Characterization of Pseudomonas stutzeri NT-I capable of removing soluble selenium from the aqueous phase under aerobic conditions

Pseudomonas stutzeri strain NT-I was isolated from the drainage wastewater of a selenium refinery plant. This bacterium efficiently reduced selenate to elemental selenium without prolonged accumulation of selenite under aerobic conditions. Strain NT-I was able to reduce selenate completely at high concentrations (up to 10 mM) and selenite almost completely (up to 9 mM). In addition, higher concentrations of selenate and selenite were substantially reduced. Activity was observed under the following experimental conditions: 20–50°C, pH 7–9, and 0.05–20 g L^–1 NaCl for selenate reduction, and 20–50°C, pH 6–9, and 0.05–50 g L^–1 NaCl for selenite reduction. Under anaerobic conditions, selenate was reduced more rapidly, whereas selenite was not reduced at all. The high selenate- and selenite-reducing capability at high concentrations suggested that strain NT-I is suitable for the removal of selenium from high-strength industrial wastewater.     

Phenolic profiles of cultivated, in vitro cultured and commercial samples of Melissa officinalis L. infusions

Melissa officinalis L. (lemon balm) is normally consumed as an infusion and presents therapeutic properties, such as sedative, carminative and antispasmodic, also being included in some pharmaceutical preparations. The phenolic profiles of different samples of lemon balm, prepared as infusions, were evaluated by HPLC-DAD-ESI/MS. The profiles were compared in order to understand the differences between cultivated, in vitro cultured and commercial (bags and granulated) samples. All the samples showed a similar phenolic profile, presenting differences only in the quantities found of each compound. Rosmarinic acid was the most abundant compound, being higher in commercial samples, especially in tea bag sample (55.68 mg/g of infusion) and lower in in vitro cultured sample (15.46 mg/g). Moreover, dimers, trimers and tetramers of caffeic acid were identified and quantified for the first time in lemon balm. Only one flavonoid, luteolin-3′-O-glucuronide was found in all the samples, ranging from 8.43 mg/g in commercial granulate sample to 1.22 mg/g in in vitro cultured sample. Overall, cultivated and in vitro cultured samples presented the lowest amounts of phenolic compounds (59.59 and 30.21 mg/g, respectively); otherwise, commercial samples showed the highest contents (109.24 mg/g for tea bag and 101.03 mg/g for granulate sample). The present study shows that infusion of lemon balm can be a source of phenolic compounds, known for their bioactive effects.     

Enhancement of the nitrogen fixation efficiency of genetically-engineered Rhizobium with high catalase activity

The vktA catalase gene, which had been cloned from Vibrio rumoiensis S-1^T having extraordinarily high catalase activity, was introduced into the root nodule bacterium, Rhizobium leguminosarum bv. phaseoli USDA 2676. The catalase activity of the vktA-transformed R. leguminosarum cells (free-living) was three orders in magnitude higher than that of the parent cells and this transformant could grow in a higher concentration of exogenous hydrogen peroxide (H₂O₂). The vktA-transformant was inoculated to the host plant (Phaseolus vulgaris L.) and the nodulation efficiency was evaluated. The results showed that the nitrogen-fixing activity of nodules was increased 1.7 to 2.3 times as compared to the parent. The levels of H₂O₂ in nodules formed by the vktA-transformant were decreased by around 73%, while those of leghemoglobins (Lba and Lbb) were increased by 1.2 (Lba) and 2.1 (Lbb) times compared with the parent. These results indicated that the increase of catalase activity in rhizobia could be useful to improve the nitrogen-fixing efficiency of nodules by the reduction of H₂O₂ content concomitantly with the enhancement of leghemoglobins contents.     

Identification and epidemiological relationships of Aeromonas isolates from patients with diarrhea, drinking water and foods

A collection of Aeromonas isolates obtained over a three-year period in the same geographic area (León, NW of Spain) was characterized by (GTG)₅-PCR fingerprinting, amplified fragment length polymorphism (AFLP) analysis and gyrB gene sequence analysis. The isolates originated from human diarrheal stools (29 isolates), potable water (13 isolates), rabbit meat (13 isolates) and marine fish (5 isolates). The distribution of Aeromonas species varied with the strain source. Aeromonas caviae HG4 and Aeromonas media HG5 were predominant in clinical and water isolates, respectively, whereas motile Aeromonas salmonicida HG3 strains were most frequently found in fish and meat. Molecular typing revealed several genotypic relationships among specific isolate subsets: (i) two clones of A. media HG5 persisted in drinking water over the study period, (ii) different patients harbored identical or closely related clones during several months, and (iii) clonal relatedness was observed in two sets of water and human isolates. The first of these sets comprised nine water isolates and two human A. media HG5 isolates, whereas the other one included a water isolate and a human isolate of A. caviae HG4. The latter finding suggests that Aeromonas transmission in the studied region followed a waterborne route. Interestingly, the three human isolates closely related to water isolates were recovered in a period of four days in June 2006 from non-related patients without underlying medical conditions that tested negative for other enteric pathogens. The data imply the transmission through contaminated water of strains of the A. caviae group that can produce disease in humans.     

Short communication: Identification and technological characterization of yeast strains isolated from samples of water buffalo Mozzarella cheese

Sixty yeast cultures were isolated from samples of water buffalo Mozzarella, a popular “pasta filata” cheese, originating on 16 farms located in the provinces of Salerno, Caserta, and Frosinone (Italy). Strains were identified by means of 5.8S internal transcribed spacer rDNA PCR-RFLP combined with 26S rRNA gene partial sequencing and characterized for their ability to exert biochemical properties of technological interest. The recorded dominance of fermenting yeasts such as the lactose-fermenting Kluyveromyces marxianus (38.3% of the total isolates) and the galactose-fermenting Saccharomyces cerevisiae (21.6% of the total isolates) suggests that these yeasts contribute to the organoleptic definition of the water buffalo Mozzarella. The speciographic analysis revealed the presence of 7 other species rarely or never reported in a dairy environment belonging to the genera Pichia and Candida, whose role in Mozzarella cheese organoleptic properties need to be further investigated.     

Characterization for enterotoxin production,virulence factors,and antibiotic susceptibility of Staphylococcus aureus isolates from various foods in Portugal

Staphylococcus aureus represents a public health challenge worldwide. The aim of this study was the characterization of different food isolates of S. aureus on the basis of their production of enterotoxins, hemolysins and resistance to antibiotics. A total of 148 coagulase-positive staphylococcal strains isolated from different food origins were identified to the species level. By multiplex PCR, 69% of the isolates were shown to be enterotoxigenic (SEs); the most common were sea seg, sea seg sei and seg sei. According to CLSI [CLSI, Clinical and Laboratory Standards Institute, 2007. Performance Standards for Antimicrobial Susceptibility Testing; Fifteenth Informational Supplement. CLSI document M100-S15. Clinical and Laboratory Standards Institute, Wayne, PA], 38% of the isolates were resistant to oxacillin (≥6 μg/mL; MRSA positives) but only 0.68% showed the presence of mecA gene. 70 and 73% of the S. aureus strains were resistant to β-lactams, ampicillin and penicillin, respectively. The virulence pattern was demonstrated to be origin and strain dependent. These findings emphasise the need to prevent the presence of S. aureus strains and SEs production in foods.     

Antioxidant properties of methanolic extracts from Agaricus blazei with various doses of γ-irradiation

Agaricus blazei Murrill (Agariaceae) was irradiated with gamma rays at doses of 2.5, 5, 10, 15 and 20 kGy and the antioxidant properties of its methanolic extracts were studied. At 7.5 and 10.0 mg/ml, antioxidant activities of methanolic extracts from 2.5 to 20 kGy γ-irradiated A. blazei were significantly higher than those of methanolic extract from the nonirradiated control. At 0.5-7.5 mg/ml, reducing powers of methanolic extracts from A. blazei with 2.5, 10, 15 and 20 kGy of irradiation and without irradiation were comparable. All methanolic extracts showed excellent scavenging abilities of 95.2-100.7% against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals at 0.5 mg/ml. With regard to the scavenging ability against hydroxyl radicals, unirradiated and γ-irradiated A. blazei were comparable. Total phenols were the major naturally occurring antioxidant components found in the range of 18.77-21.48 mg/g. All EC₅₀ values were below 10 mg/ml, except values in reducing power, scavenging ability against DPPH radicals and chelating ability against ferrous ions were below 1 mg/ml. That indicates the unirradiated and irradiated A. blazei were good in antioxidant properties. Summarily, up to 20 kGy of irradiation did not remarkably affect the amounts of total antioxidant components in A. blazei.     

Inhibition of Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) development with essential oil extracted from Cymbopogon schoenanthus L. Spreng. (Poaceae), and the wasp Dinarmus basalis (Rondani) (Hymenoptera: Pteromalidae)

The control of the development of Callosobruchus maculatus was studied using a method that combined exposure to essential oil extracted from Cymbopogon schoenanthus and the introduction of a pteromalid natural enemy of the bruchid, Dinarmus basalis. The effect of the essential oil used was evaluated on all developmental stages of C. maculatus and on adults of D. basalis. At the highest concentration tested (33.3 μl/l) all adults of C. maculatus were killed within 24 h of exposure to the oil and the development of newly laid eggs and neonate larvae was also inhibited. However, the oil had variable efficacy against the bruchid instars developing inside the seeds: 5-day-old larvae (63% LI and 37% LII) of C. maculatus developing inside the seeds proving to be highly susceptible while 15-day-old insects (84% of pupae and 16% of larvae) were tolerant. Under the same conditions (33.3 μl/l), adults of D. basalis were very susceptible to oil vapours and to the residual activity of the oil after 3 or 6 days. However, the introduction of 10 pairs of adult D. basalis into a jar containing 100 hosts aged 10 days, 3 or 6 days before the oil application, gave respectively an emergence of 26 or 18 adults of the parasitoid compared to 28 in the control and there was no adult emergence of the host. The possibility of an integrated pest management strategy by using allelochemicals such as essential oils and indigenous natural enemies to control C. maculatus development in cowpea stocks is discussed.     

Evaluation of reducing power and radical scavenging activities of water and ethanol extracts from sumac (Rhus coriaria L.)

The purpose of this study was to determine the reducing power, metal chelating, and radical scavenging capabilities of water and ethanol extracts of sumac (Rhus coriaria L.), comparatively. The water and ethanol extracts of sumac were evaluated for their radical scavenging activities by means of the DPPH and DMPD assays. Water extract of sumac (R. coriaria L.) scavenged radicals effectively with EC₅₀ values of 36.4 ??g/ml for DPPH free radical and 44.7 ??g/ml for DMPD cation radical. Similarly, the total reducing power of water extract was found higher than ethanol extract in both potassium ferricyanide reduction (FRAP) and cupric ions reduction capacity methods (CUPRAC). 2,2′-Bipyridine was used to determine the metal chelating activity and the result of water extract was found higher than ethanol extract. Total phenolic and total flavonoid contents of both extracts were studied as well. The values of water extract were found to be higher than that of ethanol extract. The present study found that water extracts of sumac (R. coriaria L.) have effective antioxidant and radical scavenging activities as compared to ethanol extracts.     

Prevalence of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) in food samples associated with foodborne illness in Alberta, Canada from 2007 to 2010

Consumption of foods containing Staphylococcus aureus can cause severe gastro-intestinal illness. Given the fact that over the past decade, Canada has seen increasing rates of methicillin-resistant S. aureus (MRSA) carriage and infection, the objective of this study was to investigate the impact of methicillin-susceptible S. aureus (MSSA) and MRSA on foodborne illness in Alberta, Canada. Between January 2007 and December 2010, there were 693 food samples associated with foodborne investigations submitted to the Alberta Provincial Laboratory for Public Health (ProvLab). These foods were screened for: Bacillus cereus, Clostridium perfringens, S. aureus, Aeromonas spp., Campylobacter spp., Escherichia coli O157:H7, Salmonella, Shigella spp., and Yersinia spp. S. aureus was identified in 10.5% (73/693) of samples, and of these, 59% (43/73) were co-contaminated with at least one other organism on the screening panel. The S. aureus positive samples included 29 meat, 20 prepared foods containing meat, 11 prepared foods not containing meat, 10 dairy, and three produce. Methicillin-resistance was not detected in any isolates tested. These findings indicate that the presence of S. aureus in food associated with foodborne investigations is a cause for concern, and although MRSA was not found, the potential for outbreaks exists, and ongoing surveillance should be sustained.     

Effects of various solvents on the extraction of antioxidant phenolics from the leaves, seeds, veins and skins of Tamarindus indica L.

The effects of solvents, of varying polarities, on the extraction of antioxidant phenolics from the leaves, seeds, veins and skins of Tamarindus indica (T. indica) were studied. The efficiencies of the solvents for extraction of the antioxidant phenolics were in the order: methanol > ethyl acetate > hexane. Phenolic content ranged from 3.17 to 309 mg of gallic acid equivalents/g. Methanol leaf extract (MEL) had the highest phenolic content and was the most potent scavenger of DPPH and superoxide radicals. Methanol vein extract had the highest ferric reducing activity whereas methanol seed extract was the most potent ABTS radical-scavenger. A positive correlation existed between phenolic content and antioxidant activities of the plant parts. HPLC analyses of MEL revealed the presence of catechin, epicatechin, quercetin and isorhamnetin. Overall, methanol was the most effective solvent for extraction of antioxidant phenolics from T. indica. T. indica, particularly the leaf, can be a useful source of natural antioxidants.     

Phytochemical composition and antioxidant capacity of various botanical parts of the fruits of Prunus × domestica L. from the Lorraine region of Europe

Mirabelle plums represent a famous fruit from the Lorraine region, however little is known about their phytochemical composition. The oil of the fruit contained predominantly oleic acid (59%) and linoleic acid (29%). The total content of phenolic antioxidants in the whole fruits was 5.338 g/kg with 456 mg/kg (9%), 701 mg/kg (13%) and 4159 mg/kg (78%) detected in the peels, flesh and pits respectively. The peels contained solely 3,4-dihydroxybenzoic acid (270 mg/kg) and rutin (186 mg/kg), the flesh exclusively echinoids (723 mg/kg), whereas the pits contained a rich variety of phenolic compounds (4.2 g/kg) dominated by amygdalin (3.8 g/kg), but with significant contributions from vanillin (102 mg/kg), guajacyl-glycerin-coniferyl aldehyde isomers (87 mg/kg), dehydro-diconiferyl aldehyde (52 mg/kg), and vanillin diglucoside (48 mg/kg). Of the major phenolic compounds tested across a range of in vitro assays, rutin was the superior antioxidant.     

Antioxidant properties of methanolic extracts from Antrodia camphorata with various doses of γ-irradiation

The mycelia of Antrodia camphorata (Chang & Chou) Wu, Ryvarden & Chang were irradiated with gamma rays at doses of 2.5, 5, 10, 15 and 20 kGy and the antioxidant properties of the methanolic extracts were studied. At 2.5 mg/ml, antioxidant activities of methanolic extracts from 10 to 20 kGy-irradiated mycelia were significantly higher than those of the non-irradiated control. Reducing powers of methanolic extracts from unirradiated and 0.5–7.5 kGy-irradiated mycelia were comparable except for the 20 kGy-irradiated mycelia. At 2.5 mg/ml, all methanolic extracts showed excellent scavenging abilities of 92.3–103% on DPPH radicals. Scavenging abilities of methanolic extracts from 2.5 to 20 kGy irradiated mycelia were better than that of the unirradiated control at 10 mg/ml. With irradiation at 5–20 kGy, mycelia possessed higher chelating ability on ferrous ions than did the unirradiated control. The EC₅₀ values were below 15 mg/ml, except for values of scavenging ability of the unirradiated control on hydroxyl radicals. Total phenols were the major naturally occurring antioxidant components found in the range of 13.0–15.5 mg/g. In summary, γ-irradiation not only maintained the antioxidant properties of mycelia but also enhanced the antioxidant properties to some extent.     

Characterization of pLAC1, a cryptic plasmid isolated from Lactobacillus acidipiscis and comparative analysis with its related plasmids

The pLAC1 plasmid of Lactobacillus acidipiscis ACA-DC 1533, a strain isolated from traditional Kopanisti cheese, was characterised. Nucleotide sequence analysis revealed a circular molecule of 3478 bp with a G + C content of 37.2%. Ab initio annotation indicated four putative open reading frames (orfs). orf1 and orf4 were found to encode a replication initiation protein (Rep) and a mobilization protein (Mob), respectively. The deduced products of orf2 and orf3 revealed no significant homology to other known proteins. However, in silico examination of the plasmid sequence supported the existence of a novel operon that includes rep, orf2 and orf3 in pLAC1 and that this operon is highly conserved also in plasmids pLB925A02, pSMA23, pLC88 and pC7. RT-PCR experiments allowed us to verify that these three genes are co-transcribed as a single polycistronic mRNA species. Furthermore, phylogenetic analysis of pLAC1 Rep and Mob proteins demonstrated that they may have derived from different plasmid origins, suggesting that pLAC1 is a product of a modular evolution process. Comparative analysis of full length nucleotide sequences of pLAC1 and related Lactobacillus plasmids showed that pLAC1 shares a very similar replication backbone with pLB925A02, pSMA23 and pLC88. In contrast, mob of pLAC1 was almost identical with the respective gene of plasmids pLAB1000, pLB4 and pPB1. These findings lead to the conclusion that pLAC1 acquired mob probably via an ancestral recombination event. Our overall work highlights the importance of characterizing plasmids deriving from non-starter ‘wild’ isolates in order to better appreciate plasmid divergence and evolution of lactic acid bacteria.     

Enrichment of stirred yogurt with soluble dietary fiber from Pachyrhizus erosus L. Urban: Effect on syneresis, microstructure and rheological properties

Yam soluble fiber (YSF) extracted from Pachyrhizus erosus was added (1 g per 100 mL) to a stirred yogurt (SY_YSF). Its syneresis and microstructure properties were evaluated and compared to those of a stirred yogurt (SY_C) without added YSF. The SY_C yogurt exhibited a more compact casein micelle aggregates network than that of the SY_YSF yogurt which was more open, relaxed and covered with fibrous structures attributed to the YSF components. The rheological analysis showed that the SY_YSF yogurt had lower storage modulus (G′) and loss modulus (G″) values in the linear viscoelastic region than the SY_C yogurt, but its flow behavior was characterized by a lower flow index (n), higher consistency index (k), and higher yield stress (τ₀) than the SY_C yogurt. Incorporation of the YSF reduced significantly the syneresis and produced a more acceptable mouthfeel in the SY_YSF yogurt in comparison to the SY_C yogurt, indicating the viability of the process to obtain a commercial product.     

Characterization of NAD-dependent alcohol dehydrogenase enzymes of strawberry's achenes (Fragaria x ananassa cv. Elsanta) and comparison with respective enzymes from Methylobacterium extorquens

Achenes were isolated from strawberries fruits, while the methylotroph Methylobacterium extorquens (strain with CABI registration number IMI 369321), which has been isolated from strawberry (Fragaria x ananassa cv. Elsanta) callus cultures, was grown on a mixture of methanol (0.25% v/v) and 1,2-propanediol (0.75% v/v). The Alcohol Dehydrogenase (ADH) enzymatic activities of the achenes were assessed and the optimum pH for ADH activity was found to be pH 10.0. Enzyme assays were carried out in order to define the best substrate specificity at pH = 10.0. The best substrates were found to be ethanol (Km = 5.950 mM) and methanol (Km = 12.610 mM). Only enzymes from the bacterium showed capability of using aldehydes - especially formaldehyde - as substrates. A wide variety of metals as well as EDTA and NaN₃ were shown to decrease the enzymatic activity. Furthermore, SDS-PAGE Electrophoresis experiments showed Molecular Weight of 47.0 kDa for the Alcohol Dehydrogenase from achenes of strawberries (Fragaria x ananassa). Additional experiments were conducted in order to define certain thermodynamic properties of the enzymes, by using the dehydrogenation activities of these three enzyme sources which were calculated by measuring the absorption of NADH at 340 nm, in the Arrhenius equation.