Crab-meat-isolated psychrophilic spore forming bacteria inactivation by electron beam ionizing radiation.

The present work was performed to evaluate the potential of electron beam ionizing radiation for the inactivation of three psychrophilic spore forming bacteria (Bacillus mycoides, Bacillus weihenstephanensis and Psychrobacillus psychrodurans) isolated from ready-to-eat brown crab (Cancer pagurus). Inactivation curves for the three spores were performed in both types of crab meat, brown and white. Also the effect of pH and water activity (aw) on the lethal efficacy of ionizing radiation, for the three different psychrophilic spore forming bacteria, was evaluated. The effects of pH, aw and their possible interactions were assessed in citrate-phosphate buffers of different pH, ranging between 7 and 4, and aw, ranging from <0.99 to 0.80. A reduction of aw increased the spores resistance between >0.99 and 0.90, while an aw reduction from 0.90 to 0.80 had a minor impact on their resistance. In contrast to aw, the effect of pH showed a greater variability depending on the spore species. While pH did not affect the resistance of B. weihenstephanensis at any aw, B. mycoides showed slightly higher resistance at pH 5.5 at aw of 0.90 and 0.80. pH showed a significant effect on the resistance of P. psychrodurans. For the two types of crab meat, slightly differences were observed in 6D values. B. weihenstephanensis was the most resistant, requiring 7.3-7.6 kGy to inactivate 6 Log10-cycles of this spore forming bacterium, while for B. mycoides and P. psychrodurans 6.1-6.3 and 5.4-5.3 kGy respectively were necessary to reach the same inactivation level in crab meat. An agreement between spore resistance in crab meats and lab media, with similar characteristics in pH and aw, was also observed. The results obtained in this research demonstrated the potential for ionizing radiation to achieve an appropriate inactivation level of spores naturally present in brown crab with the application of doses lower than 10 kGy.

Microbiological aspects related to the feasibility of PEF technology for food pasteurization.

Processing unit operations that seek to inactivate harmful microorganisms are of primary importance in ascertaining the safety of food. The capability of pulsed electric fields (PEF) to inactivate vegetative cells of microorganisms at temperatures below those used in thermal processing makes this technology very attractive as a nonthermal pasteurization process for the food industry. Commercial exploitation of this technology for food pasteurization requires the identification of the most PEF-resistant microorganisms that are of concern to public health. Then, the treatment conditions applicable at industrial scale that would reduce the population of these microorganisms to a level that guarantees food safety must be defined. The objective of this paper is to critically compile recent, relevant knowledge with the purpose of enhancing the feasibility of using PEF technology for food pasteurization and underlining the required research for designing PEF pasteurization processes.

Cryptic speciation of Greek populations of the freshwater shrimp genus Atyaephyra de Brito Capello, 1867 (Crustacea, Decapoda), evidence from mitochondrial DNA.

The freshwater shrimp genus Atyaephyra de Brito Capello, 1867 is a complex of cryptic species with morphologically overlapping features, which has led to invalid identifications until recently. The genus is distributed around the entire Mediterranean Basin and surrounding areas. In 2009 we used a molecular approach with 16S and Cox1 genes to identify the European and African populations and to show the existence of different species and groups of haplotypes (García Muñoz et al. 2009). Christodoulou et al. (2012) recently identified four species in Greece (Atyaephyra stankoi, A. thyamisensis, A. strymonensis and A. acheronensis) based on morphology and some partial sequences of Cox1. In the present study we analyse several Greek populations of Atyaephyra spp. using morphology and two mitochondrial markers (16S and Cox1). Our molecular data confirm the taxonomic validity of A. stankoi, A. thyamisensis and A. strymonensis but show that A. acheronensis should be considered a synonym of A. desmarestii. The limited distribution of A. desmarestii in only a few rivers in the Ionian region is probably the result of a posterior introduction from neighbouring European areas. After the genetic identification and as a consequence of the high variability observed within species and between rivers, mainly in A. thyamisensis, we re-describe this species and provide morphological data for the other species. We also give information on the chromatophore pattern in larvae, which could be very useful in the future for recognizing species and their distribution. The distribution of Greek Atyaephyra species seems to be related to the very complex paleogeographical history of the Paratethys and Mediterranean Sea in the Balkan Peninsula. Three areas can be recognized in relation to the distribution of these species: a global region, a western region separated from the eastern region by the Pindos Mountains, and a Macedonia-Thracian region. 

Taxonomic study of the Pagurus forbesii "complex" (Crustacea: Decapoda: Paguridae). Description of Pagurus pseudosculptimanus sp. nov. from Alborán Sea (Southern Spain, Western Mediterranean Sea).

Study of hermit crabs from Alboran Sea has allowed recognition of two different morphological forms under what had been understood as Pagurus forbesii. Based on morphological observations with various species of Pagurus, and molecular studies, a new species is described as P. pseudosculptimanus. An overview of species of Pagurus from the eastern Atlantic and Mediterranean Sea is provided.

Inactivation of Salmonella enterica by UV-C light alone and in combination with mild temperatures.

The aim of this investigation was to study the efficacy of the combined processes of UV light and mild temperatures for the inactivation of Salmonella enterica subsp. enterica and to explore the mechanism of inactivation. The doses to inactivate the 99.99% (4D) of the initial population ranged from 18.03 (Salmonella enterica serovar Typhimurium STCC 878) to 12.75 J ml(-1) (Salmonella enterica serovar Enteritidis ATCC 13076). The pH and water activity of the treatment medium did not change the UV tolerance, but it decreased exponentially by increasing the absorption coefficient. An inactivating synergistic effect was observed by applying simultaneous UV light and heat treatment (UV-H). A less synergistic effect was observed by applying UV light first and heat subsequently. UV did not damage cell envelopes, but the number of injured cells was higher after a UV-H treatment than after heating. The synergistic effect observed by combining simultaneous UV and heat treatment opens the possibility to design combined treatments for pasteurization of liquid food with high UV absorptivity, such as fruit juices.

Inactivation of Salmonella spp. in liquid whole egg using pulsed electric fields, heat, and additives.

This paper evaluates the lethal effectiveness on 7 different Salmonella serovars of the application, in static and continuous conditions, of pulsed electric fields (PEF) followed by heat treatments in liquid whole egg (LWE) with additives (EDTA or triethyl citrate-TC-). Compared to heat treatments, the PEF (25 kV/cm and 75-100 kJ/kg) followed by heat (52°C/3.5', 55°C/2', or 60°C/1') in LWE with 2% TC permitted the reduction of heat treatment time from 92 fold at 52°C to 3.4 fold at 60°C, and 4.8 fold at 52°C in LWE with EDTA for a 9-Log(10) reduction of the population of Salmonella Enteritidis. The new designed treatments inactivated more than 5 Log(10) cycles of Salmonella serovars Dublin, Enteritidis 4300, Enteritidis 4396, Typhimurium, Typhi, Senftenberg, and Virchow, both in static and continuous conditions. Conversely, current heat pasteurization treatments of 60°C/3.5' and 64°C/2.5' reduced 5 Log(10) cycles of various serovars of Salmonella but only 2 and 3-4 Log(10) cycles of Salmonella Senftenberg and Salmonella Enteritidis 4396, respectively. Soluble protein content (SPC) decreased 1.8%, 1.3%, and 5.0% after the successive application of PEF followed by heat at 52, 55, and 60°C in the presence of 2% TC, respectively, whereas 1.6% and 9.4% of SPC were reduced after heat pasteurization at 60 and 64°C, respectively. Results indicate that designed treatments could be an alternative to current heat pasteurization of LWE as showed higher lethal effectiveness against Salmonella serovars with a similar or even lower decrement of the soluble protein content.

Two new hermit crab species of Diogenes (Crustacea: Decapoda: Diogenidae) from Atlanto-Mediterranean coasts of Iberian Peninsula: Poleward migrants or merely overlooked indigenous species?

A new hermit crab species of the genus Diogenes with reddish-orange cheliped, Diogenes erythromanus sp. nov., is described and illustrated based on specimens from the Mediterranean coasts of the Iberian Peninsula, southern Spain. In addition, a second morphotype originating from Mauritanian waters and morphologically very close to D. erythromanus sp. nov. is described as a different species, D. arguinensis sp. nov. The new species are here compared to morphologically similar congeners, especially to those inhabiting the same geographical range. Diogenes erythromanus sp. nov. is distinguishable from other Diogenes primarily by the shape and armature of the left cheliped, with a palm slightly higher than long, with a ridge of spines running along the proximal lower margin that continues with a series of spinose rows forming a central band parallel to the upper margin of the palm. The palm in D. arguinensis sp. nov. is longer than high and shows similar proximal ridge, but without central spinose ridge. The shape of the cheliped is also different in D. arguinensis sp. nov., with long dactylus, which is also flattened and twisted. Sequences from two mitochondrial and one nuclear genes, and comparative analyses with other available sequences for the genus, are also included. Molecular phylogenetic analyses support the morphological delimitation, with D. erythromanus sp. nov. and D. arguinensis sp. nov. forming a separate group, more related to other tropical species, which raises different possible explanations for its presence in the Iberian Peninsula.

A new species of hermit crab of the genus Pagurus Fabricius, 1775 (Crustacea: Anomura: Paguridae) from north-western Africa.

One male and two female specimens of an undescribed species of the heterogeneous hermit crab genus Pagurus Fabricius, 1775, were found during the study of the collections of the Oceanographic Institute of Cádiz, containing specimens from African and European coasts collected during the scientific cruises carried out between in years 2008-2013. The new species is named Pagurus pectinidactylus n. sp., in reference to the presence of a well-developed toothless pectinate masticatory border in the dactylus of the left cheliped, composed by a row of tightly placed bristles forming a brush-like structure. The new species is compared to morphologically similar congeners, especially those that are genetically closest. Molecular phylogenetic analyses support the morphological delimitation, with P. pectinidactylus n. sp. forming a separate clade, more closely related to a group of South African species belonging to the Pagurus anachoretus group.

Changes in phenolic compounds of Aragon red wines during alcoholic fermentation.

Changes in the principal phenolic compounds during the maceration-fermentation process of Garnacha, Merlot and Syrah grapes cultivated in Aragón region (northeast of Spain) have been investigated. While Garnacha is a traditional grape variety cultivated in this region, Merlot and Syrah have been introduced recently. During fermentation, Syrah showed the highest concentration in anthocyanins, flavan-3-ols and flavonols (802.7 ± 0.5 mg/L, 74.7 ± 2.4 mg/L and 37.1 ± 1.5 mg/L at the end of fermentation, respectively). Unexpectedly, Garnacha, a variety with lower phenolic content, showed the highest amount of hydroxycinnamic acids (83.1 ± 5.6 mg/L at the end of fermentation). The overall results also indicated that the evolution during maceration-fermentation process of the different phenolic compounds and their concentrations were influenced by the varietal factor.

Validation of a novel handheld 3D ultrasound system for imaging scoliosis - phantom study.

Use of 3D ultrasound (US) scanners to detect and monitor scoliosis have been validated. The Cobb angle, axial vertebral rotation, spinal flexibility, curvatures in the sagittal profile and the Cobb angle on the plane of maximum curvature (PMC) can be measured from coronal, transverse and sagittal planes of ultrasound images. However, traditional 3D ultrasound scanners are relatively bulky and expensive. 2D US handheld and low-cost scanners are widely available. To adapt the 2D scanners for scoliosis applications, a position and orientation system is integrated with the scanner. The objective of this study was to validate a newly developed 3D handheld US system to image the spine. The wireless handheld US scanner (C3-HD, Clarius, Canada) was selected because of its high resolution and availability of raw data. A wireless tracking system based on electromagnetic (G4 system, Polhemus, USA) was integrated with the Clarius ultrasound. During scanning, the ultrasound information was synchronized with the scanner's position and orientation by using custom developed software. Both information were streamed wirelessly to a laptop. Custom software reconstructed and displayed the 3D spinal image in real-time. A single 3D printed vertebra, two full plastic spine phantoms from T1-T12 vertebrae and a non-scoliotic volunteer were scanned. The 3D reconstruction process of a spine image was less than 3 seconds. The dimensional and the angle errors were 1 mm and 3°, respectively. This study demonstrated that a low-cost ($11,000 USD) handheld 3D ultrasound system was developed and validated. Clinical trials on subjects attending will be the next step.

Direct contact ultrasound assisted freezing of chicken breast samples.

Nowadays, rapid freezing is sought to favor the formation of small ice crystals. Several studies have shown that the application of ultrasounds (US) accelerates the processes of energy and mass transfer when they are applied through immersion systems. However, there are hardly any studies on its application in direct systems without the use of a liquid medium for its transmission. Therefore, the objective of this work was to evaluate the potential of the application of US for improving the freezing process of chicken breast samples. First, the application of intermittent US treatments at different net sonication times of 7, 17, 37, 50 and 67% during the freezing of distilled water samples in a conventional freezer was evaluated. It was observed that net sonication times of 37, 50 and 67% reduced the phase change period by 30.0, 21.4, 27.0%, respectively. The effective freezing time was also reduced by 12.4 and 12.8% by applying net sonication times of 37 and 50%. Considering these results, an intermittent US treatment with a net sonication time of 37% was chosen for chicken breast freezing in an air-forced cooling tunnel at ambient temperatures from -13 to -22 °C. The length of all the freezing phases was reduced upon application of US, leading to an overall process time reduction of approx. 11%. On the other hand, no significant differences were found either in the Water Holding Capacity (WHC) or Cooking Loss (CL) values between control and US assisted frozen chicken breast samples. Furthermore, in vitro experiments showed that US-assisted freezing did not influence protein digestibility of chicken meat samples. This study demonstrates the potential of the application of US by direct contact to favor energy transfer processes during freezing of water and chicken breasts samples. However, its effect on the quality of the frozen products should be further studied.

Evaluation of pulsed electric fields technology for liquid whole egg pasteurization.

This investigation evaluated the lethal efficiency of pulsed electric fields (PEFs) to pasteurize liquid whole egg (LWE). To achieve this aim, we describe the inactivation of Salmonella Enteritidis and the heat resistant Salmonella Senftenberg 775 W in terms of treatment time and specific energy at electric field strengths ranging from 20 to 45 kV/cm. Based on our results, the target microorganism for this technology in LWE varied with intensity of the PEF treatment. For electric field strengths greater than 25 kV/cm, Salmonella Enteritidis was the most PEF-resistant strain. For this Salmonella serovar the level of inactivation depended only on the specific energy applied: i.e., 106, 272, and 472 kJ/kg for 1, 2, and 3 Log(10) reductions, respectively. The developed mathematical equations based on the Weibull distribution permit estimations of maximum inactivation level of 1.9 Log(10) cycles of the target Salmonella serovar in the best-case scenario: 250 kJ/kg and 25 kV/cm. This level of inactivation indicates that PEF technology by itself cannot guarantee the security of LWE based on USDA and European regulations. The occurrence of cell damage due to PEF in the Salmonella population opens the possibility of designing combined processes enabling increased microbial lethality in LWE.

Inactivation kinetics of pulsed electric field-resistant strains of Listeria monocytogenes and Staphylococcus aureus in media of different pH.

A study of the effect of pulsed electric fields (PEF) on the inactivation of Listeria monocytogenes STCC 5672 and Staphylococcus aureus STCC 4459 in McIlvaine buffer covering a range from pH 3.5 to 7.0 was conducted. Mathematical models based on the Weibull distribution were developed to describe the influence of the electric field strength, treatment time and pH of the treatment medium on the lethality of both Gram positive pathogenic bacteria after PEF treatments. Both microorganisms were more sensitive to PEF in media of low pH, although the influence of the pH on the PEF resistance was more significant in S. aureus. In the best cases scenario, the highest inactivation levels achieved were 3.3 and 6.1 log(10) cycles for L. monocytogenes and S. aureus respectively in pH 3.5 after 500 micros of 35 kV/cm. Based on these results and those observed in literature, L. monocytogenes STCC 5672 at any pH investigated has been shown as one of the most PEF resistant microorganism. Therefore, this microorganism should be considered as a possible target microorganism to define process criterion for PEF pasteurization.

Evaluation of pulsed electric fields technology for the improvement of subsequent carotenoid extraction from dried Rhodotorula glutinis yeast.

This research aims to evaluate whether the electroporation of Rhodotorula glutinis fresh biomass improved the subsequent extraction of carotenoids from dry biomass using supercritical CO2 and traditional solvent extraction. Supercritical CO2 extraction yields were low after all treatments assayed. Similarly, solvent extraction of carotenoids from untreated or PEF treated cells that were immediately freeze-dried after the pre-treatment was neither effective (extraction yield < 20% total content). Conversely, PEF-treatment and subsequent intermediate incubation in aqueous buffer for 24 h, followed by freeze-drying and extraction, led to a large improvement with the three solvents assayed (acetone, hexane, ethanol). Ethanol was the most efficient, reaching an extraction yield of 80% of total carotenoid, which represents a recovery of 267 µg/gdw. Torularhodin esters constituted the main carotenoid found in the extracts. This is of great interest, as ethanol is eco-friendly solvent and potential applications of torularhodin range from food to medical purposes.

A novel solution for registration of stereo digital torso images of scoliosis patients.

This paper presents a procedure for registration of a pair of stereo digital images giving an improvement in accuracy and speed over existing methods. It does so by a novel approach combining color based image segmentation and differential geometry. It involves three stages: image segmentation, adaptive local pixel matching, and deferential geometry in a tree weighted belief propagation procedure. The registration was compared to 2 existing registration procedures, segment-based adaptive belief propagation (adaptive BP) and color-weighted hierarchical belief propagation (hierarchical BP). A 3D scan of a mannequin was obtained and errors in reconstruction were measured for each of the 360 cross sections of the mannequin. The proposed procedure outperforms existing methods, particularly for high curvature regions and significantly large cross sections. Its accuracy of reconstruction ranged from 85-100% compared to 75-100% for other existing methods. It was 35% to 40% faster. This work provides a solution to the registration problem and is an important step in developing a cost effective technique for measuring torso shape and symmetry of scoliosis patients using stereo digital cameras.

Constrained intensity-based image registration: application to aligning human back images.

In this work, an accurate method to register multi-view images of the human torso is developed. In particular, a new framework that incorporates prior statistical knowledge about the registration is developed and tested. This framework leads to a computationally efficient procedure to accurately align images of the human torso. An intensity based image registration procedure is used to obtain the deformation fields by modelling them as both locally affine and globally smooth. Next, the estimated geometric deformation fields are analyzed in order to construct a prior deformation model. Two subspace analysis projection techniques are used to construct the subspaces of plausible deformations, namely principal component analysis (PCA) and independent component analysis (ICA). Accurate deformations are now guaranteed by projecting the locally computed geometric transformations onto the subspaces of plausible deformations. The proposed registration method was validated using high resolution images of the human torso. In order to handle the high resolution images, a multi-resolution framework was employed in the registration process. Experiments demonstrate promising performance in terms of mean square error and in the computational complexity. The main contribution of this work is the development of image registration method that uses subspace constraints to align images of the human torso. This method did not use the intra and inter image constraints used in most intensity based image registration algorithms in the literature.

Computer-assisted Cobb angle measurement on posteroanterior radiographs.

The Cobb angle method is the gold standard to assess severity of scoliosis. A computer-aided method was developed to provide a semi-automatic Cobb angle measurement during a scoliosis clinic. This study was to evaluate the reliability and accuracy of the developed method. Curve types were also tested. The computer method required enhancement of the contrast, normalization of the image size, and selection of the end-vertebrae on the radiographs before the automatic measurement started. The computer-aided process automatically identified the line segments that fitted to the endplates of the end-vertebrae. The Cobb angle was then calculated from the slopes of these lines. Seventy-six radiographs were randomly selected and categorized with Lenke's classification. Among them, 75 cases were used and categorized into 4 types: 1, 3, 5 and 6. One type 2 case was excluded. An orthopedic spine surgeon measured the radiographs manually, serving as the reference standard. Two observers used the developed method and measured twice. For each curve type, the inter-method, inter-observer, and intra-observer variability were analyzed by Intraclass correlation coefficients (ICC[2,1]). The ICC values were higher than 0.90 in all these types. The developed method was reliable to measure the Cobb angle and was not dependent on the curve type.

Preliminary validation of curve progression model for brace treatment.

Brace treatment is the most commonly used non-surgical treatment for adolescent idiopathic scoliosis (AIS). A brace compliance monitoring system consisting of a microcomputer and a force transducer was used to monitor how brace candidates used their braces during daily activates. A prediction model of the brace treatment outcome was developed based on 20 AIS subjects. Six subjects (1M, 5F) with AIS who had worn their braces for six weeks participated into this study. One month data was recorded during the study period. Knowing the risk progression at the beginning of brace treatment plus how brace subjects used their braces in terms of brace tightness and wear time during brace treatment yielded a predicted outcome which was compared to the final treatment outcomes with 2 years followed-up. This preliminary result demonstrated that the prediction model was able to predict the treatment outcome within +/-3.5 degrees.

Evaluation of the potential of ultrasound technology combined with mild temperatures to reduce cadmium content of edible crab (Cancer pagurus).

The consumption of crustaceans is correlated with certain health risks, particularly due to several highly toxic elements they contain, including cadmium (Cd). Although Cd content in one sole crab generally exceeds the total weekly recommended intake of cadmium as established by EFSA (especially in brown meat), efficient modern strategies to reduce Cd content in crabs still have not yet been developed. The objective of this research was therefore to evaluate the potential use of ultrasound technology in combination with temperature (50°-80 °C) with the purpose of releasing Cd from brown crab (Cancer pagurus), thereby reducing the Cd content in its meat. Female crabs were immersed in a water bath at 50, 65, and 80 °C in presence or absence of ultrasound; Cd concentration in the water was monitored along time. At the end of the process, Cd content in brown and white crab meat was likewise quantified. Treatment temperature did not bear an influence on the release of Cd in absence of ultrasound, but proved to be an important variable when ultrasound assisted the process. Ultrasound increased Cd release rates 8.7-, 2.1- and 2.7-fold in conjunction with the treatments at 50, 65 and 80 °C, respectively. The maximum percentage of Cd extracted (22.8%) was observed at 50 °C for an ultrasound input power of 200 W. These results have demonstrated for the first time that the application of ultrasound during the crab-cooking process could serve as an effective physical procedure for reducing the Cd content of crabs, thereby improving the product's safety for consumers.

Pulsed electric fields cause bacterial envelopes permeabilization depending on the treatment intensity, the treatment medium pH and the microorganism investigated.

The relationship between membrane permeabilization and loss of viability by pulsed electric fields (PEF) depending on the treatment intensity and the treatment media pH in two gram-positive (Lactobacillus plantarum, Listeria monocytogenes) and two gram-negative (Escherichia coli, Salmonella senftenberg 775W) bacterial species has been investigated. Loss of membrane integrity was measured as increased uptake of the fluorescent dye propidium iodide (PI). Non-permanent/reversible permeabilization was detected when cells stained with PI during PEF resulted in higher fluorescence than that measured in cells stained after PEF. Whereas loss of viability of the two gram-negative bacteria was correlated with the sum of non-permanent and permanent membrane permeabilization when treated at pH 7.0, in the case of the two gram-positives, loss of viability was correlated with a permanent loss of membrane integrity. At pH 7.0, the four bacteria exhibited reversible permeabilization. However, whereas the gram-positives capable of reversing permeabilization survived, the gram-negative cells died, despite their capacity to reverse permeabilization immediately after PEF. Thus, resealing is not necessarily related to the survival of PEF-treated cells. In contrast, when cells were PEF-treated at pH 4.0 a more complicated picture emerged. Whereas loss of viability was correlated with a permanent loss of membrane integrity in L. monocytogenes cells, in L. plantarum the degree of permeabilization was higher, and in the gram-negative strains, much lower than the percentage of inactivated cells. These results support the view that membrane permeabilization is involved in the mechanism of bacterial inactivation by PEF, but the nature of membrane damage and its relationship with cell death depends on the bacterial species and the treatment medium pH.