Anisakis antigens detected in fish muscle infested with Anisakis simplex L3

Anisakis simplex is 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 Anisakis larvae 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.     

Alterations of phenolic compounds in red raspberry juice induced by high-hydrostatic-pressure and high-temperature short-time processing

Most studies on health-promoting attributes of red raspberries have been conducted using extracts or freeze-dried powders instead of commercially processed products. This work aimed to trace the alterations of phenolic compounds in HHP- and HTST-treated juices. In total, 151 phenolic compounds were identified: 13 and 26 differential phenolics were discriminated in HHP- and HTST-treated juices, respectively. The alterations of phenolic compounds could be related to the increased oxidative stress derived from the indirect oxidation and thermal degradation. HTST processing promoted the release of free phenolic acids from their conjugated forms, while HHP processing could maximize the flavonol glycosides. Furthermore, morin was observed with a 7400-fold increase in HTST-treated juice, indicating the potential use for juice authenticity. Generally, HHP processing showed less impact on phenolic profiles in comparison with HTST processing. These findings provide novel insights into the impacts of sterilization processes on phenolic compounds in red raspberries.     

High hydrostatic pressure treatment of finfish to inactivate Anisakis simplex

High hydrostatic pressure has been demonstrated to be a useful technique for treating food to reduce the number of pathogenic organisms and to extend shelf life. Most research in this area has focused on bacteria. However, a concern in the sashimi (raw fish) industry is that nematode worms such as Anisakis simplex occur naturally in cold-water marine fish. The objectives of this research were to perform a pilot study to determine the effect of high hydrostatic pressure on the viability of Anisakis simplex larvae, commonly found in king salmon and arrowtooth flounder, and to evaluate the effects of high hydrostatic pressure on the color and texture of the fish fillets. Pieces of fish (ca. 100 g per bag) containing 13 to 118 larvae were exposed to pressures of up to 80,000 lb/in2 (552 MPa) for up to 180 s. The times and pressures required to kill 100% of the larvae were as follows: 30 to 60 s at 60,000 lb/in2 (414 MPa), 90 to 180 s at 40,000 lb/in2 (276 MPa), and 180 s at 30,000 lb/in2 (207 MPa). For all salmon treatments that killed 100% of the larvae, a significant increase in the whiteness of the flesh was observed. Although high hydrostatic pressure was effective in killing A. simplex larvae in raw fish fillets, its significant effect on the color and overall appearance of the fillet may limit its application to the processing of fish for raw-fish markets.     

Survival of Anisakis simplex in microwave-processed arrowtooth flounder (Atheresthes stomias).

The purpose of this study was to define the relationship between survival and temperature of nematodes of the species Anisakis simplex in microwave-processed arrowtooth flounder (Atheresthes stomias). Ten fillets (each 126 to 467 g, 0.5 to 1.75 cm thick), with an average of five larvae of Anisakis simplex per fillet, were processed to target temperatures on high (100%) power using a commercial 700-W microwave oven. Fillets were neither covered nor rotated and had a temperature probe inserted to two-thirds depth into the thickest portion. After the fillet was digested using a 1% pepsin solution, the viability of nematodes was determined by viewing them under a dissecting microscope. Survival rates were 31% at 140 degrees F (60 degrees C), 11% at 150 degrees F (65 degrees C), 2% at 160 degrees F (71 degrees C), 3% at 165 degrees F (74 degrees C), and 0% at 170 degrees F (77 degrees C). Microwave processing of standardized fillet "sandwiches," 14 cm long, 4.5 cm wide, and approximately 1.75 cm high, each of which was preinoculated with 10 live nematodes, resulted in no survival at either 160 degrees F or 170 degrees F. Using ultraviolet light to detect both viable and nonviable nematodes in fillet sandwiches as an alternative method to pepsin digestion resulted in survival rates of 1% at 140 degrees F (60 degrees C), 3% at 145 degrees F (63 degrees C), and 0% at 150 degrees F (65 degrees C). Smaller fillet sandwiches, which most likely had fewer cold spots during microwave processing, required 150 degrees F (65 degrees C), whereas larger whole fillets required 170 degrees F (77 degrees C) to kill larvae of Anisakis simplex. The parasites were most likely inactivated by a thermal mechanism of microwave treatment. Damage to the nematodes was often evident from ruptured cuticles that were no longer resistant to digestive enzymes. The high hydrostatic pressure and low chloride content of the pseudocoelomic fluid probably contributed greatly to the damage incurred by the larvae.     

恒温实时荧光法快速检测简单异尖线虫方法的建立

为了快速鉴定简单异尖线虫,本研究建立了一套检测简单异尖线虫DNA的恒温实时荧光环介导等温扩增（loop-mediated isothermal amplification,LAMP）方法,根据LAMP方法原理,针对简单异尖线虫ITS2区域设计引物特异性识别靶标基因。进行了特异性、灵敏度、重复性和实际样品的测试,并与传统的PCR方法进行比较。结果表明,该方法能够特异性扩增简单异尖线虫DNA,对含有简单异尖线虫ITS2目的基因片段的质粒DNA检测限为1 fg/μL,灵敏度比传统的PCR方法高100倍,重复性良好,对实际样品进行检测,与传统的PCR测序方法结果相符。本研究建立的恒温实时荧光快速检测方法适用于特异性检测简单异尖线虫。      

Survival of Anisakis simplex in arrowtooth flounder (Atheresthes stomia) during frozen storage

Survival of naturally occurring larvae of Anisakis simplex in fresh arrowtooth flounder (Atheresthes stomia) was determined after storage for specified periods at four freezing temperatures. All larvae were killed by 96, 60, 12, and 9 h at temperatures of -15, -20, -30, and -40 degrees C, respectively. The average percentages of live larvae per fillet at the next shortest holding time were as follows: 72 h at -15 degrees C, 0 to 3%; 48 h at -20 degrees C, 11 to 30%; 9 h at -30 degrees C, 5%; and 6 h at -40 degrees C, 0 to 3%. Larval survival was directly related to fillet thickness or weight (P < or = 0.05). Larval death was directly correlated to freezing temperatures. Holding time necessary to kill larval nematodes decreased as storage temperature decreased.     

Inactivation of Escherichia coli in milk by high-hydrostatic-pressure treatment in combination with antimicrobial peptides

We studied the inactivation in milk of four Escherichia coli strains (MG1655 and three pressure-resistant mutants isolated from MG1655) by high hydrostatic pressure, alone or in combination with the natural antimicrobial peptides lysozyme and nisin and at different temperatures (10 to 50 degrees C). Compared with that of phosphate buffer, the complex physicochemical environment of milk exerted a strong protective effect on E. coli MG1655 against high-hydrostatic-pressure inactivation, reducing inactivation from 7 logs at 400 MPa to only 3 logs at 700 MPa in 15 min at 20 degrees C. An increase in lethality was achieved by addition of high concentrations of lysozyme (400 microg/ml) and nisin (400 IU/ml) to the milk before pressure treatment. The additional reduction amounted maximally to 3 logs in skim milk at 550 MPa but was strain dependent and significantly reduced in 1.55% fat and whole milk. An increase of the process temperature to 50 degrees C also enhanced inactivation, particularly for the parental strain, but even in the presence of lysozyme and nisin, a 15-min treatment at 550 MPa and 50 degrees C in skim milk allowed decimal reductions of only 4.5 to 6.9 for the pressure-resistant mutants. A substantial improvement of inactivation efficiency at ambient temperature was achieved by application of consecutive, short pressure treatments interrupted by brief decompressions. Interestingly, this pulsed-pressure treatment enhanced the sensitivity of the cells not only to high pressure but also to the action of lysozyme and nisin.     

Anisakis simplex allergens remain active after conventional or microwave heating and pepsin treatments of chilled and frozen L3 larvae

BACKGROUND: Some Anisakis simplex allergens, Ani s 4 among them, are reported to be resistant to freezing, heat and pepsin. However, the effect of conventional and microwave heating on live and frozen larvae, common conditions for fish preparation, and consecutive pepsin treatment have not been studied previously. In this study, live and frozen/thawed A. simplex larvae were subjected to conventional or microwave heating during time–temperature sufficient to kill live larvae, and digested with pepsin in the strong conditions used in fish inspection. The antigenicity of A. simplex in the larvae extracts and in the incubation media filtrates after all treatments was studied. RESULTS: The immunoblotting assay showed the presence of Ani s 4 in all the larvae extracts and all the incubation media filtrates. A. simplex crude antigens were detected in all conditions; nevertheless, differences were observed among treatments, with lower values detected in the filtrates obtained after the strong acidic pepsin treatment. CONCLUSION: The results indicate that ingestion of A. simplex larvae can cause allergy in consumers already sensitised to this allergen, even if the parasitised fish is consumed well‐cooked and after freezing in the recommended conditions selected for killing the larvae to avoid human anisakiasis. Copyright © 2009 Society of Chemical Industry     

Inactivation of Anisakis simplex L3 in the flesh of white spotted conger (Conger myriaster) by high hydrostatic pressure and its effect on quality

Koreans consume much seafood; the country is surrounded on the east, west and south by the sea. Koreans have eaten raw sashimi for a long time. However, a concern in the raw sea food industry is that the parasitic nematode Anisakis simplex L3 occurs naturally in marine fish. Thus, the fishery industry needs a non-thermal processing method. High hydrostatic pressure (HPP) has been demonstrated to be effective. White spotted conger flesh containing 20 live larvae was exposed to different pressures (150 and 200 MPa for 1 and 5 min; 250 and 300 MPa each for 1 min). The viability of A. simplex L3 was significantly (p < 0.05) reduced in the flesh of white spotted conger by the stepwise increase of high pressure and time. The conditions required to eliminate A. simplex L3 were as follows: 200 MPa for 5 min or 300 MPa for 1 min. The flesh of the white spotted conger treated at 300 MPa for 1 min was whiter and yellower than untreated controls or that treated at 200 MPa for 5 min. No significant changes (p > 0.05) in any of the Hunter colour (‘L’, ‘a’ and ‘b’) values were found after HPP at 200 MPa for 5 min. The fresh treated at 300 MPa for 1 min scored < 4.0 (the defect limit of quality) of flavour, texture and overall acceptability in untrained sensory evaluation using a seven-point hedonic scale. However, the flesh treated at 200 MPa for 5 min scored > 5.0 (‘like’) for all sensory parameters. This study suggested that HPP at 200 MPa for 5 min could potentially be used for the inactivation of A. simplex L3 in raw fishery food products without any concomitant changes in their colour or sensory qualities.     

Effect of high hydrostatic pressure on mortality and allergenicity of Anisakis simplex L3 and on muscle properties of infested hake

BACKGROUND: High pressure (HP) ranging from 100 to 350 MPa (1–15 min) was applied to Anisakis simplex larvae and parasitised hake (Merluccius merluccius) muscle. The aim of the study was to kill the larvae to prevent human anisakidosis, to evaluate the effect on A. simplex allergens and to minimally alter fish muscle quality. RESULTS: The larvae were killed at pressures ≥ 200 MPa and times ≥ 1 min, producing alterations in the larva body and ruptures in the cuticle when observed by scanning electron microscopy. Nevertheless, Ani s 4 and A. simplex crude antigens were recognised by immunoblotting and immunohistochemistry at all HPs assayed. Small changes in colour and texture were observed in fish muscle under all pressure/time conditions. Major changes were observed visually at 300 MPa, where the muscle appeared as slightly cooked. Apparent viscosity of muscle homogenates decreased significantly at longer times or higher applied pressure. No changes were detected at 200 MPa in the electrophoretic pattern of proteins treated with or without β‐mercaptoethanol, suggesting that disulfide bonds were not formed. CONCLUSION: Application of HP at 200 MPa for up to 5 min would kill A. simplex larvae, avoiding infestation of the consumer and causing small changes in the hake muscle perceived sensorially. However, HP‐treated A. simplex‐parasitised fish would still be a potential hazard for consumers allergic to the larvae. Copyright © 2009 Society of Chemical Industry     

Anisakis Infection and Allergy in Humans

Compared with other well-studied parasitic diseases, fish-borne parasitic zoonoses do not get enough attention, especially because these zoonoses have been limited for the most part to populations living in low- and middle-income countries in Europe. Human fishery product-borne parasitic diseases caused by nematodes are the results of infection following ingestion of viable parasites, or as allergic reactions against parasite antigens. With the globalization of the seafood industry, the risk of humans acquiring anisakiasis in developed countries appears to be underestimated. For allergy, the only implicated parasite in fishery products is the nematode Anisakis simplex.     

Prevalence and mean intensity of Anisakis simplex (sensu stricto) in European sea bass (Dicentrarchus labrax) from Northeast Atlantic Ocean

Viscera and muscle of a total of 40 wild 1-2 kg European sea bass (Dicentrarchus labrax) from Northeast Atlantic (FAO area 27) were examined for Anisakidae larvae detection by digestion method. Extracted parasites were counted and mean intensity was calculated. Parasites were identified by genetic/molecular markers (allozymes and sequences analysis of the mtDNA cox2 gene) as belonging to the species Anisakis simplex (sensu stricto). In viscera, the main localisations of the larvae were under the gastric serosa, where several parasites alive and dead were found, and intestinal serosa. The visceral prevalence was 0.950 and the mean intensity was 96.39. The main localisation of A. simplex (s.s.) in edible parts was in belly muscles, with a prevalence of 0.425 and a mean intensity of 1.9. This is the first record on the prevalence and mean intensity of A. simplex (s.s.) in European sea bass muscle. This finding has an important consequence on epidemiology of anisakiasis and public health risk assessment.     

Anisakis simplex s.l. parasitization in mackerel (Scomber japonicus) caught in the North of Morocco--prevalence and analysis of risk factors

Consumption of mackerel (Scomber japonicus) is very widespread in Morocco, where its production is an important economic pillar. We investigated the prevalence of infection by Anisakis spp. in mackerel caught in Moroccan waters, analyzing infection risk factors. The prevalence was generally higher in fish from the Atlantic (67.9%) than from the Mediterranean (57.0%), but they did not differ in the mean abundance, intensity, or prevalence of muscle parasitization. A. pegreffii was the predominant species (82.6% of larvae) followed by the hybrid A. simplex s.s./A. pegreffii (16.3%). Only one L3 of A. simplex s.s. was found in a specimen of S. japonicus from the Atlantic. Mackerel infection was associated with total fish weight, gonad weight, catch area, and catch season. However, muscle infection was associated solely with total weight and parasite loads. The consumption of lower-weight mackerel may be a good prophylactic measure against human anisakiasis.     

Survival of Anisakis larvae in marinated herring fillets

The survival of Anisakis sp. larvae in herring fillets marinated in brines with different concentrations of salt (NaCl) and acetic acid was examined. Traditional German and Danish procedures for marinating herring fillets require a storage time of at least 5 and 6 weeks, respectively, for all larvae to be killed. Other concentrations of salt and acetic acid were also studied. A reduction of the salt content in the water phase of the fish tissue from 9% (w/w) to 4.3% (w/w), while keeping the concentration of acetic acid constant at 2.6%, increased the survival time of the nematodes from at least 35 days to more than 119 days. Increases in the acetic acid concentration or the addition of potassium sorbate had little or no effect on the viability of the larvae.     

核桃加工副产物的综合利用及精深加工

我国核桃资源丰富,核桃加工后会产生大量的副产物。对核桃加工副产物的营养成分提取及精深加工研究进行综述,旨在为进一步开发核桃精深加工系列产品与有效利用核桃副产物资源提供理论依据与技术指导。     

丝光绿蝇幼虫油脂提取方法的响应曲面优化

为确定丝光绿蝇幼虫油脂的提取方法和性质,为其利用奠定基础,以石油醚为溶剂,采用浸提法和超声波协同微波提取法提取丝光绿蝇幼虫油脂,用响应曲面对2种方法进行优化,得出最佳提取条件,并对油脂的理化性质进行了分析。得到浸提法的最佳提取条件为:温度40℃,时间7.93h,料液比1∶19.71,理论得率28.27%,实际得率为27.89%;得到超声波协同微波提取法的最佳提取条件为:时间10 min,料液比1∶12,超声波功率339.10 W,微波功率446.64 W,温度57.77℃,理论得率24.69%,实际得率为24.67%。2种方法提取油脂的碘值相近,超声波协同微波提取法油脂的酸值(KOH)较低,为6.55 mg/g;浸提法油脂的过氧化值为6.22 meq/kg;2种油脂颜色饱和值S分别为0.72、0.95,超声波协同微波提取法油脂颜色较深。浸提法提取油脂得率较高,超声波协同微波提取法耗时最短,品质较好。综合分析,超声波协同微波提取法是丝光绿蝇幼虫油脂的适宜提取方法。     

核桃仁的水剂法加工技术

核桃仁中的粗脂肪含量60%以上,其中不饱和脂肪酸可达到90%,是一种优质的油脂原料,适合水剂法加工。核桃仁水剂法加工的技术要点为研磨、分离、破乳及产品包装,其加工的产品有核桃油、核桃粉和核桃饮料。核桃仁的水剂法加工技术具有良好的推广应用前景,其中分离机是关键设备。