HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF BARLEY PROTEINS: RELATIVE QUANTITIES OF HORDEIN FRACTIONS CORRELATE WITH MALT

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1 /. Inst. Bew., Januay-Febuay, 99, Vol. 99, pp HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF BARLEY PROTEINS: RELATIVE QUANTITIES OF HORDEIN FRACTIONS CORRELATE WITH MALT EXTRACT By Pete W. Janes and John H. Skeitt (CSIRO Gain Quality Reseach Laboatoy, Division of Plant Industy, PO Box 7, Noth Ryde, N.S.W. 2 Austalia) Received 4 June 992 Seveal high pefomance liquid chomatogaphy (HPLC) appoaches have been used to study the elationships between amounts of paticula goups of gain stoage poteins (hodeins) and malt extact, using two sets of baley samples. The aeas of seveal chomatogam peaks vaied between samples in a manne that coelated with malt extact. Size-exclusion HPLC of potein aggegates extacted by sonication gave negative coelations of the elative aeas off the thee lagest chomatog am peaks with malt extact in a set of 9 samples of the one vaiety, but not in a set of samples of 8 vaieties gown at 4-5 sites. Unde educing conditions, disulphide-bonded hodeins wee quantitatively extacted and the popotion of hodein in the lagest peak, compised of D- and B-hodeins, coelated well with malt extact in both sets of samples. The hodeins sequentially extacted into 50% (v/v) - popanol and 50% (v/v) -popanol-50 mm dithiotheitoi (DTT) wee also analysed by evesed-phase HPLC, and could be esolved into seveal peaks of B-, C- and D- hodeins. Relationships between the amount of cetain C- hodeins in the popanol extact, and cetain B- hodeins in the popanol-dtt extact and malt extact wee seen. As well as poviding a bette undestanding of the elationship between baley gain potein composition and malting quality, these HPLC methods ae useful additional methods to enable patial pediction of malt extact using un-malted baley. Key Wods: Potein, malt extact, hodein, chomatogaphy Intoduction Baley malting quality has fo many yeas been ecognised as being highly dependent on the total amount of gain potein4-8. Compaed with studies on enzyme activities and cabohydate composition5-0, thee has been somewhat less wok pefomed on elating baley o malt potein compo sition to malting quality. Smith and Liste9 found vaietal diffeences in the amounts of an aggegated hodein faction they temed "gel potein', and by solubility factionation and potein detemination, they obseved that at high nitogen levels, poo vaieties tended to have moe gel potein. Eal ie, quality-elated diffeences in hodein distibution between factions extactable in aqueous popanol in the pesence and in the absence of educing agents had been noted2. While these studies povided valuable infomation on the elationship between amounts of two hodein solu bility factions and malting quality, the methods used wee not amenable eithe to apid pediction of malt extact o to sceening of lage numbes of small baley samples, such as in a beeding pogamme. High-Pefomance Liquid Chomatogaphy (HPLC), like electophoesis has the advantage of poviding infomation on potein composition but it can moe eadily quantify specific factions. With automated samples and compute analysis, HPLC is a less labou-intensive method than potein electophoesis. Finally, since it is moe likely to be diffe ences in the amounts of specific polypeptides athe than the pesence o absence of paticula polypeptides that influences malt extact2-20-2, the ability of HPLC to quantify paticu la poteins o goups of poteins is especially valuable. Revesed-phase HPLC methods have been used by a numbe of goups fo the vaietal identification of baleys o malts- 726, while we have ealie used size-exclusion HPLC analy sis of low-molecula weight polypeptides in malt to assess the degee of endospem modification5. Peliminay HPLC studies", using RP-HPLC and potein factionation methods, found a diffeence between the amounts of hodein factions in a good-malting and in a poo-malting vaiety. Howeve, systematic studies with lage numbes of baley samples dif feing in quality wee not undetaken. In the pesent study, we have analysed two sets of baley vaieties using one evesed-phase and two size-exclusion HPLC methods. Significant coelations between the po potions of potein pesent in specific peaks and quality paametes such as malt extact wee found with each method, and wee not just seconday effects aising fom the negative elationship between potein content and malt extact. Examination of the composition of these peaks by sodium dodecyl sulphatc-polyacylamide gel electophoesis (SDS-PAGE) identified the potein factions that had geat est coelation with degee of malt modification. Mateials and Methods Chemicals and eagents All chemicals used wee of eagent gade o bette. Acetonitile and tifluoacetic acid wee obtained fom Ajax (Sydney, Austalia) and Piece Chemical Co. (Rockfod, IL, USA) espectively. Baley Samples A set of 9 Schoone (malting vaiety) baley samples fom the 987 havest was obtained fom diffeent fam sites thoughout South Austalia. Thity-gam samples wee micomalted at the Waite Agicultual Reseach Institute, Adelaide, South Austalia. The method based on a mico pocesso-contolled micomalting facility (manufactued by Phoenix Systems, Adelaide, Austalia) as descibed ealie2 was followed, except that the steeping stages wee both lengthened to 8 hous and the ai est to 0 hous. In addition, 7 samples of eight genetically-unelated baley vaieties (Clippe, Covette, Gimmett, Schoone, Stiling, Lada, HIY 9, HIHI 429 and RAN 60) wee obtained fom eithe fou o five sites (including Ackland, Biloela, Hemitage, Hemitage (iigated) and Toobeah) in the 985 Queensland Regional baley tials. These wee micomalted at the Queensland Wheat Reseach Institute, Toowoomba, Aus talia. Eighty gam samples wee malted without the use of This document is povided compliments of the Institute of Bewing and Distilling Copyight - Jounal of the Institute of Bewing

2 78 BARI.F.Y PROTEINS [J. Inst. Bew. additives and malt extact detemined afte extaction of gound malt fo hou at 65. This method anks lines in an appopiate ode, but gives values that ae lowe and with geate spead than commecial malt extact data6. Baley gain was gound in a UDY Cyclone mill with a 0.8 mm sceen (UDY, Fot Collins, CO, USA) befoe extaction fo HPLC analysis. A summay of some of the malt analytical data fo the two sets of samples is povided in Table I. High-Pefomance Liquid Chomatogaphy HPLC analyses wee pefomed on a Wates (Milfod, MA, USA) system compising two model 50 pumps, a WISP 72 automatic sample, and a model 48 UV-visible detecto set at 24 nm. Pump contol and data acquisition was achi eved using a model 840 chomatogaphy station.. Size-Exclusion Chomatogaphy of Uneduced Hodein Aggegates The method used was adapted fom that of Singh et a/.4, who developed a technique fo the analysis of wheat glutenin aggegates following extaction by sonication in buffeed SDS solutions. A Banson sonifie (Banson, Danbuy, CT, USA) was used with a mm diamete tip pobe, at 0 W powe. Baley meal (20 mg) was suspended in ml of 2% (w/v) SDS in 0.05 M sodium phosphate buffe ph 6.9, then sonified 0 sec, befoe centifugation in a micocentifuge at 5,600 g fo 20 minutes. In some expeiments, sonication times and buffes wee alteed as indicated. The supenatants (20 u.) wee analysed using a Wates Potein-Pak 00 column, which was eluted at 0.5 ml/min using acetonitile (50%) TFA (0.%). The factionation ange fo SDS-denatued "andom coil" poteins is M 2,000-50,000 (Potein Pak Manual, Millipoe-Wates, Milfod, MA, USA). Some extacts wee also un on a Potein-Pak 25 column, which factionates ove a lowe molecula weight ange (M,000-0,000). 2. Size-Exclusion Chomatogaphy of Reduced Hodein Polypeptides This was pefomed using the same column and eluant as descibed above, but with diffeent gain extacts. Baley also equied pealing befoe extaction, since extacts of gound whole gain clogged the column afte only a few analyses. In these expeiments, baley (50 mg) was initially extacted by votex mixing fo hou at 20 C with ml SDS (.5%, w/v), the extact was centifuged, and the supenatant was discaded. Disulphide-bonded poteins wee then extacted fom the esidue using ml SDS (2%, w/v)- 2- mecaptoethanol (0%, v/v) by incubation ovenight at 45 C. Mecaptoethanol at 0 pe cent is likely to be acting as a solvent as well as a educing agent, since disulphide eduction is complete at somewhat lowe concentations (Skeitt and Janes, unpublished). Using SDS-PAGE, this extactant has been shown to extact hodein quantitatively fom unmalted gain6-2.. Revesed-Phase Chomatogaphy Two hunded mg samples of baley meal wee extacted sequentially at 60 C with: a) 0.5 M NaCI (twice), b) wate, c) 50% (v/v) -popanol (twice), and finally in d) 50% (v/v) l-popanol-50 mm dithiotheitol (DTT). All extactions wee fo 0 minutes using.2 ml solvent. Factions c) and d) wee filteed though a Nucleopoe 0.45 u,m filte then analysed by RP-HPLC. A 25 cm x 0.46 cm Vydac 28 TP 54 C-8 column, poe size 00 Angstoms (The Sepaations Goup, Hespeia, CA, USA) was used. Column tempeatue was maintained at C, and poteins wee eluted using a gadient of acetonitile in 0.05% (v/v) tifluoacetic acid-wate. Initial conditions wee 0% acetonitile, then a linea gadient to 55% acetonitile ove 50 minutes, followed by a 5 minute wash with 70% acetonitile. The total un time was 70 minutes, including 5 minutes fo e-equilibation. Hodein Composition of Chomatogam Peaks The hodein composition of the HPLC peaks was analysed by SDS-PAGE. Factions fom -0 identical uns wee collected manually, and solvent was emoved by eithe cen tifugal evapoation using a Speedvac (Savant, Famingdale, NY, USA) o dialysis against 0.% SDS and feeze-dying. The factions wee then analysed by SDS-PAGE, using a gadient (0-5% w/v,.% cosslinking) of polyacylamide, using the buffe systems descibed ealie24. Gels wee inten tionally oveloaded, with some loss of esolution, in ode to identify mino polypeptide species. Data Analysis Whee appopiate, individual peaks o goups of peaks wee integated manually using the scanne mode of the Wates 840 data contol station. Simple linea egession analyses wee pefomed to examine coelations between chaacteistics of individual peaks and malt quality pa- TABLE I. Malting quality chaacteistics fo baley samples used in this study Set Potein ME FAN DP stach BG Schoone (n = 9) mean ± SD ange 0.9 ± ± ± ± ± ± coelation with: malt extact potein * ** (.0) (.0) * ** -0.56*** ** * ** * ** +0.6* " vaiety set (n = 7) mean ± SD ange.± ± ± ± coelation with: malt extact potein * ** (.0) (.0) * ** " abbeviations and units as follows: baley potein (N x 5.7, %), malt extact (ME, %), malt fee amino nitogen (FAN, mg/l). malt diastatic powe (DP, mm/scc/kg), baley stach (%), and baley beta-glucan (%. fo Schoone samples only), (not detemined). b data ae linea coelation coefficients (). * P 0.05, ** P 0.0, * P This document is povided compliments of the Institute of Bewing and Distilling Copyight - Jounal of the Institute of Bewing

3 Vol. 99, 99] BARLEY PROTEINS b) elutfon time (min) Fig.. Size-exclusion hplc pofiles of uneduced hodeins extacted, espectively, fom good and poo malting baleys: ) samples of Schoone baley fom two sites (Cystal Book and Pcakc, a and b); 2) samples of baley fom the Toobeah, QLD site (Stiling and HIHI-426, c and d). 0 ametes. Relative peak aeas wee calculated by dividing pecentage peak aeas by the mean peak aeas fo the set. This enabled a compaison of the elative changes in choma togam peak aea that occu with a given incement in malt extact value. Geate slopes of the elative peak aea vesus malt extact plots indicate a moe significant effect of the chomatogam peak unde study on malt quality, thus the slope of the plot is as impotant as the egession coelation coefficient between peak aea and malt extact. Data shown thoughout this pape epesents the mean of duplicate extactions, which diffeed by less than 0%. Results and Discussion Malting chaacteistics Some of the majo malt quality paametes of the samples6-2 ae summaised in Table I. As indicated in the methods, the malting methods fo the two sets of samples diffeed, thus the mean malt extact values wee lowe and the spead of values geate fo the 8-vaiety set. Howeve, it is not possible to state whethe o not the aveage mean quality of the two sets would have diffeed, had they been malted unde identical conditions. Samples of baley wee available fo the 8-vaiety set fom each of eithe 4 o 5 sites, since complete sets fo the same goups of 4 o 5 sites wee not available thoughout, the 7 samples fom 8-vaiety set have been teated as a goup athe than subjected to analysis of vaiance in a "genotype-by-envionment" study. Nonethe less, they seve to identify elationships between chomatog am peak aeas and malting quality that pesist on analysis of diffeent genotypes. Potein content was coelated with malt extact fo the Schoone set of baleys ( = , P 0.00), but moe weakly fo the 8-vaiety set ( = , P 0.0). Diastatic powe was coelated quite closely with malt extact in the Schoone set, but not fo the 8-vaiety set (Table I), while fee amino nitogen was positively coelated with total po tein (and negatively coelated with malt extact) in the Schoone set but not the 8-vaiety set. Measuements of baley stach and beta-glucan content wee only made with the Schoone set; the fome was weakly positively and the latte weakly negatively coelated with malt extact. Thus A. T 2 4 B. 2 4 T Fig. 2. SDS-PAGE analysis of chomatogam peaks fom SE-hpIc of Schoone baley. A. Uneduced poteins extacted by sonication; left to ight: molecula weight makes (92000, 68000, 4000, 0000, 2000, 4400), total baley gain extact (T), peak, peak 2, peak, peak 4. B. Reduced SDS-insolublc potein; left to ight: molecula weight makes, peak, peak 2, peak, mecaptoethanol peak (not shown in Fig. 4) and total baley gain extact. Majo hodein clustes ae labelled. This document is povided compliments of the Institute of Bewing and Distilling Copyight - Jounal of the Institute of Bewing

4 80 BARLEY PROTEINS [J. Inst. Bew. the only measued paamete that coelated with malt extact in both sets was potein content. While malting qual ity is a multifactoial chaacteistic, malt extact is one of the most impotant single measues of suitability of a paticula malt fo use in the bewing pocess. Theefoe, in this pape the focus has been made on elationships between elative chomatogam peak aea (epesenting quantification of specific potein factions), total gain potein and malt extact. Size-Exclusion (SE)-HPLC of Uneduced Hodeins Chomatogaphy of sonicated SDS extacts of baley gave a pofile somewhat simila to those obtained with wheat flou2, with seven peaks being noted (Fig. ). Although son ication fo 0 seconds in 2% SDS- 50 mm sodium phosphate, ph 6.9 was found to quantitatively extact potein fom wheat flou4, SDS-PAGE analysis of the esidue fom baley gain sonicated unde simila conditions then extacted using 0% (v/v) mecaptoethanol-6% (v/v) N.N'-dimethylfomamide 4% (w/v) SDS showed that some hodein emained. Initial expeiments wee pefomed in ode to identify whethe conditions could be modified to extact hodein aggegates quantitatively (in the absence of educing agents). Howeve, ) use of eithe o both 0. M acetic acid o 8 M uea with the 2% SDS extactant, o 2) inceasing the SDS concentation to 4%, failed to extact significantly moe potein. Analysis of potein extacts using the bicinchinoic acid method22 indicated that less than 0% moe potein was extacted by lengthening the sonication time fom 0 seconds to 2 minutes. Also, the size of the fist (aggegated) chomatogam peak was educed, indicating that this exta sonication was causing beakdown of aggegates. Theefoe, the conditions fo potein extaction used by Singh and cowokes" fo wheat wee used in the pesent study. An SDS-PAGE analysis of the majo peaks is shown in Fig. 2A. The numbeed peaks coesponded to:. D- and B- hodeins, 2. mainly C- hodeins,. B- and gamma- ho deins, and 4. low molecula weight poteins, especially polypeptides of M 6,000. These pobably coesponded to the majo CM-poteinsv. The last thee peaks contained only low-molecula weight ultaviolet-absobing mateial, pob ably oligopeptides and amino acids. Use of the popotion of aea unde each peak in the total chomatogam (elative peak aea), which is automatically povided by many HPLC softwae analysis packages, was especially useful in the cuent study, which involved analysis of sample extacts diffeing in potein content. This was because diect effects of diffeences in potein content on peak size wee accounted fo, minimising effects of vaiation in total potein content on the elationships between peak aeas and malt quality paametes. Diffeences in peak aeas of the same peak between samples wee noted, with cetain peaks vaying moe than othes. Fo the Schoone baleys, the elative aeas of the fist two peaks coelated significantly and negatively with malt extact and positively with baley gain potein (Table II, Fig. ). Thus as extact pecentage deceased, the amounts of aggegating potein (fist peak) and C- hodein (second peak) inceased. The coelation between malt extact and elative peak aea was geatest (and the elative peak height-malt extact plot steepest (Fig. B, Table II)) fo the second peak, which compised mostly monomeic C- hodeins athe than the fist peak, which compised mostly aggegated B- and D- hodeins. While the latte have been TABLE II. SE-HPLC of baley extacts: elationships between elative aeas of chomatogam peaks, malt extact and potein Peak malt extact Uneduced baley extacts * * * ** ** Schoone slope" potein 0.466" 0.870*** **' Reduced SDS-insoluble poteins ** 0.50* * ** * * " malt extact vaiety set slope 0.526*** * *** -6.0 potein *** " pecentage change in elative peak aea fo a % incease in malt extact (Schoone baleys) o a 5% incease in malt extact (8-vaiety set). = not calculated since linea egession coelation coefficient () did not achieve statistical significance. * P 0.05, ** P 0.0, ** P 'o o UJ HI Q. 5 0 LU t ui Q MALT EXTRACT (% MALT EXTRACT (%) 82 Fig.. Relationships between SE-hplc pecentage peak aeas and malt extact fo the set of uneduced extacts of Schoone baleys. A. Peak (aggegated hodcins). Baleys ove (O) and unde % potein ( ). The solid egession line is fo the full set of baleys, the dashed line fo baleys unde % potein. B. Peak 2 (C-hodcins). This document is povided compliments of the Institute of Bewing and Distilling Copyight - Jounal of the Institute of Bewing

5 Vol. 99, 99 BARLEY PROTEINS 8 moe consistently associated with poo malt extact, they wee not quantitatively extacted by the sonication po cedue. Indeed, moe potein was left in the esidue in pooe-malting vaieties (see below). Howeve, despite extaction being incomplete, it was vey epoducible, with excellent coelations being noted between chomatogam peak aeas obtained fom eplicate extactions. Examination of the elationship between aggegated po tein extacted and malt extact showed that the elationship was a bell-shaped cuve (Fig. A). This could be explained in tems of a balance between deceasing extactability and inceasing amounts of aggegated potein as malt extact deceased. On analysis of only the Schoone baleys of acceptable malting gade (i.e. ove 76% malt extact, o less than % potein espectively, Fig. ), impoved negative coelations ( = , n = 0, P 0.00; and = , n = 2, P espectively) wee obtained between the amount of aggegated potein and malt extact, and the slopes of the elative peak aea vesus malt extact plots doubled. Howeve, attempts wee not made to tans fom the data to fit a paabola, as it is pefeable fo the puposes of malt quality pediction to select othe peaks, that exhibited a simple linea elationship with malt extact. Ou ealie wok" had shown that the C-hodeins ae moe pooly modified in malting than the othe stoage poteins. Thus a ole fo these moe eadily-extactable po teins as makes of poo malt modification cannot be dis counted. While the aea of the thid peak did not vay much between baleys, the elative aea of the fouth peak coelated positively with malt extact and negatively with potein. This eflects diection of potein synthesis towads lowe molecula weight, possibly metabolic poteins at lowe gain potein contents. The elationships between elative peak aeas and malt extact wee less stong fo the 8-vaiety set (Table II), pesumably because of genotype vaiation adding to the envionmental effects. The elationship between the elative aea of the second peak with malt extact did not each statistical significance, although the elative heights of the second and fouth peaks wee coelated with malt extact (fo Peak 2, = , P 0.00, fo Peak 4, = 0., P 0.05), possibly because of diffeences in the shape of the peak between vaieties. Also, in two sets of vaieties (Clippe and Covette), potein content had a positive effect on malt extact. This may be due to enzyme activity and cabohydate composition having the main effects on malt quality in these sets. SE-HPLC of Reduced Hodein Polypeptides A shotcoming of the method descibed above is that poo extactability and high levels of aggegation among hodein poteins would have conflicting effects in the quantification of effects of diffeences in hodein composition. Theefoe, elative amounts of size factions of educed hodeins wee measued by size-exclusion chomatogaphy of polypeptides eluted fom the esidue following extaction with SDS. Pe liminay expeiments compaed two columns with diffeent factionation anges the Potein Pak 00 (Fig. 4) and the I- 25 (Wates Chomatogaphy). Both columns poduced thee main potein peaks, although fo some samples the second peak an as a shoulde on the othes. Using the -25 column, significant ovelap of the potein and mecaptoethanol peak (fom the extactant) occued. Analysis of the mecapto ethanol peak, (which eluted at minutes) showed that it contained two mino bands on SDS-PAGE analysis, which ae commonly-noted atifacts25. Theefoe, the Potein-Pak 00 column was used outinely, as shown in Fig. 4. The peak compositions wee analysed by SDS-PAGE (Fig. 2B). The fist peak contained some lage B- hodeins of mobility simila to C- hodein2 as well as some smalle B-hodeins, the second was highly eniched in D- hodein and the thid B- and D- hodeins. The elative aeas of the fist two peaks wee coelated positively with malt extact and the thid and lagest peak negatively with malt extact (Table II). None of the slopes of the elative peak aea vesus malt extact plots was flat, which suggests that each peak aea changes significantly as malt extact vaies. Simila tends wee seen with the 8-vaiety set, and in this case the coelation coefficients in the two sets wee simila. With the latte set, elative peak aeas and potein wee not coelated, which indicates that diffeences in the amounts of specific hodein goups wee not a simple esult of diffeences in gain potein content (Table II). These esults suggest that the highe-molecula weight B- hodein cluste2 may not negatively influence malt extact. Secondly, the D- hodeins wee distibuted between two well-esolved peaks. This could eithe epesent sepaate polypeptide species o D- hodein with diffeent degees of unfolding unde the chomatogaphic conditions used. It is notewothy that the goup of polypeptides that eluted fist on the SE-HPLC column did not have the highest appaent molecula weight on SDS- PAGE. Revesed-Phase (RP)-HPLC of Baley Poteins Following emoval of wate- and salt-soluble poteins, ho deins wee extacted using fistly 50% popanol then 50% popanol- % DTT. These extacts wee analysed by RP- HPLC. The popanol extact was analysed both as is (uneduced) and also afte the addition of DTT (to % final concentation) to the extact. This enabled diect compaison of esults obtained with the two extactants. Chomatogams of each extact ae shown in Fig. 5. E 0.8 a) b) * CM ^** ) 'c a o c (0 n o (0 JQ Q C) 2 j\ i-j\j\ J ^ d) s 0 IS 20 O elution time (min) Fig. 4. Size-exdusion hplc pofiles of educed hodein polypeptides fom good and poo-malting baleys. Details as fo legend to Fig.. This document is povided compliments of the Institute of Bewing and Distilling Copyight - Jounal of the Institute of Bewing

6 82 BARLEY PROTEINS [J. Inst. Bew. GOOD POOR 0.60 A) 50% -popanol extacts B) educed 50% popanol extacts 2a C) 50% -popanol -% DTT extacts elution time (min) Fig. 5. Revesed-phase hplc of potein extacts fom Schoone baleys: Cystal Book, SA site (good-malting) and Peake, SA site (poomalting). A) 50% -popanol extacts (uneduced). B) educed 50% -popanol extacts, and C) 50% -popanol % DTT extacts. The uneduced popanol extacts esolved into thee main peak clustes. Analysis of the clustes evealed that the fist peak was C- hodeins, the second (majo) one, B- hodeins and the thid, poteins of gamma-hodein mobility (Fig. 6). Upon eduction of the extact, the etention times of the fist peak and the small thid peak wee unalteed, but the second cluste esolved into thee main goups of peaks. These changes ae consistent with the confomation of C- hodein, a goup of poteins lacking inte- o inta-molecula disulphides", being unaffected by teatment with educing agents. The thee clustes of B- hodeins had diffeing compositions on SDS-PAGE. The fist cluste compised Bl- and POH POH-DTT T 2 2a 2b 2c hmw-b- Fig. 6. Analysis of chomatogam peaks fom evcsed-phasc HPLC of Schoone baley extacts. Fom left to ight: molecula weight makes, total baley gain polcin extact (T), 50% popanol (POH) extact, peak clustes, 2 and, educed popanol extact (POH-DTT), peak clustes, 2a, 2b, 2c and. B-, the second cluste mainly B- and some B2- and the thid cluste B2- and some B-hodeins. Seveal significant coelations wee found between the elative aeas of the hodein clustes and malt extact (Fig. 7, Table III). In both the uneduced and educed 50% popanol extacts, signifi cant coelations between the size of each peak and gain potein content wee found fo the Schoone set but not the 8-vaiety set of baleys. Despite this diffeence, in both sets of baleys the elative amount of the C- hodein clustes deceased makedly with inceasing malt extact values, con fiming the esult obtained with size-exclusion HPLC. Analysis of the 50% popanol-dtt extact of the esidue esulting afte extaction with 50% popanol, indicated 4 main goups of peaks, in two clustes (Fig. 5). The fist peak compised D-hodein, while the second cluste of peaks wee B-hodeins peaks of diffeing composition. Howeve, consist ent elationships between peak aea and malt extact in the two sets wee not obseved. This may elate to the finding that the popanol-dtt extactant did not completely extact the B- and D-hodeins; these could be detected in 6M uea- % DTT extacts of the esidue fom popanol-dtt extac tion. Conclusions We have ealie used size-exclusion HPLC analyses of lowmolecula weight poteins in 2-day malts5, as well as a method based on quantification of a specific goup of ho deins by tubidity fomation in cabonate buffes to athe accuately pedict final malt extact values of the malts7. Howeve, pediction of final malt quality fom analysis of unmalted baley would be expected to be somewhat moe difficult than fom analysis of malts ealy in the malting pocess. This is because malt modification depends not only on the composition of the oiginal baley gain, but on the activities of hydolytic enzymes fomed duing malting, and thei access to thei substates in the modifying gain. Despite these limitations, we have obtained coelations between the elative amounts of a numbe of gain potein factions as assessed using eithe RP- o SE-HPLC methods, and malt extact. While total potein content was negatively coelated with This document is povided compliments of the Institute of Bewing and Distilling Copyight - Jounal of the Institute of Bewing

7 Vol. 99, 99] BARLEY PROTEINS UJ 0 70 LU oo LLJ MALT EXTRACT (%) MALT EXTRACT (%) Fig. 7. Relationships between evesed-phase hplc pecentage peak aeas and malt extact fo 50% -popanol extacts of the set of Schoone baleys. A) Peak (C-hodeins), B) Peak 2 (soluble B-hodeins). TABLE III. RP-HPLC of alcohol-soluble baley gain poteins: elationship between elative chomatogam peak aeas, malt extact and potein Peak 50% popanol extacts 2 malt extact *** 0.785*** 0.446" Schoonei slope" educed popanol extacts 2a 2b 2c 50% popanol/% DTT extacts 2a 2b 2c *" 0.647*" 0.766*" 0.57*** * potein 0.757*" *** -0.52*" 0.875*" *" -0.8"* "* -0.84* " malt extact "* 0.52*" " 0.572*" -0.4* * 0.425" - 0.6* 8-vaiety set slope" potein " pecentage change in elative peak aea fo a % incease in malt extact (Schoone baleys) o a 5% incease in malt extact (8-vaiety set). = not calculated since linea egession coelation coefficient () did not achieve statistical significance. * P 0.05, " P 0.0, *** P malt extact, the magnitude of the changes in the elative amounts of cetain goups of hodeins in baleys that diffeed in malt extact was somewhat geate than the coesponding changes in total potein content. In geneal, the coelations between amounts of potein factions and malt extact wee good within diffeent samples of the cultiva, Schoone, and a little less consistent when data fom sets of diffeent cultivas was pooled. This is likely to be due to the influence of vaietal diffeences in polypeptide composition, along with genotype diffeences influencing enzyme activities and betaglucan levels. Howeve significant coelations between the elative amounts of seveal hodein factions and malt extact wee seen in the 8-vaiety set. The poo extactability of baley seed poteins in the absence of educing agents complicated seveal of the analy ses, although extaction fom a given sample was epoduc ible. Unlike wheat gain poteins4, it was not possible to completely extact all hodeins by sonication in the absence of educing agent. Theefoe, an HPLC method fo analysis of educed hodein polypeptides was also developed. The latte method was elatively low in esolution. Even though vaious B-, C- and D- hodeins factionate as distinct clustes on SDS-PAGE, and on the column used, sepaation of thee majo peaks was obtained, but they did not coespond to the size classes denned by electophoesis. Nonetheless, SE- HPLC of eithe educed o uneduced hodein could be used to pedict malt extact. The most useful pedictions fo both sets of baleys wee based on:. quantification of C-hodeins in eithe the second peak of SE-HPLC chomatogams of uneduced sonicated SDS extacts o in the fist peak of 50% popanol extacts analysed by RP-HPLC, o 2. quantification of B- and D-hodeins in the thid (majo) peak of SE-chomatogams of SDS-mecaptoethanol extacts. The C-hodeins ae consideed to play less of a diect ole in bewing poblems associated with high gain potein, but ae vey slow to modify in malting. Othe studies have shown the quantity of C-hodeins to be makedly affected by gain potein content27. Evidence was also obtained fo the exist ence of two goups of hodeins one that is eadily extactable in the absence of educing agents and one that patici pates in disulphide-bonded gel potein. Howeve, factionation of B- hodein goups by RP-HPLC did not isolate a cluste consistently associated with poo malt extact. In conclusion, while malting quality of baley is makedly affected by seveal paametes, including enzyme activity potential, cabohydate composition and potein con tent, analysis of potein composition is a useful additional tool in quality pediction studies. Acknowledgements. The authos ae gateful to Ds R. Heny (Queensland Wheat Reseach Institute, Toowoomba) and R. C. M. Lance and L.C. McLeod (Waite Institute, Adelaide) fo povision of baley samples and malt analyses. D I. L. Batey is thanked fo advice on HPLC pocedues and Ms J. Haington fo help with some peliminay expe- This document is povided compliments of the Institute of Bewing and Distilling Copyight - Jounal of the Institute of Bewing

8 84 BARLEY PROTEINS [J. Inst. Bew. iments. The financial suppot of the Gains R & D Copo ation of Austalia is acknowledged. Refeences. Allison, M. J. & Bain, H. Euphytica, 986, 5, Batcy. I. L., Gupta, R. & MacRitchie, F. Ceeal Chemisty, , Baxte, E. D. & Wainwight, T. Jounal of the Ameican Society of Bewing Chemists, 979, 7, Bishop, L. R. Jounal of the Institute of Bewing, 90, 6, Biggs, D. E. Bewing Science, Vol. (J. R. A. Pollock, cd.), London: Academic Pess. 987, pp Heny. R. J. & McLean, B.T. Jounalofthe Institute of Bewing, , Machylo. B. A. & Kuge, J. E. Ceeal Chemisty, 984, 6, Machylo, B. A., Kugc, J. E. & Hatche, D. Ceeal Chemisty, 986, 6, Paz-Acs, J., Henandez-Lucas, C, Salcedo, G., Aagoncillo, C, Ponz, F. & Gacia-Olmedo, F. Jounal of Expeimental Botany, 98, 4, Pcece, I. A. Jounal of the Institute of Bewing, 96, 69, 47.. Schmitt, N.. Gille. P.. Gauche. J. & Montcmbault. A. Euo pean Bewey Convention Poceedings of the 22nd Congess, Zuich, 989, 22, Shcwy, P. R., Faulks, A. J., Pama, S. & Miflin, B. J. Jounal of the Institute of Bewing, 980, 86, 8.. Shewy, P. R., Field, J. M., Kikman, M. A.. Faulks, A. J. & Miflin, B. J., Jounal of Expeimental Botany, 980,, Singh, N. K., Donovan, G. R., Batey, I. L. & MacRitchie. R. Ceeal Chemisty, 990, 67, Skeitt, J. H. & Collings, D. Poceedings of the Institute of Bewing Convention (Austalia and New Zealand Section), 988, 20, Skeitt. J. H. & Heny, R. J. Jounal of Ceeal Science, 988, 7, Skeitt. J. H., Hayes, G. & Heny, R. J. Jounal of The Institute of Bewing, 987, 9, Smith, D. B. Plant Vaieties and Seeds, 990,, Smith, D. B. & Liste, P. R. Jounal of Ceeal Science, 98,, Smith, D. B. & Simpson, P. A. Jounal of Ceeal Science, 98. I, Smith. D. B.. Liste, P. R. & Hanson, P. R. Jounal of Ceeal Science, 986, 4, Smith. P. K., Cohn, R. I.. Hemanson, G. T.. Mallia, A. K.. Gatne. F. H., Povenzano, M. D., Fujimoto, E. K.. Goeke, N. M., Olson, B. J. & Klenk, D. C. Analytical Biochemisty, 985, 50, Spaow, D. B., Lance, R. C. M. & Cleay, J. G. Poceedings of the V Baley Genetics Symposium, edited by Yasuda, S. and Konishi, T. 987, Sanyo Pess. Okayama, pp Spence. D., Higgins, T. J. V.. Button, S. C. & Davey. R. A. Plant Physiology, 980, 66, Tasheva, B. & Dessev, G. Analytical Biochemisty, Wingad, C. E., Iqbal, M., Giffin, M. & Smith, F. J. Chomatogaphia, 986, 2, Wynne Giffiths, D. Jounal of the Science of Food and Agicul tue, 987, 8, 229. This document is povided compliments of the Institute of Bewing and Distilling Copyight - Jounal of the Institute of Bewing

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