Rapid method for controlling the correct labeling of products containing common octopus (Octopus vulgaris) and main substitute species (Eledone cirrhosa and Dosidicus gigas) by fast real-time PCR

The TaqMan real-time PCR has the highest potential for automation, therefore representing the currently most suitable method for screening, allowing the detection of fraudulent or unintentional mislabeling of species. This work describes the development of a real-time polymerase chain reaction (RT-PCR) system for the detection and identification of common octopus (Octopus vulgaris) and main substitute species (Eledone cirrhosa and Dosidicus gigas). This technique is notable for the combination of simplicity, speed, sensitivity and specificity in an homogeneous assay.     

Identification of Cephalopod Species (Ommastrephidae and Loliginidae) in Seafood Products by Forensically Informative Nucleotide Sequencing (FINS)

ABSTRACT: An identification technique of commercially important cephalopods based on 16s RNA analysis was developed. A set of primers was designed to amplify a fragment of approximately 200 bp that presents enough variability for reliable species identification. Sequences from this fragment of 9 different authentic species were studied, genetic distances were measured, and a phylogenetic tree was constructed, with individuals of the same species grouped within the same cluster. Eight different types of commercial seafood products, mainly labelled as "squid rings", were analyzed and sequences were employed for species identification showing that FINS is a suitable technique for identification of processed cephalopods.     

Seasonal changes of fatty acids of cuttlefish Sepia officinalis L. (Mollusca: Cephalopoda) in the north eastern Mediterranean sea

The total lipids and seasonal variations in the fatty acids of the mantle of cuttlefish (Sepia officinalis) captured in the north eastern Mediterranean were investigated and the mantle was found to be a good source of polyunsaturated fatty acids (ω3 PUFAs, in particular). In all seasons, the major fatty acids in the cuttlefish mantle were observed to be palmitic acid (16:0), stearic acid (18:0), eicosapentaenoic acid (EPA, 20:5ω3) and docosahexaenoic acid (DHA, 22:6ω3). A comparison of the saturated fatty acid (29.5–36.8%), monounsaturated fatty acids (7.81–9.84%) and polyunsaturated fatty acids (43.7–49.6%) of the cuttlefish mantle revealed that polyunsaturated fatty acids (PUFA) constituted the highest proportion. The levels of DHA in the cuttlefish mantle in autumn, winter, spring and summer were 27.6%, 28.5%, 29.5% and 23.9%, while those of EPA were 16.8%, 15.4%, 14.7% and 13.9%, respectively.     

A heat-stable trypsin from the hepatopancreas of the cuttlefish (Sepia officinalis): Purification and characterisation

Thermostable trypsin from the hepatopancreas of Sepia officinalis was purified by fractionation with ammonium sulphate, Sephadex G-100 gel filtration, DEAE-cellulose an ion-exchange chromatography, Sephadex G-75 gel filtration and Q-Sepharose anion-exchange chromatography, with a 26.7-fold increase in specific activity and 21.8% recovery. The molecular weight of the purified enzyme was estimated to be 24,000 Da by SDS-PAGE and size exclusion chromatography. The purified enzyme showed esterase specific activity on Nα -benzoyl-L-arginine ethyl ester (BAEE) and amidase activity on Nα -benzoyl-DL-arginine-p-nitroanilide (BAPNA). The optimum pH and temperature for the enzyme activity were pH 8.0 and 70 °C, respectively, using BAPNA as a substrate. The enzyme was extremely stable in the pH range 6.0–10.0 and highly stable up to 50 °C after 1 h of incubation. The purified enzyme was inhibited by soybean trypsin inhibitor (SBTI) and phenylmethylsulphonyl fluoride (PMSF), a serine-protease inhibitor. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGKESSPYNQ. S. officinalis trypsin, which showed high homology with trypsins from marine vertebrates and invertebrates, had a charged Lys residue at position 5 and a Ser residue at position 7, where Tyr and Cys are common in all marine vertebrates and mammalian trypsins. Further, the enzyme had an Asn at position 11, not found in any other trypsins.     

Development of a genetic method for the identification of salmon,trout, and bream in seafood products by means of PCR–RFLP and FINS methodologies

In the present study, two alternative methods for identifying 13 salmon, trout and bream species were developed. Both of them are based on polymerase chain reaction (PCR) amplification of a cytochrome b gene fragment. Subsequently, different techniques were assayed to assign the PCR amplicons previously obtained to particular species. The first one is based on the restriction fragment length polymorphism (RFLP) and includes three endonucleases for generating species-specific restriction profiles, while the second one is based on the phylogenetic analysis of DNA sequences. The main novelty of this work lies in the applicability of the developed methods to all kinds of processed products, including those undergoing intensive processes of transformation, as for instance canned foods. Finally, the methods were applied to 25 commercial samples including some that had been subjected to intensive thermal treatment, allowing the detection of those incorrectly labeled (16%). Therefore, these methods are useful to check the fulfillment of labeling regulations for seafood products, verify the correct traceability in commercial trade, and for fisheries control.     

Improvement of functional properties and antioxidant activities of cuttlefish (Sepia officinalis) muscle proteins hydrolyzed by Bacillus mojavensis A21 proteases

Functional properties and antioxidant activities of cuttlefish (Sepia officinalis) muscle protein hydrolysates, with different degrees of hydrolysis (DH from 7.3% to 18.8%), obtained by treatment with Bacillus mojavensis A21 alkaline proteases were investigated. Protein contents for all freeze-dried cuttlefish muscle protein hydrolysates (CMPHs) ranged from 80% to 86%. For the functional properties, hydrolysis by A21 proteases increased (p < 0.05) protein solubility to above 78% over a wide pH range (2.0–11.0). However, the interfacial activities (emulsion activity index, emulsion stability index, foaming capacity and foaming stability) decreased with the increase of the DH. All CMPHs exhibited significant metal chelating activity and DPPH free radical-scavenging activity, and inhibited linoleic acid peroxidation. Antioxidant properties of protein hydrolysates increased with protein hydrolysis and the highest activities were obtained at DH of 16%. The IC₅₀ values for DPPH radical-scavenging and metal chelating activities were found to be 0.52 ± 0.01 mg/ml and 0.67 ± 0.13 mg/ml. The obtained results suggested that functional properties and antioxidant activities of cuttlefish muscle protein hydrolysates were influenced by the degree of hydrolysis.The composition of amino acids of undigested and hydrolyzed proteins was determined. CMPHs have a high percentage of essential amino acids such as arginine, lysine, histidine and leucine. They have a high nutritional value and could be used as supplement to poorly balanced dietary proteins.     

Occurrence of psocids and natural predators on organic rice in Calasparra (Murcia, Spain)

Five Psocoptera species: Lepinotus reticulatus, Liposcelis entomophila, Liposcelis mendax, Liposcelis bostrychophila and Liposcelis paeta were identified on organic paddy stored in south-east Spain. The main natural predator associated with the psocids in the study area was the pseudoscorpion Withius piger, reported for the first time within the Iberian Peninsula.     

Low and high temperatures for the control of cowpea beetle, callosobruchus maculatus (F.) (coleoptera: Bruchidae) in chickpeas

Chickpea (Cicer arietinum L.; Leguminasae) is an important pulse crop grown, around the world. The whole grain of chickpea is damaged by the cowpea seed beetle, Callosobruchus maculatus (Coleoptera: Bruchidae), which is the most important field-carry-over storage pest of pulses. The management of this insect in storage using chemicals leads to insecticide residues in grains and insecticide resistance development in insects. Thermal disinfestation is one of the means of physical insect control. Eggs, larvae, pupae and adults were held at 42 or 0 °C for varying durations. Pupae and adults were equally heat tolerant. The lethal time to reduce survival by 50% (LT₅₀) at 42 °C for eggs, larvae, pupae and adults were 18, 57, 78 and 71 h, respectively. Pupa was the most cold-tolerant stage. The LT₅₀ at 0 °C for eggs, larvae, pupae and adults were 3, 8, 10 and 4 d, respectively. The LT₅₀ for pupae were 4907, 4262, 336, 36 and 13 min at the grain temperature of 42, 45, 50, 55 and 60 °C, respectively. The LT₅₀ of pupae at 0,−5,−10 and −15 °C were 274, 122, 7 and 2 h, respectively.     

Production of salami from beef, horse, mutton, Blesbok (Damaliscus dorcas phillipsi) and Springbok (Antidorcas marsupialis) with bacteriocinogenic strains of Lactobacillus plantarum and Lactobacillus curvatus

Lactobacillus plantarum 423, producer of bacteriocin 423, Lactobacillus curvatus DF38, producer of curvacin DF38, and a bacteriocin-negative mutant of L. plantarum 423 (423m) were evaluated as starter cultures in the production of salami from beef, horse, mutton, Blesbok (Damaliscus dorcas phillipsi) and Springbok (Antidorcas marsupialis). Growth of L. plantarum 423 and L. curvatus DF38 was best supported in Blesbok salami, as revealed by the highest growth rate during sweating, cold smoking and maturation, and final cell numbers after 70 days (1 × 10⁸ and 5 × 10⁷ cfu/g, respectively). Growth of Listeria innocua was the best suppressed in Blesbok salami fermented with L. plantarum 423 and L. curvatus DF38. Growth of L. innocua in horse salami was best suppressed when fermented with L. curvatus DF38. The final pH of salami fermented with L. plantarum 423 and L. plantarum 423m was slightly lower (4.4) compared to the pH of salami produced with L. curvatus DF38 (pH 4.7). No significant differences (P > 0.05) were recorded by a trained sensory taste panel amongst the three starter cultures regarding colour and venison like aroma. Horse, Blesbok and Springbok salami were rated significantly higher (P ? 0.05) in salami flavour than mutton salami, which was rated the lowest for this attribute. Blesbok salami was rated the highest for sour meat aroma, while beef salami was rated the lowest. Springbok salami was rated the highest in terms of oily mouth feel. Beef salami had the most compact structure and horse salami the softest structure of all meat types fermented. In general, salami produced with L. plantarum 423 yielded the best sour meat aroma, colour, texture, venison like flavour, sour meat flavour and oily mouthfeel and is considered superior to the L. plantarum mutant (strain 423m) and L. curvatus DF38.     

Effectiveness of products from four locally grown plants for the management of Acanthoscelides obtectus (Say) and Zabrotes subfasciatus (Boheman) (both Coleoptera: Bruchidae) in stored beans under laboratory and farm conditions in Northern Tanzania

The effectiveness of whole or powdered leaves (botanicals) from four locally grown plant species applied at a rate of 1.5 kg per 100 kg beans (Phaseolus vulgaris) against Acanthoscelides obtectus and Zabrotes subfasciatus was compared under laboratory and farm conditions. In the laboratory, Chenopodium ambrosioides, applied as powder or as whole leaves, was the most effective, with 100% mortality of adult insects in less than three days and no progeny. Less C. ambrosioides (about 200 g per 100 kg beans) still resulted in 100% mortality within 24 h. Tagetes minuta applied as powder also increased mortality and reduced oviposition and progeny production significantly. The other treatments - T. minuta applied as leaves, and Azadirachta indica or Cupressus lusitanica applied as powder or as whole leaves - had no significant effects upon mortalities, oviposition rate, or progeny production compared with control treatments. When the rate of application was increased to about 8.3 kg per 100 kg beans, there was a slight increase in mortality using T. minuta and A. indica, but not with C. lusitanica. An additional trial with C. ambrosioides from different collections and with plants at different stages of development revealed considerable variations in the efficacy of the treatment.In the on-farm trials, A. indica-seed powder was the most effective treatment, followed respectively by leaf powders of C. ambrosioides, C. lusitanica and T. minuta. All treatments were significantly more effective than the control in reducing the numbers of live insects; they also reduced numbers of damaged beans and maintained germination rates after 5 months of storage. The results of evaluations of the treatments made by farmers just after the trials and five years later are reported.     

Resistance in maize to the Larger Grain Borer, Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae)

Host plant resistance in maize can be a useful component of Integrated Pest Management (IPM) of the Larger Grain Borer (LGB), Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae). The germplasm bank of the International Maize and Wheat Improvement Centre (CIMMYT) has more than 14,000 maize accessions from all over the world. In 1993, from the seed regeneration nursery of Caribbean land races, 105 land races were selected using good plant type as the criterion. When grains of these land races were infested with LGB in the laboratory, 19 showed resistance as evidenced by the low amount of powder (chewed uneaten maize plus feces) formed 140 days after the infestation. Through a series of infestation, selection and inbreeding (self-pollination of land races) over four generations, S₃ maize ears were generated. These ears showed a high level of resistance as indicated by a low powder production relative to the susceptible control. The LGB reproduction was adversely affected on resistant ears as indicated by the small size of adult populations. Thus, antibiosis could be the mechanism of resistance operating within the S₃ progenies of selected land races. Such sources of resistance can be very useful in developing maize populations/hybrids for use in IPM of LGB.     

Development of a method for the identification of scombroid and common substitute species in seafood products by FINS

In the present work, a method for the authentication of scombroid products was developed, by means of FINS (Forensically Informative Nucleotide Sequencing) technique (Polymerase Chain Reaction (PCR) followed by phylogenetic analysis). The methodology developed allows the identification of most important scombroid species using the mitochondrial cytochrome b as molecular marker. Due to the different commercial value of the species belonging to this family, substitutions between species in seafood products can take place.     

Insecticidal effect of Steinernema feltiae (Filipjev) (Nematoda: Steinernematidae) against Tribolium confusum du Val (Coleoptera: Tenebrionidae) and Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) in stored wheat

The confused flour beetle, Tribolium confusum, and the Mediterranean flour moth, Ephestia kuehniella, are important pests of stored grain products. The insecticidal effect of three strains (UK 76 [=Nemasys], USA/SC, and Hawaii) of the entomopathogenic nematode Steinernema feltiae was determined in the laboratory, in wheat, against these pests. The nematodes were applied at three dose rates: 100, 300 and 900 nematodes/insect individual. The Hawaii strain was most virulent against T. confusum adults and larvae, with a significant dose effect in the case of the larvae. Larval mortality of this species reached 79% and 100% after 7 and 14 d of exposure to the nematodes, respectively, at the highest dose applied. On the other hand, adult mortality of T. confusum did not exceed 66%. In the case of E. kuehniella larvae, USA/SC performed best causing 52% and 69% mortality after 7 and 14 d exposure, respectively, at the highest dose tested. Since very few data are available on the effect of entomopathogenic nematodes against these pests, it is concluded that the Hawaii and USA/SC strains of S. feltiae should be further investigated as promising biological control agents for T. confusum and E. kuehniella.     

Effects of hydrogen peroxide and Fenton’s reagent on the properties of film from cuttlefish (Sepia pharaonis) skin gelatin

Properties of film from cuttlefish (Sepia pharaonis) ventral skin gelatin without and with partial hydrolysis (1.2% degree of hydrolysis), as influenced by H₂O₂ and Fenton’s reagent at different levels, were investigated. Films treated with H₂O₂ (0.01–0.04 M) and Fenton’s reagent [H₂O₂ (0.01–0.04 M) + FeSO₄ (0.001–0.004 M)] had higher tensile strengths (TS) but similar or lower elongations at break (EAB), compared with the control film (p < 0.05). Slight differences in water vapour permeability (WVP) were observed for all films. Films treated with Fenton’s reagent had a lower L^∗-value but higher a^∗-, b^∗- and ΔE^∗-values, while films treated with H₂O₂ had lower b^∗-values (p < 0.05), than had the control film. Cross-linking was pronounced in films treated with H₂O₂ or Fenton’s reagent and was associated with increased heat stability. Films treated with Fenton’s reagent had the lowest solubility in water (p < 0.05). However, fragmentation more likely took place when Fenton’s reagent (at a higher level) was used. Generally, similar results were noticeable between films from gelatin with and without partial hydrolysis. Thus, H₂O₂ and Fenton’s reagent directly affected the properties of film from cuttlefish skin gelatin, regardless of hydrolysis.     

A laboratory assessment of the potential of different strains of the entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) to control Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) in stored cowpea

Twelve indigenous and exotic isolates of Beauveria bassiana and Metarhizium anisopliae were evaluated for their virulence and their ability to suppress populations of Callosobruchus maculatus in stored cowpea. LT₅₀ values ranged from 3.11 to 6.12 days following immersion in aqueous suspensions containing 1×10⁸ conidia ml⁻¹. Indigenous isolates that had been recovered from C. maculatus were more virulent in laboratory bioassays than exotic isolates from other insects. The two isolates with the shortest LT₅₀ values were compared in dose-response assays by immersion and by exposure to cowpea grains treated with dry conidia. In both assays B. bassiana 0362 was consistently more virulent than M. anisopliae 0351. By immersion, LC₅₀ values on day 6 post-treatment were 9.10×10⁴ and 7.10×10⁵ conidia ml⁻¹ for B. bassiana 0362 and M. anisopliae 0351, respectively. Exposure to treated grains gave LC₅₀ values of 1.15×10⁷ and 4.44×10⁷ conidia g⁻¹ grain for B. bassiana 0362 and M. anisopliae 0351, respectively. In 1 kg batches of cowpea stocked with 50 adult C. maculatus, B. bassiana 0362 at both 1×10⁷ and 1×10⁸ conidia g⁻¹ grain led to significant adult mortality and reduced F₁ emergence relative to untreated populations. At 1×10⁸ conidia g⁻¹ the effect of the fungus persisted into the F₁ generation. The net reproductive rates, R₀, measured 26 days after insects were released were 5.16 and 7.32 for the high and low doses compared to 9.52 for the untreated control.     

Study of the content of cadmium,chromium and lead in bivalve molluscs of the Pacific Ocean (Maule Region,Chile)

The concentration of Cd, Cr and Pb in the following species Ameghinomya antiqua, Aulacomya atra and Mytilus chilensis, bivalves from Iloca, Constitución and Pelluhue, coastal towns located in the Maule Region, Chile was determined. Representative tissue samples of each species were analysed by using atomic absorption spectroscopy with flame. Validation of methodology was carried out using a certified reference material (TORT-1). The ranges of concentrations found for Cd, Cr and Pb in the 3 species were, respectively, 0.21–4.32, 0.38–12.62 and 0.43–31.10 mg kg⁻¹ dry weight. In relation to the results obtained and the average of each individual metal in the different species of bivalves, Pb concentrations were found in higher levels rather than those of other metals reported by other authors in a similar work, this element constitutes a potential health hazard for the consumers of shellfish. The results were statistically evaluated for possible correlations in the content of metals in different species and in different sites of origin.     

Co-amplification and sequencing of a cytochrome b fragment affecting the identification of cattle in PCR-RFLP food authentication studies

Food authentication studies based on PCR-RFLP analysis are frequently targeted to well-conserved mitochondrial sequences, such as certain regions of the cytochrome b (cytb) gene. The use of mitochondrial L14841/H15149 universal cytb-targeted primers in PCR-RFLP assays revealed the existence of a complex restriction pattern in three genetically-unrelated Iberian (Northern Spain) cows, this being due to the simultaneous co-amplification of two 359 bp cytb fragments. Microsatellite analysis of 11 bovine-specific loci confirmed no familiar linkage among the animals investigated. Both co-amplification products were successfully separated by specific cleavage with endonucleases RsaI and MvaI, which allowed the recovery of each amplification product, respectively. The new co-amplified cytb fragment described in this study was successfully sequenced, and exhibited a significantly high homology (95.1–99.3% range) with respect to mitochondrial sequences previously described for a Bos indicus specimen and for another two Asian Bos taurus, this underlining that its presence in cattle may be more extended than initially thought. In contrast, the homology with the cytb sequence widely accepted for B. taurus was only 89.6%. The results presented in this work imply that food authentication studies by PCR-RFLP analysis may be complicated in the case of cattle by the co-amplification of two different cytb fragments.     

Direct characterisation of phenolic antioxidants in infusions from four Mapuche medicinal plants by liquid chromatography with diode array detection (HPLC-DAD) and electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS)

A simple, fast and direct method was developed for the qualitative analysis of phenolic constituents from infusions of Mapuche medicinal plants. Teas made of Linum chamissonis Schiede, Quinchamalium chilensis Mol., Adesmia emarginata Clos. and Escallonia illinita K. Presl. were analysed by high-performance liquid chromatography with diode array detector (HPLC-DAD) and electrospray mass spectrometry (ESI-MS). This technique allowed for the first time the tentative identification of 16 phenolic compounds in E. illinita, 27 in Q. chilensis, 10 in L. chamissonis and 19 in A. emarginata. The compounds were mainly phenolic acids, flavonoid glycosides, anthocyanins and tannins. The total phenolic and flavonoid content of the infusions was assessed as well as the free radical scavenging capacity measured by the bleaching of a solution of the 2,2-diphenyl-1-picrylhydrazyl (DPPH⁻) radical. From the four species, Q. chilensis exhibited the strongest antioxidant activity with highest total phenolic and flavonoid content.     

Sensory,microbiological and chemical assessment of the freshness of red mullet (Mullus barbatus) and goldband goatfish (Upeneus moluccensis) during storage in ice

The shelf life of red mullet and goldband goatfish during ice storage were studied in terms of sensory, microbiological and chemical changes. The sensory acceptability limit was 8 days for goldband goatfish (Upeneus moluccensis) and 11 days for red mullet (Mullus barbatus) stored in ice. The TVC level was correlated with sensory assessment. The TVC exceeded 7 log cfu g⁻¹ after 8 days for goldband goatfish, and 11 days for red mullet. At the end of storage period, pH, TVB-N, TBA, FFA and PV for red mullet were 7.84, 47.19 mg/100 g, 0.69 mg MA kg⁻¹, 1.17% oleic acid and 1.58 meq O₂/kg and for goldband goatfish they were 7.53, 43.97 mg/100 g, 0.74 mg MA kg⁻¹, 1.62% oleic acid and 1.68 meq O₂/kg, respectively. In red mullet, agmatine, serotonin, histamine and dopamine became the dominant amines, reaching 7.30, 5.97, 2.52 and 2.31 mg/100 g, respectively. Also the dominant amines for goldband goatfish were 4.37, 3.88, 3.38 and 2.00 mg/100 g for histamine, agmatine, dopamine and putrescine, respectively.     

Identification of female adult Lasioderma serricorne (F.) by simple external observation of the abdomen

Lasioderma serricorne (F.) adults were examined under a stereomicroscope after being placed in 70% alcohol or benzene for about 5 min. The urosternites were thus rendered transparent and the V-shaped apodeme (important sexual characteristic of the female) was made visible through simple external observation, without dissection of their abdomen.