Effect of Grain Sorghum Protein Digestibility on Starch Gelatinization and Enzymatic Conversion to Glucose

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1 An ASABE Meeting Presenttion Pper Number: Effet of Grin Sorghum Protein Digestibility on Strh Geltiniztion nd Enzymti Conversion to Gluose Jon R. Hernndez, Grdute Reserh Assistnt Biologil nd Agriulturl Engineering Deprtment, Texs A&M University, College Sttion, Texs 77843, E-mil: Sergio C. Cpred, Assistnt Professor Biologil nd Agriulturl Engineering Deprtment, Texs A&M University, College Sttion, Texs 77843, E-mil: Dirk B. Hys, Assistnt Professor Cerel Grin Developmentl Genetis Deprtment of Soil & Crop Sienes, Texs A&M University, College Sttion, Texs 77843, E-mil: Ostilio R. Portillo, Grdute Reserh Assistnt Deprtment of Soil & Crop Sienes, Texs A&M University Affilition, College Sttion, Texs E-mil: Willim L. Rooney, Assoite Professor Sorghum Breeding nd Genetis Deprtment of Soil & Crop Sienes, Texs A&M University, College Sttion, Texs E-mil: Written for presenttion t the 2008 ASABE Annul Interntionl Meeting Sponsored by ASABE Rhode Islnd Convention Center Providene, Rhode Islnd June 29 July 2, 2008 Abstrt. The objetive of this study is to selet the best high digestible grin sorghum (HDGS) lines tht breeders hve developed for low energy input liqueftion, shrifition nd fermenttion for bio-ethnol prodution. The prentl lines nd offspring generted by rosses of two HD lines (P nd P851171) with three wild type (WT) sorghum lines (B.Tx635, R.Tx436 nd The uthors re solely responsible for the ontent of this tehnil presenttion. The tehnil presenttion does not neessrily reflet the offiil position of the Amerin Soiety of Agriulturl nd Biologil Engineers (ASABE), nd its printing nd distribution does not onstitute n endorsement of views whih my be expressed. Tehnil presenttions re not subjet to the forml peer review proess by ASABE editoril ommittees; therefore, they re not to be presented s refereed publitions. Cittion of this work should stte tht it is from n ASABE meeting pper. EXAMPLE: Author's Lst Nme, Initils Title of Presenttion. ASABE Pper No St. Joseph, Mih.: ASABE. For informtion bout seuring permission to reprint or reprodue tehnil presenttion, plese ontt ASABE t rutter@sbe.org or (2950 Niles Rod, St. Joseph, MI USA).

2 96GCPOB124) were used in this study. After the identifition of reombinnt inbred lines (RILs) s high, medium nd norml protein digestible grin sorghum using the protese turbidity ssy, the flour psting properties of the sorghum smples were exmined using rpid viso-nlyzer (RVA). Results showed tht the HDGS lines with modified endosperm mtries hs redued temperture nd durtion needed to solubilize the grin strh for hydrolyti enzyme ess nd onversion to fermentble sugrs. Keywords. enzymti hydrolysis, grin sorghum, high protein digestibility, high strh digestibility, shrifition (The ASABE dislimer is on footer on this pge, nd will show in Print Preview or Pge Lyout view.) The uthors re solely responsible for the ontent of this tehnil presenttion. The tehnil presenttion does not neessrily reflet the offiil position of the Amerin Soiety of Agriulturl nd Biologil Engineers (ASABE), nd its printing nd distribution does not onstitute n endorsement of views whih my be expressed. Tehnil presenttions re not subjet to the forml peer review proess by ASABE editoril ommittees; therefore, they re not to be presented s refereed publitions. Cittion of this work should stte tht it is from n ASABE meeting pper. EXAMPLE: Author's Lst Nme, Initils Title of Presenttion. ASABE Pper No St. Joseph, Mih.: ASABE. For informtion bout seuring permission to reprint or reprodue tehnil presenttion, plese ontt ASABE t rutter@sbe.org or (2950 Niles Rod, St. Joseph, MI USA).

3 Introdution On well mnge plnting dtes, grin sorghum offers exeptionl rottion rop tht provides mple residue for onservtion-tillge system nd potentil yield for South Texs frmers (Smith, 2007). For estblishing rop under onservtion tillge system, previous rop s residues re purposely left on the soil surfe to onserve soil nd wter. More effiient wter use n turn into higher yields for rops grown without irrigtion in drought-prone soils like in semirid regions. With onservtion tillge, other benefits inlude redued fuel onsumption, redued soil omption nd lbor requirements, plnting nd hrvesting flexibility, nd improved soil tilth (Sullivn, 2003). Sorghum grin hs strh-rih endosperm but it hs been underutilized for bio-bsed produts nd bio-energy prodution due to its poor wet-milling properties (Zhn et l., 2003). Approximtely 82 perent of the seed struture is the grin endosperm (Hoseney, 1994) whih is omprised of ells ontining protein bodies trpped in protein mtrix tht surrounds the lrger strh grnules (Kulp nd Ponte, 2000). The strh being imbedded in the protein body (kfirin) mtries, whih restrit geltiniztion, is the predominnt theory why norml grin sorghum hs low protein nd strh digestibility. The Sorghum Breeding Progrm t the Soil nd Crop Siene Deprtment, Texs A&M University hs developed nd identified high digestible grin sorghum (HDGS) genotypes with modified endosperm mtries tht lk the kfirin protein body highwys. We hypothesized severl dded benefits of these genotypes, like low energy input during geltiniztion prior to enzymti hydrolysis nd higher lysine protein ontent thn orn, therefore, mking these ultivrs suitble lterntive for orn in the typil bio-ethnol-feed supplement system. The objetive of this study is to selet the best sorghum lines tht breeders hve developed for the development of new low energy input liqueftion, shrifition nd fermenttion methodologies to produe ethnol. The objetives of this reserh re s follow: ) investigte the effet of grin sorghum protein digestibility on the temperture nd time of strh geltiniztion; nd b) evlute enzymti shrifition effiieny. Mterils nd Methods Mterils Using two prent lines of highly digestible protein (HD) grin sorghum (P nd P851171) rossed with three prent lines of wild type (WT) grin sorghum (B.Tx635, R.Tx436 nd 96GCPOB124) with high grin mold disese resistne, three fmilies with four distint reombinnt inbred lines (RILs) were developed. They were first phenotyped s high, medium nd norml protein digestibility using the protese turbidity ssy developed by Aboubr et l., (2003). After two suessive extrtion of protein from sorghum flour smples with pepsin solution, the remining protein from the reovered solids were further removed using extrtion buffer. The extrtion buffer ws then nlyzed for bsorbne fter 60 min of dilution in 72% TCA. The digestible group ws defined s one or more RILs whose bsorbnes (fter 60 min of dilution in 72% TCA) re not signifintly different ording to Tukey s HSD t 0.05 level of signifine. Using ner-infrred refletne (NIR) spetrophotometry, the totl strh, rude protein nd moisture ontent of these whole grin smples were nlyzed in three seprte replites. The RILs grown nd hrvested from Welso,Texs in 2006 were used in this study. Approximtely 2 kg seeds of eh prents 2

4 nd RILs were olleted, lened nd then dry-milled in Jy Bee 1647 Hmmer Mill using sreen of bout 1 mm openings. Sorghum Flour Psting The psting properties of sorghum flours were determined within 13 min intervl using Rpid- Viso-Anlyzer (RVA). About 28 g mixture (15% w/w db) of sorghum flour nd wter were prepred nd held t 50 C temperture for 1 min nd then heted from 50 C to 95 C. After holding the hot pste t 95 C for 2.5 min, the slurry ws gin ooled to 50 C, nd then held t tht temperture for 2 min. Shrifition of Sorghum Strh Using Enzyme The lph-mylse nd gluo-mylse, nmely SPEZYME XTRA nd G-ZYME 480 Ethnol respetively, used in this study were smples given by Genenor Interntionl, Inorported. One Alph Amylse Units (AAU) of bteril α-mylse ws the mount of enzyme required to hydrolyze 10 mg strh per minute while one Glio-mylse Unit (GAU) is the mount of enzyme needed to relese one grm of gluose per hour from soluble strh substrtes. The method for enzymti onversion of strh to fermentble gluose units ws bsed on NREL LAP-016 (Ntionl Renewble Energy Lbortory Lbortory Anlytil Proedure, 2005). Two sets of inubtion time nd enzyme onentrtion were used to determine the onversion rte nd onversion effiieny of strh to gluose with the use of enzyme. The smples were prepred in duplite for onversion rte determintion nd triplite for the onversion effiieny nlysis. Using tred 15 ml plsti entrifuge with tightly fitting srew p, 100 mg of ground smples from eh RIL ws mixed with 0.2 ml 190 proof ethnol nd 2 ml dimethyl sulfoxide (DMSO). The smple mixture ws vortexed vigorously to id strh dispersion nd then inubted in briskly boiling wter bth for 5 min to geltinize the strh. The mixture idified with 2.9 ml sodium ette buffer (ph 4.5 ontining 11.8 ml glil eti id per liter solution) nd dded with 0.1 ml thermostble lph-mylse (6.82 AAU nd AAU) ws inubted t 80 o C wter bth (for 6 min nd 10 min) to liquefy the strh. Another 4 ml buffer nd 0.1 ml gluomylse (0.19 GAU nd 4.12 GAU) were dded nd then inubted t 50 o C (for 30 min nd 60 min) to shrify the hydrolyzed strh. The smple tht is tightly pped throughout the nlysis hs finl volume of 9.3 ml. Sugr Anlysis Using 2 ml Eppendorf tubes, bout 1.5 ml of the shrified liquot ws heted for 2 min in briskly boiling wter nd then entrifuged t 3000 rpm for 10 min. The smples were filtered through 0.22 µm membrne filter prior to HPLC nlysis. The Wters Alline HPLC system with 2690 Seprtion Modules (integrtes five 24-vil rrousel, solvent delivery system, onbord ontroller, omprtment for olumn nd olumn heter) nd Wters 2410 RI detetor were used for the nlysis of gluose onversion. The Shodex SP0810 olumn (8.0 mm id x 300 mm) equipped with SP-G gurd olumn (6.0 mm id x 50 mm) ws used t olumn temperture of 60 o C using filtered nd degssed deionized wter s the eluent t 0.7 ml/min. Eh smple ws nlyzed for 20 min nd stndrds were run t the strt, middle nd end of smple nlysis. For qulity ssurne purposes, 20% of the smples were nlyzed in duplite nd blnk ws run every 10 smple injetions. One-wy ANOVA nd liner regression in SPSS 15 were used to nlyze the dt for determining signifint differenes mong mens nd orreltion between protein nd strh digestibility. 3

5 Results nd Disussion Highly Digestible Protein (HD) Sorghum Line The grin sorghum tegorized s high protein digestibility using the protese turbidity ssy hs n bsorbne rnge of to wheres norml digestible lines hve rnge of to bsorbne (Figure1). In highly digestible lines, more proteins were esily removed during pepsin solution extrtion, leving less extrtble protein in the extrtion buffer for the bsorbne nlysis. b Figure 1. Digestible group bsed on 60 min turbidity ssy. RILs with sme letter (: highly digestible protein; b: medium digestible protein; nd : norml digestible protein) re not different t 0.05 signifine level. 4

6 Tble 1. NIR nlysis of the totl strh, rude protein nd moisture ontent of these whole grin smples Protein Grin Sorghum Lines Digestibility %Moisture(wb) % Protein (wb) % Strh (wb)* 1 P High BTx 635 Norml b 3 BTx 635 * P High BTx 635 * P850029b Norml b 5 BTx 635 * P Medium d 6 BTx 635 * P850029d Norml RTx 436 Norml d 8 RTx 436 * P Norml d 9 RTx 436 * P850029b High b 10 RTx 436 * P Norml d 11 RTx 436 * P850029d Norml GCPOB124 * P High b 13 96GCPOB124 * P851171b Norml GCPOB124 * P Norml d 15 96GCPOB124 * P851171d Norml d 16 P High GCPOB124 Norml *Aording to Tukey's HSD, reombinnt inbred lines with the sme letter re not different t 0.05 level of signifine. Bsed on the NIR nlysis shown on Tble 1, HD RILs hve the lowest strh ontent. This suggests tht s the protein digestibility of the sorghum line inreses the strh ontent dereses. Sorghum Flour Psting The strh visosity nlysis of high digestible protein modified endosperm nd norml endosperm RILs is shown in Figure 2. The solid stright line refers to the strh visosity temperture urve. The pek visosity signifintly inresed with highly digestible vriety. HD RILs (P85009, RTx 436 * P850029b, P851171, 96GCPOB124 * P nd BTx 635 * P850029) begn to geltinize nd liquefy t lower tempertures (93.3 to 94.5 o C), in less time (4.5 to 4.7 min), nd to greter degree (440.9 to P) thn norml endosperm RILs. The exmples of ND RILs, tht showed the rnge of pek visosity (237.5 to P), time (5.4 to 5.7 min) nd temperture (94.8 to 95.0 o C) were BTx 365 nd 96GCP0B124. In ontrst, ND RILs sorghum did show grdul inrese in the ourse of heting, holding, nd ooling, resulting to finl visosity equl or muh higher thn tht of HD RILs. These results imply tht HD protein endosperm lines hve redued strh geltiniztion tempertures nd time whih signify lower energy input needed for enzyme hydrolysis. 5

7 Figure 2. Psting properties of sorghum flours using Rpid-Viso-Anlyzer (RVA) Enzymti Shrifition of Sorghum Strh Initil rte of onversion ws nlyzed to illustrte how fst the strh is being onverted to gluose. Dilute enzyme onentrtion nd redued retion time were used s the first tretment to ensure tht onversion will not pproh ompletion upon gluose nlysis. To shrify nd hydrolyze 100 mg strh smple, 6.82 AAU nd 0.19 GAU were used for 30 min. Perent strh onversion on the bsis of dry flour weight orrelted with protein digestibility nd strh ontent using regression nlysis re shown in Figure 3. Results showed tht strh ontent of the smple hd no signifint orreltion with rte of strh onversion to gluose (R 2 = nd P = 0.803) wheres bsorbne ssy hd negtive orreltion to rte of strh onversion (R 2 = nd P = 0.011). This only shows tht n inresed rte of strh onversion is gretly ffeted by protein digestibility rther thn the strh ontent of the smple. The higher is the protein digestibility of the RILs, the more essible is the strh to the enzyme during shrifition. Thus fster onversion of strh to gluose would tke ple whih is desirble during ethnol fermenttion. The seond tretment using AAU nd 4.12 GAU were used to shrify the strh for 60 min. Allowing the onversion to pproh ompletion, the % effiieny of strh onversion using enzyme ws lulted vi dividing the strh onverted to gluose by the strh ontent dry bsis. Results showed tht ll of the highly digestible lines hve signifintly higher onversion effiieny (79.16% to 87.92%) thn the norml digestible lines (Figure 4). 6

8 Figure 3. Correltions between % strh onversion to gluose, protein digestibility nd % strh ontent (db). Figure 4. verge strh onversion effiieny of different sorghum lines. RILs with sme letter re not different t 0.05 signifine level ( HD; MD;nd ND). 7

9 Conlusion Protein digestibility of grin sorghum reombinnt inbred lines hs signifint effet on the effiieny of strh onversion to fermentble sugrs. Using turbidity ssy, RTx 436 * P850029b, 96GCPOB124 * P nd BTx 635 * P850029, from the HD prent lines P nd P85009, were phenotyped s high protein digestible RILs. HD RILs begin to geltinize nd liquefy t lower tempertures nd to greter degree thn ND RIL. This indites tht HD protein endosperm lines would need lower energy input during geltiniztion for enzymti hydrolysis ompred to ND lines. Fster onversion of strh to gluose would tke ple when strh is more essible to the enzyme during shrifition. Further reserh is needed to eluidte effet of protein digestibility on onversion effiieny in ethnol fermenttion using these designer sorghums to optimize grin for bio-ethnol onversion. Aknowledgements This projet ws supported by the Sun Grnt Inititive, ntionl progrm estblished to rete new solutions for future U.S. energy needs. We would like to thnk Genenor Interntionl, Inorported for providing SPEZYME XTRA nd G-ZYME 480 Ethnol in this reserh. Referenes Aboubr A., Axtell J. D., Nduulu L., & Hmker B. R., Turbidity ssy for rpid nd effiient identifition of high protein digestibility sorghum lines. Cerel Chemistry. 80(1): Hoseney, R. C., Priniples of Cerel Siene nd Tehnology, 2nd edition. Pge 21 in: Struture of Cerels. Amerin Assoition of Cerel Chemist. St. Pul, Minnesot, USA. Kulp K., & Ponte, J.G., Hndbook of Cerel Siene nd Tehnology. 2nd edition. Pge 151. Mrel Dekker. In. New York, USA. Sluiter, Amie nd Justin Sluiter NREL Lbortory Anlytil Proedure (LAP-016): Determintion of Strh in Solid Biomss Smples by HPLC. Golden, Colordo: Ntionl Renewble Energy Lbortory. Avilble t pdfs/42624.pdf. Aessed 11 Jnury Smith, Ron Plnting Dte: Critil Deision in Grin Sorghum Yield Potentil. Southwest Frm Press. Avilble t Aessed 8 Mrh Sullivn, Preston Conservtion Tillge. Approprite Tehnology Trnsfer for Rurl Are Publition # CT105. Avilble t Aessed 28 April 2008 USDA Whet Prodution in the Upper Plins: Ntionl Agriulturl Sttistis Dtbse. Wshington, D.C.: USDA Ntionl Agriulturl Sttistis Servie. Avilble t: Aessed 23 April 2000 Zhn, X., D. Wng, X. S. Sun, S. Kim, nd D. Y. C. Fung Lti id prodution using extrusion-ooked grin sorghum. Trns. ASAE. 46(2):