Biodiversity of the microbial community in a Spanish farmhouse cheese as revealed by culture-dependent and culture-independent methods

The microbial diversity within Alberquilla cheese, made from a spontaneously fermented mixture of raw goats' and sheep's milk in the Alpujarra mountains (Granada, south-east Spain), has been studied by the classical culturing method and also by molecular analysis of community DNA. A collection of 206 isolates was obtained from the cheese on different selective/differential media, which were then re-grouped to 52 after randomly amplified polymorphic DNA (RAPD)-PCR analyses. Isolates on Man-Rogosa and Sharpe-agar (MRS), M17-glucose agar and Kenner Fecal (KF)-agar medium were identified by specific PCR or 16S rRNA gene sequencing and belonged mainly to the lactic-acid bacteria group. The predominant genus was Lactobacillus, which accounted for more than 50% of the isolates, the most abundant species being Lactobacillus paracasei, followed by considerably less quantities of Lb. plantarum and Lb. brevis. Other lactic-acid bacteria identified were Pediococcus urinaequi, Leuconostoc pseudomesenteroides, Leuc. mesenteroides, Lactococcus lactis and even the enterococci Enterococcus faecium and E. devriesei. Cluster analyses of RAPD-PCR patterns revealed a high degree of diversity among the lactobacilli. The Gram-negative bacterial strains belonged mainly to Hafnia alvei species. The microbes occurring in Alberquilla cheese were also studied by PCR temporal temperature-gradient gel electrophoresis (TTGE) of the 16S rRNA V3 region and partial 16S rRNA sequencing of the TTGE bands. The results showed a major presence of lactic-acid bacteria closely related to Lc. lactis, Lb. paracasei, Lb. plantarum, Lb. brevis, Lb. acidophilus and Enterococcus sp. The non-lactic-acid bacterium detected was identified as Escherichia coli. All the Enterococcus strains showed great susceptibility to the most clinically relevant antibiotics, harbouring only the virulence gene efaAfm. On the basis of their antimicrobial activity against Listeria monocytogenes we chose two strains of Ln. mesenteroides that produced mesenterocin B105 and mesenterocin Y105, as revealed by PCR techniques.     

Modeling the effects and uncertainties of contaminated sediment remediation scenarios in a Norwegian fjord by Markov chain Monte Carlo simulation

Multimedia environmental fate models are useful tools to investigate the long-term impacts of remediation measures designed to alleviate potential ecological and human health concerns in contaminated areas. Estimating and communicating the uncertainties associated with the model simulations is a critical task for demonstrating the transparency and reliability of the results. The Extended Fourier Amplitude Sensitivity Test(Extended FAST) method for sensitivity analysis and Bayesian Markov chain Monte Carlo (MCMC) method for uncertainty analysis and model calibration have several advantages over methods typically applied for multimedia environmental fate models. Most importantly, the simulation results and their uncertainties can be anchored to the available observations and their uncertainties. We apply these techniques for simulating the historical fate of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in the Grenland fjords, Norway, and for predicting the effects of different contaminated sediment remediation (capping) scenarios on the future levels of PCDD/Fs in cod and crab therein. The remediation scenario simulations show that a significant remediation effect can first be seen when significant portions of the contaminated sediment areas are cleaned up, and that increase in capping area leads to both earlier achievement of good fjord status and narrower uncertainty in the predicted timing for this.     

The bioavailability of an omega‐3‐rich algal oil is improved by nanoemulsion technology using yogurt as a food vehicle

Global trends show that habitual omega‐3 intakes are short of recommended guidelines, particularly among vegetarians. Subsequently, alternative dietary sources of long‐chain omega‐3 polyunsaturated fatty acids (LC3PUFA) from vegetarian sources are needed. Food technology methods are advancing, and nanoemulsion technologies have improved the bioavailability of certain lipid‐based nutrients. This study examined whether ingestion of an omega‐3 algal oil nanoemulsion led to improved bioavailability compared to the bulk oil. Eleven subjects completed a single‐blind, randomised crossover trial, with a 21‐day washout between interventions. Results demonstrated that LC3PUFA absorption from the nanoemulsion was significantly higher than the bulk oil. Percentage blood fatty acids were significantly increased for docosahexaenoic acid (DHA; P ≤ 0.05) while LC3PUFA: PUFA ratios increased (P ≤ 0.05) and omega‐6:omega‐3 ratios were reduced (P = 0.028). Larger and longer intervention studies are now needed, but these preliminary findings demonstrate that nanoemulsion technology may improve the absorption of omega‐3 fatty acids.     

Lipid rafts in the bovine milk fat globule membrane revealed by the lateral segregation of phospholipids and heterogeneous distribution of glycoproteins

This study reveals the lateral organisation of the milk fat globule membrane (MFGM). Using confocal laser scanning microscopy (CLSM) and a lipid soluble molecule, an exogenous phospholipid and two lectins as fluorescent probes we located triacylglycerols in the core of fat globules and investigated the organisation of the polar lipids and glycoproteins of the MFGM, in situ in milk. Lipid rafts corresponding to the lateral segregation of sphingolipids in liquid-ordered phases surrounded by liquid-disordered domains composed by the glycerophospholipids were observed in the MFGM. These lipid rafts which correspond to rigid sphingolipid-rich domains have a circular shape at room temperature. CLSM experiments revealed that glycoproteins and glycolipids are heterogeneously distributed around fat globules and that they are not located in the lipid rafts. The characterisations performed by in depth thin sectioning of fat globules and in dynamic as a function of time revealed chemical and structural heterogeneities in the MFGM. Schematic 3D and 2D representations of the MFGM are proposed and discussed. The physiological and nutritional consequences of the lateral organisation of polar lipids and glycoproteins in the MFGM are discussed but remain to be elucidated.     

Measurement of dissolved reactive phosphorus using the diffusive gradients in thin films technique with a high-capacity binding phase

Measurement of dissolved reactive phosphorus (DRP) by the diffusive gradients in thin films (DGT) technique was investigated using a new binding phase. Half-dried amorphous zirconium oxide (with 50 ± 5% of water content) was mixed with acrylamide solution for the preparation of the new binding phase. The resulting binding gel had a high binding capacity (223 μg P cm(-2)) for phosphate. The solution of NaOH (1 M) was used for elution of phosphate from the gel, and an elution efficiency of 0.95 was obtained. A test of DGT uptake with this gel showed its dependence on temperature, and there was no influence of pH (3 to 10) and ionic strength (10 nM to 0.1 M). Its capacity for DGT response exceeded 100 μg P cm(-2), corresponding to a DRP concentration of more than 20 mg L(-1) for a 24 h deployment with a standard DGT device at 25 °C, which was at least 50 times of the Fe-oxide gel commonly used in the present DGT technique. Measurements with this high-capacity DGT technique in a laboratory microcosm of homogeneously mixed sediments gave smooth and reproducible mass-depth profiles. This technique was well demonstrated by in situ measurements in algal- and macrophyte-dominated regions of Lake Taihu. The DGT-measured concentrations of DRP were on average 20% and 40% of the DRP concentrations in pore waters, respectively, indicating a partial resupply of the sediments to the pore waters with DRP.     

Surfactants as bubble surface modifiers in the flotation of algae: dissolved air flotation that utilizes a chemically modified bubble surface

In this paper we present an investigation into the use of bubbles modified with surfactants in dissolved air flotation (DAF). Bubble modification was investigated by dosing surfactants of varying character into the saturator of a DAF unit in turn. The cell removal efficiency only improved when using a cationic surfactant where optimum removal of Microcystis aeruginosa cells was obtained when using 0.0022-0.004 mequiv L(-1) surfactant. However, the magnitude of the removal differed according to the hydrophobicity of the surfactant. Typically, the more efficiently the surfactant adsorbed at the bubble interface, the better the removal efficiency. When the dose to saturator ratio was kept constant and the recycle ratio varied, the removal efficiency improved with increasing recycle ratio, reaching a maximum removal efficiency of 87% for M. aeruginosa. This value was comparable with that predicted by a theoretical model. The bubble collection efficiency of a maximum of two cells per bubble was constant irrespective of the influent cell number or recycle ratio. Treatment of additional species in this way revealed a relationship between increasing size and both increasing removal efficiency and decreasing surfactant dose, which is supported by theoretical relationships.     

High-resolution, two-dimensional measurement of dissolved reactive phosphorus in sediments using the diffusive gradients in thin films technique in combination with a routine procedure

Dissolved reactive phosphorus (DRP) is the most available P form in sediments and often directly controls phytoplankton blooms in aquatic systems. In this study, a novel procedure was developed for two-dimensional (2D) measurement of DRP in sediments at a spatial resolution of 0.45 mm using the diffusive gradients in thin films (DGT) technique with a revised high-capacity binding phase (Zr oxide gel). This procedure involves DGT uptake of P in sediments, 2D slicing of the binding gel on a 0.45 × 0.45-mm grid system, elution of P from each gel square with 1 M NaOH, and microcolorimetric determination of DRP in each eluted solution using 384-microwell plates. Measurements of DRP via this procedure were tested in homogeneous solutions and sediments and produced an acceptable error (<20% relative standard deviation) for the analysis once the accumulated mass of P in each gel square reached 1.2 μg cm(-2). This method was successfully applied to produce 2D images of the DRP distribution in sediments with and without the influence of tubificid worm bioturbation, revealing a much more pronounced and localized impact from tubificid worms than that found using a one-dimensional measurement of pore water DRP concentrations at 1-cm resolution.     

Formulation and development of a patch containing tamarind fruit extract by using the blended chitosan-starch as a rate-controlling matrix

A cosmetic patch containing tamarind fruit extract was formulated and developed by blending two types of natural polymers: chitosan with molecular weight of 100 000 and starch such as corn, potato or tapioca starch. The physicochemical characteristics, i.e. flexibility, colour, transparency, integrity, gloss, water sorption and bioadhesion property and the stability of the patch without tamarind content were investigated. Stability test was performed by keeping the prepared patches at 4 degrees C, at room temperature or at 45 degrees C for 2 weeks. The results showed that the formulations composed of chitosan:corn starch ratio of 4.5 : 0.5 (CC(4.5 : 0.5)) and chitosan:tapioca starch ratios of 4.5 : 0.5 (CT(4.5 : 0.5)) and 4.0 : 1.0 (CT(4 : 1)) provide patches with favourable physical characteristics, high water sorption, good bioadhesion ability and good stability. After the lyophilized tamarind extract in an amount corresponding to 5% of tartaric acid was incorporated into the formulations of CC(4.5 : 0.5), CT(4.5 : 0.5) and CT(4 : 1), the ability of the patches to adhere to skin was improved. However, after keeping the test patches at room temperature or at 45 degrees C for 6 weeks, their colours were intensified while their flexibilities and skin adhesion properties decreased. A 12-h in vitro permeation was investigated by studying the cumulative amount of tartaric acid permeated through the Silastic membrane (Dow-Coming, Midland, MI, USA). The CC(4.5 : 0.5) patch tended to give the highest amount of tartaric acid released. The release pattern of all the blended polymeric matrices was exhibited in two distinct phases: the rapid phase, where the flux averaged 3.61 microg min(-1) mm(-2); and the slow phase, where the flux averaged 1.89 microg min(-1) mm(-2).     

Determination of polychlorinated dibenzo-p-dioxins and dibenzofurans and comparison of extraction methods for edible freshwater fish and frogs in South Korea by using a high-resolution GC/MS.

Three different extraction methods were evaluated: Soxhlet extraction, accelerated solvent extractor extraction and alkali digestion with high-resolution gas chromatography/high-resolution mass spectrometry, for the analysis of edible freshwater fish and frogs. The recovery efficiencies for the different extraction methods were also compared. Using these extraction methods, the concentrations and spatial distributions of these compounds in freshwater fish and frogs (n = 99), collected from 31 locations on four major river systems (Han, Nakdong, Kum and Youngsan) and two wetlands in South Korea, were examined. The concentrations of total polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in all the freshwater fish and frog samples were analysed. The toxic equivalency (TEQ) concentration of PCDD/Fs in fish ranged from 0 (n.d.) to 1.309 pg I-TEQ g(-1) wet weight. Detailed concentrations for different types of fish were as follows (pg I-TEQ g(-1) wet weight): 0 (n.d.)-1.309 for crucian (mean 0.331, n = 28), 0.004-0.743 for carp (0.244, n = 10), 0.004-0.592 for dace (0.198, n = 8), 0.187-0.400 for cornet fish (0.287, n = 3), 0.164-0.410 for Korean piscivorous chub (0.303, n = 6), 0.105-0.516 for bass (0.310, n = 2), 0.451-0.812 for mullet (0.631, n = 2), 0.082 for long-nosed barbell, 0.100 for common Korean bitterling and 0.995 for dark chub. The total TEQ concentration ranged from 0 (n.d.) to 1.258 pg I-TEQ g(-1) wet weight for the two kinds of frogs (n = 36) investigated: 0 (n.d.)-1.258 pg I-TEQ g(-1) (mean 0.346, n = 29) for the bullfrog and from 0 (n.d.)-0.474 pg I-TEQ g(-1) (0.185, n = 7) for the mountain frog.     

Flushing history as a hydrogeological control on the regional distribution of arsenic in shallow groundwater of the Bengal Basin

Whereas serious health consequences of widespread consumption of groundwater elevated in As have been documented in several South Asian countries, the mechanisms responsible for As mobilization in reducing aquifers remain poorly understood. We document here a previously unrecognized and consistent relationship between dissolved As concentrations in reducing groundwater and the phosphate-mobilizable As content of aquifer sediment for a set of precisely depth-matched samples from across Bangladesh. The relationship holds across nearly 3 orders of magnitude in As concentrations and suggests that regional as well as local patterns of dissolved As in shallow groundwater are set by the solid phase according to a remarkably constant ratio of approximately 250 microg/L dissolved As per 1 mg/kg P-mobilizable As. We use this relationship in a simple model of groundwater recharge to propose that the distribution of groundwater As in shallow aquifers of the Bengal Basin could primarily reflect the different flushing histories of sand formations deposited in the region over the past several thousand years.     

In search of a global model of cultivation: using remote sensing to examine the characteristics and constraints of agricultural production in the developing world

In most developing countries, people are heavily reliant on inexpensive, locally grown food. However, while dependence on cropping crosses national and continental boundaries, the selection of land for cropping has adapted to the available conditions. Recent analyses conducted by the Famine Early Warning System Network (FEWS NET) show that the characteristics of cropped area differ in different countries, indicating that the critical variables influencing the selection of location for the establishment of agriculture also vary. This study looks at a selection of FEWS NET work using high resolution remotely sensed imagery to analyze cropped areas in Afghanistan, Eritrea, Guatemala, Haiti, Mali, Mozambique, South Sudan, Burkina-Faso and Tajikistan. This analysis identifies similarities and differences in the significant factors impacting cropped area in each country. Furthermore, the effectiveness of the application of high-resolution imagery to estimating cultivation is assessed. The results highlight the context-specific nature of cultivation and the effectiveness of very high-resolution satellite imagery for crop estimation. The results also suggest that a single, generally applicable model of cultivation will require complex interactions between economic, governmental and population characteristics in addition to local landscape/geophysical properties.     

Sugars and amino acids as factors affecting the synthesis of fumonisins in liquid cultures by isolates of the Gibberella fujikuroi complex

The capacity of four isolates belonging to the Gibberella fujikuroi complex to produce fumonisin B1 and fumonisin B2 when grown in liquid medium supplemented with one sugar and one amino acid at various concentration levels has been investigated. The sugars used for supplementing the medium were glucose, fructose, rhamnose, sucrose, maltose, and trehalose at 5, 10 or 20 g/l. The amino acids used were serine, threonine, glutamic acid, aspartic acid, valine, isoleucine, methionine, glycine, alanine, and cystine at 1 or 10 g/l. Fumonisins were extracted from culture filtrates, purified by SAX column and determined by reversed-phase C18 HPLC with fluorescence detection of the o-phthaldialdehyde derivatives. Two isolates produced very low concentrations of fumonisins with all sugars. The remaining isolates provided increased contents of fumonisins when sugar level increased. Concerning the amino acids, production of fumonisins was also dependent on the isolate, although at 1 g/l, the production of fumonisins was greater than at 10 g/l. The results indicate that the sugar-amino acid-isolate combination is basic in fumonisin biosynthesis and that the particular behaviour of each isolate in the different nutritional conditions may constitute a piece of interesting information in the fields of the Taxonomy, Physiology and Toxicology of these fungi. This is the first report on the influence of the carbon and nitrogen sources on fumonisin production by isolates of the G. fujikuroi complex.     

Observation of the distribution of Zn protoporphyrin IX (ZPP) in Parma ham by using purple LED and image analysis

We investigated the distribution of Zn protoporphyrin IX (ZPP) in Parma ham by using purple LED light and image analysis in order to elucidate the mechanism of ZPP formation. Autofluorescence spectra of Parma ham revealed that ZPP was present in both lean meat and fat, while red emission other than that of ZPP was hardly detected. Although ZPP was found to be distributed widely in Parma ham, it was more abundant in intermuscular fat and subcutaneous fat than in lean meat. The intensity of red emission was weak in muscles that were exposed during the processing. ZPP in both lean meat and subcutaneous fat tended to be more abundant in the inner region than in the outer region. It was thought that ZPP is transferred from lean meat to fat tissue during the processing, resulting in the small amount of ZPP in the lean meat adjacent to subcutaneous fat. Our results led to a completely new hypothesis that ZPP is formed in lean meat and transferred to fat tissue.     

Changes in the calcium cluster distribution of ultrafiltered and diafiltered fresh skim milk as observed by Small Angle Neutron Scattering

The effect of ultrafiltration and diafiltration on the distribution of the calcium phosphate clusters of the casein micelle was investigated using Small Angle Neutron Scattering (SANS). In the case of ultrafiltration, fresh skim milk was subjected to concentration using membrane filtration up to 5× its original volume, the retentate was rediluted with its corresponding serum and subsequently dialyzed against reconstituted milk powder dispersed in D2O/H2O (UF 5×(D)). In the case of diafiltered samples, the samples were concentrated adding water (diafiltration) at two different levels (DF 2·5× or DF 5×) and then redispersed with D2O/H2O. In the DF 5× case, the serum components were diluted to less than 1% of their original concentration. For analysis, all samples had the same volume fraction of dispersed casein micelles (φ=0·1), which is that of the control, unprocessed skim milk. A peak in the SANS data was observed in fresh skim milk at a scattering vector, qo, of 0·034 ?-1 (directly proportional to the reciprocal characteristic length), in agreement with previous literature results. Neutron data on the ultrafiltered, UF 5×(D) and diafiltered, DF 2·5× and DF 5× milk samples showed a progressive decrease in the intensity of the peak but invariance in qo. These results, combined with the determination of soluble and insoluble calcium in the samples, suggest a progressive and irreversible removal of calcium from within the micelle during membrane filtration of milk. Using SANS it was possible to clearly show changes in the micellar calcium clusters that may not otherwise be fully determined by only measuring the amount of total and insoluble calcium in milk.     

Community structure, abundance, and in situ activity of nitrifying bacteria in river sediments as determined by the combined use of molecular techniques and microelectrodes

The community structure, spatial distributions, and in situ activity of ammonia-oxidizing bacteria (AOB) representing the Betaproteobacteria and nitrite-oxidizing bacteria (NOB) representing the genus Nitrospira in three different river sediments with different pollution sources and levels along the Niida River, Hachinohe, Japan, were investigated by the combined use of 16S rRNA gene-cloning analysis, real-time quantitative polymerase chain reaction (RTQ-PCR) assays, and microelectrodes. The goal of this research was to evaluate the contribution of nitrifying activity in the sediment to the overall nitrogen elimination rate in this river. The 16S rRNA gene-cloning analysis revealed that the community structures of AOB and Nitrospira-like NOB are present in three sediments. On the basis of the results of 16S rRNA gene-cloning analysis, the RTQ-PCR assay using a TaqMan probe was developed and optimized for the quantification of the Nitrospira-like NOB. In the sediments, AOB specific 16S rRNA genes were detected in the range of 10(6) to 10(7) copies/cm3 and evenly distributed over the sampled sediment depth (0-5 mm), whereas the Nitrospira-like NOB 16S rRNA gene copy numbers per cm3 were 1-2 orders of magnitude higher than the AOB copy numbers. Under light conditions, intensive oxygenic photosynthesis occurred in the surface and increased the maximal O2 concentration and O2 penetration depth in all sediments. This concomitantly stimulated nitrifying bacteria present in diurnally anoxic deeper zones and expanded nitrification zones, which consequently increased the total NH4+ consumption rate in the sediment (i.e., total NH4+ flux into the sediment). The results suggested that the in situ nitrifying activity was restricted mainly to the surface 2 mm of the sediment and linked with photosynthetic activity, which obviously plays an important role in nitrogen elimination in this river.     

Changes in rumen microbiota composition and in situ degradation kinetics during the dry period and early lactation as affected by rate of increase of concentrate allowance

Changes in rumen microbiota and in situ degradation kinetics were studied in 12 rumen-cannulated Holstein Friesian dairy cows during the dry period and early lactation. The effect of a rapid (RAP) or gradual (GRAD) postpartum (pp) rate of increase of concentrate allowance was also investigated. Cows were fed for ad libitum intake and had free access to a mixed ration consisting of chopped wheat straw (dry period only), grass silage, corn silage, and soybean meal. Treatment consisted of either a rapid (1.0 kg of dry matter/d; n = 6) or gradual (0.25 kg of dry matter/d; n = 6) increase of concentrate allowance (up to 10.9 kg of dry matter/d), starting at 4 d pp. In whole rumen contents, bacterial community composition was assessed using samples from 50, 30, and 10 d antepartum (ap), and 3, 9, 16, 30, 44, 60, and 80 d pp, and protozoal and archaeal community composition using samples from 10 d ap, and 16 and 44 d pp. Intake of fermentable organic matter, starch, and sugar was temporarily greater in RAP than GRAD at 16 d pp. Bacterial community richness was higher during the dry period than during the lactation. A rapid increase in concentrate allowance decreased bacterial community richness at 9 and 16 d pp compared with a gradual increase in concentrate allowance, whereas from 30 d pp onward richness of RAP and GRAD was similar. In general, the relative abundances of Bacteroidales and Aeromonadales were greater, and those of Clostridiales, Fibrobacterales, and Spirochaetales were smaller, during the lactation compared with the dry period. An interaction between treatment and sampling day was observed for some bacterial community members, and most of the protozoal and archaeal community members. Transition to lactation increased the relative abundance of Epidinium and Entodinium, but reduced the relative abundance of Ostracodinium. Archaea from genus Methanobrevibacter dominated during both the dry period and lactation. However, during lactation the abundance of the methylotrophic Methanomassiliicoccaceae and Methanosphaera increased. The in situ degradation of organic matter, neutral detergent fiber, starch, and crude protein was neither affected by treatment nor by transition from the dry period to lactation. Results show that the composition of the rumen microbiota can change quickly from the dry period to the lactation period, in particular with a rapid increase in fermentable substrate supply postpartum, but this was not associated with changes in rumen degradation kinetics.