Agriculture CLASSI- FICATION 8
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A09NCY FOR INTERNATIONAL DEVELOPMENT FOR AID USE ONLY WASIIdOTON, 0. C. 20623 BIBLIOGRAPHIC INPUT SHEET A. PRIMARY I. SUBJECT Agriculture CLASSI- FICATION 8. SECONDARY Cereal Crops 2. TITLE AND SUBTITLE Simple chemical and biological methods used at Purdue University to evaluate - r pgngte fnr rrntdain u,,-1lity 3. AUTNOR(S) Mertz, E.T.; Jambunathan, R.; Misra, P.S. 4. DOCUMENT DATE 15. NUMBER OF PAGES 6. ARC NUMBER 1975 25 p. ARC 7. REFERENCE ORGANIZATION NAME AND ADDRESS Agronomy Department, Purdue University, Lafayette, Indiana 47907 S. NOT) a 0 Izal.n Pub.liherp AvaIlabit Su se inT nernaiona1 programs ,i agricuiural station Bul. No. 70 9. ABSTRACT A lack of the amino acid lycine in cereals of economic importance makes them inferior to other high protein foods. The recent discovery that cereal proteins can be improved thrcugh genetic manipulation has made determina tion of the protein quality of these grains essential. This report details the simple methods by which even the most elementary equipped laboratory can produce reliable data for a plant-breeding program for protein qualtiy. Chemical reagents and procedures are described for nitrogen content analysis, tryptophan determination in maize, lysine levels in non-piymented cereals, lysine screening using a ninhydrin reagent, and dye binding for high lysine cereals. Procedures for biological assay, based on the diets of young rats who receive all their protein from cereals, is also provided. Both the ninhydrin and dye binding tests are recommended for mass screening of large populations. 10. CONTROL NUMBER 11. PRICE OF DOCUMENT PN-AAB-490 12. rESCRIPTORS -iS, PROJECT NUMBtR 14. CONTRACT NUMBER CSD-2809 Res. I. TYPE OF DOCUMENT AID $90,l 14741 SIMPLE CHEMICAL AND BIOLOGICAL METHODS USED AT PURDUE UNIVERSITY TO EVALUATE CEREALS FOR proiteinquality IF4 Edwin T. Mertz, Department of Biochemistry, Purdue University R. Jambunathan, ICRISAT, Hyderabad, India Prem S. Misra, Department of Biochemistry, Purdue University Supported by Agency for International Development Contract CSD/28090 "Inheritance and Improvement of Protein Quality and Content in Haist," and Contract CSD/1175, "Inheritance and Improvement of Protein Quality and Content in Sorghum." -2- CONTENTS INTRODUCTION 3 SAMPLE PREPARATION 4 NITROGEN DETERMINATION (PROTEIN) 4 Reagents Procedure NINHYDr.IN TEST 6 Mass Screening Ninhydrin Reagent Floury Endosperm Cereals Non-Destructive Method for Single Seeds Destructive Method Vicreous Etdoperm Cereals Non-Destructive Method for Single Seeds Confirmatory Ninhydrin Test Reagents Procedure TRYPTOPHAN IN MAIZE 9 Sample Preparation for Maize Endosperm Analysis Reagents Procedure LYSINE IN 14ON-PIGMENTED CEREALS 11 Reagents Copper Phosphate Reagent Pyridine Reagent Amino Acid Mixture Papain SoliitiOn Procedure DYE BINDING MASS' SCREENING TEST 14 Reagent Proc edu re BIOLOGICAL ASSAY IN RATS is Hat Feeding Trtls SUI4ARY AND CONCLUSIONS 21 -3- INTRODUCTION The different cereals of economic importance, i.e., rice, wheat, maize, sorghum, barley, oats, and millets, all have one characteristic in common they are low in the essential amino acid, lysine. Primerily because of the lack of adequate lysine, cereal proteins are inferior nutritionally to the proteins found in milk, meat and eggs. With the discovery by Mertz, Bates and Nelson that the maize mutant opaque-2 had near'y twice as much lysine as ordinary maize, scientists realized that cereal grain pr:teins could be improved in 3uality by genetic manipulation. The recent discovery by Ingversen and co-workurs in Denmark 2 and by Axtell and co-work.rs 3 1n the United :tatt ,f higIh lysine mutant-; of barley and sorghum, respectively, rai-.ies th, hope that all cereals of economic impx)rtance may eventually ho improved in prot eii quality b' em't ic teil oction. The protein quality of a cereal grain can bo ltrmi ned only Iy chemical or biological , notht, . In a compl|emontary rtak+ lt w rker s at Pujrdue di Pcuss the statun of uofv. Io|E'tient f mai.r with i.lr,)v.e1 protoein qua lity , arid briefly review t he eht-mi : 1 and i ol (3 ret+it oli a l l o t f,, I ie pl1 ant I reole r to *valu4to pst-atri qtuaitllty. II thls rapl ,rt W11 havt o11t I llio]n 1Alt a1 | t h" Gimlapo metu It, titd at Portlti, t hat arty tit t of, e ftjr a 1-rat oryt verely Iimitod in eq"Ulite-It 'awI t ali 1,146tmioiel. Wo Au~2] .;t etirochla I4u-atorls-y to riqueat attinlas=l] clheck ::aftpj| (,f ljorfial 41 t+mit+! ritnal qliatit wtth "iown contnito (it l4 'tIu i ly)+liin Aiilb f'-! itpt uphati $tu a pii, t I s ato riit ry in Nip ftiit liti fte-4' Iba'] tan-lit r c ti clcii iltatm, t" 'Ji v tel lal'ti 'l46t i for a plant Iiotee1"Ji j ',i tt forl1it, ertii+1i Ilty wbcora )-*I.j44tuty fatrilmositar* . iited, bIIorata..r, mcts--w as-a l'dla!ta4 lay jnurpiert raumb re, QIsPl.at by nulra I. psrettheutul. rinferti.nOt -4- SAMPLE PREPARATION For all of the tests described below, except the split seed ninhydrini use test, the grain sample must be ground to a fine powder. If available, are 10-30 grams of a represertative sample. Where quantities of kernels limited, as little as 500 mg of sample will be sufficient for a single determination with each of the various methods. The sample is first ground in a Wiley mill to pass a sieve of 90 mesh in (1) (or an ordinary hand grincier (2)). The ground material is placed cellulose tlimbles and defatted with hexane overnight in a Soxhiet apparatus (3). The sample is defatted to imp'ove storage qualities and precision ground to of analysis. The defatted sample is allowed to air dry and then a fine powder in a small ball mill (4). NI'MOGIRN DETERMINATION (PROTEIN) The most reliable met'od for nitroqen is the well-known micro-Kjeldahl method. It is usedl in a vari.ty of forms. The nimpleslt is the Association of Offirial Analytical Cht~mist (A.O.A.C.) mthod which invcilvS di pqestion oxidation of the nam|pi) with . lufuri : acid ald at!arm distillinIq th( ammonia formed at htfih lollititt a lxIrloha cd ao1 ut 10. '11 un boratotaMK)un1 formed il thri titr4atei-d with utarildr,1 hyilrochloric acid. Tho 1 ,ercttnta(I of protein 10 obti ni-d bty mu lti plyin per cnt ilt rulie blythi! fatfor E6.25 (maize, (barley, oats, ryd and wheat) 7 millet and riolr.jm), 5.'Th (rice), and S.Utt The ratlents and procedure are described bolow. Reaej ent o Sulphuric acid -- ap. qr. 1.5, N-free. Catalyst mixtuto -- 190 q of Y05 4 and 4 q KgO. 000 9 of Sodium hydroxde -- sodium thionuitate solutio, DiOtlV. -5- NaOH in 1500 ml of cold distilled water, adding small increments of the alkali at a time with constant stirring. Dissolve 100 g Na2S2 03.5H 20 in distilled water 'use 200 ml or less). Mix the two solutions and dilute to 2 liters, Indicator dye -- Mix one part of 0.2% methyl red in ethanol with 5 parts of 0.2% bromocresol green in ethanol. Standard hydrochloric acid -- 0.02N. Boric acid -- 4% solution. Procedure Accurately weigh (5) an amount (40-50 mg) of sample and place in a 30 ml ijeldahl digestion flask. Add 2 g of catalyst mixture and 2 ml of sulphuric acid. Gently heat the flask on a rotary digestor (6)until the frothing ceases. Raise the temperature to boiling and boil for 1 hour after the contents of the fIa3k are clear. Coolp and add 8 -.d distilled water. Transfer the dlqg nt to a steam distillation apparatus (7) via the sample funnel and jinse the flask 5-6 times with 1-2 ml portions of distilled water. Place a 50 n I Erlenmoyer flask containing 5 ml of boric acid and 4 drops of mixed inuicator dye beneath the condenser, with the dolivery tip immersed in the solut ion. Add 15 ml of th, nodium hydroxide-sodium thionulphato solution to the digest, rinan tho nample funnol with water and start steam distillation. Collect dlistill4t. for 4-5 minutes, then rinao the coridanser tip with water and titrate the diatillate plus rinsinq with standard hydrochloric -6 acid in a microburette (8) to a grey or blue end-point. Run a blank containing the same quantities of all reagents but without sample for every set of nitrogen determinations. Also run a check sample of cereal grain of known protein content. Calculate the percentage of nitrogen. % N - (ml HCl-ml blank) x normality x 14.007 x 100 mg sample NINHYDRIN TEST Mass Screening Test Mertz, et al. 8 have rho-sn that high lysine mutants of maize, sorghum, and barley give a more intense blue color with ninhydrin than their normal counterparts because (- higher levels of free amino acids. Mass screening of floury endosperm types can be made rapidly with this method. Mass screening of vitreous endospei'm types is slower because of lack of penetration of the ninhydrin reagent into the exposed surface of the edosperm. With vitreous endosperm the kernels carL be ground before testing. The procedures are described here. Ninhydrin |eaqent Grind a mixture containing 16 parts (by weight) of ninhydrin (practical), 58 parts of sodium citrate and 26 parts of citric acid to a fine powder with a murtar and pestle. Store the mixture in a brown glass bottle and protect from moisture. Under dry conditions in darkness the mixture does not deteriorate, Floury Endosperm Cereals Non-bestructive Method for Sinle Seeds. Expose the endosperm near the crown by rubbing the crowt on sandpapor.