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1.
Food Chem ; 330: 127246, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32526647

RESUMO

Previously we purified and characterized a sarcoplasmic serine proteinase (SSP) from the belly muscle of the threadfin bream as a modori-inducing proteinase. In our attempt to clarify the structure and physiological functions of SSP, we successfully cloned the full-length cDNA of SSP (ORF 726 bp). The deduced amino acid sequence of SSP (241 residues) was highly homologous to fish trypsinogen. The distribution of SSP mRNA and the proteinase activity in the tissue indicated that SSP was mainly synthesized and existed in the digestive system under physiological conditions. After ice storage of the threadfin bream without gutting, a high SSP activity was detected only in the belly muscle because of SSP leaked from the viscera. Therefore, it is desirable to use edible proteinase inhibitor to inactivate the leaked SSP during production of surimi-based products or to take effective measures to prevent the proteinase leakage during post-harvest storage.


Assuntos
Serina Proteases/metabolismo , Vísceras/enzimologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , Peixes/metabolismo , Gelo , Músculo Esquelético/enzimologia , Proteólise , Alimentos Marinhos , Distribuição Tecidual
2.
Food Chem ; 284: 198-204, 2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-30744846

RESUMO

A sarcoplasmic serine proteinase (SSP) was purified from threadfin bream (Nemipterus virgatus) belly muscle by ammonium sulfate precipitation and a series of chromatographies including Q-Sepharose, Phenyl Sepharose and Superdex 200. The SSP was purified 1967 folds with a yield of 4.8%. The molecular weight of the SSP was estimated to be 43.5 kDa and 22.5 kDa on SDS-PAGE under non-reducing and reducing conditions, respectively. The N-terminal amino acid sequence of the two protein bands were determined as IVGGYEXQPYSQAHQVSLNSGY and corresponded. It is suggested that the SSP exists as a homodimer. Optimum pH and temperature were 9.5 and 50 °C, using Boc-Val-Pro-Arg-MCA as a substrate. Substrate specificity and effects of inhibitors indicated that the SSP was a trypsin-like serine proteinase. The SSP was responsible for hydrolyzing myosin heavy chain (MHC) and inducing modori phenomenon in the threadfin bream surimi gel. Thus, the SSP was considered as a modori-inducing proteinase.


Assuntos
Peixes , Músculo Esquelético/enzimologia , Serina Proteases/isolamento & purificação , Serina Proteases/metabolismo , Sequência de Aminoácidos , Animais , Cumarínicos/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas de Peixes da Dieta/química , Proteínas de Peixes da Dieta/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cadeias Pesadas de Miosina/metabolismo , Oligopeptídeos/metabolismo , Inibidores de Serina Proteinase/farmacologia , Especificidade por Substrato , Temperatura , Tripsina/metabolismo
3.
J AOAC Int ; 101(3): 798-804, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29187265

RESUMO

Crustacean proteins are food allergens that cause severe allergic reactions in patients with food allergies; therefore, the identification of crustaceans such as shrimp, crab, and lobster as ingredients in processed food products is mandatory in Japan. We previously developed and validated an ELISA method coupled with an extraction process using the surfactant sodium dodecyl sulfate and the reductant 2-mercaptoethanol (2-ME) to quantify crustacean protein. However, 2-ME was designated as poisonous in Japan in 2008. Therefore, in this study, we developed and evaluated an ELISA method for detecting and quantifying crustacean protein that uses sodium sulfite (Na2SO3) in place of 2-ME for extraction. The proposed ELISA method showed high sensitivity, with an LOQ of 0.66 µg protein/g food sample. Furthermore, the proposed method showed high specificity for the Decapoda order within the subphylum Crustacea, with recoveries ranging from 83.8 to 100.8% for model processed foods, as well as high reproducibility (intra- and interassay CVs of ≤8.2%) and high correlation with our previously validated ELISA method for processed foods (correlation coefficient of 0.996). The proposed ELISA method does not require the use of poisonous reagents, provides acceptable accuracy, and is useful for the routine monitoring of food products.


Assuntos
Proteínas de Artrópodes/análise , Ensaio de Imunoadsorção Enzimática/métodos , Análise de Alimentos , Animais , Proteínas de Artrópodes/isolamento & purificação , Calibragem , Galinhas , Peixes , Sucos de Frutas e Vegetais/análise , Limite de Detecção , Produtos da Carne/análise , Penaeidae/química , Reprodutibilidade dos Testes , Dodecilsulfato de Sódio/química , Extração em Fase Sólida/métodos , Sulfitos/química
4.
Food Chem ; 150: 348-52, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24360461

RESUMO

We developed and validated a novel lateral flow assay for the detection of crustacean protein in processed foods. This assay had high sensitivity; the visual detection limit for shrimp protein extract was 25µg/L, equivalent to 1µg/g protein in a food sample, and results could be obtained within 20min without sophisticated procedures or expensive equipment. Concordance between our assay and another validated quantitative enzyme-linked immunosorbent assay was 97% for commercially processed foods. This assay is rapid, simple, reliable, and highly correlated with validated enzyme-linked immunosorbent assays and is thus suitable for monitoring of food products, especially in food-processing facilities.


Assuntos
Proteínas de Artrópodes/análise , Crustáceos/química , Contaminação de Alimentos/análise , Imunoensaio/métodos , Alérgenos/análise , Alérgenos/imunologia , Animais , Proteínas de Artrópodes/imunologia , Crustáceos/imunologia , Fast Foods/análise , Imunoensaio/instrumentação , Limite de Detecção
5.
Biosci Biotechnol Biochem ; 72(12): 3091-9, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19060408

RESUMO

Three seaweed chitinase isozymes (Chi-A, B, and C) were purified from a red algae, Chondrus verrucosus. The molecular weights and isoelectric points were 24.5 kDa and 3.5 for Chi-A, 25.5 kDa and 4.6 for Chi-B, and 24.5 kDa and <3.5 for Chi-C. Optimum pH and temperature were observed at pH 2.0 at 80 degrees C for Chi-A and Chi-C, and at pH 1.0 and 70 degrees C for Chi-B. Toward N-acetylchitooligosaccharide (GlcNAc(n)) (n=2 to 6), Chi-A, B, and C hydrolyzed GlcNAc(5) and GlcNAc(6) and produced GlcNAc(n) (n=2 to 4). GlcNAc(n) (n=3, 4) with the reducing end-side of beta anomer was detected in the hydrolysis products. These results indicate that the reactions of Chi-A, B, and C for GlcNAc(n) were a retaining mechanism similar to that of family 18 chitinase. Toward crystalline chitins, Chi-A, B, and C degraded squid pen beta-chitin more than crab shell or shrimp shell alpha-chitin.


Assuntos
Quitinases/isolamento & purificação , Quitinases/metabolismo , Chondrus/enzimologia , Sequência de Aminoácidos , Quitinases/química , Eletroforese , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Solubilidade , Especificidade por Substrato , Temperatura
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