Animal-derived food allergen: A review on the available crystal structure and new insights into structural epitope.

Immunoglobulin E (IgE)-mediated food allergy is a rapidly growing public health problem. The interaction between allergens and IgE is at the core of the allergic response. One of the best ways to understand this interaction is through structural characterization. This review focuses on animal-derived food allergens, overviews allergen structures determined by X-ray crystallography, presents an update on IgE conformational epitopes, and explores the structural features of these epitopes. The structural determinants of allergenicity and cross-reactivity are also discussed. Animal-derived food allergens are classified into limited protein families according to structural features, with the calcium-binding protein and actin-binding protein families dominating. Progress in epitope characterization has provided useful information on the structural properties of the IgE recognition region. The data reveals that epitopes are located in relatively protruding areas with negative surface electrostatic potential. Ligand binding and disulfide bonds are two intrinsic characteristics that influence protein structure and impact allergenicity. Shared structures, local motifs, and shared epitopes are factors that lead to cross-reactivity. The structural properties of epitope regions and structural determinants of allergenicity and cross-reactivity may provide directions for the prevention, diagnosis, and treatment of food allergies. Experimentally determined structure, especially that of antigen-antibody complexes, remains limited, and the identification of epitopes continues to be a bottleneck in the study of animal-derived food allergens. A combination of traditional immunological techniques and emerging bioinformatics technology will revolutionize how protein interactions are characterized.

Dietary Phospholipids Alleviate Diet-Induced Obesity in Mice: Which Fatty Acids and Which Polar Head.

The weight loss effects of dietary phospholipids have been extensively studied. However, little attention has been paid to the influence of phospholipids (PLs) with different fatty acids and polar headgroups on the development of obesity. High-fat-diet-fed mice were administrated with different kinds of PLs (2%, w/w) with specific fatty acids and headgroups, including EPA-enriched phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine (EPA-PC/PE/PS), DHA-PC/PE/PS, Egg-PC/PE/PS, and Soy-PC/PE/PS for eight weeks. Body weight, white adipose tissue weight, and the levels of serum lipid and inflammatory markers were measured. The expression of genes related to lipid metabolism in the liver were determined. The results showed that PLs decreased body weight, fat storage, and circulating lipid levels, and EPA-PLs had the best efficiency. Serum TNF-α, MCP-1 levels were significantly reduced via treatment with DHA-PLs and PS groups. Mechanistic investigation revealed that PLs, especially EPA-PLs and PSs, reduced fat accumulation through enhancing the expression of genes involved in fatty acid ß-oxidation (Cpt1a, Cpt2, Cd36, and Acaa1a) and downregulating lipogenesis gene (Srebp1c, Scd1, Fas, and Acc) expression. These data suggest that EPA-PS exhibits the best effects among other PLs in terms of ameliorating obesity, which might be attributed to the fatty acid composition of phospholipids, as well as their headgroup.

Several natural phytochemicals from Chinese traditional fermented food-pickled Raphanus sativus L.: Purification and characterization.

In this study, we aimed to isolate and identify the bioactive compounds from 5-year pickled radish. The pickled radish was extracted with methanol or ethyl acetate. Sephadex LH-20, normal phase and reverse phase silica gel column chromatography were used for separation and purification, combined with thin layer chromatography (TLC), high performance liquid chromatography (HPLC), electrospray mass spectrometry (ESI-MS), nuclear magnetic resonance spectroscopy (NMR) technology for structural identification. The results showed that 6 compounds were separated and purified from methanol and ethyl acetate extracts of 5-year-old pickled radish. The structures were identified as 5-hydroxymethylfurfural, ß-sitosterol, ß-sitosterol-3-O-glucose glycosides, α-linolenic acid, 1-monopalmitin and chaenomic acid A. Using molecular docking, it was determined that ß-sitosterol and its derivative ß-sitosterol-3-O-glucose glycosides have high affinity for five antioxidant enzymes, and there were multiple hydrogen bonds between them. These results indicated that pickled radishes might be used as an important source of natural chemical substances.

Two allergens from Scylla paramamosain share common epitopes showed different allergenic potential in Balb/c mice.

Filamin C (FLN c) and triosephosphate isomerase (TIM) are novel allergens of crab (Scylla paramamosain) which are sharing common epitopes. This work aimed to assess their contributions to the induction and elicitation of allergenic responses. Balb/c mice were sensitized by intraperitoneal injections and challenged by intragastric gavage with purified proteins. Upon oral challenge, FLN c triggered more severe anaphylactic symptoms, higher levels of specific antibodies and histamine in serum than TIM, while TIM was a more active promotor of early specific antibody production and stimulated stronger Th2-biased responses. Combined with the results of in vitro assays, the data demonstrated that though with common epitopes, the two allergens showed a different allergenicity, TIM favored Th2 polarization in sensitization stage, while FLN c had a better ability to stimulate B cells and is highly immunogenic in oral challenge stage. The findings can help with the better understanding of allergenicity of crab allergens.

Assessment of genetic diversity and variety identification based on developed retrotransposon-based insertion polymorphism (RBIP) markers in sweet potato (Ipomoea batatas (L.) Lam.).

Sweet potato, a dicotyledonous and perennial plant, is the third tuber/root crop species behind potato and cassava in terms of production. Long terminal repeat (LTR) retrotransposons are highly abundant in sweet potato, contributing to genetic diversity. Retrotransposon-based insertion polymorphism (RBIP) is a high-throughput marker system to study the genetic diversity of plant species. To date, there have been no transposon marker-based genetic diversity analyses of sweet potato. Here, we reported a structure-based analysis of the sweet potato genome, a total of 21555 LTR retrotransposons, which belonged to the main LTR-retrotransposon subfamilies Ty3-gypsy and Ty1-copia were identified. After searching and selecting using Hidden Markov Models (HMMs), 1616 LTR retrotransposon sequences containing at least two models were screened. A total of 48 RBIP primers were synthesized based on the high copy numbers of conserved LTR sequences. Fifty-six amplicons with an average polymorphism of 91.07% were generated in 105 sweet potato germplasm resources based on RBIP markers. A Unweighted Pair Group Method with Arithmatic Mean (UPGMA) dendrogram, a model-based genetic structure and principal component analysis divided the sweet potato germplasms into 3 groups containing 8, 53, and 44 germplasms. All the three analyses produced significant groupwise consensus. However, almost all the germplasms contained only one primary locus. The analysis of molecular variance (AMOVA) among the groups indicated higher intergroup genetic variation (53%) than intrapopulation genetic variation. In addition, long-term self-retention may cause some germplasm resources to exhibit variable segregation. These results suggest that these sweet potato germplasms are not well evolutionarily diversified, although geographic speciation could have occurred at a limited level. This study highlights the utility of RBIP markers for determining the intraspecies variability of sweet potato and have the potential to be used as core primer pairs for variety identification, genetic diversity assessment and linkage map construction. The results could provide a good theoretical reference and guidance for germplasm research and breeding.

Colorimetric detection of neomycin sulfate in tilapia based on plasmonic core-shell Au@PVP nanoparticles.

The rapid colorimetric detection of neomycin sulfate has been achieved using polyvinyl pyrrolidone shell coated gold nanoparticle (Au@PVP NPs) sol. We also observed that, the aggregation of Au@PVP NPs, possibly caused by the hydrogen bonds formed between neomycin sulfate and PVP shell, generates a new surface plasmon resonance absorption in the wavelength of 600 ~ 700 nm. The proposed method showed an excellent performance towards the determination of neomycin sulfate in wide linear range from 0.01 ~ 10 µM with a correlation coefficient of 0.99 and low detection limit of 1 nM. After extracted with trichloroacetic acid and treated with hot chloroform, neomycin sulfate in the tilapia fish samples was detected with satisfied recovery. Additionally, the high selectivity of Au@PVP NPs sol towards neomycin sulfate has been achieved even in presence of common interfering agents. This method has the advantages of high sensitivity, rapidity, specificity, low cost and no complicated pretreatment procedure.

Integrated transcriptome, small RNA and degradome sequencing approaches proffer insights into chlorogenic acid biosynthesis in leafy sweet potato.

Leafy sweet potato is rich in total phenolics (TP) which play key roles in health protection, the chlorogenic acid (CGA) constitutes the major components of phenolic compounds in leafy sweet potato. Unfortunately, the mechanism of CGA biosynthesis in leafy sweet potato is unclear. To dissect the mechanisms of CGA biosynthesis, we performed transcriptome, small RNA (sRNA) and degradome sequencing of one low-CGA content and one high-CGA content genotype at two stages. A total of 2,333 common differentially expressed genes (DEGs) were identified, and the enriched DEGs were related to photosynthesis, starch and sucrose metabolism and phenylpropanoid biosynthesis. The functional genes, such as CCR, CCoAOMT and HCT in the CGA biosynthetic pathway were down-regulated, indicating that the way to lignin was altered, and two possible CGA biosynthetic routes were hypothesized. A total of 38 DE miRNAs were identified, and 1,799 targets were predicated for 38 DE miRNAs by using in silico approaches. The target genes were enriched in lignin and phenylpropanoid catabolic processes. Transcription factors (TFs) such as apetala2/ethylene response factor (AP2/ERF) and Squamosa promoter binding protein-like (SPL) predicated in silico were validated by degradome sequencing. Association analysis of the DE miRNAs and transcriptome datasets identified that miR156 family negatively targeted AP2/ERF and SPL. Six mRNAs and six miRNAs were validated by qRT-PCR, and the results showed that the expression levels of the mRNAs and miRNAs were consistent with the sequencing data. This study established comprehensive functional genomic resources for the CGA biosynthesis, and provided insights into the molecular mechanisms involving in this process. The results also enabled the first perceptions of the regulatory roles of mRNAs and miRNAs, and offered candidate genes for leafy sweet potato improvements.

Sulfated oligosaccharide of Gracilaria lemaneiformis protect against food allergic response in mice by up-regulating immunosuppression.

Sulfated oligosaccharide of Gracilaria lemaneiformis (GLSO) was prepared from sulfated polysaccharides which possessed antiallergic activity by degradation with high temperature and pressure combined with vitamin C treatment. The present study demonstrated that GLSO could attenuate food anaphylaxis, and inhibit the production of immunoglobulin E, histamine, and related cytokines in both prevention and therapy ovalbumin-induced mice model. Additionally, the gut microbiota analysis revealed that GLSO markedly rescued OVA-induced changes in the Firmicutes to Bacteroidetes ratio. Following flow cytometry, GLSO was found to suppress the subpopulation of T helper 2 and B cells, and significantly up-regulate regulatory T cells (Tregs) differentiation. Furthermore, GLSO-mediated immunosuppression could be verified by co-culturing Tregs sorted from GLSO-treated mice and CD4+ T cells or mast cells. In a word, GLSO attenuated food anaphylaxis through the regulation of gut microbiota and induction of immunosuppression. GLSO had the potential to be used as a nutrient component against food allergy.

Phenotypic diversity of pea (Pisum sativum L.) varieties and the polyphenols, flavonoids, and antioxidant activity of their seeds / Diversidade fenotípica de variedades de ervilha (Pisum sativum L.) e polifenóis, flavonóides e atividade antioxidante de suas sementes

ABSTRACT: Pea (Pisum sativum L.) is the fourth leading legume crop in the world, and its demand is increasing. In this study, the morphological characteristics (seed shape, seed surface, seed coat color, hilum color, cotyledon color, 100-seed weight and color values), total phenolic content (TPC), total flavonoid content (TFC), 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) free radical scavenging capacity and ferric reducing antioxidant power (FRAP) of 75 pea cultivars were investigated. Results showed rich genetic diversity and a wide range of phenolic contents and antioxidant activities. Sixteen varieties enriched with phenolic contents and high antioxidant activities were screened out. A significant correlation was reported among color values, TPC, TFC, ABTS and FRAP. Principal component analysis (PCA) extracted four principal components with a total cumulative contribution of 81.29%. Hierarchical cluster analysis based on the four extracted principal components resulted in a dendrogram dividing the peas into three groups. In addition, dark pea seeds have potential as a functional food in addition to their traditional role in providing dietary protein and fibre. This study provided a scientific basis for the breeding of pea varieties, development of new products and improvement of pea resource utilization.

RESUMO: A ervilha (Pisum sativum L.) é a quarta cultura de leguminosas líder no mundo e sua demanda está aumentando. Neste estudo, as características morfológicas (forma da semente, superfície da semente, cor da casca, cor do hilo, cor do cotilédone, peso de 100 sementes e valores de cor), teor fenólico total (TPC), teor de flavonóides totais (TFC), 2,2. A capacidade de eliminação de radicais livres de '-azinobis- (3-etilbenzotiazolino-6-sulfonato) (ABTS) e o poder antioxidante redutor de ferro (FRAP) de 75 cultivares de ervilha foram investigados. Os resultados mostraram rica diversidade genética e uma ampla gama de conteúdos fenólicos e atividades antioxidantes. Dezesseis variedades enriquecidas com conteúdo fenólico e alta atividade antioxidante foram descartadas. Uma correlação significativa foi encontrada entre os valores de cores, TPC, TFC, ABTS e FRAP. A análise de componentes principais (PCA) extraiu quatro componentes principais com uma contribuição total acumulada de 81, 29%. A análise hierárquica de agrupamento foi baseada nos quatro componentes principais extraídos resultou em um dendrograma dividindo as ervilhas em três grupos. Assim, as sementes de ervilha escura têm potencial como alimento funcional, além de seu papel tradicional no fornecimento de proteína e fibra dietética. Este estudo fornece uma base científica para a criação de variedades de ervilha, desenvolvimento de novos produtos e melhoria da utilização de recursos de ervilha.

Coumarin alleviates ovalbumin-induced food anaphylaxis in a mouse model by affecting mast cell function.

Coumarin is an important organic heterocyclic compound with a wide range of sources in nature. It plays an important role in the drug discovery process due to its existence in diverse biologically active compounds and its broad bioactivity. In this study, the anti-allergic activity of coumarin was evaluated using an ovalbumin (OVA)-induced mouse food allergy model and an immunoglobulin (Ig)E mediated mouse bone marrow-derived mast cell (BMMC) model. Coumarin could alleviate the OVA-induced allergic symptoms, decrease the diarrhea rates, and promote the rectal temperature rise in allergic mice. Moreover, coumarin had the ability to reduce the levels of histamine and mouse mast cell proteinases, inhibit OVA-specific IgE, and significantly decrease the population of mast cells in the spleen and mesenteric lymph nodes. Coumarin could also significantly suppress mast cell-dependent passive cutaneous anaphylaxis. Additionally, the number of mature BMMCs was decreased as coumarin caused the suppression of c-KIT receptors. Furthermore, coumarin up-regulated the apoptosis of OVA-activated BMMCs in a concentration-dependent manner. In conclusion, coumarin displayed effective anti-food allergy activity via the regulation of mast cell function and numbers. Coumarin and its derivatives provide a new direction for the development of anti-food allergic drug components.

Crystal structure determination of Scylla paramamosain arginine kinase, an allergen that may cause cross-reactivity among invertebrates.

Shellfish are one of the most common causes of food allergy. Arginine kinase (AK) is known as an important allergen in shellfish. In the present study, AK from crab (Scylla paramamosain) was purified and its crystal structure was determined. A comparison of AK from S. paramamosain to AKs of other species showed high amino acid sequence and secondary structure identity, while the superposition of crystal structures of AKs from different species revealed only slight differences. Similarity of the linear epitope regions among species was observed in the epitope alignment of AKs; conformational epitopes were located in the regions where secondary structure was conserved. The structure of S. paramamosain AK is an accurate template for the analysis of the IgE binding pattern, and the structure conservation and epitope similarity of AKs among species could help to inform our understanding of the cross-reactivity and contribute to the prediction of cross-reactivity related epitopes.

Deep-Sea-Derived Butyrolactone I Suppresses Ovalbumin-Induced Anaphylaxis by Regulating Mast Cell Function in a Murine Model.

Deep-sea-derived butyrolactone I (BTL-I), which was identified as a type of butanolide, was isolated from Aspergillus sp. Ovalbumin (OVA)-induced BALB/c anaphylaxis was established to explore the antifood allergic activity of BTL-I. As a result, BTL-I was able to alleviate OVA-induced allergy symptoms, reduce the levels of histamine and mouse mast cell proteinases, inhibit OVA-specific IgE, and decrease the population of mast cells in the spleen and mesenteric lymph nodes. BTL-I also significantly suppressed mast-dependent passive cutaneous anaphylaxis. Additionally, the maturation of bone marrow-derived mast cells (BMMCs) declined as BTL-I caused down-regulation of c-KIT receptors. Furthermore, molecular docking analyses revealed that BTL-I interacted with the inhibitory receptor, FcγRIIB. In conclusion, the reduction of mast cell function by deep-sea-derived BTL-I as well as its interactions with the inhibitory receptor, FcγRIIB, may contribute to BTL-I-related protection against food anaphylaxis.

[Progress in digital PCR technology and application].

Digital PCR is an emerging analysis technology for absolute quantification after realtime-PCR. Through digital PCR, single DNA molecules are distributed into isolated reactions, and the product with fluorescence signal can be detected and analyzed after amplification. With the advantages of higher sensitivity and accuracy, digital PCR, independent of a standard curve, is developing rapidly and applied widely to the next generation sequencing and detection fields, such as gene mutation, copy number variation, microorganism, and genetically modified food. In this article, we reviewed the quantitative method and research progress of digital PCR technology in the main application fields.

In vitro and in vivo immunomodulatory activity of sulfated polysaccharide from Porphyra haitanensis.

The immunoregulatory activity of sulfated polysaccharide from Porphyra haitanensis (PHPS) was investigated in a RAW264.7 macrophages cell model and a BALB/c murine model. The subpopulation of dendritic cells (DCs) and regulatory T cells (Tregs) from PHPS-treated mice splenocytes were also measured by flow cytometry. Consistent with previous reports, we showed that PHPS increased the phagocytosis of RAW264.7 macrophages, and enhanced the secretion of interleukin (IL)-6, IL-10 and tumor necrosis factor-α (TNF-α). Meanwhile, PHPS induced the production of nitric oxide via the Jun N-terminal kinase (JNK) and the Janus kinase (JAK2) signaling pathways in RAW264.7 macrophages. Furthermore, PHPS promoted the proliferation of mice lymphocytes, inducing the generation of TNF-α and IL-10 in vivo, as well as the subpopulation of CD4+ splenic T lymphocytes, DCs, and Tregs. These results indicated that PHPS plays key roles in immunoregulation and may be apply to develop new health foods.

Genome-wide assessment of population structure and genetic diversity and development of a core germplasm set for sweet potato based on specific length amplified fragment (SLAF) sequencing.

Sweet potato, Ipomoea batatas (L.) Lam., is an important food crop that is cultivated worldwide. However, no genome-wide assessment of the genetic diversity of sweet potato has been reported to date. In the present study, the population structure and genetic diversity of 197 sweet potato accessions most of which were from China were assessed using 62,363 SNPs. A model-based structure analysis divided the accessions into three groups: group 1, group 2 and group 3. The genetic relationships among the accessions were evaluated using a phylogenetic tree, which clustered all the accessions into three major groups. A principal component analysis (PCA) showed that the accessions were distributed according to their population structure. The mean genetic distance among accessions ranged from 0.290 for group 1 to 0.311 for group 3, and the mean polymorphic information content (PIC) ranged from 0.232 for group 1 to 0.251 for group 3. The mean minor allele frequency (MAF) ranged from 0.207 for group 1 to 0.222 for group 3. Analysis of molecular variance (AMOVA) showed that the maximum diversity was within accessions (89.569%). Using CoreHunter software, a core set of 39 accessions was obtained, which accounted for approximately 19.8% of the total collection. The core germplasm set of sweet potato developed will be a valuable resource for future sweet potato improvement strategies.

Triosephosphate Isomerase and Filamin C Share Common Epitopes as Novel Allergens of Procambarus clarkii.

Triosephosphate isomerase (TIM) is a key enzyme in glycolysis and has been identified as an allergen in saltwater products. In this study, TIM with a molecular mass of 28 kDa was purified from the freshwater crayfish (Procambarus clarkii) muscle. A 90-kDa protein that showed IgG/IgE cross-reactivity with TIM was purified and identified as filamin C (FLN c), which is an actin-binding protein. TIM showed similar thermal and pH stability with better digestion resistance compared with FLN c. The result of the surface plasmon resonance (SPR) experiment demonstrated the infinity of anti-TIM polyclonal antibody (pAb) to both TIM and FLN c. Five linear and 3 conformational epitopes of TIM, as well as 9 linear and 10 conformational epitopes of FLN c, were mapped by phage display. Epitopes of TIM and FLN c demonstrated the sharing of certain residues; the occurrence of common epitopes in the two allergens accounts for their cross-reactivity.

Polyoxomatelate functionalized tris(2,2-bipyridyl)dichlororuthenium(II) as the probe for electrochemiluminescence sensing of histamine.

A novel electrochemiluminescence (ECL) sensing system was developed to determine histamine in aquatic food with tris(2,2-bipyridyl)dichlororuthenium(II) (Ru(bpy)3(2+)) and Keggin-type polyoxomatelate (H3PMo12O40). A hybrid material was synthesized by mixing Keggin H3PMo12O40 (PMo12) and Ru(bpy)3(2+) and it was applied in capillary electrophoresis-electrochemiluminescence (CE-ECL) as a histamine probe for the first time. Some factors which affected the performances of separation and detection were investigated. Under the optimized conditions, one single quantitative analysis of histamine was achieved at a separation voltage of 16kV, and the limit of detection (LOD, S/N=3) of histamine was 1.0×10(-3)mg/L. The linear concentration range was between 0.01 and 1mg/L and the relative standard deviation (RSD) of peak height was between 0.27% and 1.29%, while the RSD of migration time was between 0.96% and 1.87%. The results indicated that the proposed probe presented good characteristics in terms of higher sensitivity and better reproducibility for histamine detection than those of the Ru(bpy)3(2+) ECL system.

Inhibitory effects of Na7PMo11CuO40 on mushroom tyrosinase and melanin formation and its antimicrobial activities.

Keggin-type Cu-substituted phosphomolybdic acid (Na7PMo11CuO40, abbreviated as PMo11Cu) was synthesized and characterized. The inhibitory effects of PMo11Cu on mushroom tyrosinase and melanin formation in B16 melanoma cells were studied. The results showed that PMo11Cu could strongly inhibit the activity of tyrosinase, and it was reversible and competitive inhibitor. The IC50 value was estimated to be 0.48 mM for diphenolase activity. PMo11Cu also exhibited inhibitory effects on cell viability, cellular tyrosinase activity and melanin formation in B16 melanoma cells at concentrations ranging from 0 to 200 µM for 24 h. Furthermore, the antimicrobial activities of PMo11Cu against Sarcina lutea, Staphylococcus aureus, Bacillus subtilis and Escherichia coli were investigated. The results showed that PMo11Cu had an obvious antimicrobial activities, and it was more effective against two kinds of coccus than two kinds of bacillus. This study may provide theoretical basis for designing novel effective mushroom tyrosinase inhibitors and extend the use of polyoxometalates in the fields of food preservation and depigmentation.

Preparation, characterisation and use for antioxidant oligosaccharides of a cellulase from abalone (Haliotis discus hannai) viscera.

BACKGROUND: In China, abalone (Haliotis discus hannai) production is growing annually. During industrial processing, the viscera, which are abundant of cellulase, are usually discarded or processed into low-value feedstuff. Thus, it is of interest to obtain cellulase from abalone viscera and investigate its application for preparation of functional oligosaccharides. RESULTS: A cellulase was purified from the hepatopancreas of abalone by ammonium sulfate precipitation and two-steps column chromatography. The molecular weight of the cellulase was 45 kDa on SDS-PAGE. Peptide mass fingerprinting analysis yielded 103 amino acid residues, which were identical to cellulases from other species of abalone. Substrate specificity analysis indicated that the cellulase is an endo-1,4-ß-glucanase. Hydrolysis of seaweed Porphyra haitanensis polysaccharides by the enzyme produced oligosaccharides with degree of polymerisation of two to four, whose monosaccharide composition was 58% galactose, 4% glucose and 38% xylose. The oligosaccharides revealed 2,2'-diphenyl-1-picrylhydrazyl free radical as well as hydrogen peroxide scavenging activity. CONCLUSION: It is feasible and meaningful to utilise cellulase from the viscera of abalone for preparation of functional oligosaccharides. © 2015 Society of Chemical Industry.

Matrix Metalloproteinase 2 (MMP-2) Plays a Critical Role in the Softening of Common Carp Muscle during Chilled Storage by Degradation of Type I and V Collagens.

Matrix metalloproteinases (MMPs) are proposed to play important roles in the degradation of collagens, thus causing the post-mortem softening of fish muscle, although the specific mechanism remains largely unresolved. Previously, we reported the existence of gelatinase-like proteinases in common carp (Cyprinus carpio) muscle. The primary structures of these proteinases, however, have never been investigated. In the present study, two MMPs with molecular masses of 66 and 65 kDa were purified to homogeneity from common carp muscle by ammonium sulfate fractionation and a series of column chromatographies. Matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS) analysis indicated that they are completely identical to MMP-2 from common carp. During chilled storage of common carp at 4 °C, the enzymatic activity of MMP-2 increased to 212% in 12 h while the texture profile increased over the first 2 h and gradually decreased. On the other hand, type V collagen was purified to homogeneity and a specific polyclonal antibody against this protein was prepared. Both type I and V collagens were effectively hydrolyzed by MMP-2 at 30 °C and even at 4 °C. Furthermore, injection of metalloproteinase proteinase inhibitor EDTA into the blood vessel of live common carp suppressed post-mortem tenderization significantly. All of these results confirmed that MMP-2 is a major proteinase responsible for the degradation of collagens, resulting in the softening of fish muscle during chilled storage.