Antibacterial activity of lysozyme after association with carboxymethyl konjac glucomannan.

The unique high isoelectric point of lysozyme (LYZ) restricts its application in composite antibacterial coating due to the unfavorable liability to electrostatic interaction with other components. In this work, the antibacterial activity of a dispersible LYZ-carboxymethyl konjac glucomannan (CMKGM) polyelectrolyte complex was evaluated. Kinetic analysis revealed that, compared with free LYZ, the complexed enzyme exhibited decreased affinity (Km) but markedly increased Vmax against Micrococcus lysodeikticus, and QCM and dynamic light scattering analysis confirmed that the complex could bind with the substrate but in a much lower ratio. The complexation with CMKGM did not alter the antibacterial spectrum of LYZ, and the complex exerted antibacterial function by delaying the logarithmic growth phase and impairing the cell integrity of Staphylococcus aureus. Since the LYZ-CMKGM complex is dispersible in water and could be assembled easily, it has great potential as an edible coating in food preservation.

Phytoremediation of cadmium-polluted soil by Chlorophytum laxum combined with chitosan-immobilized cadmium-resistant bacteria.

This study examined the performance of the chitosan-immobilized cadmium-resistant bacteria Arthrobacter sp. and Micrococcus sp. on cadmium phytoremediation by Chlorophytum laxum in cadmium-polluted soil. These immobilized cadmium-resistant bacteria can survive in cadmium-contaminated soil and significantly increased soil cadmium solubility, but the ability of chitosan-immobilized cells to increase cadmium solubility was lower than that of free cells. A pot experiment demonstrated that chitosan-immobilized Micrococcus sp. promoted the growth of C. laxum planted in cadmium-contaminated soil. A significant increase in the cadmium concentration in the roots and aboveground parts of C. laxum was found in plants inoculated with free and chitosan-immobilized cells of these bacteria. The performance of Arthrobacter sp. free cells to augment cadmium accumulation in C. laxum was a little bit better than that of chitosan-immobilized Arthrobacter sp., except at 9 weeks after planting. The phytoextraction coefficient, bioaccumulation factor, and translocation factor of C. laxum inoculated with free and chitosan-immobilized cells of cadmium-resistant bacteria were higher than those of the uninoculated control and increased with time. Our findings suggest that chitosan-immobilized cells can be exploited to enhance the efficiency of cadmium phytoremediation by C. laxum.

[THE MYCOBIOTA OF TUNICA MUCOSA OF MOUTH AND SURFACE OF REMOVABLE ACRYLIC LAMINAR DENTAL PROSTHESES UNDER ORTHOPEDIC REHABILITATION].

The analysis was carried out to detect mycobiota of tunica mucosa of mouth and surface of dental prostheses under orthopedic rehabilitation using removable acrylic laminar dental prostheses. The inoculation of biosamples received from examined patients permitted to isolate Candida albicans. The C. albicans from tunica mucosa of mouth of patients before prosthetics inoculated in low concentration making up 0.33±0.23 CFU/ml in comparison with concentration of 1.92±0.53 CFU/ml after prosthetics. The highest content of C. albicans was marked in biosample from surface of dental prostheses in comparison with biotope of tunica mucosa of mouth of patients. The concentration of microbiota from surface of dental prostheses signicantly surpassed the same on tunica mucosa of mouth of patients prior prosthetics. In patients with removable acrylic laminar dental prostheses under orthopedic rehabilitation various spectrum of representatives of microbiota was detected From biosamples from surface of dentalprostheses of patients the most frequently were inoculated such representatives of gram-positive microbiota as S. aureus, Micrococcus spp., S.haemolyticus, and of gram-negative microbiota Klebsiella pneumonae, Pseudomonas aeruginosa. The cultural analysis of biosamples from patients with removable acrylic laminar dental prostheses detected Candida albicans on tunica mucosa of mouth before and after prosthetics as well as on surfaces of prostheses. The highest concentration of C.albicans is established in case of colonization of removable acrylic laminar dental prostheses. The received data testifies possible involvement of fungi capable of expressed potential ofpathogenicity, in development and maintenance of inflammatory process of tunica mucosa of mouth under orthopedic rehabilitation using removable acrylic laminar dental prostheses.

Comparative analysis of ampoules and vials in sterile and conventional packaging as to microbial load and sterility test / Análise comparativa de ampolas e frascos-ampolas em embalagens estéreis e convencionais quanto a carga microbiana e teste de esterilidade

ABSTRACT Objective To compare sterility and microbial (bacteria and fungi) load in the outer part of hyperbaric bupivacaine (Neocaína®) in ampoule and bupivacaine in vial, in conventional and sterile pack formulations. Methods The sterile packs were divided into two groups: G1 (n=16) with ampoules and G2 (n=16) with vials. Conventional formulations were divided into two groups, being G3 (n=16) with ampoules and G4 (n=16) with vials. The ampoules and vials were opened and had their content drawn. The empty bottles were then placed in sterile plastic bags and sent for analysis of microbial load (bacteria and fungi) and sterility testing. Data were analyzed using the χ2 test with Yates correction, and 95% confidence interval. Results G1 and G2 showed no bacterial growth when compared to conventional groups (p<0.001). The most common agent in conventional microbiological samples was Staphylococcus aureus. There was no fungal growth in both groups. Conclusion The use of (sterile pack) reduces the microbial load of bottles, and would decrease the chance of exposure to potential contamination of the anesthetic solution.

RESUMO Objetivo Comparar a esterilidade e a carga microbiana (bactérias e fungos) da parte externa dos frascos de envasamento de bupivacaína hiperbárica (Neocaína®) em ampola e bupivacaína em frasco-ampola das apresentações convencional e estéril (sterile pack). Métodos As apresentações estéreis (sterile pack) foram distribuídas em dois grupos, sendo que o G1 (n=16) continha as ampolas e o G2 (n=16), os frascos-ampola. As apresentações convencionais foram distribuídas em dois grupos, a saber G3 (n=16) com as ampolas e G4 (n=16) com os frascos-ampola. As ampolas e os frascos-ampolas eram abertos e tinham seu conteúdo aspirado. Os frascos vazios eram, então, acondicionados em sacos plásticos estéreis e enviados para análise quanto à carga microbiana (bactérias e fungos), bem como para o teste de esterilidade. Os dados foram analisados por meio do teste χ2 com correção Yates com intervalo de confiança de 95%. Resultados Os grupos G1 e G2 não apresentaram crescimento bacteriano quando comparado aos grupos convencionais (p<0,001). O microbiano mais comum nas amostras convencionais foi o Staphylococcus aureus. Não houve crescimento de fungos em nenhum dos grupos. Conclusão O uso de embalagens estéreis (sterile pack) diminui a carga microbiana dos frascos de envasamentos, o que diminuiria a chance de exposição a uma potencial contaminação da solução anestésica.

Comparative analysis of ampoules and vials in sterile and conventional packaging as to microbial load and sterility test.

OBJECTIVE: To compare sterility and microbial (bacteria and fungi) load in the outer part of hyperbaric bupivacaine (Neocaína®) in ampoule and bupivacaine in vial, in conventional and sterile pack formulations. METHODS: The sterile packs were divided into two groups: G1 (n=16) with ampoules and G2 (n=16) with vials. Conventional formulations were divided into two groups, being G3 (n=16) with ampoules and G4 (n=16) with vials. The ampoules and vials were opened and had their content drawn. The empty bottles were then placed in sterile plastic bags and sent for analysis of microbial load (bacteria and fungi) and sterility testing. Data were analyzed using the χ2 test with Yates correction, and 95% confidence interval. RESULTS: G1 and G2 showed no bacterial growth when compared to conventional groups (p<0.001). The most common agent in conventional microbiological samples was Staphylococcus aureus. There was no fungal growth in both groups. CONCLUSION: The use of (sterile pack) reduces the microbial load of bottles, and would decrease the chance of exposure to potential contamination of the anesthetic solution. OBJETIVO: Comparar a esterilidade e a carga microbiana (bactérias e fungos) da parte externa dos frascos de envasamento de bupivacaína hiperbárica (Neocaína®) em ampola e bupivacaína em frasco-ampola das apresentações convencional e estéril (sterile pack). MÉTODOS: As apresentações estéreis (sterile pack) foram distribuídas em dois grupos, sendo que o G1 (n=16) continha as ampolas e o G2 (n=16), os frascos-ampola. As apresentações convencionais foram distribuídas em dois grupos, a saber G3 (n=16) com as ampolas e G4 (n=16) com os frascos-ampola. As ampolas e os frascos-ampolas eram abertos e tinham seu conteúdo aspirado. Os frascos vazios eram, então, acondicionados em sacos plásticos estéreis e enviados para análise quanto à carga microbiana (bactérias e fungos), bem como para o teste de esterilidade. Os dados foram analisados por meio do teste χ2 com correção Yates com intervalo de confiança de 95%. RESULTADOS: Os grupos G1 e G2 não apresentaram crescimento bacteriano quando comparado aos grupos convencionais (p<0,001). O microbiano mais comum nas amostras convencionais foi o Staphylococcus aureus. Não houve crescimento de fungos em nenhum dos grupos. CONCLUSÃO: O uso de embalagens estéreis (sterile pack) diminui a carga microbiana dos frascos de envasamentos, o que diminuiria a chance de exposição a uma potencial contaminação da solução anestésica.

Effect of cooking temperatures on characteristics and microstructure of camel meat emulsion sausages.

BACKGROUND: The camel is an excellent source of high quality meat and camel meat might be a potential alternative for beef. This study aimed to manipulate the raw camel meat for the production of stable and acceptable emulsion sausage, as well as to study the effect of cooking at different core temperatures on the tenderness, sensory quality and microstructure of produced sausage. RESULTS: Increasing the cooking temperature of sausages resulted in reduction of the shear force values from 2.67 kgf after cooking at 85 °C to 1.57 kgf after cooking at 105 °C. The sensory scores of sausages have been improved by increasing the cooking core temperature of meat batter. The light and scanning electron microscope micrographs revealed solubilisation of the high quantity of connective tissue of camel meat. High emulsion stability values for the camel meat batter associated with high values of water-holding capacity for raw camel meat and meat batter have been recorded. CONCLUSION: Stable and acceptable camel meat emulsion can be developed from camel meat. Increasing the cooking core temperature of meat batter improved the quality of produced sausages. Therefore, camel meat emulsion sausages might be a potential alternative for beef particularly in Asian and African countries. © 2015 Society of Chemical Industry.

Lvserpin3 is involved in shrimp innate immunity via the inhibition of bacterial proteases and proteases involved in prophenoloxidase system.

Serine protease inhibitor, represented by serpin, plays an important inhibitory role on proteases involved in the immune responses. To clarify the immune characterizations of serpin, a novel serpin (Lvserpin3) encoding for 410 amino acids with a 23-amino acid signal peptide and a serpin domain was identified from the Pacific white shrimp Litopenaeus vannamei. Lvserpin3 expressed strongest in hepatopancreas, and was significantly up-regulated in the early stage upon Vibrio anguillarum, Micrococcus lysodeikticus or White Spot Syndrome Virus (WSSV) infection. Suppression of Lvserpin3 by dsRNA led to a significant increase in the transcripts of LvPPAF, LvproPO and phenoloxidase (PO) activity, and also led to the high cumulative mortality. The recombinant Lvserpin3 protein (rLvserpin3) inhibited the proteases secreted by M. lysodeikticus and Bacillus subtilis, and further exhibited inhibitory role on the growth of B. subtilis and M. lysodeikticu. Moreover, rLvserpin3 was found to be able to block the activation of prophenoloxidase system. Taken together, the results imply that Lvserpin3 may be involved in shrimp innate immunity via the inhibition of bacterial proteases and proteases involved in prophenoloxidase system.

Beneficial Bacteria Isolated from Grapevine Inner Tissues Shape Arabidopsis thaliana Roots.

We investigated the potential plant growth-promoting traits of 377 culturable endophytic bacteria, isolated from Vitis vinifera cv. Glera, as good biofertilizer candidates in vineyard management. Endophyte ability in promoting plant growth was assessed in vitro by testing ammonia production, phosphate solubilization, indole-3-acetic acid (IAA) and IAA-like molecule biosynthesis, siderophore and lytic enzyme secretion. Many of the isolates were able to mobilize phosphate (33%), release ammonium (39%), secrete siderophores (38%) and a limited part of them synthetized IAA and IAA-like molecules (5%). Effects of each of the 377 grapevine beneficial bacteria on Arabidopsis thaliana root development were also analyzed to discern plant growth-promoting abilities (PGP) of the different strains, that often exhibit more than one PGP trait. A supervised model-based clustering analysis highlighted six different classes of PGP effects on root architecture. A. thaliana DR5::GUS plantlets, inoculated with IAA-producing endophytes, resulted in altered root growth and enhanced auxin response. Overall, the results indicate that the Glera PGP endospheric culturable microbiome could contribute, by structural root changes, to obtain water and nutrients increasing plant adaptation and survival. From the complete cultivable collection, twelve promising endophytes mainly belonging to the Bacillus but also to Micrococcus and Pantoea genera, were selected for further investigations in the grapevine host plants towards future application in sustainable management of vineyards.

Extracellular polymeric substances govern the development of biofilm and mass transfer of polycyclic aromatic hydrocarbons for improved biodegradation.

The hypothesis that extracellular polymeric substances (EPS) affect the formation of biofilms for subsequent enhanced biodegradation of polycyclic aromatic hydrocarbons was tested. Controlled formation of biofilms on humin particles and biodegradation of phenanthrene and pyrene were performed with bacteria and EPS-extracted bacteria of Micrococcus sp. PHE9 and Mycobacterium sp. NJS-P. Bacteria without EPS extraction developed biofilms on humin, in contrast the EPS-extracted bacteria could not attach to humin particles. In the subsequent biodegradation of phenanthrene and pyrene, the biodegradation rates by biofilms were significantly higher than those of EPS-extracted bacteria. Although, both the biofilms and EPS-extracted bacteria showed increases in EPS contents, only the EPS contents in biofilms displayed significant correlations with the biodegradation efficiencies of phenanthrene and pyrene. It is proposed that the bacterial-produced EPS was a key factor to mediate bacterial attachment to other surfaces and develop biofilms, thereby increasing the bioavailability of poorly soluble PAH for enhanced biodegradation.

Antibacterial activity of ruthenium(II) polypyridyl complex manipulated by membrane permeability and cell morphology.

This study investigates the antibacterial effects of the ruthenium(II) complex RuBP and the mechanism of RuBP action on bacteria. Results show that RuBP can inhibit the growth of Gram-positive bacteria, such as Staphylococcus aureus and Micrococcus tetragenus. Cellular uptake and laser confocal microscopic studies reveal the efficient uptake of RuBP by M. tetragenus cells. Scanning electron microscopic observations of the morphologies of M. tetragenus and S. aureus treated with RuBP further confirm that direct contact of both bacteria with RuBP can damage the cell membrane and membrane integrity, which may eventually induce growth inhibition and bacterial death. After RuBP treatment, the electrical conductivity of the bacterial suspensions increases. Spectroscopic studies and agarose gel electrophoresis indicate that intact DNA and RNA decrease or disappear in RuBP-treated bacterial cells, thus demonstrating that RuBP performs its antibacterial function by increasing the permeability of cell membranes. This study provides new insights for understanding the antibacterial actions of RuBP and designing metal complex antibiotics for other biomedical applications.

Microbial load and safety of paper currencies from some food vendors in Jimma Town, Southwest Ethiopia.

BACKGROUND: Paper currency is used for every type of commerce and plays an important role in the life of human beings. However, the combination of its widespread use and constant exchange make paper currency a likely agent for disease transmission. Thus, the aim of this study was to evaluate the microbial load and safety of Ethiopian paper currencies collected from some food vendors in Jimma town. METHODS: Standard microbiological methods were used for the enumeration of various microbial groups, isolation and characterization of pathogenic bacteria and their growth potential in selected weaning foods. A total of 100 samples of Ethiopian paper currencies, consisting of five denominations, from street food venders, hotels and cafeterias in Jimma town were collected aseptically. Sterile cotton swabs moistened with buffered peptone water solution were used for swabbing and the swabs were separately soaked into 10 ml sterile buffered peptone water solution. RESULTS: Mean microbial counts of Aerobic mesophilic bacteria, Staphylococci, Enterobacteriaceae, coliforms and Aerobic bacterial spores were (log CFU/cm2) 6.32, 4.43, 3.14, 2.98 and 3.78, respectively. However, mean counts of Yeasts and Moulds were below detectable levels. There was statistically significant variation (p<0.05) among the mean counts of microbes isolated from samples of paper currencies. The predominantly isolated microbial groups were Staphylococcus spp. (34.06%) followed by Bacillus spp. (31.88%), Enterobacteraceae (13.39%), Micrococcus spp. (9.55%) and Streptococcus spp. (9.03%). Overall, 25% and 10% of the samples were positive for S. aureus and Salmonella spp, respectively. In challenge study, Salmonella spp. and S. aureus reached the infective dose within 12 to 18 hours of inoculation. CONCLUSION: Thus, paper currencies could be considered as one of the possible vehicles for transmission of disease causing microorganisms. Poor handling practices and personal hygiene of the food vendors could contribute to the observed microbial counts. Thus, it calls for awareness development on the potential risks associated with poor handling of paper currencies at all level of the food establishments.

Synergistic activity of biocides and antibiotics on resistant bacteria from organically produced foods.

Synergism between biocides and antibiotics was investigated in 20 biocide and antibiotic-resistant bacterial strains that were previously isolated from organically produced foods, according to their antimicrobial resistance profiles. Most of the antibiotic/biocide combinations yielded synergistic interactions, reducing the inhibitory concentrations of biocides and antibiotics by 4- to 16-fold. Among enterococci, synergism with biocides was detected for amoxicillin (AM), cefuroxime (CX), erythromycin (EM), ciprofloxacin (CP), and trimethoprim/sulphametoxazol (T/S). Among staphylococci, interactions were synergistic (AM) and either synergistic or indifferent (CX and EM, depending on biocide). Among the three methicillin-resistant Staphylococcus aureus clinical strains included in the study, the combinations of methicillin and triclosan or hexachlorophene acted synergistically in all strains, but interactions were either synergistic or indifferent for the other biocides, depending on the strain. All combinations tested were synergistic for Lactobacillus (AM, CX, EM, and CP) and Micrococcus (AM, EM). In Salmonella, interactions were indifferent (AM, CX, EM, and CP) or synergistic (T/S). Synergism with biocides was also detected in Klebsiella isolates (AM, CX, and T/S), Enterobacter sp. (AM, CX, EM, and T/S), Pantoea (AM, CX, EM, CP, and T/S), and Chryseobacterium sp. (EM). These results suggest that combinations of biocides and antibiotics may open new possibilities to combat antimicrobial resistance.

Draft genome comparison of representatives of the three dominant genotype groups of dairy Bacillus licheniformis strains.

The spore-forming bacterium Bacillus licheniformis is a common contaminant of milk and milk products. Strains of this species isolated from dairy products can be differentiated into three major groups, namely, G, F1, and F2, using random amplification of polymorphic DNA (RAPD) analysis; however, little is known about the genomic differences between these groups and the identity of the fragments that make up their RAPD profiles. In this work we obtained high-quality draft genomes of representative strains from each of the three RAPD groups (designated strain G-1, strain F1-1, and strain F2-1) and compared them to each other and to B. licheniformis ATCC 14580 and Bacillus subtilis 168. Whole-genome comparison and multilocus sequence typing revealed that strain G-1 contains significant sequence variability and belongs to a lineage distinct from the group F strains. Strain G-1 was found to contain genes coding for a type I restriction modification system, urease production, and bacitracin synthesis, as well as the 8-kbp plasmid pFL7, and these genes were not present in strains F1-1 and F2-1. In agreement with this, all isolates of group G, but no group F isolates, were found to possess urease activity and antimicrobial activity against Micrococcus. Identification of RAPD band sequences revealed that differences in the RAPD profiles were due to differences in gene lengths, 3' ends of predicted primer binding sites, or gene presence or absence. This work provides a greater understanding of the phylogenetic and phenotypic differences observed within the B. licheniformis species.

Novel-porous-Ag0 nanocomposite hydrogels via green process for advanced antibacterial applications.

Silver nanoparticles (NPs) antibacterial characteristics were depends on its particle stabilization, particles size and nucleation agent. In this study, we report on green process of porous silver nanocomposite hydrogels for advanced antibacterial applications. The porous poly(acrylamide) (PAM) hydrogels were developed employing sucrose as porogenator. Silver NPs were nucleated with natural biomass Neem (Azadirachta indica) leaf extracts within the porous hydrogel networks. The formation of silver NPs in the porous hydrogels was confirmed by ultraviolet-visible spectroscopy, fourier transform infrared spectroscopy, X-ray diffraction, and thermo gravimetric analysis. Morphological studies done by scanning electron microscopy and transmission electron microscopy showed that the hydrogels were porous in nature and stabilization of NPs, size, and particles shape. The porous PAM silver nanoparticle hydrogels demonstrated excellent antimicrobial activity with significant effect against Escherichia coli, Micrococcus, and Candida albicus. Hence, it was clear that the developed hydrogels can be used effectively for preventing and treating infections.

Covalent immobilization of lysozyme on ethylene vinyl alcohol films for nonmigrating antimicrobial packaging applications.

The objective of this study was to develop a new antimicrobial film, in which lysozyme was covalently attached onto two different ethylene vinyl alcohol copolymers (EVOH 29 and EVOH 44). The EVOH surface was modified with UV irradiation treatment to generate carboxylic acid groups, and lysozyme was covalently attached to the functionalized polymer surface. Surface characterization of control and modified films was performed using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and dye assay. The value of protein loading after attachment on the surface was 8.49 µg protein/cm(2) and 5.74 µg protein/cm(2) for EVOH 29 and EVOH 44, respectively, after 10 min UV irradiation and bioconjugation. The efficacy of the EVOH-lysozyme films was assessed using Micrococcus lysodeikticus. The antimicrobial activity of the films was tested against Listeria monocytogenes and was similar to an equivalent amount of free enzyme. The reduction was 1.08 log for EVOH 29-lysozyme, 0.95 log for EVOH 44-lysozyme, and 1.34 log for free lysozyme. This work confirmed the successful use of lysozyme immobilization on the EVOH surface for antimicrobial packaging.

[Metabolism features of bacteria resistant to high concentrations of chromate].

Twenty strains of bacteria resistant to high concentrations of chromate were isolated from different ecological niches. They were able to reduce chromate to compounds of trivalent chromium--nonsoluble chromium hydroxide or soluble crystalline hydrates of trivalent chromium. The growth features of these microorganisms on media containing chromate at high concentrations (up to 20.0 g/l) are described. Besides chromate bacteria can reduce vanadate to compounds of V(4+) and Mo(6+) to Mo(5+). The best reduction takes place on the media where MPB. glucose or ethanol serves as the source of carbon. The growth and reduction of anion-in-study did not occur on organic acids. It was shown that tungstate, chlorate or perchlorate were not toxic for the studied bacteria up to concentrations of 10.0 g/l, however were not reduced by these microorganisms. The most active strains belong to genera Pseudomonas, Oerskovia, Bacillus, Micrococcus.

Changes in biogenic amine levels during storage of Mexican-style soft and Spanish-style dry-ripened sausages with different a(w) values under modified atmosphere.

Two raw sausages were prepared: a soft and a dry-ripened one, both by local traditional and industrial manufacturing practices. Sausages were packaged under a CO2/N2 atmosphere at different targeted activity water (aw) values: 0.96 and 0.92 (soft sausages) and 0.88 and 0.82 (dry-ripened sausages). Sausages were then stored at 5 °C for 42 days or at 12 °C for 240 days (soft and a dry-ripened sausages, respectively). The time-related changes in dominant microbiota, pH and biogenic amine contents during storage were determined. Tyramine was the most abundant biogenic amine in all the sausages. Biogenic amine levels were higher in dry-ripened sausages than in soft sausages at packaging. However, during refrigerated storage soft sausages were fermented and the levels of biogenic amines increased (P<0.05). At the end of storage, traditional soft sausages with 0.96 aw presented comparable levels of biogenic amines to traditional dry-ripened sausages.

Role of transition metals in UV-B-induced damage to bacteria.

The purpose of this study was to explore the possible link between metals and UV-B-induced damage in bacteria. The effect of growth in the presence of enhanced concentrations of different transition metals (Co, Cu, Fe, Mn and Zn) on the UV-B sensitivity of a set of bacterial isolates was explored in terms of survival, activity and oxidative stress biomarkers (ROS generation, damage to DNA, lipid and proteins and activity of antioxidant enzymes). Metal amendment, particularly Fe, Cu and Mn, enhanced bacterial inactivation during irradiation by up to 35.8%. Amendment with Fe increased ROS generation during irradiation by 1.2-13.3%, DNA damage by 10.8-37.4% and lipid oxidative damage by 9.6-68.7%. Lipid damage during irradiation also increased after incubation with Cu and Co by up to 66.8% and 56.5% respectively. Mn amendment decreased protein carbonylation during irradiation by up to 44.2%. These results suggest a role of Fe, Co, Cu and Mn in UV-B-induced bacterial inactivation and the importance of metal homeostasis to limit the detrimental effects of ROS generated during irradiation.

Putrescine biosensor based on putrescine oxidase from Kocuria rosea.

The novel putrescine oxidase based amperometric biosensor selectively measures putrescine, which can be considered as an indicator of microbial spoilage. Putrescine oxidase (PUOX, EC 1.4.3.10) was isolated from Kocuria rosea (Micrococcus rubens) by an improved and simplified purification process. Cells were grown on brain heart infusion medium supplemented with putrescine. Cell-free extract was prepared in Tris buffer (pH 8.0) by Bead-beater. A newly elaborated step based on three-phase partitioning (TPP) was applied in the purification protocol of PUOX. The purified enzyme was immobilized on the surface of a spectroscopic graphite electrode in redox hydrogel with horseradish peroxidase, Os mediator and poly(ethylene glycol) (400) diglycidyl ether (PEGDGE) as crosslinking agent. This modified working electrode was used in wall-jet type amperometric cell together with the Ag/AgCl (0.1M KCl) reference electrode and a platinum wire as auxiliary electrode in flow injection analysis system (FIA). Hydrogel composition, pH and potential dependence were studied. Optimal working conditions were 0.45 mLmin(-1) flow rate of phosphate buffer (66 mM, pH 8.0) and +50 mV polarizing potential vs. Ag/AgCl. The linear measuring range of the method was 0.01-0.25 mM putrescine, while the detection limit was 5 µM. Beer samples were investigated by the putrescine biosensor and the results were compared by those of HPLC reference method.

High-level expression of bioactive recombinant human lysozyme in the milk of transgenic mice using a modified human lactoferrin BAC.

Transgenesis has been used for expressing human lysozyme (hLZ) in the milk of livestock to improve their disease resistance. Here we describe a human lactoferrin (hLF) BAC as a candidate vector for high-level expression of hLZ in the milk of transgenic mice. Using recombineering, hLF genomic DNA in the hLF BAC was replaced by the hLZ gene (from the ATG start codon to the TAA stop codon), and flanking regions of the hLF gene (a 90-kb 5' and a 30-kb 3') were used as transcriptional control elements for hLZ expression. When this construct was used to generate transgenic mice, rhLZ was highly expressed in the milk of four transgenic mouse lines (1.20-1.76 g/L), was expressed at a lower level in one additional line (0.21 g/L). rhLZ from the milk of these transgenic mice exhibited the same antibacterial activity as native hLZ. Our results suggest a potential approach for producing large amounts of hLZ in the milk of livestock.