Purification, immunological properties and molecular cloning of two allergenic parvalbumins from the crimson sea bream, Evynnis japonica

Parvalbumin, a calcium-binding sarcoplasmic protein of 12 kDa, represents a major allergen of fish. Previous immunoblotting experiments suggested the presence of a 14 kDa allergen, together with parvalbumin, in sea breams. In this study, the 14 kDa allergen (PA I) and parvalbumin (PA II) were purified from the white muscle of crimson sea bream, Evynnis japonica, by gel filtration and reverse-phase HPLC. Amino acid sequencing of lysylendopeptidase peptide fragments demonstrated that both PA I and PA II were isoforms of parvalbumin. As analysed by ELISA, PA I showed weaker reactivities with IgG (monoclonal or polyclonal antibody) and serum IgE in fish-allergic patients than did other fish parvalbumins, including PA II. The amino acid sequences of PA I and PA II were elucidated by cDNA cloning. PA I was found to have more specific amino acid residues at several positions compared to the other fish parvalbumins.     

Seasonal changes in the proximate chemical compositions and fatty acids of chub mackerel (Scomber japonicus) and horse mackerel (Trachurus trachurus) from the north eastern Mediterranean Sea

The effects of seasonal variations on the proximate chemical compositions and fatty acid profiles of chub mackerel (Scomber japonicus) and horse mackerel (Trachurus trachurus) captured in the north‐eastern Mediterranean Sea were investigated. Protein fluctuations were observed in two species for all seasons. The lipid content of both species was lower in winter than in autumn and spring. In all seasons, the major fatty acids in both species were observed to be palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1 ω9), eicosapentaenoic acid (20:5 ω3) and docosahexaenoic acid (20:6 ω3). Chub mackerel and horse mackerel exhibited seasonal fluctuations in their fatty acid contents. The fatty acid profile of the two species had a higher degree of unsaturation during winter. The levels of EPA in chub mackerel in winter, spring and autumn were 5.96%, 4.86% and 4.33%, respectively, while those of DHA were 24.94%, 18.75% and 17.12%, respectively. The levels of EPA in horse mackerel in winter, spring and autumn were 5.42%, 5.03% and 4.86%, respectively, while those of DHA were 14.96%, 13.31% and 11.10%, respectively. The PUFA (polyunsaturated fatty acids) values and ω3/ω6 ratios in the two species were highest in winter. The results indicate that chub mackerel and horse mackerel captured in the north‐eastern Mediterranean Sea, which are among the most important fish in Turkey and of international commercial value, are a good source of nutrition for human consumption in terms of their proximate chemical composition and fatty acids.     

Amino Acid and Vitamin Composition of Raw and Cooked Horse Mackerel

Amino acid, vitamin (A, E, B₁, B₂, B₃ and B₆), and proximate composition were determined in raw and cooked horse mackerel. The changes in amino acid, vitamin, and proximate content were found to be significant for all cooking methods (frying, grilling, and steaming). Cooking did in general significantly increase the contents of essential, semi-essential, and other amino acids compared to raw fish species. Amino acid contents of grilled mackerel were significantly higher (p < 0.05) than those found in fried and steamed mackerel. The A, E, B₂, and B₆ vitamin content of fried horse mackerel was found to be significantly (p < 0.05) higher than the grilled and steamed samples. The B₁ content of steamed and B₃ content of grilled were found higher than the other cooked samples. Moisture, protein, fat, ash, and carbohydrate contents of cooked fish ranged between 56.52% to 61.34%, 20.79% and 23.93%, 13.44% and 19.61%, 1.70% and 2.47%, and 1.02% and 4.36 %, respectively. Fried fish had intermediate fat values, while grilled and steamed fishes had a comparatively low value.     

as1-酪蛋白IgE抗原决定簇的识别

以αs1- 酪蛋白氨基酸序列为模板,错位合成αs1- 酪蛋白多肽,以收集到的牛乳过敏患者血清为抗体,鉴定αs1- 酪蛋白IgE 抗原决定簇,分析影响致敏性的关键氨基酸,探讨牛乳过敏机理。结果表明：αs1- 酪蛋白IgE 抗原决定簇有5 条,它们在αs1- 酪蛋白的定位分别为aa21～35、aa26～40、aa91～105、aa141～155、aa186～200。影响αs1- 酪蛋白致敏性的关键氨基酸为组氨酸、谷氨酸、脯氨酸。说明特异性水解抗原决定簇或定点修饰过敏原氨基酸实现脱敏的方法是可行的。     

β-乳球蛋白IgE抗原决定簇的识别

以β- 乳球蛋白氨基酸序列为模板,错位合成β- 乳球蛋白多肽,以收集到的牛乳过敏患者血清为抗体,鉴定β- 乳球蛋白IgE 抗原决定簇,分析影响致敏性的关键氨基酸,探讨牛乳过敏机理。结果表明：β- 乳球蛋白IgE抗原决定簇有4 条,氨基酸序列分别为17～31、72～86、92～106、152～166,并且苏氨酸(AA20)、蛋氨酸(AA23)、天冬氨酸(AA27)是影响β- 乳球蛋白致敏性的关键氨基酸。说明特异性水解抗原决定簇或定点修饰氨基酸可以实现过敏原脱敏的目的。     

Heavy metal contents and chemical compositions of atlantic (<Emphasis Type="Italic">Scomber scombrus</Emphasis>), blue (<Emphasis Type="Italic">Scomber australasicus</Emphasis>), and chub (<Emphasis Type="Italic">Scomber japonicus</Emphasis>) mackerel muscles

The heavy metal contents and chemical compositions among mackerel species were investigated. Atlantic and chub mackerel had higher mercury, but lower lead than blue mackerel. Chub mackerel had the highest crude fat (18.62%) and the lowest moisture (58.29%), whereas blue mackerel had the highest moisture (70.10%) and the lowest crude fat (2.89%). Atlantic and blue mackerel had total saturated fatty acids (SFA)>polyunsaturated fatty acids (PUFA)>monounsaturated fatty acids (MUFA), whereas chub mackerel had PUFA>SFA>MUFA. The main SFA of the 3 mackerels were 16:0 and the main PUFAs were 22:6n-3 and 20:5n-3. The total amino acid contents in Atlantic, blue, and chub mackerel were 190.63, 169.49, and 172.97 mg/g, respectively. The major amino acids of the 3 mackerels were glutamic acid, aspartic acid, lysine, and leucine.     

Comparison of natural and recombinant forms of the major fish allergen parvalbumin from cod and carp

Allergic reaction following fish consumption can trigger life-threatening reactions in predisposed individuals. Parvalbumins from different species have been identified as the major fish allergens. There are two distinct phylogenetic lineages of parvalbumins, alpha and beta. Most allergic reactions are caused by beta-parvalbumins. We cloned and expressed cDNAs encoding cod (Gadus morhua) and carp (Cyprinus carpio) beta-parvalbumins and purified natural cod beta-parvalbumin. CD spectra of the purified proteins showed that their overall secondary structure contents were very similar. No differences in thermal stability were monitored in the calcium-bound or calcium-depleted form of natural cod parvalbumin. IgE reactivity was assessed using 26 sera of fish allergic patients from Spain, The Netherlands, and Greece in immunoblot and ELISA experiments. Twenty-five of the 26 patients with IgE reactivity to native and recombinant cod parvalbumin also reacted to the recombinant carp parvalbumin. IgE inhibition assays were performed using cod and carp extracts and purified recombinant parvalbumin of cod and carp. High crossreactivity among cod and carp parvalbumins was observed in immunoblots as well as in fluid phase assays. Natural and recombinant parvalbumins gave comparable results when performing various in vitro diagnostic assays.     

Proteins, fatty acids and nutritional value in the muscle of the fish species Mora moro (Risso, 1810)

Deep-water fish are becoming an interesting object of studies and research due to the development of deep fishery activities. This paper analyses the chemical composition and nutritional value of the fish species Mora moro (Risso, 1810) inhabiting deep Mediterranean waters. The fatty acid profile and the principal water-soluble proteins present in the white muscle of this fish species have also been determined. The major fatty acids were 22 : 6n-3, 16 : 0, 18 : 1n-9, 20 : 4n-6 and 20 : 5n-3. The polyunsaturated fatty acid (PUFA) content was higher than that of saturated (SFA) and monounsaturated fatty acids, but the ratio PUFA/SFA was lower than the value reported in other studies. Both the atherogenic index and thrombogenic index were very low. Water-soluble proteins were characterised by monodimensional native PAGE and 2-D SDS-gel electrophoresis. Protein patterns showed the presence of parvalbumins and of the principal myofibrillar proteins. Therefore, the deep-water fish M. moro could represent an interesting target for deep-sea fishery and commercial exploitation.     

Isolation and characterization of acid soluble collagens and pepsin soluble collagens from the skin and bone of Spanish mackerel (Scomberomorous niphonius)

The objective of this study was to extract and characterize acid soluble collagens (ASCs) and pepsin soluble collagens (PSCs) from the skin and bone of Spanish mackerel (Scomberomorous niphonius) and to provide a simultaneous comparison of the four collagens. The yields of ASC-S (ASC from skin), PSC-S (PSC from skin), ASC-B (ASC from bone) and PSC-B (PSC from bone) were 13.68 ± 0.35, 3.49 ± 0.24, 12.54 ± 0.83 and 14.27 ± 0.66% (on wet weight basis), respectively. The four collagens contained glycine (341.6–352.6 residues/1000 residues) as the major amino acid and the contents of imino acids were between 177.1 and 184.3 residues/1000 residues. Amino acid composition, SDS-PAGE and FTIR confirmed that ASC-S, ASC-B and PSC-B were mainly composed of type I collagen with slight molecular structure differences, and PSCs had lower content of high-molecular weight cross-links than that of ASCs. The denaturation temperatures of ASC-S, PSC-S, ASC-B and PSC-B were 15.12, 14.66, 18.02 and 16.85 °C, respectively, which were much lower than those of collagens from the mammalian and tropical fish species due to low imino acid contents. All the collagens were soluble at acidic pH (1–4) and lost their solubility when the NaCl concentrations were above 2% (w/v). The four lyophilized collagens displayed a uniform and regular network ultrastructure based on the ultrastructural analysis. The isolated collagens from Spanish mackerel could serve as an alternative source of collagens for further application in food and neutraceutical industries.     

核桃中主要过敏原Jug r 1 B细胞线性表位中关键氨基酸的免疫信息学预测和识别

为确定影响Jug r 1线性表位致敏性的关键氨基酸,研究2 种免疫信息学方法预测Jug r 1线性表位关键氨基酸的准确率和效率,以进一步认识核桃主要过敏原Jug r 1的B细胞线性表位。本研究通过2 种免疫信息学的方法对Jug r 1中3 个线性表位上的关键氨基酸进行预测,采用丙氨酸扫描诱变法对关键氨基酸进行识别,采用固相合成技术合成Jug r 1系列突变多肽,以部分中国核桃过敏患者血清为探针,识别Jug r 1关键氨基酸。结果表明：表位1（4LVALLFVANAAA15）中的第4位亮氨酸、第7位亮氨酸、第8位亮氨酸和表位2（16FRTTITTMEIDEDID30）中的第20位异亮氨酸、第21位苏氨酸、第22位异亮氨酸以及表位3（125CGISSQRCEIRRSWF139）中的第127位异亮氨酸、第129位丝氨酸、第130位谷氨酰胺为关键氨基酸。使用两种免疫信息学结合预测的关键氨基酸准确率达到100%,但预测效率仅为11%。因此,使用2 种免疫信息学结合的方式预测表位关键氨基酸可提高准确性,但会忽略一些关键氨基酸。免疫信息学结合丙氨酸扫描诱变法是识别表位关键氨基酸的重要研究思路。     

Effects of Vacuum Packaging, Glazing, and Erythorbic Acid on the Shelf-Life of Frozen White Hake and Mackerel

The quality changes during frozen storage of two underutilized species of fish: mackerel (Scomber scombrus), a fatty species, and white hake (Urophycis tenuis) a nonfatty, gadoid species, with or without a bag, vacuum, and/or erythorbic acid, were measured using the dimethy-lamine (DMA) test for hake and the thiobarbituric acid (TBA) test for mackerel, and by Instron deformation, expressible moisture, thaw drip, cook loss, and sensory evaluation for both species. Texture deterioration and lipid oxidation limited the shelf-life of hake and mackerel, respectively. Air (oxygen) prolonged the shelf-life of hake but lessened that of mackerel. Erythorbic acid accelerated the rate of texture deterioration in hake but inhibited the rate of lipid oxidation in mackerel.     

Sensory and Chemical Characteristics of Selected Gulf of Mexico Coastal Herring Species

Gulf of Mexico coastal herrings may become an important alternative fisheries resource for U.S. commercial fisherman. Effects of on board handling on sensory characteristics of chub mackerel (Scomber japonicus) and rough scad (Trachurus lathami) were investigated. Proximate and fatty acid composition of chub mackerel, rough scad, round scad (Decapturus punctatus), round herring (Etrumeus teres), and silver rag (Ariomma bondi) were determined to provide nutritional and product formulation data. Chub mackerel and rough scad had similar sensory characteristics found in other commercial species, which, with proper on board handling, could be maintained during frozen storage. Proximate and fatty acid compositions of the fish varied by species, season, and year.     

Structural properties of trypsin from cold-adapted fish,arabesque greenling (<Emphasis Type="Italic">Pleurogrammus azonus</Emphasis>)

A cDNA clone encoding trypsin (AG-T) was isolated from the pyloric ceca of cold-adapted fish, arabesque greenling (Pleurogrammus azonus). The cDNA was composed of 892 bp with an open reading frame of 729 bp at nucleotide positions 25–753. Similar to all the known trypsin, the AG-T seemed to be synthesized as preproenzyme that contains a hydrophobic signal peptide, an activation pentapeptide and a mature trypsin of 222 amino acid residues. The AG-T also completely conserved the major structural features common to trypsin such as the catalytic triad (His57, Asp102, and Ser195), the obligatory Asp189 and twelve Cys residues. On the other hand, the AG-T possessed the deletion of Tyr151 and substitution of Pro152 for Gly in the autolysis loop when aligned with the sequence of tropical-zone fish and bovine trypsins. In addition, Val75 concerned in a combination with calcium ion was exchanged for Ala in the AG-T, and the content of positively charged amino acid residues at the calcium-binding site of the AG-T was three times higher than those of tropical-zone fish trypsins. Moreover, the ratio between charged and hydrophobic amino acid residues in the N-terminal region of the AG-T was also higher than those of temperate-zone fish and tropical-zone fish trypsins. Such structural properties of the AG-T would contribute to its low thermostability.     

Properties of Gelatin Film from Horse Mackerel (Trachurus japonicus) Scale

Optimal conditions for extracting gelatin and preparing gelatin film from horse mackerel scale, such as extraction temperature and time, as well as the protein concentration of film‐forming solutions were investigated. Yields of extracted gelatin at 70 °C, 80 °C, and 90 °C for 15 min to 3 h were 1.08% to 3.45%, depending on the extraction conditions. Among the various extraction times and temperatures, the film from gelatin extracted at 70 °C for 1 h showed the highest tensile strength and elongation at break. Horse mackerel scale gelatin film showed the greatly low water vapor permeability (WVP) compared with mammalian or fish gelatin films, maybe due to its containing a slightly higher level of hydrophobic amino acids (total 653 residues per 1000 residues) than that of mammalian, cold‐water fish and warm‐water fish gelatins. Gelatin films from different preparation conditions showed excellent UV barrier properties at wavelength of 200 nm, although the films were transparent at visible wavelength. As a consequence, it can be suggested that gelatin film from horse mackerel scale extracted at 70 °C for 1 h can be applied to food packaging material due to its lowest WVP value and excellent UV barrier properties.     

Effect of brine freezing on the rancidity development during the frozen storage of small pelagic fish species

Brine freezing was applied to two small pelagic underutilised fish species (mackerel, Scomber scombrus; horse mackerel Trachurus trachurus). Rancidity development was studied during their frozen (–18 °C) storage up to 9 months, and quality change results were compared to common freezing conditions (control treatment). Fish samples treated under brine freezing conditions showed a higher lipid oxidation development (peroxide value and thiobarbituric acid index) and worse marks on some sensory attributes (general aspect, odour and colour) than control fish. However, samples treated under brine freezing conditions provided a lower lipid hydrolysis development (free fatty acid formation) and better scores for consistency. Comparison between both fish species led to a higher secondary lipid oxidation formation (thiobarbituric acid index) for mackerel, while horse mackerel showed to be more prone to interaction compound formation (fluorescence detection); however, both fish species showed the same shelf-life times (3 and 5 months for brine and control freezing conditions, respectively). As a result of the brine freezing conditions, an increase in NaCl content in white muscle of both species was observed. According to the results obtained in the present work, the brine freezing treatment is not recommended for these two small pelagic fish species.     

Microbial Activity Inhibition in Chilled Mackerel (Scomber Scombrus) by Employment of an Organic Acid‐Icing System

Abstract: The present study concerns Atlantic mackerel (Scomber scombrus) traded as a chilled product. The study was aimed to investigate the effect of including a mixture of organic acids (citric, ascorbic, and lactic) in the icing medium employed during the fish chilled storage. To this end and according to preliminary trials results, an aqueous solution including 0.050% (w/v) of each acid was employed as icing medium; its effect on the microbial activity development in mackerel muscle was monitored for up to 13 d of chilled storage and compared to a counterpart‐fish batch kept under traditional water ice considered as control. Results indicated a lower bacterial growth in mackerel muscle subjected to storage in the organic acid‐icing system by comparison with control fish. Thus, statistically‐significant (P < 0.05) differences between both batches for all 6 microbial groups investigated (aerobes, anaerobes, psychrotrophes, Enterobacteriaceae, lipolytics, and proteolytics) and for 2 chemical indices related to microbial activity development (total volatile bases and trimethylamine) were obtained. The surface wash caused by the melting of the ice during storage and the subsequent antimicrobial effect of such acids on skin microflora of the fish can be invoked as the main reasons for the limited bacterial growth found in the corresponding mackerel muscle. Practical Application: Among natural antioxidants, citric, ascorbic, and lactic acids are low molecular weight organic compounds that represent a relevant choice because of their easy availability, low commercial cost, and wide range of permitted concentrations for their use in foods. Present results obtained by their inclusion in a novel icing system have led to a lower microbial development in chilled mackerel when compared to its counterpart fish kept under traditional icing conditions. Such a finding indicated that inclusion of this acid mixture in the icing medium can lead to a marked quality and safety enhancement as well as to profitable commercial value increases.     

Cold-adapted structural properties of trypsins from walleye pollock (<Emphasis Type="Italic">Theragra chalcogramma</Emphasis>) and Arctic cod (<Emphasis Type="Italic">Boreogadus saida</Emphasis>)

Complementary DNA clones encoding trypsins were isolated from pyloric ceca of cold-adapted fish, walleye pollock (Theragra chalcogramma) (WP-T) and Arctic cod (Boreogadus saida) (AC-T). The isolated full-length cDNA clones of WP-T and AC-T were 852 and 860 bp, respectively, and both cDNAs were contained an open reading frame of 726 bp. WP-T and AC-T seemed to be synthesized as preproenzyme that contains a signal peptide, an activation peptide, and a mature trypsin. Although the amino acid sequence identities of WP-T and AC-T to that of bovine trypsin were 64 and 63%, respectively, they completely conserved the structural features for catalytic function of trypsin. On the other hand, WP-T and AC-T possessed the four Met residues (Met135, Met145, Met175 and Met242) in their molecules and the deletion of Tyr151 and substitution of Pro152 for Gly in their autolysis loops when aligned with the sequences of tropical-zone fish and bovine trypsins. In addition, the contents of charged amino acid residues at the N-terminal regions (positions 20–50) of WP-T and AC-T were extremely higher than those of other fish and bovine trypsins. Moreover, one amino acid (Asn72) and two amino acids (Asn72 and Val75) coordinating with Ca²⁺ in bovine trypsin were exchanged for another amino acids in WP-T (His) and AC-T (His and Glu), respectively, and the contents of negative charged amino acids at their Ca²⁺-binding regions were lower than those of tropical-zone fish and bovine trypsins. Therefore, it was considered that these structural characteristics of WP-T and AC-T are closely related to their lower thermostability.     

Electron spin resonance (ESR) study on free radical transfer in fish lipid–protein interaction

Oxidised lipids interact with proteins causing undersirable changes in the nutritional and functional properties, including a loss of amino acids, cross-linking and damage to proteins and DNA. Proteins including egg lysozyme, egg ovalbumin, fish myosin and amino acids arginine, lysine and histidine were exposed to oxidised lipids methyl linoleate and oil extracted from Atlantic mackerel (Scomber scombrus). A strong central singlet signal was induced in the proteins and amino acids which was detected by ESR spectroscopy and assigned to the carbon radical (g value range 2.0021–2.0049); with ovalbumin and fish myosin a downfield shoulder, attributed to the sulphydryl group (g value 2.014–2.017), was also observed. The above changes in the proteins were accompanied by an increase in fluorescence indicating the formation of cross-links. Synthetic antioxidants such as butyl hydroxytoluene (BHT) and butyl hydroxyanisole (BHA) as well as natural antioxidants ascorbic acid and α-tocopherol inhibited the development of both the free radical signal and fluorescence when added to the proteins prior to incubation with oxidised lipids; the central singlet signal attributed to the carbon radical was reduced by 10–50%. This paper clearly indicates direct free radical transfer from oxidised lipids to amino acids and proteins which results in protein denaturation and the formation of insoluble aggregates in fish flesh on storage. © 1999 Society of Chemical Industry     

Proteolytic processing of Atlantic mackerel (Scomber scombrus) and biochemical characterisation of hydrolysates

Research on fish composition has demonstrated that they constitute a promising source of molecules with health benefits. In the present study, Atlantic mackerel ( Scomber scombrus ) was processed using Protamex® and the distribution of nutrients in various fractions obtained following membrane filtration was analysed. The fish starting material was approximately 84% water and the dry matter contained 42.7% proteins, 45.2% lipids and 5.6% minerals. The recovery of fish dry matter in the liquid hydrolysate was 77.8%. Most of the fractions were protein-enriched and characterised by a well-balanced amino acid composition, notably in terms of the most essential amino acids and by molecules of relatively low molecular weight (≤42 kDa). Even before process optimising, the biochemical and nutritional analyses indicate that the Atlantic mackerel may provide high-value products for future applications in the health and food sectors.     

STABILITY OF FISH MUSCLE PARVALBUMINS AT DIFFERENT pH VALUES

Parvalbumins of herring (Clupea harengus) and carp (Cyprinus carpio) are essential components of the pattern ofsarcoplasmic proteins used for fish species identification by isolelectric focusing. In fishery products oflowpH, like marinated herring (pH ～ 4), parvalbumins were not detectable. It was demonstrated for herring and carp that parvalbumins were not precipitated at pH 4, but were degraded by fish muscle proteases.