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

Yazarlar kendi teknik destek için Thomas Mison minnettarız ve yazının gözden geçirilmesi için Rachel Kopec için.

1. Bakteri kültürleri <ol><li> LB agar ortamı bir plaka yüzeyi üzerine, LB (Luria-Bertani ortamında) içinde 30 ° C&#39;de inkübe soyun bir gecelik kültüründen 100 ul yayarak bakteriler (Bacillus cereus ATCC 14579) &#39;in bir çim hazırlayın. 30 ° C&#39;de gece boyunca inkübasyona bırakılır. </li></ol> 2. ECL: Eşdeğer Zincir uzunluğu <ol><li> şöyle ECL hesaplayın: <img alt="denklem" src="/files/ftp_upload/54960/54960eq1.jpg" /> ile: Ben ilgi çözünen; N, çözünen önce elüsyon düz zincirli, doymuş yağlı asit metil ester karbon sayısı I; N + 1, çözünen sonra elüsyon, düz zincirli doymuş yağlı asit metil ester karbon sayısı I; RTI, RTN R (n + 1), yukarıda tarif edilen FAME tepe tutma süreleri verir. Not: (aykırı bir karışımının enjeksiyonla B, düz zincirli doymuş yağ asitleri metil esterlerin tutma süreleri elde edilirAME). </li></ol> 3. FAME Hazırlama ve Analiz <ol><li> Agar plakasından koloniler kazınarak lipit yağlı asitler, hasat bakteriyel hücreler elde etmek ve vidalı kapaklar ve PTFE keçe 10 ml&#39;lik bir cam tüp içine bakteri (10 9 canlı hücre eşdeğer taze ağırlık), 40 mg aktarmak için. </li><li> Aşağıda ayrıntıları verilen ester bağlantı yöntemiyle 14,15 aracılığıyla transesterifikasyon gerçekleştirin. <ol><li> Taze bakteri hücreleri için metanol içinde 0.2 M KOH 5 ml ilave edilir ve 1 saat boyunca 37 ° C&#39;de inkübe edin. Bu reaksiyon, lipid ester bağlantısı kırma ve yağlı asit metil esterlerinin üretilmesi, alkalin metanoliz oluşur. </li><li> 7.0 pH değerini düşürmek için 1 M asetik asit 1 ml ilave edilir. pH test şeritleri ile pH&#39;ı kontrol edin. </li><li> FAMEler çıkarmak için heksan 3 ml ekleyin. </li><li> yaklaşık 200 ul elde etmek üzere bir nitrojen sürekli akış altında oda sıcaklığında buharlaştırma ile temiz bir tüp içine süpernatant (organik faz) aktarın ve konsantreekstresi. ekleme ile GC şişe içine örnek aktarın. </li><li> Bir gaz kromatografisi-kütle spektrometrisi (GC-MS) sistemi hülasalarının enjekte edilir. </li></ol></li></ol> 4. GC / MS koşulları <ol><li> bir kılcal kolon ZB-balmumu (çapı, 0,25 mm, film kalınlığı 0.25 um uzunluğunda, 30 m) ile donatılmış bir GC-MS cihazı içine FAME örnekleri enjekte edilir. </li><li> (Yarıksız modunda) enjeksiyon ağzının 250 ° C&#39;ye sıcaklığa ayarlayın. 37 cm / sn arasında bir doğrusal hız ile, taşıyıcı gaz olarak helyum kullanın. 20 ° C / dk bir oranda 190 ° C&#39;ye arttırmak ve 2 ° C / dk bir oranda 230 ° C&#39;lik bir son sıcaklığa kadar daha da artması, 1 dakika boyunca 50 ° C de fırın sıcaklığı tutun. </li><li> MS için 70 eV elektron iyonizasyon (EI) kütle spektrumları kaydedebilir ve 50 ve 400 atomik kütle birimi (akb) (2 tarama / sn) arasındaki toplam iyon akımı satın ayarlayın. </li><li> Gerektiğinde, aynı durum İstisnai altında DMOX ve pikolinil türevleri enjekteaşağıdaki gibi sıcaklık programı fırın t: DMOX: 50 ° C (1 dakika), 20 ° C 210 ° C ye kadar / dk ve 2 ° C / dakika, 240 ° C (5 dakika) kadar; Pikolinil: 6 ° C (1 dakika), 220 ° C&#39;ye kadar 20 ° C / dakika ve 250 ° C (20 dakika kadar) 2 ° C / dak. </li></ol> FAME 16 5. Pikolinil ester Preparasyon <ol><li> bir azot akımı (en az 10 mg kuru madde) ile Kısım 3&#39;ten FAME özü buharlaştınn ve kuru diklorometan 1 ml içinde çözülür. </li><li> Tetrahidrofuran içerisinde potasyum tert t-butoksit içindeki bir 1.0 M çözelti hazırlayın. </li><li> FAME özü ve aşama 5.2&#39;de yapılan çözeltinin 0.2 ml 3-piridinmetanol ila 0.1 ml ilave edilir. </li><li> kapalı bir şişede 30 dakika boyunca 40 ° C&#39;da, çözelti ısıtılır. </li><li> Oda sıcaklığına kadar soğutulduktan sonra, (Malzeme bakınız Tablo, 2 mL) ve heksan (4 mi) arıtılmış, iyonu giderilmiş su ilave edilir. Bir girdap ile karıştırın faz ayrılması için izin ve organik faz toplamak. </li><li> drorganik faz kadar susuz sodyum sülfat ilave edilerek y bunu çok açıktır. Temiz bir tüp içine aktarın. Daha sonra 200 ul buharlaştırılmaktadır. ekleme ile GC şişe içine örnek aktarın. </li></ol> FAME 17 6. DMOX hazırlanması <ol><li> nitrojen (en az 10 mg kuru madde) içindeki bir akışı ile Bölüm 3&#39;ten FAME özü buharlaştırın. </li><li> FAME kuru ekstre etmek için, 2-amino-2-metil-1-propanol, 250 mg ekleyin. Nitrojen ile kap yıkayın bir stoper ekleyin ve 190 ° C de bir gece boyunca ısıtma bloğu içine koyun. </li><li> Oda sıcaklığına kadar soğutulduktan sonra, tüp, 3 ml diklorometan ekleyin ve 5 ml deiyonize edilmiş su (Malzeme Tablosu) arıtıldı. </li><li> Faz ayrımı için çalkalanır ve daha sonra sulu faz çıkarın. </li><li> 5 ml su ile organik faz yıkanır. Faz ayrımı için çalkalanır ve daha sonra sulu faz çıkarın. </li><li> organik faz kadar susuz sodyum sülfat eklenerek kuru çok açıktır veTemiz bir tüp içine aktarın. 200 ul bir hacim elde edilene kadar bir azot akımı altında buharlaştırılır. ekleme ile GC şişe içine örnek aktarın. </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.

Tablo 1 &#39;de gösterilen FAs kromatogram profilleri bir agar plakası yüzeyi üzerinde büyütülmüş, B. cereus ATCC 14579 karşılık gelmektedir. Bakteri, aynı sıcaklıkta 8 de gaz sıvı ortam içinde büyütülmüştür da benzer profilleri elde edilmiştir. Sıvı ortamda yetişen bakteri durumunda, bakteriyel biyokütlenin büyüme ortamının santrifüj ile toplanır ve büyüme koşulları 8,19 bağlı olarak daha önce tarif edilen protokollere göre yıka...

Yağlı asit metil ester (FAME), gaz kromatografisi (GC), lipid karakterizasyonu için gerekli bir yöntemdir. Bu hızlı bir şekilde ayırır ve kısa bir ekstraksiyon aşamasından sonra bir numune, çeşitli yağlı asitleri (FAS) ifade etmektedir. metil ester türevleri, kromatografik kolona doğru yüksek, uçucu istikrarlı ve inert böylece atık doruklarına kaçınarak. kromatografik profiller yayımlanmış veya standartlarla karşılaştırıldığında, çünkü örnek tanınmış finansman kurumu oluşuyorsa kendi kimlik oldukça basittir. Buna ek olarak, çeşitli Fas&#39;ın ölçümü için bir kalibrasyon standartları tekrar enjeksiyon alev iyonizasyon saptamasıyla (FID) 1 kendi hemen hemen sabit tepki verilir, gerekli değildir. FID ek olarak, kütle spektrometrisi (MS) tespit FAMEler teyit etmek için bir bilgi tamamlayıcı bir dizi sağlar. Kareleri elektron iyonizasyonu (El) ile şarj edilir, ancak elde edilen spektrumlar her th için izin vermeFA ince yapısının e tanımlanması. Teşhis iyonları 1 tespit edilmesi zor olan ve hedef iyon bolca karakteristik bir değişiklik kütle spektrumu kütüphaneleri 2 kullanımını engelleyen, makineye bağlı olduğu için, örneğin, konum dallanma (yani, metil dallı grup) tahmin etmek zordur. Başka bir sorun EI çift bağ göçünü neden olduğundan çift bağ pozisyonunu belirleme yatıyor. Böylece, çift bağ pozisyonlarını değişen FA izomerleri kendi kütle spektrumları ayırt edilemez. Neyse ki, başka araçlar FA tanımlanması için geliştirilmiştir. Örneğin, durum ve fasıl&#39;da çift bağların ya da dallanma konumu eş zincir uzunluğuna sahip (ECL) 3 hesaplanarak conjectured edilebilir. Diğer türetme yöntemleri bir çift bağ ya da kollara ayrılmış bir metil grubunun konumuna bağlı olarak, farklı kütle spektrumları ile sonuçlanır. 4,4-Dimetil oksazolin türevleri (DMOX) 4 EAS izintekli doymamış yağ asidi çift bağların pozisyon y tanımlanması. Ester (pikolinil ester) türevleridir metil yerini kesin tanımlanması için izin veren 3-pyridylcarbinyl FAs 5 dallanmış. Nükleer manyetik rezonans (NMR) spektrumu, yapısal karakterizasyon 1 tartışılmaz bir yöntem için gerekli olan, saflaştırma karmaşık yöntemler kullanmaya gerek kalmadan kromatografik (ECL) ile kütle spektrumları (DMOX ve pikolinil) bilgi çoğu Fas&#39;ın saptanmasına olanak birleştirme . Bazı insan ve hayvan patojenleri içeren Bacillus, bakteriler, çok çeşitli bir niş kolonize edebiliyoruz ve bu nedenle yaygın olarak çevre 6 dağıtılır. Bacillus cinsi içinde, FA bileşimi (çevresel değişiklikler geniş bir uyum FA desen modülasyonlar türlerin ekolojik niş etkilenir, örneğin, büyüme ortamı, sıcaklık,pH, vs.) 7-9. Çünkü standart koşullarda büyüme sırasında Bacillus türleri arasında FA model göreli homojenlik FA bileşimin belirlenmesi Bacillus türleri tanımlamak için kullanılan temel kriterlerden biridir. Bacillus cinsinin eşsiz bir nitelik 12-17 karbon iso ve çevre koşullarına uyum önemli bir belirleyicisi olan anteiso- izomerler arasındaki oran ile 10-12 ihtiva eden dallı zincirli finansman kurumu bolluğu olduğunu. Bacillus türleri de doymamış yağ asitlerinin oranının değiştirilmesiyle çevresel değişimlerin uyum. Bacillus cereus gibi bazı türler, iki yağ asidi desatürazlar uyum 9 farklı rollere sahip alkil zincirinin 13 farklı pozisyonlarda çift bağ oluşturur. Bacillus cinsinin örnek tam çift bağ pozisyonunu belirleme ve FA dallanma önemini göstermektedir. Toplamakively, Bacillus FA desen kimlik birçok yararlı uygulamalar vardır. Bu yazıda, klasik bir GC-MS analizi doğasında kısıtlamaların üstesinden Bacillus FA desen tanımlanması için yeni bir GC-MS yaklaşımı öneriyoruz. Bu yenilikçi yaklaşım, ham biyolojik bir malzeme üzerinde doğrudan kullanılan ve mevcut tekniklerin bir kombinasyonundan oluşur edilebilir: tutma süreleri (ECL) ve farklı FAs türevleri (FAME, DMOX ve pikolinil-ester) kütle spektrumları modülleri. Biz şu FA isimlendirme kullanın. a ve n, iso gösterir I, anteiso- metil sırasıyla dallanmış ve düz zincirli yağlı asit. Doymamış FAs C olarak seçilmiştir: Cı karbonlu yağlı asit atomuna ve D sayısı D ikili bağların sayısı. Δ X bir çift bağ karboksilik asit uç sayarak, X. karbon-karbon bağı bulunmaktadır çift bağ pozisyonunu gösterir.

<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

Bacillus türleri metil dalının farklı pozisyonları ile zincirli ve doymamış yağ asitleri (FAS) dallanmış (izo ya anteiso-) ve çift bağın. FA kompozisyonundaki değişikliklerin çevreye bakterilerin adaptasyon önemli bir rol oynamaktadır. Bu değişiklikler anteiso- fas dallı karşı iso oranında bir değişiklik gerektirir ve belirli pozisyonlarda oluşturulan ikili bağlar ile doyurulmuş Fas&#39;a doymamış FAs göreli, oranında. FA profilinin kesin kimlik Bacillus türlerinin adaptasyon mekanizmalarının anlaşılması gereklidir. Bacillus&#39;dan Fas&#39;ı çoğu ticari olarak temin edilebilir değildir. yağlı asit metil esterleri (FAME), 4,4-dimetil oksazolin: Bu tarifnamede önerilen strateji üç FA türevlerinin tiplerine kütle spektrumları ile (eş zincir uzunluğuna sahip (ECL) hesaplanması ile) alıkoyma süresi hakkında bilgi birleştirerek Fas tanımlar türevleri (DMOX) ve3-pyridylcarbinyl ester (pikolinil). Bu yöntem, bilinmeyen fas arındırmak için gerek kalmadan fas belirleyebilir. Standartları ticari bir karışım olan, Bacillus cereus hazırlanabilir FAME kromatografik profillerini karşılaştırmak için diğer Fas&#39;ın kimliğine fas, ECL hesaplama ve hipotez, doymuş, düz zincirli tanımlanmasına izin verir. ve Fames karbona aynı sayıda doğrusal doymuş finansman kurumu göre FAS, iso veya anteiso-, ECL sürekli bir negatif kayma gösterir doymuş dallı. Doymamış Fas&#39;ı kareleri moleküler iyon kütlesi ile tespit ve karşılık gelen doymuş Fas&#39;ına göre ECL olumlu bir artışa neden olabilir. Fas&#39;ı dallanma konumu ve doymamış Fas&#39;ın çift bağ pozisyonu, sırasıyla, pikolinil ve DMOX türevlerinin elektron iyonizasyon kütle spektrumları ile tespit edilebilir. Bu yaklaşım, tüm bilinmeyen doymuş şube tanımlarEd fasının doymamış, düz zincirli Fas ve B. cereus özü doymamış dallı FASB.

Bakteriyel hücrelerden FA kimlik stratejisi Şekil 1 &#39;de sunulmuştur. Her adım tamamlayıcı spektral bilgi veya kromatografik tutma hakkında bilgi sağlar. Aşama 1, standart çözeltisi kullanılarak, ön FA belirlenmesinden oluşur. Adım 2 geçici ürünlerini belirlemek için, FAME EI spektrumları ve ECL yorumlanması için izin verir. Adım 3 dallı zincirli-Fas kesin dallanma konumunu tanımlar. Son olarak, aşama 4, doymamış fasıl&#39;da çift bağlar...

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).

Yağ Asitlerinin Tanımlanması içinde<em&gt; Bacillus cereus</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

Research

JoVE Journal

Biochemistry

Identification of Fatty Acids in Bacillus cereus

9.5K Görüntüleme.  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). 

Video: 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.

Mikroalglerden yağlı asit içeriği ve bileşimi analizi

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

Çevre

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.

Yağ asidi 13C profil oluşturma Isotopologue trofik karbon Transfer ve Lipid metabolizması, omurgasız tüketicilerin sağlar

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

Biyokimya

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.

Membran fosfolipidler içine bakteriyel patojen yağ asidi kuruluş çalışması için tavuk yumurtası sarısı lipoprotein parçacıklarının yalıtım

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.

Tandem Kütle Spektrometresi Ile Birleşen Sıvı Kromatografi kullanarak Saccharomyces Cerevisiae'nin Hücresel Lipidomukan Analizi

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

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.

Metabolik Etiketleme ve İnce Tabaka Kromatografisi ile Saccharomyces serevizisinde Nötr Lipid Sentezinin Analizi

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.

MALDI-TOF-TOF-MS/MS ve Yukarıdan Aşağıya Proteomik Analiz kullanılarak Escherichia coli'de Antibakteriyel Bağışıklık Proteinlerinin Tanımlanması

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

Kimya

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.

Enterik patojenler safra tuzu kaynaklı biyofilm oluşumu: kimlik ve miktar teknikleri

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

İmmünoloji ve Enfeksiyon

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.

İnce Tabaka Kromatografisi ile Mikobakterilerin Lipid Bileşiminin Analizi

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

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

Çoklu doymamış yağ asitlerinin Diyet takviyesi<em&gt; Caenorhabditis elegans</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

Biyoloji

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>Sağlıklı Domuzların Taze Dışkısından Bacitracin Üreten Bacillus licheniformis'in İzolasyonu, Saflaştırılması ve Tanımlanması

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.

Sağlıklı Domuzların Taze Dışkısından Bacitracin Üreten Bacillus licheniformis'in İzolasyonu, Saflaştırılması ve Tanımlanması

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

Yağ Asitlerinin Tanımlanması içinde

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