Name: identification of fatty acids in bacillus cereus
Uploaded: 2016-12-05T15:00:00.000+00:00
Duration: 8 min 41 s
Description: Watch this Scientific Journal Video about Identification of Fatty Acids in Bacillus cereus at JoVE.com

作者感谢MISON托马斯对他的技术支持，并藉由完整雷切尔修订的手稿。

1.细菌培养<ol><li>通过扩频将100μl的LB（Luria液体-Bertani培养基）在30℃培养菌株的过夜培养物，LB琼脂培养基的板的表面上制备的细菌（ 蜡状芽孢杆菌菌株ATCC 14579）的草坪。过夜孵育所述板在30℃。 </li></ol> 2. ECL:等效链长<ol><li>计算ECL如下: <img alt="方程" src="/files/ftp_upload/54960/54960eq1.jpg" />搭配: 我，感兴趣的溶质; n中，直链饱和脂肪酸甲酯溶质之前洗脱的碳原子数我; 的n + 1中，直链饱和脂肪酸甲酯的溶质后洗脱的碳原子数我; RTI，RTN，RT（N + 1）上述的FAME峰的保留时间。 注意:通过标准的混合物的注射获得直链饱和脂肪酸的甲酯的保留时间（BAME）。 </li></ol> 3. FAME制备及分析<ol><li>为了通过从琼脂平板刮落，以获得脂质的脂肪酸，收获细菌细胞和细菌的40毫克（鲜重，相当于10 9活细胞）转移到10ml玻璃管螺丝帽和PTFE密封。 </li><li>通过下文详述的酯键的方法14,15进行酯交换反应。 <ol><li>在甲醇加入5毫升的0.2M的KOH的新鲜细菌细胞，并在37℃下孵育1小时。这个反应由碱性甲醇分解的，打破在脂的酯链接并产生脂肪酸甲酯。 </li><li>加入1毫升1M乙酸将pH值降低至7.0。检查其pH值试纸。 </li><li>加入3毫升己烷提取脂肪酸甲酯。 </li><li>在室温下转移上清液（有机相）转化为清洁管和浓缩通过蒸发在氮气下的连续流，以获得约200微升的提取物。将样品转移到与插入一个GC小瓶。 </li><li>注入提取成气相色谱 - 质谱（GC-MS）系统。 </li></ol></li></ol> 4. GC / MS条件<ol><li>注入FAME样品到装有毛细管柱ZB-WAX（长度30米;直径0.25毫米;膜厚为0.25μm）的GC-MS仪器。 </li><li>设置注入口（在无分流模式）温度至250℃。使用氦作为载气，用37厘米/秒的线速度。保持在50℃的烘箱温度下进行1分钟，增加至190℃以20℃/分钟的速度，并以2℃/分钟的速率进一步提高到230℃的最终温度。 </li><li>为MS，在70伏特记录由电子电离（EI）的质谱，并设置50和400原子质量单位（AMU）（2次扫描/秒）之间的采集的总离子电流。 </li><li>当需要时，注射在相同的条件下excep DMOX和甲代吡啶基衍生物t是温度程序烘箱如下: DMOX:50℃（1分钟），20℃/分钟直到210℃和2℃/分钟直到240℃（5分钟）; 甲代吡啶:6℃（1分钟），20℃/分钟直到220℃和2℃/分钟直到250℃（20分钟）。 </li></ol> 5.代吡啶酯准备从16 FAME <ol><li>蒸发从第3节的FAME提取与氮气流（至少10mg干基料）和在1毫升无水二氯甲烷溶解。 </li><li>制备钾叔叔丁醇在四氢呋喃中的1.0M溶液。 </li><li>添加FAME提取和0.2毫升3吡啶甲醇0.1毫升溶液在步骤5.2制成。 </li><li>加热在40℃下在密闭的小瓶中的溶液30分钟。 </li><li>在冷却至室温后，加入精制去离子水（2毫升，见材料表 ）和己烷（4ml）中。用涡旋混合，让相分离，并收集有机相。 </li><li>博士ý它加入无水硫酸钠，直至有机相是完全清楚的。它转移到一个干净的试管中。然后蒸发至200微升。将样品转移到与插入一个GC小瓶。 </li></ol> 6. DMOX准备从17 FAME <ol><li>蒸发从第3节的FAME提取用氮气（至少10mg干基料）的流动。 </li><li>向FAME干提取物，加入250毫克的2-氨基-2-甲基-1-丙醇。用氮气冲洗容器，添加一个塞子，并将其放置在一个加热块过夜，在190℃。 </li><li>在冷却至室温后，加入3毫升二氯甲烷的管，和5ml纯化的去离子水（见材料表 ）。 </li><li>摇相分离，然后取出水相。 </li><li>洗涤有机相用5毫升水。摇相分离，然后取出水相。 </li><li>加入无水硫酸钠，直至有机相干燥是完全清楚，将其转移到一个干净的试管中。在氮气流中蒸发，直至达到200微升的体积。将样品转移到与插入一个GC小瓶。 </li></ol>

<ol>
	<li>Christie, W. W., Han, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lipid Analysis 4th Edition. , (2010).</li><li>H&#220;bschmann, H. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of GC-MS: fundamental and application. Third edition. , (2015).</li><li>Sasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Bacteria+by+Gas+Chromatography+of+Cellular+Fatty+Acids.">Identification of Bacteria by Gas Chromatography of Cellular Fatty Acids.</a> MIDI Technical note. 101, 1-6 (1990).</li><li>Spitzer, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+analysis+of+fatty+acids+by+gas+chromatography+-+Low+resolution+electron+impact+mass+spectrometry+of+their+4,4-dimethyloxazoline+derivatives+-+A+review.">Structure analysis of fatty acids by gas chromatography - Low resolution electron impact mass spectrometry of their 4,4-dimethyloxazoline derivatives - A review.</a> Prog Lipid Res. 35 (4), 387-408 (1996).</li><li>Harvey, D. J., Christie, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in lipid methodology. Volume 1. , 19-80 (1992).</li><li>Diomande, S. E., Nguyen-The, C., Guinebreti&#232;re, M. -. H., Broussolle, V., Brillard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+fatty+acids+in+Bacillus+environmental+adaptation.">Role of fatty acids in Bacillus environmental adaptation.</a> Front Microbiol. 6, (2015).</li><li>Brillard, J., 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=Identification+of+Bacillus+cereus+Genes+Specifically+Expressed+during+Growth+at+Low+Temperatures.">Identification of Bacillus cereus Genes Specifically Expressed during Growth at Low Temperatures.</a> Appl Environ Microbiol. 76 (8), 2562-2573 (2010).</li><li>de Sarrau, B., 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=Influence+of+Anaerobiosis+and+Low+Temperature+on+Bacillus+cereus+Growth,+Metabolism,+and+Membrane+Properties.">Influence of Anaerobiosis and Low Temperature on Bacillus cereus Growth, Metabolism, and Membrane Properties.</a> Appl Environ Microbiol. 78 (6), 1715-1723 (2012).</li><li>Diomand&#233;, S. E., 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=Involvement+of+the+CasK/R+two-component+system+in+optimal+unsaturation+of+the+Bacillus+cereus+fatty+acids+during+low-temperature+growth.">Involvement of the CasK/R two-component system in optimal unsaturation of the Bacillus cereus fatty acids during low-temperature growth.</a> Int J Food Microbiol. 213, 110-117 (2015).</li><li>Berkeley, R. C. W., Heyndrickx, M., Logan, N., De Vos, P., Berkeley, R. C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Applications and Systematics of Bacillus and Relatives. , 1-7 (2002).</li><li>K&#228;mpfer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limits+and+Possibilities+of+Total+Fatty+Acid+Analysis+for+Classification+and+Identification+of+Bacillus+Species.">Limits and Possibilities of Total Fatty Acid Analysis for Classification and Identification of Bacillus Species.</a> System. Appl. Microbiol. 17 (1), 86-98 (1994).</li><li>Kaneda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty-acids+of+genus+bacillus+-+example+of+branched-chain+preference.">Fatty-acids of genus bacillus - example of branched-chain preference.</a> Bacteriol Rev. 41 (2), 391-418 (1977).</li><li>Chazarreta Cifre, L., Alemany, M., de Mendoza, D., Altabe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+Biosynthesis+of+Unsaturated+Fatty+Acids+in+Bacillus+cereus+ATCC+14579+and+Functional+Characterization+of+Novel+Acyl-Lipid+Desaturases.">Exploring the Biosynthesis of Unsaturated Fatty Acids in Bacillus cereus ATCC 14579 and Functional Characterization of Novel Acyl-Lipid Desaturases.</a> Appl Environ Microbiol. 79 (20), 6271-6279 (2013).</li><li>Sasser, M., 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=Identification+of+Bacillus+anthracis+from+culture+using+gas+chromatographic+analysis+of+fatty+acid+methyl+esters.">Identification of Bacillus anthracis from culture using gas chromatographic analysis of fatty acid methyl esters.</a> J AOAC Int. 88 (1), 178-181 (2005).</li><li>Schutter, M. E., Dick, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+fatty+acid+methyl+ester+(FAME)+methods+for+characterizing+microbial+communities.">Comparison of fatty acid methyl ester (FAME) methods for characterizing microbial communities.</a> Soil Sci Soc Am J. 64 (5), 1659-1668 (2000).</li><li>Destaillats, F., Angers, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+methodology+for+the+synthesis+of+FA+picolinyl+esters+from+intact+lipids.">One-step methodology for the synthesis of FA picolinyl esters from intact lipids.</a> J Am Oil Chem Soc. 79 (3), 253-256 (2002).</li><li>Fay, L., Richli, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Location+of+double-bonds+in+polyunsaturated+fatty-acids+by+gas-chromatography+mass-spectrometry+after+4,4-dimethyloxazoline+derivatization.">Location of double-bonds in polyunsaturated fatty-acids by gas-chromatography mass-spectrometry after 4,4-dimethyloxazoline derivatization.</a> J Chromatogr. 541 (1-2), 89-98 (1991).</li><li>Zhang, J. Y., Yu, Q. T., Liu, B. N., Huang, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+modification+in+mass+spectrometry+IV-2-alkenyl-4,4-dimethyloxazolines+as+derivatives+for+the+double+bond+location+of+long-chain+olefinic+acids.">Chemical modification in mass spectrometry IV-2-alkenyl-4,4-dimethyloxazolines as derivatives for the double bond location of long-chain olefinic acids.</a> Biol Mass Spect. 15 (1), 33-44 (1988).</li><li>de Sarrau, B., 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=Unsaturated+fatty+acids+from+food+and+in+the+growth+medium+improve+growth+of+Bacillus+cereus+under+cold+and+anaerobic+conditions.">Unsaturated fatty acids from food and in the growth medium improve growth of Bacillus cereus under cold and anaerobic conditions.</a> Food Microbiol. 36 (2), 113-122 (2013).</li><li>Miwa, T. K., Mikolajczak, K. L., Earle, F. R., Wolff, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+chromatographic+characterization+of+fatty+acids.Identification+constants+for+mono-+and+dicarboxylic+methyl+esters.">Gas chromatographic characterization of fatty acids.Identification constants for mono- and dicarboxylic methyl esters.</a> Anal Chem. 32 (13), 1739-1742 (1960).</li><li>van Den Dool, H., Kratz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+generalization+of+the+retention+index+system+including+linear+temperature+programmed+gas-liquid+partition+chromatography.">A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography.</a> J Chromatogr A. 11, 463-471 (1963).</li><li>Stransky, K., Jursik, T., Vitek, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+equivalent+chain+length+values+of+monoenic+and+polyenic+(methylene+interrupted)+fatty+acids.">Standard equivalent chain length values of monoenic and polyenic (methylene interrupted) fatty acids.</a> J High Res Chromatogr. 20 (3), 143-158 (1997).</li></ol>

The authors declare that they have no competing financial interests.

在表1所示的的FA色谱轮廓对应蜡状芽孢杆菌 ATCC 14579生长在琼脂平板的表面。当细菌在充气液体介质在相同温度下8生长得到类似的轮廓。在液体培养基中生长的细菌的情况下，细菌的生物量是通过生长培养基的离心收集，并可以根据取决于生长条件8,19先前描述协议来洗涤。由蜡状芽孢杆菌产生的各种的FA的识别允许在的FA的比例细变化突变对低温7,9-</...

脂肪酸甲基酯（FAME）气相色谱（GC）为脂质表征的基本方法。它迅速分离和短提取步骤后量化的样品的各种脂肪酸（FAS）。酸甲酯衍生物是极不稳定的，稳定的和惰性朝色谱柱，从而避免拖尾峰。他们的身份是相当简单的，当样本包括知名的FA，因为色谱配置文件登载或比较的标准。此外，校准标准关于各种的FA的量化的重复注射是不需要的，因为它们对火焰离子化检测器（FID）1几乎恒定的响应。 除了FID，质谱（MS）检测提供了一个互补组的信息，以确认脂肪酸甲酯。然而，当脂肪酸甲酯使用电子电离（EI）充电，由此产生的光谱并不总是允许日FA精细结构的E识别。例如，分支位置（ 即，支甲基）很难预测，因为诊断离子是很难检测1和目标离子丰度的特性的改变是依赖于机器的，防止使用质谱库2。另一个挑战在于确定双键的位置，因为EI使双键迁移。因此，具有不同双键位置的FA的异构体不能由它们的质谱鉴别。幸运的是，其他的工具已经开发了FA鉴定。例如，存在和支化或中的FA双键的位置可以通过计算等效链长（ECL）3进行推测。 其它衍生方法导致不同的质谱，依赖于一个双键或支链甲基的位置。 4,4-二甲基唑啉衍生物（DMOX）4允许EASŸ识别单不饱和脂肪酸双键的位置。 3- pyridylcarbinyl酯（甲代吡啶酯）衍生物允许甲基的位置的明确识别支链的FA 5。结合色谱保留（ECL）和质谱（DMOX和甲代吡啶）的信息允许对大部分的FA的鉴定，而不需要使用纯化的复杂的方法，因为需要用于核磁共振（NMR）谱，进行结构表征1无可争辩方法。 芽孢杆菌属，包括一些人类和动物病原体的细菌，都能够定殖高度多样化龛，因此被广泛分布于环境6。间的芽孢杆菌属，FA组成是由物种的生态位的影响，在FA模式调制，以适应范围广泛的环境变化（ 例如 ，生长培养基，温度，pH 等 ）7-9。因为在标准化条件生长期间通过芽孢杆菌属物种的FA图案的相对同质性，FA组成的测定是用来定义芽孢杆菌物种的主要标准之一。 芽孢杆菌属的一个独特属性是含有12-17个碳10-12用异和反异异构体是适应的对环境条件的关键决定因素之间的比支链的FA的丰度。 杆菌物种还通过改变不饱和脂肪酸的比例适应环境的波动。在一些物种中，如蜡状芽孢杆菌，二脂肪酸去饱和酶中创建与适应9不同的角色烷基链13的不同位置上的双键。 芽孢杆菌属的实施例说明的精确识别所述双键位置和FA分支的重要性。搜集ively， 芽孢杆菌 FA模式的识别有几个有用的应用程序。这里，我们提出了一种克服经典GC-MS分析的固有局限性芽孢 FA图案识别的新颖GC-MS的方法。 这种创新的方法，可以直接使用原料的生物材料，和由现有技术的组合的:保留时间（ECL）和不同的FA衍生物（FAME，DMOX和甲代吡啶酯）的质谱信息。 我们用下面的FA命名。 I，A和n表示异，反异甲基支链的，直链脂肪酸，分别。不饱和的FA由名为C:d，其中C是在脂肪酸的碳原子数以及d的数目的双键的数量。 Δx表示双键，其中双键位于第x个碳-碳键，从该羧酸端计数的位置。

<table><tbody><tr><td>GC/MS</td><td>Shimadzu</td><td>QP2010</td><td></td></tr><tr><td>capillary column ZB WAX</td><td>Phenomenex</td><td>7HG-G007-11</td><td>30 m x 0.25 mm x 0.25&amp;nbsp;&amp;micro;m</td></tr><tr><td>Methanol Lichrosolv</td><td>VWR</td><td>1.06018.2500</td><td></td></tr><tr><td>potassium hydroxide</td><td>Aldrich</td><td>P1767</td><td></td></tr><tr><td>THF</td><td>Hipersolv Chromanorm</td><td>28559.320</td><td></td></tr><tr><td>Dichloromethane</td><td>Hipersolv Chromanorm</td><td>23373.320</td><td></td></tr><tr><td>Hexane</td><td>Hipersolv Chromanorm</td><td>24575.320</td><td></td></tr><tr><td>3-pyridinemethanol</td><td>Aldrich</td><td>P6-680-7</td><td></td></tr><tr><td>potassium tertiobutoxide</td><td>Aldrich</td><td>156671</td><td></td></tr><tr><td>2-amino-2-methyl-1-propanol</td><td></td><td>A-9879</td><td></td></tr><tr><td>MilliQ Academic</td><td>Millipore</td><td>ZMQS50001</td><td></td></tr><tr><td>Bacterial Acid Methyl Ester (BAME) Mix</td><td>Sigma-Aldrich</td><td>47080-U Supelco</td><td></td></tr></tbody></table>

identification of fatty acids in bacillus cereus

的芽孢杆菌种含有支链和不饱和脂肪酸（FAS）的甲基支链的多样的位置（异或反异）和双键。在FA组成的变化发挥细菌的适应了至关重要的作用，以他们的环境。这些修改意味着在异相对于反异支链的FA的比例的变化，并在的FA相对不饱和与饱和的FA，以在特定位置产生双键的比例。在FA轮廓的精确识别是必要了解芽孢杆菌物种的适应机制。 许多从芽孢杆菌的FA都没有可商购的。这里提出的策略识别的FA通过与三种类型的FA衍生物的质谱相结合上的保留时间（由等效链长（ECL）的计算）信息:脂肪酸甲酯（脂肪酸甲酯），4,4-二甲基恶唑啉衍生物（DMOX），和3 pyridylcarbinyl酯（皮考）。该方法可以识别的FA，而不需要净化未知的FA。 比较从蜡样芽胞杆菌制备的标准商业混合物FAME色谱型材允许直链的识别饱和的FA时，ECL的计算，并假设在其他的FA的身份。的脂肪酸甲酯支链饱和的FA，异或反异，显示在ECL恒定负移位相比，具有相同数目的碳的直链饱和的FA。不饱和的FA的脂肪酸甲酯可以通过它们的分子离子的质量进行检测，并导致在ECL正移位相比相应的饱和的FA。 的FA的和分支位置的不饱和的FA的双键位置可以通过甲代吡啶并DMOX衍生物的电子电离质谱分别进行识别，。这种方法确定所有未知的饱和支ED FAS，不饱和直链FAS和从蜡样芽孢杆菌提取物的不饱和支的FA。

从细菌细胞FA识别该战略提出了如图1所示 。每一步都提供了互补的光谱信息或有关色谱保留的信息。步骤1包括使用标准溶液初步FA鉴定。第2步允许FAME EI谱和ECL的解释，以初步确定的产品。步骤3识别支链-FAS的确切分布位置。最后，第4步确定的不饱和的FA双键的精确位置。 FAME和ECL <p class="jove_content" fo:keep-together.w...

Watch this Scientific Journal Video about Identification of Fatty Acids in Bacillus cereus at JoVE.com

Identification of Fatty Acids in Bacillus cereus

We propose a protocol to identify fatty acids without the need to purify them. It combines information on the retention times with the mass spectra of three types of fatty acid derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl esters (picolinyl).

脂肪酸鉴定<em&gt;蜡状芽孢杆菌</em

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the ...

identification-of-fatty-acids-in-bacillus-cereus

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

Identification of Fatty Acids in Bacillus cereus

9.5K 次观看.  INRA, Université d'Avignon. We propose a protocol to identify fatty acids without the need to purify them. It combines information on the retention times with the mass spectra of three types of fatty acid derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl esters (picolinyl). 

视频: Identification of Fatty Acids in Bacillus cereus

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (&omega;-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.
With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.
This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.
The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete transesterification.

脂肪酸组分和含量微藻分析

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

科研

环境科学

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer tissue with minor or no modification, allowing the dietary routing between different trophic levels. However, the FA trophic marker approach is still hampered by the limited knowledge in lipid metabolism of the soil fauna. This study used entirely labelled palmitic acid (13C16:0, 99 atom%) as a tracer in fatty acid metabolism pathways of two widespread soil Collembola, Protaphorura fimata and Heteromurus nitidus. In order to investigate the fate and metabolic modifications of this precursor, a method of isotopologue profiling is presented, performed by mass spectrometry using single ion monitoring. Moreover, the upstream laboratory feeding experiment is described, as well as the extraction and methylation of dominant lipid fractions (neutral lipids, phospholipids) and the related formula and calculations. Isotopologue profiling does not only yield the overall 13C enrichment in fatty acids derived from the 13C labeled precursor but also produces the pattern of isotopologues exceeding the mass of the parent ion (i.e., the FA molecular ion M+) of each labeled FA by one or more mass units (M+1, M+2, M+3, etc.). This knowledge allows conclusions on the ratio of dietary routing of an entirely consumed FA in comparison to de novo biosynthesis. The isotopologue profiling is suggested as a useful tool for evaluation of fatty acid metabolism in soil animals to disentangle trophic interactions.

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

The fatty acid trophic marker approach, i.e., the assimilation of fatty acids as entire molecule and transfer into consumer tissue with no or minor modification, is hampered by knowledge gaps in fatty acid metabolism of small soil invertebrates. Isotopologue profiling is provided as a valuable tool to disentangle trophic interactions.

脂肪酸13C Isotopologue 分析提供了对无脊椎动物消费者营养碳转移和脂质代谢的洞察力

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer ...

生物化学

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

从鸡蛋黄中分离脂蛋白颗粒,研究细菌病原体脂肪酸并入膜磷脂

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

使用液色谱结合串联质谱法对糖皮质酶细胞脂质的定量分析

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in addition to its implications in cancer and neurodegenerative diseases. Recent developments in fatty acylation detection methods have enabled efficient and non-hazardous detection of fatty acylated proteins, particularly through the use of click chemistry with bio-orthogonal labeling. However, click chemistry detection can be limited by the poor solubility and potential toxic effects of adding long chain fatty acids to cell culture. Described here is a labeling approach with optimized delivery using saponified fatty acids in combination with fatty-acid free BSA, as well as delipidated media, which can improve detection of hard to detect fatty acylated proteins. This effect was most pronounced with the alkynyl-stearate analog, 17-ODYA, which has been the most commonly used fatty acid analog in click chemistry detection of acylated proteins. This modification will improve cellular incorporation and increase sensitivity to acylated protein detection. In addition, this approach can be applied in a variety of cell types and combined with other assays such as pulse-chase analysis, stable isotope labeling with amino acids in cell culture, and mass spectrometry for quantitative profiling of fatty acylated proteins.

The study of fatty acylation has important implications in cellular protein interactions and diseases. Presented here is a modified protocol to improve click chemistry detection of fatty acylated proteins, which can be applied in various cell types and combined with other assays, including pulse-chase and mass spectrometry.

优化了炔基脂肪酸类似物的掺入，用于使用点击化学检测脂肪酰化蛋白

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in ...

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de novo&#160;from acetyl-CoA and are primarily present in eukaryotes in the form of triglycerides (TGs) and sterol-esters (SEs). The enzymes responsible for the synthesis of NLs are highly conserved from Saccharomyces cerevisiae (yeast) to humans, making yeast a useful model organism to dissect the function and regulation of NL metabolism enzymes. While much is known about how acetyl-CoA is converted into a diverse set of NL species, mechanisms for regulating NL metabolism enzymes, and how mis-regulation can contribute to cellular pathologies, are still being discovered. Numerous methods for the isolation and characterization of NL species have been developed and used over decades of research; however, a quantitative and simple protocol for the comprehensive characterization of major NL species has not been discussed. Here, a simple and adaptable method to quantify the de novo&#160;synthesis of major NL species in yeast is presented. We apply 14C-acetic acid metabolic labeling coupled with thin layer chromatography to separate and quantify a diverse range of physiologically important NLs. Additionally, this method can be easily applied to study in vivo reaction rates of NL enzymes or degradation of NL species over time.

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.

通过代谢标记和薄层色谱分析 糖浆 中中性脂质合成

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain using antibiotic induction, and identified by MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. The immunity protein of colicin E3 (Im3) and the immunity protein of bacteriocin (Im-Bac) were identified from prominent b- and/or y-type fragment ions generated by the polypeptide backbone cleavage (PBC) on the C-terminal side of aspartic acid, glutamic acid, and asparagine residues by the aspartic acid effect fragmentation mechanism. The software rapidly scans in silico protein sequences derived from the whole genome sequencing of the bacterial strain. The software also iteratively removes amino acid residues of a protein sequence in the event that the mature protein sequence is truncated. A single protein sequence possessed mass and fragment ions consistent with those detected for each immunity protein. The candidate sequence was then manually inspected to confirm that all detected fragment ions could be assigned. The N-terminal methionine of Im3 was post-translationally removed, whereas Im-Bac had the complete sequence. In addition, we found that only two or three non-complementary fragment ions formed by PBC are necessary to identify the correct protein sequence. Finally, a promoter (SOS box) was identified upstream of the antibacterial and immunity genes in a plasmid genome of the bacterial strain.

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

Here we present a protocol for the rapid identification of proteins produced by genomically sequenced pathogenic bacteria using MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. Metastable protein ions fragment because of the aspartic acid effect and this specificity is exploited for protein identification.

使用MALDI-TOF-TOF-MS/MS和自上而下的蛋白质组学分析鉴定 大肠杆菌 中的抗菌免疫蛋白

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain ...

化学

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

胆汁盐诱导肠道病原菌生物膜形成的鉴别和定量技术

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

免疫与感染

Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well as other phenotypic characteristics. However, they all have in common the most relevant character of mycobacteria: its unique and highly hydrophobic cell wall. Mycobacteria species contain a membrane-covalent linked complex that includes arabinogalactan, peptidoglycan, and long-chains of mycolic acids with types that differ between mycobacteria species. Additionally, mycobacteria can also produce lipids that are located, non-covalently linked, on their cell surfaces, such as phthiocerol dimycocerosates (PDIM), phenolic glycolipids (PGL), glycopeptidolipids (GPL), acyltrehaloses (AT), or phosphatidil-inositol mannosides (PIM), among others. Some of them are considered virulence factors in pathogenic mycobacteria, or critical antigenic lipids in host-mycobacteria interaction. For these reasons, there is a significant interest in the study of mycobacterial lipids due to their application in several fields, from understanding their role in the pathogenicity of mycobacteria infections, to a possible implication as immunomodulatory agents for the treatment of infectious diseases and other pathologies such as cancer. Here, a simple approach to extract and analyze the total lipid content and the mycolic acid composition of mycobacteria cells grown in a solid medium using mixtures of organic solvents is presented. Once the lipid extracts are obtained, thin-layer chromatography (TLC) is performed to monitor the extracted compounds. The example experiment is performed with four different mycobacteria: the environmental fast-growing Mycolicibacterium brumae and Mycolicibacterium fortuitum, the attenuated slow-growing Mycobacterium bovis bacillus Calmette-Gu&#233;rin (BCG), and the opportunistic pathogen fast-growing Mycobacterium abscessus, demonstrating that methods shown in the present protocol can be used to a wide range of mycobacteria.

A protocol is presented to extract the total lipid content of the cell wall of a wide range of mycobacteria. Moreover, extraction and analytical protocols of the different types of mycolic acids are shown. A thin-layer chromatographic protocol to monitor these mycobacterial compounds is also provided.

薄层色谱分析分枝杆菌的脂质成分

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well ...

Isolation, Purification, and Identification of Bacitracin-producing Bacillus licheniformis from Fresh Feces of Healthy Pigs

Bacillus licheniformis and bacitracin have a huge application market and value in the fields of medicine, chemistry, aquaculture, agricultural, and sideline products. Therefore, the selection of B. licheniformis with high production of bacitracin is of great importance. In this experimental protocol, Bacillus with a high yield of bacitracin was isolated, purified, and identified from the fresh feces of healthy pigs. The inhibitory effect of secondary metabolite bacitracin on Micrococcus luteus was also tested. Thin-layer chromatography and high-performance liquid chromatography were used for the qualitative and quantitative detection of bacitracin. The physiological and biochemical characteristics of B. licheniformis were determined by relevant kits. The phylogenetic relationships of B. licheniformis were determined and constructed using gene sequence detection. This protocol describes and introduces the standard isolation, purification, and identification process of B. licheniformis from animal fresh feces from multiple perspectives, providing a method for the large-scale utilization of B. licheniformis and bacitracin in factories.

<meta charset="utf8"></head><body>从健康猪的新鲜粪便中分离、纯化和鉴定产酸杆菌肽的地衣芽孢杆菌

This protocol provides a complete and comprehensive description of the detailed process for the isolation, purification, and identification of bacitracin-producing Bacillus licheniformis from healthy pig feces.

从健康猪的新鲜粪便中分离、纯化和鉴定产酸杆菌肽的地衣芽孢杆菌

Bacillus licheniformis and bacitracin have a huge application market and value in the fields of medicine, chemistry, aquaculture, agricultural, and ...

脂肪酸鉴定

摘要

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此视频中的章节

脂肪酸组分和含量微藻分析

脂肪酸¹³C Isotopologue 分析提供了对无脊椎动物消费者营养碳转移和脂质代谢的洞察力

从鸡蛋黄中分离脂蛋白颗粒,研究细菌病原体脂肪酸并入膜磷脂

使用液色谱结合串联质谱法对糖皮质酶细胞脂质的定量分析

优化了炔基脂肪酸类似物的掺入，用于使用点击化学检测脂肪酰化蛋白

通过代谢标记和薄层色谱分析糖浆中中性脂质合成

使用MALDI-TOF-TOF-MS/MS和自上而下的蛋白质组学分析鉴定大肠杆菌中的抗菌免疫蛋白

胆汁盐诱导肠道病原菌生物膜形成的鉴别和定量技术

薄层色谱分析分枝杆菌的脂质成分

从健康猪的新鲜粪便中分离、纯化和鉴定产酸杆菌肽的地衣芽孢杆菌

脂肪酸鉴定

摘要

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此视频中的章节

脂肪酸组分和含量微藻分析

脂肪酸13C Isotopologue 分析提供了对无脊椎动物消费者营养碳转移和脂质代谢的洞察力

从鸡蛋黄中分离脂蛋白颗粒,研究细菌病原体脂肪酸并入膜磷脂

使用液色谱结合串联质谱法对糖皮质酶细胞脂质的定量分析

优化了炔基脂肪酸类似物的掺入，用于使用点击化学检测脂肪酰化蛋白

通过代谢标记和薄层色谱分析 糖浆 中中性脂质合成

使用MALDI-TOF-TOF-MS/MS和自上而下的蛋白质组学分析鉴定 大肠杆菌 中的抗菌免疫蛋白

胆汁盐诱导肠道病原菌生物膜形成的鉴别和定量技术

薄层色谱分析分枝杆菌的脂质成分

从健康猪的新鲜粪便中分离、纯化和鉴定产酸杆菌肽的地衣芽孢杆菌

脂肪酸¹³C Isotopologue 分析提供了对无脊椎动物消费者营养碳转移和脂质代谢的洞察力

通过代谢标记和薄层色谱分析糖浆中中性脂质合成

使用MALDI-TOF-TOF-MS/MS和自上而下的蛋白质组学分析鉴定大肠杆菌中的抗菌免疫蛋白