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Anisakis simplexis a fish parasite able to induce allergic reactions in humans infected when eating raw or undercooked fish parasitized with viable third-stage larvae. Some authors claim that exposure to nonviable Anisakismaterial can result in allergic symptoms in previously sensitized patients, indicating that parasite allergens are resistant to the thermal treatments of usual cooking procedures. Furthermore, some patients report symptoms after eating canned fish. The aim of this work was the analysis of parasite allergen stability in heating to 121°C in an autoclave to simulate the thermal process applied to canned fish. Third-stage larvae were subjected to autoclaving for 20, 40, and 80 min, and parasite crude extracts were analyzed by electrophoresis, immunoblotting, and a flow-cytometric basophil activation test. Allergens resistant to autoclaving were separated by reversed-phase high-performance liquid chromatography and identified by ion trap mass spectrometry. Protein analysis by sodium dodecyl sulfate—polyacrylamide gel electrophoresis showed that autoclaving considerably reduced the number and intensity of identifiable protein bands in a time-dependent manner. Several allergens were detected by immunoblotting with a pool of A. simplexallergic patients' sera after autoclaving. Allergens of 9 and 14 kDa resistant to autoclaving were identified as Ani s 4 and Ani s 1 allergens, respectively. Functional analysis showed that allergens retain their capacity to activate basophils even after autoclaving for 80 min. In conclusion, some relevant A. simplexallergens retain their capacity to bind immunoglobulin E and activate basophils after being subjected to autoclaving, which is a method equivalent to that used in industrial canning processes.

Inactivation of parasites in food by microwave treatment may vary due to differences in the characteristics of microwave ovens and food properties. Microwave treatment in standard domestic ovens results in hot and cold spots, and the microwaves do not penetrate all areas of the samples depending on the thickness, which makes it difficult to compare microwave with conventional heat treatments. The viability of Anisakis simplex(isolated larvae and infected fish muscle) heated in a microwave oven with precise temperature control was compared with that of larvae heated in a water bath to investigate any additional effect of the microwaves. At a given temperature, less time was required to kill the larvae by microwaves than by heated water. Microwave treatment killed A. simplexlarvae faster than did conventional cooking when the microwaves fully penetrated the samples and resulted in fewer changes in the fish muscle. However, the heat-stable allergen Ani s 4 was detected by immunohistochemistry in the fish muscle after both heat treatments, even at 70°C, suggesting that Ani s 4 allergens were released from the larvae into the surrounding tissue and that the tissues retained their allergenicity even after the larvae were killed by both heat treatments. Thus, microwave cooking will not render fish safe for individuals already sensitized to A. simplexheat-resistant allergens.

AbstractFish-borne parasitic zoonoses such as Anisakiasis were once limited to people living in countries where raw or undercooked fish is traditionally consumed. Nowadays, several factors, such as the growing international markets, the improved transportation systems, the population movements, and the expansion of ethnic ways of cooking in developed countries, have increased the population exposed to these parasites. Improved diagnosis technology and a better knowledge of the symptoms by clinicians have increased the Anisakiasis cases worldwide. Dietary recommendations to Anisakis-sensitized patients include the consumption of frozen or well-cooked fish, but these probably do not defend sensitized patients from allergen exposure. The aim of our work was to develop a sensitive and specific method to detect and quantify Anisakis simplexallergens in fish muscle and its derivatives. Protein extraction was made in saline buffer followed by preparation under acid conditions. A. simplexantigens were detected by IgG immunoblot and quantified by dot blot. The allergenic properties of the extracts were assessed by IgE immunoblotting and basophil activation test. We were able to detect less than 1 ppm of A. simplexantigens, among them the allergen Ani s 4, in fish muscle with no cross-reactions and with a recovery rate of 82.5%. A. simplexantigens were detected in hakes and anchovies but not in sardines, red mullets, or shellfish. We detected A. simplexallergens in cooked hakes and also in hake stock. We proved that A. simplexallergens are preserved in long-term frozen storage (−20°C ± 2°C for 11 months) of parasitized hakes. Basophil activation tests have proven the capability of the A. simplex–positive fish extracts to induce allergic symptoms.

Heat treatments (40 to 94°C, 30 s to 60 min) were applied to different batches of Anisakis simplexL3 larvae isolated from hake ovaries and viscera to study the effect of heat on the viability of the larvae measured as mobility, emission of fluorescence under UV light, and changes in color after staining with specific dyes, and on A. simplexantigenic proteins. The aim was to determine the lowest time-temperature conditions needed to kill the larvae to avoid anisakiasis in consumers, and to evaluate whether high temperature modifies the antigenicity of A. simplexextracts. Heating at 60°C for 10 min (recommended by some authors) was considered unsafe, as differences in viability between batches were found, with some larvae presenting spontaneous movements in one batch. At higher temperatures (≥70°C for ≥1 min), no movement of the larvae was observed. Antigenic protein Ani s 4 and A. simplexcrude antigens were detected in the larvae heated at 94 ± 1°C for 3 min. This indicates that allergic symptoms could be provoked in previously sensitized consumers, even if the larvae were killed by heat treatment.

This article examines the viability of and the alterations to the larval cuticle and the pattern of the antigens released when live or frozen Anisakis simplexlarvae were treated with acid and pepsin. The results showed that freezing did not greatly alter the larva body. If ruptures were observed, the antigen release to the incubation media was not enhanced, and most of the antigenic content was retained inside the bodies of the larvae. The immunoblotting assay demonstrated that most of the antigens released, including the allergen Ani s 4, were resistant to pepsin. Freezing killed the larvae, but their survival was not compromised by acid treatment or pepsin digestion when kept chilled. All these findings support recommendations about freezing fish for consumption raw or undercooked to prevent human infection by A. simplexlarvae. However, our data show that the antigenicity of the larvae is preserved after freezing and may explain why some sensitized patients develop symptoms after ingestion of infested frozen fish.

Anisakis simplexis a fish parasite that is a public health risk to those consuming raw or poorly cooked marine fish and cephalopods because of the possibility of becoming infested with live larvae. In humans, penetration of the larvae into the gastrointestinal track can cause acute and chronic symptoms and allergic anisakiasis. Excretion and secretion products released by the larvae are thought to play a role in migration through the tissues and induce an immunoglobulin E–mediated immune response. The aim of this preliminary study was to detect parasite antigens and allergens in fish tissues surrounding the migrating larvae. Hake and anchovy fillets were artificially parasitized with Anisakislarvae and stored in chilled conditions for 5 days. Larvae were evaluated for fluorescence, fish muscle tissue was examined with transmission electron microscopy, and immunohistochemical reactions of two rabbit polyclonal antisera against a parasite crude extract and the allergen Ani s 4 were recorded. Larvae immediately migrated into the fish muscle, and no emission of bluish fluorescence was observed. Fish muscle areas in contact with the parasite showed disruptions in the structure and inclusion of granules within sarcomeres. Both parasite antigens and the Ani s 4 allergen were located in areas close to the larvae and where sarcomere structure was preserved. These findings indicate that parasite antigens and allergens are dispersed into the muscle and might cause allergic symptoms such as dyspnea, vomiting, diarrhea, urticaria, angioedema, or anaphylaxis in some individuals sensitive to A. simplex.

Ingestion of fish parasitized with Anisakislarvae can produce infestation and/or allergy in consumers. Technological and food processing treatments have been applied to parasitized fish in order to kill the larvae and avoid the infestation; however, their influence on allergenicity has not been studied. Four lots of hake (Merluccius merluccius) steaks artificially parasitized with Anisakislarvae were subjected to two storage chilling (5°C ± 1°C) and freezing (−20°C ± 1°C) treatments and two food processing treatments of heat (final temperature 86.3°C) and microwave (final temperature 66.9°C) and studied by scanning electron microscopy, environmental scanning electron microscopy (ESEM) (acid [pH = 2] and water preparations), and emission of fluorescence. Anisakislarvae were resistant to acid conditions, remaining alive after treatment. Larvae in the heat- and microwave-treated lots presented coagulated and disrupted zones in the cuticle with release of fluids. The cylindrical shape changed to a dehydrated appearance mainly observed by ESEM. Fluorescence was only noticeable in the frozen larvae. Larvae without apparent changes, together with dehydrated ones, were observed by ESEM in the frozen lot; nevertheless, no disruptions in the cuticle were perceptible. Further studies are needed in order to elucidate if the changes observed in the cuticle reduce the resistance of the parasites to the action of gastric enzymes in the gastrointestinal tract and to determine the release of allergens to the flesh by the live larvae during chilled storage of the fish.

Abstract. The microflora of farmed gilthead seabream stored in ice and the effect of washing of the fish with tap water were examined in different culture media and incubation temperatures. Washing reduced the number of microorganisms in all culture conditions studied. For unwashed and washed lots the highest number of bacteria were obtained on spread plates in Iron Agar + 0.5% NaCl incubated at 15 °C, H₂S-producing microorganisms constituting a high percentage of the total flora. The correlation, up to the sensory rejection point, between bacterial counts and sensory score for the raw fish varied with washing and with the culture conditions, and was higher for the H₂S-producing microorganisms.

Abstract. This paper examines the influence of the slaughter method and the post-mortem treatment (gutting) on the quality during ice storage of cultured gilthead seabream collected under industrial conditions. The choice of slaughter method had no great effect on the main biochemical indices, physicochemical parameters or sensory assessment, although there were slight microbiological differences. The expected improvement in quality using "humane" as opposed to commercial methods was not observed and was probably masked by the pre-slaughter stress affecting industrially harvested fish. The influence of gutting materialised as a decrease in the intensity of rigor mortis and a drop in microbial load, largely at the outset of shelf life. All the other parameters considered were almost unaffected by gutting. The physicochemical quality indices normally used to determine fish spoilage during ice storage are not suitable for establishing the quality of immature farmed gilthead seabream.

This work studied the performance of the artificial digestion method in terms of recovery and viability of Anisakis simplexthird-stage larvae (L3) when previous treatments given to the infected fish muscle may accidentally render viable larvae. For that: a) hake mince was spiked with 10 L3/75g mince, frozen at −10, −15, −20, and −30°C and immediately thawed, or stored for 12 or 24h, and subjected to pepsin digestion; b) the mince was spiked under the same conditions, frozen at the above temperatures and thawed immediately. After manual recovery, L3 were assessed for viability, used to spike again the minced fish and subjected to pepsin digestion; c) the mince was spiked with 10 L3 which were: i) living (i.e. chilled), ii) freeze-surviving (live L3 had been previously recovered after freezing at −10°C), or iii) dead (frozen at −30°C or−80°C), and then subjected to pepsin digestion. Results showed that the artificial digestion method kills a significant number of larvae that may have survived freezing and thus may underestimate the number of viable larvae in a given batch. The method may also underestimate the infection level of fish batches containing dead larvae. It is suggested to take these limitations into account when designing digestion protocols for specific applications, especially when there is a risk of insufficiently treated or cooked fish batches or ready-to-eat foods.

In a previous work it was suggested that the texture of kamaboko (set and cooked) gels made from sardine surimi under varying setting conditions was predetermined by the specific matrix forming in each suwari (set) gel. This paper describes the microstructure of the networks formed in suwari and kamaboko gels set at 25, 35 and 40 °C for 30 or 60 min as examined by scanning electron microscopy (SEM). Cooking conditions for kamaboko gels were fixed at 90 °C for 30 min; other preparation conditions were invariable. At low magnification (≤×500) the gel matrixes were compact, with practically no differences among lots. At higher magnification (×20 000), the suwari gel matrixes formed at low temperature consisted of globules. At higher temperatures the globules joined up to form fibrillar structures (fibres) and zones of disordered globule aggregation (coagula); at longer setting times, lateral bonding of the fibres became apparent. Kamaboko gels produced from unstructured globular matrixes exhibited only a few fibrillar zones and large areas of coagula. Where there was already an incipient fibrous formation, these developed into individual fibres or bundles of fibres that correlated with the best texture characteristics. Suwari gels with extensive lateral bonded fibres gave rise to kamaboko gels with a highly compact appearance under SEM; this correlated with a decline in texture values. These different structures suggest that the protein–protein bonds in the suwari networks have different levels of stability to heat, and these levels determine whether or not the proteins can subsequently be reorganised when the kamaboko gel forms. © 1999 Society of Chemical Industry

Abstract:  Aggregation in minced hake muscle (Merluccius merluccius) during storage at –20  C was studied in conditions where there is progressive deterioration of functionality and texture as measured by apparent viscosity and shear resistance. Natural actomyosin was extracted with 0.6 M NaCl over a period of 49 weeks. Insoluble residue was extracted successively with 2% sodium dodecyl sulphate (SDS) and 2% SDS plus 5% β-mercaptoethanol (ME) solutions. SDS–polyacrylamide gel electrophoresis was performed on the extracted fractions. The results showed a 75% decrease in 0.6 M NaCl extractability by the end of the storage period. Initially the remaining precipitate was all extracted in 2% SDS and although the amount extracted in this solution increased as storage time progressed, its proportion decreased, accounting for as little as 40–50% of the protein aggregate by the end of storage. The proteins most involved in formation of the aggregate not extracted in 0.6 M NaCl were myosin and actin. Neither of these proteins was fully recovered in the fractions extracted with 0.6 M NaCl, 2% SDS, or 2% SDS plus 5% ME, and therefore it was inferred that they were forming part of the aggregates, bound by covalent bonds.

Zusammenfassung In einigen Ländern Südeuropas ist es üblich, verschiedene Mengen an Salz in das erforderliche Eis zu geben für die Konservierung der Sardinen. Dadurch wird die Textur verbessert und die Lagerzeit verlängert. Die so behandelten Sardinen kann man zur Herstellung verschiedener Produkte verwenden. Aus diesem Grund sollte in der Untersuchung festgestellt werden, ob Salz die Eigenschaften der gekühlten Sardinen beeinflußt, und zwar die Proteinlöslichkeit, die Emulsionsfähigkeit und die scheinbare Viscosität. Der Grund dieser Untersuchung ist die Eignung des Fleisches für gewisse technologische Prozesse zu prüfen. Das Salz im Eis vermindert am Anfang die Proteinlöslichkeit; bei zunehmender Lagerzeit nimmt die Emulsionsfähigkeit und die scheinbare Viscosität ab. Wenn Salz im Eis nicht vorhanden ist, dann bleiben diese Eigenschaften unverändert oder sie erhöhen sich. Als Endergebnis verlängert das Salz die Lagerzeit, und gleichzeitig wird die Protein-Funktion stärker reduziert als bei nicht behandelten Sardinen. Deswegen sind die unbehandelten Sardinen für technologische Prozesse weniger geeignet.

Zusammenfassung Der Einfluß von zugesetztem Kabeljauleberöl (CLO) und oxidiertem Kabeljauleberöl (OCLO) auf die Konsistenz von zerkleinertem Fischfleisch aus Seehecht (Merlucdus merlucdus L.), Flügelbutt (Lepidorhombus whiffiagonis W.) und Sardine (Sardina pilchardus W.) wurde während der Gefrierlagerung (bei-18 °C) gemessen. Die Ergebnisse zeigen, daß weder der Zusatz von neutralem noch der von oxidiertem Öl, selbst bei erheblicher Ranzigkeit, den Scherwiderstand im Fisch beeinflußt, wenn kein Formaldehyd gebildet wurde. Bei der Fischart aber, die während der Gefrierlagerung Formaldehyd bildet, wie z. B. Seehecht, können die Werte des Scherwiderstandes in Gegenwart von neutralen und oxidierten Lipiden gegen Ende der Lagerperiode niedriger sein als bei Abwesenheit der Lipide; wahrscheinlich wegen verminderter Formaldehydbildung. Obwohl es sehr schwierig ist, die Bedeutung einer einzelnen Verbindung zu bewerten, scheinen diese Ergebnisse zu zeigen, daß Formaldehyd für die Konsistenzveränderungen in tiefgefrorenem Fisch einen viel wichtigeren Faktor darstellt als oxidierte Lipide.

Zusammenfassung Es wurden die Veränderungen im elektrophoretischen Profil myofibrillärer Proteine in Muskeln von Blauem Wittling (Micromesistius poutassou R), Stöcker (Trachurus trachurus L) und Makrele (Scomber scombrus L) untersucht — Arten, deren Zustand sich während der Gefrierlagerung auf unterschiedliche Weise verschlechtert. Diese Fische wurden in zwei verschiedenen Jahreszeiten (Winter und Sommer) gefangen, in denen auch zu erwarten ist, daß sich der physiologische Zustand verändert und ein Jahr lang bei -18°C tiefgefroren gelagert. Die elektrophoretischen Muster änderten sich je nach Fischart während der Tiefkühllagerang. Aus dem Vergleich der MHC/A-Verhältnisse ist ersichtlich, daß Blauer Wittling die Art ist, die sich am meisten verändert und dies im Sommer intensiver als im Winter stattfindet. Bei Stöcker und Makrele ändert sich das MHC/A-Verhältnis auf ähnliche Weise im Winter, während sich Stöcker im Sommer als die stabilste Spezies zeigte. In allen Fällen lag die MHC/A-Verringerung grundsätzlich an der MHC-Veränderung und diese Wirkung war im Blauer Wittling besonders deutlich zu sehen. Bei allen untersuchten Arten blieb das Tropomyosin während der Lagerzeit stabil.

Zusammenfassung Während der Lagerung gewisser Magerfische in gefrorenem Zustand bildet sich Formaldehyd (FA), was die Bildung von Aggregaten myofibrilärer Proteine verursacht. Diese Aggregate wurden im Modell mit Actomyosin (NAM) (5 mg/mL) und wachsenden Formaldehydkonzentrationen untersucht. Die Muster wurden zwei Monate in gefrorenem Zustand bei -20 °C gelagert. Während dieses Zeitraums wurden die Löslichkeit in 0,6 M NaCl, die Ca2+ATPase-Aktivität, die cis-Parinarsäure-(CPA)-Hydrophobie und SH-Gruppen gemessen sowie SDS-Polyacrylamid-Gel Elektrophoresen (PAGE) durchgeführt. — FA verursachte einen sofortigen Verlust von Ca2+ATPase-Aktivität sowie einen Rückgang löslicher Proteine und in der CPA-Hydrophobie. Dieser Effekt verstärkte sich bei eingefrorenen Mustern. Die elektrophoretischen Profile derjenigen Proteine, die löslich blieben, zeigten sowohl in frischen als auch in gefrorenen Mustern, daß das erste Protein, das bei einer Reaktion von FA mit NAM gelöst wird, Myosin ist, darauf folgen Actin, die Troponine und leichten Myosinketten und schließlich Tropomyosin, was wiederum von der FA-Menge und Reaktionszeit abhängt. In den Anfangsstadien wurden hochmolekulare Aggregate gefunden, die wahrscheinlich das Ergebnis gleichwertiger Bindung von Myosinmolekülen sind. Bei Erhöhung der FA-Menge oder Verlängerung der Lagerzeit bildeten sich hochmolekulare Strukturen, die im löslichen Teil nicht gefunden werden konnten.

Zusammenfassung Es werden drei unterschiedliche Methoden zur Aufbewahrung des Sardinenfleisches untersucht: 1. Aufbewahrung in Salz nach dem Fang auf dem Schiff und dann in Eis nach der Landung; 2. Aufbewahung in Eis nach dem Fanz und 3. Einlagerung im Schiff bei Raumtemperatur und in Eis nach der Landung im Hafen. Der Salzzusatz veränderte drastisch den Muskel, der seine Fähigkeit, Gel zu bilden, verlor. Die Aufbewahrung in Eis unmittelbar nach dem Fang erlaubte eine längere Aufrechterhaltung der Gel-bildenden Fähigkeit als bei der Aufbewahrung in Eis nach der Landung. Die elektrophoretische Profile der myofibrillären Proteine, die aus dem in Salz aufbewahrten Muskel extrahiert worden waren, unterschieden sich sehr von denen der Proteine des durch die zwei anderen Methoden aufbewahrten Muskel; alle Profile aber änderten sich im Laufe der Aufbewahrungszeit.

The textural characteristics and water holding capacity of suwari (set) and kamaboko (set and cooked) sardine surimi gels were examined in order to clarify the influence of the initial network formed in setting conditions (25, 35 and 40°C for 30 and 60 min) on the texture of the kamaboko gels. Although the texture of suwari gels set at 35°C improved with longer setting, both setting times ensured kamaboko gels with the highest gel strength. Suwari gels set at 25°C also improved with longer setting but the gel strength of both suwari and kamaboko gels was lower than at 35°C. For gels set at 40°C prolonged setting weakened the suwari networks formed, leading to kamaboko gels with poorer textural characteristics. ©1997 SCI