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

这项研究由江苏省自然科学基金专项资金（BK20170185）、江苏省水产厅项目（Y2017-30）、国家自然科学基金（NFSC31801575）、中国农业研究资助。系统（CARS-45-26）、国家一级食品科技学科项目（JUFSTR20180201）和易通江苏博士后项目。

1. 样品制备
<ol><li>在低于 20°C 的流水下解冻冷冻的鱼肉。然后，清洁，剥皮，将鱼切成立方体（2厘米×2厘米×1.5厘米）。 注：原料可以是新鲜或冷冻的鱼肉。</li>
	<li>将鱼块与 6-12%（w/v）盐溶液混合，速率为 1：1（鱼立方质量到溶液体积），在 10°C 下，1-3 小时。</li>
	<li>将腌制鱼块放在不锈钢网格板和干鱼块上，在 40-60 °C 下用热空气干燥约 6-10 小时，或在真空中干燥鱼块，直到水含量降至 ...

<ol>
	<li>Riebroy, S., Benjakul, S., Visessanguan, W., Tanaka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+during+fermentation+and+properties+of+som-fug+produced+from+different+marine+fish.">Changes during fermentation and properties of som-fug produced from different marine fish.</a> Journal of Food Processing and Preservation. 31 (6), 751-770 (2007).</li><li>Xu, Y., Xia, W., Yang, F., Jinmoon, K., Nie, X. <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+fermentation+temperature+on+the+microbial+and+physicochemical+properties+of+silver+carp+sausages+inoculated+with+Pediococcus+pentosaceus.">Effect of fermentation temperature on the microbial and physicochemical properties of silver carp sausages inoculated with Pediococcus pentosaceus.</a> Food Chemistry. 118 (3), 512-518 (2010).</li><li>Cano-Garcia, L., Flores, M., Belloch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+and+aromatic+potential+of+Debaryomyces+hansenii+strains+isolated+from+naturally+fermented+sausages.">Molecular characterization and aromatic potential of Debaryomyces hansenii strains isolated from naturally fermented sausages.</a> Food Research International. 52 (1), 42-49 (2013).</li><li>Chen, Q., Liu, Q., Sun, Q., Kong, B., Xiong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavour+formation+from+hydrolysis+of+pork+sarcoplasmic+protein+extract+by+a+unique+LAB+culture+isolated+from+Harbin+dry+sausage.">Flavour formation from hydrolysis of pork sarcoplasmic protein extract by a unique LAB culture isolated from Harbin dry sausage.</a> Meat Science. 100, 110-117 (2015).</li><li>Saithong, P., Panthavee, W., Boonyaratanakornkit, M., Sikkhamondhol, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+starter+culture+of+lactic+acid+bacteria+in+plaa-som,+a+Thai+fermented+fish.">Use of a starter culture of lactic acid bacteria in plaa-som, a Thai fermented fish.</a> Journal of Bioscience &amp; Bioengineering. 110 (5), 553-557 (2010).</li><li>Zeng, X., Xia, W., Jiang, Q., Guan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+Sensory+Characteristics+of+Whole+Carp+Inoculated+With+Autochthonous+Starter+Cultures.">Biochemical and Sensory Characteristics of Whole Carp Inoculated With Autochthonous Starter Cultures.</a> Journal of Aquatic Food Product Technology. 24 (1), 52-67 (2015).</li><li>Casaburi, A., 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=Biochemical+and+sensory+characteristics+of+traditional+fermented+sausages+of+Vallo+di+Diano+(Southern+Italy)+as+affected+by+the+use+of+starter+cultures.">Biochemical and sensory characteristics of traditional fermented sausages of Vallo di Diano (Southern Italy) as affected by the use of starter cultures.</a> Meat Science. 76 (2), 295-307 (2007).</li><li>Gao, P., 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=Effect+of+autochthonous+starter+cultures+on+the+volatile+flavour+compounds+of+Chinese+traditional+fermented+fish+(Suan+yu).">Effect of autochthonous starter cultures on the volatile flavour compounds of Chinese traditional fermented fish (Suan yu).</a> International Journal of Food Science and Technology. 51 (7), 1630-1637 (2016).</li><li>Zeng, X. 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. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ripening+of+salted+anchovy+(Engraulis+anchoita):+development+of+lipid+oxidation,+colour+and+other+sensorial+characteristics.">Ripening of salted anchovy (Engraulis anchoita): development of lipid oxidation, colour and other sensorial characteristics.</a> Journal of the Science of Food and Agriculture. 91 (4), 609-615 (2011).</li></ol>

本研究提供了一种生产具有葡萄酒香气的优质发酵鱼产品的新技术，并测试了感官特性。该技术的关键工艺是腌制、干燥、发酵和灭菌。在腌制过程中，盐的浓度、温度和时间都影响着鱼的纹理特性。随着盐浓度（0-12%，w/v）和腌制时间延长（0-2.5 h），产品的硬度和咀嚼性逐渐增加;但 2 小时后的增加并不显著。同时，当温度高于10°C时，咀嚼性随腌汁温度的增加而增加，而硬度降低。在干燥过程?...

目前我国商业腌制鱼产品存在咸味重、葡萄酒香气不足、弹性差、色泽淡等问题，降低了消费者的接受度。因此，需要创造和优化具有葡萄酒香气的高品质鱼肉产品的新技术。
近年来，现代发酵技术在肉鱼中的应用越来越受到研究者的关注。通过向肉鱼中接种起动机，提高了食品安全，缩短了加工时间;产品感官特性已被修改。Saithong等人5日从天然乳酸杆菌（LAB）中分离出来，并利用该LAB作为启动培养物，诱导高酸度和抑制致病菌。Zeng等人6日报告说，用自体启动剂培养物接种可缩短发酵时间，改善样品的感官特性。Casaburi等人7日声称，使用微生物启动培养物会影响发酵肉类中香气的发展。在这些启动培养物中，S.cerevisae可以通过酒精发酵产生葡萄酒香气，还可以使产品具有其他改进的感官品质。因此，S. cerevisae是一种适合葡萄酒香气产品8、9、10 和葡萄酒香鱼产品的起动机，可由S. cerevisae制成。
在制作葡萄酒香鱼产品的过程中，肉和鱼的质地会受盐含量、含水量、pH、蛋白质变性等影响。因此，腌制、干燥、发酵和灭菌都会影响质地特性。风味和味觉的形成是复杂的，主要受腌制和发酵的影响，因为它与碳水化合物、蛋白质和脂质的水解以及轻度脂质氧化11、12密切相关。它也可能受到添加香料13的影响。为了发展颜色，梅拉德反应发生，这是涉及发酵和灭菌的过程10。
本文可为葡萄酒香气发酵鱼产品的产业化提供技术支持，对鱼类加工业的发展具有重要意义。该技术可以通过增加蛋白酶（更多的游离氨基酸和TCA-可溶性肽），主要通过酒精（乙醇，1-Octen-3-ol，2-甲基-1-丙醇和3-甲基-1-丁醇），酯（乙酸乙酯）和醛（非肛门，3-甲基丁酸和苯甲醛），增加口感通过更高的硬度，弹簧，软胶和咀嚼性，并给予更有吸引力的红组颜色和明亮的表面14。它还为消费者提供了便利，因为产品可以在室温下储存。正如其他先前研究所描述，15、16、17、与S.cerevisae发酵也已被证明可以显著改善其他肉类或鱼类产品的感官质量。
值得注意的是，引入的协议也可以应用于其他鱼类物种，如草鲤、银鲤、黑鲤、大头鲤、鳕鱼、鲑鱼等。对于高质量的鱼产品，应使用未经加工的鱼，如鲜鱼、冰鱼或储存不到1年的冷冻鱼。此外，由于轻度脂质氧化可以增强风味，而广泛的脂质氧化带来令人不快的味道，因此推荐脂肪较少的鱼或脱脂鱼。

<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

该协议提供了一种制备工业化发酵鱼产品的方法，程序是：（1）对养殖的鱼苗进行预处理，包括斩首、脱皮、剥皮、清洗和切割;（2） 在 6-12% （w/v） 盐溶液（1：1，鱼立方质量到溶液体积）中腌制鱼块;（3）通过热空气（40-60°C）或真空干燥鱼块，水含量为50-60%;（4） 发酵涉及在鱼块的风味溶液中加入0.4-1.6%（w）S.cerevisiae的乳化鱼块，并在25-35°C下发酵6-10小时; （5） 用腌制和发酵溶液在真空包装中密封鱼块;（6） 在115-121°C下消毒10-20分钟。用这种方法制备的鱼肉制品味道鲜美，醇厚，有各种类型和大量挥发性香料化合物，如醇和酯类，可以掩盖鱼的霉味和难闻的气味，具有适量的盐分内容，但良好的纹理性能，如高弹簧，软胶和咀嚼性，具有明亮的红花色和有吸引力的外观。这项新技术也可应用于其他鱼类的加工，提供方便的鱼类零食，可在室温下储存。它适用于海洋和淡水鱼。

合适的盐浓度、腌制时间和温度使产品的质感质量更好。最佳腌制条件如下：腌制溶液中盐浓度为8%;腌制时间为2小时;腌制温度为10°C。参见图 1。
在干燥过程中，最终的水分含量和干燥温度会影响质地和感官质量。最佳干燥条件如下：干鱼块含水量55%，干燥温度50°C。参见图2。
在发酵过程中，加入S.cerevisae...

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

Preparation of High-Quality Fermented Fish Product

该协议的目标是提供基于接种沙沙霉菌的工业化鱼类发酵技术。

优质发酵鱼产品的制备

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

本工艺生产出具有葡萄酒香气的优质发酵鱼产品，可应用于工业生产。该技术赋予鱼产品一个有吸引力的外观，独特的味道，美味的味道，良好的质地属性。本产品可储存在室温下至少一年。要开始此过程，请解冻、清洁和皮肤冷冻鱼肉，如文本协议中所述。把鱼切成块。接下来，在10摄氏度的1比1下，将鱼块与6至12%的盐溶液混合，一至三个小时。然后，将腌制的鱼块放在不锈钢光栅板上，在40至60摄氏度的高温下用热空气干燥6至10小时。为了准备香料液，混合香料，包括1.2个单位的葱，1个单位的生姜，0.6个单位的星光，0.6个单位的芬芳，0.3单位的绿茶，和0.3单位的胡椒与40个单位的水。煮半小时，收集滤物。要准备调味液，请将香料液、黄葡萄酒、白葡萄酒、甘蔗糖、谷氨酸钠和醋混合到此处所示的质量分数中。在此之后，将干鱼块与风味溶液混合，以一比一的比例接种0.4至1.6%的糖精。将混合物密封在容器中，在25至35摄氏度的温度下发酵6至10小时。将发酵的鱼块放入真空包装中，采用腌制和发酵溶液，以0.085兆帕的真空强度密封真空包装，密封时间为3.6秒，冷却时间为5.5秒。然后用115至121摄氏度的灭菌温度对包装的鱼块进行消毒，10至20分钟。本研究准备了高质量的发酵鱼产品。使用合适的盐浓度、腌制时间和温度使产品纹理质量更好。最好的腌制条件似乎是盐浓度为8%和腌制溶液，腌制时间为两小时。和腌制温度10摄氏度。在干燥过程中，最终水分含量和干燥温度会影响质地和传感器质量。最佳干燥条件似乎是干鱼块的水分含量为55%，干燥温度为50摄氏度。在发酵过程中，糖精的加入、温度、时间都影响鱼的味道、味道、颜色和质地特性。成功的发酵鱼在口感、风味、颜色、质地方面得分高，产生和谐口感、发酵香、酒色、红味、亮面、焦糖和质地。失败的鱼产品在味道、味道、颜色和质地方面得分低，导致味道差、苦味、酸味、鱼味、色色苍白、表面粗糙、口感粗糙、质地变质。适当的灭菌和强度似乎是4.5分钟。而适当的灭菌温度似乎是121摄氏度。然后按照文本协议中概述的存储注意事项进行计算和确定。对保质期的预测是264天，在25摄氏度，或在35摄氏度下205天。执行此程序时，必须注意发酵和灭菌步骤对于该技术的成功至关重要。学习此程序后，可用于其他鱼种的发酵。将来，可以研究与其他菌株的发酵。如乳酸杆菌、葡萄球菌等。区域，在消毒鱼产品时一定要采取预防措施，因为极高的温度可能会造成伤害。

preparation-of-high-quality-fermented-fish-product

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.

定量应用同质MALDI样品的制备

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

科研

生物化学

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.

从交联骨骼肌制备高质量核的基于过滤的方法进行染色质免疫沉淀

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.

通过建立单罐双酶级联,从弗拉瓦内生物合成一种黄酮

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.

通过基因表达分析的改良方法从谷物种子获取高质量的转录数据

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.

单分子鱼的转录活性等位基因单细胞分析

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.

纳米微分扫描荧光仪，用于在基于碎片的铅发现中筛选

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.

冷冻电镜高温样品网格的制备

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.

肝糖原分子的提取用于糖原结构测定

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.

用于线粒体DNA合成的siRNA混合物的制备

用于原位细胞冷冻断层扫描的电子显微镜网格的制备

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.

作者聚焦：优化增强型冷冻电子断层扫描的网格制备 

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).

哺乳动物细胞 原位 冷冻断层扫描的样品制备

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