Structure of Starch Granules

Transcription

1 31 J. Appl. Glycosci., 54, (2007) C 2007 The Jpnese Society of Applied Glycoscience Awrd Address: The Awrd of Merit of the 2006 s JSAG Structure of Strch Grnules (Received September 28, 2006) Jy lin Jne 1, 1 Deprtment of Food Science nd Humn Nutrition, Iow Stte University (Ames, IA 50011, USA) Abstrct: Strch grnules re synthesized in semicrystlline structure nd hve chrcteristic morphology nd properties depending on the botnicl origin nd the orgn of the plnt. Surfce geltiniztion studies hve shown tht the internl structures of strch grnules re not homogeneous nd vry with rdil loctions, from the hilum to the periphery. The internl structure of mize strch grnule coincides with the biosynthesis nd development of strch grnules nd the expression of strch synthse ctivities during the mturtion of mize kernels. The enzyme digestibility of strch grnules reltes to the internl structure nd the internl surfce re of the strch grnule s reveled by confocl lser-scttering microgrphs. Short brnchchins of mylopectin (DP 6 12) destbilize the grnule structures, resulting in lobed or compound strch grnules nd smll grnules. Rtios of long brnch-chins to short brnch-chins of mylopectin molecules ffect the shpe of the molecule, e.g., cylindricl versus conicl shpes. The shpe of the mylopectin molecule my ffect the pcking of the molecules in the grnule nd, in turn, determines the morphology nd the size of the strch grnule. Key words: strch, grnule, structure, biosynthesis, enzyme digestibility Strch is synthesized in semicrystlline grnulr structures, which is in contrst to glycogen tht hs n morphous structure. The different structures of these two glucns could be relted to the ptterns of how these two storge glucns re utilized. Glycogen is present in muscle nd liver, nd it needs to be redily vilble to provide energy for muscle functions. Therefore, glycogen molecules re morphous nd re redily hydrolyzed to produce energy in nimls. 1) Strch, however, is minly synthesized in storge orgns, such s seeds, tubers, nd roots. To store glucn molecules in n effective nd stble condition in those storge orgns, strch is pcked in the semicrystlline grnulr form to reserve energy for use when the seeds re redy to germinte nd grow. 2) Strch grnules of different botnicl origins disply different morphology, 3) crystlline structures, 4,5) nd susceptibility to enzymtic 6) nd chemicl rections. 7) Experimentl results hve shown tht these grnulr structures nd properties re relted to the structures of mylose nd mylopectin synthesized in different plnts, 4,5,8) different orgns, 9,10,11) nd t different stges of development. 9,12) In this lecture, the internl structures of strch grnules nd the concepts for how the molecules in the strch grnule re synthesized will be reviewed. Also, dt on how the structures of strch molecules ffect the geometricl pcking rrngements nd, in turn, determine the shpes of strch grnules will be discussed. Structures nd biosynthesis of strch molecules in the grnule. To gin better understnding of the internl structures of strch grnules, strch molecules on the surfce of Corresponding uthor (Tel , Fx , E mil: jjne@istte.edu). strch grnules cn be geltinized using sturted lithium chloride solution 13) or sturted clcium chloride solution 14) nd then seprted from the inner prt of the strch grnule. Results obtined from the studies of surfce geltiniztion of potto 14) nd norml mize 13) strches show tht mylose is more concentrted on the periphery thn in the core of the grnule nd the brnch chins of mylopectin re shorter in the surfce strch thn tht of the mylopectin in the remining strch grnule (Fig. 1). These results reflect tht strch grnules do not hve homogeneous internl structures nd suggest tht strch biosynthesis is not consistent throughout the development of strch grnules. The structures of mylose nd mylopectin vry in the rdil loctions, from the hilum to the periphery. 13,14) Recent studies 9) on structures of strch molecules during mize kernel development hve shown tht smll strch grnule nuclei (dimeter 1 4 µm) first pper in the endosperm on 5 dys fter pollintion (DAP), nd the number of strch grnule nuclei increses but the size remins similr up to 12 DAP. 9) On the 14 DAP, the sizes of strch grnules pper lrger (dimeter 7 µm) nd the sizes of the grnules continue to increse up to 23 µm on 30 DAP. 9) These results re in greement with the strch grnule development in rice endosperm 12) nd reported increses in the size of strch grnules with kernel mturtion of rice, 15) brley, 16) mong other plnts. These results indicte tht the development of individul strch grnule is prllel to the development of the kernels. During the mize kernel development, the strch content of the kernel increses from 1% on 8 DAP to 88.9% on 30 DAP, nd the mylose content of the strch increses from 9.2% for the kernels hrvested on 12 DAP to 24.2% on 30 DAP nd remins similr for the mture nd field dried kernels hrvested on 45 DAP (Tble 1). 9) A substntil increse in

2 32 J. Appl. Glycosci., Vol. 54, No. 1 (2007) Tble 1. Strch nd mylose contents of the endosperm t different developmentl stges of mize kernels. Dys fter pollintion Strch content (% dry weight) Amylose content (%) 0 ND ND 8 1.0±0.1 ND ±0.8 ND ± ± ± ± ± ± ± ±0.8 Not determined. Fig. 1. Brnch chin lengths of the long chins of mylopectin nd the mylose contents of ntive lrge grnule cornstrch (dimeter >5 µm), 65 nd 84% surfce geltinized nd removed remining grnules from the lrge grnule cornstrch nd ntive smll grnule cornstrch (dimeter < 5 µm). The surfce geltiniztion is conducted using sturted LiCl solution reported in Ref. 13). Fig. 2. Brnch chin length distributions of mize mylopectin isolted from strch t different mize kernel developmentl stges, 10 dys fter pollintion (DAP) to mture (45 DAP nd dried). The brnch chin length of mylopectin is determined using isomylse debrnched mylopectin nd nlyzed using high performnce nion exchnge chromtogrphy equipped with n on line glucomylse rector nd pulse mprometric detector. A PA 100 nion exchnge column is used for the seprtion. Printed with permission from Ref. 9). the strch content of mize kernels between 12 DAP nd 14 DAP coincides with the increse in strch synthetic enzymes, including ADP glucose pyrophosphorylse, UDP glucose pyrophosphorylse, hexokinse, grnule bound strch synthses, nd soluble strch synthses. 17) The increse in mylose content during the mturtion of the kernel nd strch grnules coincides with the increse in the ctivity of grnule bound strch synthse (GBSSI). 10) Becuse strch grnules re known to grow by pposition, 18,19) incresing the concentrtion of mylose would occur t the periphery of the strch grnule. This result is in greement with tht obtined using surfce geltiniztion method. 13,14) Brnch chin length distributions of mylopectin molecules isolted from strch grnules hrvested t different mize kernel developmentl stges show tht the verge brnch chin lengths increse from DP 23.6 (10 DAP) to DP 26.7 (14 DAP) nd then grdully decrese until mture (DP 24.9) (Tble 2). 9) Short brnch chins (DP 12) of mylopectin lso decrese from 10 DAP to 20 DAP nd then increse until mture (Tble 2 nd Fig. 2). 9) Amylopectin molecules isolted from mize strch of 10 DAP show lrge proportion of very short chins (DP 5 8), which diminish or reduce during the mturtion of the mize kernels. The unusully lrge proportion of DP 5 8 my reflect the lck of SSI ctivity. 20) The mylopectin of 10 DAP lso lcks long brnch chins lrger thn DP 55 (Fig. 2), indicting the deficiency of soluble strch synthse ctivities. As the mize kernels develop to 12 DAP, the short brnch chins of DP 4 8 decrese, wheres the long brnch chins (DP>55) increse nd eventully rech the mximum length on 14 DAP (Fig. 2). There is n obvious shoulder shown t DP 17 in the brnch chin length distribution of mylopectin hrvested on 12 DAP. This my suggest deficiency in the ctivities of SSII nd/or SSIII. 6,20) When the mize kernels further develop nd become more mture, the mylopectin brnch chins of round DP 48 increse, but the brnch chins of DP>50 decrese s shown for the brnch chin length distribution of mylopectin of 20 DAP until mture (Fig. 2). The brnch chin length distribution of mylopectin hrvested on 20 DAP nd lter dtes show typicl bimodl brnch chin length distribution with the pek chin lengths t DP 14 nd DP 48 for the short nd long brnch chins, respectively. The increse in brnch chins DP 24 fter 12 DAP (Tble 2) cn result from the decrese in the rtio of BEI/BEIIb fter 12 DAP. 21) The mize brnching enzyme IIb (BEIIb) is known for trnsferring short brnch chins, wheres BEI is for trnsferring long brnch chins. 22,23) And the increse in brnch chins of round DP 48 (Fig. 2) cn result from the increse in SSIII ctivities nd double helix formtion. The onset geltiniztion temperture of the mize strch isolted from kernels of different development stges increses from 61.3 C (8 DAP) to the mximum of 69.0 C

3 Structure of Strch Grnules 33 Tble 2. Brnch chin length distributions of endosperm mylopectins. Smples Percent distribution Dys fter pollintion DP 12 DP DP DP 37 Averge CL ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± (mture nd dried) 19.4± ± ± ± ±0.5 Vlues given re mens±sd obtined from two replictes. Tble 3. Smples Therml properties of ntive endosperm strches. Ntive strch Dys fter pollintion To ( C) Tp ( C) Tc ( C) H (J/g) 8 b b ±0.2 c 67.8± ± ± ± ± ± ± ± ± ± ± ± ± ± ± (mture nd dried) 62.8± ± ± ±0.1 Smples ( 3.0 mg, dsb) nd deionized wter ( 9.0 mg) were used for the nlysis; To, Tp, Tc nd H re onset, pek, conclusion temperture, nd enthlpy chnge, respectively. b Vlue ws mesured once. c Vlues were clculted form three replictes; ±SD. (14 DAP), nd then decreses to 67.4 C (30 DAP) nd 62.8 C (45 DAP) (Tble 3). 9) The mture nd field nd ir dried strch grnules (45 DAP), consisting of 19.4% short brnch chins of DP 12, disply substntilly lower onset geltiniztion temperture (62.8 C). This substntil decrese in the geltiniztion temperture is interesting. Becuse the mize strch isolted from the mture nd dried kernel of 45 DAP consists of lrge number of pinholes on the surfce of strch grnules, further studies re needed to revel if enzyme degrdtion of the strch during mturtion my contribute to the reduction in the geltiniztion temperture in ddition to the presence of lrge proportion of the short brnch chins of DP 12. 9) Effects of the internl structure of strch grnule on its enzyme digestibility. Strch of different botnicl origins is known to hve different enzyme digestibility ) A feeding study with bby chickens using ground kernels of mize mutnts, norml, wxy, nd sugry 2 with isogenic bckground, show tht the growth rte of the bby chickens fed with these three mize mutnts decreses in the order of su 2 mize>wxy mize>norml mize. 6) The in vitro enzyme hydrolysis of the strch grnules using porcine pncretic α mylse displys the sme order (Fig. 3). 9) The differences in the enzyme digestibility between the three mize strch mutnts cn be ttributed to their different moleculr structures of mylopectin (Tble 4) nd mylose contents. Among the three mize strch mutnts, the su 2 strch consists of mylopectin with lrge proportion of short brnch chins of DP 6 12 nd lrge content of pprent mylose (33.5%); 6) the wxy strch nd the norml Fig. 3. Enzyme hydrolysis rtes of mize mutnt strches, norml, wxy, nd sugry 2 mize strch, by porcine pncretic lph mylse. Printed with permission from Ref. 6). strch consist of mylopectin of similr structure, but the wxy mize strch hs no mylose, wheres the norml mize strch hs 21.6% mylose. 6) Becuse of the lrge content of short brnch chins of mylopectin nd reltively lrge concentrtion of mylose, the strch grnules of the su 2 mize strch disply lobe nd compound grnule morphology, s shown by scnning electron microgrphs (SEM) (Fig. 4). 6) The su 2 mize strch lso displys wek crystllinity nd very low geltiniztion temperture, 47.8 C versus 64.4 C for norml mize strch nd 64.2 C for wxy mize strch. 6) The mize su 2 mutnt is known for missing soluble strch synthse II (SSII) ctivity, which results in very lrge proportion of short brnch chins of DP 6 12 (22.2%) nd fewer chins of DP The lrge proportion of short brnch chins (A nd B1 chins) nd lcking of B2 chins to connect between two clusters result in mylopectin molecules with brush like cluster structure, which do not form extended nd stble double helices nd, thus, disply low crystllinity, low geltiniztion temperture, nd even lose the integrity of strch grnules. Potto strch of SSII nd SSIII ctivity missing lso show lobe nd compound strch grnules, 27,28) but when both SSIII nd GBSS ctivities re missing, the potto strch grnules develop into regulr shpe. 28) Amylopectin of nturlly present compound strches, such s rice nd ots strches, hve lrge proportions of short brnch chins of DP ) The reltionship between the grnulr structure formtion nd the short brnch chins of DP 6 12 is in-

4 34 J. Appl. Glycosci., Vol. 54, No. 1 (2007) Tble 4. Brnch chin length distribution of mylopectin. Strch Pek 1 Pek 2 % Distribution (DP) Norml corn ± ± ± ± ±0.2 Wxy corn ± ± ± ± ±0.5 Sugry ± ± ± ± ±0.3 Results were obtined by using high performnce nion exchnge chromtogrph equipped with post column myloglucosidse rector nd pulsed mperometric detector (HPAEC ENZ PAD), men±sd. Fig. 4. Scnning Electron Microgrphs (SEM) nd confocl lser scttering microgrphs (CLSM) of mize mutnt strches. A nd D re SEM of norml mize strch t different mgnifiction; B nd E, SEM of wxy mize strch; nd C nd F, SEM of sugry 2 mize strch. G, H, nd I re CLSM of norml, wxy, nd sugry 2 mize strch, respectively. Reprint with permissions from Refs. 6) nd 29). triguing nd needs further studies. Confocl lser scttering microgrphs (CLSM) of su 2 mize strch grnule stined with Rhodmin B fluorescent dye nd extr dye removed show seprte ggregtes of strch sub grnules in the grnule. 29,30) The smll sub grnules hold the dye tightly (Fig. 4). Wheres tht of the norml mize strch grnules disply chnnel like voids nd tht of wxy mize strch grnules show loosely pcked structure t the periphery. The difference in the CLSM imges between the norml mize nd wxy mize strch cn be ttributed to the presence of mylose in the norml mize strch, but not in the wxy mize strch. Amylose is known to be more concentrted t the periphery of the strch grnule. The mylose in the norml mize strch intertwines with mylopectin nd holds the molecules together, prticulrly t the periphery of the grnule where mylose is more concentrted. The intertwining between mylose nd mylopectin mintins the integrity of the strch grnules. 31) Wxy mize strch does not hve mylose to intertwine with mylopectin nd, thus, displys loosely pcked structure t the periphery. Consequently, wxy mize strch grnules tend to disperse more esily 31) nd show fission when it is suspended in DMSO. All three mize mutnt strch grnules show internl surfce, 29,30) which results in greter susceptibility to enzyme hydrolysis 6) nd chemicl penetrtion 7) when compred with the strches of the B type polymorphism nd some C type polymorphism. 30) Among these three strches, su 2 strch consists of the lrgest internl surfce, which displys the gretest enzyme digestibility, followed by wxy mize strch nd norml mize strch, s shown in Fig. 3. 6) The internl voids shown in CLSM imges coincide with the internl structures of strch grnules reveled fter surfce strch hs been removed by surfce geltiniztion. The remining strch grnules of norml mize strch show porous nd loosely pcked or split structure, wheres tht of the potto strch show solid structure. 30) Reltionships between strch moleculr structures nd grnulr shpes. Strch of different botnicl origins disply chrcteristic nd diverse grnule shpes nd sizes. 3) For exmples, whet strch consists of bimodl grnules: lrge nd disk shped A grnules nd smll nd sphericl B grnules

5 Structure of Strch Grnules 35 (Fig. 5). 32) It is not known how strch grnules re synthesized into specific nd chrcteristic shpes for different plnts nd in different orgns. Some recent studies hve shown tht the brnch structures of mylopectin molecules of the whet A grnule differ from tht of the B grnule. 32) The mylopectin of the lrge nd disk shped A grnules consists of more B2 chins tht extend through two clusters, wheres tht of the sphericl B grnules consists of more short chins, i.e., A chins nd B1 chins tht extend only through one cluster (Fig. 6). When there re more B2 chins present in the molecules, the mylopectin molecules hve cylindricl shpes nd re likely to rrnge in prllel pttern nd developed into disk shped grnule s reveled by n opticl density mp. 33) In contrst, when n mylopectin molecule consists of more short chins, A nd B1 chins, nd fewer B2 chins, the molecule hs conicl shpe nd is better rrnged into sphericl grnule, the B grnule. Further studies re needed to fully understnd the effects of moleculr structures on the grnulr structures. The uthor thnks Dr. S. Srichuwong for criticl review of the mnuscript. REFERENCES Fig. 5. Scnning Electron Microgrphs (SEM) of frctionted whet lrge grnule strch (A) nd whet smll grnule strch (B). 1 ) M. Mtsui, M. Kkut nd A. Miski: Fine structurl fetures of oyster glycogen: Mode of multiple brnching. Crbohydr. Polym., 31, (1997). 2 ) D. French: Orgniztion of strch grnules. in Strch: Chemistry nd Technology. R.L. Whistler, J.N. BeMiller nd E.F. Pschll, eds., 2nd Ed., Acdemic Press, New York, NY, pp. Fig. 6. Brnch chin length distribution of mylopectin isolted from whet lrge grnule strch (A), nd whet smll grnule strch (B), the difference in brnch chin length between the lrge nd the smll grnule strch (B A) (C), nd proposed models of the mylopectin molecules of the A nd the B grnules. The brnch chin length nlysis is conducted using the sme method described in Fig. 2. Printed with permission from Ref. 32).

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