, citric acid (CT), tartaric acid (TA) and boric acid (BA) on the degradation of ascorbic acid (AH(2) ) in oil-in-water cream formulations exposed to the UV light and stored in the dark. The apparent first-order rate constants (0.34-0.95 × 10(-3) min(-1) in light, 0.38-1.24 × 10(-2) day(-1) in dark) for the degradation reactions in the presence of the stabilizers have been determined. These rate constants have been used to derive the second-order rate constants (0.26-1.45 × 10(-2) M(-1) min(-1) in light, 3.75-8.50 × 10(-3) M(-1) day(-1) in dark) for the interaction of AH(2) and the individual stabilizers. These stabilizers are effective in causing the inhibition of the rate of degradation of AH(2) both in the light and in the dark. The inhibitory effect of the stabilizers is in the order of CT > TA > BA. The rate of degradation of AH(2) in the presence of these stabilizers in the light is about 120 times higher than that in the dark. This could be explained on the basis of the deactivation of AH(2) -excited triplet state by CT and TA and by the inhibition of AH(2) degradation through complex formation with BA. AH(2) leads to the formation of dehydroascorbic acid (A) by chemical and photooxidation in cream formulations.     

Simultaneous Determination of 18 Bioactive Compounds in Italian Bitter Liqueurs by Reversed-Phase High-Performance Liquid Chromatography–Diode Array Detection

A simple, fast and accurate method has been developed to simultaneously determine 18 bioactive compounds in Italian bitter liqueurs containing gentian, cinchona, cinnamon, rhubarb, clove, star anise or orange, by reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with diode array detection (DAD). HPLC analysis was performed with a C18 column using methanol and aqueous phosphoric acid (pH 2.5) as mobile phase. Selected wavelengths, i.e. 210, 232, 275, 285, 291, 310 and 368 nm, were used for quantification of compounds. The column temperature was controlled at 30 °C. The correlation coefficients (R ²) of the calibration curves of the analysed compounds were ≥0.9999 in a relatively wide concentration range (0.5–50 μg/ml). The proposed method proved successful in simultaneously analysing 18 bitter liqueurs produced in Italy. The concentration of the most important bitter principles, gentiopicroside, amarogentin, quinine and naringin, ranged as follows: 1.17–299.20, 0.25–32.24, 1.44–6.93 and 0.28–39.99 μg/ml, respectively.     

Use of phenolic compounds for sensitizing Listeria monocytogenes to high-pressure processing

Three Listeria monocytogenes strains (Scott A, OSY-8578, and OSY-328) that differ considerably in barotolerance were grown to stationary phase and suspended individually in phosphate buffer (pH 7.0). Twelve phenolic compounds, including commercially used food additives, were screened for the ability to sensitize L. monocytogenes to high-pressure processing (HPP). Each L. monocytogenes strain was exposed to each of the 12 phenolic compounds (100 ppm each) for 60 min; this was followed by a pressure treatment at 400 MPa for 5 min. Six phenolic compounds increased the efficacy of HPP against L. monocytogenes but tert-butylhydroquinone (TBHQ) was the most effective. The additives alone at 100 ppm were not lethal for L. monocytogenes. Subsequently, the three L. monocytogenes strains were exposed to TBHQ before or after pressure treatments at 400 or 500 MPa for 5 min. When TBHQ was added after the pressure treatment, the combined treatment was more lethal than was pressure alone. However, the lethality attributable to TBHQ was greater when the additive was applied before rather than after pressure treatment. The inactivation kinetics of the L. monocytogenes strains at 300, 500, and 700 MPa, in the presence or absence of TBHQ, was investigated. All survivor plots showed non-linear inactivation kinetics, but tailing behavior was most pronounced when HPP was used alone. Combinations of TBHQ and HPP eliminated tailing behavior when survivors were monitored by direct plating or an enrichment procedure. Pressure and phenolic additives are apparently a potent bactericidal combination against L. monocytogenes.     

A coagulase-negative variant of Staphylococcus aureus from bovine mastitis milk

Bacteriological analysis of milk samples from quarters of a dairy cow suffering from subclinical mastitis yielded two isolates of Staphylococcus aureus which gave a negative reaction in the standard coagulase test. Both isolates were also clumping factor and thermonuclease negative, and gave a negative reaction in the Staphaurex? test. The isolates were identified by using commercial biochemical systems, and by PCR analysis of different staphylococcal cell surface protein and exoprotein genes. Further molecular identification of the isolates, which included sequencing of the 16S rRNA gene and RT-PCR of coagulase (coa), clumping-factor (clfA) and thermonuclease (nuc) genes, was consistent with the diagnosis phenotypically 'coagulase-negative variant of Staph. aureus'. The fact that coagulase-negative Staph. aureus variants can occur in the context of intramammary infections in cattle may result in the incorrect diagnosis 'coagulase-negative staphylococci (CNS)' in routine mastitis diagnostic, at least in rare cases. To fully ensure correct species diagnosis, sequencing of the 16S rRNA gene and amplification of specific genes such as coa is necessary in these cases.     

Complex regulation of the aflatoxin biosynthesis gene cluster of Aspergillus flavus in relation to various combinations of water activity and temperature

A microarray analysis was performed to study the effect of varying combinations of water activity and temperature on the activation of aflatoxin biosynthesis genes in Aspergillus flavus grown on YES medium. Generally A. flavus showed expression of the aflatoxin biosynthetic genes at all parameter combinations tested. Certain combinations of a_w and temperature, especially combinations which imposed stress on the fungus resulted in a significant reduction of the growth rate. At these conditions induction of the whole aflatoxin biosynthesis gene cluster occurred, however the produced aflatoxin B₁ was low. At all other combinations (25 °C/0.95 and 0.99; 30 °C/0.95 and 0.99; 35 °C/0.95 and 0.99) a reduced basal level of cluster gene expression occurred. At these combinations a high growth rate was obtained as well as high aflatoxin production. When single genes were compared, two groups with different expression profiles in relation to water activity/temperature combinations occurred. These two groups were co-ordinately localized within the aflatoxin gene cluster. The ratio of aflR/aflJ expression was correlated with increased aflatoxin biosynthesis.