Name: rapid high throughput amylose determination in freeze dried potato tuber samples
Uploaded: 2013-10-14T21:00:00
Description: Watch this Scientific Journal Video about Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples at JoVE.com

Finansieringen af ​​denne forskning blev leveret delvist af USDA-ARS Research Associate Program og USDA Crop germplasma udvalget.

1.. Amylose Bestemmelse fra kartoffelknolden Stivelse <ol><li> Skræl og skær friske knolde i små tern. </li><li> Placer kartoffel kuber i små brune poser og opbevares natten over ved -80 ° C. </li><li> Overfør kartoffel terninger til nylon poser og til frysetørring. </li><li> Efter frysetørret, slibe kartoffel prøven i pulver ved hjælp af morter og støder eller et Wiley Mølle. </li><li> I et 50 ml rør, tilsættes 20-30 mg frysetørret, formalet tuber prøve. </li><li> Der fremstilles en opløsning af 45% ...

<ol>
	<li>Composition Hoover, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=molecular+structure,+and+physicochemical+properties+of+tuber+and+root+starches:+a+review.">molecular structure, and physicochemical properties of tuber and root starches: a review.</a> Carbohydr. 45 (3), 253-267 (2001).</li><li>Bertoft, E., Blennow, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Potato+Starch.">Structure of Potato Starch.</a> Advances in Potato Chemistry and Technology. , 83-98 (2009).</li><li>Campbell, M. R., Yeager, H., Abdubek, N., Pollak, L., Glover, D. <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+methods+for+amylose+screening+in+maize+starches+from+exotic+backgrounds.">Comparison of methods for amylose screening in maize starches from exotic backgrounds.</a> Cereal Chem. 79, 317-321 (2002).</li><li>Zhu, T., Jackson, D. S., Wehling, R. L., Geera, B. <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+amylose+determination+methods+and+the+development+of+a+dual+wavelength+iodine+binding+technique.">Comparison of amylose determination methods and the development of a dual wavelength iodine binding technique.</a> Cereal Chem. 85, 51-58 (2008).</li><li>Hovenkamp-Hermelink, J., Devries, J., Adamse, P., Jacobsen, E., Witholt, B., Feenstra, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+estimation+of+the+amylose+amylopectin+ratio+in+small+amounts+of+tuber+and+leaf+tissue+of+the+potato.">Rapid estimation of the amylose amylopectin ratio in small amounts of tuber and leaf tissue of the potato.</a> Potato Res. 31, 241-246 (1988).</li><li>Shannon, J. C., Garwood, D. L., Boyer, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+and+Physiology+of+Starch+Development.">Genetics and Physiology of Starch Development.</a> Starch. , 23-82 (2009).</li><li>Haase, N. U., Plate, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+potato+starch+in+relation+to+varieties+and+environmental+factors.">Properties of potato starch in relation to varieties and environmental factors.</a> St&#228;rke. 48 (5), 167-171 (1996).</li><li>Johnston, F. B., Urbas, B., Khanzada, G. <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+storage+on+the+size+distribution+and+amylose/amylopectin+ratio+in+potato+starch+granules.">Effect of storage on the size distribution and amylose/amylopectin ratio in potato starch granules.</a> Am. Pot. J. 45, 315-321 (1968).</li><li>Gibson, T. S., Solah, V. A., McCleary, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+procedure+to+measure+amylose+in+cereal+starches+and+flours+with+concanavalin+A.">A procedure to measure amylose in cereal starches and flours with concanavalin A.</a> J. Cereal Sci. 25 (2), 111-119 (1997).</li><li>Sun, S., Matheson, N., Yun, S. -. H. <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+amylose+content+of+starches+after+precipitation+of+amylopectin+by+concanavalin+A.">Estimation of amylose content of starches after precipitation of amylopectin by concanavalin A.</a> Starch. 42, 302-305 (1990).</li></ol>

Amyloseindhold i kartoffelknolde typisk fra 20-35% 7,8. Hvis de fleste værdier er uden for dette område, overveje disse mulige fejl: 1) rå data blev anvendt i stedet for slukkede data for konstruktionen af ​​amylose / amylopektin standardkurve, 2) rå data blev anvendt i stedet for slukkede data til bestemmelse af amylose / amylopektin nøgletal fra kartoffel prøver, eller 3) det vand, der anvendes til analysen var ikke ren nok. Vi har bemærket, at ved anvendelse af destilleret vand og ikke ultra-ren...

Ca. 80% af den friske vægt af en kartoffelknold er vand, og næsten al den tilbageværende tørstof er stivelse 1. De fleste af stivelse (70%) består af amylopectin, mens resten er amylose. Forholdet mellem amylose og amylopectin er den vigtigste egenskab at påvirke de fysiske egenskaber af stivelse. Amylose er en lineær alfa 1-4 glucose kæde, mens amylopectin er en lineær alfa 1-4 kæde med alpha 1-6 grene 2. Fremgangsmåder, såsom iod binding, differentiel scanningskalorimetri (DSC), højtydende gelpermeationskromatografi (HPSEC) og concanavalin A-interaktioner er blevet udviklet for stivelse bestemmelse i forskellige afgrødearter 3,4. Hver protokol kræver særlige færdigheder og udstyr, hvilket gør det svært at skalere op at kvantificere mange prøver ad gangen. Den metode, vi præsenterer her er en modificeret protokol fra Hovenkamp-Hermelink et al. 5, som er en dobbelt bølgelængde jod bindendemetode baseret på farvede stivelsesgranuler. Fordelene ved denne fremgangsmåde i forhold til andre omfatter amylose bestemmelse af rå stivelse uden oprensning deraf og anvendelse af den dobbelte bølgelængde system til at øge præcisionen af fremgangsmåden 4,6.

<table border="1">
<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td> Reagents</td>
 </tr>
 <tr>
 <td>Amylose </td>
 <td>Sigma</td>
 <td>859656</td>
 <td></td>
 </tr>
 <tr>
 <td>Amylopectin </td>
 <td>Sigma</td>
 <td>A8515</td>
 <td></td>
 </tr>
 <tr>
 <td>Perchloric Acid 60%</td>
 <td>Sigma</td>
 <td>311413</td>
 <td>Very hazardous to skin and eyes</td>
 </tr>
 <tr>
 <td>Iodine</td>
 <td>Sigma</td>
 <td>23214TD</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium iodide</td>
 <td>Sigma</td>
 <td>08625JE</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Plate Reader</td>
 <td>Bio Tek</td>
 <td>ELx800</td>
 <td></td>
 </tr>
 </tbody>
</table>

rapid high throughput amylose determination in freeze dried potato tuber samples

Denne protokol beskriver en højt gennemløb kolorimetrisk metode, der er baseret på dannelsen af ​​et kompleks mellem iod og kæder af glucosemolekyler i stivelse. Jod danner komplekser med både amylose og lange kæder indenfor amylopektin. Efter tilsætning af jod til en stivelse prøve forekommer den maksimale absorption af amylose og amylopectin ved 620 og 550 nm. Den amylose / amylopektin-forholdet kan estimeres ud fra forholdet mellem de 620 og 550 nm absorbansværdier og sammenligne dem med en standardkurve i hvilke specifikke kendte koncentrationer afbildes mod absorptionsværdier. Dette høje gennemløb og billig metode er pålidelig og reproducerbar, tillader evaluering af store populationer af kartoffel kloner.

Den standardkurve konstrueret fra slukkede vedrørende forskellige amylose / amylopectin koncentrations løsninger viser de omtrentlige absorbans forhold i intervallet fra 0,7 til 1,6 (tabel 1). En lineær regression tendens linje fra disse oplysninger senere bruges til at udlede amyloseindhold i frysetørrede kartoffel prøver (figur 1). Produktionsanlægget miljø har en vis indvirkning på den amyloseindhold i kartoffelknolden 7. Replikeret amy...

Watch this Scientific Journal Video about Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples at JoVE.com

Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples

Rapid High Throughput Amylose Bestemmelse i frysetørret kartoffelknolden Prøver

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in ...

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