MATERIALS AND METHODS

Transcription

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2 form, they mght have propertes that would allow a manufacturer to use them and avod the term "modfed starch" on the label, and they would provde the raw materal for modfcaton to provde starch wth mproved physcal behavor. The best-known effect of the mutant maze genotypes s on the proporton of amylose and amylopectn: hgh-amylose maze starches have 50-70% apparent amylose, whereas waxy maze starch has essentally no amylose (Shannon and Garwood 1984). What s less commonly apprecated s that the structure of the amylopectn s also affected n the mutant genotypes. n fact, the apparent amylose values for hgh-amylose starch are probably not good estmates of the actual amylose content because odne bndng by the amylopectn n hgh-amylose starches contrbutes to the apparent amylose values (Boyer and Lu 1985). t s not clear how much the altered physcal behavor of hgh-amylose starch s due to the proporton of amylose and how much s due to the altered amylopectn structure. Average chan lengths of amylopectns from varous plant speces have been known to dffer for some tme (L and Lneback 1977, Whstler and Danel 1984), but the specfc nature of the branched structure has proved a formdable problem. The elucdaton of amylopectn fne structure has evolved as analytcal technques have mproved. The Haworth (Haworth et al 1937), Staudnger (Staudnger and Huseman 1937), and Meyer (Meyer and Bernfeld 1940) models were based on the observed proporton of 1-4 and 1-6 glycosdc lnkages. Peat ntroduced the A, B, and C termnology for the types of lnear chans (Peat et al 1952). The Meyer model was refned by Nkun (1969) and French (1972) to a cluster model, whch was further refned by Robn et al (1974). Recent mprovements n the resoluton of sze excluson hgh-performance lqud chromatography (HPLC) have led to a refnement of the model based on nterpretaton of the dstrbuton of lnear chans followng soamylase dgeston (Hzukur 1986, nouch et al 1987). n Hzukur's model (1986) the B chans are consdered accordng to how many clusters they span: Bl chans are localzed to one cluster, B2 chans span two clusters, B3 chans span three clusters, and so forth. A chans are tactly assumed to be localzed to one cluster. The mportant physcal propertes of starch materals nclude the thermal requrements for gelatnzaton, the vscosty generated by gelatnzaton, stablty of gelatnzed granules to contnued heat and shearng acton, and susceptblty of gelatnzed starch to retrogradaton. The physcal behavor most readly studed on a small scale s the thermal behavor durng gelatnzaton. The temperature of gelatnzaton may be studed by dfferental scannng calormetry (DSC) or by loss of brefrngence under a polarzed lght mcroscope equpped wth a hot stage (Lund 1984). DSC has the advantage that n addton to the gelatnzaton temperature, the enthalpy of gelatnzaton may also be obtaned. Donovan (1979) has descrbed the theoretcal nterpretaton of DSC data for starch gelatnzaton. From the Pennsylvana State Unversty maze breedng collecton we have avalable starches from the endosperm of several mutant genotypes as expressed n four nbred lnes (three sweet lnes [1a5125, la453, and S3-61] and a dent lne [W64A]). One objectve of the present work was to screen a selecton of these samples for unque physcal propertes as observed by DSC. Among the avalable mutant genotypes are several contanng the gene, whch s thought to result n a lack of amylose (Shannon and Garwood 1984). Assumng that the amylopectn fne structure of these samples dffers, DSC analyss of these samples should provde nsght nto the magntude of the potental contrbuton of amylopectn to granule physcal behavor. A second objectve was to examne the varaton n thermal behavor of starches comprsed of only amylopectn. A thrd objectve was to begn the elucdaton of the amylopectn fne structure of these samples to nvestgate the hypothess that the physcal behavor observed by DSC s related to some aspect of the amylopectn fne structure. For the second and thrd objectves, four contanng genotypes (,,, and ) were examned as expressed n the four nbred lnes. MATERALS AND METHODS Starch Samples Backcross conversons for the endosperm mutants waxy (), amylose-extender (ae), sugary (su), and dull (du) were produced by standard procedures n the maze genetcs program at the Pennsylvana State Unversty. Multple mutant combnatons were produced by crossng the backcross conversons of the sngle mutants, selfng, and selecton for multple mutant lnes. All genotypes were verfed by approprate allele tests. Genotypes wll be dentfed by those gene loc homozygous for recessve mutant alleles. Other gene loc not lsted are therefore homozygous for the domnant allele. Mature kernels (20) were soaked n 50 ml of a 20 mm sodum acetate, ph 6.5, 10 mm mercurc chlorde soluton. After 24 hr. the germ and percarp were removed and 50 ml of fresh soluton was added for an addtonal 24 hr (Boyer et al 1976). Endosperm was repeatedly homogenzed for 1-mn ntervals n a mortar and pestle, and freed starch granules were collected by washng through a 4 4 -nm mesh. Starch granules were purfed by multple extractons (8-12) wth salne and toluene (Boyer et al 1976). Granular starch was washed three tmes wth double dstlled water, once wth acetone, and dred at 400 C. soamylase soamylase was solated from Pseudomonas amyloderamosa by the procedure of Yokobayash et al (1970). No contamnatng amylase actvtes were observed. Thermal Analyss by Dfferental Scannng Calormetry Thermal analyss was performed usng a dfferental scannng calormeter (model DSC-4, Perkn-Elmer, Norwalk, CT). Starch powder samples of 1.5 mg were placed n preweghed, coated alumnum hermetc pans (no , DuPont, Wlmngton, DE), and the precse weght was determned (Autobalance, Perkn- Elmer). Deonzed water (15 Ml) was added and the pan was sealed. The reference pan contaned 15,ul of water. The heatng rate was 100C/mn, from 15 to 1150C. Temperature and enthalpy were calbrated usng ndum. Endotherms were analyzed (Thermal Analyss Data Staton, Perkn-Elmer) to calculate peak onset temperature (TO), peak maxmum temperature (Tmax), and peak enthalpy (AH). Enthalpes were calculated on a starch weght bass. DSC data for fve starch samples (from a5125 normal,, ae, du, and ) were reported prevously (Brockett et al 1988) based on estmaton of starch content by total carbohydrate analyss. For all data reported n the present work, t was assumed that samples were 90% starch. Amylopectn Fne Structure Analyss soamylase dgeston. Starch samples were dspersed n 90:10 (v/v) dmethyl sulfoxde/deonzed water (hereafter termed 90% DMSO) by heatng 10 mn n a bolng water bath, coolng to room temperature, and heatng agan for 10 mn. Dspersons were 10 mg/ml, and alquots of ml were subsequently dgested. Alquots were precptated wth three volumes of 1% KCl n 75% methanol, and centrfuged 5 mn at 900 X g. The supernatant was dscarded and the precptate was resuspended n the orgnal volume of 90% DMSO by heatng n bolng water for 10 mn. The methanol precptaton was repeated, and the precptate was taken up n 1.8 tmes the orgnal volume of 0.5M sodum acetate buffer at ph 3.5. An alquot of soamylase (1 mg/ml n 0.5M sodum acetate buffer at ph 3.5) was added to brng the total volume to two tmes the orgnal volume. Dgeston was performed at 350C. After one week, addtonal soamylase soluton was added. Dgeston was montored by szeexcluson chromatography. Dgeston was consdered complete when the areas of the two major chromatographc peaks dd not change by more than 2% n 24 hr. Sze-excluson chromatography of enzyme dgests. The methodology was adapted from that of Kobayash et al (1986). The HPLC system conssted of a pump (model 510, Waters, Mlford, MA), an njector (U6K, Waters), a 2-cm guard column Vol. 67, No. 6,

3 (LC-18 Pellguard, Supelco, Bellefonte, PA), two 30-cm sze excluson columns (Zorbax PSM 60S, DuPont, Wlmngton, DE) n seres, a dfferental refractometer (model 410), and an ntegrator (model 745, Waters). The moble phase was DMSO and the flow rate was 0.4 ml/mn. The columns and detector were mantaned at 400C. A ml alquot was removed from the soamylase dgeston mxture and added to ml of DMSO. The mxture was heated n bolng water for 10 mn n a capped 1.5-ml tube. and then centrfuged for 5 mn at 2,500 X g. A 100-A.l porton of the clear superrratant was njected nto the HPLC system. Chromatograms were nterpreted as havng two major peaks separated by the mnmum dentfed by the ntegrator, as ndcated n the fgures. Estmaton of chan lengths of debranched materal. A sample of partally acd-hydrolyzed starch contanng a range of chan lengths was made to 5 mg/ ml n 90% DMSO. A 20-tl alquot was njected nto the HPLC system. The retenton tmes for degree of polymerzaton (DP) 1-5 could be determned drectly from the chromatogram. Ths partally hydrolyzed starch was also made up n 0.01N NaOH (5 mg/ ml), and 2 ml was appled to a sze-excluson column (Sephadex G-75, 1.5 X 100 cm). The moble phase was 0.01N NaOH and the flow rate was ml/mn. Eghty 2.8-ml fractons were collected and analyzed for total carbohydrate by the phenol-sulfurc acd test (Hodge and Hofreter 1962) and the Park Johnson test for reducng ends (Porro et al 1981). Based on these two analyses, the average chan length for each fracton was calculated. To calbrate the Sephadex G-75 column, lnear regresson was done on a plot of the log chan length versus fracton number. Alquots (2 ml of 5 mg/ ml) from two debranched starch samples were also appled to the column, and for each sample the DP of the two peaks was calculated. These peaks were assumed to correspond to the two peaks observed by HPLC for each sample. A sample of commercally prepared potato amylose (no. A05 12, Sgma Chemcal Co., St. Lous, MO) was analyzed for total carbohydrate and reducng ends as above; the DP was calculated to be 330. Ths sample was made to 20 mg/ml of 90% DMSO and njected nto the HPLC system. Ths retenton tme was combned wth the retenton tmes for DP 1-5 and for the debranched amylopectn samples above. The relatonshp between the natural logarthm of the degree of polymerzaton and retenton tme was ftted to a lnear equaton (y = x, r = 0.98), whch was used to estmate the degree of polymerzaton of the observed HPLC peaks. Statstcs Analyss of varance was done on the DSC data and on the HPLC data. Peak onset temperatures were not compared statstcally due to the varable nature of ths value when the endotherm was broad. Multple comparsons were done by least sgnfcant dfference (LSD) after a prelmnary F test. Threeway analyss of varance was performed on the data for the four -contanng genotypes from the four nbred lnes. n ths 2 X 2 X 4 factoral desgn, the two gene factors were the absence or presence of ether du or ae; the thrd factor was the nbred lne. Correlaton analyss was performed on the means of HPLC chromatogram data and of DSC thermogram data. RESULTS AND DSCUSSON The 1a5125 nbred Lne Thermal analyss. For the la5125 nbred lne, the thermal behavor durng gelatnzaton n excess water was studed for the normal starch and the sngle, double, and trple mutants based on the, ae, du, and su genotypes. The data are presented n Table and thermograms are presented n Fgure 1. For the sngle mutants, the Tmax was hghest for the ae starch at 77.60C. Prevous work wth dent maze starch showed that Tmax for the ae starch was hgher than for other sngle mutants, but the temperature was much hgher (Zobel 1984) than for ths genotype n the a5125 lne n the present work. n addton, the endotherm was much broader n the dent lnes prevously studed (Zobel 1984, Wooten and Bamunuarachch 1979). The dfference s at least partally due to lne dfferences but may also nvolve annealng of those commercally steeped dent starches (Krueger et al 1987). Based on the peak heght ndex crteron of Krueger et al (1987), starches n the present work were not annealed (Brockett et al 1988). Enthalpy values for all sngle mutant starches n the present work were about 3 cal/g, slghtly hgher than observed for the normal. Starches from four of the sx double mutant genotypes had smlar Tmax values to that of the normal starch. However, the genotype gelatnzed at a hgher temperature. Ths result mght suggest that the ae genotype controls the gelatnzaton temperature; however, no elevated gelatnzaton temperature was observed for the ae du or the ae su genotypes. Furthermore, the double mutant wth by far the hghest Tmax was du su at C. Enthalpy values vared consderably for the double mutants; agan the enthalpy of gelatnzaton for the du su starch was far hgher than the others, at 4.7 cal/g. Although t appears plausble that the gelatnzaton temperature and enthalpy would be related, support for ths relatonshp n Table s equvocal at best. The thermal behavor of the trple mutant starches cannot be predcted from that of the double and sngle mutants. The lowest Tmax for any of the starches (64.30C) was for the ae du su, despte the observaton that the du su combnaton showed the hghest Tmax and the presence of the ae gene that also was responsble for a relatvely hgh Tmax. Agan, the hghest enthalpy and Tmax were observed for the same sample, ae su. t s nstructve to observe the DSC thermograms for these starches (Fg. 1). The peak s relatvely sharp and well defned for the normal and for and du starches. The peak s much broader for the ae starch. The thermogram for the su starch s a hybrd of the two types of peak: a sharp peak at the low temperature and a low broad peak at a hgher temperature. For the double mutants, the second peak was observed at the hgher temperature for two starches: ae du and du su. For the latter, TABLE Thermal Analyss of Gelatnzaton of Starches from Several Mutant Genotypes n the a5125 nbred Lnea Starchb To ( 0 C) Tmax( 0 C) /AH (cal/g) Normal (3) 64.2 ± ± 0.1 fg 2.7 ± 0.1 Sngle mutants (4)c 61.0 ± ef ef ae (7) 68.8 ± ± 0.8 b 3.1 ± 0.2 g du (4) 61.0 ± ± 0.2 h 2.9 ± 0.0 hd su (3) 61.3 ± ± 0.2 h 3.3 ± 0.0 f Double mutants ae(4)c b d (3)c c c su (3) cd c ae du (3) 59.9 ± ± 0.9 fgh 2.4 ± 0.0 j ae su (3) 55.7 ± de de du su (3) 53.6 ± a a Trple mutants du su (4) gh ae du su (3) g (3)c ae su (3) 54.8 ± ± cd ±0.2 b f ±0.2 b avalues are mean ± SD. n the same column, values followed by a common letter are not sgnfcantly dfferent at P < 0.05, by LSD analyss. Pooled standard errors are not reported due to unequal n. Expermental error mean square for Tmax ; for AH = bgenotype (number of replcate analyses). 'Data for ths sample are also found n Table. denthalpy for ths sample was prevously reported (Brockett et al 1988) based only on the man endothermc peak. The value here s slghtly hgher due to ncluson of the area of the secondary peak, as was done n the present work for other starches when a secondary peak was observed (Fgs. 1 and 3). 596 CEREAL CHEMSTRY

4 ths hgh-temperature peak was the domnant peak. For the trple mutants, only the ae du su starch showed even a hnt of the hgh-temperature peak. t s of nterest that only those starches lackng the gene showed the hgher temperature peak. Both the ae du su starch and the gave a very broad gelatnzaton endotherm. The ae su starch showed a strong low-temperature shoulder to the man gelatnzaton peak. Amylopectn fne structure. soamylase dgestons of the WaS125 starches were only done for those starches contanng the gene. The data are presented n Table, and representatve HPLC chromatograms are presented n Fgure 2A. Each of the two major peaks was apparently made up of more than one component. By the model of Hzukur (1986) the low molecular weght peak would be composed of A chans (slghtly shorter) and B1 chans (slghtly longer). The hgh molecular weght peak would be composed of B2, B3, and longer chans. Varaton was observed n the proporton of the total area n the two major peaks and n the relatve proporton of the components wthn each major peak. The latter was dffcult to quantfy but could be observed qualtatvely. For example, the debranched starch dffered from the starch n two respects (Fg. 2A): the proporton of materal n the hgh molecular weght peak was greater, and the hgher molecular weght component of the low molecular weght peak was more pronounced. n all but two starches only the lower molecular weght component of the low molecular weght peak was consstently TABLE Dstrbuton of Lnear Chans After soamylase Debranchng and Sze- Excluson Hgh-Performance Lqud Chromatography Analyss for -Contanng Starches n the a5125 lne' Hgh-MWc Peak Retenton Tme (mn) Retenton Percent Man Secondary Tme of Total Low-MW Low-MW Starchb (mn) Area Peak Peak WXd 19.7±0.1 d 41±1 d 23.5±0.1 b nonee d 20.0±0.2 bcd 50±1 b 23.3±0.1 c 22.4±0.0 d 19.8±0.3 cd 38±1 e 23.7±0.2 ab nonee su 21.0±0.3 a 48±0 b 23.7±0.0 a nonee d 20.3±0.3 bc 45±0 c 23.7±0.1 ab 22.2±0.1 aesu 20.5±0.3 b 56±1 a 23.3±0.1 c nonef du su 20.4±0.4 b 37±2 e 23.8±0.1 a nonee avalues are mean ± SD. n the same column, values followed by a common letter are not sgnfcantly dfferent at P< 0.05, by LSD analyss. Pooled standard errors: hgh-mw retenton tme, 0.027; hgh-mw percent of total area, 0.346; low-mw retenton tme, bgenotype. Three replcate analyses for each genotype. CMW = Molecular weght. ddata also contaned n Table V. eno peak was dentfed by the ntegrator, but a shoulder was present n ths regon. fa peak was dentfed on two of the three chromatograms; retenton tmes were 22.3 mn LL H- w CE H- 0 z uj Dt 0.1 mcal/sect ae su du su ae du su TEMPERATURE (OC) Fg. 1. Dfferental scannng calormetry thermograms for selected mutant maze genotypes n the WaS125 nbred lne: sngle mutants (A), -contanng double mutants (B), other double mutants (C), trple mutants (D). Vol. 67, No. 6,

5 detected by the ntegrator as a peak (Table ), although t s evdent n Fgure 2A that the hgher molecular weght component of the low molecular weght peak was present n all samples. For the and starches, a secondary low molecular weght peak was also consstently detected (Table ). Of the WaS125 starches analyzed by both DSC and HPLC, t s only for the and the ae su starches that a hgher Tma, was observed. These two starches also had the hghest proporton A DP B DP T -- x LLJ z su LLJ ae su -- LL du su CE -j H- z C 15 3 DP, D 15 3 DP ll LL5 30 RETENTON TME (mn) 15 3 Fg. 2. Sze-excluson chromatograms for soamylase-debranched starch from selected maze mutant genotypes as expressed n four nbred lnes: a5125 (A), a453 (B), S3-61 (C), and W64A (D). 598 CEREAL CHEMSTRY 1 5

6 of the total materal n the hgh molecular weght peak. The su starch also had a relatvely hgh proporton of materal n the hgh molecular weght peak, but for ths starch the lower molecular weght component of the low molecular weght peak was proportonately large. Although the starch does have a detectable secondary low molecular weght peak, the Tmax s not hgh. However, for ths starch the percent of the total area n the hgh molecular weght peak s relatvely lower than for the and ae su starches. t s plausble to suppose that the proporton of two lnear components wthn the low molecular weght peak, as well as the overall percent of the branches n the hgh molecular weght peak, may be mportant n determnng the gelatnzaton Tmax n ths sweet lne. Starches from Four -Contanng Genotypes n the Four Lnes Thermal analyss. DSC data for thermograms for gelatnzaton of starches from the,,, and genotypes s summarzed n Table. Representatve thermograms are shown n Fgure 3. Tmax for these starches ranged from 64.40C for the n 1a453 to 85.30C for the n W64A. Wthn each lne the hghest Tmax was for the starch from the genotype; two of these were above 800C (80.5 and 85.30C for the S3-61 and W64A, respectvely). For the 16 starches, enthalpes ranged from 3.0 cal/g for the starch n W64A to 5.6 cal/g for n W64A. n three lnes, the hghest enthalpy was for starch from the genotype. As wth Tmax, the two hghest enthalpes were for n the S3-61 and W64A lnes (4.9 and 5.6 cal/g, respectvely). The shapes of the thermograms vared among the 16 contanng starches (Fg. 3). Wthn each lne, the and du starches produced the sharpest peaks. The peak for the ae starch was usually the broadest. The peak for the starch was ntermedate. The thermogram for the starch from the W64A lne apparently has two components. The dfferences n thermal behavor seen among these 16 starches llustrate the complex role that amylopectn can play n determnng gelatnzaton behavor (Table ). n all four lnes, thermograms for the and starches show relatvely broad peaks. These broad peaks may be due to ether a lack of cooperatvty among crystalltes wthn smlar granules, to varaton among granules, or both. Some of the broad thermograms appear to have two components, whch mght be TABLE Thermal Analyss of Gelatnzaton of Starches from -Contanng Genotypes from Four nbred Lnesa Starchb To ( C) Tmx( 0 C) /AH (cal/g) a5125 (4) 61.0 ± ± 0.2 h gh (4) 69.8 ± ± 0.2 c 3.5 ±0.1 f (3) 64.4 ± ± 0.0 ef e (3) 54.8 ± ± 0.7 ef h a453 (4) 56.0 ± ± 0.2j j (3) 69.0 ± ± 0.7 c 3.9 ± 0.0 cd (3) 64.1 ± ± 0.0 efg fg ae (3) 58.8 ± ± 0.3 e j S3-61 (4) 60.1 ± ± h (3) 71.0 ± ± 0.5 b b (3) 59.5 ± ± 0.7 gh 3.8 ± 0.1 de (3) 54.2 ± ± 0.9 d j W64A (4) 66.7± ±0.8fg j (3) 66.9 ± ± 1.2 a a (3) 60.9 ± ± 0.4 fg c (3) 53.6 ± h 3.6 A 0.1 f avalues are mean ± SD. n the same column, values followed by a common letter are not sgnfcantly dfferent at P< 0.05, by LSD analyss. Pooled standard errors not reported due to unequal n. Expermental error mean square for Tmax = 0.308; for AH = bnbred lne and genotype (number of replcate analyses). nterpreted as two overlappng peaks. Such an nterpretaton s consstent wth the possblty that two populatons of granule are present. By 2 X 2 X 4 factoral analyss, the two gene treatments (±ae and ±du) the four lnes, and the nteracton terms were all hghly sgnfcant (P < ) wth respect to Tmax and enthalpy. Consequently, both genotype and lne nfluence thermal behavor durng gelatnzaton, but the nfluence of a partcular gene vares accordng to the presence of other mutant genes and accordng to the lne. Thus wth respect to gelatnzaton, the nfluence of genotype and lne s hghly complex and deserves further study. Amylopectn fne structure. HPLC chromatograms of debranched starches from all four lnes are presented n Fgure 2. The retenton tmes for the two major peaks and the percent of the total area n the hgh molecular weght peak are presented n Table V. For the 16 samples, retenton tmes for the two peaks were generally smlar, although sgnfcant dfferences were observed. The mean retenton tme for the hgh molecular weght peak for all 16 starches was 20.0 mn, whch corresponds to a DP of 48. The ndvdual retenton tmes corresponded to a range of DP (except for the su starch, whch had a much lower DP of 33). The hgh molecular weght peak maxma are nterpreted to result from a domnant populaton of B2 chans. Most of the hgh molecular weght peak maxma are smlar to those reported by Takeda (1988) for three dent maze samples (DP 45, 47, and 48). For 14 of the samples, the mean retenton tme for the low molecular weght peak was 23.5 mn, whch corresponds to a DP of 10. The range was DP Two values were not ncluded n these calculatons because the retenton tmes of the man low molecular weght peaks were well below those for the other 14 starches. These values were for the starches from the S3-61 and W64A lnes, whch had retenton tmes of 22.7 and 22.5 mn, respectvely, correspondng to DP 15 and 16. These latter values correspond well to those for Bl chans, as reported by Hzukur (1986) for varous nonmaze starches (DP 15-19). Chromatograms of the starches n the a453 and as125 lnes contaned a shoulder strong enough to be recognzed by the ntegrator at approxmately the same retenton tme as the man low molecular weght peak for the other starches. Furthermore, chromatograms for all starches contaned ths promnent, recognzed shoulder. Thus, putatve B1 chans were strongly evdent for all and starches. t s noteworthy that for these two -contanng genotypes, but not other -contanng genotypes, Boyer and Lu (1985) observed that a consderable fracton of the amylopectn was of lower than usual molecular weght. n the present work, the observed DP of correspond to those observed by Hzukur (1986) for the A chans for the nonmaze starches studed but are apparently shorter than those observed by Takeda et al (1988) for normal maze. Although no attempt was made to dscuss A and Bl chans n that work, the chromatograms publshed by Takeda et al (1988) ndcate that the Bl chans mght be DP > 20 and the A chans - DP The relatve areas of the two major chromatographc peaks vared consderably. Wthn each lne, the starches had the hghest proporton as the hgh molecular weght peak, at 50-64%. Wthn each lne, the starches had the lowest proporton as the hgh molecular weght peak (but not sgnfcantly so for the S3-61 lne) at 29-38% from the Oh43 nbred lne. Fuwa et al (1987) also observed a low proporton of materal n the hgh molecular weght peak for debranched starch. Most of the chromatograms had shoulders wthn one or both of the major peaks. Wthn each lne, the and starches had hgher molecular weght shoulders n the hgh molecular weght peak. The prmary low molecular weght peak was accompaned by a secondary peak at slghtly hgher molecular weght n ae starches from all four lnes (Table V) as well as n the starches from the WaS125 and a453 lnes. For the starches from the S3-61 and W64A lnes, the low molecular weght peak was broad, spannng the regon of the prmary peak and Vol. 67, No. 6,

7 the shoulder or secondary peak n the other starch samples. t appears that varaton n the proporton of chans between and wthn the two major peaks, rather than translocaton of the entre chan length dstrbuton, may be responsble for varaton n amylopectn structure. Varaton n these proportons may help explan dfferences n physcal propertes of the starches. By factoral analyss, the effect of genotype and lne on the pattern of debranched materal s hghly complex, wth all man effects and nteractons nfluencng the percentage of the total area n the man peaks and the low molecular weght retenton tme. Although the man effects of ae and of nbred lne on the hgh molecular weght retenton tme were hghly sgnfcant, the man effect of du was not sgnfcant, possbly due to a hghly sgnfcant nteracton term, du X lne. By examnaton of Table V, t would appear that ths sgnfcant nteracton term results prmarly from the data n the S3-61 lne. Relatonshp Between Gelatnzaton Behavor and Amylopectn Fne Structure Wthn the as125 lne. Only the -contanng genotypes were subjected to soamylase debranchng as well as to thermal analyss; consequently, dscusson must be lmted to seven starches from the WaM125 lne. For these starches the hghest enthalpy, and one of the two hghest Tmax values, was observed for the ae su genotype. Ths sample also had the greatest proporton of chans n the hgh molecular weght peak (56%). The other sample wth a hgh T was, yet enthalpy for ths sample was 0.8 cal/g lower than for the ae su. t too had a relatvely large proporton of chans n the hgh molecular weght peak (50%). Both samples had a slghtly shorter retenton tme for the low molecular weght peak than for the others, correspondng to a slghtly larger DP. For one of the two starches wth the lowest proporton of area n the hgh molecular weght peak, du su, enthalpy was the lowest. For the other,, enthalpy was consderably hgher. Other -contanng samples wthn the a5125 lne showed no consstent relatonshp between ther gelatnzaton behavor and chan dstrbuton. Overall, for samples from the as 125 lne, there may be a weak relatonshp between fne structure parameters and thermal analyss parameters. Starches from the four -contanng genotypes from the four lnes. Among these 16 starch samples, a pattern s observed n two respects. Wthn each lne, the starches had the greatest proporton of area n the hgh molecular weght peak as well as the hghest T.ax. Secondly, the two samples wth much shorter retenton tmes for the prmary low molecular weght peak, the n S3-61 and W64A, were the samples wth the two hghest enthalpes. These observatons are consstent wth the noton that a hgher proporton of B2 and B3 chans to Bl and A chans may be responsble for a hgher Tmax but not necessarly a hgher enthalpy. Because the much shorter retenton tme for the low molecular weght peak may be nterpreted as due to a predomnance of Bl over A chans (a reversal of the stuaton for most starches analyzed), t s plausble that the proporton of Bl to A chans s a strong determnant of enthalpy, possbly n combnaton wth the proporton of the chans n the two major A,, ~~~~0. 1 mcal/sec C _ 0.1 mcal/sec A / 3: 0 11 LL 0 a: 0 z LU B B 0.1 mcal/sec AL. D At 0.1 mcal/secl ae du Aft TEMPERATURE (OC) Fg. 3. Dfferental scannng calormetry thermograms for four -contanng maze genotypes as expressed n four nbred lnes: la5125 (A), 1a453 (B), S3-61 (C), and W64A (D). 600 CEREAL CHEMSTRY l

8 TABLE V Dstrbuton of Lnear Chans After soamylase Debranchng and Sze-Excluson Hgh-Performance Lqud Chromatography Analyss for -Contanng Genotypes from Four nbred Lnesa Hgh-MWc Peak Retenton Tme (mn) Retenton Tme Percent of Total Man Low-MW Secondary Low- Starchb (mn) Area Peak MV Peak a5125 d 19.7 ± 0.1 fg 41 ± 1 e 23.5 ± 0.1 ab nonee d bcde 50 ± c 23.3 ± 0.1 c 22.4 ± 0.0 d 19.8 ± 0.3 defg 38 ± 1 f 23.7 ± 0.2 a nonee ae du WXd b 45 ± 0 d 23.7 ± 0.1 a 22.2 ± 0.1 a ± 0.2 cdef 42 ± 2 e 23.6 ± 0.0 ab nonee a 64 ± 0 a c 22.6 ± fg ab nonee b 36 gh ab 22.3 ± 0.1 S ± 0.0 efg 41 ± 2 e 23.6 ± 0.0 ab nonee defg 55 0 b d nonef du lbcd 35± lh 23.7±0.2a nonee 20.2 ± 0.0 bc 36 ± 0 h 23.6 ± 0.1 ab 22.3 ± 0.0 W64A 19.8 ± 0.2 defg 41 ± 2 e 23.5 ± 0.1 b none' bcd 62 ± a 22.5 ± 0.0 e nonef g 29 j ab nonee bc 38 fg ab 22.4 ± 0.1 avalues are mean ± SD. n the same column, values followed by a common letter are not sgnfcantly dfferent at P < 0.05, by LSD analyss. Pooled standard errors: hgh-mw retenton tme, 0.011; hgh-mw percent of total area, 0.399; low-mw retenton tme, bgenotype. Three replcate analyses for each genotype. CMW = Molecular weght. ddata also contaned n Table. 'No peak was dentfed by the ntegrator, but a shoulder was present n ths regon. ffor ths sample, the prmary peak occurred at a retenton tme approxmately that observed for the secondary peak for 14 of the 16 samples. TABLE V Correlaton Between HPLC' Sze-Excluson Chromatogram Parameters for Debranched Starch and DSCb Thermal Behavor Parameters for -Contanng Genotypes from Four nbred Lnes Parameters Correlaton Hgh-MWc peak retenton tme and To Tmax 0.28 AH Hgh-MW peak percent of total area and T Tmax 0.71 AH Low-MW peak retenton tme and T Tmax A\H ahgh-performance lqud chromatography. bdfferental scannng calormetry. 'MW = Molecular weght. peaks. Varaton among amylopectn molecules may also contrbute to observed thermal behavor of these starches. For the genotype, the lne (las125) wth the greatest proporton of low molecular weght amylopectn (Boyer and Lu 1985) had the lowest enthalpy. Correlaton analyses between the three HPLC chromatogram parameters and the three DSC thermogram parameters are shown n Table V. t s clear that the retenton tme of the hgh molecular weght peak dd not correlate well wth any DSC parameter. On the other hand, the percent of total area n the hgh molecular weght peak correlated postvely wth each DSC parameter, and a negatve correlaton of smlar magntude was observed between the retenton tme of the low molecular weght peak and each DSC parameter. Thus, t would appear that the thermal behavor of the starches mght be related to two aspects of the amylopectn structure: more chans of hgher molecular weght, and a slghtly longer populaton of lower molecular weght chans. Both would contrbute to the prevously observed longer average chan lengths for starches. t should be noted that the strength of the above correlatons results dsproportonately from the dfferent behavor of the starches relatve to that for the other starches. That Tmax and enthalpy mght be a functon of both aspects of amylopectn structure may be explaned based on the crystallte structure of the granule. Accordng to the several cluster models of amylopectn, many crystalltes are lnked due to the ncluson of at least one chan n contguous crystalltes. f the proporton of chans common to contguous crystalltes were greater (.e., more B2 chans), the stablty of each crystallte would become ncreasngly nfluenced by the behavor of ts partner. Due to the ncreased supracrystallte order, t would take more thermal energy to overcome the knetc barrer to gelatnzaton. The hgher enthalpy would result from the ncrease n the total noncovalent forces of attracton of the lnear chans. n Hzukur's model (1986) the Bl chans are thought to be longer than the A chans, and both are thought to be localzed to one crystallte regon. The length of a crystallte would be longer (n the axs of the 1-4 lnkages) f t were made up solely of Bl chans than f t were solely A chans. A hgher proporton of Bl chans would result n slghtly longer crystalltes and at the same tme ncrease the effcency of the packng wthn the crystallte buldng blocks, leadng to a proportonately greater fracton of the starch n crystallne form. On ths bass as well, gelatnzaton T and enthalpy would be hgher. These conclusons appear reasonable for all four genotypes examned n the four lnes; however, wthn the as125 lne for a greater varety of genotypes, ncludng several contanng the su gene, the relatonshp may be more complex. Contnued research s needed to understand the way genotype and lne mght nfluence amylopectn structure and consequently alter starch physcal behavor. One aspect of amylopectn structure not addressed n the present work s molecular weght, whch has been shown to be unusually dsperse for starches from the ae and genotypes (Boyer and Lu 1985). On another level, the nfluence of genetc background on granule behavor may be partally medated through varaton n granule Vol. 67, No. 6,

9 archtecture, sze, or morphology. Consderable research remans to be done to address the complex relatonshp between genetc background and starch physcal behavor. ACKNOWLEDGMENTS The techncal assstance of Ronne Yuan n determnng the degree of polymerzaton of the amylose s apprecated. Fundng for HPLC columns was kndly provded by Kraft, nc. LTERATURE CTED BOYER, C. D., GARWOOD, D. L., and SHANNON, J. C The nteracton of the amylose-extender and waxy mutants of maze (Zea mays L). Fne structure of amylose-extender waxy starch. Staerke 28:405. BOYER, C. D. and LU, K. C The nteracton of endosperm genotype and genetc background. Part. Dfferences n chromatographc profles of starches from nonmutant and mutant endosperms. Starch/ Staerke 37:73. BROCKETT, E., THOMPSON, D., DAVS, T., and BOYER, C. D Gelatnzaton characterstcs of starch from du,, ae, and ae endosperm of sweet corn nbred a5125. Starch/Staerke 40:175. DONOVAN, J. W Phase transtons of the starch-water system. Bopolymers 18:263. FRENCH, D Fne structure of starch and ts relatonshp to the organzaton of starch granules. J. Jpn. Soc. Starch Sc. 19:8. FUWA, H., GLOVER, D. V., MYAURA, K., NOUCH, N., and SUGMOTO, Y Chan length dstrbuton of amylopectns of double- and trple-mutants contanng the waxy gene n the nbred Oh43 maze background. Starch/ Staerke 9:295. HAWORTH, W. N., HRST, E. L., and SHERWOOD, F. A Polysacchardes, Part XX. Determnaton of the chan length of glycogen. J. Chem. Soc. (London) p HZUKUR, S Polymodal dstrbuton of the chan lengths of amylopectns, and ts sgnfcance. Carbohydr. Res. 147:342. HODGE, J. C., and HOFRETER, B. T Determnaton of reducng sugars and carbohydrates. Page 380 n: Methods n Carbohydrate Chemstry, vol 1. R. L. Whstler and M. L. Wolfrom, eds. Academc Press: New York. NOUCH, N., GLOVER, D. V., and FUWA, H Chan length dstrbuton of amylopectns of several sngle mutants and the normal counterpart, and sugary- phytoglycogen n maze (Zea mays L.). Starch/ Staerke 39:259. KOBAYASH, S., SCHWARTZ, S. J., and LNEBACK, D. R Comparson of the structures of amylopectns from dfferent wheat varetes. Cereal Chem. 63:71. KRUEGER, B. R., KNUTSON, C. A., NGLETT, G. E., and WALKER, C. E A dfferental scannng calormetry study on the effect of annealng on gelatnzaton behavor of corn starch. J. Food Sc. 52:715. L, C. Y., and LNEBACK, D. R Characterzaton and comparson of cereal starches. Cereal Chem. 54:138. LUND, D nfluence of tme, temperature, mosture, ngredents, and processng condtons on starch gelatnzaton. CRC Crt. Rev. Food Sc. Nutr. 20:249. MEYER, K. H., and BERNFELD, P Recherches sur l'amdon V. L'amylopectne. Helv. Chm. Acta 23:875. NKUN, Z Scence of Cookery (n Japanese) 2:6. (Orgnal not seen, cted n NKUN 1978). NKUN, Z Studes on starch granules. Starch/Staerke 30:105. PEAT, S., WHELAN, W. J., and THOMAS, G. J Evdence of multple branchng n waxy maze starch. J. Chem. Soc. (London) p PORRO, M., VT, S., ANTON, G., and NER, P Modfcatons of the Park-Johnson ferrcyande submcromethod for the assay of reducng groups n carbohydrates. Anal. Bochem. 118:301. ROBN, J. P., MERCER, C., and GULBOT, A Lntnerzed starches. Gel fltraton and enzymatc studes of nsoluble resdues from prolonged acd treatment of potato starch. Cereal Chem. 51:389. SHANNON, J. C., and GARWOOD, D. L Genetcs and physology of starch development. n: Starch: Chemstry and Technology. R. L. Whstler, J. N. BeMller, and E. F. Paschall, eds. Academc Press: Orlando, FL. STAUDNGER, H., and HUSEMAN, E Uber hochpolymere verbndungen. Uber de konsttuton der starke. Justus Lebgs Annal. Chem. 527:195. TAKEDA, Y., SHTAOZONO, T., and HZUKUR, S Molecular structure of corn starch. Starch/ Staerke 40:51. WHSTLER, R. L., and DANEL, J. R Molecular structure of starch. n: Starch: Chemstry and Technology. R. L. Whstler, J. N. BeMller, and E. F. Paschall, eds. Academc Press: Orlando, FL. WOOTEN, M., and BAMUNUARACHCH, A Applcaton of dfferental scannng calormetry to starch gelatnzaton.. Commercal natve and modfed starches. Starch/ Staerke 31:201. YOKOBAYASH, K., MSAK, A., and HARADA, T Purfcaton and propertes of Pseudomonas soamylase. Bochm. Bophys. Acta 212:458. ZOBEL, H. F Gelatnzaton of starch and mechancal propertes of starch pastes. n: Starch: Chemstry and Technology. R. L. Whstler, J. N. BeMller, and E. F. Paschall, eds. Academc Press: Orlando, FL. [Receved October 5, Revson receved June 8, Accepted June 11, 1990.] 602 CEREAL CHEMSTRY