Fish and seafood traceability based on AFLP markers: Elaboration of a species database

Several sociological, health and conservation arguments request a correct labelling of seafood products. Nowadays, molecular genetics is a useful tool for food chain traceability, particularly in regards to species identification. Among the variety of PCR-based molecular markers, AFLPs (Amplified Fragment Length Polymorphisms) have recently been used to investigate genomes of different complexities. This paper assesses the potential use of the AFLP technology to determine fish and seafood species in processed commercial products and domestic stocks. In particular a species database of fish, molluscs and crustaceans has been created with the aim to identify species of origin of seafood products by previously defined AFLP patterns. Different EcoRI and TaqI primer combinations were selected from 20 screened combinations in relation to the total number of detected fragments and polymorphic ones. Most informative combinations were E32/T32, E32/T33, E33/T33, E33/T37, E33/T38, E40/T33, E40/T37, E42/T32, E42/T37. The comparison of informative markers between unknown frozen or fresh products and reference samples has enabled the accurate identification of 32 different species. The taxonomic characterization has been performed either at the species or at the population level depending on the number of available individuals. AFLP variation at the population level is particularly helpful for the stock traceability of domestic strains. Size homoplasy was also investigated in one species to assess the rate of non-homologous comigrating fragments and to detect additional polymorphic markers to be used in stock identification. Results of Band Sharing Index (BSI) and percentage of polymorphic fragments are presented and are discussed in relation to the wide applicability of AFLPs both for fish and seafood safety and authenticity testing in such fields as food traceability and restocking management. The database, available upon request at nonnis@biol.unipr.it, will be continuously updated.     

Interactions of high methoxyl pectin with whey proteins at oil/water interfaces at acid pH

The interactions between whey protein isolate (WPI) and high methoxyl pectin (HMP) at pH 3.5 were investigated in situ using ultrasound (US) and diffusing wave spectroscopy (DWS). HMP was added to 10% oil-in-water emulsions containing 1% WPI. At neutral pH, no protein-pectin interactions were observed as both molecules are negatively charged, while at pH 3.5 bridging flocculation occurred via electrostatic interactions. Four different stages were distinguished during the addition of HMP in WPI-stabilized emulsions at pH 3.5. At a concentration below a critical value, no interactions were observed. At concentrations >0.02% HMP, a change in the l factor indicated a change in the ordering of the emulsion droplets, influenced by long-range interactions. At higher concentrations (in the range between 0.04 and 0.06% HMP), attenuation showed significant changes in the surface of the oil droplets, changes which affected the droplet-droplet interactions. At pectin concentrations >0.05%, attenuation of sound and 1/l* decreased, while velocity of sound and particle size increased, as a result of bridging flocculation. These results demonstrated for the first time that methods such as US and DWS combined permit the observation of the early stages of the interactions between two biopolymers at the interface. This is significant in light of increasing efforts in engineering complex interfacial layers.     

Results of the international aqueous mercury speciation intercomparison exercise

Twenty-seven laboratories from around the world agreed to participate in an intercomparison exercise for total Hg (Hg_t) and methyl Hg (MMHg) in pristine lake water. Unfiltered samples from a remote brown water lake in northern Wisconsin (USA) were collected into acid cleaned Teflon^® bottles using ultra-clean sample handling techniques. The samples were acidified in the field with 0.4% by volume of pre-analyzed HCl (12N; <5 pg Hg/mL), and sent to the primary reference laboratory (PRL) by overnight mail. Within one week of receipt, the samples were randomized, and 10% analyzed for Hgt and MMHg at the PRL to verify the homogeneity of the set. Each participating laboratory was then sent 3 randomly selected 1 L bottles, while the PRL retained 30, and the secondary reference laboratory (SRL) retained 12 samples. The participating laboratories were asked to analyze each bottle in triplicate for both Hgt and MMHg, reporting all QA data including blanks, spike recoveries, and detection limits. The PRL analyzed samples in triplicate at both the beginning and the end of the analytical window, to provide a controlled estimate of any changes in concentration or speciation over that time. Of the 23 laboratories that returned results, 18 utilized BrCl oxidation, gold trapping, and cold vapor atomic fluorescence (CVAFS) detection for Hg_t. Four laboratories reported similar techniques, varying either in detector (cold vapor atomic absorption), or wet chemistry. Only 16 laboratories reported MMHg results, with 15 using a variation of the aqueous phase ethylation, GC separation, and CVAFS detection technique. The results show good convergence between the participating labs for both Hg_t and MMHg. For Hgt 18 of 23 labs reported means within 20% of the consensus value and PRL results (1.27±0.18 ng/L and 1.27±0.14 ng/L respectively). For MMHg, 13 of the 16 labs reported results within 20% of either the consensus value (0.420±0.055 ng/L) or the PRL value (0.446±0.041 ng/L).     

MR properties of water in saturated soils and resulting loss of MRI signal in water content detection at 2 tesla

This paper reports a systematic MRI study at 2 tesla of 23 soils, each separately saturated with a known amount of water. The percentage of that water which could be detected using various MR methods was determined by comparison with a liquid reference sample. A pulse-acquire sequence gave a bulk detection of between 47 and 94% of the known water content of saturated soil. Also for bulk measurements, the inversion-recovery sequence used for determining T₁ values detected a range of 0.7–75% of the existing soil water. The CPMG sequence with an echo time (TE) of 1 ms used for determining the bulk T₂ values gave lower values, in the range of 0.4–66% overall. A spin-echo MRI sequence with a TE of 2.9 ms gave an even lower bulk detection, ranging from 0.2 to 57%. These low values for the detectable water content of bulk saturated soil water reflect the loss of water magnetization which occurs even during short echo time MR sequence at 2 tesla field strength. The source of the above findings was investigated by measurements of the longitudinal (T₁) and transverse (T₂) relaxation times and spectral linewidths of the soil-water protons, and by conventional analysis of soil properties. The MR parameters of critical importance to water quantification are T₂ and T₂^*, shorter values of which lead to a progressively greater loss of signal intensity for all MR protocols. Those parameters are affected by the following soil chemical and physical features: soil magnetic susceptibility, and the content of free iron oxides, clay, sand, exchangeable cations (K, Na and Ca), and organic matter. The implication of this work is that the only soil water which can be detected quantitatively at 2 tesla using a conventional spin-echo MRI protocol with echo times of 3 ms or longer is that located in the relatively large soil pores. Using the protocols investigated in this work, water in smaller pores will only be detected accurately for soils which have relatively low paramagnetic-metal impurities and/or have low clay content. Future MR studies of soil water should consider the use of other MRI protocols (e.g. for solid state), and measurement at low magnetic fields.     

Characterization of the antifungal activity on Botrytis cinerea of the natural diterpenoids kaurenoic acid and 3beta-hydroxy-kaurenoic acid

The antifungal activity on Botrytis cinerea of the diterpenoids 3beta-hydroxy-kaurenoic acid and kaurenoic acid, obtained from the resinous exudates of Pseudognaphalium vira vira, was determined. 3beta-Hydroxy-kaurenoic acid reduced the mycelial growth of B. cinerea in solid and liquid media. Additionally, the damage produced by the fungus on the surface of tomato leaves in the presence of the diterpenoids was evaluated. A higher protective effect was observed in the presence of the hydroxylated diterpene. On the other hand, the effect of the diterpenoids on the production of enzymes that participate in the plant infection by B. cinerea was analyzed. p-Nitrophenylbutyrate esterase production was induced by both diterpenoids, whereas laccase production was only induced by the hydroxylated diterpene. In the study of the mechanism of action of these compounds, it was determined that 3beta-hydroxy-kaurenoic acid would produce permeabilization of the cell membrane of B. cinerea.     

Ecology of mucilage production by Amphora coffeaeformis var. perpusilla blooms of Adriatic Sea

Diatoms isolated from mucilage blooms which emerged in the Adriatic Sea during summer 1988, were put in culture and then later, identified as Amphora coffeaeformis var. perpusilla (GRUN.) Cleve. Experiments of growth in orthophosphate and nitrate enrichment led to noticeable variations in the mucilage production. In this paper we propose a benthonic origin for the Adriatic mucilages, due to the presence of considerably more nitrates rather than phosphates.     

Heat-induced soy-whey proteins interactions: formation of soluble and insoluble protein complexes

The aggregation behavior during heating of a solution containing soy protein and whey protein isolate (WPI) was studied using rheology, confocal microscopy, gel filtration, and electrophoresis. Soy/WPI mixtures formed gels at 6% total protein concentration with a high elastic modulus (G') and no apparent phase separation. The ratio of soy to WPI was fundamental in determining the type of network formed. Systems containing a high soy to WPI ratio (>70% soy protein) showed a different evolution of the elastic modulus during heat treatment, with two apparent stages of network development. Whey proteins formed disulfide bridges with soy proteins during heating, and at low ratios of soy/WPI, the aggregates seemed to be predominantly formed by 7S, the basic subunits of 11S and beta-lactoglobulin. Size exclusion chromatography indicated the presence of high molecular weight soluble complexes in mixtures containing high soy/WPI ratios. Results presented are the first evidence of interactions between soy proteins and whey proteins and show the potential for the creation of a new group of functional ingredients.     

Biomass-Size Distribution of the Aquatic Community in Limed, Circumneutral and Acidified Reference Lakes

Semi-quantitative biomass-size distributions (BSD's) along a joint axis of individual size provided an integrated illustration of aquatic communities sampled at different taxonomic and trophic levels. The approach was applied within the Swedish ISELAW-programme (integrated studies of the effects of liming acidified waters) to test the general hypothesis that aquatic communities in limed lakes are not systematically different from communities in comparable non-limed circumneutral lakes. Input data included pelagic phytoplankton and zooplankton, sublittoral/profundal macroinvertebrates, and benthic fish, within twelve Swedish lakes (six limed, two acidic and four circumneutral reference lakes). The four compartments were sampled on different spatial scales, but each designed for between-lakes comparisons. There were no clear-cut differences in overall size distribution between the three categories of lakes. The mean BSD of limed lakes was indeed more similar to the mean BSD of circumneutral lakes than to that of two acidic lakes. Due to high variation within categories, however, acifidication status alone can not be used for reliable prediction of BSD in a certain lake.     

Mercury in Small Freshwater Lakes: A Case Study: Lake Velenje, Slovenia

Lake Velenje is located in one of the most polluted regions ofSlovenia, the ?alek Valley. The major source of pollution is the coal-fired thermal power plant in ?o?tanj (?TPP). In the past, dumping of coal ash directly into Lake Velenje and drainage water from the ash disposal site resulted in unique chemical characteristics of the lake water, such as very high pH (10–12) and high concentrations of heavy metals. The introduction of a closed cycle ash transport system in 1995resulted in a very fast recovery of the lake water quality. The aim of our study was to establish the sources, fate and distribution of mercury in Lake Velenje. In order to establishrecent sources of mercury, total mercury and methylmercury concentrations were measured in various environmental samples(lake inflows, outflow, rainwater, sediments, etc.). Total mercury and methylmercury concentrations were measured at thesurface and at different depths to establish mercury cycling, its transport and chemical transformations in the lake. Generalwater quality parameters (such as pH, Eh, O₂, temperature,TDS, conductivity) were also determined. The results show that the major sources of mercury in Lake Velenje are lake inflows and wet deposition. Total mercury andmethylmercury concentrations in the water column are very low (total mercury: 0.2–2.7 ng L^-1; methylmercury: 20–86 pg L^-1) and can be compared to other non-contaminated freshwater lakes. These results suggest that the major form of mercury emitted from thermal power plant stacks is volatile Hg⁰, whichhas no or very little influence on the nearby surroundings, but rather is subject to long-range atmospheric transport.     

Assessing Small-Scale Forestry by Data Base for Forest Owner Analysis

In Sweden, as in other countries with a growing and increasingly diverse population of forest owners, there is an apparent need for more detailed quantitative data of high quality in order to describe and understand present forest conditions and predict and explain future trends. Therefore, the Swedish University of Agricultural Sciences has developed a Data Base for Forest Owner Analysis (DBFOA) by combining existing forest measurement statistics, gathered on a regular basis by the Swedish Forest Agency since 1992, with records of the individual forest owners. The database consists of self-reported measurement statistics in terms of cuttings, cleaning, scarification and planting from about 30,000 forest management units. It includes information on the owner age, gender, residential proximity to the management unit and the extent of work undertaken by the owner. From 1999 it also indicates whether the forest is certified. This paper demonstrates the use of the database by presenting results from (1) a comparison of management practices on properties that are certified with those that are not, and (2) an examination of how the area of planting and final felling have changed from 1999 to 2006 in total and between male and female forest owners. Results from the first analysis show that the willingness to certify increases with the size of the forest property and also that harvesting activities are more frequent on certified than non-certified properties. The second analysis, show a higher ratio of final felling during 2003–2006 on properties owned by women than properties owned by men.