Sonication-assisted extraction of chitin from North Atlantic shrimps (Pandalus borealis)

The influence of sonication during extraction of chitin from North Atlantic shrimp (NAS) shells (Pandalus borealis) on chitin yield, purity, and crystallinity was investigated. Shells were peeled, washed, lyophilized, ground, and suspended for 4 h in 0.25 M HCl (1:40) at 40 degrees C followed by ultrasonication at 41 W/cm(2) for 0, 1, and 4 h, respectively. Demineralized shells were lyophilized, resuspended in 0.25 M NaOH (1:40), and ultrasonicated at 41 W/cm(2) for 0, 1, and 4 h to remove proteins. The yield and mineral and protein contents were determined after each processing step. The purity of extracted chitin was determined from the total amount of glucosamine. The crystallinity index and size of crystals were calculated from wide-angle X-ray scattering measurements. Scanning electron microscope images were recorded to evaluate morphological changes in samples. The yield of chitin from NAS decreased from 16.5 to 11.4% for 0 and 1 h sonicated samples, respectively, which was attributed to increased concentrations of depolymerized materials in the wash water. Sonication did not enhance the removal of minerals. The application of ultrasound enhanced the removal of proteins from 39.8 to 10.6, 8.3, and 7.3% after 0, 1, and 4 h of sonication treatments. The crystallinity index of chitin decreased from 87.6 to 79.1 and 78.5% after 1 and 4 h of sonication, yielding chitosans with crystallinity indices of 76.7, 79.5, and 74.8% after deacetylation, respectively. Fourier transform infrared spectroscopy scans indicated that the degree of acetylation of chitins was unaffected by sonication. Comparison of the extraction results of NAS with that from freshwater prawns indicated that more impurities were left in NAS chitin, suggesting that composition and structural arrangement of chitin in shells influence the efficiency of ultrasound-assisted extraction.     

Sonication-assisted extraction of chitin from shells of fresh water prawns (Macrobrachium rosenbergii)

The effect of sonication during chitin extraction from freshwater prawn shells on yield, purity, and crystallinity of chitin was investigated. Dry prawn shells were suspended for 4 h in 0.25 M HCl at 40 degrees C while they were sonicated for 0, 1, and 4 h. Demineralized shells were lyophilized, resuspended in 0.25 M NaOH, and sonicated again for 0, 1, and 4 h for protein removal. The yield of chitin decreased from 8.28 to 5.02% for nonsonicated and sonicated samples, respectively, which was attributed to losses of depolymerized materials in the wash water. The application of ultrasound enhanced the removal of proteins. In nontreated shells, the amount of protein was 44.01% and was reduced to 12.55, 10.59, and 7.45% after 0, 1, and 4 h of sonication treatments. The glucosamine content slightly decreased with sonication probably because of losses due to depolymerization. The crystallinity indices of chitins decreased as the time of sonication increased. The degree of acetylation of chitins was unaffected by sonication, but the degree of acetylation of chitosans produced from sonicated chitin decreased from 70.0 to 68.7 and 61.4% for 1 and 4 h sonicated samples, respectively.     

Detection of avian rotaviruses of groups A, D, F and G in diseased chickens and turkeys from Europe and Bangladesh

Avian rotaviruses (AvRVs) represent a diverse group of intestinal viruses, which are suspected as the cause of several diseases in poultry with symptoms of diarrhoea, growth retardation or runting and stunting syndrome (RSS). To assess the distribution of AvRVs in chickens and turkeys, we have developed specific PCR protocols. These protocols were applied in two field studies investigating faecal samples or intestinal contents of diseased birds derived from several European countries and Bangladesh. In the first study, samples of 166 chickens and 33 turkeys collected between 2005 and 2008 were tested by PAGE and conventional RT-PCR and AvRVs were detected in 46.2%. In detail, 16.1% and 39.2% were positive for AvRVs of groups A or D, respectively. 11.1% of the samples contained both of them and only four samples (2.0%) contained rotaviruses showing a PAGE pattern typical for groups F and G. In the second study, samples from 375 chickens and 18 turkeys collected between 2009 and 2010 were analyzed using a more sensitive group A-specific and a new group D-specific real-time RT-PCR. In this survey, 85.0% were AvRV-positive, 58.8% for group A AvRVs, 65.9% for group D AvRVs and 38.9% for both of them. Although geographical differences exist, the results generally indicate a very high prevalence of group A and D rotaviruses in chicken and turkey flocks with cases of diarrhoea, growth retardation or RSS. The newly developed diagnostic tools will help to investigate the epidemiology and clinical significance of AvRV infections in poultry.     

Landscape occupancy and local population size depends on host plant distribution in the butterfly Cupido minimus

We tested whether the landscape occupancy and local population size of the monophagous butterfly Cupido minimus can be predicted by patch size and isolation of its host plant or by other habitat characteristics. C. minimus and its larval food plant Anthyllis vulneraria are classified as rare and endangered in northern Germany. Adults of C. minimus are ranked as the most sedentary butterfly species in northern Europe.Around the city of Göttingen (Germany), we checked all known locations of A. vulneraria (n=70) in June 2002 for butterfly eggs (in blooming flowerheads) and adult butterflies (within 20-min transects).We found eggs of C. minimus or a high number of adults (>7) in all habitats with A. vulneraria (which are calcareous grasslands) even when isolated up to 2-4 km. In multiple regression analyses, local population size of adult butterflies was positively related to the cover of its larval food plant A. vulneraria explaining 65% of variance. Cover of A. vulneraria increased with increasing habitat area and increasing cover of plant species in flower and decreased with increasing cover of shrub layer. Habitat isolation and further factors describing habitat quality were not related to C. minimus population size or cover of its larval food plant.The results suggest that dispersal ability of C. minimus is greater than expected and that management should focus to increase A. vulneraria patches. For conservation, low impact grazing once a year and removing of excessive shrubs in winter seems to be the most appropriate strategies.     

Evaluation of serological tests for detecting tick-borne encephalitis virus (TBEV) antibodies in animals

Tick-borne encephalitis (TBE) in animals is not well understood yet. TBE virus (TBEV) serology in several host species could be valuable for epidemiological analyses in the field as well as for the detection of clinical cases. However, performance and suitability of the available test systems are not well assessed. Therefore, we evaluated two commercial TBEV-ELISA kits in a pilot study and compared them for their suitability in veterinary applications. For this purpose, we tested 163 field collected goat sera and evaluated the results by serum neutralization test (SNT) as "gold standard". Twenty-eight SNT positive sera (17.2%) were detected. The best suited ELISA kit was used for determination of a species-specific cutoff for horses, cattle, sheep, goats, pigs, mice, dogs, rabbits and monkeys with defined sera from animals without known or with improbable contact to TBEV. The level of non-specific ELISA results does not only differ between animal species but may also be influenced by the age of the tested animals. The number of sera which tested false positive by ELISA was higher in older than in young sheep. In order to obtain defined polyclonal sera as references, two dogs, cattle, goats, sheep, rabbits and pigs each, as well as one horse and 90 mice were immunized four times with a commercially available TBEV vaccine. In conclusion, our results demonstrated that commercial TBEV-ELISA kits are suitable for application in veterinary medicine for both, verification of clinical TBE cases and epidemiological screening. However, positive ELISA results should be verified by SNT. Only a very low number of false negative ELISA-results were found.     

Isolation of scFv fragments specific to OmpD of Salmonella Typhimurium

Pork meat is one of the major sources for human infections with Salmonella enterica subspecies enterica serovars. Further, zoonoses caused by S. enterica subspecies enterica serovars are responsible for substantial economical losses in industrial countries. Quick and reliable detection of this infection is urgently needed to improve consumer security. Due to its capability to identify infections independent of the species, a competitive ELISA is the preferable method for the detection of anti-Salmonella antibodies in serum. Recombinant antibody fragments (scFvs) were isolated from the naive human antibody gene library HAL7 by phage display. Recombinant produced outer membrane protein D (OmpD) of Salmonella Typhimurium was used as antigen. The characterization of the isolated single chain Fv (scFv) antibodies was done by enzyme-linked immunosorbent assay (ELISA), immunoblot, sequencing, epitope mapping and size exclusion chromatography (SEC). The detection of anti-OmpD IgGs in swine sera by competitive ELISA was shown in a proof of principle concept. Furthermore, the developed competitive ELISA would be compatible to a recently published DIVA vaccine, allow to distinguish between infected and vaccinated pigs.     

Investigation of the volatile composition of pinotage wines fermented with different malolactic starter cultures using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOF-MS)

Headspace solid phase microextraction in combination with comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (HS-SPME-GC × GC-TOF-MS) was used for the detailed investigation of the impact of malolactic fermentation (MLF) using three commercial Oenococcus oeni strains on the volatile composition of Vitis vinifera cv. Pinotage wines. GC × GC allowed the identification of 115 volatile compounds, including both major constituents and trace-level compounds, in a single analysis. A number of compounds differing in mean concentration levels between the control wines and those fermented with different starter cultures were shown for the first time to be influenced by MLF and/or the bacterial strain. Principal component analysis (PCA) provided excellent separation between the wines fermented with different MLF starter cultures and the control wine. Significantly different levels for some volatile compounds in wines fermented with one of the LAB starter cultures could be indicative of metabolic differences of this strain.     

Cross-linking of interfacial layers affects the salt and temperature stability of multilayered emulsions consisting of fish gelatin and sugar beet pectin

This study assessed the stabilizing effect of enzymatic cross-linking on double-coated emulsions (beet pectin-fish gelatin). The beet pectin layer was cross-linked via ferulic acid groups using laccase (an enzyme that is known to catalyze the oxidation of phenolic groups). Fish gelatin-coated oil droplets (primary emulsion) were mixed at pH 3.5 to promote electrostatic deposition of the beet pectin molecules onto the surfaces of the oil droplets (secondary emulsion). Laccase was then added to promote cross-linking of the adsorbed beet pectin layer. Cross-linked pectin-coated oil droplets had similar or significantly better stability (p < 0.05) than oil droplets of primary or secondary emulsions to NaCl addition (0-500 mM), CaCl(2) addition (0-250 mM), and thermal processing (30-90 °C for 30 min). Freeze-thaw stability and creaming behavior of enzyme-treated, secondary emulsions after two cycles (-8 °C for 22 h; 25 °C for 2 h) were significantly improved (p < 0.05). These results may have important implications for food manufacturers that are in need of emulsions with improved physical stability, for example, emulsions used in frozen foods for sauces or dips.     

Ultrasonic spectroscopy and differential scanning calorimetry of liposomal-encapsulated nisin

The thermal stability of phosphatidylcholine (PC) liposomes (colloidal dispersions of bilayer-forming polar lipids in aqueous solvents) in the presence and absence of the antimicrobial polypeptide nisin was evaluated using differential scanning calorimetry (DSC) and low-intensity ultrasonic spectroscopy (US). PC liposome mixtures with varying acyl chain lengths (C16:0 and C18:0) were formed in buffer with or without entrapped nisin. Gel-to-liquid crystalline phase transition temperatures (T(M)) of liposomes determined from DSC thermograms were in excellent agreement with those determined by ultrasonic velocity and attenuation coefficient measurements recorded at 5 MHz. The dipalmitoylphosphatidylcholine (DPPC) T(M) measured by DSC was approximately 41.3 and approximately 40.7 degrees C when measured by ultrasonic spectroscopy. The T(M) of distearoylphosphatidylcholine (DSPC) and DPPC/DSPC 1:1 liposomes was 54.3 and 54.9 degrees C and approximately 44.8 and approximately 47.3 degrees C when measured by DSC and US, respectively. The thermotropic stability generally increased upon addition of nisin. Analysis of the stepwise decrease in ultrasonic velocity with temperature indicated an increased compressibility corresponding to a loss of structure upon heating.