Research On Method For Determination Of Amylose Content In Rice

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Proceedings of the 7 th InternationalWorktng Conference on Stored-product Protection - Volume 2Research on method for determination of amylose content inriceYuan jian, Yang Xiaorong, and Wang Zhaoci1AbstractA simple and rapid methodfor determinationof amylosecontent m nee was establishedin this research.The effectsof ondition (time,reagenttemperature,refluxingincludingWith methanol(brownneefor 4hr)dispersedalkalinityRound-grainedand theether)for 2 hrin a Soxhlex extractor,chromophoriciodine solution as chromophoriccondi tions, 0.01299X (rwas 0.3%(n reagent.the linear regression 0.9999),Ynon-glutinous1. N.rice (P.MethodsThe1.0 mlUnder the selectedequation waslong-rice (G.rice (P. R. N.non-glutinousR. ), published long-grainedreaction acidityof 0.5 ml 1 mol/l acetic acid (or hydrochloric acid),N. R.),glutinousR. ), published glutinous nee (P G. R. )dispersedsample at 85'C for 10 minutes in a 100 ml beaker by 9.0 mlof 1 mol/l sodium hydroxide,(R.ricerice (1. N. R. ),R. ), published round-grainedthe polished rice bypetroleumnon-glutinousgrained non-glutinousThe optimal n,method,analytical results were investigated.were determined,chromophoricMaterials and Methodsrecommendedmethodwascomparing with the 1806647 -1987E,advancedbyGB7648 -1987,andetc.optirmzmg operation condition. 0.0015Results and Discussionthe relative standard deviationWIth the coefficient of variation less than 1.61 %10)ThisresearchprovidedscientificbaSIS forComparisonconditionsrevising the quality standard of the state.andselectionofSelection of the defatting method,In this researchstudied.theIntroductiondifferentthereagent and timecategoriesof samplesTables 1 and 2 were t of defattingreagentsbydifferentmethods for different times.Amylose content is a important index for appraising the foodqualityand theedibletechnologicalqualityItof rice.Table 1. Companson of defatting method and reagents.provides Important scientific basis for putting nee resourcesMethod *to rational use as well as selecting and breeding rice variety.The researchwas based on the ISO 6647 and m whichcomprehensivecomparison tests were carried out among ISO6647 and GB 7648, etc.conditionof thespecified.Accordingfor determinationThe optimizationdeterminationABCDof the operationof amylosecontentto the result a recommendedwasP R. N. RIce51SzReagentmethanol 0.380Petroleum 0.360petroleum 0 .3020.342* A - dagatted before dispersed,methodof amylose content was advanced and theC - defatted after dispersed,scientific basis for revising the quality standard of state wasprovided.P. 1. N. Rice51Sz0.3850.3700 .3550.3600.3200.3100 .2700.2700.3250.3200 .2600.275P. G. Rice510.1000.100O.1000.123B - defatted before dispersed,D - undefattedSome conclusions were obtained from Table 1 as follows:(a) The absorbance was on the low SIde obviously because oftheinterferencedeterminedINanjlng Uruversrty of Econorrucs,Chma128 Trelubeure,Nanjmg 210003,offatwhenthetestsamplesweredirectly.(b) The result was on the low side obviously,because ofvolumetricchangeto mutualdissolutionwhen the test samples defattedof the testsolutionowingether twice after dispersed were determined.1710with petroleum

Proceedmqe of the 7 th InternationalWorking Conference on Stored-product Protection - Volume 2(c) The result by defatting WIth petroleum ether was a httlewhen the rice was defatted by refluxing with methanol for 2less than with methanol. So methanoldefatting reagent In this researchhr in a Soxhlet extractorTable 2 shows that the interferencewas taken for thehr , it was unnecessaryor brown rice with methanol for 4for 16 hr.of fat was eliminatedTable 2. Comparison tests of different times and reagents.2 ice0.3180.310Brown 0.3120.3200.3100.2800.2700.2700.260directly in amollL sodium hydroxide solution used for dispersing the testthe result is shown in Table 5.samples,flask, another was that the test samplesin a beaker,thenthesuspensionto a 100 ml volumetricquantitativelywasTable 5. Influence of volumes of alkali (Imol/LThe determineddeviationflask directlyof dispersingwerebiggerAbsorbancesamplesthanNaOH) forthe test sample dispersing.flask anddiluted to volume mark with water and mixed fully.volumetricether0.325Comparison was carried out that different volumes of the 1One was that the test samples were dispersedtransferredPetroleum0.355Two methods of dispersion were compared (see Table 3) .wereMethanolether0.320Selectum of dispereum. method and condition100 ml volumetric24 hours4 hoursPetroleumSamplein thedispersing5.0 ml9.0 ml15.0 ml70.3540.3580.36080.2850.2900.290inbeaker.The reason is that amount of the test sample is few andthe test sample is apt to lose or adhere to wall when it wastransferredinto volumetric flask. So this method is adopted.Table 5 showed that the resultswhole of accordantTable 3. Comparison of dispersion methods.No. Method123Absorbancewere Identical on thewhen 9.0 ml or 15.0 ml of 1.0 mollLNaOH was added as dispersing 3600.3590.0051 CH3CROOB2 11:1In this researchthe test samples were dispersedat 70,85, or 100'C for 10 min. , as well as at ambient temperaturefor 24 hrs. The result is shownInTable 4.500In accordance WIth Table 4, the results were Identical onthe whole of samples dispersed at 85 and 100 C for 10 to 15min. and at ambient temperatureTable4.ComparisonofAbsorption spectrum under different acidity,Fig. 1.for 24 hrs.dispersiontemperatureanddispersion times.No.800100600AbsorbanceSelection of the acid and the acidity m. chrorrwphoricreaciuni1 .0 ml of 1 mol/L acetic acid or 1.0 ml of 1 mol/Lhydrochloricacidis addedto potatoAmbientt 24 rption spectrum was determined.50.2900.2900.2800.290their absorption spectrum was arryoutwere re 1 showed thatout that differentvolumes of

Proceedittgs of the 7th InternatwnalWorkittg Conference on Stored-product Protectum - Volume 2acetic acid was added respectively to test samples solution,and hydrochloric acid to amylose and amylopectm standardsolutions (Figure 2).FIgure 2 show that the absorbance of test samples solutionand standard solution are basically steady when 0.3 ml to1.0 ml 1mollL aceticacid or 1 mollL HCI IS added mchromophonc reaction. Therefore, 0.5 ml of 1 mollL aceticacid or hydrochlonc acid was used.0.8Hel0.60.40.2rtubes, then color developed and colorimetric analysis,Prepare the calibration curve by plotting absorbance vs. theamylose content Result was expressed as a percentage bymass, in the nulled rice on the dry baSIS. The linearregression equation was Y 0.0015 0.01299X (r 0.9999). Result was shown m FIg. 2.In addition, the method that defatted milled nee flours ofpredetermmed amylose content may be used for thecalibration m place of amylose and amylopectm suspensionswas adopted m tlus research. That can omit tedIOUSandtime-consummg preparation and punfication of amylose andamylopectm. The cahbration curve was prepared by roundgramed non-glutinous rice suspension (amylose content was18.0%) and the linear regression equation was Y 0.0018 0.0127 X (r 0.9996). The other four samples'amylose contents (on the dry baSIS) calculated by thiscalibration curve were listed m Table 6Table 6. Results by different calibration graph.Amylose content (%)Method * aFig. 2.0.20.40.60.81.0mlEffects of the volumes of acids.Selection of the volurne of iodine solution as thechromophoric reagentDifferent volumes of iodme solution were addedrespectively to mixture WIth 1.0 ml of 1 mg/ml amylosestandard solution and 1.5 ml of 1 mg/ml amylopectmstandard solution. Then these solutions were diluted to 50ml. The results of companson are shown m FIg 3 FIgure 3shows that the aosorbance was basically steady when thevolumes of iodine solution changed from 0.8 to 1.5 ml.Therefore, 1.0 ml of Iodine solution as chromophoncreagent ISadopted m tlus research.0.4Fig. 3.0.81.2mlEffects of the volumes of iodine solution.Preparation of the calibration. curveMix amylose and amylopectin standard suspensions (seeBlank test in Procedure) and the O. 09molIL NaOH 2.50 mlof each calibration solution was pipetted mto 50ml testSample 9Sample 10 Sample 11 Sample 12A23.816.817.817.5B24.017 518.018.0Deviation0.20.70.20.5* Method A is preparation cahbration curve by amylose andamylopectm standard suspension, and method B by nee flours ofpredetermmed amylose contentPrecision of the methodThe deviation of amylose content mainly ongmated fromtest sample's weighmg, piping solution and standardsolution, and samples' adhesion to the walls of the flask mdispersmg.In this study, each of 10 samples of round-grained nonglutmous nee or long-gramed non-glutmous nee weredetermined repeatedly for eight times by different operatorsm the same laboratory and different laboratories (NanjmgUniversity of Economics, Sichuan Research Institute ofGram Storage of the Mnustry of Internal Trade and theCentral Laboratory of Gram Bureau, Jiangsu province). Theresults were listed in Table 7.Table 7 showed that the deviation determmed by differentoperators in the same laboratory (NanJmg Institute ofEconomics) was less than 0.4 % was 80 %. The maximumdeviation was 0.6 %, the maximum of relative deviation was3.2%, mean of relative deviation less than 1.0 % was 70% ,the standard deviation repeatability (SD) was less than0.3%, and the coefficient of variation of repeatability (CV)was 1 61%. 70% of the deviations determmed by threelaboratones were less than 1 0%, the repeatability wasfrom 0.20 to 0.29, and the repeatability was less than0.27. Therefore, the allowable error of two determmation1712

Proceedmas of the 7 th Iniemaiumaiof rice amylose contentwas providedWorktng Conference on Stored-product Protection. - Yolurne 2m this research,itthan 10.0%,should not exceed 0.5 % while amylose content of nee IS lessTable 7. Precision of the method for determmationwlule amylosecontent of nee more than 10.0 % .of amylose content.R.G.NLaboratoryand It should not exceed 1.0%1. G N. RiceRiceItemsMeanStandard deviation (SD)ACoefficient of 0.518.619.40.180.20o ax. of relative deviation ( % )1.51.92.43.22.11.71.61.41.51.6Mean of relative deviation (%)0.80.81.11 028.827.325.1Standard deviation (SD)0.430.360.280.320.370.330.220.361. 750.341.36MaxBof differenceCoeffrcient of vanation2.281.661.411. 741.861.470.911.260.64of .120.920.022.125.128.226.225.1Standard deviation icien t of vana non1.600.941.591. 721.411.171.161.121.91Max. of 20.518.519.722.624.528.627.025.2error among ea tabih tyReproducibility* A - Nanjmg Universityof Economics.(%)B - Central Labof Gram Bureau,33Iiangsu,C - Sichuan Gram Storage Research InstituteApparatusMethod Recommendedas a State StandardRIce huller,spectrometerReagentsnee null, micro-null WIth SIeve of 80 mesh,and other usual laboratory equipment.Procedure(a) Methanol,Preparation. of test sample(b) Ethanol,Gnnd appropnatemilled nee m a micro-mill to very fme(c) Sodium hydroxide solution (1 mollL and 0 09 mol/L) ,powder that Will pass through the SIeve of 80 mesh(d) Acetic acid or hydrochlonckept m a bottle fitted WIth a stopper.acid solution (1 mol/L) ,(e) Iodme solution (0.2%),before nulling.(0 Amylose standard (1. Omg/ml suspension) :Weigh 100 (0. 5mg of defattedand equilibriumpotato amylose mto a 100 ml beaker,Defat the flour by refluxmgconditionedSoxhlex extractorcarefully add 1.0 ml ofAfter defattmg,WIth methanolfor 2 hrThen,methanol and to make ItS moisture content equihbnum.mamtamedat 85 C. After solution was cold, transfer It quantitativelya 100 ml volumetncandmIXtofor 2 daysto allowWeigh 100 ( 0.5VIgorouslybeaker,carefullyadd l.ml of ethanolwashing wall of the beaker,Prepare from milled defatted and conditioned glutmous neehydroxide solutionIts amylopectmmamtamed at 85 C for 10 -Accordmg to thesuspension.proceduregivenm(f)to prepareof residualmg of the test sample mto a 100 ml(g) Amylopectin standard (1.0 mg/ml suspension}.was known to be at least 99%.evaporationPreparatwn of the test solutionflask, dilute to volume mark WIth watercontentaspread the flour m a thm layer on a dishand leavem a water-bathin(brown nee for 4 hr ):ethanol and 9.0 ml of 1 mollL sodium hydroxide solution.heat It for 10 to 15 minIt wasRIce should be hulledroom temperatureat room1713usinga pipette,pipe 9.0 ml of 1molIL sodiumHeat the test solution in a water-bathrapidlytemperature15 nunThen cool solution toAlternatively,for15-24leave tlus solutionhrTransferIt

Proceedings of the 7 th InternationalWorking Conference on Stored-product Protection - Volume 2quantitatively to a 100 ml volumetric flask and make up tovolume with water and mix.Blank testCarry out a blank test in parallel With the determmationby the procedure mentioned above, using the samequantities of all the reagents as in the determination exceptusing 2.5 ml of 0.09 mollL sodium hydroxide solutioninstead of the test solution.Preparation of the calibration graphMix the amylose and amylopectin standard suspensionsand 2.0 ml of the o. 09molIL NaOH solution in accordancewith the table as follows:Amylose in milled rice(m/m)%Composition of mixture (ml)dry basis Amylose 222DeterminationPipe a 2.5 ml aliquot of the test solution into a 50 ml testtube, and proceed according to the procedure mentionedabove, starting with the addition of acetic acid. Measure theabsorbance at 620 nm against the blank, using thespectrometer.Expression of resultsThe content of amylose was expressed as percentage bymass on a dry baSIS and could be obtained by checking theabsorbance from the calibration curve.Take as the result the arithmetic mean of duphcatedeterminations to one place of decimals.The permissible error of result of the twin determinationsmust not exceed 1.0% when amylose content of rice ISabove 10.0%.AcknowledgmentsThese values have been calculated on the basis of anaverage starch content of 90% (rn/m) m milled rice on thedry basis.Pipe a 2.5 ml aliquot of each calibration solution into aseries of five 50 ml test tubes. Add 0 .5 ml of acetic acid andmix fully. Then add 1.0 ml of iodine solution, then make upto the mark with water and mix. Leave mixed solutions tostand for 20 min. Measure the absorbance at 620 nm againstthe blank using the spectrometer. Prepare a calibrationcurve by plotting absorbance vs. the amylose content.Result was expressed as a percentage by mass, in the milledrice on the dry basis.The authors are grateful to the Center Laboratory of GrainBureau, Jiangsu province and Shichuan Research Institute ofGRain storage for their assistance m the performance ofverified work.ReferencesGB 7648-87, Determination of amylose in rice, corn andmillet.ISO 6647-1987 (E), Determination of amylose content inrice.He Zhaofan, 1985. Quality of Grain & oil seeds and analyticaltechnology, the Agricultural Press, China.Wang Zhaoci, et al., 1994. Quality analysis of grain, oiland foods, the Light Industry Press, China.1714

when the rice was defatted by refluxing with methanol for 2 hr in a Soxhlet extractor or brown rice with methanol for 4 hr , it was unnecessary for 16 hr. Sample 2 hours Petroleum ether 4 hours Methanol Petroleum ether 24 hours Methanol Methanol Petroleum ether P.R.N.Rice P.L.N Rice Brown Rice 0.323 0.318 0.230 0.310 0.310 0.250

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