Name: preparation of high-quality fermented fish product
Uploaded: 2019-08-23T15:00:00
Description: Watch this Scientific Journal Video about Preparation of High-Quality Fermented Fish Product at JoVE.com

Esta pesquisa foi apoiada financeiramente pelo fundo destinado a Jiangsu natural Science Fund (BK20170185), projeto de Jiangsu Fisheries Administrator (Y2017-30), National natural Science Foundation of China (NFSC31801575), China agriculturas Research Sistema (CARS-45-26), programa nacional de disciplina de primeira classe de ciência e tecnologia de alimentos (JUFSTR20180201), e Yi Tong-Jiangsu programa de pós-doutorado.

1. preparação da amostra
<ol><li>Descongelar a carne congelada do esturjão a água de fluxo abaixo de 20 ° c. Em seguida, limpe, descasque e corte o peixe em cubos (2 cm × 2 cm × 1,5 cm). Nota: a matéria-prima podia ser carne fresca ou congelada do esturjão.</li>
	<li>Misture os cubos de peixe com 6-12% (w/v) solução de sal a uma taxa de 1:1 (massa de cubo de peixe para volume de solução), a 10 ° c para 1-3 h.</li>
	<li>Coloque os cubos de peixe marinado em uma chapa de aço inoxidável gridding e cubos...

<ol>
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F., Xia, W. S., Jiang, Q. X., Xu, Y. S., Fan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+Mixed+Starter+Cultures+to+Flavor+Profile+of+Suanyu+-+A+Traditional+Chinese+Low-Salt+Fermented+Whole+Fish.">Contribution of Mixed Starter Cultures to Flavor Profile of Suanyu - A Traditional Chinese Low-Salt Fermented Whole Fish.</a> Journal of Food Processing and Preservation. 41 (5), 15 (2017).</li><li>Anihouvi, V. B., Sakyi-Dawson, E., Ayernor, G. S., Hounhouigan, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiological+changes+in+naturally+fermented+cassava+fish+(Pseudotolithus+sp.)+for+lanhouin+production.">Microbiological changes in naturally fermented cassava fish (Pseudotolithus sp.) for lanhouin production.</a> International Journal of Food Microbiology. 116 (2), 287-291 (2007).</li><li>Corral, S., Salvador, A., Flores, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salt+reduction+in+slow+fermented+sausages+affects+the+generation+of+aroma+active+compounds.">Salt reduction in slow fermented sausages affects the generation of aroma active compounds.</a> Meat Science. 93 (3), 776-785 (2013).</li><li>Wang, W., Xia, W., Pei, G., Xu, Y., Jiang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolysis+during+fermentation+of+Suanyu+as+a+traditional+fermented+fish+product+of+China.">Proteolysis during fermentation of Suanyu as a traditional fermented fish product of China.</a> International Journal of Food Properties. 20 (90), 29-38 (2017).</li><li>Coventry, M. J., Hickey, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+spices+and+manganese+on+meat+starter+culture+activity.">The effect of spices and manganese on meat starter culture activity.</a> Meat Science. 33 (3), 391-399 (1993).</li><li>Zhu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+on+quality+characteristics+of+pickled+and+fermented+sturgeon+(Acipenser+sinensis)+meat+in+retort+cooking.">Comparative study on quality characteristics of pickled and fermented sturgeon (Acipenser sinensis) meat in retort cooking.</a> International Journal of Food Science &amp; Technology. , (2019).</li><li>Mendonca, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+yeast+flora+in+artisanal+country+style+and+industrial;dry+cured+sausage+(yeast+in+fermented+sausage).">Dynamics of the yeast flora in artisanal country style and industrial;dry cured sausage (yeast in fermented sausage).</a> Food Control. 29 (1), 143-148 (2013).</li><li>Zeng, X. F., Xia, W. S., Jiang, Q. X., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+autochthonous+starter+cultures+on+microbiological+and+physico-chemical+characteristics+of+Suan+yu,+a+traditional+Chinese+low+salt+fermented+fish.">Effect of autochthonous starter cultures on microbiological and physico-chemical characteristics of Suan yu, a traditional Chinese low salt fermented fish.</a> Food Control. 33 (2), 344-351 (2013).</li><li>Gao, P., Jiang, Q., Xu, Y., Xia, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Esterase+activities+of+autochthonous+starter+cultures+to+increase+volatile+flavor+compounds+in+Chinese+traditional+fermented+fish+(Suan+yu).">Esterase activities of autochthonous starter cultures to increase volatile flavor compounds in Chinese traditional fermented fish (Suan yu).</a> International Journal of Food Properties. 20 (1), 663-672 (2017).</li><li>Wahyuni, S., Holilah, , Asranudin, , Noviyanti, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+shelf+life+of+wikau+maombo+brownies+cake+using+Accelerated+Shelf+Life+Testing+(ASLT)+method+with+Arrhenius+model.">Estimation of shelf life of wikau maombo brownies cake using Accelerated Shelf Life Testing (ASLT) method with Arrhenius model.</a> IOP Conference Series Earth and Environmental Science. 122 (1), 12-22 (2018).</li><li>Zeng, X. F., Xia, W. S., Jiang, Q. X., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+microbial+properties+of+Chinese+traditional+low-salt+fermented+whole+fish+product+Suan+yu.">Chemical and microbial properties of Chinese traditional low-salt fermented whole fish product Suan yu.</a> Food Control. 30 (2), 590-595 (2013).</li><li>Yu, D., Jiang, Q., Xu, Y., Xia, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+shelf+life+extension+of+refrigerated+grass+carp+(Ctenopharyngodon+idellus)+fillets+by+chitosan+coating+combined+with+glycerol+monolaurate.">The shelf life extension of refrigerated grass carp (Ctenopharyngodon idellus) fillets by chitosan coating combined with glycerol monolaurate.</a> International Journal of Biological Macromolecules. 101, 448-454 (2017).</li><li>Czerner, M., Tomas, M. C., Yeannes, M. 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Neste estudo, uma nova técnica para a produção de produtos de peixe fermentado de alta qualidade com aroma de vinho e testes para propriedades sensoriais são fornecidos. Os principais processos desta técnica são a marinação, secagem, fermentação e esterilização. No processo de marinação, a concentração de sal, a temperatura e o tempo todos afetam as propriedades texturais do peixe. A dureza e o mastigabilidade do produto aumentam gradualmente com o aumento da concentração de sal (0-12%, w/v) e o prolong...

O produto de peixe marinado comercial atual em China tem o problema do gosto salgado pesado, do aroma insuficiente do vinho, da elasticidade pobre e da cor pálida, que diminui a aceitabilidade aos consumidores. Portanto, uma nova técnica para um produto de carne de peixe de alta qualidade com aroma de vinho precisa ser criada e otimizada.
Nos últimos anos, a aplicação de técnicas modernas de fermentação em carnes e peixes atraiu a atenção de mais e mais pesquisadores1,2,3,4. Por inoculação de culturas iniciais em carne e peixe, a segurança alimentar foi reforçada, o tempo de processamento foi encurtado; e as propriedades sensoriais do produto foram modificadas. Saithong et al.5 bactérias isoladas de LACTOBACILLUS (Lab) de Plaa-som natural e usaram este Lab como uma cultura inicial, que induziu alta acidez e suprimiu bactérias patogênicas. Zeng et al.6 relataram que a inoculação com as culturas autóctones de partida reduziu o tempo de fermentação e melhorou as propriedades sensoriais das amostras. Casaburi et al.7 afirmaram que o uso de culturas microbianas de partida influencia o desenvolvimento do aroma em carnes fermentadas. Nestas culturas iniciais, S. cerevisiae poderia produzir aroma de vinho por fermentação alcoólica e também poderia dar ao produto outras qualidades organolépticas melhoradas. Conseqüentemente, s. cerevisiae é uma cultura apropriada do acionador de partida para produtos do vinho-aroma8,9,10 e o produto do peixe do vinho-aroma poderia ser feito por S. cerevisiae.
No processo de fazer vinho-aroma de produtos de peixe, a textura de carne e peixe pode ser afetada pelo teor de sal, teor de água, pH, desnaturação de proteínas, etc. Portanto, a marinação, secagem, fermentação e esterilização podem influenciar as características de textura. A formação de sabor e sabor é complicada e é afetada principalmente pela marinação e fermentação, pois é altamente relacionada à hidrólise de carboidratos, proteínas e lipídios, e oxidação lipídica leve11,12. Também pode ser afetado pela adição de especiarias13. Para o desenvolvimento da cor, a reação de Maillard ocorre que é envolvida no processo de fermentação e de esterilização10.
Este artigo poderia fornecer suporte técnico para a industrialização do produto de peixe fermentado com aroma de vinho, que é de grande importância para o desenvolvimento da indústria de processamento de peixe. Esta técnica pode melhorar o sabor do produto pelo aumento da proteólise (mais aminoácidos livres e peptídeos solúveis em TCA), modificar o sabor principalmente por álcoois (etanol, 1-octen-3-OL, 2-metil-1-propanol e 3-metil-1-butanol), ésteres (acetato de etila) e aldeídos (nonanal, 3-methylbutanal e benzaldehyde), aumento bucal por maior dureza, Springiness, gomosidade e chewiness, e dar mais atraente russet cor por e uma superfície brilhante14. Igualmente dá a conveniência dos consumidores porque o produto pode ser armazenado na temperatura ambiente. Como descrito por outros estudos prévios15,16,17, a fermentação com S. cerevisiae também tem sido comprovada para melhorar significativamente as qualidades organolépticas em outros produtos de carne ou peixe.
Vale ressaltar que o protocolo introduzido também pode ser aplicado em outras espécies de peixes, como carpa de capim, carpa de prata, carpa preta, carpa bighead, bacalhau, salmão, etc. Para a alta qualidade de produtos de peixe, os peixes sem processamento devem ser usados, como peixe fresco, peixe no gelo ou peixe congelado armazenado por menos de 1 ano. Além disso, como a oxidação lipídica suave poderia aumentar o sabor enquanto a oxidação lipídica extensa traz sabor desagradável, peixe com menos gordura é preferido ou peixe desnatado é recomendado.

<table>
	<tbody>
		<tr>
			<td>2,4,6-trimethylpyridine</td>
			<td>Tokyo Chemical Industry Co., Ltd.</td>
			<td>Purity 98%</td>
			<td></td>
		</tr>
		<tr>
			<td>Colorimeter</td>
			<td>Hunterlab</td>
			<td>UltraScan Pro1166</td>
			<td></td>
		</tr>
		<tr>
			<td>DB-WAX column</td>
			<td>Agilent</td>
			<td>30 m &#215; 0.25 mm &#215; 0.25&#956;m</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital pH meter</td>
			<td>Mettler toledo Instrument (Shanghai) Co., Ltd.</td>
			<td>DELTA-320</td>
			<td></td>
		</tr>
		<tr>
			<td>Drying oven</td>
			<td>Shanghai Yiheng Scientific Instrument Co., Ltd.</td>
			<td>DHG-9070A</td>
			<td></td>
		</tr>
		<tr>
			<td>Frozen sturgeon</td>
			<td>Huada Marine Industry Group Co., Ltd</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas chromatograph-mass spectrometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>TSQ Quantum XLS</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidities incubator</td>
			<td>Shanghai Yiheng Scientific Instrument Co., Ltd.</td>
			<td>LHS-250HC-II</td>
			<td></td>
		</tr>
		<tr>
			<td>Saccharomyces cerevisiae</td>
			<td>Angel Yeast Co., Ltd</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Spices</td>
			<td>Auchan Supermarket</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilization pot</td>
			<td>Longqiang Machinery Technology Co., Ltd.</td>
			<td>RHS-03-700</td>
			<td></td>
		</tr>
		<tr>
			<td>Supelco</td>
			<td>Sigma</td>
			<td>65&#956;m, PDMS/DVB</td>
			<td></td>
		</tr>
		<tr>
			<td>Texture analyzer</td>
			<td>Stable Micro Systems, Ltd.</td>
			<td>TA-XT2i</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum package machine</td>
			<td>Quanzhou Yiminxin Electromechanical Co., Ltd.</td>
			<td>YMX-958-10L</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of high-quality fermented fish product

Este protocolo fornece um método para a preparação do produto de peixe fermentado industrializado com o produto de carne de esturjão (aquilaria sinensis). Os procedimentos foram: (1) pré-tratamento de esturjão de viveiro, incluindo decapitação, evisceração, esfolamento, limpeza e corte; (2) marinada cubos de peixe em 6-12% (w/v) solução de sal (1:1, massa de cubo de peixe para o volume da solução); (3) secagem de cubos de peixe para um teor de água de 50-60% por ar quente (40-60 ° c) ou por vácuo; (4) fermentação envolvendo cubos de peixes inoculados com 0,4-1,6% (p/p) S. cerevisiae em solução de sabor a cubos de peixe e fermentando a 25-35 ° c por 6-10 h; (5) vedação de cubos de peixe em embalagens a vácuo com soluções de marinagem e fermentação; (6) esterilizando a 115-121 ° c por 10-20 min. O produto da carne do esturjão preparado por este método tem o gosto delicioso que é maduro e grosso, tem vários tipos e grandes quantidades de compostos voláteis do sabor tais como álcoois e ésteres que poderiam mascarar o odor e desagradável dos peixes, tem o sal moderado o índice mas as boas propriedades da textura tais como o Springiness elevado, o gomosidade e o chewiness, e têm a cor russet brilhante e a aparência atrativa. Esta nova técnica também poderia ser aplicada no processamento de outros peixes para fornecer alimentos convenientes de lanches de peixe que poderiam ser armazenados à temperatura ambiente. É apropriado para peixes marinhos e de água doce.

A concentração de sal apropriada, o tempo e a temperatura de marinada fazem a qualidade textural do produto melhor. As melhores condições de marinação foram as seguintes: a concentração de sal de 8% na solução marinadora; tempo de marinação de 2 h; e temperatura de marinada de 10 ° c. Consulte a Figura 1.
No processo de secagem, o teor de umidade final e a temperatura de secagem podem influenciar a textura e a qualidade sensorial. As melhores condiç?...

Watch this Scientific Journal Video about Preparation of High-Quality Fermented Fish Product at JoVE.com

Preparation of High-Quality Fermented Fish Product

O objetivo do protocolo é fornecer uma técnica de fermentação de peixes industrializada com base na inoculação de Saccharomyces cerevisiae.

Preparação de produtos de peixe fermentado de alta qualidade

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures ...

Este procedimento produz um produto de peixe fermentado de alta qualidade com aroma de vinho, e poderia ser aplicado na produção da indústria. Esta técnica dá ao produto de peixe uma aparência atraente, sabor único, sabor delicioso e boas propriedades de textura. E este produto pode ser armazenado à temperatura ambiente por pelo menos um ano.Para iniciar este procedimento, descongele, limpe e congele a carne de esturjão congelada, conforme descrito no protocolo de texto. Corte o peixe em cubos. Em seguida, misture os cubos de peixe com solução de 6 a 12% de sal em uma proporção de um para um a 10 graus Celsius por uma a três horas.Em seguida, coloque os cubos de peixe marinado em uma placa de grade de aço inoxidável e seque-os com ar quente a uma temperatura entre 40 e 60 graus Celsius por seis a 10 horas. Para preparar o líquido de especiarias, misture especiarias incluindo 1,2 unidades de cebolinha, uma unidade de gengibre, 0,6 unidades de anis estrelado, 0,6 unidades de erva-doce, 0,3 unidades de chá verde e 0,3 unidades de pimenta com 40 unidades de água. Ferva por meia hora, e pegue o filtrado.Para preparar a solução de sabor, misture especiarias líquidas, vinho amarelo, vinho branco, açúcar de cana, glutamato monossódico e vinagre nas frações de massa aqui mostradas. Depois disso, misture os cubos de peixe seco com a solução de sabor, inoculando 0,4 a 1,6% de cerevisiae de saccharomyces em uma proporção de um para um. Sele a mistura em um recipiente e fermente a uma temperatura entre 25 e 35 graus Celsius, por seis a 10 horas.Coloque os cubos de peixe fermentados em uma embalagem de vácuo com soluções de marinação e fermentação e sele o pacote de vácuo com uma força de vácuo de 0,085 megapascals, um tempo de vedação de 3,6 segundos e um tempo de resfriamento de 5,5 segundos. Em seguida, esterilize os cubos de peixe embalados usando uma temperatura de esterilização entre 115 e 121 graus Celsius por 10 a 20 minutos. Neste estudo, é preparado um produto de peixe fermentado de alta qualidade.Usar uma concentração de sal adequada, tempo de marinação e temperatura torna a qualidade textural dos produtos melhor. As melhores condições de marinação parecem ser uma concentração de sal de 8% E a solução marinando, um tempo de marinação de duas horas. E uma temperatura marinando de 10 graus Celsius.No processo de secagem, o teor final de umidade e a temperatura de secagem podem influenciar a textura e a qualidade do sensor. As melhores condições de secagem parecem ser um teor de umidade dos cubos de peixe seco de 55% e temperatura de secagem de 50 graus Celsius. No processo de fermentação, a adição de saccharomyces cerevisiae, a temperatura e o tempo todos afetam as propriedades de sabor, sabor, cor e textura dos peixes.O peixe fermentado bem sucedido tem altas pontuações para sabor, sabor, cor e textura, resultando em sabor harmonioso, fragrância fermentativa, vinícola, cor russet, superfície brilhante, caramelo e textura al dente. O produto de peixe fracassado tem as pontuações baixas para sabor, sabor, cor e textura, resultando no mau gosto, amargura, azeite, peixaria, cor pálida, superfície áspera, sensação bucal grossa e textura deteriorada. A esterilização e intensidade adequadas é de 4,5 minutos.E uma temperatura de esterilização adequada parece ser de 121 graus Celsius. As considerações de armazenamento são então calculadas e determinadas conforme descrito no protocolo de texto. E a previsão para o prazo de validade é de 264 dias a 25 graus Celsius ou 205 dias a 35 graus Celsius.Ao realizar este procedimento, é importante notar que tanto a fermentação quanto as etapas de esterilização são vitais para o sucesso desta técnica. Após o aprendizado deste procedimento, pode ser usado na fermentação de outras espécies de peixes. No futuro, a fermentação com outras cepas poderia ser investigada.Como lactobacillus, staphylococcus, et cetera. regiões, certifique-se de tomar precauções ao esterilizar produtos de peixe, pois a temperatura extremamente alta pode causar danos.

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Research

JoVE Journal

Biochemistry

Preparation of Homogeneous MALDI Samples for Quantitative Applications

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The heterogeneity of ion signals is a severe problem in MALDI, which results in poor data reproducibility and makes MALDI unsuitable for quantitative analysis. By regulating sample plate temperature during sample preparation, thermal-induced hydrodynamic flows inside droplets of sample solution are able to reduce the heterogeneity problem. A room-temperature sample preparation chamber equipped with a temperature-regulated copper base block that holds MALDI sample plates facilitates precise control of the sample drying condition. After drying of sample droplets, the temperature of sample plates is returned to room temperature and removed from the chamber for subsequent mass spectrometric analysis. The areas of samples are examined with MALDI-imaging mass spectrometry to obtain the spatial distribution of all components in the sample. In comparison with the conventional dried-droplet method that prepares samples under ambient conditions without temperature control, the samples prepared with the method demonstrated herein show significantly better spatial distribution and signal intensity. According to observations using carbohydrate and peptide samples, decreasing substrate temperature while maintaining the surroundings at ambient temperature during the drying process can effectively reduce the heterogeneity of ion signals. This method is generally applicable to various combinations of samples and matrices.

A protocol for reducing spatial heterogeneities of ion signals in MALDI mass spectrometry by regulating substrate temperature during sample drying processes is demonstrated.

Preparação de Amostras de MALDI homogênea para Aplicações quantitativas

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization ...

Bioquímica

A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a challenge for ChIP due to technical difficulty in isolation of high-quality nuclei with minimal contamination of myofibrils. Previous protocols have attempted to purify nuclei before cross-linking, which incurs the risk of altered DNA-protein interaction during the prolonged nuclei preparation process. In the current protocol, we first cross-linked the skeletal muscle tissue collected from mice, and the tissues were minced and sonicated. Since we found that ultracentrifugation was not able to separate nuclei from myofibrils using cross-linked muscle tissue, we devised a sequential filtration procedure to obtain high-quality nuclei devoid of significant myofibril contamination. We subsequently prepared chromatin by using an ultrasonicator, and ChIP assays with anti-BMAL1 antibody revealed robust circadian binding pattern of BMAL1 to target gene promoters. This filtration protocol constitutes an easily applicable method to isolate high-quality nuclei from cross-linked skeletal muscle tissue, allowing consistent sample processing for circadian and other time-sensitive studies. In combination with next-generation sequencing (NGS), our method can be deployed for various mechanistic and genomic studies focusing on skeletal muscle function.

We present a filtration-based protocol to isolate high-quality nuclei from cross-linked mouse skeletal muscle wherein we removed the need for ultracentrifugation, making it easily applicable. We show that chromatin prepared from the nuclei is suitable for chromatin immunoprecipitation and likely chromatin immunoprecipitation sequencing studies.

Um método baseado na filtração de preparação de núcleos de alta qualidade a partir de músculo esquelético reticulado para imunoprecipitação com cromatina

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a ...

Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro enzymatic synthesis of a flavonol. In this method, Atf3h and Atfls1, two key genes in the biosynthetic pathway of the flavonols, are cloned and overexpressed in Escherichia coli. The recombinant enzymes are purified via an affinity column and then a bienzymatic cascade is established in a specific synthetic buffer. Two flavonols are synthesized in this system as examples and determined by TLC and HPLC/LC/MS analyses. The method displays obvious advantages in the derivation of flavonols over other approaches. It is time- and labor-saving and highly cost-effective. The reaction is easy to be accurately controlled and thus scaled up for mass production. The target product can be purified easily due to the simple components in the system. However, this system is usually restricted to the production of a flavonol from a flavanone.

The derivation of a flavonol is crucial for its application in healthcare and the food industry. Here, we provide a detailed protocol for the biosynthesis of a flavonol from a flavanone and discuss the crucial steps and its advantages over other approaches.

Biossíntese de um flavonol de um Flavanone estabelecendo uma cascata Bienzimática de um pote

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro ...

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality RNA is often hindered by high starch and sugar content. These compounds can reduce both the yield and the quality of the extracted total RNA. The deterioration in quantity and quality of total RNA can subsequently have a significant impact on the downstream transcriptomic analyses, which may not accurately reflect the spatial and/or temporal variation in the gene expression profile of the samples being tested. In this protocol, we describe an optimized method for extraction of total RNA with sufficient quantity and quality to be used for whole transcriptome analysis of cereal grains. The described method is suitable for several downstream applications used for transcriptomic profiling of developing, germinating, and mature cereal seeds. The method of transcriptome profiling using a microarray platform is shown. This method is specifically designed for gene expression profiling of cereals with described genome sequences. The detailed procedure from microarray handling to final quality control is described. This includes cDNA synthesis, cRNA labelling, microarray hybridization, slide scanning, feature extraction, and data quality validation. The data generated by this method can be used to characterize the transcriptome of cereals during germination, in various stages of grain development, or at different biotic or abiotic stress conditions. The results presented here exemplify high-quality transcriptome data amenable for downstream bioinformatics analyses, such as the determination of differentially expressed genes (DEGs), characterisation of gene regulatory networks, and conducting transcriptome-wide association study (TWAS).

A method for transcriptome profiling of cereals is presented. The microarray-based gene expression profiling starts with the isolation of high-quality total RNA from cereal grains and continues with the generation of cDNA. After cRNA labelling and microarray hybridization, recommendations are given for signal detection and quality control.

Obtenção de dados de transcriptome de alta qualidade de sementes de cereal por um método modificado para criação de perfil de expressão genética

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Análise celular única de alelos ativos transcricionralmente por peixe de molécula única

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

Fluormetria de varredura nano-diferencial para triagem em descoberta de chumbo baseada em fragmentos

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer ...

Preparation of High-Temperature Sample Grids for Cryo-EM

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, mostly at 4 &#176;C and occasionally at room temperature. Recently, we discovered that the protein structure solved at low temperature may not be functionally relevant, particularly for proteins from thermophilic archaea. A procedure was developed to prepare protein samples at higher temperatures (up to 70 &#176;C) for cryo-EM analysis. We showed that the structures from samples prepared at higher temperatures are functionally relevant and temperature dependent. Here we describe a detailed protocol for preparing sample grids at high temperature, using 55 &#176;C as an example. The experiment made use of a vitrification apparatus modified using an additional centrifuge tube, and samples were incubated at 55 &#176;C. The detailed procedures were fine-tuned to minimize vapor condensation and obtain a thin layer of ice on the grid. Examples of successful and unsuccessful experiments are provided.

This paper provides a detailed protocol for preparing sample grids at temperatures as high as 70 &#176;C, prior to plunge freezing for cryo-EM experiments.

Preparação de grades de amostras de alta temperatura para crio-EM

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, ...

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are influenced by its structure. Hence, a suitable extraction method that isolates representative samples of glycogen is crucial to the study of this macromolecule. Compared to other extraction methods, a method that employs a sucrose density gradient centrifugation step can minimize molecular damage. Based on this method, a recent publication describes how the density of the sucrose solution used during centrifugation was varied (30%, 50%, 72.5%) to find the most suitable concentration to extract glycogen particles of a wide variety of sizes, limiting the loss of smaller particles. A 10 min boiling step was introduced to test its ability to denature glycogen degrading enzymes, thus preserving glycogen. The lowest sucrose concentration (30%) and the addition of the boiling step were shown to extract the most representative samples of glycogen.

An optimal sucrose concentration was determined for the extraction of liver glycogen using sucrose density gradient centrifugation. The addition of a 10 min boiling step to inhibit glycogen-degrading enzymes proved beneficial.

A extração de moléculas de glicogênio hepático para determinação da estrutura do glicogênio

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are ...

Preparation of siRNA Mixture for Mitochondrial DNA Synthesis

To begin, prepare a 140 nano molar mix of small interfering RNA or siRNA by diluting the siRNA in optimum medium. Add the five microliters of the diluted mix to the wells of a black 384 well cell culture microplate. Prepare the appropriate amount of RNAiMAX transfection reagent solution in optimum medium and add 10 microliters to each well of the 384 well plate with a reagent dispenser.

Preparação de Mistura de SiRNA para Síntese de DNA Mitocondrial

Preparação de Grelhas de Microscopia Eletrónica para Criotomografia Celular In Situ

Sample Preparation for In Situ Cryotomography of Mammalian Cells

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly relevant to cellular biology and virology. The potential of combining cryotomography with other microscopy modalities makes it a perfect technique for integrative and correlative imaging. However, sample preparation for in situ cellular tomography is not straightforward, as cells do not readily attach and stretch over the electron microscopy grid. Additionally, the grids themselves are fragile and can break if handled too forcefully, resulting in the loss of imageable areas. The geometry of tissue culture dishes can also pose a challenge when manipulating the grids with tweezers. Here, we describe the tips and tricks to overcome these (and other) challenges and prepare good-quality samples for in situ cellular cryotomography and correlative imaging of adherent mammalian cells. With continued advances in cryomicroscopy technology, this technique holds enormous promise for advancing our understanding of complex biological systems.

Autor em destaque: Otimizando a preparação da grade para tomografia crioeletrônica aprimorada 

This method provides an accessible and flexible protocol for the preparation of electron microscopy (EM) grids for in situ cellular cryotomography and correlative light and electron microscopy (CLEM).

Preparo de Amostras para Criotomografia In Situ de Células de Mamíferos

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly ...